Bacteriophages in Health and Disease

Bacteriophages in Health and Disease
Bacteriophage T4 seen obliquely from the base plate end

Picture created by Steven McQuinn using protein structure data from Rossmann Lab,
furnished by the RCSB Protein Databank. The various software utilized includes UCSF
Chimera, Accelrys DS Visualizer, MeshLab and DAZ Carrara.
Bacteriophages in Health and Disease
Edited by
Paul Hyman
Department of Biology/Toxicology, Ashland University
and
Stephen T. Abedon
Department of Microbiology, The Ohio State University
Advances in Molecular and Cellular Microbiology 24
Advances in Molecular and
Cellular Microbiology
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Cover image. The electron micrograph on the cover is of Salmonella
phage bound to cell wall residues forming a phage bouquet. The phage
were negatively stained with uranyl acetate. This image was prepared by
Jochen Klumpp at the Institute of Food, Nutrition and Health, Zurich,
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A catalogue record for this book is available from the British Library,
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Library of Congress Cataloging-in-Publication Data
Bacteriophages in health and disease / [edited by] Paul Hyman,
Stephen T. Abedon.
p. ; cm. -- (Advances in molecular and cellular microbiology ; 24)
Includes bibliographical references and index.
ISBN 978-1-84593-984-7 (alk. paper)
I. Hyman, Paul (Paul Lawrence) II. Abedon, Stephen T. III. C.A.B.
International.
IV. Series: Advances in molecular and cellular microbiology ; 24.
[DNLM: 1. Bacteriophages. 2. Biological Therapy--methods. QW 161]

579.26--dc23
2012007325
ISBN-13: 978 1 84593 984 7
Commissioning editor: Rachel Cuts
Editorial assistant: Alexandra Lainsbury
Production editor: Simon Hill
Typeset by Columns Design XML, Reading.
Printed and bound in the UK by MPG Books Ltd.

vii
Contents
Contributors ix
Foreword xiii
Preface xv
1. Phages 1
Stephen T. Abedon*
Part I: Phages, Bacterial Disease and Normal Flora
2. Bacteriophages as a Part of the Human Microbiome 6
Andrey V. Letarov*
3. Diseases Caused by Phages 21
Sarah Kuhl*, Stephen T. Abedon and Paul Hyman
4. Prophage-induced Changes in Cellular Cytochemistry and Virulence 33
Gail E. Christie, Heather E. Allison, John Kuzio, W. Michael McShan, Mathew K.
Waldor and Andrew M. Kropinski*
5. The Lion and the Mouse: How Bacteriophages Create, Liberate and
Decimate Bacterial Pathogens 61
Heather Hendrickson*
6. Phages and Bacterial Epidemiology 76
Michele L. Williams and Jerey T. LeJeune*
Part II: Phage-based Biomedical Technology
7. Phages as Therapeutic Delivery Vehicles 86
Jason Clark, Stephen T. Abedon and Paul Hyman*
8. Clinical Applications of Phage Display 101
Don L. Siegel*
viii Contents
9. Phages and Their Hosts: a Web of Interactions Applications to Drug Design 119
Jeroen Wagemans and Rob Lavigne*
10. Bacteriophage-based Methods of Bacterial Detection and Identication 134
Christopher R. Cox*
11. Phage Detection as an Indication of Faecal Contamination 153
Lawrence D. Goodridge* and Travis Steiner
Part III: Phage-based Antibacterial Strategies
12. Phage Translocation, Safety and Immunomodulation 168
Natasza Olszowska-Zaremba*, Jan Borysowski, Krystyna Dbrowska and Andrzej Grski
13. Phage Therapy of Wounds and Related Purulent Infections 185
Catherine Loc-Carrillo*, Sia Wu and James Peter Beck
14. Phage Therapy of Non-wound Infections 203
Ben Burrowes and David R. Harper*
15. Phage-based Enzybiotics 217
Yang Shen, Michael S. Mitchell, David M. Donovan and Daniel C. Nelson*
16. Role of Phages in the Control of Bacterial Pathogens in Food 240
Yan D. Niu, Kim Stanford, Tim A. McAllister and Todd R. Callaway*
17. Phage-therapy Best Practices 256
Stephen T. Abedon*
Index 273
* Corresponding author

ix
Contributors
Stephen T. Abedon, Ph.D., Department of Microbiology, The Ohio State University, 1680
University Drive, Manseld, OH 44906, USA; [email protected]
Heather E. Allison, Ph.D., Department of Functional and Comparative Genomics, Institute of
Integrative Biology, University of Liverpool, Liverpool L69 7ZB, UK.
James Peter Beck, M.D., Department of Orthopaedics, The University of Utah, 590 Wakara
Way, Salt Lake City, UT 84108, USA; George E. Wahlen Department of Veterans Aairs
Medical Center, VA Salt Lake City Health Care System, Salt Lake City, UT 84148, USA.
Jan Borysowski, M.D., Department of Clinical Immunology, Institute of Transplantology,
Medical University of Warsaw, ul. Nowogrodzka 59, 02-006 Warsaw, Poland.
Ben Burrowes, Ph.D., Ampliphi Biosciences Corporation, Colworth Science Park, Sharnbrook,
Bedfordshire, MK44 1LQ, UK.
Todd R. Callaway, Ph.D., Food and Feed Safety Research Unit Agricultural Research Service/
USDA, TX, USA; [email protected]
Gail E. Christie, Ph.D., Molecular Biology and Genetics, School of Medicine, Virginia
Commonwealth University, Richmond, VA 23298-0678, USA.
Jason Clark, Ph.D., Novolytics Ltd, ITAC-BIO Daresbury Laboratory, Daresbury Science and
Innovation Campus, Warrington, WA4 4AD, UK; BigDNA Ltd, Wallace Building, Roslin
BioCentre, Roslin, Midlothian EH25 9PP, UK.
Christopher R. Cox, Ph.D., Colorado School of Mines, Department of Chemistry and
Geochemistry, Golden, CO 80401, USA; [email protected]
Krystyna Dbrowska, Ph.D., Phage Laboratory and Therapy Unit, Institute of Immunology
and Experimental Therapy, Polish Academy of Sciences, ul. Rudolfa Weigla 12, 53-114
Wroclaw, Poland.
David M. Donovan, Ph.D., Animal Biosciences and Biotechnology Lab, ANRI, ARS, USDA,
Bldg 230, Room 104, BARC-East 10300 Baltimore Ave, Beltsville, MD 20705, USA.
Lawrence D. Goodridge, Ph.D., Center for Meat Safety and Quality, Department of Animal
Sciences, Colorado State University, Fort Collins, CO 80523, USA; Lawrence.Goodridge@
ColoState.edu
Andrzej Grski, M.D., Ph.D., Phage Laboratory and Therapy Unit, Institute of Immunology
and Experimental Therapy, Polish Academy of Sciences, ul. Rudolfa Weigla 12, 53-114
Wroclaw, Poland; Department of Clinical Immunology, Institute of Transplantology,
Medical University of Warsaw, ul. Nowogrodzka 59, 02-006 Warsaw, Poland.
x Contributors
David R. Harper, Ph.D., Ampliphi Biosciences Corporation, Colworth Science Park,
Sharnbrook, Bedfordshire, MK44 1LQ, UK; [email protected]
Heather Hendrickson, Ph.D., New Zealand Institute for Advanced Study, Massey University,
Private Bag 102 904, North Shore Mail Centre, Auckland, New Zealand; former address:
Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Rd,
Oxford, OX1 3QU, UK; [email protected]
Paul Hyman, Ph.D., Department of Biology/Toxicology, Ashland University, 401 College
Avenue, Ashland, OH 44805, USA; [email protected]
Andrew M. Kropinski, Ph.D., Department of Molecular and Cellular Biology, University of
Guelph, Guelph, ON N1G 2W1, Canada; Public Health Agency of Canada, Laboratory for
Foodborne Zoonoses, Guelph, ON N1G 3W4, Canada; [email protected]
Sarah Kuhl, M.D., Ph.D., VA Northern California Health Care System, Martinez, CA 94553,
USA; Contra Costa Regional Medical Center, Martinez, CA 94553, USA; sarah.kuhl@va.
gov
John Kuzio, Ph.D., Department of Microbiology and Immunology, Queens University,
Kingston, ON K7L 3N6, Canada.
Rob Lavigne, Ph.D., Division of Gene Technology, Department of Biosystems, Katholieke
Universiteit Leuven, Kasteelpark Arenberg 21 Box 2462, B-3001 Leuven, Belgium; rob.
[email protected]
Jerey T. LeJeune, D.V.M., Ph.D., Food Animal Health Research Program, The Ohio
Agricultural Research and Development Center, Wooster, OH 44691, USA; lejeune.3@osu.
edu
Andrey V. Letarov, Ph.D., Winogradsky Institute of Microbiology, Russian Academy of
Sciences, Moscow, Russia; [email protected]
Catherine Loc-Carrillo, Ph.D., Department of Orthopaedics, The University of Utah, 590
Wakara Way, Salt Lake City, UT 84108, USA; George E. Wahlen Department of Veterans
Aairs Medical Center, VA Salt Lake City Health Care System, Salt Lake City, UT 84148,
USA; [email protected]
Tim A. McAllister, Ph.D., Agriculture and Agri-Food Canada, Lethbridge, AB, Canada.
W. Michael McShan, Ph.D., Department of Pharmaceutical Sciences, College of Pharmacy, The
University of Oklahoma, Oklahoma City, OK 73126-0901, USA.
Carl R. Merril, M.D., 6840 Capri Place, Bethesda, MD 20817, USA.
Michael S. Mitchell, Ph.D., Institute for Bioscience and Biotechnology Research, University of
Maryland, Rockville, MD 20850, USA.
Daniel C. Nelson, Ph.D., Institute for Bioscience and Biotechnology Research, University of
Maryland, Rockville, MD 20850, USA; Department of Veterinary Medicine, University of
Maryland, and Virginia-Maryland Regional College of Veterinary Medicine, College Park,
MD 20742, USA; [email protected]
Yan D. Niu, Ph.D., Agriculture and Agri-Food Canada, Lethbridge, AB, Canada.
Natasza Olszowska-Zaremba, M.S., Department of Clinical Immunology, Institute of
Transplantology, Medical University of Warsaw, ul. Nowogrodzka 59, 02-006 Warsaw,
Poland; [email protected]
Yang Shen, Ph.D., Institute for Bioscience and Biotechnology Research, University of Maryland,
Rockville, MD 20850, USA.
Don L. Siegel, Ph.D., M.D., Division of Transfusion Medicine, Department of Pathology and
Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, 510
Stellar-Chance Laboratories, 422 Curie Blvd, Philadelphia, PA 19104, USA; siegeld@mail.
med.upenn.edu
Kim Stanford, Ph.D., Alberta Agriculture and Rural Development, Lethbridge, AB, Canada.
Travis Steiner, B.S., Center for Meat Safety and Quality, Department of Animal Sciences,
Colorado State University, Fort Collins CO 80523, USA.
Contributors xi
Jeroen Wagemans, M.S., Division of Gene Technology, Department of Biosystems, Katholieke
Universiteit Leuven, Kasteelpark Arenberg 21 box 2462, B-3001 Leuven, Belgium.
Mathew K. Waldor, Ph.D., Department of Medicine, Brigham and Womens Hospital, Boston,
MA 02115, USA; and, Channing Laboratory and HHMI, Harvard University, Boston, MA
02115, USA.
Michele L. Williams, D.V.M., Ph.D., Food Animal Health Research Program, The Ohio
Agricultural Research and Development Center, Wooster, OH 44691, USA.
Sia Wu, Department of Orthopaedics, The University of Utah, 590 Wakara Way, Salt Lake
City, UT 84108. USA; George E. Wahlen Department of Veterans Aairs Medical Center,
VA Salt Lake City Health Care System, Salt Lake City, UT 84148, USA.
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xiii
Foreword
It has been personally gratifying to examine the sections and chapters that make up this book,
Bacteriophages in Health and Disease. The book represents real progress since the now
almost half a century when I was rst introduced to the bacteriophages at a Cold Spring
Harbor Phage Course in 1965. At that time, the course concentrated on just a few phage strains,
as these viruses and their bacterial hosts were seen as a potential gateway for the understanding
of basic life processes. As I had recently graduated from medical school, I raised the question
as to why these bacterial viruses were not employed in antibacterial therapy. One of the course
mentors answered that question by suggesting that I read Sinclair Lewis, 1925, book
Arrowsmith. On reading the book, it soon became apparent that the ctionalized clinical trials
described in the book failed not because of deciencies in the antibacterial phage strains but
because the hero/antihero chose to break the double-blind code, so that he could treat all the
inhabitants on the ctional island who were in the midst of a major plague epidemic. In other
words, the actions of the ctionalized character, Arrowsmith, destroyed the capacity of the
clinical trial to determine the e cacy of phage therapy, so the book really could not address
whether the phage were useful as an antibacterial therapy or not.
At the time of the 1965 course, mouth pipeting represented the state of the art of laboratory
technology, so I asked whether the bacteriophage could have an adverse eect on the students
in the course. The reply, by one of the instructors, was that they could not because: they are
bacteriophage, which means bacteria eaters. I found this reply unsatisfactory, as it occurred to
me that the viruses might not be restricted in their actions by a name (bacteriophage) that we
had given them. Following the phage course, I returned to my laboratory at the NIH, where,
with the help of colleagues, I was able to screen for eects of phage strains on mammalian cells
in culture. We found no clear-cut gross eects with the phage strains we tested, such as cell
killing or alterations in cellular growth paterns. When transducing phages carrying the
bacterial galactose genes were used with human cells defective in certain galactose genes,
however, we found evidence for restoration of the defective galactose pathway. In addition, in
control experiments we discovered that most of our stocks of fetal calf serum used as an
ingredient in mammalian cell cultures were contaminated by bacteriophage. At rst we
thought that this contamination was limited to our lab, but we soon found similar contamination
in all of the fetal calf serum samples that we were able to sample from our colleagues at the
NIH.
As we realized that many human vaccines are produced by mammalian cell cultures grown
in fetal calf serum, we tested some vaccines that we purchased at a local drugstore and found
that they were also contaminated by phages. The FDA conrmed our results. To permit the
continued use of the vaccines they obtained a Presidential Executive Order permiting the sale
xiv Foreword
and use of bacteriophage-contaminated vaccines, since bacteriophage were known to be
specic for bacteria. In a subsequent presentation, at the NIH, I noted that phage are not
entirely innocuous, such as in the case of diphtheria, a disease which is caused by a toxin gene
that is carried into and expressed in the infecting bacteria by a phage. Lest we forget, this toxin
has enzymatic activity that results in the inactivation of a critical mammalian protein synthesis
factor and which has no known eects in bacteria.
We also initiated experiments to study phages in germ-free animals, to simplify the system
and to look for interactions between phage and animals. It was in these experiments that we
recognized the immediate ability of animals to respond to phages even before antibody
production could be initiated. Most phages are destroyed in the liver while a small number
remained intact in the spleen (they could still infect bacteria when isolated from the spleens of
these animals). It was these experiments that gave us ideas that we used in subsequent
experiments to select phage strains that could remain in the circulation to optimize their
capacity to serve as antibacterial therapeutic agents.
While our carefully controlled studies using phages to treat animals with experimental
systemic infections were very encouraging they did not generate support from our
administrators, who ofen cited the book, Arrowsmith, which they clearly hadnt read, as
their evidence against phage therapy. In addition, an argument that was raised repeatedly was
that anyone who worked with phages in a lab should know that phage therapy would never
be useful because the development of phage-resistant bacterial strains is inevitable. I guess
they didnt know that this is true for most antibacterial agents including most antibiotics, but
a number of lives can be saved in the meantime. Besides, with our capacity to isolate new
phage strains and engineer existing ones it should be possible to overcome most phage-
resistant bacteria. Of course, we should always be aware of possible phage-mediated
detrimental eects such as the ability of phage to carry toxin genes, as in the case of diphtheria.
In addition, as noted in the last chapter in this book, on Phage-therapy Best Practices (see
Abedon, Chapter 17, this volume), careful considerations also need to be used in developing
phage protocols and formulations.
For the above reasons and experiences I am personally pleased to see that despite the
naysayers, eorts to use phages as therapeutic agents, both as antibacterial agents and as
therapeutic delivery vehicles (see Clark et al., Chapter 7, this volume), have substantially
progressed. Studies of phages in the human microbiome may facilitate the development of
new therapeutic phage strains and therapeutic strategies. Most importantly, careful clinical
trials are being designed and conducted as described in several chapters to help us avoid
falling into the trap that undid Arrowsmith. In time, truth will overcome the shadow of that
ction.
Carl R. Merril

xv
Preface
If all things have to have a beginning, then the beginning of this monograph, arguably, can be
traced to the 1980s in the laboratory of Harris Bernstein (Department of Microbiology and
Immunology, University of Arizona). That was the lab that both of us joined to do our Ph.D.
research and where we met. Though we worked on separate projects, both involved phages
and this led to many discussions. Thus the team began. Afer graduating we went our separate
ways. While S.T.A. dabbled in what eventually would be all things phage ecological, P.H.
followed a much more molecular, then medical, then applied route, but ultimately returned to
phage biology and what would be a series of collaboration with S.T.A., starting in 2001. The
story of how this monograph came to be is slightly more complicated, however.
S.T.A. at the time at the beginning of his career at the Ohio State University, in 1995
founded what would become the Bacteriophage Ecology Group. His management of the
associated web site (phage.org, though see also archaealviruses.org) began to lead to chapter
invitations in the early 2000s, resulting in part in his contributing to the editing of Rich
Calendars The Bacteriophages 2/e, which was published in 2006. Very soon afer S.T.A.s
contribution to that monograph ended he was invited to edit a monograph on phage ecology,
which would become the 2008 Bacteriophage Ecology (Cambridge University Press), included in
which was a chapter we coauthored.
Meanwhile, speculators brought down the worlds economy, hiting academic publishers
quite hard. The series that Bacteriophage Ecology had been a part of, Advances in Molecular and
Cellular Microbiology, was sold to CABI Press (cabi.org), a not-for-prot international organization
that improves peoples lives by providing information and applying scientic expertise to solve problems
in agriculture and the environment. It was 100% their idea to do the current monograph and,
based on his experience with the Bacteriophage Ecology volume, S.T.A. was recruited to edit it.
S.T.A., though, was busy at the time, pulling together an edited volume on phage therapy (see
Current Pharmaceutical Biotechnology, volume 11, issue 1), so recruited P.H. to join in on yet
another collaboration. We developed a formal proposal including recruiting the authors of the
many chapters found herein and the result is this monograph.
Bacteriophages in Health and Disease, an early working title that stuck, is an eort to provide
an introduction to the breadth of roles that phages play or can play in our everyday lives. They
can serve as causes of disease, treatments of disease, indicators of the potential for disease,
preventers of disease, and even contribute in various ways to the evolution of bacterial
pathogens. To capture this variety of phage roles in human conditions, both natural and
applied, we have divided the text into three parts. Weve also provided a brief introduction to
various concepts and terminology associated with phages (chapter 1); for a glossary covering
these basics of phage biology, and much more, see phage.org/terms.
xvi Preface
Part I considers the role of phages in the natural state. That is, where phages are, how they
contribute directly to disease, the underlying mechanism by which phages do this, and then,
especially, how they can contribute indirectly to disease, that is, to pathogen evolution. The
three basic themes are ones of phage presence, phage genes, and phage-mediated transduction
of bacterial DNA between bacteria, though a common thread is that they touch upon, in
various ways, issues of lysogeny, which is the long-term incorporation of phages as prophages
into the genomes of their bacterial hosts. These issues are covered by chapters 2 through 6.
Part II, chapters 7 through 11, considers various phage-based technologies other than the
use of whole phages to combat bacterial infections (i.e., besides phage therapy). This includes
in particular the use of both modied and disembodied phage parts. Phages thus can serve as
carriers and delivery vehicles of especially DNA but also of other chemicals, including serving
as vectors for either gene therapy or DNA vaccines. The potential of phages to serve in these
functions stems greatly from their relative safety as well as their genetic malleability, such as
one sees for example in phage display technologies. Phage antibacterial properties too can be
used as a means of discovering novel targets for action by traditional small-molecule
antibacterial chemotherapeutics. In addition, phages can be employed in bacterial detection
and identication including as indicators of faecal contamination.
Phages typically in a relatively unaltered state can be employed directly as antibacterials,
that is, in phage therapy, a theme covered by Part III and chapters 12 through 17. Here we
provide a chapter introducing various phage properties as medicinals, including the relative
safety associated with phage application to bodies. Phage therapy of humans is then considered
in two chapters, which we have divided up roughly into treatment of wounds and non-wound
infections. Certain phage parts can be used, on their own, as antibacterial drugs, most notably
phage lysins which are covered in a subsequent chapter. The potential phage role in food
safety is considered, and we then end the monograph with a chapter targeted to would-be
phage therapy experimentalists, one that considers, in light of phage properties, especially
how phage therapy protocols may be developed in terms of the use of animal models of
bacterial disease.
If your primary interest is in learning about the myriad roles that phages can and do play in
human health and disease, read all of the chapters perhaps up to this last one. If you already
know all there is to know about phages such that much of this book is superuous, you may
still want to at least read this last chapter! In any case, we see this monograph as appealing to
the needs of two not-so-disparate groups, those with a primary interest in phage biology,
though from an especially applied perspective, and those who are curious as to how phage
biology can impact the practice of medicine. In particular, we provide a snap shot of the current
state of the eld of phage biology, applied as well as basic, concentrating on the roles that
phages can play in both health and disease.
We thank our editors at CAB International, Rachel Cuts and Alex Lainsbury, for their
support and help bringing this volume to fruition and dedicate this volume to our common
but nonetheless not-so-common mentor, Harris Bernstein.
CAB International 2012. Bacteriophages in Health and Disease
(eds P. Hyman and S.T. Abedon) 1
1 Phages
Stephen T. Abedon
1
1
Department of Microbiology, The Ohio State University
In a phrase such as Bacteriophages in Health
and Disease, the least familiar term to anyone
will be that of bacteriophages. It is the
purpose of this introductory chapter to make
sure that this term is less unfamiliar even to
those working in the eld so that greater
emphasis may be made, in this monograph,
on issues of health and disease. In this
chapter, I thus will provide a brief and also
not terribly molecular overview of the viruses
of bacteria. Greater detail as applicable to
particular topics will be found in appropriate
chapters. For additional discussion of con-
cepts, see www.phage.org/terms/, as well as
the resources list presented by www.ISVM.
org. For general references, see www.phage.
org/terms/phages.html and www.phage.org/
terms/phage_history.html.
Bacteriophages, or phages for short, are
viruses that infect members of what is known
as the domain Bacteria, which are the
common prokaryotic organisms associated
with, for example, the human body. To help
orient readers towards understanding the
role that phages can play as part of the human
microbiome (see Letarov, Chapter 2, this
volume), in health as well as disease (see
Kuhl et al., Chapter 3, this volume), in this
chapter I provide an overview of basic phage
properties. These include, in particular, what
phage virions look like, the basic character-
istics of phage infections and, because it can
play such a large role in bacterial evolution,
the potential for phages to move DNA
between bacteria, a process known as
transduction. I begin, however, with a brief
glimpse at the history of phage research.
History
Although a number of publications in the late
19th and early 20th century may hint at the
observation of phage-like phenomena, the
discovery of phages is unambiguously traced
to the work of Twort and, independently, that
of dHrelle (see the rst volume of the
journal, Bacteriophage, 2011, particularly issues
1 and 3, for discussion and references). Early
phage work by necessity addressed issues of
basic phage biology, although these eorts
were based on both primitive techniques and
minimal understanding of just what the
phage phenomenon actually entailed. The
promise of phage therapy, with the use of
phages to combat bacterial infections in
particular, was an important driver of early
phage research (see Burrowes and Harper,
Chapter 14, this volume, for additional
historical consideration of this issue).
S.T. Abedon
2 S.T. Abedon
It was in the late 1930s and early 1940s
that phage research began to make what
today we view as its most signicant impact
on basic biological research. These eorts are
associated in large part with the physicist-by-
training Max Delbrck and associated
members of what became known as the
Phage Group. It was within this context that
the eld of molecular genetics and the related
discipline of molecular biology were forged.
James Watson, for example, was a member of
the Phage Group, and Francis Crick too
became an important contributor to phage
research following their co-discovery of the
structure of DNA.
By contributing to the development of
techniques that ultimately would greatly
simplify the study of the biology of organisms
far more complicated than either phages or
their bacterial hosts, in a sense phage research
set the stage for its own eclipse, perhaps
particularly in terms of funding opportunities.
Changes in this situation involved an
increased focus on areas other than purely
the molecular aspects of phages: consideration
of aquatic phage ecology, growing appre-
ciation of the role of phages in horizontal
gene transfer and increasing emphasis on the
application of phage-based biotechnologies.
The later includes in particular techniques
known as phage display (see Siegel, Chapter
8, this volume), the use of phages in bacterial
detection as well as identication (see
Williams and LeJeune, Cox, and Goodridge
and Steiner, Chapters 6, 10 and 11, this
volume) and, of course, phage therapy (see
Olszowska-Zaremba et al., Loc-Carrillo et al.,
Burrowes and Harper, Shen et al., Niu et al.
and Abedon, Chapters 1217, this volume).
All of these issues are considered in this
monograph, particularly in the last two parts.
The association of phages with bacterial
disease is a primary emphasis of Part I (see
Kuhl et al., Christie et al. and Hendrickson,
Chapters 35, this volume).
Types of phages
Virus particles consist essentially of two
components, what is on the inside and what
is on the outside. What is on the inside
primarily is nucleic acid and what is on the
outside is responsible for transporting that
nucleic acid between cells. The nucleic acid
component varies substantially between
phage types, both in terms of size and
structure, and can consist of either RNA
or DNA, but not both, with DNA being
more common. It can be single-stranded or
double-stranded, although for most phages
it is double-stranded. It also can be multi-
segmented, particularly tripartite (e.g.
Pseudo monas phage |6), although the vast
majority of phage genomes are monopartite.
The outside portion of virions consists of
a combination of the capsid, which surrounds
the nucleic acid, and various appendages,
most of which are involved in virion
adsorption to bacteria. While some phages
have capsids that contain lipids (again such
as phage |6), for most phages capsids consist
solely of multiple units of proteins known as
capsomeres. For simple phage virions, cap-
sids are either helical/lamentous or ico-
sahedral, which in both cases surround
single-stranded nucleic acids (usually DNA,
but not always). These phages fall into the
phage families Leviviridae, Microviridae and
Inoviridae. More specically, these are single-
stranded RNA icosahedral, single-stranded
DNA icosahedral and ssDNA lamentous
phages (e.g. phages MS2, |X174 and M13,
respectively). A very small number of phages
have dsRNA genomes (such as |6).
The vast majority of phages by contrast
possess complex rather than simple virion
morphologies; dsDNA, monopartite genomes;
and protein capsids lacking lipids. The
dening feature of these phages all members
of phage order Caudovirales are their tails,
however. Tails are multi-protein appendages
involved in virion adsorption (see the cover
and prelims for illustration). Members of the
phage family Podoviridae, such as coliphage
T7, possess short, non-contractile tails. The
tails of members of the family Siphoviridae,
including coliphage , can be quite long and
are also non-contractile. Finally, the tails of
members of the family Myoviridae, coliphage
T4 being the most familiar, are approximately
intermediate in length while possessing an
ability to contract in the course of virion
adsorption to bacteria. All tailed phages are
Phages 3
lytic and some are also temperate. Discussion
of basic phage morphologies in greater detail
can be found in a number of reviews that
have been published by Hans-Wolfgang
Ackermann.
Virus-like particles
The concept of virus-like particle, or VLP,
has at least two distinct meanings as used in
this monograph. First are entities that appear
to be virus-like as viewed in an electron
micrograph (see Letarov, Chapter 2, this
volume). Secondly, the term is used to
describe phages that lack genomes and which
therefore are virus-like rather than actually
viruses (see Clark et al., Chapter 7, this
volume). The term ghost has also been
employed in the phage literature to describe
these later entities, including those generated
from particles that initially possess genomes.
Types of infection
A phage infection begins with virion
atachment to a potential host cell, the
culmination of a multi-step process known as
adsorption. It then proceeds through the
translocation of phage nucleic acid into the
cell variously described as both ejection and
injection, as well as uptake but begins in
earnest only once the phage genome has
made its way into the bacterial cytoplasm.
The results of phage adsorption can vary
depending on the characteristics of the phage,
the bacterium and circumstances. At its most
basic level, the phage may either live or die
(that is, produce or not produce replicative
products afer infecting a host cell), and the
same is true for the infected bacterium.
Furthermore, and depending on infection
type, all four combinations of living versus
dying are possible. See Cox (Chapter 10, this
volume) for additional discussion of phage
infections.
In an abortive infection, both the infecting
phage and infected bacterium die. In a lytic
infection, the infecting phage lives, producing
phage virions, while the infected bacterium
both dies and is lysed. In various other
circumstances, the infecting phage can die but
not the infected bacterium, which is seen
particularly when bacteria carry re striction
endonucleases, although also when they carry
the CRISPR/cas systems. Lastly, under certain
circumstances both phage and bacterium can
live. While one means by which this co-
survival can occur is seen with the chronic
infections of members of the phage family
Inoviridae, most commonly simultaneous
survival of both infecting phage and infected
bacterium is seen with phage exhibition of
lysogeny (below). Productive infections, those
quickly leading to the release of infectious
viral progeny, normally occur afer infection
by both lytic and chronic phages. Overall, the
majority of phages are lytic phages.
Once the virion genome of a lytic virus
gets into the bacterial cytoplasm, the phage
genes are expressed. This gene expression
has the eect of taking over the bacterial
metabolism so that phage virion particles are
produced, the proteins of which are encoded
by additional phage genes (see Wagemans
and Lavigne, Chapter 9, this volume, for
greater consideration of what goes on, at a
molecular level, within a phage-infected
bacterium). The products of yet more phage
genes contribute, at the end of what is known
as the phage latent period, to the destruction
of the bacterial cell envelope so that phage
progeny can leak into the extracellular
environment. For tailed phages, this lysis
process involves coordinated action by at
least two phage gene products, the holin and
endolysin or lysin proteins. In particular, the
holin protein is responsible for controlling
the timing of host lysis, stimulating the action
of the lysin, and, as a by-product, shuting
down infection and therefore host meta-
bolism. Lysin is the enzyme that is responsible
for degradation of the bacterial cell wall. See
Shen et al. (Chapter 15, this volume) for
additional discussion of phage lysins.
Lysogeny
The other major state that involves both
successful phage infection and bacterial
survival is known as the lysogenic cycle, a
phenomenon, as noted in the preface, that
4 S.T. Abedon
serves to unite the chapters found in Part I of
this volume. All phages that can display
lysogeny are described as temperate, and the
majority of temperate phages are tailed
(although a small number are instead
lamentous, i.e. members of the phage family
Inoviridae; see Christie et al., Chapter 4, this
volume, for discussion of a prominent
member of the later, Vibrio cholerae phage
CTX|). Lysogenic cycles are char acterized by
two features. First, the phage genome, now
called a prophage, is replicated su ciently
rapidly within infected bacteria that daughter
bacteria, following binary ssion, each inherit
at least one prophage copy. Secondly, the
infections are not pro ductive, that is, no
virions are produced.
The prophage can exist either integrated
into the bacterial chromosome or as a
plasmid. Integration is typically accomplished
by the action of a phage protein termed an
integrase. Integrases generally bind to a
specic site on the bacterial genome and a
corresponding, partially homologous site in
the phage genome. The result is site-specic
recombination to integrate the phage genome
into the bacterial genome.
A bacterium that can undergo a lysogenic
cycle is described as a lysogen, the process of
lysogen formation is called lysogenization
and the conversion of a lysogenic infection
into a productive one (typically a lytic one) is
called induction. In some lysogens, the
prophage only expresses genes whose pro-
teins are needed to prevent induction or,
instead, trigger induction upon receiving an
appropriate signal. Expression of the re-
pressor proteins in particular prevents such
induction but also has the eect of blocking
the infection of lysogens by similar (known as
homoimmune) phages, a process known as
superinfection immunity. Other prophages
also express genes that can alter the phenotype
of the lysogenic bacterium, a process called
lysogenic conversion (see Kuhl et al. and
Christie et al., Chapters 3 and 4, this volume).
Common terms
To avoid ambiguity when considering
dierent types of phage infection, I provide
explicit denitions of the following terms:
- Temperate description of a phage that is
capable of displaying a lysogenic cycle; all
temperate phages and indeed all phages
also display productive cycles at some
point in their life cycles. Note that
lysogenic phage is not a synonym for
temperate phage but instead is a
misnomer (bacteria can be lysogenic,
while phages are temperate).
- Lytic description of a phage that lyses its
host in the course of productive infection;
note that most temperate as well as most
non-temperate phages are lytic phages.
- Chronic description of a phage that does
not lyse its host in the course of produc-
tive infection; these phages are released by
crossing relatively intact bacterial cell
envelopes.
- Obligately lytic description of a func-
tional phage that is not capable of display-
ing either lysogenic or chronic infections.
- Professionally lytic description of a phage
that is both obligately lytic and not recently
descended from temperate phages.
- Virulent a common synonym of obli-
gately lytic, although it can also describe
the potential for a phage isolate to bring a
population of target bacteria under
control, particularly through infection that
is followed by bacterial lysis and associ-
ated phage population growth.
- Phage titre (or just titre) a measure of the
number of phages per millilitre in a liquid
stock, and typically a measure of viable
phages as determined via plaque counts
rather than of virion particles as deter-
mined by various forms of microscopy.
- Plaque a visible clearing on a bacterial
lawn, growing in or on agar found in a
Petri dish, which is the result of localized
inhibition of bacterial growth such as can
be mediated by phages.
To this list one might add additional
terms such as PFU standing for plaque-
forming unit (just as CFU stands for colony-
forming unit in bacteria). Note that the
lamentous coliphage M13 is a prominent
chronically infecting phage. See Clark et al.
and Siegel (Chapters 7 and 8, this volume) for
discussion of the utility especially of this
phage to biotechnology and also Goodridge
Phages 5
and Steiner (Chapter 11, this volume)
for further discussion of chronically in fecting
phages (i.e. members of the family Inoviridae).
Transduction
Although covered in greater detail by Kuhl et
al., Christie et al. and Hendrickson (Chapters
35, this volume), I provide here a brief
introduction to the idea of phage-mediated
movement of bacterial DNA. Any time that
phages are identied within an environment,
this means that a potential exists for phage-
mediated horizontal gene transfer of bacterial
DNA between bacteria. Generally, we can
dierentiate phage-mediated horizontal gene
transfer into four categories: generalized
transduction, specialized transduction, phage
morons and a category of gene movement
that technically is not transduction at all but
instead is the movement of phage genes by
temperate phages. I will discuss these briey.
Generalized transduction is the move-
ment of bacterial DNA that has been packed
into phage capsids without accompanying
phage DNA. Generalized transduction has
the property of being able to transfer large
segments of DNA, that is, many tens of
thousands of base pairs, such as those
associated with bacterial pathogenicity
islands (see Hendrickson, Chapter 5, this
volume). Specialized transduction, by con-
trast and as narrowly dened, is the in-
corporation of bacterial genes that are found
adjacent to prophage integration sites into the
bacterial chromosome. Typically, only
relatively few genes are transferred. As phage
genomes tend to be limited in their size by
constraints on their packaging into phage
capsids (heads), this transduction of even
relatively few bacterial genes can result in
impairment of phage functioning. This type
of specialized transduction is by denition
limited to temperate phages that integrate
their genomes into the host chromosome in
the course of infection.
Specialized transduction, as more
broadly dened, involves simply the inte-
gration of bacterial genes into phage genomes.
An aspect of this form of transduction has
become associated with the term moron,
meaning more DNA. Morons are typically
considered to be bacterial genes that have
become incorporated into phage genomes via
processes of illegitimate recombination and
which do not encode a mechanism for their
removal. Lastly are seemingly legitimate
phage genes, ones associated with temperate
phages that modify the phenotypes of
lysogens. Included under this heading are a
number of virulence factor genes. The dif-
ference between a moron and these lysogenic
converting genes is one of degree of integration
into the phages genetic structure, with
morons more evidently newly acquired by
the transferring phage. See Christie et al.
(Chapter 4, this volume) for consideration of
lysogenic conversion in general as it applies
to the encoding of bacterial virulence factors
by phages.
Conclusion
It is important to keep in mind that phages
are highly diverse. This diversity is seen in
terms of genotype, phenotype, the proteins
produced and interactions with hosts.
Phages also vary in terms of their host range
(which bacteria they infect), their trans-
ducing ability, their virion morphology, and
also with respect to their general infection
character istics. Thus, whenever mention is
made of a specic phage, it should be kept in
mind that substantial eort may be necessary
to elucidate the specic properties, especially
phenotypic, that are associated with that
phage or phagehost combination. On the
other hand, various generalizations may
also be made. This monograph will present a
mix of both generalizations and specics in
considering phage presence in bodies
without disease, their role in both bacterial
disease and pathogen evolution, and how
phages can be employed to combat infectious
diseases, in particular of humans.
6 (eds P. Hyman and S.T. Abedon)
Human beings, our problems, our triumphs
and our everyday lives, have through the
ages been central to philosophy, literature,
arts and religion. This anthropocentric
comprehension of the Universe has led to
numerous, dramatic and sometimes quite
controversial considerations in the natural
sciences as well. These include, for example,
resistance to acceptation of the heliocentric
model of the Universe (16th century), the
di culties that the theory of evolution met
before gaining wide acceptance (19th century)
and, more recently, the numerous problems
ranging from underestimation to over-
estimation in interpreting the mental
capacities of dierent animal species, ranging
from bees to dogs, dolphins, or even to our-
selves (Alcock, 2001).
The anthropocentric view has led to a
perception by many modern biologists,
whose work is not directly aimed at species-
specic aspects of animal biology, to regard at
rst glance the human animal as a valid
model for mammals in general or vice versa.
Indeed, almost everyone, scientist as well as
non-scientist, is biased towards the idea that
humans or indeed any animal or plant
consists exclusively of cells, which can be
described unambiguously as animal or plant
cells. Such a perspective, however, is not
even close to accurate, as each macroscopic
organism hosts many types of microorganisms
as part of its normal state: the microbiome of
the organism. In this chapter, I emphasize
essentially this aspect of the human body
that traditionally one wouldn't regard as the
human body. Furthermore, my emphasis will
not even be on all of the microbiome but
instead on the viruses that infect most of
those non-human cells, the bacteriophages or
phages. See Loc-Carrillo et al. (Chapter 13,
this volume) for a discussion focusing in part
instead on the bacterial aspects of the human
normal ora.
This discussion serves as the beginning of
an exploration of copious connections
between humanity, the most powerful of
organisms, and phages, which are the most
numerous. Indeed, in this monograph the
numerous roles that phages can play in
healthy humans, in contributing to human
disease (see Kuhl et al., Christie et al. and
Hendrickson, Chapters 35, this volume), in
the treatment of human disease (see Siegel,
Wagemans and Lavigne, Olszowska-Zaremba
et al., Loc-Carrillo et al., Burrowes and Harper,
Shen et al. and Abedon, Chapters 8, 9, 1215
and 17, this volume) and in the prevention of
human disease (see Clark et al., Cox,
Goodridge and Steiner, and Niu et al.,
2 Bacteriophages as a Part of the Human
Microbiome
Andrey V. Letarov
1
1
Winogradsky Institute of Microbiology, Russian Academy of Sciences
A.V. Letarov
Bacteriophages as a Part of the Human Microbiome 7
Chapters 7, 10, 11 and 16; see also Williams
and LeJeune, Chapter 6, this volume) all
will be discussed. First, and the emphasis of
this chapter, will be the normal state of aairs,
which is the phage contribution to the
collection of cells and microorganisms that
together make up the human body; that is,
ourselves and our microbiome, especially as
seen during the normal, healthy state.
Phages and the Human Microbiome:
an Overview
From the point of view of the microorganism,
the human or animal body is merely a system
of connected colonizable ecotopes, that is,
ecologically distinct features of environments.
Each ecotope varies in terms of host factors
(the colon versus the lung, for example), in
terms of its history (dictating in part what
organisms can be present), in terms of
interactions between the microorganisms
that are there and as a consequence of feed-
back mechanisms between micro organisms
and host. The result is a high potential for
variation in microorganism types, including
virus types, going from organ to organ, tissue
to tissue, individual to individual, and also
over time both within individuals and
through the generations (both humans and
our ancestors). Adding further to these
complications is the potential for at least
some microorganisms to move between
species. For phages, we can also add an
ability to modify their bacterial hosts (see
Christie et al. and Hendrickson (Chapters 4
and 5, this volume), including in terms of
phage susceptibility (see Williams and
LeJeune, Chapter 6, this volume).
The result of all of these factors is the
potential for a high degree of individual
variation in the composition of, for example,
intestinal bacterial populations in humans, as
was reported by Costello et al. (2009). The
individuality of the associated phage
populations can be even higher than that of
bacteria (Reyes et al., 2010; Caporaso et al.,
2011). Consistently, divergence of both
bacterial and phage communities at the same
body sites but in dierent individuals were
found to be higher than in the same
individuals over time (Costello et al., 2009;
Reyes et al., 2010; Caporaso et al., 2011). On
the other hand, communities of gut bacteria
were much more related in closely connected
people such as monozygotic twins and their
mothers, although this consistency applies
less so to the phage component, which
appears to be highly individual but none the
less quite stable over time (Reyes et al., 2010).
These data highlight the eect of ampli-
cation of slight dierences in physiology (or
conditions) by the complex events of micro-
bial interactions as well as exposure history,
resulting in signicantly dierent states of
microbial systems in dierent subjects, and
indicate that phage communities may be
more sensitive to colonization history than
bacterial microora (Costello et al., 2009; see
also Nemergut et al., 2011). The data on the
phage prevalence and activity in dierent
sites of the human body, analysed in this
chapter, thus may not represent any para-
digmal model for phages in animal-associated
systems but could instead reect particular
features of our species, and maybe even of
individuals or subpopulations included in
the studies cited. By comparison, we can con-
sider the current understanding of bac-
teriophage ecology in other animal-associated
systems, as recently reviewed elsewhere
(Letarov and Kulikov, 2009).
Despite early work with phages where
there was a strong emphasis on the impact of
phages on the human antibacterial immunity,
along with a strong medical orientation of
basic research in microbiology and virology
in recent decades, the phage ecology of the
human body was substantially neglected
until recently. Even now it is poorly under-
stood if compared, for example, with phage
ecology in aquatic systems. The rst
observations of human-associated bacterio-
phages, however, were published by one of
the discoverers of these viruses, Felix
dHrelle, who demonstrated phages lysing
enterobacteria in faeces (dHrelle, 1921).
Nevertheless, and despite two periods of
high interest in phage therapy in the 1920s
to 1930s and from the 1990s until now
(Abedon, 2011) these endogenous phages
have been subject to systemic research only
over the past few years. Thus, our knowledge
8 A.V. Letarov
of bacteriophage impact on microbial ecology
and on macro-host homeostasis in humans
(and, more widely, in animals) is highly
mosaic, with many important parts of this
puzzle still missing.
Here, I focus exclusively on the available
data on phages in human symbiotic
microbiota, presenting a limited subset of
data for other species only for comparison. I
consider the phage component following
standard biogeographical principles, that is,
based on body site. As has been evident since
early electron-microscopy based studies of
non-cultured viral communities from human
faeces and other body sites, and later con-
rmed by metagenomic analysis (reviewed
below), phages in fact appear to dominate the
human body virome, being much more
prevalent than any eukaryotic viruses.
Skin
The skin is the largest organ of the human
body and is a reservoir for multiple and
highly diverse habitats for symbiotic bacterial
communities. The diversity of bacteria on the
skin is comparable to that of the gut, although
the total microbial biomass is much lower.
The variation in species composition of the
skin microora of individual people appears
to be highest among all the body sites that
have been analysed (Costello et al., 2009). The
data on skin-associated bacteriophages,
however, is almost non-existent for humans,
as well as for other animals.
Alternatively, phages potentially associ-
ated with skin microora have been isolated
from downstream habitats such as Staphy-
lococcus aureus phages isolated from sewage.
Generally, such occurrence of phages in-
fecting human-associated (and animal-
associated) hosts from sewage and other
downstream sources is a well-known
phenomenon. Moreover, this kind of samp-
ling is widely used in many works on the
isolation of phages of pathogenic bacteria for
phage therapy or diagnostic applications (see
Gill and Hyman, 2010, for review). There is
always a question, however, of whether these
phages come from human or animal micro-
biomes or instead are indigenous for specic
systems (waste-water drains, for example). In
numerous cases, it has been di cult to isolate
phages directly from animals, but they are
easily found in farm waste water. Further-
more, T-even-related bacteriophages are
seldom isolated from healthy humans or
animals (see below), but they can frequently
be isolated from sewage samples (where T4
and T6 were originally found; Abedon, 2000).
The details of the life strategy that make a
phage beter adapted to macro-host-
associated or downstream habitats are not
yet clear.
Respiratory tract
The respiratory tract of healthy subjects is
believed to be poorly colonized by any
microora, thus suggesting that no stable
bacteriophage population should be present.
In good agreement with this conclusion are
the results of a recent metagenomic study of
the viruses contained in the sputum samples
from ve cystic brosis (CF) patients and ve
non-CF subjects (Willner et al., 2009). The
diversity of both viral communities was at the
level of about 175 viral genotypes in both
metagenomes. The metagenomes of CF
patients shared signicant similarity and it
was possible therefore to evaluate the core of
the CF-specic phage community (Willner et
al., 2009; Willner and Furlan, 2010). These
phages were proposed to play signicant
roles in the pathological microbial ecology in
CF patients lungs, encoding numerous
bacterial virulence factors including adhesins,
biolm-formation genes and quorum-sensing
genes (Willner and Furlan, 2010; see Christie
et al., Chapter 4, this volume, for a general
overview of phage encoding of bacterial
virulence factors). At the same time, the
phages present in the lungs of non-CF
persons were found to vary considerably and
most likely represented the random samples
of environmental viruses. Two non-CF
sputum metagenomes collected from people
sharing the environment with CF patients
were more similar to the CF prole than the
other three non-CF samples, and this
observation was in good agreement with the
assumption that the viral community of the
respiratory tract in healthy people is repre-
sented by transitionally captured particles
originating from the air.
Gastrointestinal tract
The human gastrointestinal tract includes the
mouth cavity, throat, oesophagus, stomach
and gut, the later comprising the small
intestine, large intestine and rectum. Due to
the bactericidal activity of high acidity along
with the proteolytic enzymes found in gastric
juices, the stomach is poorly colonized by
microorganisms and contains only about 10
3
bacterial cells ml
1
. In the small intestine, the
bacterial population is also limited by the
rapid peristalsis combined with the action of
the bile and pancreatic secretions and reaches
about 10
5
cells ml
1
(up to 10
8
ml
1
in the
ileum; Baranovsky and Kondrashina, 2008).
The main reservoir of the gut microbial
biomass is the large intestine, harbouring up
to a total of 10
14
bacterial cells per individual
(Savage, 1977) and corresponding to
hundreds of grams of bacterial biomass.
Below, I consider the phage populations in
the oral cavity and in the large intestine that
represent the major ecotopes of the human
gastrointestinal tract colonized by bacteria.
Oral cavity and pharynx
Studies assessing phage presence in the
human oral cavity and pharynx are not
extensive. Direct electron microscopic ob-
servations indicate the presence of a large
number of VLPs in some but not in all
samples of the dental plaque material (Brady
et al., 1977). The presence of VLPs in the
matrix of complex multi-species biolms
seems logical, as the data suggest that in
some bacterial species, such as Pseudomonas
aeruginosa, induction of the prophage and
production of viral particles may be a normal
programmed stage of the biolm develop-
ment (Rice et al., 2009).
These observations of high concentrations
of VLPs in dental plaque to my knowledge
have not been corroborated. Nevertheless,
the existence of bacteriophages in dental
plaque material was recently conrmed by a
small viral metagenomic survey of the dental
plaque uncultured viral community collected
from a single individual (Al-Jarbou, 2012). Of
the 80 sequences obtained, only a total of 21
phage-related sequences were discovered.
Despite the paucity of the data set obtained in
this study, it indicates clearly the low
complexity of the dental plaque viral com-
munity.
Another recently published metagenomic
study of viral communities of pooled oro-
pharyngeal swabs from 19 healthy individuals
(Willner et al., 2011) also indicated that they
are formed almost exclusively by phages a
mix of virulent and temperate phages. The
sole exception was the eukaryotic Epstein
Barr virus. Of interest, the complete genomes
of three phages were assembled and among
them was Escherichia coli phage T3. The
natural reservoir for this virus was never
identified, although the data of Willner et al.
(2011) indicated that it may be a normal
inhabitant of the human pharynx. The
authors also detected a number of phages of
Propionobacterium, Lactobacillus and other
lactic acid bacteria, streptococci and other
hosts as well as non-host-attributed
bacteriophage-related sequences. The SM1
bacteriophages encoding the platelet-binding
factors of Streptococcus mitis were also found,
suggesting possible involvement of the
oropharyngeal microbiota in development of
endocarditis. The total abundance of the
bacteriophages was not directly determined
in this study but would appear not to be very
high, as the authors applied an amplication
procedure prior to sequencing. The diversity
was estimated to be about 236 different viral
genomes from the 19 pooled samples.
A much more profound metagenomic
study of human saliva viromes has recently
been published by Pride et al. (2011). The
authors collected saliva samples from several
healthy subjects at dierent time points. The
viruses were quantied by epiuorescence
microscopy and the morphology of VLPs
was investigated by electron microscopy.
Profound sequencing of the virome meta-
genomes was performed accompanied by
10 A.V. Letarov
sequencing of bacterial 16S rRNA gene
libraries from the same samples. This study
conrmed the presence of a robust indigenous
phage population dominating the saliva
microbiome, comprising up to 10
8
VLPs ml
1

of saliva. The composition of the viromes in
dierent subjects was highly individual, but
two viromes collected from members of the
same household showed much greater
relatedness than the others, indicating the
impact of externally acquired phages on this
system (which makes a striking contrast to
the data of Reyes et al., 2010, which demon-
strated that faecal viromes are markedly
distinct in closely connected subjects such as
monozygotic twins and their mothers see
below). The composition of the viromes
within subjects at dierent time points was
highly related but nevertheless exhibited
signicant changes over time, especially at
6090-day time intervals. The saliva virus
population thus appears to be quite dense
and at the same time dynamic. The presence
of phage integrases in 10% of all contigs and
the identication of virus contigs matching
distinct regions in sequenced bacterial
genomes indicated a high prevalence of
lysogeny. At the present time, however, the
data are insu cient to estimate the relative
signicance of virulent and temperate phages
(or of phage multiplication in the lytic cycle
versus lysogen induction in this system).
Overall, the substantial numbers of phage
particles detected by Brady et al. (1977), Al-
Jarbou (2012) and Pride et al. (2011) in oral
cavity-derived samples is inconsistent with
published negative results of atempts to
isolate phages from the human oral cavity
active against normal indigenous bacteria
present in that site (Hitch et al., 2004); these
authors were, however, able to isolate a phage
of a non-oral pathogen, Proteus mirabilis.
A very limited number of successful
phage isolations from the oral cavity have
been described. For example, Tylenda et al.
(1985) reported the isolation of actinobacterial
phages from ten out of 336 samples of human
dental plaque material. The isolation of
Veillonella bacteriophage from the oral
samples was also published by Hiroki et al.
(1976). More recently, phages for Enterococcus
faecalis were cultured from human saliva
(Bachrach et al., 2003), but it should be noted
that in this later study the authors failed to
detect bacteriophages for a number of other
species of bacteria of the normal oral
microbiota. Temperate E. faecalis phages were
also successfully induced from root canal
isolates of this bacterium (Stevens et al., 2009).
At the moment, it is di cult to build a
comprehensive concept of phage ecology in
the oral cavity. Summarizing the data, one
could conclude that the bacterial community
of the human oral cavity and probably also of
the pharynx are not substantially impacted
by phages. The metagenomic data of Pride et
al. (2011), however, suggests that the viral
community in this site is dynamic and is able
to incorporate externally acquired phage
strains and to maintain quite an elevated
density. No coherent explanation of these
contradictions was suggested. Given the
limited amount of data on oral bacteriophages
from non-human species that has been
published, it is di cult to speculate on how
these human traits compare with the phage
ecology of the oral cavity in mammals.
The gut
The gut, especially the lower intestine, is
believed to be the main habitat of the human-
associated microbiota including bacterio-
phages. Being the natural habitat of the
worlds best-studied bacterium E. coli the
intestinal microbial system of humans and
animals has served as a subject of multiple
studies of coliphage ecology (see below),
starting from dHrelles pioneering work in
1921.
Total viral counts
Transmission electron microscopy-based
studies have repeatedly demonstrated a high
abundance of VLPs in the faeces and intestinal
contents of humans (Flewet et al., 1974), as
well as in other species. The later include
catle and sheep (Paynter et al., 1969;
Hoogenraad et al., 1967), the rumen of
reindeer (Tarakanov, 1971), the forestomachs
of Australian marsupials (Hoogenraad and
Hird, 1970; Klieve, 1991) and the large
intestine contents and faeces of horses
(Alexander et al., 1970, Kulikov et al., 2007). In
all of these cases, the vast majority of observed
VLPs belonged to tailed bacteriophages.
No quantitative data characterizing the
morphological diversity of the human gut
bacteriophages, however, has been published
(in contrast to some animal-associated com-
munities, as recently reviewed by Letarov
and Kulikov, 2009).
Metagenomic studies
Currently available data on the diversity of
the non-cultured viral community of the
human gut has been based mainly on
metagenomic data. The rst metagenomic
analysis of the virome of a single specimen of
human faeces, collected from a 30-year-old
male subject, was published by Breitbart et al.
(2003). In their study, about two-thirds of the
sequences obtained were database orphans;
among the rest, known viral sequences
constituted 27%. The predominant viral
group, judged by database hits, in human
faeces were siphoviruses (bacteriophages
with long, non-contractile tails), which are
probably most prevalent in the majority of
natural habitats (Weinbauer, 2004). The
estimated diversity of bacteriophages was
about 1200 viral genotypes present in the
sample.
Sequences related to eukaryotic viruses
comprised only a minor fraction. This is
consistent with the fact that such particles are
rarely seen in transmission electron micro-
scopy images of faecal viral comm unities. It is
interesting that, in metagenome analyses of
RNA-containing VLPs extracted from human
faeces, the sequences of plant viruses were
highly predominant (Zhang et al., 2006),
indicating that ingestion of these particles
with food is much higher than the internal
production of RNA-containing phages. The
later meta genomic analysis of multiple
samples of human faeces used high-
throughput sequenc ing technology (pyro-
sequencing) and re vealed very interesting
features of these communities.
In order to determine the impact of the
genetic background of the macro host on the
composition of the human intestinal virome
and the stability of the individual phage
populations, Reyes et al. (2010) collected
samples of faeces from four pairs of twins
and their mothers over a 1-year period. VLPs
were extracted from these samples (32
viromes in total were characterized) and the
associated viral metagenomes were se-
quenced. About 85% of the sequences from
the VLP metagenomes did not correspond to
any known viruses, while most of the rest
matched various known prophages and
temperate phages.
The bacterial diversity of the same set of
samples was analysed by 16S rRNA gene
library sequencing. The diversity of bacterial
communities was estimated to be about 800
species-level bacterial phylotypes, while the
complexity of the phage community was
measured by two dierent approaches as 52
2773 (median 346) or 10984 (median 35)
predicted virotypes. Both the abundance of
phages and the number of phage species per
bacterial species was therefore quite low in
comparison with known, free-living bacterial
communities. The idea of a temperate nature
of predominant phage types in the analysed
viromes was strongly supported by the
identication of a signicant number of
sequences related to known bacteriophage
integrases, the phage-encoded site-specic
recombinases responsible for integration of
the temperate phage genome into the host
chromosome.
The similarities seen in the bacterial
communities studied by Reyes et al. (2010)
strongly correlated with family links between
the subjects. In contrast, the VLP metagenome
composition was highly individual and
varied considerably between the subjects,
regardless of family relationships. At the
same time, the variability was low within
individuals: over the time of the study, the
sequences of the VLP metagenomes were
almost stable. Moreover, the authors were
able to detect the dominant phage that
persisted at high levels in one of the
individuals for an extended period of time
but showed no signicant divergence or
mutations in its genome.
The proposed low dependence of the
phage populations in this environment on
12 A.V. Letarov
their success in competition for host bacteria
might facilitate long-term persistence of those
phage populations that happen to colonize a
particular niche rst. Alternatively, if the
assumption of high prevalence of temperate
phages made in the above-cited studies is
correct, then those temperate phages that
colonized the genomes of bacteria that have
established their populations in an individual
gut ecosystem may predominate. The high
individuality of the phage community in
dierent subjects therefore may reect the
history of colonization of the infant gut
by bacteria, phages and phage lysogens
(Breitbart et al., 2008).
What does not seem to be present is
substantial selective pressure acting on either
bacteria or phages, at least over the intervals
analysed by metagenomic studies. The data
of Reyes et al. (2010), in particular, provided
no indication of Red Queen dynamics
(continuing change and adaptation of host
and parasite; Weitz et al., 2005) in human
faecal microbial communities, although
perhaps the resolution of metagenomic
studies could be unable to provide such a
signal. Thus, the bacteriophages in this
environment do not seem to exert a su cient
inuence on the dynamics of bacterial
populations in the gut to result in bacterial
resistance. Abedon (2011) suggested that this
sort of low phage pressure despite ongoing
phage presence may be the norm, given
bacterial persistence in environments pre-
dominantly as biolms (see also MacFarlane
et al., 2011).
This concept of human intestinal phage
ecology is in good agreement with the data of
Caporaso et al. (2011) who used a somewhat
dierent approach for analysis of the
metagenomic data. These researchers studied
26 viral metagenomes of human faeces
collected from 12 individuals (one to four
samples per individual). They compared the
composition of these viromes with the
viromes obtained from a variety of free-living
communities. The authors found that the
distances between the individual viromes of
human faeces were higher than between the
samples of related free-living communities.
They also conrmed the predominance of
temperate phages and the absence of
observable phage/bacteria co-evolution in the
human gut.
In agreement with these observations,
Minot et al. (2011) reported that, following
analysis of metagenomic sequences of human
faecal viromes collected from ve people
over a 1-week period, they did not detect any
signs of Red Queen dynamics in these
systems. The viromes studied also showed
high individual variability; however, 1-week
diet interventions (as low fat/high bre or
high fat/low bre diets) led to an increase in
similarity of the viromes between subjects fed
the same diet. This may indicate that, in
humans, alterations in virome composition
follow the composition of the microbiome.
This may also be interpreted as an argument
in favour of the hypothesis of a high impact
of lysogen induction in generations of phage
VLPs in the human gut. Minot et al. (2011)
analysed the viral (phage) contigs assembled
from their data for genetic relatedness (using
various criteria, described in detail in the
paper) for temperate phages or prophages
and found about 14% of them to be potentially
temperate. However, this value was only the
lowest estimate, as not all fragments of the
temperate phage genomes would t the
criteria applied.
Culture-based analyses
When considering the culture-based data
dealing with phage indication and quanti-
cation in natural environments, one must
always remember that the majority of these
viruses tend to be specic for only a subset of
the strains found within bacterial species
(Hyman and Abedon, 2010). Thus, the phage
titre obtained with any given indicator
bacterium reects only a fraction of the phage
particles, that is, those able to infect that
particular bacterial strain (the phenomenon is
known in phage ecology as the great plaque
count anomaly; see Weinbauer, 2004).
Moreover, some of the phages that are able to
infect the bacterial strain used nevertheless
may display reduced plating e ciency, at
least during the rst passage on a host strain.
This eect may be due to the action of
restriction-modication systems, as well as of
many other host resistance mechanisms
(Labrie et al., 2010), and can lead to serious
several orders of magnitude under-
estimations of the real abundance of a
particular phage strain in a sample. In each
case, one therefore has to consider the choice
of the bacterial host used for phage
quantication, especially if comparison of
phage titres in samples collected from
dierent subjects is involved.
Notwithstanding these caveats, the data
of culture-based studies are in general
agreement with the hypothesis of a low
phage impact on bacterial populations in
human intestinal ecology. Furuse et al. (1983),
for example, found that faecal coliphage titres
in healthy humans are low, and the pools of
free virions in faeces are represented mainly
by temperate phages. In contrast to healthy
people, the phage populations in some
patients with internal and leukaemic diseases
contained a substantial fraction of virulent
phages (including T-even-related phages), as
well as an increased faecal coliphage back-
ground (Furuse et al., 1983). In several
patients, phage titres increased when the
severity of the clinical symptoms increased.
Consistent results of phage isolation
from Bangladesh paediatric patients with
diarrhoea were reported by Chibani-
Chennou et al. (2004b). About 19% of acute
diarrhoeal stools yielded quite divergent T4-
related phages infecting the E. coli K803
laboratory indicator strain. The detection of
phages in the stools from convalescent
patients was less frequent. It is interesting
that other E. coli strains used for phage
isolation from the same set of samples
yielded completely dierent Siphoviridae
coliphages. The occasional presence of both
temperate and virulent coliphages as well as
some culturable phages infecting other hosts
has also been reported in the literature. In
most cases, these viruses were present in
low titres in healthy subjects (Dhillon et al.,
1976; Havelaar et al., 1986; Cornax et al.,
1994; Grabow et al., 1995; Calci et al., 1998;
Gantzer et al., 2002; Schaper et al., 2002; Cole
et al., 2003; Lusiak-Szelachowska et al., 2006;
see also Letarov and Kulikov, 2009, for
review).
Temperate phages in the gut
The results of metagenomic analysis per-
formed by Reyes et al. (2010) suggested that
the main source of free phage particles in the
human intestine are not productive cycles
immediately following phage adsorption, as
seen in the majority of other natural
communities (see Weinbauer, 2004 for
review), but instead are the induction of
bacterial lysogens that can occur due to
starvation, such as of bacteria in faeces. This
conclusion is similar to that of Furuse et al.
(1983) who rst suggested that, in healthy
people, most of the released phage particles
are produced by induced lysogenic cells and
therefore that phage multiplication may have
a limited impact on the intestinal coliform
microora.
Interestingly, the vast majority of tem-
perate coliphages isolated by Dhillon et al.
(1980) from free phage particles of human
and animal faeces belonged to the lambdoid
group, while phages obtained from cultured
bacterial lysogens were all immunologically
P2-related. These data may be explained by a
higher frequency of induction of the lambdoid
prophages present in the studied microbiomes
than that of P2-like prophages. Alternatively,
conditions favourable for phage multi-
plication in the lytic cycle may occur in the
healthy human gut, perhaps in spatially
limited sites or over short periods of time,
and this multiplication could allow the
reproduction of some phages independent of
the induction of lysogenic bacteria. The
above-cited results could thus reect the
preferential success of the lambdoid phages
within hypothetical windows for lytic cycle
multiplication.
F-specic phages
RNA-containing F-pilus-specic (F-RNA)
coliphages (members of the family Leviviridae)
have been found in human as well as in some
animal wastes and show certain species
specicities. These phages can be subdivided
into multiple genetic groups that can be also
distinguished serologically. The incidence of
these serotypes varies signicantly among
species: horse faeces, for example, rarely
14 A.V. Letarov
contain them, while the faeces of more than
70% of chickens contain high titres of these
phages (10
5
10
7
plaque-forming units (PFU)
g
1
). Only about 1020% of human faeces
contains F-RNA coliphages, but the occurrence
of group II and III F-RNA phages in these
samples is much higher (>80% of all isolates)
than in animals, where groups I and IV are
prevalent at the same level (Furuse et al., 1978;
Havelaar et al., 1986; Schaper et al., 2002; Cole
et al., 2003). No coherent explanation for this
group specicity has yet been suggested. See
Goodridge and Steiner (Chapter 11, this
volume) for further discussion.
Possible limitations on phage replication in
the gut
Among the environmental factors that may
contribute to the protection of bacteria from
phage atack in the human gut are chemicals
that can inhibit phage infection as well as the
specic physiological state of bacterial
populations. Bile salts and carbohydrates
have been shown to inhibit the adsorption of
a variety of coliphages (Gabig et al., 2002).
This eect is suppressed if Ag43 protein,
mediating cell aggregation and atachment, is
present on the surface of E. coli cells. The
expression of Ag43 is regulated in a phase-
variation manner. Thus, phages may, under
some circumstances, select against increased
biolm formation. For Bacteroides phages, the
addition of bile salts to the medium had an
opposite eect, that is, its addition improved
phage plating e ciency (Araujo et al., 2001).
Some food-derived phage inhibitors may also
be present in the intestine, at least occasionally.
For example, Swain et al. (1996) demonstrated
that tannic acid at physiological concen-
trations may inhibit bacteriophage replication
in the rumen of ruminants. This is consistent
with the observations of de Siqueira et al.
(2006) that tea infusions can inactivate
phages. Similar compounds are also ingested
by humans with certain types of food and
could therefore contribute to low phage lytic
activity in their gastrointestinal tract.
Growth in biolms and on the surfaces
of mucosa and food particles may also
contribute to bacterial anti-phage protection,
although this potential has at best only been
demonstrated inconclusively (Abedon, 2011).
There is also some evidence that the E. coli
population in the mouse gut lumen may be
starving and the actively replicating popu-
lation in fact may be limited to microcolonies
found on the mucosal surface (see Chibani-
Chennou et al., 2004a, and references
therein). It is not clear, however, if this model
of E. coli ecology in the gut can be extended to
humans and other large animals (Letarov and
Kulikov, 2009).
Phage propagation predominantly via
lysogenic cycles or associated prophage
induction, resulting in a reduced impact of
phage infections on the microbial ecology of
the human gut, may not be unique among
mammalian species (reviewed in detail by
Letarov and Kulikov, 2009; Clokie et al., 2011).
For example, atempts to isolate bacterio-
phages from dog faeces using indigenous
coliform strains (Ricca and Cooney, 2000)
were largely unsuccessful. Over 500 in-
digenous coliform strains isolated from six
specimens of dog kept in private homes did
not detect phages in the same samples, and
only one of these samples yielded phages on
the laboratory E. coli C strain. In 16 dogs from
a kennel, however, coliphages were detected
at variable titres from 0 to 10
7
PFU g
-1
. The
authors suggested that a low abundance of
coliphages in home-kept dogs may be due to
isolation from other dogs and too clean
living conditions. Recontamination by faecal
microbes is also limited in humans, which
could suggest that phages able to overcome
existing barriers for replication in the gut are
seldom acquired by humans. Perhaps the
occurrence and possible impact of phages in
humans would be higher if we lived in a less
civilized manner. It would be interesting to
compare the phage prevalence in human
subpopulations living in the same area but
diering in their quotidian hygienic practice
(for example, in Bangladeshi state employees
and peasants).
The opposite situation to that of highly
domesticated animals seems to exist in the
horse intestinal ecosystem. In our studies
(Golomidova et al., 2007; Kulikov et al., 2007;
reviewed by Letarov and Kulikov, 2009), the
structure of the indigenous coliform com-
munity was compatible with high phage
pressure. Of the dozens of horse coliphage
isolates that we were able to identify, all
belonged to known groups of virulent phages
(T-even-related, T5-related, rv5-related, N-
related, similar to Salmonella SETP3 phage,
Felix 01-related, similar to Caulobacter phage
Cd1, coliphage K1F-related and others;
Golomidova et al., 2007, and our unpublished
data). The investigation of morphological
diversity of uncultured horse faecal VLPs
(Kulikov et al., 2007) has demonstrated the
high abundances of a diversity of large
bacteriophages that are very likely to be
virulent. The ecology of intestinal bacterio-
phages thus appears to be highly dependent
on host-species-specic features of digestive
tract physiology along with the general
environment.
The vagina
The vaginal microbiota of healthy, fertile
women is normally dominated by vaginal
lactobacilli (Hillier, 2008), usually comprising
more than 70% of the total bacterial count in
this environment. These bacteria are believed
to play an essential role in colonization
resistance against pathogenic bacteria and
fungi by contributing to the establishment of
a low vaginal uid pH (Servin, 2008; Linhares
et al., 2011), along with frequently producing
hydrogen peroxide (Martin and Suarez,
2010). Lactobacilli also possess more-specic
antagonistic activities against pathogenic
bacteria (Servin, 2008). Partial loss of the
indigenous vaginal lactobacilli in combination
with polymicrobial anaerobic overgrowth on
the vaginal mucosa are characteristic features
of bacterial vaginosis (BV) one of the most
common reasons women seek medical help
(Sobel, 2000). The density of Lactobacillus
colonization of the vaginal mucosa is quite
high at 10
6
10
7
CFU per vaginal swab. Thus,
one can assume that episodes of mass killing
of lactobacilli by phages may occur, although
atempts to detect free bacteriophages in
vaginal swabs have been unsuccessful (Kili
et al., 2001). At the same time, however,
lysogenic strains of lactobacilli were shown to
be prevalent in this environment (Kili et al.,
2001).
Although the vagina is subject to
relatively low levels of exchange of bacteria
and viruses with the external environment,
except as mediated by sexual activity, a
sudden breakdown of Lact obacillus popu-
lations is frequently observed in examinations
of vaginal swabs from clinically healthy
women. Some individuals also develop
anaerobic bacterial vaginosis syndrome, when
lactobacilli are replaced by anaerobic bacteria
such as Gard nerella vaginalis, Prev otella,
Porphyromonas and Mobi luncus species. This
condition has the epidemiology of a sexually
transmited disease (Verstraelen et al., 2010). It
was proposed by Blackwell (1999) that the
causative agent triggering the breakdown of
the normal vaginal microbiota might be a
bacteriophage atack. I speculate that such
events may take place if a lysogenic strain able
to produce a phage infectious for the major
resident strain(s) is acquired (Letarov and
Kulikov, 2009). A similar scenario was
modelled on in vitro E. coli populations both
experimentally and mathe matically by Brown
et al. (2006).
The results of our recent study of the
diversity of individual populations of vaginal
lactobacilli at the strain level indicate that
individual populations of these bacteria are
normally dominated by a single strain, which
could be resolved by repetitive element PCR
ngerprinting (A. Isaeva, E. Ilina, A.
Bordovskaya, A. Ankirskaya, V. Muraviouva
and A. Letarov, unpublished data). This is
compatible with the proposed mechanism of
the rapid drop of lactobacilli counts due to
mass phage-mediated lysis, as almost all of
the cells in individual populations would
(presumably) represent the same phage-
susceptibility type. In such a case, a single
phage liberated by an invading lysogenic
strain potentially could reduce the resident
population density and make enough room
for a newly acquired lysogenic strain (and
secondary lysogens formed in some cells of
the resident strain). In contrast to the results
of Kili et al. (2001) described above, however,
the search for inducible lysogens in our
collection of vaginal isolates yielded no
strains producing a phage viable on any other
isolate tested (A. Isaeva and A. Letarov,
unpublished data). An observation similar to
16 A.V. Letarov
ours was recently reported for a Spanish
subpopulation of women (Martin et al., 2009).
These authors suggested that high con-
centrations of H
2
O
2
produced by vaginal
lactobacilli select for prophage-cured lineages
that do not suer from the mortality induced
by elevated phage induction due to
permanent oxidative stress characteristic of
this environment. Thus, the vaginal eco-
system may be considered as one in which
phages exert, ecologically, a low impact on
the resident bacterial populations. The
possible exceptions, however, require further
investigation.
Direct Interaction of the Virome with
the Macro Host
The phage ecology of human-associated
microbial systems generally should be
considered as a tripartite interplay between
the bacteriophage, the host bacteria and the
macroorganism (i.e. ourselves). In this inter-
play, the macroorganism inuences both
bacteria and phages. For example, phage
destruction can occur due to the action of
digestive enzymes as well as that of macro-
phages, phage transportation by blood and
p hage transmission between individuals that
are facilitated or restricted due to specic
macroorganism behaviour. The most striking
phenomenon may be the inuence n phage
infection by some compounds secreted by the
macro host such as bile salts (see above).
The macroorganism seems to be directly
and indirectly inuenced by both phages and
microbes. Direct interactions, once phage
particles have reached systemic circulation,
include immunomodulatory activity (see
Olszowska-Zaremba et al., Chapter 12, this
volume). Lepage et al. (2008) observed in-
creased concentrations of VLPs on ulcerated
intestinal mucosa and speculated that phage-
mediated immune eects may play some role
in the pathology of inammatory bowel
disease. Phages occurring in vivo instead may
contribute to a downregulation of the in-
ammatory response in the gut, as well as
regulation of the complex interactions of the
gut-associated lymphoid tissue with massive
amounts of antigens provided by intestinal
microorganisms (Grski et al., 2006).
Experimental data on direct interactions of
naturally occurring bacteriophages with the
immune system, however, are currently
almost non-existent.
The direct inuence of the macro host on
phages within the normal microora has not
yet been studied in any detail. It has been
reported that phages administrated orally or
rectally in the course of phage therapy
penetrated to the bloodstream from the
gastrointestinal tract (reviewed in detail in
Letarov et al., 2010; Grski et al., 2006;
Dabrowska et al., 2005). In recent rigorously
controlled studies on animal models (Olivera
et al., 2009), however, the penetration of
externally administered phages was very
low (if at all). The proposed hypothesis of
constant translocation of intestinal phages into
the bloodstream so-called physiological
phagaemia (Grski et al., 2006) was based on
reports of phage contamination of commercial
blood sera from the 1970s (see Grski et al.,
2006, for a detailed review and Olszowska-
Zaremba et al., Chapter 12, this volume). The
presence of natural phages in the blood of
humans or animals, by contrast, has not been
demonstrated. In our recent work (Letarova et
al., 2012), we were unable to detect coliphages
in the blood of horses that harboured these
viruses at levels of 10
4
10
7
PFU g
1
of faeces on
the day of blood sampling. The rate of phage
translocation from the gastrointestinal tract to
the bloodstream (similar to bacteria), however,
can vary signicantly depending on the
physiological conditions and perhaps also on
phage characteristics.
Phages delivered into the bloodstream
can be rapidly sequestered by the lymphoid
organs, spleen and liver and gradually
inactivated by the reticuloendothelial system
(see Sulakvelidze and Kuter, 2005; Uchiyama
et al., 2009) and in some cases by phage-
specic antibodies, creating a barrier for
phage travel within the blood and, at least
within the context of the blood, selecting for
viruses with altered surface proteins (Vitiello
et al., 2005; Capparelli et al., 2006, 2007). The
role of these processes in the ecology of
natural phages in humans, however, is not
clear, as the concept of penetration of phages
into the bloodstream remains elusive.
Conclusions
The permanent presence of a variety of
bacteriophages in the normal human
microbiome, especially in the intestinal
microbial complex, is now well established.
Nevertheless, it remains to be seen whether
the ecological relationships between intestinal
phage and host populations in other species
may be similar to humans or dogs, or instead
completely dierent (as in horses), thus
highlighting the need for comparative studies
of the phage component in the microora of
dierent animal species. Furthermore,
composition of the viromes of dierent
subjects appears to be highly individual in
comparison with the bacterial diversity in the
same habitats. This makes it di cult to dene
any prole of the normal composition of the
virome of any particular site of the human
body, as can be done for bacterial ora. This
may be distinct from the pathological
composition, as some links between phage
diversity and pathology can be established;
for example, the presence of T-even-related
phages in high titres in the gut is frequently
associated with diarrhoea and some other
disturbances of the gut physiology. The data
available on this subject, however, are scarce
and much work is required to create a
comprehensive description of the human
phage community in health and disease.
Acknowledgements
The work of my laboratory is supported by
RFBR grant no. 06-04-48651-, by a grant of
the program of Presidium of RAS Basic
science to medicine and by contracts with the
Russian Ministry for Education and Science.
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Genomic sequencing of bacterial pathogens
has demonstrated that many of the virulence
factor molecules (also called pathogenicity
factors) that allow bacteria to be more
eective pathogens are encoded by pro-
phages; in other words, these otherwise
seemingly bacterial genes are found within
the genomes of temperate phages. Temperate
phage genomes can, in turn, reside semi-
permanently within bacteria as prophages,
forming what are commonly described as
bacterial lysogens, or simply lysogens. In
some cases, these prophages have only
recently been acquired by the bacteria, while
in other cases they have resided within the
bacterial lineage for many generations.
Prophages can also be lost from bacteria,
wholly or in part. The acquisition step itself
represents a form of horizontal gene transfer
(see Hendrickson, Chapter 5, this volume),
one that can loosely be described as a kind of
transduction (see Abedon, Chapter 1, this
volume). Overall, the number of virulence
factors that are prophage associated is both
large and diverse (see Christie et al., Chapter
3, this volume). The evolution of many
bacterial pathogens thus appears to be as
greatly mediated by phages as by plasmid-
mediated horizontal gene transfer (Relman,
2011). Many of the resulting lysogen-
associated diseases are in fact explicitly
caused by phage-encoded exotoxins, for
example diphtheria, cholera and Shiga toxin-
induced diarrhoeas (see below). In a very real
sense, then, there exist diseases that can be
atributed, to a large extent, to the action of
phages.
These various phage-associated bacterial
diseases historically have contributed to
substantial mortality and morbidity. With
clean water, adequate nutrition, vaccinations
and eective antibiotics, however, in many
cases we have all but forgoten what many of
these diseases are like, although that is not
necessarily true in countries or regions that
lack these health-promoting services. This
relative lack of rampant infectious disease
indeed is only relatively recently the case,
even in what we today describe as the
developed world. The grandmother of one of
us (S.K.), for example, had six siblings die
before the age of ve in rural Pennsylvania,
several apparently from the deadly scourge
of childhood, diphtheria, a lysogen-mediated
disease that had incited fear for decades. By
contrast, few American physicians of more
recent generations have even seen diphtheria,
although the same cannot be said for Russian
physicians practising in the afermath of the
break up of the Soviet Union (Vitek et al.,
1999). In Haiti, a cholera epidemic, also a
lysogen-mediated disease, followed the
breakdown in public heath afer the 2010
earthquake. Cholera, which had not been
seen in Haiti for 100 years, incited a panic
similar to that seen during diphtheria or
3 Diseases Caused by Phages
Sarah Kuhl
1
, Stephen T. Abedon
2
and Paul Hyman
3
1
VA Northern California Health Care System, Martinez;
2
Department of
Microbiology, The Ohio State University;
3
Department of Biology/Toxicology,
Ashland University
22 S. Kuhl et al.
cholera outbreaks in the past. The dreaded
bad Escherichia coli, E. coli O157:H7, too is
bad particularly because of the prophage-
encoded Shiga toxin. Lysogen-mediated
diseases thus exist as more than just historical
oddities. Rather, they can have both real and
recent impacts, resulting in substantial
morbidity and mortality, even in our other-
wise modern world.
What do these diseases look like? To
orient the reader, in this chapter we provide
an overview of various diseases that are
associated with virulence factors that are
prophage encoded. Table 3.1 contains a list of
diseases in which prophage-encoded viru-
lence factors play a role. This table is not
exhaustive but provides an overview to show
the scope of illnesses caused by these virulence
factors. The goal is to bring dis cussions within
this monograph into the realm of the purely
medical before returning to emphases that are
more abstract or molecular. This chapter
additionally serves as a counterbalance to the
preceding chapter by Letarov (Chapter 2, this
volume), which places an emphasis on virions
rather than prophages and the normal,
healthy state rather than diseases. It also
provides a prologue of sorts to the chapter by
Christie et al. (Chapter 4, this volume), where
the variety of toxins at the molecular level is
deeply explored. This then is an introduction
to the dark side of phages: diseases that are
aided and abeted especially by temperate
phages and the lysogenic-converting genes
that they carry.
Diphtheria
The classical example of a phage-mediated as
well as exotoxin-mediated disease is diph-
theria. Indeed, this was the rst disease for
which a linkage between bacterial virulence
and the presence of a prophage was rmly
established (Freeman, 1951). Diphtheria is
caused by infection with Corynebacterium
diphtheriae, a Gram-positive, facultative
anaerobic bacterium. A clinically similar
disease can be caused by Corynebacterium
ulcerans strains that also produce diphtheria
toxin, presumably afer lysogenic conversion
by a toxin-encoding phage (CDC, 2011a).
Prior to the introduction of the rst eective
vaccines in the 1920s, diphtheria was endemic
worldwide. Today, it is still seen in parts of
Africa, South and Central America, Eastern
Europe, Asia and the Middle East (CDC,
2011b). See Christie et al. (Chapter 4, this
Table 3.1. Bacteria containing toxigenic prophages and associated diseases.
Organisms with toxigenic prophage(s) Disease/syndrome
Clostridium botulinum Botulism
Corynebacterium diphtheriae and
Corynebacterium ulcerans
Diphtheria, cutaneous infections
Escherichia coli, especially Shiga-toxigenic E.
coli (STEC) including E. coli O157:H7
Diarrhoeal disease of varying severity including
enterohaemorrhagic diarrhoeas, haemolytic
uraemic syndromes
Pasteurella multocida Cellulitis and other wound infections
Pseudomonas aeruginosa Opportunistic pathogen causing wound infections,
pneumonias and chronic lung infections of cystic
brosis patients
Salmonella enterica serovar Typhimurium and
related S. enterica strains
Food-associated diarrhoeal diseases
Staphylococcus aureus (including MRSA and
multidrug-resistant S. aureus)
Cellulitis, impetigo, osteomyelitis, pneumonia,
endocarditis, septicaemia, toxic shock syndrome
(TSS), staphylococcal scalded skin syndrome,
food poisoning
Streptococcus pyogenes (group A) and some
group C and G Streptococcus species
Scarlet fever, necrotizing fasciitis, streptococcal TSS,
septicaemia, myositis
Vibrio cholerae Cholera
Not all of the listed diseases are directly associated with a prophage-encoded virulence factor.
Diseases Caused by Phages 23
volume) for a more detailed discussion of the
phages and conversion factors involved.
Diphtheria, as an upper respiratory tract
infection, typically starts as a fever and sore
throat but progresses to a swollen neck and a
grey pseudomembrane that adheres to the
tonsils and throat. The pseudomembrane can
block the airway and suocate a child,
particularly a small child with a small air-
way. Diphtheria toxin can also enter the
bloodstream and cause damage to the heart
and other organs. Occasionally, a cutaneous
infection is seen, causing skin lesions that
vary in appearance. Transmission is usually
via contact with respiratory secretions,
although the exudates in cutaneous lesions
are also infectious. The mortality rate of the
respiratory form of diphtheria is 510%
(CDC, 2011a), with children most severely
aected. Morbidity and mortality are largely
a function of the diphtheria exotoxin, with
the phage-encoded virulence factor synthe-
sized particularly in response to iron
starvation (Tao et al., 1994).
Both treatment and prevention of
diphtheria involve the presence of anti-toxin
antibodies, which serve to neutralize the
toxin. Prevention is eective via the
administration of a toxoid vaccine the D or
d in the DT, dT, DTaP and Tdap vaccines
combining diphtheria, tetanus and pertussis
toxoids in concentrations appropriate to
children and adults. These vaccines can be
viewed as anti-phage or at least anti-phage
gene-product medical treatments, as well as
anti-toxin prophylaxes. Historically, passive
immunity to diphtheria, and therefore treat-
ment, was achieved via administration of
horse antiserum or anti-toxin (Mortimer,
2011), for which von Behring was awarded
the Nobel Prize in 1891. Such treatments,
however, are associated with serum sickness,
as the patients immune system reacts to the
horse serum. Administration of horse-serum
anti-toxin none the less continues to be a
treatment option (Ciok, 2000).
Botulism
The clostridia are a group of Gram-positive,
spore-forming, obligately anaerobic bacteria
that are found especially in soil environ-
ments. Clostridium-associated diseases in-
clude tet anus, gas gangrene and botulism
which are caused by Clostridium tetani,
Clostridium perfringens and Clostridium botu-
linum, respectively along with Clostridium
di cile-associated diarrhoea and pseudo-
membranous colitis. Only in the case of some
(but not all) C. botulinum toxins are any of
these species pathogenicity factors known to
be phage encoded. Further discussion of the
molecular aspects of the later is found in
Christie et al. (Chapter 4, this volume).
The predominant symptom of botulism
is paralysis, rst aecting the head (eyes and
mouth), resulting in vision problems and
di culty in speaking and swallowing. As the
disease progresses, the paralysis descends,
aecting neck, arm and hand movements,
and may also involve the legs. Eventually, the
breathing muscles become impaired, leading
to death due to respiratory failure (Sobel,
2005). The rate and extent of paralysis is
variable, presumably depending on the
toxin dose. While the toxin molecules bind
irreversibly to neurons, the cells will re-
generate over time if the patient survives, and
patients will regain movement over weeks or
months with proper supportive and re-
habilitation treatment.
Botulism typically presents as a food
poisoning. Common sources include home-
canned foods that have not been successfully
heat treated to kill spores (Sobel, 2005).
Despite this common association with
improperly canned foods, however, botulism
is in fact most ofen seen in the USA in infants
who have been fed honey, which is a
consequence of both the presence of C.
botulinum spores in honey and the relatively
high pH of an infants gastric secretions,
which allows the spores to survive and
germinate in the infant. Wound botulism can
also occur when spores from soil contaminate
and then grow within a wound, resulting in
paralysis. Thus, unlike botulism food
poisoning, which is a consequence of in-
gestion of toxin rather than spores, botulism
in infants as well as wound botulism are
consequences of direct exposure to spores.
Although the botulinum toxin is con-
sidered to be the most potent of the known
24 S. Kuhl et al.
neurotoxins, Botox treatments involve the
local injection of the toxin to cause a localized
temporary muscle paralysis, as used for the
treatment of deep facial wrinkles associated
with facial muscle contraction. This could be
considered a medical appli cation of a phage-
encoded toxin, although there are both
phage-encoded and bacterially encoded
botulinum toxins. In 2009, the US Food and
Drug Administration issued a black box
warning that the eects of the botulinum
toxin may spread from the area of injection to
other areas of the body, causing symptoms
similar to those of botulism, including
potentially life-threatening swal lowing and
breathing di culties and even death.
Only two of the seven known botulinum
toxins are encoded by prophage genes. The
other ve are produced from genes in the C.
botulinum genome (see Christie et al., Chapter
4, this volume). This raises the question of the
utility that the toxin could provide to phages
other than supplying some useful function to
bacterial hosts upon lysogen formation
(Abedon and LeJeune, 2005). It may be that
the botulinum toxin in these phages is an
example of a moron more DNA located in
a genome by evolutionarily recent random
acquisition rather than a gene maintained by
selection and adaptation. See Hyman and
Abedon (2008) for additional discussion on
the role of pathogenicity factors in phage-
bacterial interactions.
As with diphtheria, the treatment of
botulism poisoning can involve the admini-
stration of anti-toxin derived from horses. In
addition, the California Department of Health
has developed BabyBIG
, Botulism Immune
Globulin Intravenous (Human) (BIG-IV), for
infants less than 1 year of age. BIG-IV is a
human-derived botulism anti-toxin antibody
preparation puried from pooled plasma
from adults selected for their high titres of
neutralizing antibody against botulinum
neurotoxin types A and B following immuni-
zation with pentavalent botulinum toxoid.
Even with anti-toxin, supportive care, ofen
including weeks on a ventilator for patients
with respiratory failure, is the mainstay of
care.
Unlike diphtheria, no anti-botulism
vaccine is generally available. Botulism
instead tends to be avoided through proper
food handling. The later can include proper
cooking, as the botulinum toxin is
permanently denatured at relatively low
temperatures (well below boiling). Killing
spores requires pressure cooking or auto-
claving at 121C (250F) for at least 3 min.
Providing conditions that prevent the spores
from growing such as an acidic or
osmotically unfavourable environment, e.g.
traditional sugared jams and jellies is also a
successful method of prevention, as is
avoiding feeding infants honey. Notwith-
standing this phage association and indeed
cause of some of this food-borne disease,
phages can alternatively be employed as
protective agents of food supplies, as
discussed by Niu et al. (Chapter 16, this
volume), although not specically against C.
botulinum.
Staphylococcus Infection
Gram-positive bacteria are common
components of the normal ora in humans,
especially on skin (see Loc-Carrillo et al.,
Chapter 13, this volume, for an additional
discussion of human bacterial normal ora).
Among Gram-positive pathogens possessing
phage-associated pathogenicity, perhaps the
most notorious is Staphylococcus aureus. S.
aureus has numerous pathogenicity factors, a
substantial number of which can be phage
encoded (see Christie et al., Chapter 4, this
volume, for additional discussion). Indeed,
an array of phage-encoded pathogenicity
factors can be found in this pathogen: super-
antigens, including toxic shock syndrome
(TSS) toxin; PantonValentine leukocidin
(PVL) toxin; staphylococcal enterotoxins
(associated with food poisoning and TSS)
(Novick et al., 2010) which are discussed
below. Other phage-encoded virulence
factors include staphylokinase, chemotaxis
inhibitory protein, staphylococcal comple-
ment inhibitor and factors associated with
biolm formation (Verkaik et al., 2011; see
also Christie, et al., Chapter 4, this volume).
Although not a direct consequence of
phage-encoded proteins, S. aureus strains are
becoming increasingly resistant to common
antibiotics, with some strains of methicillin-
resistant S. aureus (MRSA) presenting with
very limited antibiotic choices, sometimes
limited to intravenous vancomycin, as oral
vancomycin is not absorbed (but see Williams
and LeJeune, Chapter 6, this volume, for a
brief discussion of the potential role of phages
in antibiotic resistance horizontal gene
transfer, and Hendrickson, Chapter 5, this
volume, for additional discussion). The
coincidence of the acquisition of methicillin
resistance with at least partially phage-
mediated evolution of a virulent community-
acquired strain has focused much public and
medical concern on drug-resistant strains of
S. aureus, especially MRSA. The repeated use
of vancomycin has also resulted in the
appearance of increasingly vancomycin-
resistant and -intermediate-resistant strains.
Drug-sensitive strains of S. aureus still cause
disease and mortality. Penicillin-sensitive
strains, however, are rarely seen except in
recrudescence of infections that originally
occurred before the antibiotic era, such as in
World War II veterans.
S. aureus normally colonizes the skin and
nasopharynx of humans and animals and can
produce a variety of diseases, from relatively
benign skin infections such as folliculitis (hair
follicle infections) and small abscesses to life-
threatening infections such as deep abscesses,
osteomyelitis and bone infections (see Loc-
Carrillo et al., Chapter 13, this volume),
pneumonia, septicaemia, endocarditis and
heart-valve infections (Gordon and Lowy,
2008; Que and Moreillon, 2010). In addition,
several diseases are mediated by toxin
production including staphylococcal scalded-
skin syndrome (SSSS), staphylococcal TSS,
and food poisoning or intoxication.
Skin and lung infections
S. aureus is the major pathogenic cause of
cellulitis (skin infections with diuse
inammation) and of abscesses (collections of
pus). Most people are familiar with an
infected wound, hair follicle or ngernail
containing pus, typically caused by S. aureus.
Recently, the epidemic USA300 strain of S.
aureus has presented with a localized area of
inammation, ofen described as a spider
bite. It may eventually form a pustule, and
may progress to more serious infection
including septic shock.
S. aureus can also cause deep abscesses in
muscle or sof tissue, osteomyelitis or bone
infections, pneumonia, which is ofen
necrotizing, septicaemia, endocarditis and
heart-valve infections. Necrotizing fasciitis
(esh-eating bacteria) is typically caused by
Streptococcus pyogenes but can be caused by S.
aureus. This fasciitis typically presents with
severe pain in a moderate infection, pain that
is typically out of proportion to the physical
signs of infection. Emergency surgical
debridement is indicated in these cases. These
infections may occur due to infected
intravenous lines or intravenous drug abuse,
surgical site infection or infection of a
traumatic wound.
Two forms of disseminated skin in-
fections are associated with the production of
exfoliative toxins including prophage-
encoded exfoliative toxin A. The rst, impe-
tigo, is most commonly seen in children
between 2 and 5 years of age. It presents as an
area with small sores that may be eroded
(non-bullous) or uid lled (bullous, i.e.
consisting of uid-lled blisters) (Stulberg et
al., 2002). Each sore is a site of localized
staphylococcal infection. The blisters in
bullous impetigo are uid lled, painless and
caused by a localized lifing o of the outer
layers of the skin due to the eects of the
exfoliative toxins. Patients will ofen have
fever and malaise. In most cases, impetigo is
treatable with topical antibiotics, although
there is a small risk of systemic infection
developing if the bacteria are able to penetrate
to deeper tissue. Impetigo is sometimes
considered a localized form of SSSS.
SSSS, also known as Riters disease, is a
serious, sometimes life-threatening disease
that presents with widespread erythema
(redness) and large bullae covering large
portions of the skin (Bukowski et al., 2010).
Most commonly seen in children, it is the
bullae that give the appearance of scalded
skin. Like impetigo, patients with SSSS ofen
have fever and lethargy. In contrast to the
bullae seen in impetigo, SSSS bullae ofen do
not contain staphylococci. This can occur
26 S. Kuhl et al.
because SSSS is primarily mediated by toxin
production, ofen from a localized infection.
Further challenges occur as the patient
improves with antibiotic treatment, but
desquamation of the supercial layer of skin
typically occurs. Afer this, it is important to
protect the exposed skin layers from
secondary infections as well as from uid
loss.
The role of phage-encoded toxins in
Staphylococcus infections can be seen explicitly
with PVL. PVL-producing Staphylococcus
strains have been shown to be associated
with severe or invasive skin infections and
pneumonia since PVL was discovered in the
1930s (Gordon and Lowy, 2008; see Christie et
al., Chapter 4, this volume). In the current
epidemic of the community-acquired MRSA
strain USA300, expression of PVL appears to
correlate with more-rapid onset and increased
severity of symptoms, as seen within the rst
3 days of infection, although it does not
increase the virulence of this strain in animal
studies (Diep and Oto, 2008; Kobayashi et al.,
2011). Frequently, human skin infection with
USA300 appears as an area of redness or
erythema, followed by localized tissue
necrosis. This occurs without much pus,
unlike typical purulent staphylococcal
infections, which can cause PVL-containing
infections to present like spider bites.
Staphylococcal pneumonias are ofen ful-
minant with high fevers, and necrotizing
pneumonias are not uncommon. Pneumonia
typically presents with shortness of breath,
cough and fever. Necrotizing fasciitis has also
been described with this strain.
Gastrointestinal intoxication
Enterotoxins are associated with staphyl-
ococcal food poisoning that can occur upon
consumption of S. aureus-contaminated food
or drink. This food poisoning presents 26 h
afer eating with nausea, vomiting, diarrhoea
and abdominal cramping. Symptoms can
also include prostration, hypotension, head-
ache and muscle cramps, and typically
resolve within 612 h. Treatment is
rehydration, orally if possible, intra venously
if severe. Foods may be con taminated during
preparation especially those that are typically
prepared by hand. Milk and cheese have
also been sources of infection. Because
Staphylococcus is salt tolerant, salted foods
such as ham or sh may be a source of
infection. Even cooked foods may remain
contaminated with staphy lococcal toxins,
some of which are heat stable, although the
bacteria are killed during cooking (CDC,
2006). Staphylococcal entero toxins A and P
are typically phage encoded and are
associated with staphylococcal food poison-
ing.
Staphylococcal enterotoxin A, a prophage-
encoded protein, is also a superantigen
(Balaban and Rasooly, 2000). This means it
binds directly to MHC class II molecules in a
non-specic manner and activates T cells,
resulting in a massive cytokine response,
causing the patient to develop systemic
inammatory response syndrome (SIRS). SIRS
can occur in response to sepsis or other
methods of cytokine activation such as burns
or massive trauma.
Toxic shock syndrome
Another staphylococcal disease that is
predominantly toxin-mediated is TSS. In TSS,
as with generalized SSSS, the infection may
remain localized, but the toxaemia causes
systemic eects, such as skin exfoliation and
SIRS. TSS is associated with S. aureus-
produced exotoxins, especially the epony-
mous toxic shock syndrome toxins. In the
1980s, TSS was associated with super-
absorbent tampons, which were inadvertently
well suited to supporting S. aureus growth
(and subsequently were removed from the
marketplace). TSS continues to occur in post-
surgical and other sporadic infections.
Wounds infected with S. aureus may not be
inamed, presumably due to the toxin
inhibiting the inux of macrophages.
The presentation of TSS is variable and is
thought to relate to the specic toxins
excreted by particular infecting strains. TSS
patients typically present with localized pain,
high fever, hypotension (low blood pressure)
and sometimes fatigue and confusion
(Stevens, 1995). TSS can quickly progress to
include an erythematous rash (sometimes
described as sunburn-like in appearance, but
ofen with macules, or spots), and ofen with
the involvement of other organ systems
resulting in vomiting, diarrhoea, myalgias
(muscle pain), low platelet counts, confusion,
renal insu ciency and/or hepatic inam-
mation. Treatment includes removal of the
infection focus, antibiotics, supportive care,
ofen including 1020 l of intravenous uid,
and vasopressors to counter the hypotension
due to capillary leakage.
Streptococcus Infection
Members of the genus Streptococcus are
another Gram-positive pathogen for which
phages play important roles in pathogenicity
(see Christie et al., Chapter 4, this volume). S.
pyogenes (group A Streptococcus or GAS) is the
major pathogen, but other species also have
phage-encoded virulence factors. GAS has
multiple clinical presentations, including
streptococcal pharyngitis or sore throat,
cellulitis and occasionally abscesses, necro-
tizing fasciitis, scarlet fever and TSS
(sometimes described as toxic shock-like
syndrome to dierentiate it from the
syndrome caused by S. aureus). Other
streptococcal skin and sof-tissue infections
caused include cellulitis, erysipelas (which
presents with raised demarcated bright-red
lesions), necrotizing fasciitis, and myositis or
myonecrosis. The clinical presentation
depends on the combination of infection site
and which virulence factors are produced by
the par ticular infecting strain. Additional
strepto coccal species containing toxigenic
prophages include group C and G streptococci
and Streptococcus mitis. These species typically
colonize the respiratory and gastrointestinal
tract, but can cause infections similar to those
caused by GAS, as well as endocarditis,
osteomyelitis and infections at other body
sites.
Streptococcal virulence factors include
adhesion factors, superantigens, lipase,
DNase, streptokinase and hyaluronidase
(Hynes et al., 1995; Banks et al., 2005). The
streptococcal pyrogenic exotoxins (Spes) are
a family of superantigens that cause
symptoms of systemic shock, similar to the
superantigens in S. aureus. With only a few
exceptions, these are all encoded by genes
found in prophages (see Christie et al.,
Chapter 4, Table 4.2, this volume, for specic
references for each Spe). Spes are believed to
be particularly associated with streptococcal
TSS, necrotizing fasciitis and other severe
streptococcal infections (Bisno and Stevens,
2010).
Streptococcal TSS is toxin mediated, as is
staphylococcal TSS, and presents with a
strepto coccal infection associated with
sudden onset of SIRS. Streptococcus, like
Staphylococcus, can also present with
bacteraemia and/or superantigen-mediated
activation of a cytokine response. This is
related to the SIRS, which is ofen seen in
conjunction with sepsis or septicaemia.
Typically, these patients may also present
with fever, chills, hypotension and tachy-
cardia (rapid heart rate). The septic patient is
ofen delirious and confused, in part because
the brain is poorly perfused due to vascular
leakage and hypotension.
Additional phage-encoded proteins
include hyaluronidase, DNase, streptokinase
and SpeB (a potent protease), all of which are
thought to facilitate the liquefaction of pus
and spread of streptococci through tissue
planes. DNases are thought to also help resist
neutrophil extracellular trap (NET)-mediated
killing by neutrophils. SpeB in addition
cleaves IgG bound to GAS, interfering with
ingestion and killing by phagocytes.
SpeA and SpeC are known as the
scarlatinal toxins due to their association with
scarlet fever. Scarlet fever results from
infections with strains that secrete these
pyrogenic exotoxins. This disease is most
ofen associated with streptococcal pharyn-
gitis or sore throat, but can follow wound or
other infections. Scarlet fever usually aects
children between the ages of 5 and 18, but is
sometimes seen in adults. Typically, scarlet
fever presents with fever, strawberry tongue
and a ne erythematous rash that spares the
face but is worse in skin folds (known as
Pastia lines) (CDC, 2011c).
In contrast to S. aureus, S. pyogenes
remains exquisitely sensitive to penicillin, a
cell-wall synthesis inhibitor, which as a
28 S. Kuhl et al.
consequence ofen remains the drug of
choice, although once-daily cephalosporins
are sometimes used for ease of delivery with
a negligible decrease in e cacy. In cases of
severe streptococcal infection, such as
necrotizing fasciitis, the protein synthesis
inhibitor clindamycin is typically added to
inhibit the exotoxin production by the
bacteria. The failure of penicillin in cases of
high inoculum (the inoculum eect) is
thought to be due to the ineectiveness of
penicillin against stationary-phase bacteria.
Pasteurella Infection
Pasteurella are Gram-negative bacteria that
are responsible for zoonotic infections of
humans, including afer bites by pets or by
wild animals (Zurlo, 2010). Infections are
sometimes seen afer licking of the patients
skin by a pet, especially where there is a break
in the skin such as afer injury or surgery,
including afer knee or hip replacement
(Heydemann et al., 2010). Morbidity is also
seen in infected animals.
Infections most commonly present as a
severe inammatory cellulitis or diuse
inammation of the skin around the bite or
wound site, and are caused by the species
Pasteurella multocida. Some strains of P.
multocida produce P. multocida toxin, a phage-
encoded exotoxin that can rapidly cause
severe inammation and swelling at the site
of the bite, sometimes necessitating surgical
debridement (Pullinger et al., 2004). P.
multocida remains antibiotic sensitive, usually
treated by amoxicillin-clavulanate in mild
cases, or in severe cases with an intravenous
antibiotic such as a |-lactam/|-lactamase
inhibitor or a carbapenem.
Diarrhoeal diseases
Diarrhoeal diseases are the second leading
cause of worldwide morbidity and mortality
due to infectious diseases, afer pneumonia
and other lower respiratory tract diseases.
According to the World Health Organization
(WHO, 2008), there were over 2 million
deaths due to diarrhoeal diseases in 2004.
These typically present with loose, watery
and frequent stools that may be bloody or not
depending on the pathogen. In severe disease,
such as some cases of cholera, the profuse
amounts of watery diarrhoea can cause
profound and sometimes fatal dehydration.
Treatments range from oral rehydration with
uid and electrolytes to intravenous
rehydration, antibiotics and in some cases
renal dialysis due to toxin eects on the
kidneys.
Cholera
Cholera is considered to be one of the great
plagues that has had a tremendous impact on
human history (Bray, 1996; Sherman, 2006). In
the recent cholera epidemic in Haiti, patients
became rapidly dehydrated in as litle as 2 h,
resulting in death when rehydration was not
available. Cholera stools are described as rice
water stools because the ecks of mucous,
intestinal epithelial cells and bacteria appear
similar to rice. Phages play a direct role in the
pathology of cholera, as the major cholera
toxin, CT, is encoded by a gene in CTX|, a
temperate lamentous phage that forms a
stable lysogen in Vibrio cholerae (Waldor and
Mekalanos, 1996; see also Christie et al.,
Chapter 4, this volume).
Cholera is most commonly spread by
faecal contamination of water and therefore
is best controlled by properly functioning
water treatment plants or other water
purication systems. As a consequence, the
last cholera outbreak in the USA occurred in
1910, killing 11 people in New York City. In
parts of the world with seasonal ooding,
regular outbreaks are seen when ooding
brings a connection between water supplies
and contaminated waste water. There also
appears to be a role of phages in at least some
of these outbreaks. Seasonal epidemics of
cholera in the waters of Bangladesh, for
example, appear to be controlled by lytic
bacteriophages (Faruque et al., 2005). One of
the earliest applications of phages to
populations, rather than individuals, in fact,
took place when dHrelle and others treated
cholera in India (Morison, 1932; dHrelle,
1946).
The mainstay of cholera treatment of
individual patients is rehydration with uid
and electrolytes, orally if possible and
intravenously if not. Antibiotics such as
tetracyclines and uoroquinolones will
shorten the duration of diarrhoea by about
50% and shorten the duration of Vibrio
shedding by about 1 day. Macrolide
antibiotics are typically used in children and
pregnant women. Outside the USA, there are
two licensed commercial oral vaccines. The
killed whole-cell B-subunit vaccine, WC-rBS
(Dukoral
), contains killed whole cells of V.

cholerae O1 plus recombinant cholera toxin B
subunit. It is WHO pre-qualied, allowing it
to be licensed in over 60 countries. Dukoral
also provides some protection against
infection with enterotoxigenic E. coli. The
second type of vaccine is a modied bivalent
whole-cell-type vaccine produced in two
countries. These mORCVAX and Shanchol
vaccines contain killed whole cells of V.
cholerae O1 and V. cholerae O139. These
vaccines are licensed in India and Vietnam,
respectively, and appear to be safe and
eective in endemic populations (Lopez et al.,
2008; CDC, 2010).
Shiga toxin-associated diseases
Dysentery is diarrhoea with blood or mucous
in the stool. The two bacteria that cause
bloody diarrhoea are Shigella and E. coli
strains such as O157:H7 (see Christie et al.,
Williams and LeJeune, Goodridge and
Steiner, and Niu et al., Chapters 4, 6, 11 and
16, this volume, for further discussion,
particularly of E. coli O157:H7). Shigella
typically causes fever and acute abdominal
pain, and can also cause severe dehydration
as well as haemolyticuraemic syndrome
(HUS) due to the production and systemic
release of Shiga toxin. Severe disease in
children can cause seizures, encephalopathy
(brain disease with headache, confusion and
lethargy) and sti neck. HUS leads, in some
cases, to a problem with coagulation called
disseminated intravascular coagulation
(DIC). DIC can result in a layer of brin
forming in the glomerular capillary bed.
Acute renal failure is also seen with HUS and
DIC due to a combination of the eects of
Shiga and other toxins. HUS, DIC and renal
failure are most likely to occur in children,
the elderly and pregnant women. Renal
failure can become chronic, requiring dialysis
or kidney transplantation.
Shiga-like toxins (Stx) in E. coli are
typically located in the genome of one of
several lambdoid prophages. The bacteria
lack an export mechanism for the Stx so
release of the toxin usually follows induction
of the prophage, which then lyses the cell as
part of the phage lytic cycle. The recent
outbreak of a Stx-producing E. coli in
Germany appears to be the result of a toxin-
producing phage infecting an entero-
aggregative E. coli O104:H4 strain (Rasko et
al., 2011), which otherwise tend to be less
virulent, although still causing chronic
diarrhoea.
Treatment of shigellosis (Shigella-caused
dysentery) typically consists of hydration in
combination with supportive care along with
antibiotic treatment, typically with ampicillin
or trimethoprim-sulfamethoxazole. This
appears to shorten the duration of diarrhoea
and microbial shedding. In contrast, sup-
portive care and monitoring for HUS and
DIC is the mainstay of treatment of
enterohaemorrhagic E. coli (EHEC). Antibiotic
treatment of EHEC in particular is not
recommended, as it can induce the expression
and release of Stx (Wong et al., 2000). Targeting
the toxin directly using hyperimmune anti-
toxin antisera or oral toxin-binding agents
has been proposed but remains experimental
(Gupta et al., 2011).
Pseudomonas Infections
Pseudomonas aeruginosa contains many
prophages that appear to be involved in
serotype conversion (Christie et al., Chapter 4,
this volume). P. aeruginosa strain 158, for
example, contains a cytotoxin (a leukocidin)
that is encoded by the temperate phage CTX
(Hayashi et al., 1990). More generally,
Pseudomonas is a soil organism and tends only
to be an opportunistic pathogen, rarely
causing infection in healthy people except by
inoculation, such as with a rusty nail through
30 S. Kuhl et al.
a tennis shoe. Pseudomonas, however, can
cause severe wound and post-surgical in-
fections, as well as pneumonias. Pneumonias
are seen especially with patients undergoing
respiratory therapy or on ventilators. P.
aeruginosa in addition can infect immuno-
compromised persons and the elderly. These
infections are complicated by the innate
antibiotic resistance of P. aeruginosa and
related species, with resistance to multiple
antibiotics commonly seen in samples
isolated from patients.
P. aeruginosa is a particularly serious
pathogen for persons with the genetic defect
causing cystic brosis. The respiratory tracts
of these patients are ofen colonized rst by S.
aureus and later, afer antibiotic treatment,
with P. aeruginosa (and sometimes sub-
sequently with Burkholderia cepacia). Pseudo-
monas infections tend to develop into chronic
infections that are treated intermitently to
reduce colony counts. In these cases,
Pseudomonas usually develops a mucoid
phenotype due to increased production of
alginate, which causes a chronic lung
infection in cystic brosis. P. aeruginosa and
related species possess an innate antibiotic
resistance and are becoming increasingly
resistant to the few antibiotics that are
currently used to treat these infections.
Doctors have resorted, as a consequence, to
again using more toxic antibiotics such as
colistin. See, however, Burrowes and Harper
(Chapter 14, this volume) for discussion of
the use of phage therapy against various
Pseudomonas infections.
Conclusion
Phages can be viewed as playing at least four
roles in humans and other environments.
They are an important component of the
normal ora (see Letarov, Chapter 2, this
volume), they can strongly inuence the
evolution of bacteria (see Hendrickson,
Chapter 5, this volume), they can be employed
in various guises as antibacterial agents (i.e.
phage therapy; see the last section of this
volume), and, as considered in this chapter,
they can contribute directly to bacterial
diseases. The last is a consequence of
prophages encoding bacterial pathogenicity
factors including exotoxins (see Christie et al.,
Chapter 4, this volume). It is important to
note, however, that these bacterial pathogens
ofen possess non-phage-encoded bacterial
virulence factors as well, although the
acquisition of these too, if a consequence of
horizontal transfer, can be due to the phage-
mediated process of generalized transduction.
Respectively, and metaphorically, phages
thus may be viewed, under these dierent
circumstances, as presenting to humans a
multitude of qualities including serving as
benign neighbours (normal ora; Letarov,
Chapter 2, this volume), scheming anarchists
(Hendrickson, Chapter 5, this volume),
trusted police (phage therapy) and outright
criminals (Christie et al., Chapter 4, this
volume).
Here, we have considered the con-
sequences especially of phage criminal
behaviour in the guise of prophages
encoding bacterial virulence factors. In some
cases, such as infection by opportunistic soil
bacteria such as C. botulinum, the phages
appear to be accidental or incidental
criminals. In more professional pathogens
such as C. diphtheriae, S. aureus and V. cholerae,
the phages appear to be the major contributors
to the criminal behaviour of their bacterial
hosts, coding for major pathogenicity factors.
Indeed, a common theme throughout this
monograph is the idea that bacteria in fact
can be very bad indeed, although as recounted
particularly in Chapters 617, much good can
come instead from human harnessing of
bacteriophages for the detection, prevention
and treatment of disease.
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4 Prophage-induced Changes in Cellular
Cytochemistry and Virulence
Gail E. Christie
1
, Heather E. Allison
2
, John Kuzio
3
, W. Michael
McShan
4
, Matthew K. Waldor
5
and Andrew M. Kropinski
6
1
Molecular Biology and Genetics, School of Medicine, Virginia Commonwealth
University;
2
Department of Functional and Comparative Genomics, University of
Liverpool;
3
Department of Microbiology and Immunology, Queens University;
4
Department of Pharmaceutical Sciences, The University of Oklahoma;
5
Department
of Medicine, Brigham and Womens Hospital;
6
Department of Molecular and Cellular
Biology, University of Guelph; and, Public Health Agency of Canada, Laboratory of
foodborne Zoonoses.
Lysogenic conversion has been recently
dened as a phage-associated heritable
change in the host cells genotype and
phenotype that is independent of the eects
expected from repression and integration. In
other words, phenomena that are directly
associated with lysogenization such as
immunity to superinfection (a function of the
prophage repressor) and loss of function
resulting from insertion into a host gene (a
function of the integrase) are expected
consequences of lysogenization and should
not be dened as lysogenic conversion (Los
et al., 2010). We now realize that this denition
is inadequate, as transcriptomic analysis has
revealed that the molecular impact of
lysogenization can be considerable (Su et al.,
2010) and that even virulent phages may
induce transient changes in the cells
phenotype (Busch et al., 2011). Therefore, we
suggest that the term lysogenic conversion
or phage conversion always be used in
conjunction with an explicit description of
the phenotypic change observed, as in phage
conversion of serotype or lysogenic
conversion to toxigenicity. What we will
discuss in this chapter is the impact of non-
essential temperate phage genes, variously
described as morons (Juhala et al., 2000) or
cargo genes (Brssow et al., 2004), on host
expression that leads to a phenotypic change
in virulence or in surface chemistry. The
specic emphases will be on the production
of secreted toxins by Corynebacterium, Clostri-
dium, Escherichia, Staphylococcus, Streptococcus
and Vibrio, the synthesis of membrane
proteins, and alterations in lipopolysaccharide
(LPS) structure in Salmonella and Pseudomonas.
We will conclude with a brief discussion of
the concept of phage-mediated transfer of
bacterial genes, or transduction.
Corynebacterium
Working with bacteriophage B, isolated by S.
Toshach (School of Hygiene, Toronto,
Canada) from a virulent culture of Cory-
nebacterium diphtheriae, V.J. Freeman was able
to demonstrate that this virus plaqued on
avirulent strains of the bacterium, converting
them to stable lysogenicity and toxinogeny
34 G.E. Christie et al.
(Freeman, 1951; Freeman and Morse, 1952).
Phage is directly descended from Toshachs
isolate, and is the virus that, along with
corynebacteriophage , has received the most
atention. It is a temperate, UV-inducible
member of the family Siphoviridae (Holmes
and Barksdale, 1970). The tox gene has been
found to be located adjacent to atP (Groman,
1984).
Most strains of C. diphtheriae are
lysogenic. An extended group of prophages
capable, upon induction, of plaque formation
have been isolated from C. diphtheriae and
Corynebacterium ulcerans (phage L). One can
recognize several subgroups within the ve
dierent immunity classes: group A (o
Tox+
,
|
Tox+
, P
Tox+
, e
Tox+
), group B (
Tox-
, t
Tox+
) , group
D (o
Tox+
, L
Tox+
) and two unique Tox
phages
and K (Holmes and Barksdale, 1970). All of
these phages showed similar restriction
paterns, and, with the exception of phage o,
extensive DNA sequence homology to phage
| (Rappuoli et al., 1983; Buck et al., 1985).
The next member of this group was
phage (gamma) (Groman, 1955). This phage
is serologically related to |, displays similar
restriction paterns, and heteroduplex
analyses indicate that they are 99% similar
(Buck et al., 1978), yet they are heteroimmune
and is Tox
.
Clostridium
Botulinum neurotoxins (BoNTXs) are associ-
ated as a high-molecular-weight complex
with haemagglutinin (HA1, HA2, HA3a and
HA3b) and a non-toxic non-haemagglutinin
protein. BoNTXs are classied into seven
antigenic groups, A, B, C1, D, E, F and G (Los
et al., 2010).
Lysogenic conversion to toxin production
in Clostridium botulinum was rst shown by K.
Inoue and H. Iida (Inoue and Iida, 1970,
1971), and was conrmed by M. Eklund and
co-workers (Eklund et al., 1971, 1972). They
suggested that botulinum toxin production
following phage infection was analogous to
the C. diphtheriae system with an important
dierence botulinum toxin production was
a pseudolysogenic conversion: toxigenicity
and lysogeny were lost upon serial culti-
vation, particularly in the presence of
antiphage sera (Oguma, 1976). C. botulinum-
converting phage have been classied by the
antigenic nature of the toxin they produce
(Oguma et al., 1976; Sugiyama, 1980; Simpson,
1981). Therefore, intraspecies conversions
between antigen types is possible by infecting
cured toxigenic strains (Tox
) with the
appropriate phage (Eklund and Poysky,
1974). It was also demonstrated that
Clostridium novyi strains are capable of
producing a variety of potent, antigenically
distinguishable toxins (Eklund et al., 1976).
Again, positive correlation was observed
with specic temperate phage infections in
dierent strains of C. novyi leading to the
production of a characteristic toxin (Eklund et
al., 1974; Schallehn et al., 1980). Interspecies
conversions between C. botulinum and C.
novyi strains have been reported (Eklund et
al., 1974).
We have now the sequences of numerous
C. botulinum strains (Sebaihia et al., 2007;
Smith et al., 2007c; Skarin et al., 2011). Only in
the case of C. botulinum groups C and D is
there a demonstrable relationship between
prophages and toxigenicity. C. botulinum type
C strain Stockholm and type D strain 1873
harbour UV- and mitomycin C-inducible
serologically unrelated prophages c-st and
D-1873, respectively. Strain C-203 harbours a
prophage that is antigenically identical to
D-1873, yet carries a C-type neurotoxin
(Oguma, 1976). These viruses are unusually
large members of the family Myoviridae
(Inoue and Iida, 1968). In the lysogenic state,
these temperate phages exist as plasmid
prophages. The 185.7 kb genome of c-st has
been completely sequenced (Sakaguchi et al.,
2005), while the genome of D-1873 exists in
GenBank as eight fragments totalling 142.1 kb
(NZ_ACSJ01000012.1 NZ_ACSJ01000019.1).
These two phages show only limited DNA
sequence similarity. The 203.3 kb plasmid
p1BKT015925 (Skarin et al., 2011) also carries
the BoNTX operon and shows signicantly
more regions of sequence similarity to phage
c-st than to D-1873. In the other botulinum
groups, the BoNTX operons are not associated
with phage-like elements.
In addition to the BoNTX operons, both
of these phages encode a 25 kDa basic toxin,
Prophage-induced Changes in Cellular Cytochemistry and Virulence 35
exoenzyme C3, which possesses N-ADP-
ribosyltransferase activity. This protein is also
present in a sequence (NCBI protein accession
no. A46957) from Clostridium limosum (Just et
al., 1992), which is listed, without evidence, as
being from an unnamed phage and on
plasmid p1BKT015925 (Skarin et al., 2011).
The gene for C3 exotoxin lies almost 60 kb
downstream of the botulinum toxin gene and
is anked by two identical 1.9 kb regions
corresponding to insertion sequence (IS)
OrfAB-like transposases. Hauser and col-
leagues suggested that phage c-st contains a
large transposable element (Hauser et al.,
1993). The complete DNA sequence of c-st,
however, indicates that the intra-IS region
contains genes encoding virion structural
proteins.
Staphylococcus
The pathogenesis of Staphylococcus aureus is
complex and involves the production of an
array of virulence factors that allow the
organism to cause infection at a variety of
sites and to cause multiple disease symptoms
(see Kuhl et al., Chapter 3, this volume).
Staphylococcal virulence factors include
secreted proteins such as cytotoxins, super-
antigens and tissue-degrading enzymes, cell
surface-bound proteins that promote
adhesion to cell surfaces, internalization and
resistance to host defences, and components
of the cell-wall peptidoglycan and poly-
saccharide capsule. With the exception of the
cell-wall components, which are also essential
housekeeping functions, these virulence
factors are accessory proteins that are
dispensable for bacterial growth. The genes
encoding such accessory factors are generally
carried on mobile genetic elements, and the
evolution of new pathogenic strains of S.
aureus has been atributed to the accumulation
of mobile genetic elements encoding
virulence factors and resistance determinants
into successful lineages (Novick, 2006;
Lindsay, 2010).
Phages play a major role in staphylococcal
virulence; they not only provide a vehicle for
generalized transduction but have also been
implicated in lysogenic conversion and serve
as helpers for the high-frequency mobilization
of a family of superantigen pathogenicity
islands (SaPIs). Sequence analysis of S. aureus
genomes reveals that temperate bacterio-
phages are widespread; virtually all
sequenced strains carry one to four
prophages. S. aureus prophages have been
shown to carry and/or interrupt virulence
genes, so both prophage establishment and
prophage loss can result in increased
expression of a virulence trait. Resident
staphylococcal prophages (and the SaPIs they
mobilize) can be induced by a variety of
environmental conditions that lead to
induction of the SOS response (Wagner and
Waldor, 2002; Goerke and Wolz, 2010).
Mobilization of temperate phages has
been proposed to play an important role in
the evolution of virulent clinical isolates, and
a growing body of evidence supports the
ongoing occurrence of complex phage
dynamics. Not only do closely related
methicillin-resistant S. aureus (MRSA) isolates
exhibit dierent prophage complements
(Goerke et al., 2009; Nbel et al., 2010), but
horizontal phage transfer as well as intragenic
translocation of prophages to dierent
chromosomal integration sites appears to
occur at a higher frequency during host
infection by S. aureus (Goerke et al., 2006;
Goerke and Wolz, 2010). In addition, deletion
of resident prophages reduced the virulence
of S. aureus strain Newman in a mouse model
of infection (Bae et al., 2006), consistent with a
direct contribution of lysogenic conversion to
virulence.
A large-scale genome analysis of
staphylococcal phages dened three dierent
phage classes, distinguished by genome size
and virion morphology (Kwan et al., 2005).
The vast majority were members of a large
family of related temperate Siphoviridae,
while the remainder were either Podoviridae
or Myoviridae. All of the known staphylococcal
transducing phages and converting phages
belong to the family Siphoviridae, and many
more examples have been provided by the
sequences of prophages in staphylococcal
genomes. Despite variations in virion
morphology, sequence analysis reveals that
these phages share a common modular
genetic organization and display a mosaic
patern of relatedness at the genetic level
(Kwan et al., 2005; Kahankova et al., 2010).
Classication schemes for these siphophages
have been proposed based on conservation of
morphogenetic genes (Pantucek et al., 2004)
and the integrase (Lindsay and Holden, 2004;
Goerke et al., 2009). A recent multilocus PCR
strategy for classication is based on up to
seven discrete genomic modules, which
appear to reassort freely among the dierent
members of this group (see Fig. 4.1;
Holochova et al., 2010; Kahankova et al., 2010).
There is one recently described temperate
prophage that appears to dene an additional
class of staphylococcal phages (Holden et al.,
2010). It is similar in sequence to the large
RP62a prophage previously identied in
Staphylococcus epidermidis (Gill et al., 2005)
and carries putative virulence genes, but
whether it is capable of autonomous
replication is not known.
In most cases, the genes for phage-
encoded virulence factors lie at the ends of
the prophage genomes (Fig. 4.1). While this is
Integrase
(10)
Lysogeny
control
(5)
Replication
(4)
Transcription
regulation
(4)
Head-tail
and packaging
(8)
Tail appendages
(4)
Lysis
(4)
sau421 S sau421 RM
sek2 seg2
sea
sea
sep
sep
sea-ov
sak
sak
sak
sak
sak
sak
chp
chp
chp
chp
sch
sch
sch
sch
sch
sch
sch
ear
eta
lukS-PV lukF-PV
lukF-PV
lukF-PV
lukS-PV lukF-PV
lukM
lukM
Fig. 4.1. Location and organization of staphylococcal conversion genes. Shown across the top is a
prophage map of a typical member of the S. aureus Siphoviridae, with genes in the seven different
modules dened by Kahankova et al. (2010) distinguished by different shading. Numbers in parentheses
below each module indicate how many types of each module have been dened thus far. Expanded
views of conversion genes at the left and right ends show the integrase or holin and lysin genes,
respectively, in black, with the type of module for each specied. Genes in white encode known
conversion functions, as indicated; genes in grey encode other (or unknown) functions. The seven
different types of immune evasion cluster (IEC; see text) were dened by van Wamel et al. (2006). The
lysis region in |Sea-ov contains two open reading frames, one homologous to the N-terminal end of the
ami1 type of amidase and the other homologous to the C-terminal end of ami1 (denoted ami1* and
*ami1, respectively), separated by an unrelated open reading frame.
suggestive of acquisition by aberrant
prophage excision, similar to the generation
of specialized transducing phages, it is also
possible that these are the locations in the
phage genome where insertion of foreign
DNA is not aected by and does not interfere
with expression of essential phage lytic
functions.
Lysogenic conversion
The earliest reports of lysogenic conversion
aecting virulence gene expression in S.
aureus were the loss of the exoproteins
|-haemolysin (Winkler et al., 1965) and lipase
(Rosendal et al., 1964) as the result of prophage
integration into the hlb and geh genes,
respectively (Coleman et al., 1986; Lee and
Iandolo, 1986). While this type of negative
conversion does not strictly t the denition
of lysogenic conversion as described for this
chapter, it is mentioned here because it has
been documented extensively in staphylococci
and it leads to direct eects on exotoxin
expression. Numerous phages have an
atachment site in hlb (Coleman et al., 1986,
1989; Zabicka et al., 1993; Sumby and Waldor,
2003; Dempsey et al., 2005; Goerke et al., 2006;
van Wamel et al., 2006; Kumagai et al., 2007;
see Table 4.1). The known hlb-converting
phages share a conserved integrase and lysis
module but are otherwise quite diverse
(Kahankova et al., 2010). The precise
contribution of the hlb gene product to
staphylococcal disease has not been clearly
established, but this protein, also known as
sphingomyelinase C, is produced in large
quantities by a number of staphylococcal
strains (Dinges et al., 2000). As many
characterized hlb-converting phages also
carry phage-encoded virulence factors, it has
been suggested that the integration of phages
into this site during infection leads to spliting
of the bacterial population into subfractions
exhibiting dierent virulence potentials.
The lipase activity encoded by the geh
gene has been reported, at least in vitro, to
aect phagocytosis of S. aureus by cells
involved in the immune response (Braconier
and Rollof, 1991; Izdebska-Szymona et al.,
1992). No known virulence factors or other
lysogenic conversion genes have yet been
identied in this group of phages.
A variety of staphylococcal virulence
factors and other functions are carried on
prophages and function as typical lysogenic
conversion genes. The roles and regulation of
these prophage-encoded genes have been
reviewed in detail recently (Los et al., 2010)
and are therefore only summarized below.
Exfoliative toxin A
Exfoliative toxins (ETs) are exoproteins
encoded by S. aureus strains that cause
blistering skin diseases, including bullous
impetigo and staphylococcal scalded skin
syndrome. These toxins are glutamate-
specic serine proteases that specically
cleave a single peptide bond between
cadherin repeats in the extracellular region of
desmoglein 1 (Dsg1) (Nishifuji et al., 2008),
leading to a loss of cellcell adhesion between
keratinocytes and subsequent epidermal
spliting. Of the three isoforms of ET
implicated in human disease (ETA, ETB and
ETD), only ETA has been shown to be carried
on a phage (Ladhani et al., 1999).
Leukotoxins
Leukotoxins are two-component, |-barrel
pore-forming cytotoxins that assemble into
oligomeric transmembrane complexes in
polymorphonuclear leukocytes. This acti-
vates calcium channels and leads to an inux
of divalent cations and ultimately to cell lysis
(Finck-Barbancon et al., 1993; Staali et al.,
1998). In addition, sublytic concen trations of
leukotoxins activate neutrophils and mono-
cytes, leading to the release of inammatory
mediators (Knig et al., 1995). Lysogenic
conversion of S. aureus to leuko cidin pro-
duction was rst reported in 1972 (van der
Vver et al., 1972), and two of the leukotoxins,
PantonValentine leukocidin (PVL) and
lukM/F', are known to be phage encoded.
PVL, rst described in 1932 (Panton et al.,
1932), is comprised of subunits LukS-PV and
LukF-PV and targets phagocytic cells. It
shows specicity for cells of human and
Table 4.1. Summary of S. aureus-converting phages for which the complete phage/prophage sequence
is available. The serotype (as dened by the morphogenesis module) and type of integrase and lytic
module (Kahankova et al., 2010) is shown for each phage. Phages are grouped by the conversion
functions carried (or genes interrupted).
Phage or
prophage GenBank accession no.
Sero-
type
Int
module
Lysis
module Negative
Positive
conversion
ETA NC_003288 (Yamaguchi et al.,
2000)
B Sa1 ami4 eta
ETA2 NC_008798 B Sa1 ami4 eta
ETA3 NC_008799 B Sa1 ami4 eta
SLT NC_002661 (Narita et al.,
2001)
A Sa2 ami2 lukS-PV,
lukF-PV
Sa2mw NC_003923 (Baba et al., 2002) A Sa2 ami2 lukS-PV,
lukF-PV
2958PVL NC_011344 (Ma et al., 2008) A Sa2 ami2 lukS-PV,
lukF-PV
Sa2USA NC_007793 (Diep et al., 2006) A Sa2 ami2 lukS-PV,
lukF-PV
SLT-USA300 CP000730 (Highlander et al.,
2007)
A Sa2 ami2 lukS-PV,
lukF-PV
tp310-1 NC_009761 F Sa2 ami2 lukS-PV,
lukF-PV
PVL108 NC_008689 (Ma et al., 2006) F Sa2 ami2 lukS-PV,
lukF-PV
PVL-CN125 NC_012784 F Sa2 ami2 lukS-PV,
lukF-PV
PVL NC_002321 (Kaneko et al.,
1998)
F Sa2 ami2 lukS-PV,
lukF-PV
PV83 NC_002486 (Zou et al., 2000) F Sa5 ami3 lukM, lukF-PV
Saov3 CP001996 (Guinane et al.,
2010)
F Sa5 ami3 lukM, lukF-PV
Sa3mr(252) NC_002952 (Holden et al.,
2004)
F Sa3 ami3 hlb IEC-A
NM3 NC_008617 (Bae et al., 2006) F Sa3 ami3 hlb IEC-A
13 NC_004617 (Iandolo et al.,
2002)
F Sa3 ami3 hlb IEC-B
tp310-3 NC_009763 F Sa3 ami3 hlb IEC-B
Sa3USA NC_007793 (Diep et al., 2006) F Sa3 ami3 hlb IEC-B
|C-USA300 CP000730 (Highlander et al.,
2007)
F Sa3 ami3 hlb IEC-B
SA3(JKD6159) CP002114 (Chua et al., 2010) F Sa3 ami3 hlb IEC-B
Sa3JH1 NC_009632 F Sa3 ami3 hlb IEC-B
Sa3JH9 NC_009487 F Sa3 ami3 hlb IEC-B
Sa3(TW20) FN433596 (Holden et al., 2010) F Sa3 ami3 hlb IEC-B
Mu3A NC_009782 (Neoh et al., 2008) F Sa3 ami3 hlb IEC-D
Mu50A NC_002758 (Kuroda et al.,
2001)
F Sa3 ami3 hlb IEC-D
Sa3ms(476) NC_002953 (Holden et al.,
2004)
F Sa3 ami3 hlb IEC-D, sek2,
seg2
Sa3mw NC_003923 (Baba et al., 2002) F Sa3 ami3 hlb IEC-D, sek2,
seg2
Sa3(T0131) CP002643 F Sa3 ami3 hlb IEC-D
Mu3A NC_009782 (Neoh et al., 2008) F Sa3 ami3 hlb IEC-D
Mu50A NC_002758 (Kuroda et al.,
2001)
F Sa3 ami3 hlb IEC-D
rabbit origin but not those of mouse, rat or
catle (Szmigielski et al., 1998). The lukM/
F'toxin, comprised of subunits LukM and
LukF'-PV, is associated with mastitis in
ruminants (Choorit et al., 1995) and shows a
binding specicity for bovine leukocytes
(Fromageau et al., 2010).
PVL has been associated with strains
causing both severe skin infections and
necrotizing pneumonia (Panton et al., 1932;
Lina et al., 1999; Gillet et al., 2002). The
prevalence has been increasing due to the
spread of PVL-producing community-
associated MRSA (CA-MRSA; Holmes et al.,
2005). The ve predominant CA-MRSA clonal
lineages that are associated with widespread
disease outbreaks carry PVL, and multiple
reports over the past decade or so have noted
a strong epidemiological association between
PVL and outbreaks of disease caused by CA-
MRSA (for recent reviews, see Diep and Oto,
2008; Diep et al., 2008; Kobayashi and DeLeo,
2009).
The immune evasion cluster
Co-conversion of staphylokinase (SAK) and
enterotoxin by hlb-integrating phages has
been well documented over the past two
decades (Coleman et al., 1989; Carroll et al.,
1993; Zabicka et al., 1993; Sumby and Waldor,
2003; Dempsey et al., 2005; Kumagai et al.,
2007). Two additional prophage-encoded
innate immune modulators associated with
hlb-converting phages, staphylococcal com-
plement inhibitor (SCIN) and chemotaxis
inhibitory protein of S. aureus (CHIPS), have
been described more recently (de Haas et al.,
2004; Rooakkers et al., 2005; van Wamel et al.,
2006). All of these modulators are encoded in
an 8 kb region at the 3' end of the prophage
Phage or
prophage GenBank accession no.
Sero-
type
Int
module
Lysis
module Negative
Positive
conversion
N315 NC_004740 (Kuroda et al.,
2001)
F Sa3 ami3 hlb IEC-F
N315(ECT-R2) FR714927 F Sa3 ami3 hlb IEC-F
N315(ST5G) CP001844 (Nbel et al., 2010) F Sa3 ami3 hlb IEC-F
71 NC_007059 (Kwan et al., 2005) B Sa1 ami4 ear
Sa1JH1 NC_009632 B Sa1 ami4 ear
Sa1JH9 NC_009487 B Sa1 ami4 ear
SaST5K CP001844 (Nbel et al., 2010) B Sa1 ami4 ear
Av1 NC_013450 (Lowder et al.,
2009)
F Sa1 ami4 ear
L54a (Col) NC_002951 A Sa6 ami2 geh
Sa6S0385 AM990992 (Schijffelen et al.,
2010)
A Sa6 ami2 geh
tp310-2 NC_009762 A Sa6 ami2 geh
2010)
A Sa6 ami2 geh
ROSA NC_007058 (Kwan et al., 2005;
Liu et al., 2004)
B Sa6 ami2 geh
52A NC_007062 (Kwan et al., 2005) B Sa6 ami1 geh
Sa6 JH1 NC_009632 B Sa6 ami1 geh
Sa6 JH9 NC_009487 B Sa6 ami1 geh
Sa1 (TW20) FN433596 (Holden et al., 2010) B Sa6 ami1 geh
77 NC_005356 (Liu et al., 2004) F Sa6 ami2 geh
2010)
F Sa7 ami1 sea-ov
genome, which has been named the immune
evasion cluster (IEC; van Wamel et al., 2006).
These genes are prevalent in clinical isolates
and seven dierent variants, or IEC types,
with dierent combinations of these genes
have been dened by PCR and Southern blot
analysis (van Wamel et al., 2006; see Fig. 4.1).
The IEC phages are quite diverse; the same
IEC module is found on dierent phages,
and the four IEC modules present in
sequenced phage genomes are associated
with at least seven dierent modular phage
variants, comprising ve immunity groups
(Kahankova et al., 2010).
Conversion to SAK production was
the earliest example of a phage-encoded
virulence factor in S. aureus (Winkler et al.,
1965). SAK is a potent plasminogen activator
that binds to brin-bound plasminogen,
leading to the dissolution of brin clots
(Bokarewa et al., 2006). Disruption of the brin
net that ofen forms around an infectious
focus is thought to promote invasion of the
staphylococci into deeper host tissue
(Lahteenmaki et al., 2005). SAK also neutralizes
the bactericidal eects of o-defensins,
contributing to virulence by evasion of the
host immune system (Jin et al., 2004). The gene
for SAK (sak) is generally found on hlb-
converting phages, where it is associated with
one or more additional genes of the IEC (Fig.
4.1). Several hlb
+
sak
+
phages/lysogens have
also been described (Kondo and Fujise, 1977;
Sako and Tsuchida, 1983; Sako et al., 1983;
Goerke et al., 2006). These prophages are
integrated at other chromo somal sites. A
partial sequence for one phage conrms that
sak is located at the right end of the prophage
genome and is associated with a dierent
integrase module than the more common hlb-
converting phages carrying sak as part of the
IEC (Goerke et al., 2006, 2009).
CHIPS is a 14.1 kDa secreted protein that
helps modulate immune evasion by blocking
an early step in the inammatory response,
the chemotaxis of neutrophils and monocytes
towards the site of infection. The gene for
CHIPS (chp) lies in the opposite orientation
from the other genes in the IEC (Fig. 4.1) and
was found in over 60% of clinical S. aureus
isolates surveyed by van Wamel et al. (2006).
SCIN is another small secreted protein
that acts to interfere with an early step of the
innate immune response. The 9.3 kDa SCIN
polypeptide binds to C3 convertases on the
bacterial cell surface and blocks cleavage of
the central complement protein C3 into C3a
and C3b (Rooakkers et al., 2005), which
prevents opsonization and protects the
bacteria from phagocytosis. In addition, as
C3b is required for the formation of C5
convertases, SCIN strongly atenuates C5a-
induced neutrophil activation and chemotaxis
(Rooakkers et al., 2006). van Wamel et al.
(2006) found the gene for SCIN in 90% of the
clinical isolates they examined. This gene is
annotated in a number of prophage genomes
as brinogen binding protein. The scn gene
lies at the end of the IEC and is transcribed
divergently from chp (Fig. 4.1).
Genes for several enterotoxins are also
associated with the IEC. These belong to a
large family of related pyrogenic exotoxins
that function as superantigens, (reviewed by
Fraser and Prof, 2008). The exotoxins fall into
two groups, the enterotoxins (SEs) and toxic
shock syndrome toxins (TSSTs). Both groups
have been implicated in toxic shock (Todd et
al., 1978; McCormick et al., 2001), and the
enterotoxins also have a well-established role
in staphylococcal food poisoning (Tranter,
1990). Over 20 serologically distinct super-
antigens have been identied in S. aureus;
these are related proteins that share 1590%
amino acid identity. Enterotoxin A (SEA), the
most prevalent serotype of staphylococcal
enterotoxin in human clinical isolates
(Mathews and Novick, 2005), was shown to
be phage encoded over 25 years ago (Betley
and Mekalanos, 1985). Enterotoxin P (SEP),
which has not been extensively characterized,
shares 77% amino acid identity with SEA
(Kuroda et al., 2001). The sep gene was
reported in place of sea 21% of the time among
human clinical isolates carrying an entero-
toxin gene in the IEC (van Wamel et al., 2006;
see Fig. 4.1) and in 29% of enterotoxigenic S.
aureus food isolates (Bania et al., 2006). Unlike
the rest of the genes in the IEC, the sea (or sep)
gene is located upstream of the phage lysis
genes, which argues against initial acquisition
of this gene by aberrant prophage excision.
Two additional enterotoxin genes, seg2
and sek2, have been described in phage
|Sa3ms (Sumby and Waldor, 2003). These
genes lie at the right end of the prophage
genome, between the repressor and integrase
genes (Fig. 4.1).
The ear gene
The ear gene was rst identied in a
pathogenicity island present in S. aureus
strain COL (Yarwood et al., 2002) and is
widespread among clinical isolates. The
function of ear has not been demonstrated.
The ear gene lies at the right end of the
genome of prophages that carry the same
integrase and lysis modules as the ETA-
converting phages but are otherwise quite
mosaic (Kwan et al., 2005; Lowder et al., 2009;
Kahankova et al., 2010; Nbel et al., 2010).
The sau42I gene
Staphylococcal phage |42 is a serotype F hlb-
inactivating phage that encodes sak and entA
(Coleman et al., 1989). Like prophages
carrying seg2 and sek2, this phage carries
additional genes that map just upstream of
the int gene at the lef end of the prophage
genome (Fig. 4.1). These two slightly over-
lapping open reading frames, sau42RI S and
sau42I RM, encode a BcgI-like restriction
modication system (Dempsey et al., 2005)
that confers resistance to lysis by all phages of
the International Basic Set of S. aureus typing
phages (Carroll and Francis, 1985).
High-frequency mobilization of SaPIs
Phages of S. aureus are not only carriers of
virulence genes themselves but also play a
major role in the horizontal transmission of
genes for enterotoxins and other virulence
factors carried by the SaPI family of
pathogenicity islands. Twenty-two dierent
SaPIs, which utilize six dierent phage-like
chromosomal integration sites, have been
identied thus far in sequenced staphy-
lococcal genomes (Novick and Subedi, 2007;
Guinane et al., 2010; Novick et al., 2010; Viana
et al., 2010). These highly mobile elements,
members of a larger group of elements termed
phage-related chromosomal islands, are
dependent on helper phages for their
propagation and are therefore included in this
discussion of prophage-induced changes in
bacterial phenotype. The SaPI genome, which
is stably integrated into the host chromosome,
is derepressed following induction of a
resident helper prophage or afer helper
phage infection of a SaPI-containing strain.
SaPIs are widespread among S. aureus;
most strains carry at least one SaPI and many
carry two. As most S. aureus strains also carry
multiple resident prophages, there is
signicant potential for SaPI spread following
prophage induction. Not all phages can
transduce SaPIs at high frequency. Never-
theless, there are a number of reported
examples of SaPI mobilization by endogenous
prophages (Kwan et al., 2005; Ubeda et al.,
2005; Subedi et al., 2007). Horizontal trans-
mission of SaPIs also readily occurs, albeit at
a lower frequency, by generalized trans-
duction. The biology of SaPIs is described in
detail in a recent review (Novick et al., 2010,
and references therein).
Streptococcus
Streptococci, lysogeny and toxigenic
conversion
The prophages of Streptococcus pyogenes are an
essential source of genetic variation and
virulence factors in this bacterium. The rst
evidence that a bacterial toxin (the scarlet
fever toxin) could be transferred between
strains in sterile-ltered culture media was
demonstrated by Frobisher and Brown in the
1930s, perhaps the earliest reported example
of toxigenic conversion (Frobisher, 1927,
1934).
Fourteen published S. pyogenes genomes
have revealed a large number of prophage-
encoded toxins, superantigens and strepto-
dornases (DNases) that have proposed or
conrmed roles in pathogenesis (Table 4.2).
With the exception of SmeZ (and occasionally
SpeG and SpeJ), all of the identied strepto-
coccal superantigens are elements of endo-
genous prophages or prophage remnants
(Ferreti et al., 2001).
The ability of many of these toxins to
function as superantigens has been conrmed
(Prof et al., 1999; Beres et al., 2002; Prof and
Fraser, 2003; Prof et al., 2003a,b). Genome
analysis showed that pathogenic strains
contained multiple prophages or prophage-
like elements (ranging from two to eight per
genome). Furthermore, it is common for a
given prophage to carry more than a single
virulence gene (for example, exotoxins SpeH
and SpeI are ofen encoded by the same
prophage).
In addition to toxin genes, S. pyogenes
prophages are vectors for virulence factors to
assist the bacterium in avoiding innate
immunity, particularly DNases. Neutrophils
have been shown to release chromatin DNA
and histones along with other intracellular
components that combine to form neutrophil
extracellular traps (NETs), which can trap
and kill pathogenic bacteria, minimizing
their spread (Brinkmann et al., 2004).
However, many isolates of S. pyogenes are
resistant to NET-mediated killing, employing
prophage-encoded DNases to degrade the
NETs (Buchanan et al., 2006).
SpyCI and the mutator phenotype
Another unique group of prophage-related
mobile genetic elements are commonly found
in S. pyogenes (Scot et al., 2008). These
S. pyogenes chromosomal islands (SpyCIs)
lack any discernable virulence factors but
regulate host gene expression by integrating
into the 5' end of mutL, an essential gene for
DNA mismatch repair. The inhibition of
mismatch repair has been shown to promote
homologous recombination in other bacterial
Table 4.2. Prophage and prophage-like elements in S. pyogenes strains. The serotype (M type), number
of intact prophages with their encoded virulence factors, and associated disease or site of anatomical
isolation is listed for each of the published S. pyogenes strains. Alternative gene names of virulence
factors are shown in parentheses.
Strain M type
Number of
prophages
Prophage-encoded
virulence factors
GenBank
accession no.
Associated
disease or
isolation
a
SF370 1 4 speC, speH, speI, spd1
(mf2), spd3 (mf3)
AE004092 Severe wound
MGAS5005 1 3 speA2, spd3, sdaD2 CP000017 Cerebrospinal uid
MGAS10270 2 5 speC, spd1, spd3, speK CP000260 Pharyngeal swab
MGAS315 3 6 ssa, mf4, speK, sla,
speA3, sdn
AE14074 STSS
SSI-1 3 4 speA, speG, speK, speL,
ssa, mf4.1, sda
BA000034 STSS
MGAS10750 4 4 speJ, spd1, spd3, speA2, CP000262 Pharyngitis
Manfredo 5 5 spd2 (MF4.1), spd3,
speH, speI, speC
AM295007 ARF
MGAS10394 6 8 speA, speC, spd1, speI,
speH, spd3, speK, sla,
sdn, sda, mefA
CP000003 Pharyngitis
MGAS2096 12 2 speC, spd1, srtA, sda CP000261 APSGN
MGAS9429 12 3 speC, spd1, speH, speI,
srtA, sda
CP000259 Pharyngeal swab
MGAS8232 18 5 speA1, speC, spd1, speL,
speM, spd3, sda
AE009949 ARF
MGAS6180 28 4 srtA, speC, spd1, speK,
sla
CP000056 Invasive blood
isolate
NZ131 49 3 speH, spd3 CP000829 APSGN
a
STSS, streptococcal toxic shock syndrome; ARF, acute rheumatic fever; APSGN, acute post-streptococcal
glomerulonephritis.
species, so the SpyCI may promote
recombination between dierent prophage
chromosomes to create diversity. An in-
progress genome project of the Streptococcus
anginosus strain F0211 genome has uncovered
a SpyCI-related element integrated into the
identical mutL atachment site as in S.
pyogenes, so the distribution of these mutator
phage-like chromosomal islands may extend
to even more streptococcal species.
Recombination and the dissemination of
virulence genes
Comparison of the genomes of prophages
demonstrates how modular evolution has
contributed to the diversication and dis-
semination of toxin genes and DNases to
create novel prophages.
A conserved genetic module, containing
genes for DNA packaging, head and tail
proteins and tail bre proteins, is found
combined with an assortment of virulence
genes. Additionally, dierent integrase genes
that target a variety of bacterial atachments
sites (atB) are also associated with this
conserved structural module, leading to
additional diversity. A highly conserved open
reading frame has been identied, usually
positioned between the phage-encoded
virulence gene and the atachment site, which
may promote homologous recombination
and lead to phage diversication (Aziz et al.,
2005). This open reading frame, named
paratox, was found in 18 out of 24 S. pyogenes
prophages in genome strains SF370,
MGAS8232, MGAS315 and SSI-1, and was
always located adjacent to a toxin gene.
Homologues of paratox also exist in some
Streptococcus agalactiae and Streptococcus
thermophilus phages (Aziz et al., 2005).
Other related streptococcal species
contain toxigenic prophages. Neither of the
medically important species of S. agalactiae
(group B Streptococcus) and S. pneumoniae has
been found to be a signicant carrier for
toxigenic prophages, but the group C and
group G Streptococcus species have provided
many examples. These virulence factors are all
close homologues of S. pyogenes prophage
toxins (SlaA, SpeL, SpeM, SpeH and SpeI),
strengthening the argument that these strepto-
coccal species share a genetic pool
of prophages. The group G Streptococcus
dysgalactiae subsp. equisimilis GGS_124 con-
tains a prophage with a streptodornase-like
DNase (Shimomura et al., 2011). Surveys of
group C streptococcal isolates have found
evidence for other superantigens, some of
which may be prophage associated (Sachse et
al., 2002). Among the oral streptococci, a
prophage of Streptococcus mitis was identied
that encoded a platelet-binding protein
(Bensing et al., 2001). S. mitis is an important
cause of bacterial endocarditis, and the binding
of platelets by bacteria is a proposed central
mechanism in the pathogenesis of this disease.
Escherichia coli
It was not until the early 1980s that temperate
coliphages were really associated with the
toxigenic conversion of their hosts (OBrien et
al., 1984; Strockbine et al., 1986). Acquisition
of these toxigenic converting phages was the
end result in the evolution of a pathogen
(Feng et al., 1998) that was not formally
implicated in disease until a food-borne
outbreak in 1982 (Karmali et al., 1983; Riley et
al., 1983; OBrien et al., 1984). The phages
responsible for the conversion of pathogenic
E. coli serovars to the notorious pathotype,
Shiga toxin-producing E. coli (STEC) and the
smaller but more virulent subdivision
enterohaemorrhagic E. coli (EHEC) (reviewed
in more depth by Kaper et al., 2004), are
collectively known as Shiga toxin-encoding
phages (Stx phages), although historically
they have also gone by the nomenclature
verotoxigenic phages (VT phages) and Shiga-
like toxigenic phages (SLT-phages; Allison,
2007).
All Stx phages characterized to date have
been lambdoid phages (Allison, 2007). The
only trait shared by all Stx phages is their
carriage of an stx operon (Allison, 2007). The
genes encoding Stx, an AB
5
holotoxin related
in structure to both cholera toxin (also phage
encoded) and ricin (OLoughlin and Robins-
Browne, 2001), are located in a small,
bicistronic operon, which is associated with
the late gene regulatory region of Stx phages
and prophages (Fig. 4.2). However, all Stx
phages do not share the same genes encoding
Stx. In fact, the term Shiga toxin describes
two families of toxin (Allison, 2007).
Normally, the stx operon lies just
downstream of the late gene-associated anti-
terminator, Q, but there are instances reported
in the literature of non-inducible, apparently
remnant Stx prophages, where the Q gene
has been lost while the prophage has retained
the toxin operon in the expected location
(Teel et al., 2002). The location of the toxin-
-
-
-
-
-
Fig. 4.2. Genetic map of the late gene regulatory region of sequenced Stx phages. Genes possessing
signicant gene homology are shaded identically. The rst shaded variant of a gene is labelled
accordingly. Gene abbreviations: O, origin-specic replication initiation factor; P, replication protein; ninB
and ninG, genes comprising phages ninR region involved in both RecF and RecBCD recombination as
well as Red-mediated recombination (Tarkowski et al., 2002); ant, putative anti-repressor (Fogg et al.,
2011); roi, similar to the gene encoded on HK2022 involved in controlling lytic propagation (reduced
phage lytic propagation on integration host factor-negative cells; Clerget and Boccard, 1996); Q, anti-
terminator controlling late gene expression; stxAB, Shiga toxin operon (or its inactivated derivative,
Astx::Cat; Allison et al., 2003); hyp, hypothetical proteins with no predictable function; lys, phage lysin
(1,4-|-N- acetylmuramidase); Rz, endopeptidase; bor, gene encoding the lipoprotein Bor; cII,
transcription regulator involved in the establishment of lysogeny; cI, phage repressor involved in the
maintenance of lysogeny; cro, regulator involved in prophage induction.
encoding operon in the prophage is crucial to
its regulation. In most instances, Stx
production is linked to the phage regulatory
mechanism controlling the lytic cycle. E. coli
lacks a dedicated secretion system for Stx,
although limited secretion of Stx2 can occur
in EHEC strains (Shimizu et al., 2009), and
maximal release of Stx is linked to phage-
directed lysis of the cell (Wagner et al., 2001).
Although there is at least one exception,
P27 (Recktenwald and Schmidt, 2002), Stx
phages typically have a genome that is at
least 10 kb larger than the 48.5 kb genome of
phage (Table 4.3). The limited amount of
DNA, 1.2 kb, linked to the presence of the stx
operon in the phage genome does not solely
account for the genomic size variation of Stx
phages from phage. Genes such as lom and
bor are carried by phage that are not
essential to either its replication or its biology.
These two genes have been demonstrated to
aid the lysogen in colonization of the
mammalian host (Barondess and Beckwith,
1990). These genes are also associated with
many Stx phages (Fig. 4.2), but Stx phages
appear to encode other traits that also increase
their survival in their environment.
Expression of Stx provides the lysogen with
the ability to survive protozoan grazing
(Lainhart et al., 2009), which is an important
atribute in both the ruminant gut and in soil
environments, two crucial traits for organisms
that are associated with catle, cow pats and
farm environments. The data indicate that Stx
phages are not simply toxigenic converting
phages, but that, in addition to converting the
toxigenic phenotype of their host, these
phages are capable of altering the tness and
phenotypic traits of their lysogen.
More than 500 serogroups of E. coli as
well as other members of the Enterobacteriaceae
have been associated with Shiga toxin
production and human disease (Allison,
2007). Stx phages are driving this emergence.
It is interesting to note that no two identical
Stx phages have ever been described or
sequenced (Muniesa et al., 2003, 2004a,b;
Smith et al., 2007b; H.E. Allison, unpublished
data). These phages possess genomes that are
highly mosaic. Additionally, it has been
demonstrated that many members of the
Enterobacteriaceae can support Stx phage
adsorption where the appropriate short tail is
present, even where those bacterial species
may not support infection (Smith et al., 2007a);
it may be that only simple abortive infection
systems, restriction and modication systems
or other mechanisms (Chopin et al., 2005;
Hyman and Abedon, 2010; Labrie et al., 2010)
are all that has, up to this time, protected us
from the further emergence of new Shiga
toxin-encoding pathogens. However, the
serogroup range associated with Shiga toxin
production continues to expand. As the host
range for Stx phages grows, so too will the
diversity of these phages as they continue to
mix and form new phage mosaics with the
endogenous prophages and remnant
(defective) phages found in their extended
host range (Allison, 2007).
Table 4.3. Characteristics of fully sequenced Stx phages
Stx phage Genome size (kb) Toxin variant GenBank accession no.
BP-4795 57.9 Stx1 NC_004813
Min27 63.4 Stx2 NC_010237
Stx converting phage II 62.7 Stx2 NC_004914
Stx1-converting bacteriophage 59.9 Stx1 NC_004913
Stx2-converting phage I 61.8 Stx2 NC_003525
Stx2-converting phage 1717 62.1 Stx2c NC_011357
Stx2-converting phage 86 60.2 Stx2 NC_008464
VT2-Sakai 60.9 Stx2 NC_000902
24B 58.5 Stx2 HM_208303
933W 61.7 Stx2 NC_000924
P27 42.6 Stx2e NC_003356
One further lambdoid phage-associated
product is cytolethal distending toxin (CDT),
rst identied by Johnson and Lior in 1987.
Induction of CDT-1 cells with mitomycin C
results in enhanced toxin production and the
release of CDT-1u, a member of the
Siphoviridae (Asakura et al., 2007). This phage
possesses a 47 kb genome that is normally
integrated into the prfC gene, which encodes
peptide chain-release factor RF-3. The
prophage shows similarity to the serotype-
converting Shigella phage SfV (Allison et al.,
2002). Interestingly, unlike phage SfV and Stx
phages, the toxin genes are found upstream
of the integrase gene.
Vibrio cholerae
The lamentous phage CTX| has played a
critical role in the evolution of the cholera
pathogen, V. cholerae (Waldor and Mekalanos,
1996). The genes encoding cholera toxin
(ctxAB), an AB
5
-subunit toxin whose activity
accounts for the profuse watery diarrhoea
that is characteristic of cholera, are carried by
CTX| (Fig. 4.3). In contrast to lamentous
coliphages such as M13 which replicate as
plasmids CTX| integrates into the V. cholerae
genome, creating a stable lysogen bearing
ctxAB (Davis and Waldor, 2003; McLeod et al.,
2005).
CTX| virion production does not result
in host cell lysis; instead, similar to other
lamentous phages, CTX| particles are
secreted through an outer-membrane pore
(secretin) (Davis et al., 2000a). Both CTX|
particles and cholera toxin exit the cell
through the same secretin (Davis et al., 2000a).
Like many other temperate phages, but
in contrast to the lamentous coliphages,
environmental conditions that lead to DNA
damage and induce the host SOS response
lead to increased production of CTX| virions.
However, there are two notable dierences in
the consequences of SOS induction of the
CTX| prophage: (i) CTX| prophage induction
does not kill the host cell; and (ii) induction
does not lead to excision of the CTX| genome
from the chromosome. Instead, prophage
DNA is used as a template to generate the
single-stranded DNA copies that are packaged
into virions via a replication mechanism that
depends on the presence of tandem prophages
(Davis and Waldor, 2000; Moyer et al., 2001).
Some of the bio technological applications of
CTX| have been reviewed recently (Rakonjac
et al., 2011).
Similar to other temperate phages, CTX|
lysogeny is maintained by a phage-encoded
repressor, RstR. LexA, the host-encoded SOS
repressor, acts along with RstR to inhibit
transcription from the promoter (P
rstA
) that
controls expression of most CTX| genes
(Quinones et al., 2005). The majority of toxin
transcripts initiate from a distinct promoter
found immediately upstream of ctxA (Davis
et al., 2002; Quinones et al., 2006).
The classical and El Tor biotypes of
toxigenic V. cholerae O1 harbour distinct forms
of CTX|. Classical CTX| lysogens do not
produce virions, whereas most El Tor isolates
Core
Regulation
Replication
Integration
Coat Assembly/secretion
Fig. 4.3. Genetic structure of the CTX prophage. The genome is divided into an RS and core region. The
black triangles indicate the direct repeats that ank the integrated CTX|. The arrows indicate the direction
of transcription of the respective open reading frames and the bent arrows represent promoters.
do; the arrangement of prophages in classical
strains explains why they do not produce
phage particles (Davis et al., 2000b). The
classical and El Tor CTX|s have distinct
repressors as well as polymorphisms through-
out the phage genome, including in the
cholera toxin genes. In recent years, hybrid
CTX|s have been detected (Lee et al., 2009).
Such hybrid phages ofen contain the classical
ctxB allele, which may yield a more potent
toxin, in an otherwise El Tor strain. The
existence of such hybrid phage genomes
strongly suggests that there has been (and
may still be) recombination between CTX|s,
generating new variants of toxigenic V.
cholerae.
Salmonella
That Salmonella enterica serovar Typhimurium
carried symbiotic bacteriophages was rst
recognized by Boyd in 1950. It is noteworthy
that most salmonellae carry the genomes of
temperate phage Salmonella Typhimurium
strains usually carry four to ve prophages,
while S. enterica serovar Typhi strains Ty2
and CT18 both possess seven prophages
(Thomson et al., 2004). These prophages may
induce three phenotypic changes to the cells
that carry them serotype conversion, a
change in phage type and an impact on
virulence. The presence of ST64B, for
example, changes the phage type of Salmonella
Typhimurium from denitive (phage) type
(DT) 41 to DT 44 (Tucker and Heuzenroeder,
2004), while lysogenization by ST64T is
probably responsible for converting DT 9 to
DT 64, DT 135 to DT 16, and DT 41 to DT 29
(Mmolawa et al., 2002).
Expression of the genes associated with
lysogenic conversion can also impact on the
virulence of the host. This has been recently
reviewed (Lemire et al., 2008). For example,
Fels-1 prophage carries nanH (neuraminidase)
and sodC3 (superoxide dismutase; Figueroa-
Bossi et al., 2001; Bossi and Figueroa-Bossi,
2005), while Gifsy-1 carries a number of
potential virulence modulating genes
including gipA in what is equivalent to the
b2 region. The encoded protein is involved in
colonization of the small intestine, and its
deletion results in reduced bacterial virulence.
As phage conversion of O-antigenicity
was reviewed recently in depth (Los et al.,
2010), here we shall only touch on what is
known. Among the members of subgenus I
(S. enterica subsp. enterica), a total of 57
O-antigens can be distinguished serologically
(Grimont et al., 2007). Some of these, O1, O5,
O6, O14, O15, O20, O27 and O34, are in
certain cases known to be phage encoded.
The types of change noted in the structure of
the LPS O-side-chain polysaccharide include
O-acetylations, O-glucosylations and changes
in bonding between adjacent O-antigen
repeats. In the simplest case, this is manifest
by Salmonella phage P22, an o-linked glucosyl
residue (antigen O:1) to the 6 position

of
galactose moieties in the LPS O-antigenic
branched tetrameric repeat.
This phage carries a conversion module
consisting of three genes downstream of the
phage atachment site (atP). This consists of
GtrA (120 amino acids, 13.5 kDa; glucosylated
undecaprenyl

phosphate translocase or
ippase), GtrB (311 amino acids, 35.1 kDa;
bactoprenol glucosyl transferase) and GtrC
(486 amino acids, 55.4 kDa; glucosyl
transferase) (Vander Byl and Kropinski, 2000;
Pedulla et al., 2003). The sequence of GtrC
shows more diversity than that of GtrA or
GtrB, presumably as a result of dierences in
the chemical structure of the O-antigen (Fig.
4.4).
In certain other phages, rather than a
glucosyl residue being added, one of the side-
chain sugars gets acetylated. This has been
observed with temperate Salmonella phage
g341c (GenBank accession no. NC_013059), E.
coli phage V10 (Perry et al., 2009) and
Pseudomonas aeruginosa phage D3 (see below).
In each of these cases, like the GtrC homo-
logues, the O-acetylases are high-molecular-
weight proteins with multiple transmembrane
domains.
Pseudomonas
Lysogeny and polylysogeny are common
in P. aeruginosa (Holloway et al., 1971).
Numerous early studies have shown
O-antigenic conversion followed by infection
with a variety of phages (Liu, 1969; Castillo
and Bartell, 1974; Bergan and Midtvedt, 1975;
Lanyi and Lantos, 1976; Dimitracopoulos and
Bartell, 1979). The denitive story of serotype
conversion in this bacterium is with temperate
siphovirus D3. In 1962, Holloway and Cooper
reported that P. aeruginosa D3 lysogens lost
the ability to adsorb this phage and displayed
an antigenic shif. Kuzio and Kropinski (1983)
conrmed that the lysogens did not support
phage adsorption, and that LPS from the
convertant did not neutralize D3 or the
serologically and morphologically unrelated
phage E79. Nuclear magnetic resonance
analysis of the O-side-chain polysaccharides
from strains PAO1 and PAO1(D3) revealed
that, in a manner analogous to Salmonella
phage c15, the bonding of tetrameric
O-antigenic repeats changed from o(14) to
|(14) and the d-fucosamine (2-amino-
2,6-dideoxy-d-galactose) residues were
O-acetylated. Sequencing the genome of this
phage (Kropinski, 2000) resulted in the
discovery of the fucosamine O-acetylase, but
it was not until Newton cloned the conversion
region (Newton et al., 2001) that we fully
understood the role of each of the components
in the conversion. The conversion module
consists of the acetyltransferase, a |-LPS
polymerase and Iap (inhibitor of alpha
polymerase), which is a 31 amino acid
(3884 Da) transmembrane peptide which
inhibits the o-LPS polymerase. Interestingly,
this polypeptide is extremely di cult to
express or produce chemically due to its
inherent insolubility (J. Lam, personal
communication).
The only other phage about which some
details are known is FIZ15 (Vaca et al., 1993;
Vaca-Pacheco et al., 1999), which unfortunately
is now lost (S. Vaca, personal communication).
In the lysogenic state, this virus was found
to: (i) increase resistance to phagocytosis by
mouse peritoneal macrophages; (ii) increase
resistance to killing by normal human serum;
and (iii) increase adhesion to human buccal
epithelial cells, which are considered to be a
function of changes to the LPS.
Transduction
The three common methods of genetic
exchange within bacterial species are trans-
formation, conjugation and transduction. The
last is the transfer of genetic information
between bacterial cells mediated by phage
particles. Two distinct types have been
recognized: (i) specialized transduction; and
(ii) generalized transduction (Thierauf et al.,
2009). In the former case, which is best
exemplied by coliphage , genes adjacent to
an integrated prophage (at site) can be co-
excised with the phage upon induction. The
dening characteristics of the transducing
particles are that they carry a strictly limited
number of host markers and that they contain
host DNA physically associated with phage
DNA. Lysogenic conversion could be viewed
as a unique example of specialized trans-
duction.
Generalized transduction, in which any
gene can be transduced, is best exemplied
by coliphage P1 and Salmonella phage P22.
SfX
ENT47670
SfV
ST160
ST104
P22
SE1
ST64T
0.9
(a) (b)
Fig. 4.4. Phylogenetic analysis of GtrB (a) and GtrC (b) homologues. The diagrams were generated
using one click analysis at http://www.phylogeny.fr/ (Dereeper et al., 2008).
Here, the transducing particles are derived
through the accidental packaging of host
DNA, rather than phage DNA. With renewed
interest in the use of bacteriophages in
therapy, biosanitation, biocontrol and bio-
preservation, the ability of the phage
preparations to transduce (especially toxin
and other virulence-factor genes) is one of the
issues that those who characterize phages for
practical uses need to consider. While most of
the transducing phages are temperate, that is,
are capable of entering into a lysogenic state
with a host bacterium, lytic phages may also
transduce (Table 4.4).
Genetic exchange via all three mechan-
isms is commonly observed. In contrast,
unlike many of the bacterial species men-
tioned above, transduction is the only
mechanism of genetic exchange observed in
S. pyogenes and is mediated by both lytic and
temperate phages. It was rst reported by
Leonard and co-workers when they identied
ve phages, two temperate and three lytic,
that were able to transduce streptomycin
resistance (Leonard et al., 1968). The highest
frequency of transduction was observed for
lytic phage A25 (1 10
6
plaque-forming
units). Temperate S. pyogenes phages are also
able of mediating the transfer of antibiotic
resistance by transduction (Ubukata et al.,
1975; Hyder and Streitfeld, 1978), presumably
generalized transduction.
Transduction between streptococcal
groups has been observed and may be a
potential means for disseminating virulence
genes. Bacteriophages isolated from strepto-
coccal groups A, E and G were isolated that
could lyse streptococci from serogroups A, C,
G, H and L, and in some cases, these lytic
phages were also capable of propagation in
one or more of the other serogroups (Clon et
al., 1972; Wannamaker et al., 1973). Horizontal
transfer of genetic material in S. pyogenes has
been shown by numerous recent studies to be
important in the dissemination of genes in
natural populations (Stevens et al., 1989;
Cleary et al., 1992). The role in transduction
and bacteriophages in horizontal transfer is
assumed, yet no modern studies have directly
addressed the question. The majority of
studies into streptococcal transduction were
carried out before the advent of many of the
current techniques in molecular biology, and
so the time may have come to re-examine this
phenomenon. A beter understanding of
streptococcal transduction may prove key to
understanding the ow of genetic information
in natural populations of streptococci. This is
Table 4.4. Lytic phages that can, under controlled conditions, be demonstrated to transduce.
Host Phage Reference
Bacillus subtilis SPP1 Canosi et al. (1982); de Lencastre and Archer (1980)
Caulobacter crescentus Cr30T Bender (1981)
Citrobacter rodentium CR1 Petty et al. (2007)
Erwinia carotovora M1 Toth et al. (1997)
KP Toth et al. (1993)
Escherichia coli T1 Bendig and Drexler (1977); Drexler (1970; 1977)
T4 Roberts and Drexler (1981)
RB43 Wilson et al. (1979); Young et al. (1982)
RB49 Tianiashin et al. (2003)
Pseudomonas spp. KZ Dzhusupova et al. (1982)
PA3 Monson et al. (2011)
E79 Morgan (1979)
pf16 Rheinwald et al. (1973); Daz et al. (1976); Gorbunova et al.
(1985)
Salmonella typhi ViI Cerquetti et al. (1993)
Serratia marcescens IF3 Petty et al. (2006)
Streptococcus pyogenes A25 Leonard et al. (1968)
Xanthomonas campestris XTP1 Weiss et al. (1994)
also true for other pathogenic bacteria as our
understanding of the role of horizontal gene
transfer in pathogen development is still
quite incomplete. Future studies with
transducing phages can only add to this
understanding.
Acknowledgements
A.M.K. was supported by a Discovery Grant
from the Natural Sciences and Engineering
Research Council of Canada.
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5 The Lion and the Mouse: How
Bacteriophages Create, Liberate and
Decimate Bacterial Pathogens
Heather Hendrickson
1
1
New Zealand Institute for Advanced Study, Massey University
I had my eyes opened by that microscope, to the fact that there is a world of tiny creatures that
you cant see with the naked eye. Its like looking at an African safari park but in miniature...
Richard Dawkins (Powell, 2011)
The microscopic world is comprised of free-
roaming creatures far more complex and
impressive in their everyday interactions
than what you will observe in a wildlife park.
So interdependent are these organisms that
dening which is the proverbial lion of the
jungle and which the unassuming mouse can
be a challenge. Bacterial viruses bac-
teriophages or phages prey on bacteria. On
evolutionary timescales, however, phages
also can convert bacteria from pussy cats into
the human predators we call pathogens. In a
surprising twist, it may be the inadvertent
action of viruses and the recombination they
eect, through horizontal gene transfer
(HGT), that enables the continuing existence
of bacteria. Phages, as I will discuss below,
are the accidental architects of the genetic
combinations that we observe in the bacterial
world.
Bacteria generally utilize a simple mitotic
asexual reproductive cycle. This has profound
consequences for these organisms and their
evolution. One consequence, of interest here,
can be described as Mullers ratchet, the
tendency for an asexual organisms DNA to
irreversibly acquire increasing loads of
deleterious mutations over time (Gordo and
Campos, 2008; Allen et al., 2009). Like the
ratchet in the handymans tool box, mutation
can be thought of as only permiting change
in a single direction, that is, away from the
wild type. The absence of sexual reproduction
in bacteria means there is no opportunity to
reshu e combinations of genes that sexually
reproducing organisms have, which produce
both more or less t combinations. With
asexual organisms, by contrast, once a
mutation is present, then all future progeny
of that individual will probably inherit it, as
reversion to wild type will be much rarer
than the occurrence of the mutation itself. A
continual accumulation of deleterious
mutations over time is predicted to eventually
destroy asexual lineages and to do so at
measured rates that can be as high as 1% of
lineages afer a mere 1700 generations
(Andersson and Hughes, 1996). That bacteria
continue to exist suggests that these organ-
isms must have access to recombination of a
H. Hendrickson
62 H. Hendrickson
sort. This takes place through HGT, a
phenomenon that adds to genomes by
shu ing in new genetic material from other
bacterial lineages, both closely and distantly
related to the recipient.
In this chapter, I will illustrate the
inuence of phages in the process of HGT
and the impact that HGT has on bacterial
evolution with a special interest in the
evolution of pathogens. I will begin with a
brief word about the day-to-day struggle
between phages and bacteria before
proceeding to a description of the mechan-
isms, quantities and impact of HGT in the
microbial jungle.
Phages and Bacteria:
the Short Game
Over evolutionary timescales, phage-
mediated events promote the evolution of
bacterial species through the addition of new
DNA. The essential relationship between
these organisms is however, still properly
viewed as antagonistic. Thus phages and
bacteria are engaged day to day in a
microscopic mle, and the mechanisms by
which bacteria avoid phage infection can, at
times, be in distinguishable from mechanisms
by which pathogens avoid the immune
responses of potential hosts (Abedon, 2012).
Phages, as a consequence of these
antagonistic interactions, can have a sig-
nicant eect on the short-term evolution of
bacteria by modifying their population
dynamics. Experiments in controlled com-
petitive environments in which available
nutrients are limited have been used to
demonstrate the arrival of successive adapt-
ations on both the phage and the bacterial
sides in an evolutionary arms race (Bohannan
and Lenski, 2000a,b). In population growth
experiments where bacterial genetic diversity
would normally emerge as a result of niche
specialization and trade-os, the eect of
adding predatory phages is to decrease
within-population competition, which results
in reduced diversity (Buckling and Rainey,
2002a,b). Further studies using soil habitats,
rather than liquid cultures, have demonstrated
rapid co-evolution between bacteria and their
phages, and evidence of uctuating selection
as well as growth costs associated with
evolving phage resistance (Gomez and
Buckling 2011). Understanding the popu-
lation dynamics of co-evolution in simple
cases such as controlled soil environments
will help us to understand these processes
beter in nature.
Bacteria are engaged in a constant
struggle to avoid exploitation by phages. The
continued existence of both implies that this
eort is matched step for step by continual
counter-adaptation on both sides, and
antagonistic co-evolution. It is perhaps ironic
then that reciprocal evolution in bacteria is
mediated, in part, by phage involvement in
HGT.
The Long Game: Horizontal Gene
Transfer
The bacterial genomic era, starting in 1995
with the complete genome sequence of
Haemophilus inuenzae RD1, was the beginning
of fresh insight into the evo lutionary processes
undergone by bacteria (Fleischmann et al.,
1995). This and subsequent sequencing
studies revealed that separate genes in single,
continuous DNA molecules can have dierent
phylogenetic histories. HGT takes place
readily in prokaryotes and leads to organisms
with DNA molecules that are chimaeric, with
large swathes inherited across species
boundaries (Lawrence and Hendrickson,
2003).
A successful HGT event will lead to a
recipient bacterial cell with DNA that can be
anything from a variant of the genetic material
that it already had to a novel section of DNA
with genes that are completely new to the
bacterium. While occasional transfers of small
amounts of genomic content in some fraction
of extant lineages would be novel and notable,
there are recent suggestions that an average of
81% of bacterial genes might be acquired
horizontally (Zhaxybayeva et al., 2006; Dagan
et al., 2008). HGT both has and is having a
profound impact on the nature of the bacteria
that we see around us. The redistribution of
genetic material that is implied by such a
ubiquitous process gives us cause to reconsider
How Bacteriophages Create, Liberate and Decimate Bacterial Pathogens 63
notions that we otherwise rely on in
considering bacteria, including key concepts
such as species, pathogen and population.
Detection of HGT
There are three broad classes of HGT
detection methods: parametric mismatch,
syntenic disruption and phylogenetic in-
congruity. In this section, I provide an outline
of what these dierent approaches entail, as
well as some of their shortcomings.
Parametric methods posit that bacterial
genomes acquire characteristics over
evolutionary timescales that are distinctive,
much like tool marks acquired in a master
sculptors shop. Various metrics have been
proposed for distinguishing between old and
new DNA including G+C content, dinu-
cleotide content, higher-order sequences
(6mer and 8mer repeats) and the codon
adaptation index (CAI), as well as
combinations of all of these (Karlin, 1998;
Tsirigos and Rigoutsos, 2005). Two major
advantages in using parametric methods are
that they are computationally simple and that
multiple genomes for comparison are not
required for transferred genes to be identied.
Many parametric methods have been
developed, and these generally identify
overlapping but dierent sets of genes for
the same genome. This is due, in part, to
the confounding similarity in mutational
tendencies among near relatives. In short,
atypical regions are identied with the most
condence when donor and recipient
genomes are not closely related (Azad and
Lawrence, 2007; Arvey et al., 2009; Boc and
Makarenkov, 2011). Complicating these
determinations, as time passes in a new
genomic context the mutational tendencies of
the cognate replication machinery in
combination with natural selection (acting on
spontaneous mutations) will tend to eliminate
the distinctiveness of the acquired DNA, a
process termed amelioration (Lawrence and
Ochman, 1997). It should also be noted that
parametric measures, such as CAI, can be
misread. Developmental stages in bacterial
growth, such as sporulation in Bacillus subtilis,
for example, can lead to genes with an
atypical CAI and mistaken identication, as
recently introduced (Moszer et al., 1999).
The second class of HGT detection is
syntenic disruption. If closely related organ-
isms are available for genomic comparison,
then breaks in synteny, or gene order, can be
observed when genomes are aligned. These
breaks indicate that a region of DNA has been
introduced into or deleted from one of the
bacterial genomes (Delcher et al., 2003;
Darling et al., 2010). Breaks in synteny can
reveal that HGT as well as genomic
rearrangements have taken place since the
divergence of two organisms.
Phylogenetic incongruity is the third
class of HGT detection and involves
comparing phylogenetic trees. Homologues
in closely related organisms (orthologues)
accumulate mutational dierences, which
can be used as informative character traits.
These are in turn used to build phylogenetic
trees that represent hypotheses about the
relatedness of these genes and the organisms
that possess them. The 16S and 18S rRNAs
have been proposed as the standard for
phylogenetic comparison across the three
domains of life because these molecules are
present in all branches of life and are
molecularly constrained enough to be
informative relative to other potential sites
(Fox et al., 1977; Olsen et al., 1986). Even this
paragon of molecular metrics, however, has
been horizontally transferred in some cases
(Gogarten et al., 2002). Approaches to
discovering the extent to which bacteria are
chimaeric using phylogenetic trees have
evolved quickly and ofen lead to diering
estimates of the degree to which HGT is
inuencing the evolution of bacterial lineages
(Ochman, 2005).
HGT complicates determinations of a
true tree of life. Complete genome sequences
are a record of all of the genes that an
organism has, as well as a robust source of
phylogenetic information regarding each of
these genes. Homologous genes from
dierent organisms can therefore be used to
construct a tree that represents the history of
that gene, but where such a gene tree fails to
align with the inferred organismal tree then a
transfer event can be inferred (Snel et al.,
2005). How then can one nd a true
64 H. Hendrickson
organismal tree to use as the benchmark for
such comparisons? Multiple methods are
being developed to determine the phylo-
genetic tree of most of genes, using divergence
from this norm as the metric by which
transfer is judged (Gogarten et al., 2002;
Poptsova and Gogarten, 2007; Andam and
Gogarten, 2011).
Another route to determining a tree of
life is to try to identify a set of genes that are
present in as many organisms as possible
while avoiding including any transferred
genes. Snel and colleagues have made an
impressive eort in this regard and generated
a tree that includes the three domains of life
by using 31 genes from 191 completely
sequenced genomes (Ciccarelli et al., 2006).
By ignoring the contribution of gene transfer,
however, what does this tree represent? By
one account, 31 genes represent just 1% of the
genes that the average microbial genome
contains (Dagan and Martin, 2006). Another
method, utilized by Rivera and Lake (2004),
allowed transfer and genome fusion to enter
into the model of relatedness. This analysis
revealed a circular patern or relatedness at
deep branches, evidence of ancient and
abundant HGT (Rivera and Lake, 2004). For
these reasons, biologists are being encouraged
to stop seeking a bifurcating tree of life to
represent the nature of relatedness when
considering the prokaryotes (Dagan and
Martin, 2006; Dagan et al., 2008). Despite the
controversy generated by evidence of
widespread HGT, there is still strong support
for the assertion that all cellular life has arisen
from a single ancestor. This hypothesis
received support recently through direct
testing using a set of ubiquitously conserved
gene sequences, which suggested a high-level
tree of universal relatedness (Theobald, 2010).
Mechanisms of gene exchange
When HGT is identied in a recipient
genome, the donor genome is generally not
known, although there are new methods
being proposed to overcome this limitation
(Azad and Lawrence, 2011; Boc and
Makarenkov, 2011). HGT therefore is
generally detected solely as novel DNA in a
recipient genome, and this acquisition takes
place through three processes: transformation,
conjugation and transduction (Fig. 5.1).
Transformation is the process by which
bacteria take up DNA from the extracellular
environment and enact repair-like events that
inadvertently bring this linear DNA into the
circular bacterial genome. Many naturally
competent bacteria have mechanisms in place
to ensure that they import DNA from bacteria
they are closely related to (Krger and Stingl,
2011). Gene transfer occurs through trans-
formation as the occasional by-product of
bacteria feeding on DNA (Redeld, 2001).
Conjugation is the process by which plasmids,
small accessory chromosomes with their own
genes, control the transfer of DNA, usually
the plasmid, between bacteria. Conjugation
can be considered an HGT event whether or
not the plasmid DNA is subsequently re-
combined into the bulk of the recipient
genome.
In transduction, a virus infects a
bacterium and subsequently produces non-
infectious viral particles that have accidentally
packaged the DNA of the host bacterium
rather than the viruss own DNA. These
defective phage particles then can inject this
bacterial DNA into a recipient bacterium, and
Mechanisms of Gene Exchange
Transformation
Conjugation
Transduction
Fig. 5.1. The three major categories of events that
can lead to gene transfer: (i) transformation,
whereby naked DNA from the extracellular
environment can be taken up by bacterial cells; (ii)
conjugation, the transmittance from one cell to
another of a plasmid; and (iii) transduction, the
passage of DNA from one cell to another mediated
through the accidental incorporation of bacterial
DNA into a phage particle in a prior infection.
it can then be recombined into the recipient
genome by repair-like recombination pro-
cesses. It should be noted that the process of
lysogeny or integration of a phage genome
into a bacterial chromosome can also be
considered a special instance of HGT (see
Abedon and Christie et al., Chapters 1 and 4,
this volume). Inspection of closely related
bacterial genome sequences reveals much
alignment in gene order, followed by gaps
that can be as short as 1 kb and can range up
to many hundreds of kb. Upon more detailed
analysis, many of these gaps can be atributed
to phages that have integrated in one bacterial
genome and not the other. Integrated phage
genomes can carry genetic elements that
contribute both to the pathogenicity of the
strain and to their own retention (Brssow et
al., 2004; see also Christie et al., Chapter 4, this
volume).
Constraints on HGT
If all HGT was equally likely, then we might
expect the tree of prokaryotic life to be a web
of inter-related threads; a panmictic mess.
Our notions of bacterial relatedness, however,
has structure that is present despite HGT.
Organisms that are closely related according
to 16S rRNA gene estimates also share unique
properties and lifestyles. This family-level
cohesion, despite seemingly massive oppor-
tunities for HGT, suggests that there are
natural constraints that dene or structure
this process (Gogarten et al., 2002; Andam
and Gogarten, 2011). If organisms that are
most closely related are those that are
exchanging DNA most frequently, then
vertical inheritance and HGT may both
contribute to the observed relatedness in
bacterial families, just as sexual reproduction
in higher organisms contributes directly to
the species cohesiveness (and, indeed, denes
the biological species con cept).
Barriers to transfer
Consistent with these ideas of limitations on
likelihood of HGT, newly integrated DNA
must pass through a series of chance and
selective constraints in order to be maintained
for long periods of time. These limits to
transfer are probably concurrent and,
although all are real, many are di cult to
measure and none should be considered as a
brick wall to transfer. Many natural barriers
to transfer in particular have components
that make them likely to contribute to
enforcing tendencies whereby close relatives
exchange DNA most frequently. As such, the
following is far from an exhaustive discussion
but gives a breadth of mechanisms that
constrain gene transfer.
The rst real barrier to HGT is the
transmitance of genetic material between
donor and recipient cells. This is mediated by
multiple processes, all of which will have
natural host-range limitations to one degree
or another (Fig. 5.1). Phages ofen have limited
abilities to infect (Hyman and Abedon, 2010),
plasmids are at the mercy of the success of
conjugation mechanisms between individual
cells, and transformation mechanisms can
dierentiate between closely and distantly
related DNA upon uptake. Next, incoming
genetic material will ofen be encountered in
the cytoplasm by recipient enzymes and
degraded by nucleases including restriction
endonucleases. Recipient-cell DNA has short,
specic sequences throughout the genome
that are continuously marked by methylation.
If a span of DNA enters the cytoplasm and
lacks this mark then that DNA is taken to be
foreign and is cut. Only if DNA overcomes
these barriers can a recombination repair
pathway instead mistake the DNA for native
damaged DNA and thereby integrate it into
the recipient bacterial chromosome or other
cytoplasmic replicon such as a native plasmid.
Subsequent to incorporation, new DNA
faces a number of immediate challenges that
serve to protect the integrity of the recipient
bacteriums genome. The mismatch repair
system, for example, acts to correct mutations,
including small insertions and deletions. It
does so by determining, immediately afer
replication, which is the newly synthesized
and which is the older strand of DNA (Harfe
and Jinks-Robertson, 2000). If there are
dierences between the two, then this system
directs new strand repair in order to match
the older, and therefore trusted, template.
66 H. Hendrickson
Methylation of a common 4 bp sequence is
responsible for this distinction, with methy-
lation lagging behind DNA replication such
that new strands are unmethylated for a time.
Incoming DNA that does not harbour the
native methylation signals is similarly subject
to mismatch repair anti-recombination
processing, which can contribute to genetic
isolation of even closely related bacteria
(Berndt et al., 2003).
An additional, sequence-based dis-
crimin ation mechanism has been recognized
in the clustered, regularly interspaced, short
palindromic repeats (CRISPR) module.
Estimated to be present in up to 70% of
eubacterial genomes, CRISPR is essentially
an adaptive immune system for bacterial cells
wherein the memory of previously en-
countered foreign DNA is incorporated as
small DNA spacers in the CRISPR region of
the genome (Deveau et al., 2010). These are
used to recognize and target incoming DNA
for destruction and have been shown to
interfere with lateral gene transfer (Marra ni
and Sontheimer, 2008).
Barriers to retention
In the event that a newly inserted region of
DNA has both integrated into a replicon and
passed the inspection of these various defence
mechanisms, existence in the population
depends on the tness eect of new DNA for
the host bacterium. Fitness involves many
factors. Interruptions of important genes or
regulatory regions, for example, tend to be
detrimental to tness. Likewise, newly
inserted DNA can interfere with existing
chromosomal architecture, which can impact
the ability of replicated sister chromosomes
to segregate correctly into daughter cells
(Bigot et al., 2005; Hendrickson and Lawrence,
2006; Sivanathan et al., 2009). Early evidence
of the importance of bacterial chromosome
sequence architecture was the observation
that integration of phage into the Escherichia
coli chromosome was orientation dependent.
As a long-term resident of E. coli genomes,
phage has the same replication-mediated
repeat structure as its host genome and
integration events are sensitive to matching
the host (Corre et al., 2000; Pease et al., 2005).
Constraints on HGT that are mediated
by chromosome sequence architecture may
be avoided in two primary ways. First, HGT
between closely related organisms will tend
to bring in novel DNA with compatible
sequence structure. This will be less likely to
interfere with segregation afer integration.
Secondly, transfer of entire replicating units
such as plasmids will entirely avoid this
limitation. Transfer that involves re-
combination into plasmids will also avoid the
constraints of integrating into a structured
genome. In this way, conjugation events can
create a safe haven for HGT (Hendrickson
and Lawrence 2006).
Acquisition of new functions
Newly acquired DNA in an architecturally
structured genome is more likely to decrease
rather than increase the tness of a new
recombinant. By contrast, the acquired
functions of the inherited genes have the
potential to increase the tness of the
recombinant bacterium. In order for any
tness change to be realized, the genetic
material must either include gene operator
regions that are correctly read by the
transcription factors resident in the host
cytoplasm or must have recombined down-
stream of a native operator region, ideally
without disrupting existing functions. Thus,
if the genes cannot be transcribed and
translated, then the additional DNA is
unlikely to be anything more than neutral or
may be deleterious to the tness of the
recipient organism. Weakly expressed or mis-
expressed genes can adapt over time, driving
accelerated evolution of regulatory regions in
recently acquired DNA (Lercher and Pl,
2008).
If DNA can be transcribed and translated,
then new encoded functions will determine
whether this HGT event is advantageous in
the environments in which this bacterium
and its descendants nd themselves. If newly
acquired DNA is not su ciently advan-
tageous, then the carrying individual will
probably be lost from the population.
Alternatively, the new DNA that survives in
the population despite its poor contribution
to the organisms tness can become mutated
to the point where there is no longer any
possibility of function, forming pseudogenes
that will eventually be lost. Observations of
recently acquired genes may reect neutral or
even disadvantageous acquisitions in the
process of being purged, perhaps having
been maintained up to that point by genetic
drif. Thus, a genome sequence should be
considered to be a snapshot of a single
bacterium at some point on its evolutionary
trajectory, rather than as an organism whose
genetic repertoire necessarily represents
perfect adaptation to its environment. The
combination of gene conservation by vertical
descent, frequent HGT and subsequent
taxonomically sensitive constraints leads to
the observed cohesion between closely
related bacteria.
Levels of Horizontal Gene Transfer
Observed in Bacterial Genomes
The myriad methods available for detection
of HGT and the speed at which the eld is
advancing means that any authors
descriptions of levels of gene transfer for
extant organisms are subject to both
speculation and erce debate. Early estimates
of the xation of novel DNA into E. coli put
the rate at 16 kb per 1 million years (Lawrence
and Ochman, 1998). Today, it is generally
accepted that most bacterial genomes have
experienced recent and frequent gene transfer
and that nearly all genes can be transferred
(Ochman et al., 2000; Koonin et al., 2001;
Gogarten and Townsend, 2005; Sorek et al.,
2007).
Bacterial obligate intracellular parasites
of eukaryotic cells appear to be a telling
exception to this rule. The conditions in
which these organisms live, inside their
eukaryotic hosts, limits their exposure to the
agents of gene transfer: phages, plasmids and
extracellular DNA (Moran, 2003; Blanc et al.,
2007; Nikoh and Nakabachi, 2009). The
absence of recombination through phages or
other HGT delivery mechanisms renders
these organisms victims of Mullers ratchet
and the consequences of continual mutation
(Moran, 1996; Andersson and Kurland, 1998).
Accumulation of mutations over evolutionary
timescales eventually limits their potential to
live outside the host cells resulting in their
becoming organelles such as mitochondria
and chloroplasts (Gray and Doolitle, 1982;
Moran, 1996; Lang et al., 1997; Allen et al.,
2009). It should be noted, however, that a
recent study suggests that rare co-infection
events can support genetic exchange in some
cases between such obligately intracellular
bacteria (Kent et al., 2011).
Genes found on plasmids, in phage
genomes or that are associated with other
mobility elements are more likely to be
transferred than genes that are either
intimately involved with large numbers of
other genes or genes that are otherwise very
specic in their interactions. In addition, a
recent study suggested that the number of
functional domains a protein has may also
positively aect the likelihood of its successful
transfer (Gophna and Ofran, 2010). Initially,
the notion that some genes are more reticent
to transfer led to the classication of some
genes as mobile, while other genes are
considered to be core and therefore good
candidates for establishing phylogenetic
relationships (Ge et al., 2005). These core
genes, however, have been observed to be
distributed in non-tree-like manners, sug-
gesting that, while the idea of a set of genes
that are resistant to the action of HGT might
be appealing, this idea does not appear to be
readily demonstrated (Beauregard-Racine et
al., 2011). Lastly, it would seem that the more
we examine the scope of genes that are
present in some but not all members of a
species, the higher our estimates of the
amounts of transfer become (Dagan et al.,
2008). E. coli provides an insight into this
phenomenon, as it is an organism whose
genomic depth we have sampled to a
remarkable degree.
E. coli as an Example of Degrees of
Horizontal Gene Transfer in Bacteria
Recent genome sequencing projects have
expanded our understanding of the speed at
which novel DNA is entering and exiting the
bacteria that we observe today. E. coli is an
important bacterial species, strains of which
68 H. Hendrickson
have been dierentiating for 2540 million
years (Ochman and Jones, 2000). These
organisms are intimately associated with
humans and include both commensals and
deadly pathogens such as the O157:H7 strain
(see Kuhl et al., Christie et al., Williams and
LeJeune, Goodridge and Steiner, and Niu et
al., Chapters 3, 4, 6, 11 and 16, this volume). A
recent 20-genome sequencing project
examining E. coli diversity revealed that the
core genome, the genes that appear in nearly
all E. coli, are approximately 98.3% identical
at the sequence level between strains and are
a set of approximately 2000 genes. Each
genome on average has ~4700 genes and the
total pool of genes shared is 10,000+ (Touchon
et al., 2009). Thus, each genome on average is
~43% core E. coli genes and ~57% other, more
mobile genes that have either recently arrived
or can easily be lost. This other 57% or 10,000+
genes collectively can be referred to as the E.
coli mobile-ome (Siefert, 2009). The obvious
prophage-associated genes in this study were
ofen found in only a single genome, which
suggests that, while ux of phage-borne
genes into genomes is high, the retention of
these genes is low, perhaps due to deleterious
eects (Touchon et al., 2009). Such high levels
of uctuating genomic content imply that
the utility of examining single strains as
representative of species of bacteria is
probably short-sighted.
Another noteworthy nding involves the
locations that new DNA has been introduced
into the genomes. Touchon et al. (2009) found
evidence of hot spots for newly acquired
DNA, with 71% of the non-core transferred
genes located in 133 locations across the 21
genomes. This suggests a natural constraint
on HGT that is overcome by inserting into
genome regions that themselves have been
acquired recently. Logically, these would be
locations that are not essential to bacterial
tness and therefore that are less likely to be
disrupted upon insertion of new DNA. A
subsequent study using 61 completely
sequenced E. coli and the closely related
Shigella genomes came to the conclusion that
over 80% of the genes in a typical genome
could be considered accessory rather than
core and that 90% of all of the genes found in
these organisms (their pan-genome) were
accessory genes. The message from these
large-scale genomic studies appears to be that
bacterial species are uid due to uxes in
gene content (Lukjancenko et al., 2010).
Horizontal Gene Transfer: New
Capabilities, Pathogenicity, and
Beyond
The ultimate eect of the ow of genes into
bacterial genomes is that these genes bring
about new abilities and potentials for these
organisms. It is the sudden acquisition of
segments of genetic material that gives
bacteria access to new niches resulting, for
example, in the spread of antibiotic resistance
and the transformation of commensal bacteria
into pathogenic lifestyles. These processes
have caught our atention in recent years as
dangerous new species have arisen, with
phages ofen acting as direct mediators of
change.
Antibiotic resistance
Many microorganisms express antibiotics
that serve to either kill or slow the growth of
local bacteria. Bacteria in turn develop
capacities to resist antibiotics. Phages mediate
these processes on both sides by mobilizing
genetic material in nature that allows both
resistance to and production of antibiotics
(Fig. 5.2; Davison, 1999). Williams and
LeJeune (Chapter 6, this volume) also briey
consider the role that phages might play
especially in the transduction of antibiotic
resistance genes.
Bacterial evolution and pathogenicity as a
process
Within a single lineage of bacteria, evolving
over millions of years, we can nd the entire
spectrum of host interactions: pathogens,
commensals and mutualists. This observation
alone suggests that these identities are uid
over time, as the last common ancestor of all
of these must have fallen into only one of
these categories or did it? The designation
of pathogen, commensal or mutualist ofen
appears to be dependent on the specic host
that the bacterium is being observed with
(reviewed by Lawrence and Hendrickson,
2008). This is most likely a result of the
specic nature of the chemical signals that are
transmited between a host organism and a
bacterium.
Bacteriophages are constrained some-
what in the genes that they can carry. On
average, these genomes are 60 kb, with a
range from a lower limit of 3.5 kb to an upper
described limit of ~500 kb (see www.ncbi.
nlm.nih.gov/genomes/GenomesGroup.cgi?o
pt =vi rus&t axi d=10239&host =bact eri a;
Hendrix, 2009). Classically, bacteriophages
must carry a genomic content that encodes
structural proteins as well as genome
replication and cell-escape functions. In
addition, most phage genomes contain non-
essential or accessory genes, some of whose
functions are only needed in particular host
strains (Pope et al., 2011). Bacteriophages
none the less contribute in major ways to the
genomic uidity displayed by pathogens.
There are numerous cases where the causative
agent that makes a bacterium pathogenic is a
toxin that is encoded by a prophage (see
Christie et al., Chapter 4, this volume). These
toxins can then be secreted by the pathogen
into the extracellular environment to wreck
havoc on the host organism (reviewed by
Casas and Maloy, 2011). In addition to
mediating pathogenicity in this fashion,
bacteriophages at times carry alternative
O-antigens that aid bacteria in evading host
immune systems or other predation, promote
adhesion of cells to tissues and contribute to
generalized transduction of antibiotic
resistance genes in populations (Firth and
Skurray, 2006; Wagner and Waldor, 2006;
Wildschute and Lawrence, 2007).
If bacteria can inherit a suite of deadly
pathogenicity genes in a single phage
integration event but still look otherwise
harmless, then the medically important
dierence between an inoensive enteric
bacterium and a pathogen is, from a genetic
perspective, a small one indeed. For historical
reasons, much nomenclature in bacteria is
inherently uninformative in this respect. E.
coli K12 strain MG1655 is a harmless
laboratory strain that was obtained from a
patient in Palo Alto in 1922 (Blatner et al.,
1997; Anon, 2011). On the other hand,
Pathogen-encoded
antibiotic resistance
Naturally encoded
antibiotics
Phage-mediated
gene exchange
e g n a h c x E e c n a t s i s e R c i t o i b i t n A
Fig. 5.2. Phage involvement in cycling antibiotic production and resistance genes in the environment.
Antibiotics are part of the normal competitive regime of microorganisms in nature and are produced by a
wide range of organisms (grey arrows). Bacteria are therefore under pressure to evolve resistance to
these (black notched circles). The genes that encode resistance may also be found in organisms that
produce the antibiotics (black gene in genome) and are generally benecial when transferred through
phage or other processes. Phages are therefore taking part in the transmission of genetic material that
encodes for both antibiotics and antibiotic resistance in nature.
70 H. Hendrickson
beginning 60 years later, E. coli O157:H7 strain
EDL933 was a deadly pathogen, the causative
agent of haemolyticuraemic syndrome and
responsible for fatalities afer consumption of
contaminated meat in the USA (for more on E.
coli O157:H7 see Kuhl et al., Christie et al.,
Williams and LeJeune, Goodridge and Steiner,
and Niu et al., Chapters 3, 4, 6, 11 and 16, this
volume). These versions of the same species
had diverged over an estimated period of 4.5
million years and each had acquired large
segments of DNA through HGT in the
intervening time (Perna et al., 2001; Welch et
al., 2002). These early comparisons of strains
of the same species comfortingly led to the
conclusion that these additions had been
made on a 4.1 Mb backbone of core genes, or
roughly 89% of the MG1655 and 72% of the
EDL933 total genome size. As stated earlier,
however, more recent sequencing eorts put
genes shared by all E. coli at closer to 43% of
total genes in an average representative
(Touchon et al., 2009). These eorts miss much
of the nearly homologous re com bination
taking place between more closely related
strains, and inclusion of these re combination
events as well could send estimates of the
percentage of genes in the average genome
that have been recently transferred to 81%
(15% ; Dagan et al., 2008). It seems that the
more we learn about the amount of transfer
taking place, the less certain we can be about
species delineations and their relevance to
human disease.
Phages can also increase the patho-
genicity of bacterial communities without
changing the genetic make-up of these
bacteria. Bacterial biolms have become the
subject of much speculation recently, and the
nature and signicance of these microbial
conglomerations are being investigated.
Micro organisms living in these mature
pellicle structures of excreted polysaccharides
appear to be less susceptible to antibiotics
and phages. In fact, the biolm community of
a cystic brosis lung was recently found to be
the rst phage-free environment recorded
(Hiby et al., 2010, 2011; Willner et al., 2011).
At the same time, however, it is also becoming
clear that phages are a natural part of many
of these communities (Abedon, 2011). Biolm
formation has been demonstrated in multiple
pathogenic lineages to be bolstered by the
presence of extracellular DNA. As phages
lyse bacteria, they release this DNA into the
environment and this additional extracellular
DNA enhances biolm structural stability,
and in some cases the degree of phage-
mediated lysis appears to be regulated by the
bacteria (Carrolo et al., 2010; Kay et al., 2011;
Petrova et al., 2011).
The Why of Bacteriophages and
Pathogenicity
The consensus regarding the impact of HGT
on the evolution of bacteria has been
recognized only in phases. Immediately afer
completely sequenced bacterial genomes
started to be published (1996), HGT was
considered to be a novel or rare event. Within
a few years, gene transfer began to be
appreciated as an ongoing but nevertheless
restricted process, where some genes were
thought to be mobile but most were not
transferred. More recently, by contrast, there
has been increasing suggestion that the
majority of genes in many genomes have
been transferred and that cohesion within
families might be driven by a combination of
vertical descent and constraints on these
transfer frequencies. Continued sequencing
eorts and improved methodologies for
detection have carried us through these shifs
of paradigm. Today, we are still at an impasse
as to what observed transfer means for
meaningful taxonomy. This becomes a crucial
point when nomenclature meets medical
practice (Brssow et al., 2004; Gevers et al.,
2005; see also Kuhl et al. and Williams and
LeJeune, Chapters 3 and 6, this volume).
Evolutionary biologists are ofen
interested in the why of phenomena more
than the how. The short answer to why
bacteriophages carry genes that enhance
pathogenicity is that it helps them to make
more bacteriophages. In 1976, Richard
Dawkins wrote, in The Selsh Gene, that one
could consider genes as selsh replicators
coding for phenotypes whose sole goal it is to
increase their own numbers (Dawkins, 1976).
From this perspective, any gene can be seen
as contributing to an organism only in order
to ensure that more copies of the gene are
made. As this concept has inuenced modern
biological thought, multiple lines of evidence
have contributed to the notion that many
genetic elements can be thought of in much
the same way. Selsh genetic elements have
been dened as those having characteristics
that enhance their own transmission relative
to the rest of an individuals genome, and that
are either neutral or detrimental to the
organism as a whole (Werren et al., 1988). By
this denition, bacteriophages can be thought
of as a quintessentially selsh genetic
element. These bundles of DNA wrapped in a
protein coat travel through the natural world,
inert until they happen upon a replicator to
prey on. From this view, the success of a
temperate bacteriophage is dependent on the
genes that it can imbue to a host that it
integrates into (Abedon and LeJeune, 2005). If
the bacteriophage encodes for su ciently
niche-expanding or fortifying genes, then
it can increase its copy number by
accompanying a successful host organism in
its clonal expansion during an infectious
cycle. In some cases, this will propel otherwise
harmless commensals into more pathogenic
lifestyles but the measure of success for the
phage during this outgrowth is simply the
production of more phages.
Summary
The relationship between pathogens and
bacteriophages is a complex one and the two
entities can be thought of as interdependent
in many ways. On the one hand, phages can
serve as catalysts that impel bacteria,
particularly via HGT, into a pathogenic
lifestyle. On the other hand, pathogens as
well as bacteria in general appear to be
doomed, ultimately, to mutational collapse
under the pressure of Mullers ratchet when
phages and other mediators of HGT are not
present to mediate intra-specic re-
combination. Nevertheless, although phages
apparently are benecial when viewed over
these evolutionary timescales, over short
timescales they fundamentally serve as
predators that search out and devour
bacteria, including the same pathogens that
they can play such important roles in creating.
In the tale of the lion and the mouse, the lion
threatens the mouse one day and is freed by
the mouse from a hunters trap the next.
Given the evidence of the eminently complex
co-dependencies between pathogens and
phages in the microbial world, who is the
true king of the jungle? Bacteria perhaps owe
deference to their phage predators, which
serve simultaneously as creators, sustainers
and destroyers of bacterial pathogens?
Acknowledgements
The author would like to acknowledge the
generous support of the Human Frontier of
Science Program Organization. She is also
grateful for helpful discussion with D.I.
Andersson and J.G. Lawrence, as well as
helpful access to a manuscript in press by S.
Maloy.
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6 Phages and Bacterial Epidemiology
Michele L. Williams
1
and Jeffrey T. LeJeune
1
1
Food Animal Health Research Program, The Ohio Agricultural Research and
Development Center.
The frequent, complex and intimate inter-
actions between phages and their bacterial
hosts have inuenced the evolution of both
groups of organisms. The con sequences of
their relationship have signicantly impacted
human, animal and plant health. As discussed
in Kuhl et al., Christie et al. and Hendrickson
(Chapters 35, this volume), some organisms
have acquired genes to become more virulent.
Notwithstanding this, ever since the discovery
of phages, researchers have been atempting
to harness and exploit the biological and
genetic properties of phages to advance
science and to enhance health. The use of
phages in molecular biology studies (see
Abedon, Chapter 1, this volume) and as
agents for therapeutic (see Olszowska-
Zaremba et al., Loc-Carrillo et al., Burrowes
and Harper, and Abedon, Chapters 12, 13, 14
and 17, this volume) or diagnostic (see Cox,
Chapter 10, this volume) purposes, and for
food processing (see Niu et al., Chapter 16,
this volume) has clearly provided benets to
society. Another important way that the
association between phages and bacteria has
been exploited is in the tracking of outbreaks
and epidemiological research related to
disease.
The dierentiation of bacterial isolates at
a greater resolution than the species level, in
order to identify strains or subspecies,
provides valuable information in the investi-
gation of disease outbreaks and research on
the epidemiology and ecology of pathogens
(Moorman et al., 2010). The essential and
preferred criteria for such bacterial typing
methods have evolved litle since their advent
(Anderson and Williams, 1956; van Belkum et
al., 2007). These include inherent character-
istics of the test: (i) stability (consistency over
time); (ii) typeability (the ability of the test to
describe a unique phage type); (iii) dis-
criminatory power (the ability to dierentiate
strains, (iv) epidemiological concordance
(grouping of related isolates similarly); and
(v) reproducibility (the ability to get the same
result each time the test is run). They also
include practical aspects of use such as: (i)
ease of use; (ii) applicability across a broad
range of bacterial species (exibility); (iii)
speed or turnaround time; (iv) accessibility;
and (v) cost (van Belkum et al., 2007). In this
chapter, the methodologies and application
of phages and prophages in the tracking and
subtyping of bacterial strains is reviewed.
Phage Typing: a Foundation for
Epidemiological Investigations
In 1922, Callow reported host-range dif-
ferences among a collection of staphylococcal
phages (Callow, 1922). A few years later,
Craigie and Yen (1938) were able to develop
the rst phage-based typing method by
exploiting the receptor specicity of phages
M.L. Williams and J.T. LeJeune
Phages and Bacterial Epidemiology 77
for the Vi antigen present on the surface of
some Salmonella typhi. Briey, phage typing
requires a collection of phages lytic for at
least some of the members of the bacterial
group being typed. The principles of phage
typing reect the fundamental concepts
governing the relationship between phages
and their bacterial hosts, notably receptor
binding, restriction enzyme systems and
immunity as mediated by prophages (Hyman
and Abedon, 2010; Labrie et al., 2010). Both
obligately lytic and temperate phages may be
employed. Using the highest possible dilution
known to produce areas of lysis on lawns of
its strain type (the bacterial host used to
propagate the phage), the bacterial strain to
be typed is challenged with a panel of phages.
The type of lysis that results is recorded
(conuent, opaque, individual clear plaques,
individual opaque plaques or no lysis). It is
not expected that every phage will result in
lysis of every test strain. Instead, it is the
patern of lysis caused by the panel of phages
that denes the particular phage type (PT) of
the test strain. In eect, phage typing can be
viewed as a phenotypic assessment tool (spot
formation) that represents or is governed by
genetic elements present in the host.
The initial experiments, reported in 1938
by Craigie and Yen, became the foundation
for all subsequent phage typing schemes
including those still in use today. Many
bacteria are still commonly typed using
phages (Table 6.1). The methods and specic
phages employed are standardized for each
typing scheme. The International Federation
of Enteric Phage Typing oversees and
coordinates phage typing in 55 countries
around the globe.
Issues relevant to phage typing utility:
inherent characteristics
Despite the longstanding history and value of
phage typing in epidemiological investi-
gations, this methodology performs poorly
when evaluated for several of the afore-
mentioned desirable criteria for typing
methods.
Stability
Host susceptibility to specic phages is sub-
ject to change. Prophages, stably integrated
into the host genome, can confer immunity to
Table 6.1. Bacteria commonly typed using bacteriophages.
Bacteria Reference
Acinetobacter spp. Santos et al. (1984)
Bacillus spp. Ahmed et al. (1995)
Campylobacter spp. Frost et al. (1999)
Clostridium difcile Sell et al. (1983)
Clostridium perfringens Yan (1989)
Corynebacterium diphtheriae Andronescu et al. (1997)
Erwinia spp. Toth et al. (1999)
Escherichia coli Pearce et al. (2009)
Klebsiella spp. Sechter et al. (2000)
Listeria monocytogenes McLauchlin et al. (1996)
Mycobacterium spp. Schurch and van Soolingen (2011)
Pasteurella multocida Nielsen and Rosdahl (1990)
Proteus spp. Sekaninova et al. (1999)
Pseudomonas spp. Bergmans et al. (1997)
Salmonella spp. Kafatos et al. (2009)
Serratia spp. Hamilton and Brown (1972)
Shigella spp. Bentley et al. (1996)
Streptococcus spp. Domelier et al. (2009)
Vibrio cholera Sarkar et al. (2011)
Yersinia spp. Kawaoka et al. (1987)
78 M.L. Williams and J.T. LeJeune
superinfection. Under certain circumstances,
however, these prophages may be excised
and lost. This can result in the potential for
the phage type of a given bacterial strain to
change. Prophage excision, also known as
curing, may also occur either during the
course of an outbreak (diphtheria) or during
laboratory propagation, such as following the
initial isolation of Shiga toxin-producing
Escherichia coli (Bielaszewska et al., 2007). In
addition to the loss of prophages, bacteria
may become lysogenized by temperate
phages, again potentially changing their
phage type (Rankin and Plat, 1995). Although
it would be unusual for such an event to
happen in the laboratory afer the initial
isolation of the bacterial strain, lysogenization
occurs naturally in the environment (Revathi
et al., 2011).
In addition to phage-mediated transfer
of DNA and the loss of prophages, the gain or
loss of other mobile genetic elements, notably
plasmids, can also result in phage type
conversion (Frost et al., 1989). Loss of phage
receptors can also result in changes in phage
types. For example, the sporadic transition
from smooth to rough strains of bacteria
associated with the loss of lipopolysaccharide
can result a change in the susceptibility to
phages used in typing, resulting, for example,
in the conversion of Salmonella enterica serovar
Enteritidis PT 4 to PT 7 (Chart et al., 1989).
Typeability
Typeability refers to the ability of the testing
methodology to assign a subtype to the
organism being evaluated. Because the typing
system relies on the patern of lysis for a
collection of individual phages, it is
imperative that the reference phage typing
set contains one or more phages capable of
causing lysis in the tested organism, other-
wise the organism is considered untypeable.
Depending on the bacterial genus and species
being tested, the origin of the samples and the
diversity as well as size of the typing set of
phages, phage typeability may be as low as
0% and as high as 100% (Kuramasu et al.,
1967; Valdezate et al., 2000).
Discriminatory power
Discriminatory power refers to the ability of
the typing system to distinguish between
unrelated isolates, that is, the probability that
two organisms randomly selected from a
population of genetically unrelated organisms
will produce dierent subtypes (Hunter and
Gaston, 1988). As with other typing systems,
the discriminatory power of phage typing is
dependent on the population (genus and
species) of the bacterial strain being tested
and is highly variable, but indices between 80
and 90% are typical and can be useful for
epidemiological in vestigations. For example,
the discrimina tory power of phage typing for
S. enterica serovar Enteritidis is 85% (Boxrud et
al., 2007) and for Listeria monocytogenes it
is 87% (Boerlin et al., 1995). The problem
associated with phage typing tests of low
discriminatory power is that isolates with the
same phage type could be genetically un-
related, resulting in a misclassication of
unrelated cases into an outbreak denition.
Epidemiological concordance
It is desirable that the typing method groups
all of the outbreak strains from the same
source together. Epidemiological concordance
(E) is a measure of how well the typing
method agrees with the epidemiological data
obtained from other sources. E can be
calculated by determining the fraction of
epidemiologically linked sets (ve to ten
isolates per set) of strains recovered from
outbreaks that are indistinguishable (van
Belkum et al., 2007). This atribute can be
considered the counterbalancing factor to
discriminatory power. High discriminatory
power with low epidemiological concordance
would result in a failure to cluster
epidemiologically related isolates from the
same outbreak together. The problem
associated with low epidemiological con-
cordance is that two or more isolates of
similar clonal origin that have been modied,
for example by a recent prophage in-
corporation or phage mobilization event, will
not be considered to be part of the same
outbreak when in fact they are.
Reproducibility
Reproducibility is the ability of the typing
method to generate the same result when
repeated on the same isolate on dierent
occasions in dierent laboratories. It diers
from stability (the rst criterion, above) in
that it is inuenced by factors extrinsic to
the bacteria. Specically, intra- and inter-
laboratory inconsistencies, including operator
error, contribute to variations in analysis and
interpret ation, thereby resulting in poor
reproducibility. Phage typing can be highly
reproducible (0.985; Marquet-Van der Mee and
Audurier, 1995), but it requires standardized
panels of phages and should be conducted
only in a limited number of reference labora-
tories by experienced personnel. With out tight
controls on laboratory methods and reagents,
reproducibility is poor.
Issues relevant to phage typing utility:
practicality
In addition to the abovementioned per-
formance criteria, there are several practical
aspects of phage typing that are also
considered when evaluating the usefulness of
bacterial typing schemes. These include: (i)
ease of use; (ii) applicability; (iii) cost; (iv)
accessibility; and (v) speed. Phage typing
falls short in several of these areas when
compared with other typing methods. For
example, each bacterial species requires a
dierent set of test phages for typing, thereby
limiting its applicability or exibility in terms
of usefulness for multiple bacterial species.
Although the laboratory equipment required
for test performance is not highly specialized,
it does require a fair amount of technical
expertise to perform the analysis, ranking
phage typing lower on the ease-of-use scale.
The technical expertise required and the
maintenance of a wide range of phage
libraries add to the cost and restrict the
availability of phage typing to a limited
number of highly specialized reference
laboratories. The speed at which the assay
can be completed is dependent on the time
required to detect spots on bacterial lawns.
This may be quicker than some other typing
methods (e.g. pulsed-eld gel electro-
phoresis), similar to methods of phenotypic
characterization (e.g. biochemical tests) or
slower than some more modern molecular-
based methods such as PCR.
Choosing the most appropriate typing
method is dependent on the question at hand
and the resources available. Despite the
abovementioned limitations, the use of phage
typing is still considered useful in epidemi-
ological surveillance and investigations
(Kafatos et al., 2009; Baggesen et al., 2010). In
many instances, using a combination of
typing methods including traditional phage
typing can add valuable molecular insight
into the epidemiological investigation of
outbreaks (Bhowmick et al., 2009; Cho et al.,
2010).
The Next Frontier in Phage Typing
Fundamentally, phage susceptibility and
ultimately phage typing are phenotypic
representations of the genetic composition of
the bacterial host, although as yet unidentied
environmental factors governing phage sus-
ceptibility may also exist. Host-cell suscep-
tibility to specic phages is viewed primarily
as a reection of: (i) the encoding and
expression of specic phage receptors; and
(ii) pre-existing prophage lysogenization
potentially resulting in immunity (Stephan et
al., 2008). There additionally exist a substantial
number of anti-phage resistance mechanisms
that are encoded by bacteria and which, in
principle, could be correlated with the failure
of specic typing phages to recognize the
appropriate phage. These mechanisms include
the familiar restriction-modication mechan-
isms, as well as abortive infection systems.
Certainly, of greatest current interest are
clustered, regularly interspaced, short
palindromic repeat (CRISPR)/Cas systems,
which not only can bestow anti-phage
resistance but do so in such a manner that
resistant bacteria and resisted phages display
small regions of high genetic identity (Deveau
et al., 2010; Hyman and Abedon, 2010; Labrie et
al., 2010). Thus, although phage typing is
characterized as a phenotypic subtyping
method, like other phenotypic methods it too
could mature methodologically as more
information becomes available into genotypic
methods, that is, if the genetic elements
underlying phage-resistance or phage-
sensitivity phenotypes are resolved. A number
of investigators are exploring this line of
research.
Phages ofen use outer-membrane
proteins as receptors. Thus, characterization
of the diversity of receptors present on
bacterial hosts capable of binding a variety
of phages, including those used in phage
typing, could provide a novel phage-centric
tool to discriminate and classify bacteria.
Unfortunately, not all phage receptors are
known, and it is expected that new receptors
will be discovered. Nevertheless, this
approach has been piloted for the discrimin-
ation of S. enterica serovar Enteritidis phage
types (Preisner et al., 2010). In these studies,
Fourier transform infrared spectroscopy was
used to analyse the outer-membrane proteins
present in extracts of cell membranes of
various phage types of Salmonella Enteritidis.
Although the exact components in the extract
that were correlated with the phage typing
classication were not identied, their
analysis demonstrated that this rapid method
of dierentiation of phage types was highly
discriminatory. Presumably, the factors that
contribute to the strength of the association
were either the receptors for phages them-
selves or other membrane components that
were genetically linked with susceptibility of
host lysis by specic groups of phages.
Another approach that has been used to
move away from phenotypic methods is the
identication of correlates between phage
typing systems and genetics. In Salmonella
Typhimurium, Lan et al. (2007) identied ten
sequences using amplied fragment length
polymorphism, which could be used for
discriminating eight dierent phage types of
this organism. A similar approach, PCR of
phage-derived open reading frames, was
proposed by OSullivan et al. (2010) to
dierentiate methicillin-resistant Staphylo-
coccus aureus (MRSA) subtypes. More recently,
in experiments conducted using microarray-
based technology, specically diversity arrays
technology, Hackl et al. (2010) described a set
of DNA probes that could be used to
dierentiate between Salmonella enterica
serovar Enteritidis and Typhimurium strains
based on phage types. Many of the genetic
markers were subsequently sequenced and in
a number of cases demonstrated high identity
to hypothetical or putative proteins from
phages common to Salmonella (Fels-2, Gifsy-1
and ST64B).
Studies such as the ones described here
demonstrate the possibility of linking rapid
molecular analysis of bacterial proteins or
genome sequences with established bacterial
groups dened by phage typing. This would
allow the application of rapid analysis
methods for new strain identication with
strong epidemiological concordance to
existing groups and bypassing the limitations
of traditional phage typing described above.
See Cox (Chapter 10, this volume) for
additional consideration of phage-based
methods that may be employed to distinguish
bacterial species or strains.
Beyond Typing: Additional
Epidemiological Insights
Many bacterial genomes are pervaded by
cryptic phages and prophages. As described
above, the public health arena has exploited
this phenomenon to develop and rene tools
to dierentiate strains of bacteria to determine
which cases of illness may be atributed to a
single source during outbreak investigations.
Prophages and their respective chromosomal
insertion sites, however, may also provide
valuable insights into bacterial evolution,
ecology and the pathogenesis of disease.
Although not fully understood, lysogeny by
temperate phages may impact on the ecology
and physiology of the bacterial host (Chen et
al., 2005). Moreover, several prophages en code
cardinal virulence factors necessary for disease
(e.g. botulism toxin, diphtheria toxin, cholera
toxin and Shiga toxin) or elements for the
regulation of virulence among pathogenic
bacteria (Abedon and Lejeune, 2005; see
Christie et al., Chapter 4, this volume). Further
elucidation of the role of phage-encoded
genes, or at least identifying associations
between their presence/absence and epidemio-
logical niches, may help to explain the
distribution of pathogens in the environment
and their relevance to human disease
information that is critically needed to develop
enhanced prevention and control strategies to
improve human health.
One example of a phage-encoded gene
used for epidemiological investigation is the
phage anti-terminator Q gene present in
Shiga toxin-producing E. coli O157. A Q allele
identical to that present in bacteriophage
933W is associated with high Shiga toxin
production and with most cases of clinical
disease in humans (LeJeune et al., 2004).
Additional typing systems have been
described that dierentiate between human
clinical isolates of E. coli O157 and the larger,
more diverse pool of genotypes present in the
catle population, a predominant reservoir
host for E. coli O157. Notably, the lineage-
specic polymorphism assay (LSPA) targeted
a number of phage-encoded genes (Zhang et
al., 2007). In contrast to LSPA, the Shiga toxin-
encoding bacteriophage insertion (SBI) sites
method screens putative phage insertion sites
to determine whether the sites are accessible
for lysogeny or whether they are occupied,
and, if occupied, with what size of genetic
insertion (Shaikh and Tarr, 2003). Collectively,
all of these phage-based methods con-
cordantly identify distribution dierences in
E. coli O157 genotypes recovered from human
clinical cases and the bovine reservoir
(Whitworth et al., 2010). These genetic tools
can be used to track subtypes of bacteria that
are over- or under-represented in dierent
environments (Besser et al., 2007). Our
understanding of factors governing lysogeny
in E. coli O157, however, is incomplete.
Stephan et al. (2009) were unable to transduce
two Shiga toxin-negative E. coli O157 strains
despite the presence of intact SBI sites. Hence,
the presence of intact SBI sites in bacterial
genomes is not a clear indication of potential
for lysogenic conversion and the emergence
of novel pathogenic strains (for more on E.
coli O157:H7, see Kuhl et al., Goodridge and
Steiner, and Niu et al., Chapters 3, 11 and 16,
this volume).
Antibiotic resistance
Another important role bacteriophages may
play in the epidemiology of human, animal
and plant health is in the emergence of
antibiotic resistance. The emergence of and
increase in resistance to antibiotics among
bacterial pathogens and commensal organ-
isms are important and growing public health
concerns (Bush et al., 2011), particularly
because antimicrobial-resistant infections are
ofen di cult to treat and result in increased
healthcare costs (McGowan, 2001; Merz et al.,
2010). Although much atention has been
placed on bacterial conjugation as a mechanism
for the emergence and dissemination of anti-
microbial resistance (AMR) genes, bacterio-
phages encoding AMR genes are widespread
in the environment (Colomer-Lluch et al.,
2011a,b), and the potential for transduction of
AMR genes has been ofen overlooked.
Examples of how phages may impact on
the epidemiology of human and animal
diseases include the emergence of the penta-
resistant S. enterica serovar Typhimurium,
denite type (DT) 104 in the 1990s. The
genes that confer resistance to ampicillin,
chloramphenicol, streptomycin/pectinomycin,
sulfonamides and tetracycline in this
organism are clustered chromosomally
(Schmieger and Schicklmaier, 1999) and are
not located on a plasmid. These genes,
however, are transducible by several phages
endemic to DT104 isolates (Schmieger and
Schicklmaier, 1999). Similarly, our laboratory
has demonstrated the transduction of genes
encoding extended spectrum |-lactamase
(cmy-2) and tetracycline (tetA and tetB)
resistance from S. enterica subsp. enterica
serovar Heidelberg to S. enterica serovar
Typhimurium (Zhang and LeJeune, 2008).
There is also growing evidence that MRSA
may have also evolved by the transduction of
the mecA gene to S. aureus from other
staphylococci. For additional discussion of
transduction and the evolution of bacterial
genomes, including the genomes of bacterial
pathogens, see Christie et al. and Hendrickson
(Chapters 4 and 5, this volume).
Summary and Outlook
Clearly bacteriophages have shaped the
landscape of infectious agents with signicant
impacts on the epidemiology and ecology of
bacterial pathogens (see Hendrickson,
Chapter 5, this volume). Prophages along
with various cryptic elements present in
microorganisms provide a trail of genetic
footprints that can be used to track the
evolution of pathogens and the short-term
epidemiology of disease. At present, despite
its limitations, classical phage typing which
operates in part by phenotypically detecting
these elements provides a valuable tool in
outbreak identication and investigation.
The discovery of the specic genotypic
elements underpinning the traditional
phenotypic methods of phage typing will
provide more robust and reliable methods to
track outbreaks. As more information is
acquired and the associations among phage
types, genotypes and diseases are beter
understood, it is expected that the use of
phage-based molecular methods will
supplement, if not replace, current phage
typing in epidemiological investigations and
research.
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7 Phages as Therapeutic Delivery
Vehicles
Jason Clark
1
, Stephen T. Abedon
2
and Paul Hyman
3
1
BigDNA Ltd, Roslin BioCentre, Roslin;
2
Department of Microbiology, The Ohio
State University;
3
Department of Biology/Toxicology, Ashlands University
Because of their small genomes, relative ease
of genome isolation and the more direct link
between genotype and phenotype, bacterio-
phages were some of the rst organisms upon
which modern genetic engineering tech-
niques were performed. This relative ease of
manipulation means that phages also have a
versatility in biotechnology appli cations that
is distinct from other platforms. One aspect of
that versatility can be summarized in terms of
phages serving as delivery vehicles. When
used for this purpose, bacteriophage virions
can be considered as inert, nanoscale particles
that have specic activities when they interact
with their targets.
Examples of such applications include:
the direct use of phages as killing agents
(phage therapy; see Olszowska-Zaremba et
al., Loc-Carrillo et al., Burrowes and Harper,
and Abedon, Chapters 12, 13, 14 and 17, this
volume); delivery of nucleic acid to
mammalian cells (gene therapy and vaccines,
this chapter); immunomodulation via phage
proteins (vaccines/chemotherapy; see
volume); cell binding of phages linked to
toxins (chemotherapy, this chapter); and
linking bacteria to detectors (biosensors; see
Cox, Chapter 10, this volume). These various
processes can be categorized in two ways: (i)
by considering which physical/chemical part
of the phage is considered the cargo being
delivered; and (ii) by considering eects on
the recipient cells. Table 7.1 summarizes the
classication of some applications according
to these categories. For reason of space
limitations, we have avoided providing a
comprehensive review of the literature and
will instead concentrate on an illustrative
subset of those studies we have identied.
Variations on the Theme
The phage genome can be modied to include
heterologous sequences designed to be
expressed in or to otherwise modify a target
cell. Gene expression can be under the control
of either prokaryotic or eukaryotic sequences.
Phages can be modied to deliver proteins
directly, by displaying the proteins as coat
protein fusions (phage display; see Siegel,
Chapter 8, this volume) or by articial
conjugation of proteins to the intact phage
capsid (see Cox, Chapter 10, this volume).
Other cargos may include non-protein
molecules that are atached to phages for
delivery to targets using the phage proteins
to provide targeting via specic binding
sequences. Non-DNA cargo may be subject
to control by a human operator or modied
in response to the target entity, such as
being released (i.e. solubilized) and thereby
activated.
Phages as Therapeutic Delivery Vehicles 87
The target for phage-delivered material
can be either prokaryotic or eukaryotic. The
obvious targets for phages are bacteria and
the natural specicity of phages has been
harnessed for a variety of biotechnological
applications. Alternatively, phage display or
the conjugation of non-phage molecules can
be used to target phages to cells (see Siegel,
Chapter 8, this volume), either prokaryotic or
eukaryotic, for which there otherwise is no
natural tropism. In these instances, the
phages act as agents that atach to the cell
surface, with this atachment sometimes
followed by endocytosis if the target cell is
eukaryotic. For some applications, the
interaction can be limited to simply binding
to the target, co-locating the phages cargo
with its target. While antibodies can serve
similar purposes, and indeed may be re-
sponsible for homing in on non-host targets,
phages have the advantage of providing a
larger, more exible platform with which
cargo may be delivered.
Phage-delivered cargo can be benign or
toxic to target cells. Benign treatments can
include gene therapy as well as the use of
phages as DNA vaccines, which are intended
to leave the cell metabolically active, and
producing phage-DNA encoded protein.
Alternatively, phages may be modied
genetically, phenotypically or both so that
they deliver toxins or genes expressing
damaging products. The later can include a
form of phage therapy where prokaryotic
cells are the target. Phages can be designed to
damage or kill non-bacterial targets such as
tumour cells. Phage modication of targets
can also result in the generation of a signal for
detection (Galikowska et al., 2011; see Cox,
Chapter 10, this volume).
Phage-delivered cargo can result in only
temporary expression or impact such as for
the sake of bacterial identication (see Cox,
Chapter 10, this volume). The use of phages
as carriers of DNA vaccines is also a
temporary phenomenon that induces a long-
term eect on the host. Phage therapy or
phage carriage of toxic genes or materials to
any target also, ideally, is a short-term eect.
Employment of phages as vectors for either
gene cloning or gene therapy will be relatively
long lasting, not just in its eect but also in
terms of retention and expression of
constructs. Some therapies may also rely on
long-term expression in a relatively small
number of cells (e.g. gene therapy), whereas
Table 7.1. Characteristics of material being delivered by phage in various technologies.
Application
Type of cargo
delivered
a
Type of target
Effect on
host cell
b
Result of
modication
c
Duration of
effect
d
Whole phage
uptake needed
e
Normal phage
infection
Nucleic acid Bacteria Toxic Not applicable Permanent No
Phage therapy Nucleic acid Bacteria Toxic Manipulation Permanent No
Chemotherapy Exogenous
toxin
Cancer cell Toxic Manipulation Permanent Yes/no
(depends on
toxin type)
Vaccine Nucleic acid
or protein
Immune or
other cells
Non-toxic Manipulation Temporary
(on cells
taking up
phage)
Yes
Gene therapy Nucleic acid Multiple cell
types
Non-toxic Manipulation Permanent Yes
Imaging agent Exogenous Multiple cell
types
Non-toxic Signalling Temporary No
Bacterial
biosensor
Protein or
nucleic acid
Bacteria Non-toxic Signalling Temporary No
a
Is the phage protein, nucleic acid sequence or some molecule bound to the phage virion the active material being
transported by the phage?
b
Is the material being delivered toxic to the target cell or not?
c
Target cells may be altered in some way (manipulation) or detected (signalling).
d
Target cells may be permanently changed or some property may be altered temporarily.
e
Does the whole phage need to be taken into the target cell for the desired effect to take place?
88 J. Clark et al.
others may rely on high-level expression over
short timescales (e.g. treatment of cancers).
There are a number of technologies
involving phage applications intended to
modify mammalian physiology either by
generating immune responses (vaccines; see
volume), modifying the permanent or semi-
permanent genetic complement of human
cells (gene therapy) or killing unwanted
tissues such as tumour or white fat cells
(cytotoxic agents). Phages that can bind to
mammalian tissues can become endocytosed.
Phage clearance from circulation to sites of
antigen presentation can result in increased
expression from eukaryotic expression
cassetes (Lankes et al., 2007) in a manner that
has been shown to be antibody dependent
and Fc receptor mediated, and to enhance
transfer of genes in mammalian cells both
in vivo and in vitro (Sapinoro et al., 2008).
Phages also can serve as scaolds for alter-
ing the pharmacokinetic properties of other
molecules.
Phages as Vaccine Delivery Vehicles
The ease of production of large numbers of
phages on relatively inexpensive media, com-
bined with the proposed ease of purication
(Gill and Hyman, 2010), suggests that bac-
teriophages could serve as ideal vectors for
vaccine delivery. Phages also may have
natural adjuvant properties with, for example,
unmethylated CpG motifs in phages with
double-stranded DNA genomes being potent
stimulators of Toll-like receptor 9 (Senti et al.,
2009). Similarly, the protein component of
phages can provide T-cell epitopes that
enhance antibody response and improve
immune memory. It has also been reported
that T4 phages are potent stimulators of
interferon (IFN) production (Kleinschmidt et
al., 1970), as supported by the observation
that high levels of IFN- are produced in
mouse cells following immunization with
phage (J. Clark and J.B. March, unpublished
data). Similarly, it has been shown that
treatment of blood cultures with phages
results in increased levels of tumour necrosis
factor-o and interleukin-6 (Weber-Dabrowska
et al., 2001; see also Olszowska-Zaremba et al.,
Chapter 12, this volume). The natural
adjuvant eect of phages is also implied by
results suggesting that phage vaccines give
rise to similar immune responses in the
presence or absence of adjuvant, whether
they are protein (Greenwood et al., 1991;
Minenkova et al., 1993; Willis et al., 1993) or
DNA (i.e. genetic) vaccines (Clark and March,
2004b). We dierentiate phage-based delivery
of vaccines into two basic types: those where
DNA is the cargo and those where protein
instead serves as the cargo.
Phages as DNA vaccines
In phage-mediated DNA vaccination, a
eukaryotic expression cassete is cloned into
the genome of a phage vector and whole
phage particles are used to immunize the
host. This has several potential advantages
over standard vaccination including the
generation of strong type 1 immune responses
along with the production of vaccine antigen
within host cells so that the resultant proteins
are correctly post-translationally modied.
Additionally, the phage coat protects the
DNA from degradation and the phage
particles themselves can be stable over a wide
range of temperatures and pH values (Jepson
and March, 2004). As particulate antigens,
phages should be targeted to sites of antigen
presentation where the vaccine component
can e ciently raise an immune response.
The majority of research into phage-
delivered DNA vaccination has been
performed using phage , with the rst
published report of this method using a
gt-11 vector to deliver the hepatitis B small
surface antigen (HBsAg) gene, under the
control of the cytomegalovirus promoter, to
mice (Clark and March, 2004b). Antibody
responses against the HBsAg protein were
detected afer two immunizations by a number
of dierent methodologies and in some
instances anti-HBsAg responses were in excess
of 10 mIU ml
1
, the internationally recognized
level required for protection, with the highest
level being approximately 150 mIU ml
1
. A
phage containing an expression cassete
expressing enhanced green uorescent protein
(EGFP) also generated antibody responses in
mice afer two immunizations, with sera
recognizing the EGFP protein in Western blots
(March, 2002; March et al., 2004). Subsequent
research in rabbits conrmed that the same
phage -delivered expression cassete was
able to raise signicant immune responses in
rabbits afer intramuscular immunization
(March et al., 2004). More recently, the same
phage -delivered HBsAg expression cassete
was tested in rabbits and the responses
compared with Engerix B, a commercially
available recombinant protein vaccine (Clark
et al., 2011). In this study, three out of ve
phage-vaccinated rabbits responded afer one
immunization, with the remaining two
responding afer a booster immunization. By
contrast, afer three vaccinations, one of the
Engerix B-vaccinated rabbits had not
responded. By 2 weeks afer the second
immunization, responses in the phage-
vaccinated group were higher by all tests
compared with the Engerix B-vaccinated
group.
In an interesting modication to the
standard phage-mediated DNA vaccine
technique, March et al. (2006) screened a
whole-genome library of the bovine pathogen
Mycoplasma mycoides subsp. mycoides (small
colony biotype) for immunodominant clones
using a ZAP Express phage containing
both prokaryotic and eukaryotic promoters.
Immune sera from both rabbits and cows
were used to probe plaques with the
immunodominant clones selected. Afer
sequencing and analysis, two of these clones
were tested in a mouse challenge model.
Immunization with both clones resulted in
signicant antibody responses, with one
clone also giving signicant lymphocyte
proliferation afer stimulation with whole
bacteria. Vaccination with this same clone
gave protection, as measured by reduced
levels of mycoplasmaemia afer challenge.
This approach to vaccination is particularly
interesting as protection was obtained afer
screening with no initial knowledge of the
protective antigens.
In other studies using phage as a
vaccine delivery vehicle, Ghaemi and co-
workers demonstrated the ability of phage-
delivered human papillomavirus genes to
express both in bacteria (Ghaemi et al., 2010)
and in cell lines (Ghaemi et al., 2011). In the
later study, mice were shown to be protected
against challenge with a mouse tumour cell
line expressing the same protein. The release
of IFN- and granzyme B, along with
proliferation of spleen cells, suggested that a
cell-mediated response was at least partly
responsible for protection.
Recently, protection against challenge in
a mouse model has been demonstrated
following phage-mediated DNA vaccination
(Ling et al., 2011). Phage containing a
eukaryotic cassete expressing the major
outer-membrane protein of Chlamydophila
abortus was used to immunize mice. Three
immunizations of the phage vaccine were
compared with three immunizations of a
homologous DNA vaccine and a single
immunization of a live atenuated vaccine
strain. Although antibody responses were
higher in the group immunized with the live
atenuated strain, the phage DNA vaccine
group showed antibody responses sig-
nicantly higher than the DNA vaccine group,
and levels of IFN- and interleukin-2 were
higher in the phage-vaccinated group than
in the other two groups. Protection was
measured as clearance of chlamydiae from
spleens afer challenge. The phage-vaccinated
group gave signicantly beter protection
than the DNA-vaccinated group or the control
group, although mice immunized with the
live atenuated strain showed the highest
levels of protection.
Other phages have been tested. Hashemi
et al. (2010) used lamentous phages to
deliver a genetic vaccine against sherpes
simplex virus type 1 (HSV-1), based on HSV-1
glycoprotein D. In this case, both humoral
and cellular responses were found to be
similar to those from a plasmid DNA vaccine
control group. Contrastingly, in the published
literature, phage -delivered DNA vaccines
are reported to be more eective than plasmid
DNA vaccines. The reduced response
observed with lamentous phage may be due
to the single-stranded nature of the genome.
Additionally, there is a report of a phage-
delivered mRNA vaccine (Sun et al., 2011).
Virus-like particles (VLPs) derived from the
bacteriophage MS2, which has a positive-
sense, single-stranded RNA genome were
used. These particles self-assemble and can
90 J. Clark et al.
package foreign nucleic acid. VLPs packaging
human immunodeciency virus type 1 (HIV-
1) gag mRNAs were produced in yeast cells
and used to immunize mice. Following three
immunizations with Freunds adjuvant Gag-
specic antibody responses over 2 logs higher
than in the control groups were detected.
Phages as protein vaccines
Bacteriophages have also been used for the
delivery of vaccines in the form of proteins or
peptides displayed as capsid protein fusions
or chemically conjugated to the surface of the
phage. Phages displaying protective antigens
have been tested in animals (Irving et al.,
2001; Wang and Yu, 2004). Specic antibodies
(e.g. di Marzo et al., 1994) and protection
against challenge (e.g. Bastien et al., 1997)
have been demonstrated. Peptides/proteins
that are displayed on the surface of phage
particles can be chosen based on previous
knowledge of the specic disease, particularly
of pro tective epitopes. An alternative
approach is to screen a phage display library
and select from random peptides for those
that bind to serum raised against a given
immunogen or patho genic organism (Folgori
et al., 1994). The utility of this later approach
is that, rather than guess at which linear
epitopes are most protective, one instead can
begin with an immune response that is
known to be protective. This later approach,
however, is limited by an incomplete
knowledge of which aspects of the humoral
immune response provide protection and by
the fact that, in some cases, peptides can be
antigenic in that they react with the products
of an immune response but may not be
immunogenic, that is, they are unable to
generate an immune response themselves.
Similarly, Zhong et al. (1994) were able to
identify randomly mutagenized peptides
that bound to anti bodies known to recognize
an epitope found in its native conformation.
Meola et al. (1995) compared display of
peptides with delivery of the same peptides
as multiple antigenic peptides or fused to
hepatitis B virus core protein and human H
ferritin. They found the phage-delivered
peptides to be more immunogenic. It has
also been demonstrated that peptides
displayed on phages can reach both MHC
class I and class II compartments, suggesting
that peptides presented in this way can
stimulate both humoral and cellular
responses (Wan et al., 2001; de Berardinis et
al., 2003; Gaubin et al., 2003). Filamentous
phages have also been shown to stimulate
both CD8
+
and CD4
+
T cells inducing
both short-term and long-term cytotoxic
T-lymphocyte (CTL) responses (Mascolo et
al., 2007; del Pozzo et al., 2010).
A variety of dierent phages have been
used to deliver protein vaccines to animals,
including the lamentous phages M13 and
fd, T4, , T7 and the RNA phages Q|, MS2
and PP7. Each of these phage vectors has
advantages and disadvantages in terms of
display capacity and ease of production.
Phage display by lamentous phages is
covered in substantial detail by Siegel
(Chapter 8, this volume). While the high
density of display possible with lamentous
phages can enhance immune responses
(Minenkova et al., 1993), the limit on the size
of peptides that can be displayed can limit
immunogenicity. Such constructs may be
more suited to generating protective CTLs,
rather than antibody responses. It has been
noted that CTL responses could be improved
1000- to 10,000-fold if peptides are displayed
on large carriers (Raychaudhuri and Rock,
1998). Some researchers have been able to
show improved immune responses to vaccine
phages by co-administration of T-cell epitopes
displayed on a second lamentous phage (di
Marzo et al., 1994; Perham et al., 1995;
Guardiola et al., 2001). Although lamentous
phage-display vaccines have usually been
delivered via the intraperitoneal or sub-
cutaneous routes, some researchers have also
shown immune responses afer delivery via
oral (Delmastro et al., 1997; Zuercher et al.,
2000) or nasal (Delmastro et al., 1997) routes.
Interestingly, Zuercher et al. (2000) observed
that only clones displaying a high-copy-
number peptide remained active afer oral
delivery, with low-copy-number display
phages being rapidly inactivated. Table 7.2
gives a summary of antigens used with
lamentous phage display as vaccines.
Bacteriophage T4 is probably the second
most well-characterized phage in terms of
phage display for vaccine purposes. The
capsid of T4 contains two non-essential
proteins, both of which have been used for
display purposes; highly antigenic outer
capsid protein (HOC) 39 kDa with 155 copies
per phage and small outer capsid protein
(SOC) at 10 kDa with 810 copies per phage
(for further discussion of phage T4 and its
immunogenicity, see Olszowska-Zaremba et
al., Chapter 12, this volume). The main
advantage of T4 over lamentous phages for
display purposes is that large peptides/
proteins can be displayed at high copy
numbers, which in many cases will lead to
improved immune responses. A number of
studies have employed phage T4 as a carrier
of protein antigens for vaccination, most
recently for example by an intramuscularly
delivered T4 vaccine that conferred complete
protection to Dutch-belted rabbits against
anthrax spores (Peachman et al., 2012) and in
non-human primates, delivered with or
without a monophosphoryl lipid A adjuvant
(Rao et al., 2011). Overall, although lamentous
phages are the most well-characterized
vaccine delivery vehicles, the ability to display
large proteins and peptides with signicant
copy numbers continues to make T4 an
atractive option for vaccine development.
Several groups have used RNA phages
(e.g. MS2, Q|, AP205 and PP7) to deliver
peptide or protein vaccines. These particles
spontaneously assemble if expressed in
Escherichia coli, are highly amenable to genetic
engineering and can display peptides at a
relatively high density. They ofen do not
contain genetic material and thus are ofen
used as VLPs rather than as phages. As a
consequence, they may be safer for use in
humans, although removing the ability to
replicate may make production of vaccine
particles more di cult. Vaccine delivery
vehicles based on both display of antigens as
coat protein fusions and chemical conjugates
of antigens to coat proteins have been
described; for more recent eorts, see Zou et
al. (2010) and Tissot et al. (2010). An additional
advantage of RNA phage VLPs for the
delivery of vaccine products is that, in some
cases, the particles can be loaded with CpG
oligodeoxynucleotides, which act as potent
Table 7.2. Filamentous phage display vaccines.
Antigen Type of display Reference
Circumsporozoite protein
of Plasmodium
falciparum
pVIII by complementation on
plasmid with inducible
promoter
Greenwood et al. (1991); Willis et al. (1993)
Human immunodeciency
virus (HIV-1) Gag p17
pVIII Minenkova et al. (1993); Loktev et al. (1996)
HIV-1 gp120 V3 loop pVIII (complemented) di Marzo et al. (1994)
HIV-1 proteins pVIII (complemented) Lundin et al. (1996); Scala et al. (1999)
HIV-1 pVIII (complemented) de Berardinis et al. (1999, 2000, 2003);
Guardiola et al. (2001)
HIV-1 pVIII (complemented) Chen et al. (2001)
Chlamydia trachomatis pVIII (complemented) Zhong et al. (1994)
Hepatitis B pVIII and pIII Meola et al. (1995); Delmastro et al. (1997)
pVIII (complemented) Wan et al. (2001)
Plasmodium vivax pVIII Demangel et al. (1996)
Human respiratory
syncytial virus
pIII Bastien et al. (1997)
pVIII (complemented) Grabowska et al. (2000)
Pig cysticercosis pIII and pVIII
(complemented)
Manoutcharian et al. (2004); Morales et al.
(2008)
Enterotoxigenic
Escherichia coli
pIII van Gerven et al. (2008)
Trichinella spiralis pIII Gu et al. (2008)
Alzheimers disease pIII and pVIII
(complemented)
Frenkel et al. (2000, 2001, 2004)
Melanoma pVIII (complemented) Fang et al. (2005); Sartorius et al. (2008)
92 J. Clark et al.
immunostimulators in mammalian hosts. For
example, phage Q| VLPs were loaded with
CpG oligodeoxynucleotides and used as an
adjuvant in a co-immunization with house
dust mite allergen in trials that demonstrated
a signicant reduction in the symptoms of
rhinitis and allergic asthma (Senti et al., 2009).
Use of RNA phage VLPs as vaccine
delivery vehicles is perhaps the most
developed example of a phage-derived
product for use in humans. Cytos Bio-
technology Ltd (Schlieren, Switzerland; www.
cytos.com) is currently conducting phase II
clinical trials with a number of products. These
trials include nicotine addiction (Maurer et al.,
2005; Cornuz et al., 2008), hypertension (Tissot
et al., 2008; Maurer and Bachmann, 2010),
allergic rhinoconjunctivitis (Klimek et al.,
2011), allergic asthma (Senti et al., 2009) and
Alzheimers disease (Wiessner et al., 2011). A
full discussion of this delivery technology is
beyond the scope of this chapter, but the Cytos
website has a full list of relevant references
(www.cytos.com/?id=158#candidates).
Although Cytos Biotechnology has
targeted relatively high-value indications for
vaccine development, it has also been sug-
gested that the potential ease of pro duction of
some phages could make them particularly
atractive for the development of vaccines
against orphan diseases (Rao et al., 2011). It is
also important to note that only a very few
phages have been tested as vaccine delivery
vehicles. There is a growing under standing of
the role that shape, size and charge play in the
interaction of foreign particles with the
immune system (Wang et al., 2011), and it is
possible that other phage vectors could have
advantages in terms of tissue tropism or how
they interact with the host to generate an
immune response. Further information on
phages as vaccines is given by Benhar (2001);
Irving et al. (2001); Wang and Yu (2004); Clark
and March (2004a) and Gao et al. (2010).
Phages as Gene-Therapy Vectors
The most commonly used vectors for gene
therapy the addition of DNA to eukaryotic
cells to correct a genetic defect or augment
the cells phenotype are viruses of
eukaryotes. Although not considered here, a
description of gene therapy of prokaryotic
organisms has also been proposed to combat
antibiotic-resistant infections (e.g. Norris et
al., 2000). The problems with employing
animal viruses include di culties controlling
their inherent virulence, at worst resulting in
subject death, as well as di culties associated
with modifying tissue tropism so that the
desired cells are preferentially targeted.
Therefore, there is a strong need for gene-
delivery technologies that are both more
benign and more eective. An important
consideration is that not all gene therapy is
delivered in vivo, as it is possible in certain
instances to remove specic cell types
temporarily from the body where they may
be treated ex vivo. The now genetically
modied cells are then returned to the body
where, ideally, relatively long-term expres-
sion of the introduced gene may occur. Thus,
not all gene therapy requires exposure of
systemic body tissues to DNA-delivering
vectors.
The advantages of using phages as gene-
therapy vectors are similar to the advantages
of using phages as DNA vaccines: phages
are safe, stable, inexpensive and easy to
manipulate genotypically as well as pheno-
typically. Indeed, modication of the tissue
tropism of phages is an almost trivial exercise
involving phage display technologies (see
Siegel, Chapter 8, this volume) and cloning of
the desired genes into phages. In short, phages
are perhaps ideal cloning vectors combined
with facile trophic targeting, with short-
comings found only in terms of the size of
DNA that may be cloned, a lack of inherent
nucleus-homing mechanisms and the lack of
natural sequences in vectors able to function
in eukaryotes, although some of these short-
comings can be addressed by modication of
the phage particle. In lamentous phages,
there is the additional problem that the
genome is single stranded and therefore
presumably requires conversion to double-
stranded DNA before transcription can occur.
As Hajitou et al. (2007, p. 523) noted, ...phage-
based vectors have inherently been con-
sidered poor gene delivery vehicles. As a
working hypothesis, we proposed that the
rate-limiting step might be mechanistically
related to the post-targeting fate of the single-
stranded DNA of the phage genome. This
hypothesis is further supported by research
indicating that genotoxic treatment increases
the rate of transduction of phagemid particles
containing single-stranded DNA (Burg et al.,
2002; Liang et al., 2006).
Initial studies in transfection with phage
particles used chemical agents to facilitate in
vitro uptake. Such agents included DEAE-
dextran (Yokoyama-Kobayashi and Kato,
1993) and cationic lipids (Yokoyama-
Kobayashi and Kato, 1994; Aujame et al., 2000)
with lamentous phages and calcium
phosphate with phage (Ishiura et al., 1982;
Okayama and Berg, 1985). These initial studies
proved that phage particles can express
eukaryotic genes contained in the phage
genome if uptake is su ciently e cient.
Non-specic uptake of phage particles
has relatively limited biotechnological appli-
cations, and several groups have atempted to
modify phages to provide tissue tropism. This
specic targeting of phage particles
has been shown to increase transfection
e ciencies in vitro from 12% to as much as
45% (Larocca et al., 2002). In many cases,
relatively small peptides can give tissue
tropism to phage particles, or in some
instances antibodies displayed on phages can
be used to increase cellular uptake of phage
particles (Becerril et al., 1999; Chung et al.,
2008), and antibody libraries can also be
screened to select phage clones with tropism
for specic cell types, such as tumour cells
(Poul et al., 2000). Another advantage of
lamentous phages in the context of gene
therapy is the ability to pan phage libraries to
select for phages displaying peptides that are
selectively taken up by specic cell types
either in vitro (Ivanenkov et al., 1999) or in vivo
(Molenaar et al., 2002). See Siegel (Chapter 8,
this volume) for additional discussion of
phage display and biopanning. A number of
reviews are available that further discuss the
use of modied phages as gene-therapy
vectors (Uppala and Koivunen, 2000; Monaci
et al., 2001; Sergeeva et al., 2006; Hajitou, 2010).
Phages as cytotoxic agents
Phages may be employed not just to do good
in a gene-therapy or gene-vaccination sense
but also to do good in a cytotoxic sense.
Thus, not all cell types found within bodies
are desirable or even healthy to possess.
Undesirable cells include tumour cells but
also can include white fat cells, against which
a large number of strategies (ofen ineective)
exist aimed at reducing their size or number.
Phages as cytotoxic agents against such cells
may be dierentiated in a number of dierent
ways. Phages can deliver toxins to a cell, afer
targeting and either co-localization or endo-
cytosis. Toxins can be physically delivered by
the phage or encoded by appropriate expres-
sion cassetes cloned into the phage genome.
Alternatively, an immune response against
the phage itself (facilitated by the adjuvant
eect of the phage particle) can result in
killing of target cells by the host immune
system. In all cases, the key to proper
functioning is phage targeting to specic cell
types, which typically is eected by capsid
modication based on phage display
technologies.
The most common research using phages
as cytotoxic agents is in the targeting and
destruction of cancer cells. Generally, the
phage particles are targeted to cancerous cells
and either a toxin is released or the immune
response against the phage itself promotes a
cell-killing eect. The most concerted
programme of work in using phages as
cytotoxic agents is that of Yacoby, Benhar and
others, who initially targeted bacterial
pathogens. These authors demonstrated a
technology where a lamentous phage
displays a cell-binding ligand along with a
cytotoxin that is subject to controlled release
(Yacoby et al., 2006, 2007; Yacoby and Benhar,
2008). These phages display peptides that
specically target the bacterial pathogen of
interest, even if the bacterium is not a natural
target for the phage. Afer targeting, an
antibacterial compound is released at a
locally very high concentration, which results
in more e cient bacterial killing. The same
group has developed a related technology for
application against eukaryotic cells that is
94 J. Clark et al.
dependent on endocytosis. A lamentous
phage displaying a eukaryotic cell-binding
ligand along with a cytotoxin, which is
subject to controlled release, is used to target
a specic cell type, such as a tumour cell (Bar
et al., 2008; Yacoby and Benhar, 2008). The
phages are endocytosed, resulting in both
concentration and delivery of the cytotoxin
only within the targeted cells.
Chung et al. (2008) proposed an antibody-
targeted phage construct to target tumour
cells, specically for use in inducing apoptosis
in Hodgkins-derived cell lines. In this initial
proof-of-principle report, they used an in vitro
system based on GFP expression as a measure
of phage uptake, rather than demonstrating
apoptosis per se. In an alternative strategy,
Eriksson et al. (2007; 2009) targeted lamentous
phages to tumour cells but these phages did
not specically express or carry a cytotoxic
molecule. In this case, tumour-cell killing was
promoted by an immune response against the
phage particle, specically the activation of
tumour-associated macrophages (Eriksson et
al., 2009).
Other Therapies Associated with
Phage-mediated Delivery
Phages that display specic molecules can
also be used to eect protein distribution to
locations where the protein alone would have
di culty penetrating. This approach is
exemplied by the work of Solomon and
colleagues (e.g. Frenkel and Solomon, 2002)
who used phage display to deliver mono-
clonal antibodies to the brains of mice. The
antibodies bound to an epitope found in the
amyloid plaques associated with Alzheimers
disease, and antibody binding gave rise to
plaque clearing. While antibody alone is
di cult to deliver to the central nervous
system, this barrier apparently may readily
be overcome by phage particles. As a
consequence, the phage essentially alters the
pharmacokinetic properties of the displayed
antibody, allowing penetration to amyloid
plaques, where the therapeutic eect is
required. In addition, the same group found
that the phage construct could penetrate the
central nervous system following intranasal
delivery. Brain localization by phages has
been found by others (Pasqualini and
Ruoslahti, 1996), in this case to brain-specic
blood vessels. See Olszowska-Zaremba et al.
(Chapter 12, this volume) for additional
perspectives on this idea of phage penetration
through anatomical barriers.
Additional work by the Solomon group
targeting Alzheimers disease via the immune
system (Frenkel et al., 2000, 2003) found that
lamentous phages could serve as an
adjuvant for a vaccine consisting of an epitope
found on amyloid plaques. Here, phages
were repeatedly injected intraperitoneally
into mice at a dosage of 10
11
phages ml
1

every 2 weeks for a total of six injections. The
resulting humoral immune response pro-
duced antibody, which had the eect of
reducing amyloid plaques in the transgenic
mouse Alzheimers model.
Carrera et al. (2000, 2004) provided a
similar example of intranasal phage delivery
to the central nervous system. They produced
a phage construct, based on the lamentous
phage fd, which displayed cocaine-binding
antibodies, which can sequester cocaine
molecules penetrating the brain. Using a rat
model, the phage dosage was 5 10
12
per naris
(50 l of 10
14
phages ml
1
suspended in PBS)
delivered twice daily for 3 days. A serum
immune response (antibody titre) was not
observed against the administered phage,
while substantial phage titres were localized
to the brain, as many as 2.5 10
13
(units
indicated solely as phage titre and a total of
10
15
phages were reportedly administered to
each rat prior to this determination). In all,
administration of phages displaying anti-
cocaine antibodies reduced cocaine-associated
behavioural symptoms to a statistically sig-
nicant extent. For a more detailed discussion,
see also Dickerson et al. (2005).
As discussed previously, RNA phage
VLPs have been used to deliver CpG-
containing nucleic acids as vaccine adjuvants.
A modication of this technique is the use of
bacteriophage MS2 VLPs displaying trans-
ferrin on their surface to encapsulate anti-
sense oligonucleotides and deliver them to
leukaemia cells (Wu et al., 2005). These
targeted antisense oligonucleotide delivery
vehicles were shown to increase killing of
leukaemia cells. In related research, trans-
ferrin was also chemically conjugated to Q|
particles (Banerjee et al., 2010). It was
demonstrated that the e ciency of uptake
was dependent on ligand density, implying
that chemical conjugation (as opposed to coat
protein fusion) of some targeting molecules
to phage VLPs could increase the e ciency of
gene transfer.
Conclusions
Bacteriophages have evolved capsids that can
arguably be described as the most e cient
known delivery vehicle of a genome payload
to their natural target cells. The development
of genetic engineering techniques allows the
facile manipulation of these highly e cient
delivery systems to provide a tropism
towards non-natural targets to specically
deliver therapeutic or toxic molecules. Phages
can also be modied physically or genetically
to deliver immunogenic molecules for the
purpose of vaccination. Techniques to modify
the capsid of phages have existed for over 25
years, but their use in the various applications
described here remains largely at the proof-
of-principle and early developmental stages,
although a few systems are already in clinical
trials and others are advancing towards this
stage. Well-dened and consistent pro-
grammes of work geared towards the end
point of human clinical trials are required to
transform the technologies described here
from research into viable products.
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8 Clinical Applications of Phage Display
Don L. Siegel
1
1
Department of Pathology and Laboratory Medicine, University of Pennsylvania.
As with many of the other uses for bacterio-
phages described in the accompanying
chapters of this volume, phage display repre-
sents an ingenious application of bacterio-
phages that is completely unrelated to their
natural role as a bacterial pathogen their use
as a tool for the discovery of novel peptide
and protein binders to molecular targets of
basic and clinical interest. Since its initial
description over 20 years ago, phage-display
technology has been used to understand
complex biological signalling pathways, to
study proteinprotein interactions and to
develop diagnostic and therapeutic agents
important to human health. These methods
have been the subject of a number of recent
comprehensive reviews (Hoogenboom, 2005;
Lonberg, 2008; Bratkovic, 2010; Pande et al.,
2010; Bradbury et al., 2011), and several
excellent laboratory manuals are available
that provide step-by-step protocols for their
use (Barbas et al., 2000; Clackson and
Lowman, 2004; Sidhu, 2005; Aitken, 2009).
This chapter will focus on a description of the
technology and then provide an overview of
key clinical areas in which phage display has
had its largest impact.
What is Phage Display?
In theory, if one were interested in nding a
ligand for a particular receptor of interest,
e.g. a 7 amino acid peptide (7mer), one could
synthesize every one of the more than 1
billion possible 7mers (~20
7
) and perform a
seemingly innite series of biological assays
to determine which peptide(s) bound to the
target. Such a task would be impractical not
only because of the expense and time
required, but because of the technical dif-
culty in identifying the amino acid
sequence(s) of positive peptide binders,
especially because the binders would be all
mixed together. If, however, each peptide
were in some way physically connected to the
nucleic acid that encodes that particular
peptide, then a single receptor-binding assay
could be performed with a mixture of all 1
billion peptideDNA complexes. Unbound
complexes could be washed away, and the
nucleic acid sequences of the binders could
be cloned in bacteria. Sequencing the peptide-
encoding DNA in individual bacterial clones
would then provide the amino acid
sequence(s) of the receptor-binding ligands
one was originally interested in determining.
Conceptually, the reason why this
hypothetical approach seems feasible is
because there is a physical connection
between the genotype and phenotype of the
ligand the binding of the peptide carries
along with it the nucleic acid needed for its
unique identication (its sequence) and its
ability to replicate. This linkage of genotype
and phenotype of peptides and proteins is
the hallmark of phage display.
D.L. Siegel
102 D.L. Siegel
Although there have been a number of
dierent types of bacteriophage used for
phage display, typically the lamentous
bacteriophage M13 is employed. M13
produces virions approximately 7 nm in
diameter and up to 1 m in length (Webster,
2001). As shown in Fig. 8.1(a), the genome of
phage M13 consists of 11 genes including
those that encode the major coat protein, pVIII,
for which there are thousands of copies per
virion, and the minor coat protein, pIII, which
is required for bacterial infectivity (it binds to
the F' pilus of Escherichia coli) and is present in
three to ve copies per phage particle.
In a seminal observation made over 25
years ago, George Smith (1985) likened phage
virions (M13 or others) as biological particles
that physically link the phenotype of a protein
(i.e. the viral coat protein) with the proteins
genotype (i.e. the DNA encoding the coat
protein contained within the phage genome).
He demonstrated that foreign DNA inserted
into the M13 phage genome, just upstream
and in frame with the minor coat protein gene,
gIII, would result in the production of phage in
which each pIII copy found on the phage
virion had the expressed peptide fused to its N
terminus (Fig. 8.1b). In his original experiment,
Smith used DNA encoding a portion of a
bacterial restriction enzyme. Remarkably, the
presence of this exogenous stretch of enzyme
amino acids, at the tip of each pIII, did not
pIII
pVI
pVIII
pVII
pIX
gVII
gVI
gIX
gVIII
ss gIII
ori PS
Fig. 8.1. Diagram of simplied M13 lamentous phage genomic DNA and assembled virions in (a) a
wild-type and (b) a peptide-displaying phage. Note that, in (b), exogenous peptide DNA is inserted
upstream of the minor coat protein gene, gIII, resulting in expression of exogenous peptide as fusion
protein with all copies of coat protein pIII. SS, signal sequence; PS, phage packaging signal sequence;
ori, phage DNA origin of replication.
Peptide/pIII
fusion protein
pVI
pVIII
pVII
pIX
gVII
gVI
gIX
gVIII
ss gIII
ori PS
DNA for
displayed
peptide
(a)
(b)
Clinical Applications of Phage Display 103
adversely aect phage infectivity or other
biological properties of the phage in any
signicant way. Furthermore, antibodies that
he had in hand that recognized the native
restriction enzyme were found to bind to the
phage particles via the pIII fusion protein.
Although expressing a piece of a bacterial
enzyme on the surface of a bacteriophage in
and of itself was not necessarily of any
practical importance, what Smith did was to
demonstrate how the phenotype and geno-
type of a protein could be physically con-
nected in a self-replicating biological particle.
What this led to was the ability to construct
massive libraries of M13 phage particles
generated by the cloning of billions of
dierent random peptide sequences of the
desired length (typically seven to 20 codons)
upstream of gIII. Through a process called
panning (discussed in detail later), such
libraries could be incubated with a target,
and phage-displaying peptides of su cient
binding a nity could be eluted from the
target and amplied in bacteria. Reselection
of the propagated libraries against the target
for several additional times would eventually
yield phages, all with specicity for the
target. Phage DNA could be isolated from
individual bacterial clones, sequenced and
used to determine the amino acid sequence of
the peptide(s) that bound the target. This was
the beginning of what became known as
phage display.
Technical Aspects of Peptide and
Protein Phage-display Libraries
The peptide discovery approach described
above has become a fairly routine laboratory
procedure, and pre-made M13 peptide
phage-display libraries are commercially
available. For example, New England Biolabs
(Ipswich, MA) provides linear 7mer and
12mer or cyclic 7mer libraries (displayed
7mers are anked by cysteine residues to
provide secondary structure). Phage vectors
for designing ones own peptide library are
also commercially available and require only
basic molecular biology skills to construct.
A typical phage-display experiment with
such a peptide library comprises three or four
rounds of selection against a target, which
can be accomplished in about a week and are
technically relatively simple to perform a
round of selection with a target immobilized
to a plastic well of a microtitre plate is
essentially like performing an ELISA except
that phages are added to wells for incubation
with immobilized target, not analytes or
antibodies, and instead of enzyme-conjugated
antibodies added to the wells for ELISA
development, bound phages are eluted from
the wells with a dissociation agent (typically
low pH buer) and used to infect bacteria.
Subsequent analyses of derived peptides
through nucleic acid sequencing of individual
phage clones and then production of positive
clones as soluble peptides (i.e. unlinked to
phage) to verify binding can take an additional
few weeks depending on the nature of the
project.
Such commercially available or research
laboratory-constructed peptide libraries have
been used for epitope mapping of monoclonal
antibodies (mAbs), developing enzyme
inhibitors, mapping proteinprotein contacts,
designing vaccines, discovering peptides that
mimic non-peptide carbohydrate ligands and
many other applications (reviewed by Brissete
and Goldstein, 2007). A set of particularly
clinically relevant uses of peptide phage
display in the areas of molecular imaging,
tumour targeting and diagnosis of cancer has
been reviewed recently (Deutscher, 2010).
The ability to create M13 phages dis-
playing repertoires of molecules larger than a
peptide, such as immunoglobulin fragments
with molecular weights of 3050 kDa (~275
450 amino acids) derived from a humans or
animals immune system, has provided leads
for the development of therapeutic antibodies
for treating a number of health conditions
including cancer, auto immune disorders and
others. Afer selection of phages from such
libraries, the DNA encoding positive-binding
immuno globulin fragments can be subcloned
into other vectors for the production of
full-length IgG suitable for diagnostic or
therapeutic use. One of the major driving
forces for the development of phage-display
methods for antibody dis covery was a need
for a source of human mAbs that would not
induce anti-drug immune reactions, as can be
seen with murine or even human/murine
chimaeric antibodies (Weiner, 2006). Up until
104 D.L. Siegel
the development of phage display, the cloning
of mAbs was accomplished by hybridoma
technology, which is a viable method for the
production of murine antibodies (Kohler and
Milstein, 1975) but problematic for the
production of human antibodies because
immortalizing human B cells is technically
challenging (Winter and Milstein, 1991). As
shown below, phage display oered a way of
cloning human antibody genes in bacteria
rather than requiring the ability to immortalize
the B cells from which the anti body clones
originated.
Cloning the larger-than-peptide-sized
stretches of exogenous DNA into the M13
phage genome, such as those required to
encode antibody fragments, became tech-
nically challenging, as phages containing
longer stretches of cloned DNA have a
growth disadvantage and their expressed
proteins may be toxic to their bacterial hosts.
Furthermore, as illustrated in Fig. 8.1(b),
when cloning exogenous DNA into the M13
genome, all copies of pIII per virion display
the desired ligand. Although such multi-
valent display can be advantageous in certain
applications, for the selection of phages
displaying antibody fragments that bind to
their target through a nity alone, not
inuenced by avidity eects, monovalent
display of single-chain Fv (scFv) or Fab
fragments is most ofen preferred (Fig. 8.2).
This requires the co-expression of wild-type
pIII coat protein along with pIII coat protein
fused with the displayed antibody fragment.
Cloning libraries of antibody fragment DNA
into phagemid vectors, rather than into the
actual phage genome, can be used to achieve
monovalent display, as shown stepwise in
Fig. 8.3. Phagemids engineered for this
V
L
V
H
V
H
C
L
C
H1
C
H2
C
H3
C
L
C
H1
C
H2
C
H3
Fv fragment
V
H
V
L
scFv
display
Fab
display
Fab fragment
V
L
V
H
V
L
C
H1
C
L
Fig. 8.2. Expression of antibody fragments on a lamentous phage. Monovalent display of an scFv
fragment (left) or Fab fragment (right) is illustrated using a conventional diagram of IgG (middle) to
indicate parts of IgG expressed as a fusion with the phage pIII coat protein. An Fv fragment of IgG is
dened as an antigen-binding molecule comprising just the variable regions of the immunoglobulin heavy
(V
H
) and light (V
L
) chains. In order for V
H
and V
L
to assemble into one protein, overlap PCR is used to
assemble individual V
H
and V
L
amplication products into a single-chain Fv in which a exible glycine/
serine linker of approximately 18 amino acids permits alignment of the N termini of heavy and light
variable regions. A Fab fragment is dened as an antigen-binding molecule comprising the Fd fragment
of the heavy chain (Fd = V
H
and C
H1
domains of the heavy chain constant region) paired with the entire
light chain (V
L
plus the constant region of the light chain, C
L
). For the construction of Fab phage-display
libraries, the Fd and light-chain polypeptides are produced as separate molecules off a dicistronic
message (the stop codon in the right-hand illustration); the Fd fragment is produced as a fusion with pIII
and the light chain associates with the Fd fragment (as it would do naturally in a B lymphocyte), and a
covalent disulde bond forms between C
H1
and C
L
. Note that each phage particle comprises both wild-
type pIII and pIII antibody fusion protein, and DNAs for displayed proteins are cloned into phagemid
vectors rather than the phage genome, as described in the text.

C
l
i
n
i
c
a
l

A
p
p
l
i
c
a
t
i
o
n
s

o
f

P
h
a
g
e

D
i
s
p
l
a
y

1
0
5
V
L
V
H
Plasmid
ori
ss scFv Truncated gIII
Resistance gene
Phage
ori
Phage packaging
signal
Library of
scFv cDNAs
Phagemid
V
H
V
L
scFv
scFv/pIII
fusion protein
scFv
M13 helper phage
Wild-type
pIII
scFv
scFv
Phagemid particle
displaying scFv
V
H
V
L
scFv
scFv
Fig. 8.3. Overview of the construction of phagemid-based antibody fragment (scFv) display libraries. (a) A large array of scFv constructs (e.g. those derived
from RT-PCR of immune cells such as peripheral blood lymphocytes, splenocytes or bone marrow mononuclear cells) is introduced upstream of a truncated gIII
(see text) in a phagemid vector comprising an M13 phage origin of replication (phage ori), a phage packaging signal, a resistance gene for selection and a
plasmid origin of replication (plasmid ori). (b) The library of scFv constructs is electroporated into a culture of Escherichia coli and scFv/gIII fusion proteins are
expressed and sent to the bacterial periplasmic space by the signal sequence (ss). (c) The bacterial culture is superinfected with M13 helper phage (essentially
wild-type phage with an antibiotic resistance gene and a defective packaging signal sequence) and M13 phage proteins, including wild-type pIII and proteins
required for phage assembly, are expressed. (d) Through dual antibiotic selection for both phagemid- and helper phage-containing bacteria, phagemid particles
are released into culture that contain scFv plasmid DNA within and a mixture of wild-type and scFv-fused pIII coat proteins on the surface. Because the phage
packaging signal sequence is defective in the helper phage, the plasmid DNA is preferentially packaged by the helper phage DNA-encoded proteins. Through
the optimization of various parameters (e.g. level of expression of scFv/pIII, timing of various steps including addition of helper phage), each phage particle, on
average, will have one scFv/pIII molecule per virion to approximate monovalent display.
(a) (b)
(d)
(c)
106 D.L. Siegel
purpose have cloning sites for antibody
fragment DNAs just upstream of a form of
gIII that lacks an N-terminal domain (Scot
and Barbas, 2001). This so-called truncated
gIII lacks a string of nucleotides that encodes
a region of pIII that signals to the bacterium
to cease production of pili. This natural
control mechanism carried out by the
N-terminal portion of pIII serves to prevent
an individual bacterium from being infected
by more than one phage particle (super-
infection immunity). When working with
phage as vectors, there is no need for pilus
formation afer infection, so it does not mater
that pilus formation is inhibited. In fact,
inhibition of pilus formation is absolutely
essential when working with phage vectors,
otherwise reinfection would lead to a
scrambling of the correct linkages between
displayed peptides and their encoding DNAs.
For antibody libraries, however, as phagemid
vectors require subsequent co-infection with
helper phages to provide genes for wild-type
pIII as well as for all other phage genes
required for phage particle assembly and
secretion, continued pilus expression is
required afer infection of bacterial cultures
with phagemids. The use of the truncated gIII
allows this necessary ongoing pilus expres-
sion (Fig. 8.3c).
Selection Strategies for
Phage-display Libraries
Whether a peptide or antibody phage-display
library, selection of phages displaying a target-
binding moiety is accomplished through a
series of enrichment steps referred to as
panning. Typically, three to four rounds of
panning are required for most, if not all, of the
resulting phages to display peptides or
proteins with a nity for the target. Afer
panning rounds are completed, the resulting
preparation of polyclonal phages are titrated
on E. coli to produce either individual plaques
or bacterial colonies, each having been infected
with a single bacteriophage. Typically, several
dozen clones are randomly selected from agar
plates and grown for the purpose of preparing
phage (or phagemid) DNA from which the
peptide (or antibody) sequences can be
determined. This analysis will provide an
estimate of the diversity in binders obtained
following the given number of rounds of
selection.
Depending on the nature of the project,
there may be only one or perhaps a few
dierent peptide or antibody sequences, or
the collection may be very diverse. Typically
in a peptide phage-display library experi-
ment, a series of dierent peptide sequences
will be obtained within which one can
identify a common shorter continuous or
discontinuous amino acid motif. For an
antibody phage-display experiment (see
below), the diversity in sequence of the
resulting binders will depend on a number of
factors including whether the antibody
library was constructed from an immunized
animal, in which there may be a relatively
small number of antibodies that bind with
particularly high a nity to overshadow
(outcompete for target binding) other binders.
Conversely, if the antibody library was
constructed from a non-immunized or
synthetic source of V
H
and V
L
segments, there
may be a relatively larger number of equal
but weaker binders that may require in vitro
a nity maturation, as discussed later. Quite
ofen, one may nd a series of clonally related
antibodies comprising heavy and/or light
chains that can be shown to have been
derived in vivo from a single-original B cell,
yet may dier in their exact nucleotide
sequences due to somatic mutation that had
occurred in the human or animal from which
the library was constructed (Chang and
Siegel, 1998; Roark et al., 2002).
Panning against puried, immobilized
antigen is the most common and straight-
forward type of phage library selection and is
illustrated in Fig. 8.4 for an antibody
phagemid library. As briey described above,
this form of panning is very much like
carrying out an ELISA except that binders are
eluted from microplate wells rather than
detected with a secondary antibody. Elution
is most ofen carried out with low pH (~3.0)
buer for about 10 min, which is generally
su cient to denature the target and/or
displayed antibody, resulting in release of the
phage from the well, but remarkably does not
harm the integrity of the phage so that
reinfection and amplication of eluted phage
can take place. Elution can also be
accomplished by alkaline pH or by other
conditions that are known specically to
aect the particular antibodytarget inter-
action with which one is working, such as
reduction with dithiothreitol, exposure to
certain detergents or proteases, or an
elevation in temperature, or by competition
by the addition of excess soluble target or
other antibodies to the target. Because the
eluted phages contain phagemid DNA
capable of producing scFv/pIII fusion protein
but no other phage components, helper
phages are required during the panning
process to rescue the phagemids, as in the
process of library construction outlined in
Fig. 8.3. Afer overnight incubation, the phage
particles are harvested from the supernatant
of the bacterial culture and applied to a fresh
well of target for the subsequent round of
panning.
Although solid-phase panning shown
in Fig. 8.4 is the most straightforward method,
a number of other approaches have been
described that may eliminate potential
problems, for example if adsorption of the
particular target to plastic distorts its native
conformation or results in blocking the
binding surfaces (epitopes) of interest,
rendering them inaccessible to the displayed
ligand on the phage particles. To get around
these potential pitfalls, the use of biotinylated
targets permits incubation in solution with
the phage library and the use of streptavidin-
coated magnetic beads for separation of the
target for the washing and elution steps
(Hawkins et al., 1992). Other advantages of
the biotinylated antigen approach are that it
allows the investigator to control precisely
the eective in-solution concentration of the
target during selection and thus inuence the
expected a nity of the captured phage-
displayed ligand. In fact, a strategy that is
ofen used is to sequentially decrease the
concentration of biotinylated target from
round to round of panning so as to create
competition between binders and eventually
Phagemid
library
Add to
antigen
Wash away
unbound
Elute
bound
Infect
E. coli
Add helper
phage
Antigen-selected
phagemid library
(b) (a) (c)
(d) (e) (f)
Fig. 8.4. Antibody phage-display library selection by the solid-phage panning approach. A phage
antibody library (a) is applied to the well of a microplate coated with the target antigen of interest (b) and
unbound phage are washed away (c). Bound phage are eluted (see text) (d) and used to infect E. coli (e).
Phagemid DNA is rescued by subsequent superinfection with M13 helper phage to produce a second-
generation antibody phage-display library selected on the antigen.
108 D.L. Siegel
get those with the highest a nity to win out
during progressive rounds of selection as the
amount of target becomes limiting.
There is a wide range of other selection
approaches, illustrating the enormous
versatility of phage-display approaches for
discovering binding partners for targets of
interest (reviewed by Gri ths and Duncan,
1998; Hoogenboom, 2005). For example,
phage libraries may be panned on intact live
cell surfaces to isolate binders to an antigen
or receptor that is either unknown (e.g. in the
case of a putative tumour-specic antigen) or
unpuriable in its native state (e.g. in the case
of many transmembrane proteins that would
denature if solubilized in detergent). Most
ofen, such cell-surface panning approaches
include initial subtractive selections of
phage libraries with suspensions of cells
otherwise identical to the target cells but that
specically lack the antigen of interest (e.g.
the normal cellular counterpart of the tumour
cells). Such negative selections will adsorb
pan-reactive generic phages, leaving behind
target-specic phages that can be enriched
through positive selection on antigen-positive
cells using magnetically activated cell sorting,
as illustrated in Fig. 8.5 (Siegel et al., 1997;
Siegel, 2001; McWhirter et al., 2006). Other
methods for dierential cell-surface panning
of antibody phage-display libraries have
utilized uorescently activated cell sorting
(de Kruif et al., 1995a,b; van Ewk et al., 1997;
Huls et al., 1999; Lekkerkerker and Logten-
berg, 1999; van der Vuurst de Vries and
Logtenberg, 1999).
Panning of phage libraries can also take
place on immunoblots of SDS-PAGE-
separated polypeptides (Liu and Marks,
2000) or on tissue sections (van Ewk et al.,
1997) to isolate phage binders to particular
components within complex mixtures of
proteins or cells. For example, if all that is
known about a target of interest is its
molecular weight and thus its position as a
protein band on an SDS-PAGE gel, then a
suitable phage-display library can be
incubated with an immunoblot of the gel as
though one were performing a Western blot.
The band of interest can then be cut out of the
sheet of nitrocellulose with a razor blade, the
phages eluted with acid and the phage eluate
used to reinfect bacteria for amplication.
By analogy, the panning of phage-display
libraries on tissue sections combined with
laser microdissection techniques has per-
mited the cloning of antibodies directed
against nanogram amounts of proteins
contained within intracellular structures or
against particular cells (e.g. tumour cells)
within a heterogeneous slice of tissue (Tanaka
et al., 2002). These types of selection pro-
cedures illustrate the power of antibody
phage display when compared with
traditional tissue culture hybridoma methods
in which an impractical number (thousands)
of assays (by Western blot or immuno-
histochemistry) would need to be performed
to screen each hybridoma clone one by one.
In vivo panning is perhaps one of the
most interesting forms of panning in which
phage-display libraries have been injected
into living animals including humans
(reviewed by Sergeeva et al., 2006). Following
a brief time circulating in the bloodstream,
tissues are removed and bound phages are
eluted and then amplied in bacteria. Such
approaches have atempted to identify
peptides or other ligands that home in on
vascular structures specic to particular
organs or tumours.
Use of Phage Display for Antibody
Discovery
Overview
Figure 8.6 presents an overview of the ways
in which phage display has been used to
discover new diagnostic reagents and assays
for their use, as well as reagents that have
served as leads for the development of novel
pharmaceuticals. The process begins with a
source of immunoglobulin DNA, the
construction of a phage-display library, the
selection of binders through panning and the
isolation of target-specic phage particles
(Fig. 8.6a). Immunoglobulin DNA can then be
extracted from individual phages and
subcloned into other systems to produce full-
length IgG in which antibody Fc constant
region domains are appended (Fig. 8.6b), or
to produce antibody fragments as soluble
Couple magnetic
beads () to antigen-
positive cells
Add excess antigen-
negative cells
Add phage library
containing specific
and non-specific
binders
Incubate
Load on column
without magnetic
field
Place column in
magnetic field
and wash away
antigen-negative
cells and non-
specific phage
Flush antigen-
positive cells
and bound
phage from
column, elute
bound phage and
infect bacterial
culture
Fig. 8.5. Cell-surface panning of phage-display
libraries using magnetically activated cell sorting.
(a) Target cells (in this case, red blood cells bearing
the human Rh(D) blood group antigen) are cell-
surface biotinylated and coupled to streptavidin-
conjugated magnetic beads. (b) A large excess of
antigen-negative distractor cells (in this case,
Rh(D)-negative red cells) are added and (c, d) the
cell admixture is incubated with a phage library
constructed from the peripheral blood lymphocytes
of an Rh(D)-negative patient previously sensitized
to the Rh(D) antigen. (e) The incubation mix is
added to the top of a column used for magnetically
activated cell sorting. The volume of the incubation
mix is smaller than the included volume of the
column and the column is initially kept out of a
magnetic eld so that the cells spread out evenly
within the column. (f) The column is then placed
within a magnetic eld and antigen-negative cells
along with non-specic phage-displayed antibodies
are washed away. (g) Antigen-positive cells are then
ushed out of the column, bound phage antibodies
are eluted and clones are amplied in bacterial
culture. Amplied phage are then subjected to
several additional identical rounds of competitive
cell-surface selection, after which essentially all
eluted phage are specic for the Rh(D) antigen.
(Adapted from Siegel et al., 1997.)
110 D.L. Siegel
molecules for applications in which natural
antibody eector function or long circulation
half-life is not necessary (Fig. 8.6c).
Alternatively, the phage particles can be used
directly as highly sensitive and specic
immunological detection reagents (Fig. 8.6d).
Types of antibody phage-display
libraries
Phage-display libraries can be classied as
immune, nave or synthetic depending
on the source of immunoglobulin DNA.
Immune libraries are derived from the RT-
PCR of B-cell-containing material of an
animal (e.g. peripheral blood lymphocytes,
spleen, bone marrow) that has been
immunized with a target of interest. In theory,
a phage-display library can be made from the
immune system of any animal for which PCR
primers can be designed that will amplify the
repertoire of heavy chain and light chain
variable region sequences for that species.
Though the ability to do this at rst may seem
counterintuitive, given that variable regions
of antibodies are variable in sequence and
unknown a priori. It should be appreciated
that the very beginning 5' end of heavy and
light chain genes comprises sequences
referred to as framework regions for which
there are a relatively limited number of
possible sequences. For example, in the
human, any one of the billions of heavy-chain
antibody sequences in a given individual will
fall into one of only seven families of V
H

germline genes (de Bono et al., 2004). To PCR
amplify that repertoire of heavy chains, only
half a dozen or so forward PCR primers are
needed, each paired with the same 3' primer
that anneals to the beginning of the antibody
constant region. Human k and light-chain
variable-region genes also fall into families
with limited diversity in the 5'-most
framework region, thus permiting their PCR
Fig. 8.6. Overview of phage-display applications for antibody discovery, production and use (see text for
details).
(b)
Source of Ig heavy &
light chain DNA
Phage display
library construction
Selection on target
Target-specific
phage particles
(a)
Monoclonal
B-cell lines
(human or mouse)
(f)
Convert to conventional
full-length IgG
Convert to soluble antibody
fragments (e.g. Fab, scFv)
Retain as phage particle
detection reagents
(c)
Use in conventional
immunological assays or
as therapeutic antibodies
Use in animal models,
drug targeting, imaging
(g) (h)
Use in serological
assays detecting
phage with
anti-M13 antibody
Use in novel
reagent genotyping
approach by detecting
phage DNA
(d)
In vitro affinity
maturation
(e)
amplication with a manageable number of
amplication reactions (~2040 depending on
the phage-display system used).
Species for which phage-display libraries
have been constructed have included human,
non-human primate, mouse, rabbit, sheep,
chicken, camel, shark, dog and others (Marks
et al., 1991; Siegel et al., 1999; Andris-Widhopf
et al., 2000; Andris-Widhopf et al., 2001;
Popkov et al., 2003; Harmsen and de Haard,
2007; Braganza et al., 2011; Finlay et al., 2011).
Using human material as a source of
immunoglobulin cDNA is clearly advan-
tageous if the ultimate goal is to produce a
therapeutic antibody that will be most
compatible with the immune systems of the
patients who will be treated with that drug.
However, for obvious ethical reasons,
humans cannot be immunized at will with
any substance to which one might want to
generate an antibody. Consequently, human
immune phage-display libraries are generally
limited to those constructed from individuals
who are found to already make potentially
clinically useful antibodies. Such scenarios
include post-vaccination (e.g. with tetanus
toxoid or anthrax; Barbas et al., 1991; Wild et
al., 2003), post-infection (e.g. with human
immunodeciency virus; Barbas et al., 1993),
post-treatment for underlying disease (e.g.
from sensitization to blood group antigens
following transfusion; Chang and Siegel,
1998) or in certain autoimmune diseases in
which subjects make antibodies to human
self-antigens as part of their disorder
(McIntosh et al., 1996; Graus et al., 1997; Roark
et al., 2002; Payne et al., 2005).
Although there are phage-display
methods for humanizing mouse antibodies
(Osbourn et al., 2005), animals are most ofen
used for developing antibodies for diagnostic
or research purposes where the species of
origin is not relevant. Although a com-
prehensive discussion of the relative merits of
using one animal over another for a given
phage-display project is beyond the scope of
this chapter, chickens and camels as sources
for the generation of phage-display-derived
antibodies are worth noting. Chickens oer
two advantages. First, from a biological
perspective, as birds and being evolutionarily
distinct from mammals, their immune
systems may mount vigorous immune re-
actions to highly important and thus highly
conserved mammalian proteins to which the
immune system of a mammal (e.g. human,
rabbit or mouse) will not respond due to
tolerance mechanisms (Andris-Widhopf et al.,
2001). Secondly, from a technical perspective,
chicken immune systems create their antibody
diversity through gene conversion events
rather than through the inheritance of
multiple heavy- and light-chain immuno-
globulin genes, as described for humans and
other animals (McCormack et al., 1993). In
fact, chicken immune systems comprise only
one heavy-chain germline gene and one light-
chain germline gene, which limits the number
of PCR amplication steps to a total of two,
thus simplifying phage-display library con-
struction. Antibodies derived by phage
display from camel immune systems (Arbabi
Ghahroudi et al., 1997) have been shown to
oer certain interesting advantages over those
of other species due to their unusual stability
in the presence of detergent or extremes in
temperature. For example, phage-display
libraries made from immunized camelids
(camels and llamas) have produced anti-
dandru antibodies that are stable in
shampoo (Dolk et al., 2005), as well as dip stick
devices to detect the presence of caeine to
dierentiate between caeinated versus
decaeinated hot coee (Ladenson et al.,
2006).
In contrast to the immune phage-
display libraries discussed above, nave
and synthetic phage-display libraries are
designed to get around the ethical issues
precluding the at-will immunization of
human subjects with clinically relevant target
antigens. Nave libraries are generally
constructed from large pools of B cells
collected from normal healthy individuals
(Vaughan et al., 1996; Lloyd et al., 2009). Their
construction was originally designed to
exploit the observation, originally made with
murine hybridomas, that nave IgM anti-
bodies (i.e. those that generally have not
under gone somatic mutation) can recognize a
variety of antigens. The theoretical advantage
of these libraries is that a single universal
112 D.L. Siegel
library could be constructed and used to
provide mAbs with any desired specicity.
Although this has been the prevailing view,
the a nity of the binders may, in some cases,
be suboptimal. Nevertheless, once target-
specic phage particles have been isolated,
there are a number of methods including
chemical mutagenesis, the use of mutagenic
strains of bacteria, incorporation of de-
generate oligonucleotides and error-prone
PCR that can be used to generate random or
targeted nucleotide mutations in antibody
gene segments (Martineau, 2002; Thie et al.,
2009). Reselection of those daughter libraries
on target antigens can be used to select for
those mutations that have improved the
a nity of the original parent antibody (Fig.
8.6e). Along these lines, phage display can be
used to improve the a nity or other
properties of pre-existing mAbs whether
derived from mouse hybridomas, human
mouse heterohybridomas, or human Epstein
Barr virus-transformed B cells. By cloning the
heavy and light gene segments into a phage
vector, sequences with improved binding
properties can be selected (Fig. 8.6f).
Synthetic phage-display libraries dier
from the immune and nave types in that no B
cells are involved in the generation of library
diversity. Rather, the library is composed of
human antibody genes that have been
constructed in vitro using PCR or other
strategies to randomize the hypervariable
regions of one or more human germline-
encoded variable-region genes (Hoogenboom
and Winter, 1992; de Kruif et al., 1995a;
Knappik et al., 2000; Krebs et al., 2001).
Although antibodies isolated from such
synthetic repertoires may also suer from
suboptimal a nity and require in vitro
a nity maturation, as described above, a
signicant advantage over immune and nave
libraries is that the antibody composition of
synthetic libraries is not constrained by the
mechanisms at play in vivo that normally
prevent the generation of antibodies to
human self-antigens. Therefore, the synthetic
phage library approach inherently possesses
the ability to provide human antibodies to
clinically important human proteins, excess
production of which or expression on cells
may be responsible for human disease.
Use of phage particles as immunological
reagents
The vast majority of studies have used phage
display as a tool to isolate novel antibody
DNA that would then be subcloned into
another recombinant antibody expression
system to produce soluble immunoglobulin. A
number of studies have shown, however, that
antibody-displaying phage particles them-
selves can also be used as immunological
reagents, ones that are capable of self-
replication (Fig. 8.6d). For example, phage
particles expressing antibody fragments for
red blood cell antigens were able to phenotype
cells using an indirect agglutination assay
analogous to the traditional Coombs reaction
(which utilizes rabbit or mouse anti-human
IgG) but instead using antibodies to the phage
particles as a secondary antibody (Fig. 8.6g)
(Siegel et al., 1997). The advantages of using
phage particles for these types of assays
instead of conventional IgM- or IgG-containing
antisera is sensitivity as few as 1025 anti-red
cell phage per red cell will produce a positive
reaction and cost. Indeed, an inexpensive,
1 litre overnight bacterial culture of phage can
provide enough reagent for over 100,000
diagnostic assays. The enormous sensitivity of
these assays is due in large part to the
enormous size of a phage particle when
compared with a conventional antibody; thus,
virions provide a large surface area to which
secondary antibodies can bind (Fig. 8.7).
Similarly, antibody-displaying phage
particles can be used in other immunological
assays such as ELISAs (Steinberger et al., 2001)
and ow cytometry (Siegel and Silberstein,
1994) by covalently modifying the phage
particles with colour-developing enzymes,
uorescent molecules or biotin, or by using an
appropriately conjugated anti-M13 secondary
antibody. However, more recently, the
potential use of intact phage particles in
immunological assays has been extended
further by exploiting the fact that unique
sequences of DNA are present within the
particles (Fig. 8.6h; Jaye et al., 2003; Siegel,
2005). The concept here is that the presence of
a target antigen can be determined by binding
target-specic phages and then genotyping
the phage particle. The advantages of this
approach are that methods that use nucleic
acid detection schemes oer extraordinarily
high sensitivity and specicity, require minute
amounts of reagents and are adaptable to
automation. In addition, nucleic acid-based
assays lend themselves to multiplexing
strategies, which, in the case of phenotyping
cells, would oer the possibility of
simultaneously determining the presence or
absence of an entire set of cell-surface antigens
by adding mixtures of phage, each expressing
a dierent antibody specicity on the outside
of the phage particle and bearing a unique
DNA sequence tag that had been engineered
into the phagemid DNA (Siegel, 2005).
Current Status of Phage-display-
derived Therapeutic Antibodies
The market for therapeutic mAbs has increased
to more than US$30 billion within the last few
years with approximately two dozen mAbs
currently approved for human use (Bratkovic,
2010: Nelson et al., 2010). The majority
(approximately two-thirds) of these antibodies
comprise molecules that were derived using
conventional mouse hybrid oma methods
developed in the 1970s (Kohler and Milstein,
1975), which then underwent some form of
humanization by which either the murine
antibody variable regions were spliced on to a
human constant region (chimaeric mAbs) or
the complementarity-determining regions,
which are the most variable domains of the
variable regions, were grafed on to human
framework regions equipped with human
constant regions (humanized mAbs).
However, as noted earlier, such antibodies are
not entirely derived from human genes and
may still elicit anti-drug immune responses
that can aect their e cacy as well as cause
potentially serious allergic or anaphylactic
reactions (Weiner, 2006). With the development
during the 1990s of phage display along with
one other technology that immunizes
0.2 m
Fig. 8.7. Transmission electron micrographic images of anti-Rh(D)-expressing M13 bacteriophage (white
laments) bound to the surface of a human Rh(D)-positive red blood cell. Binding occurs at the tips of the
phage where the recombinant antibody is fused to the pIII phage coat protein. Original magnication
44,000. Bar, 0.2 m. (Adapted from Siegel and Silberstein, 1994.)
114 D.L. Siegel
transgenic mice engineered to express human
immuno globulin germline genes (Green et al.,
1994; Lonberg et al., 1994; Lonberg, 2005), it
became possible to produce so-called fully
human mAbs that in theory would be beter
tolerated when used therapeutically. Of the
approxi mately 90 mAbs currently being
evaluated for clinical use that were derived
using one of these two human methods, to
date phage-display has contributed approxi-
mately 35 candidates targeting cancer,
inammatory/autoimmune disorders, neuro-
degenerative diseases, infectious diseases and
others (Lonberg, 2008; Thie et al., 2008; Nelson
et al., 2010).
The anti-tumour necrosis factor-o (TNF-
o) antibody adalimumab (Humira
) was the
rst of approximately 35 phage-display-
developed mAbs to be approved by the US
Food and Drug Administration (FDA) in 2002,
and is indicated for a number of inammatory
conditions including rheuma toid arthritis,
psoriatic arthritis, ankylosing spondylitis and
Crohns disease (van de Pute et al., 2004).
Interestingly, this antibody was derived by
employing both conventional murine hybrid-
oma methods and phage-display approaches.
Initially, the heavy- and light-chain gene
segments of a hybridoma-derived murine
anti-human TNF-o neutralizing antibody
were cloned into the M13 phage-display
system (Fig. 8.6f). This was followed by the
use of guided selection (Jespers et al., 1994;
Osbourn et al., 2005) in which the heavy chain
of the mouse antibody was paired with a
repertoire of random human light chains to
display humanmouse antibody fragments
on phage. This phage-display library was
then panned on TNF-o. In parallel, the light
chain from the original murine antibody was
paired with a repertoire of random human
heavy chains, displayed on phage and then
panned against TNF-o. Finally, a set of
guided human heavy and light chains
selected from each chimaeric library were
combined together to form yet another phage
library (now completely human), which was
panned on TNF-o. In vitro a nity maturation
(Fig. 8.6e) of the best binder was then used to
generate a higher a nity, higher potency,
anti-TNF-o mAb (D2E7) that became the
therapeutic drug adalimumab.
In contrast to adalimumab, belimumab
(Benlysta
), approved by the FDA in 2011

for the treatment of systemic lupus
erythematosus, was discovered de novo from
a human nave phage-display library selected
on B-Lys (a B-lymphocyte stimulator) and
then a nity matured to improve binding
(Baker et al., 2003; Halpern et al., 2006). B-Lys,
a naturally occurring protein important in
B-cell maturation, has been implicated in the
development of autoantibodies leading to
certain forms of autoimmune disease, and
belimumab is thought to work, in part, by
antagonizing the action of B-Lys on B-cell
stimulation.
Future Perspectives
Currently celebrating its 25th anniversary,
phage-display technology has proved to be an
enormously powerful approach for the
discovery of new diagnostic reagents and
assays for their use, as well as leads for the
development of novel molecules for clinical
use. One of its most signicant advantages in
the design of therapeutic targeting agents is
its ability to create antibodies that appear to
be less immunogenic than those produced
using conventional methods. Although the
great majority of therapeutic antibodies
currently approved for human use have not
been derived using phage-display approaches,
it is believed that the di cult-to-navigate
intellectual property climate surrounding
phage-display technology since its inception
has been in large part responsible for
restricting its proliferation in the therapeutic
market (Bradbury et al., 2011). With a number
of core patents covering key aspects of phage
display now expiring, using these methods
for commercial use may not be a limiting
factor in exploiting the enormous capabilities
of phage display (Storz, 2010). In addition to
these applications, the visibility of phage-
display technologies in the elds of nano-
technology (Sarikaya et al., 2003; Hemminga et
al., 2010) and nanomedicine (Souza et al., 2006,
2008, 2010) will increase greatly, as will their
contribution to the interface between clinical
genomics and proteomics.
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9 Phages and Their Hosts: a Web of
Interactions Applications to Drug Design
Jeroen Wagemans
1
and Rob Lavigne
1
1
Department of Biosystems, Katholieke Universiteit Leuven
The development of antibiotic resistance in
virtually all clinically important pathogens is
driving the search for alternatives to
conventional antibiotics. Currently, drug-
resistant bacteria for almost every available
antibiotic class are seen in hospital and
community setings (Clatworthy et al., 2007;
Falconer and Brown, 2009; Fernebro, 2011;
see also Kuhl et al., Chapter 3, this volume).
The growing problem of antibiotic resistance
has been exacerbated by the use of new drugs
that are merely variants of older, overused
broad-spectrum antibiotics. While it is nave
to expect to restrain the spread of resistance
without controlling antibacterial usage, the
desperate need for drugs with novel modes
of action has been recognized by health
organizations, industry and academia (Lock
and Harry, 2008). In general, the molecular
target specicity of currently used anti-
microbials is very limited, as most interfere
with the biosynthesis of DNA, proteins or cell
walls (Brown, 2006; Zlitni and Brown, 2009).
In fact, commercially available antimicrobial
agents target fewer than 20 families of
proteins (Lange et al., 2007; Silver, 2011),
whereas genome-scale mutagenesis assays
suggest that most bacteria encode at least ten
times more essential proteins, indispensable
for in vivo growth and crucial to pathogenesis
(Akerley et al., 2002; Forsyth et al., 2002;
Mecsas, 2002; Thanassi et al., 2002; Kobayashi
et al., 2003; Knuth et al., 2004; Baba et al., 2008).
Hence, it would appear that there is a plethora
of unexploited targets for antibacterial drug
discovery. Success in exploiting these targets,
however, relies on selecting those possessing
the highest potential for commercial success
(Brown, 2004).
Large pharmaceutical companies have
put great eorts into screening small-
molecule chemical libraries against novel
essential protein targets that are widely
conserved among clinically relevant bacterial
species. There is increasing evidence, how-
ever, that this high-throughput screening has
failed to produce the inux into the anti-
bacterial discovery pipeline that was hoped
for, in contrast to screening campaigns in
other therapeutic areas, which ordinarily
yield multiple leads for each screen. Out of 70
screens conducted by GlaxoSmithKline from
1995 to 2001, for example, only ve com-
pounds were identied that were able to
inhibit target activity at a low micromolar
concentration and that had also antibacterial
activity (So et al., 2011). Two of the main
problems with antibacterial screens are the
need for a proper target selection, targets that
are not prone to rapid resistance development
and the limitation of chemical diversity in
currently used libraries. Marketed anti-
bacterials apparently do not generally follow
Lipinskis rule of ve, which denes drug-
J. Wagemans and R. Lavigne
120 J. Wagemans and R. Lavigne
likeness of chemical compounds by certain
solubility and permeability characteristics
(Lipinski et al., 2001). Nevertheless, most
modern libraries are based on this rule of
thumb. These ndings and the limited prot
and extensive timelines associated with drug
development make antibacterial high-
throughput screenings less atractive for
many large pharmaceutical companies,
although advances in small-molecule libraries
may make these screenings more cost-
eective and fruitful (Payne et al., 2007;
Gwynn et al., 2010; Silver, 2011; So et al., 2011).
Proper target-molecule selection in high-
throughput screening can be enhanced using
the knowledge we have gained from the
study of bacterial natural enemies, the
bacteriophages. Drug resistance has been an
identied issue for over 50 years (Finland et
al., 1959), and ever since its emergence, there
has been particular interest in re-examining
bacteriophages, which have shown great
promise in combating pathogenic bacteria.
Besides using phages themselves (see
Burrowes and Harper, and Abedon, Chapters
12, 13, 14 and 17, this volume) or phage
antibacterial enzymes (see Shen et al., Chapter
15, this volume) as antibacterial treatments,
their non-structural metabolic proteins could
also be used to validate new bacterial targets
and even to develop novel chemical anti-
bacterials (Liu et al., 2004), as discussed
further in this chapter.
Afer injection of phage DNA into the
host bacterium, intracellular phage develop-
ment entails a number of tightly programmed
steps (see Cox, Chapter 10, this volume, for
an overview). The transition to a phage-
oriented metabolism early in infection is
crucial (Miller et al., 2003; Gutman et al., 2005;
Roucourt and Lavigne, 2009). Bacterial
viruses consequently have evolved novel
proteins that either inhibit or co-opt critical
bacterial metabolic processes to ensure
proper viral development. Intriguingly, many
phage genes can be deleted under standard
laboratory conditions without seriously
aecting phage production, suggesting that
the corresponding phage proteins are only
necessary under specic environmental
conditions, for infecting specic hosts or have
functions that are redundant with those of
other proteins expressed by the same phage
(Miller et al., 2003).
This chapter specically addresses inter-
actions between phage-encoded and bacterial
proteins within the phage-infected cell,
hereafer referred to as (bacterio)phagehost
interactions. Although important to dierent
stages of infection, other phagehost inter-
actions, such as phagereceptor inter actions
or proteinDNA interactions, are not dis-
cussed. From known bacteriophagehost
interactions, crucial examples are described,
with emphasis placed on those that could
readily be exploited in drug design. A general
phage genomics-based strategy for drug
discovery is also discussed.
Phages and Their Host: an Intriguing
Web of Interactions
Known bacteriophagehost interactions
demon strate that phages inuence their hosts
with regard to dierent metabolic aspects
(Sau et al., 2008; Roucourt and Lavigne, 2009).
Besides protecting the bacteriophage from
bacterial defence mechanisms, such as
restriction enzymes and proteases, several
crucial interactions could be utilized in drug
discovery. For example, the replication and
transcription apparatus is a common target
among phages. Furthermore, there are also
indications that phages directly aect the
translation machinery and maybe even the
global host metabolism. The following
sections describe in more detail these dierent
phage strategies and their molecular back-
ground to hack their host-cell macro-
molecular machinery and how they can be
applied in the drug-design eld.
Phages taking over the host transcription
apparatus
All bacterial RNA polymerases are closely
related and consist of ve subunits,
designated o, |, |' and o, with o present in
two identical copies. These subunits interact
to form the active enzyme, called the RNA
Phages and Their Hosts: a Web of Interactions 121
polymerase holoenzyme, but the o specicity
factor is not as tightly bound as the others,
thus easily dissociating. RNA polymerase
minus this o factor is described as the core
enzyme, consisting of o
2
||'. This core enzyme
alone can catalyse the formation of RNA, but
a o subunit is required for recognition of the
appropriate promoter sites on the DNA and
the initiation of RNA synthesis.
The RNA polymerase holoenzyme
distinguishes between dierent types of
promoters, which consist of various con sensus
sequences, on the basis of which type of o
factor is atached. The most common
promoters are those recognized by the RNA
polymerase-o
70
holoenzyme. These promoter
sequences have two important regions: a short
AT-rich region about 10 bp upstream of the
transcription start site, known as the 10
sequence, and a region about 35 bp upstream
of the start site, called the 35 sequence. The
o
70
factor must bind to both sequences to
initiate transcription. Apart from o
70
,
Escherichia coli uses several other specicity
factors

for special circumstances: for example,
o
54
exerts its function in nitrogen metabolism-
related gene transcription, while o
32
is required
for expression of heat-shock genes (Snyder
and Champness, 2003). Bacterio phages can
also encode their own o factors to co-opt the
host RNA polymerase to express phage genes.
This is most common for genes expressed later
in the infection cycle the middle and late
phage genes. Conversely, early phage genes,
transcribed immediately afer phage genome
injection, have promoters that are more like
bacterial promoters (Kuter et al., 2005).
To begin transcription, the RNA poly-
merase binds to the promoter (forming the
closed complex) and separates the DNA
strands, exposing the bases (open complex).
Once the DNA strands have been pulled
apart, RNA synthesis begins, complementary
to the transcribed strand. As the RNA chain
begins to grow, the holoenzyme releases its o
factor, and the core enzyme continues RNA
polymerization in the 5'-to-3' direction, until
it encounters a transcription termination site
in the DNA (Snyder and Champness, 2003;
Madigan et al., 2006).
As they tend to favour their own
transcription and reduce that of their host,
most phages exploit the bacterial transcription
apparatus at some stage during the infection
cycle. Consequently, the RNA polymerase
complex is a common target for early phage
proteins. Phages especially can shut down
host transcription by: (i) modifying the
bacterial RNA polymerase, thus recruiting it
for their own transcription, as in the case of
T4; or (ii) inhibiting the host RNA polymerase
and taking over transcription with a phage-
encoded RNA polymerase, as in the case of
T7 (Nechaev and Severinov, 2003; Sau et al.,
2008). Table 9.1 lists known phagehost
interactions aecting the transcription
apparatus. For each described bacteriophage
host interaction, the bacteriophage protein
and the phage it originates from as well as the
host protein are shown. If known, the eect of
the interaction is summarized. Interactions
between coliphages T4 and T7 and the E. coli
RNA polymerase have been reviewed widely
elsewhere (Roucourt and Lavigne, 2009), and
a few additional interesting examples that
could be useful for drug discovery are
discussed below.
As a rst example, Thermus thermophilus
phage P23-45 fully depends on the host
transcription machinery for transcription of
its middle and late genes. Two P23-45
proteins, gp76 and gp39, both bind the host
RNA polymerase core enzyme as well as the
RNA polymerase-o
A
holoenzyme independ-
ently and at separate sites. As a result, both
gp76 and gp39 inhibit in vitro transcription of
the bacterial 35/10 promoters. Moreover,
the inhibitory eect is even greater when
both phage proteins are present, reducing
host transcription to a minimum. In contrast,
10 promoters, predominantly located in
front of the middle and late phage P23-45
genes, are not inhibited (Berdygulova et al.,
2011). Although this bacterium does not
cause any infections, this inhibition mechan-
ism by phage P23-45 might also be used in
other phages infecting human pathogens.
In contrast to T. thermophilus, the rice
pathogen Xanthomonas oryzae siphovirus
Xp10 was shown to encode its own RNA
polymerase (Yuzenkova et al., 2003). Afer
transcription of the early genes by the host
RNA polymerase, Xp10 transcription
regulator p7 stimulates late viral gene
Table 9.1. Phagehost interactions affecting the transcription machinery.
Phage
Phage
protein Host protein Effect Reference
G1 gp67 o
SA
factor
a
Anti-o factor Dehbi et al. (2009)
N RNA polymerase core, NusA,
NusG
Anti-termination, delayed early
transcription
Mason and Greenblatt
(1991)
Q RNA polymerase holoenzyme
(core + o
70
)
Anti-termination, late
transcription
Marr et al. (2001)
N4 SSB RNA polymerase |' subunit Activation of late transcription Miller et al. (1997)
T7 gp0.7
C-term
RNA polymerase |' subunit Host transcription shutoff Michalewicz and Nicholson
(1992)
gp0.7
N-term
RNA polymerase | and |'
subunit
Efciency of termination Severinova and Severinov
(2006)
RNase III Processing of T7 mRNA Mayer and Schweiger (1983)
RnaseE and RhlB Protection of T7 mRNA from
degradation
Marchand et al. (2001)
gp2 RNA polymerase |' subunit Inhibition of transcription
initiation
Nechaev and Severinov
(1999)
T4 Alt One RNA polymerase o subunit Preferential expression of T4
early genes
Sommer et al. (2000)
Alc RNA polymerase | subunit
(postulated)
Host transcription shutoff Kashlev et al. (1993)
ModA Both RNA polymerase o
subunits
Lower expression of T4 early
and host genes
Skorko et al. (1977)
AsiA o
70
factor Inhibits recognition of o
70

promoters
Ouhammouch et al. (1995)
MotA o
70
factor Recognition of T4 middle
promoters
Ouhammouch et al. (1995)
gp55 RNA polymerase core enzyme o factor for expression from late
promoters
Kassavetis et al. (1983)
gp33 RNA polymerase core enzyme Helper of gp55 Kassavetis et al. (1983)
RpbA RNA polymerase core enzyme Favours expression of T4 late
genes (postulated)
Kolesky et al. (1999)
Mrh o
32
factor Decoy of o
32
from RNA
polymerase core (postulated)
Mosig et al. (1998)
Srh RNA polymerase core enzyme
(postulated)
Decoy of o
32
from RNA
Mosig et al. (1998)
Srd RNA polymerase core enzyme
(postulated)
Decoy of o
70
/o
38
from RNA
Mosig et al. (1998)
P2 Org RNA polymerase o subunit Initiation of late transcription Wood et al. (1997)
P4 Psu Rho factor Inhibition of Rho-dependent
termination
Pani et al. (2009)
P23-45 gp39 RNA polymerase holoenzyme
(core + o
A
)
Shutoff of host transcription Berdygulova et al. (2011)
gp76 RNA polymerase holoenzyme
(core + o
A
)
Shutoff of host transcription Berdygulova et al. (2011)
phiEco32 gp36 RNA polymerase core enzyme o factor (recognition of phage
promoters)
Savalia et al. (2008)
gp79 RNA polymerase core enzyme Inhibition of o
70
-dependent
transcription
Savalia et al. (2008)
SPO1 gp44
(E3)
RNA polymerase | subunit Inhibition of the host RNA
polymerase
Wei and Stewart (1995)
gp28 RNA polymerase core enzyme o factor for middle transcription Losick and Pero (1981)
gp33 RNA polymerase core enzyme o factor for late transcription Losick and Pero (1981)
gp34 RNA polymerase core enzyme Helper protein for late
transcription
Losick and Pero (1981)
Unknown gp67 Component of RNA polymerase Shutoff of host transcription Liu et al. (2004)
Xp10 p7 RNA polymerase |' subunit Inhibition and anti-termination of
transcription
Nechaev et al. (2002)
YuA Unknown o
54
factor Expression of late genes Nechaev and Severinov
(2008)
a
SA = Staphylococcus aureus.
transcription in two ways: p7 not only blocks
transcription from most bacterial and early
viral promotors by binding the |' subunit of
the RNA polymerase, it also stimulates late
gene transcription by inhibiting termination
of host RNA polymerase transcription from a
p7-resistant promoter (Nechaev et al., 2002;
Djordjevic et al., 2006; Yuzenkova et al., 2008).
One last example of inhibition of host
transcription through direct interaction
between a phage-encoded protein and the
RNA polymerase complex is the anti-
staphylococcal polypeptide G1ORF67 (Dehbi
et al., 2009). This phage G1 protein binds to
the primary o factor of Staphylococcus aureus,
o
SA
. As a result, transcription of host house-
keeping genes is shut down during the
phages exponential growth phase as tran-
scription of these genes is o
SA
dependent.
Dehbi et al. (2009) concluded that G1ORF67 is
a phage-encoded anti-o factor, given both its
interaction with o
SA
and its ability to inhibit
o
SA
function. As inhibiting transcription un-
avoidably leads to cell death, non-protein-
aceous chemical analogues that mimic these
phage proteins and therefore their inhibitory
eect could serve as new antibacterial com-
pounds.
Phages hijacking host genome replication
Although less extensively documented,
bacteriophages also directly target their hosts
replication system. Table 9.2 summarizes
phagehost interactions inuencing host
replication, of which some examples will be
discussed below.
Whereas most obligately lytic phages,
such as T4, encode most proteins for their
own genome replication (Mueser et al., 2010),
temperate phages and small lytic phages
ofen depend heavily upon the host replisome
(Friedman et al., 1984). To prioritize their own
DNA biosynthesis while abolishing host
genome replication, lytic phages usually
block several host DNA metabolism proteins
through direct bacteriophagehost inter-
actions. In addition, the host nucleoid is ofen
degraded to provide precursors for phage
genome replication. Temperate phages, on
the other hand, redirect the host replisome
through phagehost interactions, thereby
recruiting it for the replication of the phage
genome (Gutman et al., 2005). These dif-
ferences between lytic and temperate phages
in completely shuting down or instead
redirecting the host replication machinery,
respectively, demonstrate the possibility of
identifying a wide diversity of susceptible
drug targets within their host and dierent
inhibition mechanisms.
Liu et al. (2004) showed that the DNA
replication pathway of bacteria is indeed very
vulnerable to inhibition. They demonstrated
that among 26 diverse Staphylococcus aureus
phages, a total of seven unrelated phage
polypeptide families target and inhibit four
proteins of the replication machinery of the
host cell. The DNA primase DnaG, which is
necessary for replication initiation, and the
uncharacterized replication protein PT-R14
are targeted by only one phage protein each,
while DnaI, which is the helicase loader, is
directed by two phage proteins from two
dierent phages. Moreover, three distinct
phage polypeptides from three dierent
phages target DnaN, the sliding clamp, which
tethers the DNA polymerase to its template
afer the clamp loading process and therefore
confers processivity on that enzyme. Except
for phage Twort gp168 and G1 gp240, the
molecular mechanisms of these interactions
have not been studied. These Twort and G1
proteins have a bactericidal eect by directly
blocking the DNA sliding clamp and therefore
may be ideal candidates for the development
of mimicking drugs (Belley et al., 2006).
Both coliphages and P2 encode
polypeptides, proteins P and B, respectively,
which interact with the DNA helicase DnaB,
which unwinds the DNA prior to replication,
of their host (Klein et al., 1980; Odegrip et al.,
2000). As a consequence, phage protein P
strongly inhibits the ATPase activity of the
helicase as well as its ability to assist the E.
coli primase, thereby blocking host DNA
replication. Moreover, both protein P and
phage P2 protein B serve as DNA helicase
loaders, directing the helicase to the phage
origin of replication for commencing repli-
cation (Mallory et al., 1990). The specic
actions of both proteins could be mimicked
by chemical analogues.
Recently, Yano and Rothman-Denes
(2011) reported a dierent mechanism of
arrest of host DNA replication, which favours
phage genome replication. E. coli phage N4
specically and eectively inhibits host
replication within 5 min of infection (Schito,
1973). Yano and Rothman-Denes (2011)
revealed the interaction between N4 gp8 and
the o-subunit of the DNA polymerase III,
which is the clamp loader of this ten-subunit
complex. Gene product 8 was demonstrated
to be the specic inhibitor of E. coli DNA
replication elongation, responsible for host
DNA synthesis shuto. N4 gp8 does not
eliminate host genome replication, but
without loading the DNA clamp, the
processivity of the DNA polymerase III
holoenzyme is extremely low. The authors
also illustrated the bacteriostatic eect of N4
gp8, which again could be exploited in
antibacterial research. Interestingly, gp8 is
not conserved between N4 and any of the
currently known N4-like phages, such as
Yersinia ruckeri phage NC10 or Pseudomonas
aeruginosa phages LIT1 and LUZ7 (Yano and
Rothman-Denes, 2011). Consequently,
application of gp8 derivatives may lead to a
narrow-host-range antibiotic.
Phages manipulating the translation
machinery
The ubiquitous availability of cellular ribo-
somes allows some phages to utilize the
unmodied host translation machinery to
Table 9.2. Phagehost interactions involved in host replication shutoff.
Phage
Phage
protein Host protein Effect Reference
77 gp104 DnaI (helicase loader) Shutoff of host replication Liu et al. (2004)
G1 gp240 DnaN (DNA polymerase III |
subunit)
Shutoff of host replication Liu et al. (2004)
P DnaB (DNA helicase) Initiation of genome
replication
Klein et al. (1980)
O RNA polymerase | subunit Initiation of DNA
replication
Szambowska et al. (2011)
Gam RecBCD Inhibits nuclease and
helicase activity of
RecBCD
Court et al. (2007)
CII DnaB (DNA helicase) and
DnaC (helicase loader)
Inhibition host replication Sau et al. (2008)
N4 gp8 HolA (DNA polymerase III o
subunit)
Shutoff host replication Yano and Rothman-Denes
(2011)
T7 gp5 Thioredoxine Processivity factor of the
DNA polymerase
Tabor et al. (1987)
gp5.9 RecBCD nuclease Inhibits nuclease and
ATPase activity
Molineux (2005)
P2 B DnaB (DNA helicase) Initiation of P2 genome
replication
Odegrip et al. (2000)
PA16 gp106 DnaN (DNA polymerase III |
subunit)
Shutoff of host replication
(postulated)
Belley et al. (2006)
Twort gp168 DnaN (DNA polymerase III |
subunit)
Unknown gp016 DnaI (helicase loader) Shutoff of host replication Liu et al. (2004)
Unknown gp025 DnaN (DNA polymerase III |
subunit)
Unknown gp078 DnaG (DNA primase) Shutoff of host replication Liu et al. (2004)
Unknown gp140 PT-R14 (involved in
replication)
translate their mRNA (Nechaev and
Severinov, 2008). Several ndings, however,
suggest a direct inuence of infection on
translation by some phages. These are
summarized in this section.
Both T4 and T7 modify proteins directly
involved in translation (Robertson et al., 1994;
Depping et al., 2005). For example, T4 ADP-
ribosyltransferases Alt and ModB interact
with 27 and eight dierent host proteins,
respectively, some of which are involved in
translation. Nevertheless, the individual
eects of these ribosylations remain to be
fully elucidated (Depping et al., 2005).
Although it might be di cult or even
impossible to imitate ribosylation or other
modications with small chemical com-
pounds, these ndings indicate that there
may well be other phage mechanisms to
modify or inhibit the host translation system.
T. thermophilus phage |YS40 also seems
to inuence the host translation machinery.
This phage might control the expression of its
genes through a novel mechanism that directs
the cellular translation apparatus towards
mRNAs lacking a leader sequence. This
leader sequence contains the ribosome-bind-
ing site and is required for correct translation
initiation. As most transcripts of |YS40
middle and late genes are leaderless, this
strategy would favour translation of bacterio-
phage mRNA over host mRNA. Sevostyanova
et al. (2007) suggested this translation
manipulation because phage |YS40 appears
to specically upregulate the levels of
translation initiation factor IF2, which is
implicated in translation of leaderless RNA
(Grill et al., 2001). The exact molecular basis of
this inuence and its possible application in
drug development is still un known.
Do phages impact on global host
metabolism?
Inhibiting host replication, transcription and
translation afer infection are clearly favour-
able for a phage in that it allows a shif to
phage processes from host ones. Similarly, it
is not unimaginable that phages also might
be able to inuence the bacterial energy
metabolism or any other essential pathway,
as superuous and energy-wasting steps
could be a burden to e cient intracellular
phage development. On the other hand,
inhibiting an important energy source might
help a phage in blocking the host response to
infection.
Examples are the interactions between
phage T4 ADP-ribosyltransferase Alt and
host pyruvate kinase I and ribose 5-phosphate
isomerase A (Depping et al., 2005). These are
important enzymes in glycolysis and the non-
oxidative branch of the pentose phosphate
pathway, respectively. Although the eect
caused by these modications remains
unclear, this provides a rst indication for the
inuence of phages on host energy
metabolism. Similarly, a systematic yeast
two-hybrid screening for bacteriophagehost
interactions between P. aeruginosa and
podovirus |KMV revealed three interactions:
again an enzyme of the central energy
metabolism (malate synthase G), a regulator
of a secretion system and a regulator of
nitrogen metabolism (Roucourt et al.,
2009a,b). These rst examples suggest that
there might also be some deleterious
inuences on global metabolic pathways due
to direct phagehost interactions, potentially
useful in drug discovery.
Indeed, recently, Rybniker et al. (2011)
reported the interaction between the cytotoxic
early protein 77 of mycobacteriophage L5
and MSMEG_3532, an l-serine dehydratase
of Mycobacterium smegmatis. Minute amounts
of intracellular protein 77 act bacteriostatically
on the host of phage L5 (Rybniker et al., 2008).
In E. coli, deciency of l -serine dehydratase
has been shown to impair growth through
the inhibition of cell division (Zhang and
Newman, 2008). Rybniker et al. (2008) could
not conrm an in vitro eect of gp77 on
dehydratase activity, however. The specic
interaction of phage protein and dehydratase
may also play a role in providing amino acids
or products of the amino acid metabolism
required for phage maturation (Rybniker et
al., 2011). In terms of drug development, the
hope would be that the host target (or targets)
whose presumed inhibition resulted in the
bacteriostatic eect would be present as well
in M. smegmatis-related pathogens such as
Mycobacterium tuberculosis.
Phage-directed inhibition of cell-wall
biosynthesis?
Cell-wall biosynthesis is a crucial step
associated with bacterial growth. Not
surprisingly, many antibiotics in the past
have been shown to inhibit this process
directly. Some ndings suggest a direct
inhibition of cell-wall biosynthesis by several
phage-encoded proteins, which could lead to
new inhibition mechanisms of this crucial
step in cell division (Sau et al., 2008). For
example, the Kil protein of defective prophage
Rac was shown to arrest E. coli cell division
severely. This detrimental eect was reversed
when both Kil and cell division protein FtsZ
were overexpressed, suggesting a direct or
indirect interaction between the two proteins
(Conter et al., 1996). Like the Kil gene product
of the Rac prophage, the Kil proteins of Mu
(Waggoner et al., 1989), P22 (Semerjian et al.,
1989), (Sergueev et al., 2001) and Sf6 (Casjens
et al., 2004) also have been reported to aect
cell division, illustrating this common Kil
mechanism among phages. So far, however,
no exclusive proof for a direct eect on cell-
wall biosynthesis or cell division has been
provided.
From interactions towards new
antibacterials
Beyond identifying new antibacterial targets,
non-lytic phage molecules could also serve as
antibacterial compounds themselves or they
can form the basis for a phage protein-derived
small-molecule antibiotic, as demonstrated
by Liu and colleagues (2004) and further
discussed in the next paragraph. Although
not all phagehost interactions are detri-
mental to the host cell, some inhibit the host
macromolecular machinery or other critical
cellular processes, leading to cell-cycle arrest
or even host lethality. These particular
bacteriophagehost interactions thus might
be used towards development of new
selectively bacteriostatic or bactericidal
agents (Liu et al., 2004; Projan, 2004).
Bacteriophagehost interaction-based
de velopment of new antibacterial compounds
is a multistage process, as depicted in Fig. 9.1.
First, bacteriophages are isolated and their
genomes sequenced and annotated. Sub-
sequently, predicted or selected genes are
cloned into an inducible expression vector
and transferred to the host cell. By inducing
expression, toxic or inhibitory phage products
are identied by inhibition of host-cell
growth. In the next phase, the bacterial
interaction partner of the phage-encoded
inhibitor is revealed by proteinprotein
interaction analysis, such as yeast two-hybrid
or a nity purication. As interaction analysis
ofen contains both false-positive and false-
negative interactions, it is important to use a
combination of dierent techniques to nd
and conrm all bacteriophagehost inter-
actions (Braun et al., 2009; Chen et al., 2010).
Once the interaction partner is known, it can
be validated as a suitable target for drug
development. Apart from identifying the
new antibacterial target, the interaction itself
between the phage protein and the bacterial
protein can serve as the basis for a screening
assay in the development of a new anti-
bacterial compound. Presumably, small
chemical molecules capable of disrupting the
phagehost interaction by binding to the host
protein would mimic the inhibitory eect
of the bacteriophage protein. These assays,
using, for example, time-resolved uorescence
resonance energy transfer (TR-FRET) to
demon strate disruption of interaction, facili-
tate high-throughput screening of large
libraries of small synthetic compounds. The
chemical equivalent of the phage protein then
could provide the basis for a novel antibacterial
(Liu et al., 2004).
Liu and colleagues (2004) demonstrated
the eectiveness of this bacteriophage-
inspired platform to identify new targets and
antibacterials in the Gram-positive S. aureus.
Out of 26 fully sequenced S. aureus-specic
phages, 31 novel polypeptide families of
early proteins were identied as abolishing
growth upon expression in the bacterial host.
Using a nity chromatography of S. aureus
lysates on phage protein-coated columns and
subsequent identication by mass spectro-
metry, the cellular targets for some of the
inhibitory proteins were identied and
several were shown to be essential com-
ponents of the host DNA replication and

P
h
a
g
e
s

a
n
d

T
h
e
i
r

H
o
s
t
s
:

a

W
e
b

o
f

I
n
t
e
r
a
c
t
i
o
n
s

1
2
7
Non-induced Induced
Protein 1
s n i e t o r p e g a h p c i x o t f o n o i t a c i f i t n e d I s e m o n e g e g a h P s e g a h p o i r e t c a B
Antibacterial compound Inhibition of interaction Identification of phagehost interaction
Sequencing and
genome annotation
Expression of proteins
in host
High-throughput
screen for inhibitors
Development of interaction-
based assay
Identification of
bacterial target using
e.g. yeast two-hybrid
system
Protein 2
Protein 3
Non-toxic
Toxic
Non-toxic
Cell growth
Reporter gene
Fig. 9.1. Bacteriophagehost interaction-based development of new antibacterial compounds.
transcription machineries. One of these was
the interaction between ORF104 of phage 77
and the helicase loader DnaI, which is
essential for DNA replication initiation. This
phagehost interaction was conrmed to be
specic and direct using four independent
assays: yeast two-hybrid analysis, far Western
analysis with immobilized DnaI and
32
P-labelled gp104, biomolecular interaction
analysis (Biacore) and TR-FRET. Moreover,
DnaI was shown to be essential for viability of
the host cell. These results were then used to
screen for small synthetic molecule inhibitors,
mimicking the phage protein. Out of 125,000
compounds from commercially available
libraries, 36 were able to inhibit the phage
host interaction with a 50% inhibitory
concentration (IC
50
) of less than 10 M.
Among these, 11 compounds were found to
inhibit bacterial growth of S. aureus on their
own with a minimum inhibitory concentration
of less than 16 g ml
1
. Furthermore, neither
compound was signicantly cytotoxic to
human primary hepatocytes or to the cell
lines Hep G2 and HeLa. These lead
compounds were proposed to form a basis for
a new phage-based antibiotic (Liu et al., 2004).
Conclusion
Although microbial resistance is probably
an unavoidable consequence of antibiotic
therapy, a bacteriophage-based platform has
great potential with respect to identifying
novel mechanisms and targets to treat
bacterial infections (Falconer and Brown,
2009). In fact, known phagehost interactions
illustrate the potential for phage systems to
be used for the identication of points in host
metabolism that may be susceptible to small-
molecule inhibitors. The most e cient and
vulnerable targets are selected and validated
through billions of years of co-evolution
between phages and their hosts (Brown,
2004). As there is no dearth of bacteriophages
in nature, the quest for lethal phage proteins
as well as their cognate bacterial targets
should be continued in order to expedite the
research on antibacterial drug discovery (Sau
et al., 2008). Despite the body of available
research and the vast untapped potential,
implementations by industry have remained
limited, however. This can be explained by a
number of factors.
First, the bacteriophage research eld
remains strongly genome oriented and only a
limited number of research groups analyse
the intracellular phagehost interactions
(several of them focusing on model hosts and
not pathogens). Secondly, the step from
fundamental research to product develop-
ment is great, as an extensive body of
evidence is necessary to obtain a suitable
bacterial target candidate and relevant phage-
based inhibitors. Eorts to crystallize and
solve the structure of phage-encoded proteins
involved in inuencing the host metabolism
should be encouraged to tackle this large and
diverse, at least at the primary sequence level,
group of proteins and provide a solid basis
for rational antibacterial design. The strategy
proposed by Liu et al. (2004) (Fig. 9.1) appears
straightforward but contains genome and
expression-based assays, protein interaction
assays, crystallography and small-molecule
design. Although high-throughput sequenc-
ing and protein expression can now be
considered commonplace, protein interaction
analyses (like yeast two-hybrid analyses,
a nity purication and FRET) and (rational)
small-molecule development remain tech-
nically challenging. Thirdly, from an industry
perspective, one could state that major
pharmaceutical companies are cautious to-
wards new antibacterial strategies in view of
the limited prot margin and highly
competitive market for antibiotics. This
implies the necessity of small biotech company
initiatives (vulnerable to economic circum-
stances) to generate a substantial (and costly)
body of material for product development.
The development of phage-inspired
antibiotics nevertheless remains a strong
option for the future. One can anticipate a
shif in the general research focus from
genome-based research to proteome- and
even metabolomics-based research. We can
anticipate that technological developments of
protein interaction and mass spectrometry
approaches will be revolutionized further, in
much the same way as we have seen at the
genomics level, with the advent of next-
generation and single-molecule sequencing
techniques. Proteome- and metabolomics-
based research will rely on these develop-
ments. In addition, the bacteriophage domain
has an extensive body of front-line researchers
with experience in the structural analysis of
virion-associated proteins, which provides a
large body of experience to tackle the dark
mater within phage genomes. From an
industry perspective, we can anticipate an
increasing need within our society towards
novel antibacterials with a related increased
prot potential, which will hopefully motivate
large pharmaceutical companies and investors
in small companies as well. It will be the
responsibility of the scientists, however, to
generate the fundamental know ledge that
will help bridge the gap towards product
development in a sus tainable manner.
Acknowledgements
J.W. holds a predoctoral fellowship of the
Agentschap voor Innovatie door Wetenschap en
Technologie in Vlaanderen (IWT, Belgium).
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10 Bacteriophage-based Methods of
Bacterial Detection and Identication
Christopher R. Cox
1
1
Department of Chemistry and Geochemistry, Colorado School of Mines.
The need for rapid and inexpensive methods
of bacterial detection and identication has
become an increasingly common theme
among the medical, agricultural, food and
water processing, military, homeland security
and academic elds. Despite the permeation
of humanity by disease-causing and resource-
contaminating bacterial agents, conventional
methods fall short of widespread practicality
for bacterial detection and identication,
particularly due to a lack of rapidity or cost-
eectiveness. Consequently, an abundance of
biochemical and genotypic methods have
been developed, although with varied levels
of success.
Some newer methodologies include 16S
23S rRNA gene sequencing, which has become
the gold standard of genotypic bacterial
identication (Botger, 1989; Fredericks and
Relman, 1996; Clarridge, 2004), but also
multilocus enzyme electrophoresis (Selander
et al., 1986; Duan et al., 1991), multilocus
sequence typing (Maiden et al., 1998), multi-
locus variable tandem repeat analysis (Ross et
al., 2011), pulsed-eld gel electro phoresis
(Durmaz et al., 2009), amplied fragment
length polymorphism (AFLP; Jayarao et al.,
1992; Koeleman et al., 1998), repetitive
sequence-based PCR (Jayarao et al., 1992;
Grisold et al., 2010) and Raman spectroscopy-
based systems (Maquelin et al., 2006; Patel et
al., 2008). All of these conventional methods,
however, rely on labour-intensive micro-
biological culturing practices or costly and
time-consuming DNA isolation, amplication
and/or sequencing protocols utilizing highly
specialized equipment. This makes them
impractical for deployment as rapid, cost-
eective point-of-care or eld detection and
identication methods. In addition, most
require 2450 h for positive bacterial
identication. While these various methods
have been used eectively as tools for
laboratory analysis, there is growing interest
in the development of much faster, simpler
approaches, ideally ones that are much more
cost-eective, that do not rely on complex
equipment and that allow on-site positive
pathogen identication without the need for
extensive personnel training.
Exploitation of the bacteriophage (or
phage) life cycle and its accompanying species
specicity can eliminate much of the time and
cost associated with these methods while
rivalling the detection capabilities of even the
most sensitive of current tech nologies. The
use of phages in combination with existing
and emerging technologies consequently has
the potential to overcome many of the
shortcomings of current detection and
identication methodologies and presents a
promising opportunity to develop the next
generation of truly rapid, tractable bacterial
detection techniques. Newly developed
C.R. Cox
Bacteriophage-based Methods of Bacterial Detection and Identication 135
applications centring on the use of phage
amplication coupled to matrix-assisted laser
desorption ionization time-of-ight mass
spectrometry (MALDI-TOF MS) or easy-to-
use lateral ow immunochromatography
now allow rapid bacterial detection with total
time for sampling and analysis in 212 h
timeframes. Advancements in techniques
using immobilized phages, labelled phages,
individual phage components or phages as
vehicles for transferring reporter genes to a
bacterial host have also opened the door for a
multitude of new phage-based bacterial
detection and identication methods.
In this chapter, both traditional and
newer approaches to phage-based bacterial
detection and identication are reviewed. For
additional consideration of traditional as well
as modern approaches to phage-based
bacterial detection technologies, see Williams
and LeJeune (Chapter 6, this volume), who in
part provide a discussion of phage typing
techniques (bacterial classication based on
susceptibility to phage infection), and
Goodridge and Steiner (Chapter 11, this
volume), who consider phage detection as a
means of inferring bacterial presence within
environments, with an emphasis on faecal
contamination of water.
Phage Amplication
Phages possess two key features that make
them ideal reagents for bacterial detection
and identication: their specicity and their
ability to amplify in number in the presence
of target bacteria. These two properties are
considered in this section within the context
of a traditional approach to bacterial identi-
cation the plaque assay. A third key feature,
phage genetic malleability, is considered in
subsequent sections.
The plaque assay
The phage plaque serves as a means of
visualizing phage amplication during
growth in a Petri dish. This technique consists
simply of infection of a suspected bacterial
host with a well-characterized and species-
specic phage, followed by plating (Cherry et
al., 1954; Stewart et al., 1998). If the bacterial
target is phage susceptible, then infection is
initiated, resulting in lysis of the host and
release or burst of progeny phage. This is
followed by continued infection of remaining
uninfected bacteria in the vicinity (Kuter et
al., 2005). The result of these actions is
observed on the bacterial lawn as areas of
lysis or clearing called phage plaques (Fig.
10.1a). Traditional plaque assays have been
used routinely to identify numerous human
bacterial pathogens including Bacillus
anthracis (Thal and Nordberg, 1968; Abshire
et al., 2005), Campylobacter jejuni (Grajewski et
al., 1985), Enterococcus faecalis (Pleceas and
Brandis, 1974), Escherichia coli (Nicolle et al.,
1952), Listeria monocytogenes (Shcheglova and
Neidbailik, 1968), Pseudomonas aeruginosa
(Postic and Finland, 1961), Salmonella
typhimurium (Felix, 1956) and Staphylococcus
aureus (Wallmark and Laurell, 1952).
Plaque development possesses a number
of limitations that can result in false negatives
during bacterial identication. Of particular
concern is the potential for bacterial strains to
dier in their specic mechanisms of phage
resistance, that is, failure of plaque formation
on hosts that otherwise are the same species
as strains that can support plaque growth. A
number of reviews of these mechanisms exist
(Abedon and Yin, 2009; Hyman and Abedon,
2010; Labrie et al., 2010; Stern and Sorek,
2011). In addition, plaques typically require
at least overnight incubation as well as
bacterial cultures that are both pure and
themselves amplied overnight, problems
also seen with more elaborate phage identi-
cation techniques (see Williams and LeJeune,
Chapter 6, this volume). These characteristics
detract from the utility of the conventional
plaque assay in most cases as an approach to
rapid bacterial identication.
An important exception is seen when
considering Mycobacteria spp. for which the
rapidly growing Mycobacterium smegmatis
host may be employed as a surrogate indicator
during phage amplication on pathogenic
Mycobacterium strains (Stanley et al., 2007).
Such an approach can be particularly useful
for characterizing the antibiotic susceptibility
of these pathogens. Notwith standing the
136 C.R. Cox
known caveats, the ability of phages to form
plaques is illustrative of the utility of phage
properties towards bacterial detection and
identication properties. These are con-
sidered below, along with more modern and
sophisticated approaches to phage-based
bacterial detection and identication.
Phage properties useful to detection
technologies
Plaque formation involves the prototypical
phage infection cycle (Fig. 10.1). Familiarity
with this infection cycle is useful for
understanding other phage-based bacterial
detection methods, some of which utilize
only certain portions of the cycle.
The phage infection cycle begins with
virion adsorption to the bacterial cell surface.
This step involves atachment, for example,
to Gram-negative outer-membrane porins
(Hashemolhosseini et al., 1994) or transporter
proteins (Hantke, 1978), or atachment to
bacterial cell-wall components such as lipo-
polysaccharide (Heller, 1992) and Gram-
positive peptidoglycan (Valyasevi et al., 1990;
Duplessis and Moineau, 2001). These virion
host interactions tend to be highly precise,
resulting in adsorption by specic phages only
to specic hosts. All phage-based bacterial
detection and identication methodo logies
consequently take advantage of the specicity
of adsorption to distinguish among dierent
bacterial types.
The next step of the infection cycle
involves transfer of phage genetic material
into the host, which occurs by a number of
possible mechanisms depending on the
phage (Letellier et al., 1999; Perez et al., 2009).
This is followed by transcription and
translation of phage genes by host cellular
(a)
(b)
(c)
(d)
Fig. 10.1. A plaque assay performed by mixing a species-specic phage with an unknown bacteria and
growing the bacteria on an agar plate (a). If the target bacteria are susceptible to infection, then phage
attachment to the host is followed by insertion of phage genetic material (b), which reprogrammes the
bacterial replication machinery to produce numerous progeny phage (c). This is followed by lysis of the
host bacterium and release of new phages for subsequent infection (d), resulting in the development of
zones of clearance in the lawn, called phage plaques, as shown in (a).
machinery (Kuter et al., 2005). Although
phage transcription and translation, as well
as other molecular processes, can be highly
dependent on the biochemistry of specic
bacterial hosts, thereby providing additional
levels of specicity, the generality of these
mechanisms also allows expression of
reporter genes from genetically engineered
phages. In particular, reporter genes, as
considered below, can be more likely to be
expressed, as well as more rapid in their
expression, than plaque formation, thereby
potentially contributing to reductions in both
detection times and the occurrence of false
negatives.
The nal steps in the phage infection
cycle are assembly of new virions and virion
release, typically via host-cell lysis. Although
not all phage-based techniques involve or
require phage release, such release is crucial
for virion amplication. In addition, lysis can
release non-phage bacterial contents, which
can also be employed as a means of bacterial
detection. Phage amplication in particular
can be coupled with modern detection
instrumentation to exploit the phage infection
process to intensify a detectable signal that is
atributable to the presence of the targeted
bacteria (Madonna et al., 2007).
Phage Amplication and
MALDI-TOF MS
MALDI-TOF MS is an eective tool for
analysis and identication of a large range of
analyte molecules including peptides,
proteins, carbohydrates and polymers. The
principal mechanism of MALDI-TOF MS
centres on the rapid transfer of target pep-
tides, proteins or other molecular targets into
the gas phase by laser ablation of samples
embedded in a UV-absorbing matrix, com-
bined with TOF MS analysis. Of par ticular
interest with regard to the application of
MALDI-TOF MS to biological detection and
identication is its potential for rapid analysis
of intact bacteria and viruses (Karas and
Hillenkamp, 1988; Tanaka et al., 1988; Holland
et al., 1996). Phage amplication-coupled
MALDI-TOF MS requires species-specic
bacterial infection and allows bacterial
detection and identication in as litle as 2 h.
When combined with disulde bond re-
duction methods to enhance MALDI-TOF MS
resolution (McAlpin et al., 2010), this method
lowers the limit of detection by several orders
of magnitude over conventional biochemical
detection methods. As such, MALDI-TOF MS
protein proling or mass ngerprinting has
emerged as a powerful method of bacterial
detection and identication (Holland et al.,
1996; Madonna et al., 2003) and is readily
combined with phage amplication for rapid
bacterial identication (Fig. 10.2).
Mechanisms of MALDI-TOF MS
Typical MALDI-TOF MS applications involve
mixing of an analyte with an organic matrix
that catalyses sample crystallization and
facilitates ionization. Samples are exposed to
a laser pulse under vacuum, resulting in the
liberation of ions. Surface-ejected ions are
accelerated by an electrical eld down a TOF
tube towards a mass analyser. Smaller
analytes are accelerated to higher velocities
than larger ones and thus reach the analyser
sooner. The TOF for a given target analyte is
then proportional to its relative mass to
charge ratio (m/z). The charge (z) of an ionized
molecule, if given a value of 1, thus makes the
m/z value equal to the mass of the molecule.
In the case of biological analysis, the output
of each detection event can then be compiled
in a series of readings to generate a spectral
prole for each analyte present in a sample.
When ploted graphically, each analyte
present within the detection limits of the
device is described by its m/z ratio along the
horizontal axis and by a peak-intensity value
on the vertical axis to arrive at a spectral
analyte prole, as exemplied in Fig. 10.3.
MALDI-TOF MS is useful for direct
analysis of enriched bacterial cultures. Phage
amplication, by virtue of the production of
many copies of the phages from each bacterial
infection, increases sensitivity by eectively
lowering the amount of bacteria necessary to
elicit a detectable signal. This concept was
used to develop tractable MALDI-TOF MS
detection methods for E. coli (Madonna et al.,
2003, 2007) and Salmonella enterica (subsp.
138 C.R. Cox
enterica serovar Typhimurium) (Rees, 2005).
Madonna et al. (2003) determined that a
typical MALDI-TOF MS limit of detection for
E. coli of 1.0 10
5
colony-forming units (CFU)
ml
1
was decreased by two orders of
magnitude when coupled with MS2 phage
amplication, while Rees and Voorhees
(2005) extended this work to demonstrate,
with similar sensitivity, the simultaneous
detection of E. coli and S. enterica with MS2
and MPSS1 phage amplication. As an
example of MALDI-TOF MS phage ampli-
cation analysis, spectra resulting from the
infection of a L. monocytogenes culture with
the Listeria typing phage A511 are shown in
Fig. 10.3.
A pure L. monocytogenes culture yields a
mass spectrum showing multiple bacterial
proteins (Fig. 10.3a). In contrast, puried
A511 phage yields a mass spectrum consisting
of one main peak representative of a
previously described 48.7 kDa major capsid
protein, which is present in many copies
(Loessner and Scherer, 1995; Fig. 10.3b).
Immediately following infection with phage
and bacterial concentrations below the limit
of MALDI-TOF MS detection, mass spectra
show no discernible bacterial or viral peaks
(Fig. 10.3c). Afer a 12 h incubation at room
temperature, however, the same infection
reveals the presence of the A511 major capsid
protein among the peaks generated by lysed
bacterial cells, eectively demonstrating the
use of phage amplication and MALDI-TOF
MS analysis for bacterial detection without
the need for overnight (1620 h) bacterial
culture (Fig. 10.3d).
Phage Amplication and Lateral Flow
Immunoassays
Lateral ow immunoassays (LFIs) have been
adapted in many formats for detection of a
large number of analytes and have proven
useful for rapid, phage amplication-based
detection and identication using an easy-to-
use handheld strip (Fig. 10.4; see also
Goodridge and Steiner, Chapter 11, this
volume). First described in 1980 and later
Mass analyser
Analyte/matrix mixture
m/z
Fig. 10.2. Phage amplication-coupled MALDI-TOF MS for bacterial detection and identication. See text
for full description. Shown is a phage-specic protein prole, seen here as a 37.8 kDa major capsid
protein peak and its 18.9 kDa doubly charged ion.
Mass (m/z)
Mass (m/z) Mass (m/z)
Mass (m/z)
(a)
(c)
(b)
(d)
Fig. 10.3. MALDI-TOF MS phage amplication analysis illustrating phage protein proling for bacterial
identication. (a) Representative Listeria monocytogenes bacterial protein prole obtained after overnight
culture. The intensity scale is expanded to compensate for a very high concentration of bacterial proteins.
(b) L. monocytogenes phage A511 spectrum showing the abundant 48.7 kDa major capsid protein. (c)
Initial infection at phage and bacterial input concentrations below the limit of MALDI-TOF MS detection.
(d) Phage amplication at 12 h results in the formation of the distinct capsid protein. This is the earliest
time this peak is reliably detected. The slight shift in the mass scale in (b) and (d) are due to inherent drift
in MALDI-TOF MS instrument calibration over the course of the experiment.
Coloured nanoparticle reporter
Fig. 10.4. Phage amplication coupled to LFI for bacterial detection and identication. See text for details.
140 C.R. Cox
developed as a commercial pregnancy test,
the technique was originally termed the sol
particle immunoassay (Leuvering et al.,
1980). Since then, LFI devices have been
utilized with various levels of success for the
detection of numerous human bacterial
pathogens, including B. anthracis (Carter and
Cary, 2007), Brucella abortus (Clavo et al.,
2003), E. coli (Aldus et al., 2003), Helicobacter
pylori (Kato et al., 2004), S. aureus (Fong et al.,
2000), Streptococcus pneumoniae (Zuiderwk et
al., 2003) and Treponema pallidum (Oku et al.,
2001). LFIs have also been successfully
applied to the detection of a number of
human (Al-Yousif et al., 2002; Kikuta et al.,
2008; Chaiyaratana et al., 2009; Li et al., 2009),
plant (Kusano et al., 2007) and animal (Joon
Tam et al., 2004; Lyoo et al., 2005; Sithigorngul
et al., 2007) viral pathogens.
Mechanisms of LFIs
While LFIs have been applied to detection of
a myriad of analytes, the basic concept
remains constant. In the case of phage
amplication-coupled LFIs, a patient sample
(e.g. urine, serum, skin swab), hospital
surface swab or other sample suspected of
containing a target bacterial species is mixed
with a previously prepared species-specic
phage and incubated to allow phage infection
and amplication. Following incubation, a
primary reporter consisting of coloured
nanoparticles conjugated to phage-specic
antibodies is added to the infection reaction.
An aliquot of this is applied to a test strip (at
S in Fig. 10.5) where the liquid sample wicks
from the sample pad into a secondary control
reporter conjugate release pad (containing a
second colour of nanoparticles conjugated to
biotin). The secondary reporter and any
phageprimary reporter complexes are
carried on to a nitrocellulose detection mem-
brane striped with a detection line composed
of immobilized, anti-phage antibodies. In
the case of a positive test, phageprimary
reporter complexes concentrate along this
line resulting in a visible, coloured line (T in
Fig. 10.5). At the same time, the secondary
control reporter is carried across a second
detection zone (consisting of a stripe of
streptavidin) and concentrates there, to
indicate that the sample was completely
transported across the detection zone (C on
the device in Fig. 10.5). A positive test is thus
indicated by the formation of two separate
coloured lines one at the detection line and
one at the control line as illustrated in Figs
10.4 and 10.5(a). It follows that a negative test
is indicated by the formation of a single
control line only.
While eective as a relatively inexpen-
sive, portable means of bacterial detection,
conventional LFI applications all depend on
lengthy pre-enrichment of target bacteria
(24 h minimum) an undesirable atribute
for truly rapid, point-of-care detection and
pathogen identication. By coupling species-
specic phage amplication with an LFI,
rather than bacterial amplication, rapid
bacterial detection can be achieved in hours
rather than days. As an e xample, Fig. 10.5
depicts a phage amplication-coupled B.
anthracis detection device.
Figure 10.5(a) shows a positive result
following the addition of B. anthracis-specic
phage to a liquid bacterial sample. A sample
(a)
(b)
(c)
Fig. 10.5. Phage amplication applied to LFI-
based detection of B. anthracis. (a) A positive test
resulting from phage amplication in the presence
of the target bacterial species as indicated by the
development of two lines. (b) A phage-free,
bacteria-free control showing a single control line,
which indicates a negative result. (c) A phage-free
bacterial culture control showing a single negative
control line. C, control line; T, test line; S, sample
applicator pad.
(in this case an aliquot of a known bacterial
solution at a concentration of 5 10
5
CFU
ml
1
) was infected with a high-titre phage.
Afer 5 h incubation, an aliquot of the phage
infection reaction was applied to the pictured
handheld cassete on the sample applicator
pad (S) and allowed to wick across the test
window. A positive result is indicated by the
formation of two lines one at the test line T
and another at the control line C. In order to
verify that this positive test was the result of
phage amplication, accompanying control
tests were run at the same time. Figure 10.5(b)
shows the result of tests run without either
phage or a bacterial target, verifying the
absence of a false positive, as indicated by the
formation of only a single control line. The
test in Fig. 10.5(c) was conducted with the
same bacterial sample used in Fig. 10.5(a) but
without the addition of phage. A single test
line in the absence of phage was observed
only at the control line. Handheld LFI devices
such as this allow rapid, user-friendly, species-
specic detection of any number of phage
bacterial host pairs. In doing so, this platform
bypasses the need for laborious microbial
culturing and/or costly DNA ampli cation
methods, or complex detection instrument-
ation associated with most com monly
employed microbial identication methods.
In 2003, Voorhees et al. patented the use
of phage amplication and LFIs for rapid
bacterial identication and antibiotic resist-
ance determination. This led to the founding
of Microphage, Inc. (Longmont, CO) for
commercialization of this technology. In
2011, Microphage gained approval from the
US Food and Drug Administration and a
European CE mark for use of phage
amplication-based LFI clinical detection
and antibiotic resistance determination of
methicillin-resistant S. aureus (MRSA). The
Microphage KeyPath
TM
MRSA/methicillin-
sensitive S. aureus (MSSA) blood culture test
utilizes phage amplication for handheld,
point-of-care S. aureus detection and methi-
cillin resistance determination with a
sensitivity of 5 10
5
CFU ml
1
(afer 5.5 h
enrichment) and a total analysis time of 5.5 h.
By comparison, conventional MRSA
identication methods require a minimum of
48 h for analysis.
Phage Immobilization for Bacterial
Detection
Phages can be atached to a solid surface such
that they can adsorb to their bacterial host
while remaining xed to that surface. In this
way, phages have been used as a species-
specic biosorbent to capture or separate
bacteria from liquid samples for further
analysis by secondary methods such as PCR,
ELISA and microscopy. The rst reported use
of immobilized phages for bacterial capture
and detection focused on separation and
concentration of Salmonella from liquid
cultures (Bennet et al., 1997). The Sapphire
phage was passively atached to microtitre
plates or polystyrene dipsticks and used to
capture Salmonella from pure and mixed
bacterial cultures. While requiring only 2 h
for concentration of bacteria from an over-
night culture, poor capture e ciency (1%)
was observed, requiring a minimum of
10
5
CFU ml
1
for post-capture PCR detection.
Non-specic binding of bacterial cells was
observed in phage-free controls, further
hindering the usefulness of this approach,
but improvements using biotinylated phages
have since followed (Fig. 10.6).
In one such approach, S. enteritidis phage
SJ2 was chemically biotinylated and atached
to streptavidin-coated magnetic micro-
particles (Sun et al., 2001). Phage-coated
particles were reacted with overnight cul-
tures of a bioluminescent lux-expressing
S. enteritidis strain, and phagemicroparticle
complexes were collected with a magnetic
particle separator and assayed with a lumino-
meter. This method allowed the capture of
19.3% of cells, with a limit of detection of
2 10
6
CFU ml
1
, requiring 20 min for post-
adsorption analysis. In a similar approach,
coliphage T4 was genetically engineered to
express a biotinylated capsid protein to allow
atachment to a streptavidin-coated gold
electrode (Gervais et al., 2007). E. coli growth
was monitored following adsorption to
immobilized phages by electric cell-substrate
impedance (ECIS) at the gold surface. This
method demonstrated an approximately 15-
fold improvement in biotinylated phage
atach ment compared with passive phage
atach ment to untreated gold. A recent
142 C.R. Cox
advance ment with T4 biotincapsid fusion
and streptavidin-coated mag netic beads was
shown to capture 99% of E. coli within 10 min,
with a detection limit of 8 10
2
CFU ml
1

measured by real-time PCR (Tolba et al.,
2010). Other approaches involve covalent
atachment of Salmonella phages to glass for
bacterial capture (Handa et al., 2008), as well
as incorporation of Salmonella and B. anthracis
phages into immobilized magnet oelastic
sensors (Guntupalli et al., 2007; Lakshmanan
et al., 2007; Huang et al., 2009), although with
varied levels of success and detection limits
in the 10
3
CFU ml
1
range. The use of
immobilized phages has also been applied to
ELISA-based bacterial detection (Galikowska
et al., 2011). By this approach, several S.
enterica and E. coli phages were investigated
as primary antibody replace ments and non-
specically bound to ELISA plates to facilitate
bacterial capture
and subsequent secondary detection by con-
ventional alkaline phosphatase-mediated
absorb ance. Assay sensitivity was reported at
10
6
CFU ml
1
and required approximately
5 h. Despite these marked improvements
over the last decade, phage immobilization as
a com ponent of bacterial detection systems
remains at the proof-of-principle stage and
has not yet been incorporated into any com-
mercial applications.
Phage-encoded Reporter Genes for
Bacterial Detection
Numerous recombinant reporter phages
have been created for visual bacterial de-
tection utilizing a range of bioluminescence,
chemiluminescence and uorescence-based
approaches. The basic principle centres on
incorporation of reporter genes such as
bacterial or rey luciferase, or green
uorescent protein (GFP) into a phage
genome. As a result of phage infection, these
genes are transferred to a bacterial host and
are ultimately expressed to produce a de-
tectible photonic emission that can in turn be
used as an indication of the presence of a
given bacterial target.
lux-based reporter phages
The well-characterized bacterial luciferase
genes, encoded by the lux operon from the
Fig. 10.6. Phage immobilization for bacterial detection. See main text for description. ECIS, electric cell-
substrate impedance.
aquatic Gram-negative Vibrio scheri (Meighen,
1994), are perhaps the most frequently used
reporters for incorporation into recombinant
phages. The lux operon is composed of seven
individual open reading frames. The genes
luxA and luxB encode the two-component
luciferase holoenzyme responsible for light
emission. The lux system, in various con-
gurations, has been utilized for construction
of recombinant reporter phages. The rst
description of its use in bacterial detection
involved insertion of the complete lux operon
(Fig. 10.7) into a phage -based cloning vector
for detection of E. coli (Ulitzur and Kuhn, 1987;
Ulitzur and Kuhn, 1989) with a reported
detection limit of as few as 10 CFU ml
1
in
contaminated milk within 1 h.
Numerous other iterations using lux
genes from either V. scheri or Vibrio harveyi
have since been reported with similar success
and include the use of recombinant luxAB (in
the absence of luxCDE) for detection of L.
monocytogenes (Loessner et al., 1996, 1997),
Salmonella spp. (Chen and Gri ths, 1996;
Kuhn et al., 2002; Thouand et al., 2008) and E.
coli (Ulitzur and Kuhn, 2000; Waddell and
Poppe, 2000). Another recent approach for E.
coli detection (Ripp et al., 2006; Birmele et al.,
2008) involves the use of a phage
recombinant encoding only luxI, which
encodes the production of acyl-homoserine
lactone (AHL), the freely diusible com-
ponent of the quorum-sensing aspect of lux
activity, and an E. coli bioreporter strain
carrying luxCDABE and luxR (also involved
in lux-associated quorum sensing). The later
strain is added in tandem with the re-
combinant phage to the test sample. By this
method, the phage-infected target bacteria
release luxI-encoded AHL, which induces
bioluminescence in the bacterial reporter
strain as an indication of the presence of
target E. coli. This approach was shown to be
particularly sensitive when used with pure
cultures, demonstrating detection of 1 CFU
ml
1
within 10 h, while higher target bacterial
concentrations reduced detection times to as
litle as 1.5 h. Because of its reliance on freely
diusible AHL for activation of reporter
bioluminescence, however, this system could
be susceptible to non-specic activation by
autoinducers produced by other bacterial
phylotypes present within mixed bacterial
samples.
luc-based reporter phages
The luc-encoded luciferase of the rey,
Photinus pyralis, is a well-characterized bio-
luminescent reporter (McElroy and DeLuca,
1983; Viviani, 2002). It has been adapted
Fig. 10.7. Phage-based bioluminescence for bacterial detection. See text for description.
luxR luxI luxC luxD luxA luxB luxE
luxR luxI luxC luxD luxA luxB luxE
144 C.R. Cox
much in the same way as the lux operon for
recombinant bioluminescence and is com-
mercially available for use in a wide range of
applications (for example, the Luc-Screen

Firey Luciferase Reporter Gene Assay
System; Applied Biosystems). Luc bio-
luminescence is brought about by ATP-
dependent, luciferase-catalysed oxidation of
a luciferin substrate (benzothiazole) resulting
in light emission (DeLuca and McElroy, 1978).
As is the case with many lux-based reporter
systems, luc relies on the exogenous appli-
cation of the luciferin substrate to a phage-
infected culture to bring about light emission.
The rst use of rey luciferase for
phage-based bacterial targeting was reported
for detection and drug susceptibility
determin ation of M. smegmatis and Myco-
bacterium tuberculosis (Jacobs et al., 1993).
Recombinant mycobacteriophages carrying
the luc gene allowed the detection of
5 10
2
5 10

CFU ml
1
of M. smegmatis within
minutes of infection and were capable of
distinguishing between antibiotic-resistant
and -susceptible M. smegmatis and M.
tuberculosis. Due to the inherently slow growth
rate of mycobacteria, however, lengthy pre-
enrichment was required to obtain su cient
bacterial concentrations for detection, result-
ing in total analysis times in excess of 9 days.
Since then, several other mycobacterio phage
constructs have been engineered to exploit
luc-based bio luminescence with the aim of
increasing assay sensitivity and reducing
analysis time (Sarkis et al., 1995; Bardarov et
al., 2003; Dusthackeer et al., 2008). While these
studies show promise for myco bacterial
detection, they are still hampered by extensive
pre-enrichment requirements, mak ing it un-
likely that these systems will be tractable for
rapid bacterial detection and identication in
a truly rapid diagnostic capacity.
Fluorescent protein-based reporter
phages
GFP and its many derivatives are used in
numerous applications and have been
expressed in organisms ranging from bacteria
and nematodes (Chale et al., 1994), insects
(Wang and Hazelrigg, 1994), yeast (Kahana et
al., 1995) and plants (Haselo and Amos,
1995) to mammalian cell cultures (Olson et al.,
1995). Originally isolated from the bio-
luminescent hydrozoan jellysh Aequorea
victoria (Shimomura et al., 1962), a plethora of
commercial GFP-based cloning and expres-
sion tools are available from multiple
manufacturers and include enhanced green,
red, cyan, yellow and blue-shifed variants.
While dependent on the presence of oxygen
(Heim et al., 1994) and ofen hampered by low
signal-to-noise ratios because of bacterial,
tissue or other sample autouorescence,
the advantages oered by GFP-based
uorescence include that it does not require
the addition of an enzyme substrate, is
relatively resistant to photobleaching in
comparison with other uorophores and
produces a signal over a fairly wide emission
timeframe.
The rst use of GFP-encoding phages for
bacterial detection involved cloning GFP into
the phage genome followed by infection,
bacterial expression and epiuorescence
microscopy-based detection of E. coli
(Funatsu et al., 2002). This strategy allowed
detection of GFP-expressing bacteria within
4 h of infection but suered from reduced
sensitivity as a result of phage-mediated cell
lysis, which resulted in dispersal of the GFP-
based signal. Several studies aimed at
addressing this and improving the target host
range followed. One strategy centred on the
use of a GFP-encoding, lysozyme-decient,
broad host range coliphage T4 mutant
designed to prevent host-cell destruction
(Tanji et al., 2004). Using similar approaches,
lytic (Oda et al., 2004) and lysozyme-decient
(Awais et al., 2006) GFP-encoding PP01
mutants were independently engineered.
These iterations allowed rapid, highly specic
detection and dierentiation between
O157:H7 and K12 strains within 10 min of
infection of 24 h cultures. Interestingly, unlike
many methods that require living target cells
for phage infection, these approaches,
because they relied upon expression of a
GFPminor capsid protein fusion (an
externally GFP-tagged phage), allowed the
detection of viable but non-culturable, as well
as heat-killed (pasteurized), E. coli by way of
phage adsorption. It should be noted,
however, that viable cells gave the most
intense uorescent signal.
Recently, GFP and yellow uorescence
protein (YFP)-encoding mycobacteriophages
were reported for uorescence-based
detection and antibiotic resistance determin-
ation of M. tuberculosis and M. smegmatis (Piuri
et al., 2009; Rondon et al., 2011). Using
epiuorescence microscopy and ow cyto-
metry, these techniques were capable of
detecting approximately 100 CFU of phage-
infected, GFP-expressing mycobacteria. Total
analysis time, including bacterial pre-en-
richment was similar to that observed for luc-
based mycobacterial detection, requiring
weeks to obtain su cient optical densities for
experimentation. Fluorescence was observ-
able within 4 h of phage infection, however,
which is a signicant improvement over con-
ventional, culture-based detection methods
commonly employed by clinical laboratories.
The use of phage-mediated uorescence for
antibiotic resistance determination of
established liquid cultures required 2448 h,
while the same analysis conducted on
mycobacteria derived from single colonies
required 23 weeks.
Phage-conjugated
Quantum Dots
Quantum dots (QDs) are colloidal, semi-
conducting nanocrystals with electronic
characteristics, such as uorescent emission
wavelengths, that are closely related to and
can be controlled by their crystal diameter
(size tunability; Murray et al., 2000). With
respect to their applicability to imaging and
detection strategies, they exhibit superior
photostability in comparison with con-
ventional uorophores, are more reactive
across a broad spectrum of excitation wave-
lengths and uoresce at narrow, size-
dependent emission spectra. These atributes,
in combination with recent advances in
surface conjugation chemistry, make QDs
ideal candidates as probes for imaging of
biological systems and bacterial detection
(Dubertret et al., 2002; Hahn et al., 2005; Li et
al., 2007). Moreover, the tunability of QDs
over a wide emission bandwidth makes
multiplexed detection of numerous targets in
a single sample a favourable possibility.
The use of QDs in combination with
species-specic phages was rst explored
using E. coli and biotinylated coliphage T7
(Edgar et al., 2006). T7 was genetically
engineered to express a small peptide fused
to its major capsid protein, which, following
infection and production of progeny phages,
was post-translationally biotinylated by host
cell biotin ligases. Once released from host
cells, biotinylated progeny phages were then
tagged by the addition of streptavidin-
conjugated QDs. PhageQD complexes
extracted from mixed bacterial cultures were
then visualized by ow cytometry and
uorescence microscopy 1 h afer infection
with detection limits of around 10 CFU ml
1
.
A similar approach used biotinylatable phage
and QDs in combination with conventional
and image-based ow cytometry to achieve
phage-based signal quantication (Yim et al.,
2009).
Fluorescent Phage Endolysin Cell
Wall-binding Domains
Phage-encoded endolysins are peptidoglycan
hydrolases that serve as one component of
the phage lytic system (Fisheti, 2005). They
are produced in the host-cell cytoplasm
during phage infection but target the host
cell-wall peptidoglycan, resulting in weaken-
ing and rupture of the bacterial cell along
with subsequent release of phage progeny.
Structurally, endolysins are com posed of an
enzymatically active N-terminal domain and
a cell wall-binding C-terminal domain (CBD)
(Moak and Molineux, 2004). To date,
numerous endolysins have been sequenced,
puried and applied from without to destroy
bacteria and explore their use as potential
alternatives to antibiotics (Loe er et al., 2001;
Nelson et al., 2001; Schuch et al., 2002; see also
Shen et al., Chapter 15, this volume). Their
use is largely limited to detecting Gram-
positive bacteria, however, because the
Gram-negative outer membrane serves as a
protective barrier against exogenously
applied endolysins. Nevertheless, endolysins
are potentially powerful tools, not only for
146 C.R. Cox
biocontrol but also for highly specic
detection.
In one of the most recent of the new
approaches to phage-based bacterial de-
tection, phage endolysin CBDs were paired
with green, blue, cyan, yellow and red
uorescent reporter proteins, magnetic
separation and uorescence microscopy to
allow the capture and detection of Listeria
(Schmelcher et al., 2010). Using several
dierent CBDuorescent protein fusion-
coated magnetic particles for bacterial
separation, multiplexed detection of Listeria
strains in mixed cultures as well as in spiked
milk and cheese samples were successfully
carried out. Bacterial recovery rates of greater
than 90% were observed following 24 h of
enrichment and 16 h of selective plating of
the captured cells.
Conclusions
Despite the growing need for rapid,
inexpensive methods of bacterial detection
and identication, most current methods fall
short as user-friendly, point-of-care diag-
nostics due to their complexity, lack of
sensitivity and reliance on expensive
equipment and highly specialized facilities
and expertise, as well as their dependence
on time-consuming micro biological pre-
enrichment. By exploiting the highly species-
specic phage life cycle and accompanying
amplication, the adsorption or atachment
a nity for their bacterial hosts, their ability
to take up and transfer reporter genes and
display surface-expressed mol ecular tags,
phage-based detection methods, depending
on the application, bypass nearly all of
these pitfalls. Phage amplication-coupled
MALDI-TOF MS, for example, allows
instrumental advances in bacterial detection
via rapid, species-specic phage protein
proling. This novel signal amplication
method allows bacterial detection and
identi cation in as litle as 2 h, while lowering
the limits of detection by several orders of
magnitude over conventional spectroscopy
and phage-typing methods.
With the goal of bypassing the need for
complex instrumentation and highly trained
personnel, methods for the use of phage
amplication-coupled LFI provide a com-
bination of both rapid and sensitive bacterial
detection in an inexpensive, portable
conguration, which is ideal for point-of-care
applications. The rst of such technologies is
the recently commercialized Microphage
KeyPath
TM
system, which takes advantage of
phage amplication for patient screening for
MRSA and provides a rapid, sensitive method
for point-of-care diagnostic bacterial identi-
cation in a fraction of the time required by
conventional laboratory-based techniques.
Newly rened phage immobilization
techniques continue to advance our ability
to quickly and specically isolate and
con centrate target bacteria for analysis
with existing detection instrumentation.
Fluorescence-based bacterial detection by
targeted infection and delivery of uorescent
reporter systems by recombinant phages
show great promise as multiplexed bio-
luminescent and uorescent detection tools,
and the emerging use of highly rened phage
endolysins show strong potential in high-
e ciency bacterial capture and identication.
In all these approaches, phage-based
bacterial detection methods have proven to
be equal, if not superior, to conventional
methods in every category and stand to
transform diagnostic microbiology in the
very near future.
Acknowledgements
Portions of this chapter describing work
conducted by the author were supported by
the Defense Threat Reduction Agency of the
United States Department of Defense.
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11 Phage Detection as an Indication of
Faecal Contamination
Lawrence D. Goodridge
1
and Travis Steiner
1
1
Department of Animal Sciences, Colorado State University.
The importance of water quality to public
health has been demonstrated for thousands
of years, as archaeological evidence of safe
water usage and sanitary practices have been
found from the Incas to the Romans (Rosen,
1993). Most current issues with water quality
primarily impact on developing countries
where there is a lack of adequate sanitation
and treatment facilities (Carr, 2001). For
example, it is estimated that there are approxi-
mately 4 billion global cases of diarrhoea
annually (Carr, 2001), and inadequate water
supply, sanitation and domestic hygiene
account for 2.2 million deaths annually (Prss
and Havelaar, 2001).
While inadequate water quality is a
major problem in developing countries,
incidences of water contamination still
remain a problem in many developed
countries. For example, an outbreak in 1988
in Sweden aected 11,000 people when a
chlorination failure occurred at a water-
treatment plant (Andersson, 1991), and 2300
individuals in Walkerton, Ontario, Canada,
were aected by a waterborne outbreak of
Escherichia coli O157 when ex posed to
contaminated drinking water from a well that
had most likely been contaminated by surface
runo water (Hrudey et al., 2003). Out of the
2300 aected persons in the Walkerton
outbreak, 65 people were hospitalized, with
27 of these developing haemolyticuraemic
syndrome and seven conrmed deaths. In the
USA, a major outbreak of cryptosporidiosis
in Milwaukee, WI, caused an estimated
403,000 people to become ill with 4400
hospitalizations (Corso et al., 2003). For more
on E. coli O157:H7, see Kuhl et al., Christie et
al., Williams and LeJeune, Goodridge and
Steiner, and Niu et al. (Chapters 3, 4, 6, 11 and
16, this volume).
Such outbreaks not only aect the health
of the citizens but also have a severe economic
impact within the community. It is estimated
that the 1993 cryptosporidiosis outbreak had
a nancial cost of nearly US$100 million
when taking into account medical costs and
productivity losses (Corso et al., 2003). These
examples of waterborne illnesses highlight
the need for rapid, sensitive and inexpensive
methods to assess water quality to mitigate
such costly outbreaks of waterborne disease
in developing and developed countries. The
quality of drinking water, wastewater
treatment and recreational-use waters must
constantly be assessed due to their potential
to serve as a vehicle for spreading disease. In
some instances, untreated wastewater is used
for the irrigation of crops, creating a potential
for widespread illness (Ensink et al., 2002;
Sears et al., 1984; Srikanth and Naik, 2004).
Water used in agriculture and food processing
L.D. Goodridge and T. Steiner
154 L.D. Goodridge and T. Steiner
can thus serve as a source for transmission of
pathogens, becoming important from a food-
safety standpoint (see Niu et al., Chapter 16,
this volume).
Assessing water quality as a consequence
of these various issues can be crucial from a
public health perspective. A number of
methods for such assessment exist and can be
dierentiated into chemical, physical or
biological approaches (as well as their use in
combination). The purpose of this chapter,
however, is to describe the scientic literature
surrounding the use of coliphages and phages
that infect other bacterial species as indicators
of water quality. For additional reading, there
are a number of earlier reviews of phage use
as faecal indicators of water quality (Leclerc
et al., 2000; Ashbolt et al., 2001; Gerba, 2006;
Pillai, 2006; see also Cox, Chapter 10, this
volume). We begin with an overview of the
concept of the use of indicator organisms in
general for assessment of water quality.
Assessing Water Quality Using
Indicator Organisms
Microbial water quality currently is deter-
mined by testing for the presence of indicator
organisms (Yates, 2007). The detection,
isolation and identication of waterborne
pathogens can be expensive, di cult and
labour-intensive (Scot et al., 2002). To
alleviate these issues with waterborne
pathogen testing, indicator microorganisms
(organisms used to determine the presence of
faecal pollution) and index microorganisms
(organisms used to indicate the presence
of specic groups of pathogenic micro-
organisms) are commonly used to determine
the possible presence of faecal pollution and
thereby the relative risk, as well as, in some
cases, the actual pathogenic microorganisms
found in a sample. As most of the microbial
pathogens present in water are of faecal
origin, the detection of faecal contamination
has been the main aim of testing methodo-
logies. Historically, coliforms, the thermo-
tolerant coliform group, enterococci and
Clostridium perfringens have been the bacterial
indicators used to detect faecal contamination
(Scot et al., 2002; Biton, 2005; Savichtcheva
and Okabe, 2006; Yates, 2007), based on the
rationale that these indicator organisms are
indigenous to faeces and that their presence
in the environment is therefore indicative of
faecal pollution. Testing for these micro-
organisms in particular is necessary because
testing for the numerous bacterial, viral and
parasitical pathogens that can cause water-
borne illness is generally considered to be
impractical, particularly as doing so can
require extensive laboratory knowledge,
specialized equipment and a large quantity of
time and other resources (Havelaar and
Hogeboom, 1983; Yates, 2007).
In addition to testing for the presence of
gross faecal pollution, it is also useful to
develop methodology that can evaluate the
possible presence of the pathogenic micro-
organisms themselves. Cabelli (1977) dened
the basic criteria for a good index organism of
water quality as the following: (i) the
organism should be present when the
pathogen is present and absent when the
pathogen is not present; (ii) the organism
must not proliferate in the environment; (iii)
the organism should be present in numbers
equal to or greater than the pathogens it
indicates; (iv) the organism should be as
resistant to environmental decay and water-
treatment strategies as the pathogens it
indicates; and (v) the organism should be
both easily assayed and non-pathogenic. In
addition to these criteria, the ideal indicator/
index organism would also be able to
discriminate between contamination sources
(animal or human), also known as microbial
source tracking, and would have the ability to
predict the presence of waterborne viral
pathogens (Scot et al., 2002; Long et al., 2005;
Savichtcheva and Okabe, 2006).
The US Environmental Protection Agency
(EPA) establishes maximum contaminant
levels (MCL) for total coliforms in drinking
water based on the number of samples taken
per month, which corresponds to the size of
the population for which the water source
serves (EPA, 1989). If samples are positive for
total coliforms, then further analyses for faecal
coliforms or E. coli are required. The MCL for
drinking water, water that has been treated
and will be released into a distribution system,
and water that is currently in the distribution
Phage Detection as an Indication of Faecal Contamination 155
system are all set at no detectable thermo-
tolerant coliform or E. coli bacteria (EPA, 1989;
World Health Organization, 2006). The MCL
standard for recreational water is much more
liberal and utilizes E. coli and enterococci as
indicators. The MCL standards for recreational
waters are set at 126 colony forming units
(CFU) per 100 ml for E. coli and 35 CFU for
enterococci (EPA, 1986). These criteria are
based on the rationale that the absence of
faecal coliforms, E. coli and enterococci
indicate that the waters sampled are at a low
risk of harbouring pathogens originating
from faecal con tamination (EPA, 1989). Many
of these bacteria, however, are routinely
isolated from soil and water environments
that have not been impacted by faecal
pollution. For example, faecal coliforms and
E. coli have been known to survive and
propagate within pristine and tropical
environments, meaning that assessment of
their initial numbers can be exaggerated
(Hurst and Crawford, 2002). Additionally, the
microbiological relation ship between index
microorganisms and the full range of
pathogens they are intended to represent is
not clearly understood, nor are the dynamics
of microbial ecology expected to be globally
homogeneous (National Research Council,
2004).
There are other di culties in using these
bacteria as markers of faecal contamination.
The bacteria are able to grow in biolms
within drinking-water distribution systems.
In addition, they are occasionally absent in
water supplies during outbreaks of water-
borne disease. Also, while the persistence of
these bacteria in water-distribution systems is
comparable to that of some bacterial
pathogens, the relationship between bacterial
index organisms and the presence of enteric
viruses and protozoa is poor, which is
important, as viruses and protozoa account
for approximately 44% of waterborne
outbreaks in the USA where the aetiological
agent has been identied (Blackburn et al.,
2004). Finally, the methods used to detect the
indicators/index bacteria are problematic.
While there are established culture and
molecular methods for the detection of most
microbial pathogens, most of these methods
have important limitations, including the
length of time required for the test result
(15 days) and the specicity and sensitivity
of detection (Scot et al., 2002). Another issue
to consider is the fact that the bacterial
indicators described above are not suited to
tracking the source of faecal pollution when a
contamination event is discovered. Microbial
source tracking is extremely important as it
identies the source of the pollution, which
enables containment and a decrease in the
chance of waterborne disease outbreaks.
Due to the limitations of the bacterial
indicators, including problems with their
rapid detection, it is clear that there remains
an acute need to identify beter indicators of
microbial quality that would determine faecal
contamination in a rapid manner and also
determine the source of that pollution so that
corrective actions could be initiated. In an
eort to nd indicators and index organisms
that t the criteria previously described,
novel alternatives to total coliform and E. coli
as well as Enterococcus spp. have been
explored. Bacteriophages as indicators of
water quality, and as index organisms for
enteric viruses, have been proposed as a
substitute for bacteria.
Bacteriophages as Indicator and
Index Microorganisms
Bacteriophages were recognized as being
present in the intestinal tract of humans in the
early 1900s (dHrelle, 1926). The use of
phages to indicate the possible presence of
pathogenic enteric bacteria was an idea that
subsequently developed in the 1930s, and
direct correlations between the presence of
certain types of bacteriophages and the
presence of faecal contamination were
reported (Scarpino, 1978).
More recently, the tendency has been to
use phages as indexes to demonstrate the
presence of and model the survival of enteric
viruses as a group. As with bacterial
pathogens, the approach to monitoring for
enteric viruses is based on the idea that it is
beter to monitor for the presence of faecal
pollution than for specic pathogens, due to
the fact that, as with bacteria, there are still
unknown (and simply too many) enteric viral
pathogens to detect individually. In addition,
enteric viruses are ofen present in low
concentrations, requiring the analysis of at
least 10 litres of water, and while diagnostic
methods have been proposed for many of the
known enteric viruses, such methodology
is labour-intensive, expensive and time-
consuming. Due to their similarities to enteric
viruses, certain phages are atractive
candidates as indexes of enteric viruses in
water.
The similarities between phages and
enteric viruses begin with the simple fact that
phages are viruses themselves. Bacteriophage
assay conditions, however, are much simpler
and cheaper than any of the enteric virus
detection methods. For example, phages can
be identied and quantied using several
methods ranging from standard micro-
biological methods such as plating to more
complex molecular methods based on genus-
or species-specic antigenic properties and
genetic identication (Metcalf et al., 1995;
Vinje et al., 2004; Kirs and Smith, 2007). All
these methods of detection can suer the
same limitations as bacterial detection, in
some cases requiring even more laboratory
expertise. Some phages are so similar in
genetic structure that identication by
antigenic activity against their capsid protein
is the most eective method of discrimination.
These antigenic tests can be performed using
phage inactivation, lateral ow testing or latex
agglutination (Love and Sobsey, 2007; see
Cox, Chapter 10, this volume, for additional
discussion of phage-detection methodologies).
As with bacterial indexes of faecal-borne
bacterial pathogens, to be suitable sentinels of
enteric viruses, phages should occur
consistently and exclusively in human and
animal faeces and sewage, should not
multiply in the environment and should be
present in greater numbers than the enteric
viruses. Additionally, they should be at least
as long-lived as the enteric viruses present in
the environment, and their survival kinetics
throughout the water-treatment process
should be similar to that of enteric viruses
(Keswick et al., 1984; Payment and Franco,
1993; Hurst et al., 1994).
Three groups of phages have been
proposed as indicators of faecal pollution, and
candidate index microorganisms for the
enteric viruses including somatic coliphages
(Kot, 1966; Hilton and Stotzky, 1973; Kot et
al., 1974; IAWPRC, 1991), male-specic
coliphages (Havelaar and Hogeboom, 1984;
IAWPRC, 1991) and phages infecting
Bacteroides fragilis (Jofre et al., 1986; Tartera and
Jofre, 1987; IAWPRC, 1991; Armon and Kot,
1993; Grabow et al., 1995; Gantzer et al., 1998).
Somatic Coliphages
The somatic coliphage group includes all
phages that require the presence of a receptor-
binding protein for infection of their host E.
coli and comprise bacteriophages from the
families Myoviridae, Siphoviridae, Podoviridae
and Microviridae (Muniesa et al., 2003). These
phages recognize outer-membrane proteins
(OMPs) such as OMP C, OMP F and OMP K,
or select lipopolysaccharide and sites within
the O side chain as receptors for infection
(Linberg, 1973). Somatic coliphages have
been proposed as indicators of faecal
contamination and guidelines currently exist
for their detection and analysis via enrichment
and plating methodologies (USEPA, 2001).
The degradation of somatic coliphage
genomes has been reported to be similar to
that of enteric viruses, demonstrating that
these phages can be used as reliable indexes
of enteric virus presence when molecular
methods are used to detect them (Skraber et
al., 2004). For example, Hot et al. (2003)
studied whether the concentrations of
somatic coliphages and infectious enteric
viruses or the detection of enteric virus
genomes were associated with the detection
of human pathogenic viruses in surface
water. The researchers tested water samples
for the presence of somatic coliphages, and
any water samples that contained phages
were subsequently tested for the enteric
viruses. Of the 68 surface-water samples
positive for somatic coliphages, only two
were positive for enteric viruses when
detected by culturable methods, while 60
samples were positive for the enteric viruses
when tested using RT-PCR.
Kot et al. (1969, 1974, 1978) were among
the rst researchers to investigate whether
coliphages were valid indexes of enteric viral
pollution and showed that the phages were
present in wastewater and other faecally
contaminated waters in numbers at least
equal to the enteric viruses. Somatic phages
also persist in wastewater and surface waters
for longer time periods that enteroviruses
(Kot et al., 1969, 1974, 1978) and are detectable
using routine methodologies (Grabow et al.,
1978). Somatic coliphages have been detected
in sewage-contaminated waters but were not
found in pristine waters (Toranzos et al.,
1988), and Suan and colleagues (1988)
observed somatic coliphages to be highly
correlated with faecal coliforms in tropical
waters.
Justication for the use of coliphages as
indicators in wastewater and other faecally
contaminated waters has been reviewed by
Gerba (1987), the International Association on
Water Pollution Research and Control
(IAWPRC) Study Group on Health Related
Water Microbiology (IAWPRC, 1991) and
Limsawat and Ohgaki (1997). Nevertheless,
detractors of the use of somatic coliphages as
indicators of faecal contamination cite data
showing that propagation within the
environment could occur whenever a host
bacterium is present, causing their numbers
to be over-represented, and somatic
coliphages have been reported to replicate at
temperatures as low as 15C (Seeley and
Primrose, 1980). Further research, however,
has shown that optimal conditions required
for replication of somatic coliphages in the
environment are unlikely to occur, as
considered by Muniesa and Jofre (2004).
Muniesa and Jofre and colleagues
studied environmental bacterial host strains
and found that, although environmental
somatic coliphages could be propagated on
laboratory E. coli strains WG5 and CN13,
bacterial strains isolated from the environ-
ment were not susceptible to these coliphages
(Muniesa et al., 2003; Muniesa and Jofre,
2004). These results suggested that environ-
mental replication of somatic coliphages is
not a signicant problem and therefore
would not cause over-representation of
somatic coliphages.
Other potential limitations exist that are
relevant to the use of somatic coliphages as
faecal indicators, including that they are not
specic to E. coli. For instance, there is
evidence that somatic coliphages may
multiply in other species of Enterobacteriaceae,
which are part of the total coliform group and
ofen found associated with vegetation and
biolms. Of these, the two most common
species are Klebsiella pneumoniae and
Enterobacter cloacae. It is therefore possible
that some coliphages might be produced that
are not only unrelated to faecal contamination
but, indeed, are unrelated to any health risk.
An additional issue is that there are
many non-faecal sources of coliform bacteria.
Accordingly, it is di cult to ascertain
specically whether an isolate of a somatic
coliphage arose from any one point of
contamination. Coliforms that can colonize
biolms may be present anywhere in the
water collection treatment or distribution
system. Furthermore, studies by Vaughn and
Metcalf (1975) on coliphages and enteric
viruses in sewage e uents, shellsh and
shellsh-growing waters showed that coli-
phages are not adequate index micro-
organisms of enteric viruses. This was due to
the replication of coliphages, the presence of
more than one dominant phage type in
estuarine and fresh waters making the results
of a test for any one phage type not denitive
(Vaughn and Metcalf, 1975; Seeley and
Primrose, 1980; Parry et al., 1981; Borrego et
al., 1990) and the lack of any correlation
between the densities of coliphages and
enteroviruses in raw sewage and farm pond
water (Joyce and Weiser, 1967; Saerman and
Morris, 1976; Havelaar, 1987; Nieuwstadt et
al., 1991; Wommack et al., 1996). In addition,
inconsistent occurrence of coliphages in raw
sewage samples with the simultaneous
isolation of enteroviruses has been observed,
while other studies have reported a high
number of enteroviruses isolated in many
treated e uents with no coliphages detected
(Vaughn and Metcalf, 1975).
Male-specic Coliphages
Male-specic coliphages from the families
Inoviridae and Leviviridae are dened by their
requirement for the expression of F pili on
their host bacteria for successful infection
(Long and Sobsey, 2004). F pili are typically
expressed from a plasmid and are used by
bacteria for the exchange of genetic material
from one F
+
or male/donor bacterium to an F

or female/recipient bacterium during
conjugation (Novotny et al., 1969). The
expression of this appendage is regulated by
several factors including existence of the F
plasmid and temperature. F pili have been
shown to be expressed at the highest numbers
on the cell surface within a temperature range
of 3742C, with expression non-existent at
25C and below (Novotny and Lavin, 1971).
The specic temperature range at which the F
pili are expressed is one of the arguments for
the use of these phages as indicators, because
normal environmental temperatures do not
support the expression of pili, thereby
limiting phage propagation (Havelaar et al.,
1986).
Male-specic DNA coliphages
Male-specic coliphages are classied accord-
ing to their nucleic acid type and divided into
subgroups according to their morphology
and serological properties (Long and Sobsey,
2004). Male-specic DNA (FDNA) coliphages
from the family Inoviridae are unique in their
morphology among phages in that they do
not have a head capsid but rather are thin,
lamentous structures that contain single-
stranded DNA (Rasched and Oberer, 1986).
The lamentous structures range from
approximately 760 to 1950 nm in length and
are approximately 6 nm wide. Their use as
indicators of faecal contamination has been
widely disputed as they are not as well
characterized as the male-specic RNA
(FRNA) coliphages and do not have
morphologies or environmental stability
similar to the pathogens they are intended to
indicate. In addition, a link between FDNA
phages and their sanitary signicance has not
been shown (Scot et al., 2002; Sinton et al.,
1996). A study by Long (1998), however,
indicated that these phages were present
during times when FRNA phages were
absent, and the author proposed the use of
both FRNA and FDNA phages as possible
water-quality indicators during dierent
seasons of the year.
The predominating FDNA coliphages
have received less atention as index
organisms of enteric viruses because they are
generally less plentiful than FRNA coliphages
(Leclerc et al., 2000), they do not resemble
human enteric viruses morphologically and
their ecology is poorly understood (Leclerc et
al., 2000). FDNA phages have been assayed
utilizing plaque assays and membrane
ltration and elution methods (Sinton et al.,
1996). As FDNA and FRNA utilize the same
pili-expressing host, the standard method for
identifying FDNA phages involves using
medium that contains RNase to inactivate
FRNA coliphages (Cole et al., 2003).
Male-specic RNA coliphages
FRNA phages have received the most
atention as indicators of faecal contamination
and especially as index microorganisms. This
prominence is due to the fact that many
studies have conrmed that, for monitoring
purposes, FRNA phages are reliable
determin ants of the possible presence of
human enteric viruses, as they behave like
waterborne viruses (Havelaar et al., 1993). As
noted, FRNA phages enter the host cell by
adsorption to F pili and are a more
homogeneous group than other micro-
organisms (Seeley and Primrose, 1980). Also
as noted, F pili are only produced by E. coli
cells at temperatures above 25C, meaning
that the phages would be unable to multiply
in most environments. Therefore, detection of
these phages indicates a recent contamination
event.
FRNA phages have also atracted interest
as useful alternatives to bacterial indexes
because their morphology and survival
characteristics closely resemble the human
enteric gastrointestinal viruses (Scot et al.,
2002), meaning that, in addition to the
usefulness of these phages as indicators of the
presence of their host bacteria (E. coli) and
therefore the presence of faecal pollution,
they could also be used as surrogate markers
for the presence of enteric viruses such as
noroviruses or rotaviruses in water. They are
also similar to enteric viruses with respect to
inactivation kinetics following exposure to
environmental factors and water treatments
(Allwood et al., 2003; Grabow, 2001). FRNA
coliphage virions are approximately 26 nm in
diameter and their capsids exhibit an
icosahedral symmetry that consists of 180
copies of a dimer coat protein containing one
copy of positive-sense, single-stranded RNA
of between 3500 and 4200 nt (Bollback and
Huelsenbeck, 2001; Stewart et al., 2006).
FRNA phages are grouped into two distinct
genera that are dened by their genomic
organization. FRNA phages from the genus
Levivirus contain a gene locus encoding a
lysis protein. Phages from the genus
Allolevivirus contain a gene with a leaky stop
codon that becomes a read-through
approximately 5% of the time (Bollback and
Huelsenbeck, 2001; Stewart et al., 2006),
encoding an extended coat protein. Unlike
phages from the genus Levivirus, Allolevivirus
phages do not produce a protein dedicated to
host-cell lysis (Bollback and Huelsenbeck,
2001; Stewart et al., 2006).
Both FRNA phage genera are further
classied into four individual serogroups (I
IV) based on serological as well as genetic
and physiochemical properties (Havelaar
and Hogeboom, 1984). They can be identied
via inactivation by antiserum, RT-PCR, latex
agglutination and nucleic acid hybridization
(Furuse et al., 1978; Vinje et al., 2004; Kirs &
Smith, 2007; Love and Sobsey, 2007; Friedman
et al., 2009). Of these four groups, groups I
and IV have been associated primarily with
animal faecal sources, while groups II and III
have been demonstrated to be associated
primarily with faecal material from human
faecal sources (Havelaar et al., 1990; Fig. 11.1).
This trait makes the FRNA coliphages
atractive as indicators of faecal pollution as
well as useful for microbial source tracking
analysis.
Serotyping of FRNA phages has been
suggested as a method to determine the
origin of faecal pollution (Furuse et al., 1978).
In this work, the authors produced antisera
against type phages from each of the four
groups, including MS2 (group I), JP34 (group
II), GA (group II), Q/ (group III), VK (group
III) and SP (group IV). Unknown phages
were incubated with antisera from each
group and then grown on their respective E.
coli host strain. If the phage was neutralized,
then it belonged to the same group as the
antisera and the phage failed to form plaques.
In this way, the authors were able to determine
that, of 52 phages that were serotyped, 46
belonged to group III (Furuse et al., 1978). The
serotyping method can be used in a source
tracking scheme to discover the origin of
faecal pollution. The serotyping method as
described by Furuse et al. (1978), however, is
Leviviridae
Levivirus Allolevivirus
I II III IV
MS2, f2, fr
M12, R17
GA, JP34,
KU1, TH1
Q, M11, ST
TW18, VK
SP, F1, ID2
NL95, TW28
Cows, dogs,
elephants,
foxes, horses,
pigs, sheep
Humans,
pigs
Humans Birds, chickens,
pigs, tigers, apes
Family
Genera
Subgroup
Representative
phages
Sources
Fig. 11.1. Taxonomy of the FRNA phages. Phages in bold represent the type phage for each particular
serotype or genogroup. Adapted from Smith (2006).
tedious, requires standard laboratory equip-
ment (e.g. Petri plates, incubators) and takes
at least 24 h to complete (Hsu et al., 1995).
Hsu et al. (1995) showed that highly
specic nucleic acid probes could be used to
genotype FRNA phages as an alternative to
serotyping. In this work, the authors
developed oligonucleotide probes that were
specic to each of the four serogroups and
used these probes to genotype unknown
phages. The genotyping method showed
excellent agreement with the serotyping
method (P<0.05) and the authors concluded
that genotyping FRNA coliphages appears to
be practical and reliable for typing unknown
phage isolates in eld samples. As with the
serotyping method, genotyping has been
demonstrated as a useful approach to source
track the origin of faecal pollution in water
samples. Using the genotyping method,
Schaper et al. (2002) conrmed that genotypes
II and III predominated in municipal sewage,
illustrating that these genotypes were of
human origin. Nevertheless, the genotyping
method is laborious, time-consuming and
requires extensive operator training. For
example, the genotyping method as described
by Hsu et al. (1995) includes isolating the
phage from a water sample, conducting a
plaque assay, obtaining a well-isolated plaque
and transferring it to a membrane. Next, the
membrane-bound phage are denatured,
followed by xation of the released nucleic
acid on to the membrane with UV light.
Finally, the genotype-specic probes are
added, allowed to hybridize with the phage
nucleic acid and detected colorimetrically
(Hsu et al., 1995).
Dor et al. (2000) investigated using
FRNA phage to indicate the likely presence of
enteric viruses in shellsh sold for con-
sumption. FRNA phage and E. coli levels were
determined over a 2-year period for oysters
harvested from four commercial sites chosen
to represent various degrees of sewage
pol lution. Three sites required cleaning
(depuration) of the oysters before sale. Oysters
from the fourth site could be sold directly
without further processing. Samples were
tested at the point of sale following commercial
processing and packag ing. All of the shellsh
complied with the mandatory European
Community E. coli standard (less than 230
CFU per 100 g of shellsh esh), and the levels
of contamination for more than 90% of the
shellsh were at or below the level of
sensitivity of the assay (20 E. coli CFU per
100 g), which indicated good quality based on
this criterion. In contrast, FRNA phage were
frequently detected at levels that exceeded 10
3

plaque-forming units (PFU) per 100 g. High
levels of FRNA phage contamination were
strongly associated with harvest area faecal
pollution and with shellsh-associated
disease outbreaks. Interestingly, FRNA phage
contamination exhibited a marked seasonal
trend that was consistent with the trend of
oyster-associated gastroenteritis in the UK.
The correlation between FRNA phage
contamination and health risk was investi-
gated further using a RT-PCR assay for
Norwalk-like virus. Norwalk-like virus
contamination of oysters was detected only at
the most polluted site and also exhibited a
seasonal trend that was consistent with the
trend of FRNA phage contamination and with
the incidence of disease. The authors
concluded that the results of this study
suggested that FRNA phages could be used as
viral index organisms for market-ready
oysters (Dor et al., 2000).
Other studies have corroborated the
nding that seasonal uctuations in FRNA
concentration mirror that of the Norwalk-like
viruses. Dor et al. (2003) collected a total of
608 shellsh samples from 49 shellsh-
harvesting areas and tested them for the
presence of E. coli and FRNA phage. The
results indicated that FRNA phage
concentration in all samples was more than
three times greater than that of E. coli
(geometric mean counts of 1800 and 538 per
100 g for FRNA phage and E. coli, respectively).
In addition, FRNA phage concentrations
were strongly inuenced by season, with a
geometric mean count of 4503 PFU per 100 g
in the winter (OctoberMarch) compared
with 910 PFU per 100 g in the summer (April
September). The elevated levels of FRNA
phages observed in the winter concur with
the known increased virus risk associated
with shellsh harvested at this time of year in
the UK. The authors suggested that data from
this study could be employed to introduce
FRNA phages as an index of the virus risk
associated with shellsh (Dor et al., 2003).
The concentrations at which these
indicators exist in the environment are fairly
low at about <10
4
PFU ml
1
(Furuse et al., 1983;
Havelaar et al., 1986). To remedy this problem,
the phages can be concentrated using
charged membranes and elution. Alterna-
tively, enrich ment is possible for certain
phages using the desired host bacterium. The
later is typically required to achieve numbers
high enough for detection (Love and Sobsey,
2007). Furthermore, it has been shown that
FRNA phages are not present in the faeces of
all animals and have been reported to be
isolated in the faeces of only about 3% of the
human population (Havelaar et al., 1990).
This would suggest that the use of these
coliphages as useful indicators of faecal
contamination may be limited to water
contamination from pooled sources such as
sewage, septic tanks or agricultural runo
water.
Assaying for male-specic coliphages
One problem encountered when assaying for
male-specic coliphages is the possibility of
also detecting somatic coliphages. This is due
to the existence of somatic receptors on the
cellular surface of some of the host strains
used to enumerate male-specic coliphages
in water samples (Havelaar and Hogeboom,
1983; Hsu et al., 1996; Rhodes and Kator,
1991). The male-specic coliphage host E. coli
F
amp
is susceptible to infection by FDNA
phages, but this may not prove to be a
detriment until the sanitary signicance of
FDNA phages is beter understood (Hsu et al.,
1996; Sinton et al., 1996). One route towards
addressing these concerns is to use host
strains besides E. coli F
amp
that contain the F
plasmid. Salmonella enterica subsp. enterica
serovar Typhimurium WG49 and E. coli
C3000 have both been proposed as candidates
for detection of FRNA phages (Leclerc et al.,
2000). The use of these bacterial host strains is
accompanied by its own set of problems,
however. For example, while E. coli C3000
produces the required F pili, the bacterium is
not antibiotic resistant and is susceptible to
infection by both somatic coliphages and
male-specic DNA phages (Hsu et al., 1996).
Furthermore, while the use of Salmonella
Typhimurium WG49 solves the problem of
atack by somatic coliphages, because, as a
Salmonella species, it lacks the receptors
required for somatic coliphage infection
(Havelaar and Hogeboom, 1983), the strain is
still susceptible to atack by somatic Salmonella
phages (Stetler and Williams, 1996).
Real-time PCR is a sensitive, rapid
method for identifying the presence of nucleic
acids. FRNA bacteriophages, however, har-
bour their genetic information in the form of
single-stranded positive-sense RNA which
must rst be transcribed into cDNA before a
detectable amplicon can be produced. Despite
this hurdle, multiplex PCR assays and real-
time RT-PCR assays have been developed
that have shown specicity for each subgroup
of FRNA phages (Vinje et al., 2004; Kirs &
Smith, 2007; Friedman et al., 2009). For
example, Kirs and Smith (2007) developed a
culture-independent multiplex real-time RT-
PCR assay to eect simultaneous detection
and quantication of the four FRNA groups.
The assay detected as few as ten copies of
isolated phage RNA and was able to quantify
FRNA phages in seawater when culture-
based methods (the double agar layer assay)
failed. The authors concluded that accurate
identication of the in situ concentration of
FRNA phages using this method will facilitate
more eective remediation strategies for
impacted environments.
Friedman et al. (2009) developed a real-
time RT-PCR for FRNA phages by rst
designing genogroup-specic primer sets
based on a minimum of ve and a maximum
of ten complete phage genome sequences
from strains in each FRNA genogroup. When
a heat-release protocol that eliminated the
need for RNA purication and the real-time
RT-PCR method for genotype identication of
FRNA phages were combined, the assay could
detect multiple environmental FRNA phage
strains. Following up on this work, Friedman
et al. (2011) designed a real-time, quantitative
RT-PCR assay to beter dierentiate the four
genogroups of FRNA phages. In this work,
primers and probes were designed using
complete genomic sequences from 29 FRNA
phages to develop a set of primer/probe sets
that were based on: (i) ability to amplify a
single, specic product; (ii) genogroup
specicity; (iii) lack of cross-reactivity; and
(iv) experimental reproducibility and sensi-
tivity over a range of target concentrations. A
custom RNA molecule was employed as an
internal, non-competitive control. The assay
was tested on a total of 49 FRNA phages
isolated from various warm-blooded animals,
sewage and combined sewage overow.
FRNA phages from animal wastes were
genotyped as 86% group I, 4% group III Q-like
and 9% group IV. Two sewage isolates typed
to genogroup I and combined sewage over-
ow isolates genotyped as 40% group II and
52% group III.
Goodridge and Du Preez (2010) de-
veloped and evaluated an integrated con-
centration and detection method to rapidly
assay for FRNA bacteriophages as indicators
of faecal contamination in river water.
Sampling for the study took place over a
3-week period during January 2010 in Phola
Township (immediately adjacent to a sewage
treatment plant) and Pretoria, South Africa.
Following tropical rain events, 4 l of river
water were collected from rivers at both
sampling sites and transported back to the
laboratory on ice. FRNA phages were
concentrated from the water by addition of
1.5 g of anionic exchange resin (Amberlite

IRA 900; Sigma-Aldrich) to each water
sample, followed by continuous agitation to
keep the resin in suspension. Afer 4 h of
concentration, the resin was recovered from
the water samples and virions bound to the
resin beads were detected by isolating viral
RNA directly from the beads. The later was
accomplished with real-time RT-PCR using a
SYBR Green assay and primers designed to
individually detect the four FRNA phage
genogroups. With an enrichment step, the
real-time RT-PCR assay detected FRNA
phages belonging to genogroups II and III
from the Phola Township samples, clearly
indicating the presence of human faecal
pollution. When real-time RT-PCR was
performed directly from the resin beads
(without enrichment), FRNA phages
belonging to genogroups II, III and IV (Phola
Township) and II and IV (Pretoria) indicated
the presence of animal faecal pollution in
addition to human pollution. The entire
concentration and detection assay (without
enrichment) was completed within 8 h. These
results suggest that anion exchange capture
and real-time RT-PCR can be used as a rapid
and sensitive assay for detection of FRNA
bacteriophages as indicators of human and
animal faecal pollution. Moreover, this
method would be especially useful as a rapid
presumptive assay to test compromised
water supplies following hurricanes,
monsoons and other similar weather events.
PCR methods have been criticized for
potentially detecting non-viable organisms as
well as free nucleic acids (Leclerc et al., 2000;
Gassilloud et al., 2003). Research has shown,
however, that free RNA from FRNA phage
Q| is degraded and undetectable by PCR
within 1 h following exposure to wastewater
(Limsawat and Ohgaki, 1997), which would
suggest that if FRNA bacteriophage nucleic
acid is detected, it would be from a viable
organism or an extremely recent RNA
contamination event.
B. fragilis Bacteriophages
B. fragilis has been suggested previously as an
alternative bacterial indicator to standard
indicators such as E. coli and total coliforms
(Fiksdal et al., 1985; Biton, 2005). Phages that
have specicity for B. fragilis infect one of the
most abundant bacteria in the gastrointestinal
tract and belong to the family Siphoviridae.
Phages that infect B. fragilis strain RYC2056
are not human specic, while phages that
infect host-strain B. fragilis HSP40, while
being less numerous than those infecting
strain RYC2056, are considered to be human
specic (Puig et al., 2000). Therefore, phages
that infect B. fragilis HSP40 have been
proposed as indicators of human faecal
contamination because of their specicity
along with their inability to propagate in the
environment. A study by Tartera et al. (1989)
replicated optimal environmental conditions
for the propagation of phages infecting B.
fragilis, including temperature, host-cell
concentration and anaerobic conditions but
excluding nutrients for host growth. Despite
the combination of these conditions, which
themselves are unlikely to be found in the
environment, researchers did not observe
replication of Bacteroides-specic bacterio-
phages in signicant numbers in slaughter-
house wastewaters. They were also not
present in faecally polluted waters containing
faecal contamination from wildlife only. In
contrast, phages active against B. fragilis
HSP40 were detected in faeces (found in 10%
of human faecal samples but not in animal
faeces), sewage and other polluted aquatic
environments (river water, seawater, ground-
water and sediments) and were absent in
non-polluted sites (Tartera and Jofre, 1987;
Cornax et al., 1990). In addition, Gantzer et al.
(1998) demonstrated that B. fragilis phages
were found to be reliable indexes of
enterovirus contamination in the three dif-
ferent types of wastewater that were tested.
These characteristics make B. fragilis HSP40
phages good candidates for microbe source
tracking of a contamination event of human
origin.
Standard methods for identication and
quantication of phages that infect B. fragilis
include double-layer agar most-probable-
number methods (Tartera and Jofre, 1987).
Several molecular and serological methods of
detection have also been explored, including
DNA hybridization and PCR. Plating-based
methods, however, remain the standard for
water assessment (Puig et al., 2000), and
certain hurdles exist that complicate the use
of B. fragilis phages as indicators of faecal
contamination, not least of which is that this
host is an obligate anaerobe, requiring very
specic growth conditions and laboratory
skills to conduct phage assays (Leclerc et al.,
2000). In addition, studies conducted by
Gantzer et al. (2002) conrmed previously
reported data indicating that phages infecting
B. fragilis are not found homogenously within
the faeces of humans worldwide (Tartera and
Jofre, 1987; Havelaar et al., 1993; Grabow et al.,
1995). Finally, B. fragilis phages are detected
in lower concentrations than other phages in
fresh water (115 PFU per 100 ml; Araujo et
al., 1997), meaning that the use of con-
centration methods to increase the numbers
of these phages prior to detection is needed,
which may complicate downstream identi-
cation.
Conclusion
Bacteriophages continue to emerge as
alternative indicators of faecal contamination
and as index organisms identifying the
presence of enteric viruses. While many peer-
reviewed studies demonstrating their
usefulness as indicators of water quality
abound, a lack of standardized methods used
for analysis of the presence of phages in
various water types makes most of the data
di cult to compare. The fact that there are
many variables that aect the incidence,
survival and behaviour of phages in dierent
water environments, including the densities
of both host bacteria and phages, temperature
and pH, further complicates comparisons.
Finally, the myriad of bacterial hosts used to
recover phages during water-quality experi-
ments has led to dierent results reported
with respect to recovery e ciencies of phages
isolated from the same environ mental
samples. Neverthelss, it is clear that phages
are viable candidates as water-quality
sentinels and, as more methods are developed
and standardized for their e cient detection,
it is expected that phages will nd increased
usefulness in assessments of microbial water
quality, particularly as indexes of the presence
of enteric viruses, the methods for detection
of which are still in their infancy.
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12 Phage Translocation, Safety and
Immunomodulation
Natasza Olszowska-Zaremba
1
, Jan Borysowski
1
, Krystyna
Dabrowska
2
and Andrzej Grski
2
1
Department of Clinical Immunology, Medical University of Warsaw;
2
Institute of
Immunology and Experimental Therapy, Polish Academy of Science.
A growing body of data shows that bac-
teriophages are omnipresent in the environ-
ment. For example, phages are present in
high numbers in dierent ecosystems
including water ecosystems, the rhizosphere
and soil. Moreover, phages have been
detected in some foodstus and drinks; they
also constitute a signicant component of the
natural microbiota of both humans and
animals (see Letarov, Chapter 2, this volume).
Interestingly, there are some data to suggest
that phages, like bacteria, might undergo
translocation from the gut to mesenteric
lymph nodes and then to the extranodular
space.
A major application of bacteriophages is
the treatment of antibiotic-resistant bacterial
infections (phage therapy; see Loc-Carrillo et
al., Burrowes and Harper, and Abedon,
Chapters 13, 14 and 17, this volume). Several
important issues need to be taken into
consideration when discussing the safety of
phage therapy. First, only virulent phages
should be used for therapeutic purposes;
temperate phages should be excluded from
therapy (see Christie et al. and Abedon,
Chapters 4 and 17, this volume). Secondly,
the antibacterial range of phages is very
narrow, so they are not likely to disturb the
balance of the natural microora. Thirdly,
dierent components of bacterial cells may
be present in phage preparations; these com-
ponents may also be released from bacteria in
vivo following phage-induced lysis of
bacterial cells. A thorough analysis of the
literature on phage therapy, however, clearly
shows that phages are safe antibacterial
agents.
Although the natural hosts of bac-
teriophages are bacterial cells, a number of
studies have shown that phages can interact
with some populations of eukaryotic cells,
especially with immune cells. In fact, phages
can aect a wide range of functions of dif-
ferent populations of immune cells involved
in both innate and adaptive immunity,
including the production of antibodies, the
proliferation of T and B cells, phagocytosis
and the respiratory burst of phagocytic cells,
and the production of cytokines.
Background
With the discovery of penicillin, a period of
intensive development of antibiotic therapy
began, resulting in a belief that the problem
of bacterial infections had been eliminated. A
number of factors, including overuse of
antibiotics in clinical medicine, animal
production and agriculture, however, have
resulted in the emergence and spread of
Phage Translocation, Safety and Immunomodulation 169
multidrug-resistant bacterial strains. In view
of the inadequate number of new antibiotics
(in 2003 only ve out of 400 drugs under
clinical trials in the USA were antibiotics), the
re-emergence of the pre-antibiotic era has
become a real prospect (Nelson, 2003).
One of the classes of antibacterial agents
that could be used to combat multidrug-
resistant bacteria is bacteriophages. A large
number of studies have been conducted on
the use of phages in humans, and of particular
importance are studies that were carried out
in Georgia and Poland. Bacteriophages have
several advantages over antibiotics, including
high specicity of their antibacterial activity,
a capacity to kill antibiotic-resistant bacteria,
a lack of any signicant side eects and the
low cost of phage preparations (Grski et al.,
2009; Housby and Mann, 2009). However, it is
essential to conduct formal clinical trials to
conrm the e cacy and safety of phage
therapy before bacteriophages become
widely available as therapeutic agents (see
Burrowes and Harper, Chapter 14, this
volume).
An understudied aspect of phage biology
is their potential eects on the immune
system. It is known that infections with
pathogenic viruses a category that does not
include bacteriophages are associated with
activation of coordinated mechanisms of
immune responses (Horst et al., 2011). At rst,
non-specic immune responses are induced
following the recognition by immune cells of
pathogen-associated molecular paterns in
virions. Primary immune responses to viral
infections are mediated by macrophages,
natural killer cells, granulocytes, complement
and interferons. Viral antigens are presented
by class I and class II major histocompatibility
complex (MHC) molecules to T cells, which
results in the activation of these cells. Humoral
and cellular specic immune responses are
induced, resulting in the neutralization of
viruses by antibodies and killing of the
infected host cells by mechanisms of cellular
cytotoxicity. In the course of evolution,
however, many pathogenic viruses have
developed mechanisms to avoid elimination
by the immune system. Pathogenic viruses
can avoid recognition by the host immune
system as a result of high antigenic diversity,
such as human immunodeciency virus
(HIV), or a decrease in the expression of class
I and class II MHC molecules on the infected
cells, as occurs in infection with cyto-
megalovirus, EpsteinBarr virus and herpes
simplex virus type 1 (HSV-1) and HSV-2
(Ploegh, 1998; Olszewska and Radkowski,
2005). Moreover, pathogenic viruses can sub-
stantially aect antiviral immune responses
by interfering with the activity of cytokines,
such as the production by adeno viruses and
EpsteinBarr virus of proteins with activity
antagonistic to cytokines, and by interfering
with the production of antibodies and
components of the complement system, as
occurs with HSV infection (Ploegh, 1998;
Olszewska and Radkowski, 2005). So far, over
50 viral genes whose products exert immuno-
modulatory activity have been identied
(Ploegh, 1998). Thus, it is not unreasonable
that phages infecting pathogenic and normal
ora bacteria would also be able to evolve
immunomodulating proteins to aid in their
retention in the body.
Prevalence of Bacteriophages and
the Safety of Phage Therapy
Prevalence of bacteriophages in the
environment
There is a surprising abundance and
diversity of bacteriophages in the environ-
ment. It is estimated that there are between
10
4
and 10
8
phages ml
1
in water ecosystems
(Weinbauer, 2004). Bacterio phages have also
been isolated from sewage (mean con-
centration 10
4
10
8
ml
1
), the rhizosphere and
soil (1.5 10
8
g
1
; Weinbauer, 2004). It was
also reported that bacteriophages are present
in yogurt (Lactobacillus phages) and sauer-
kraut, where as many as 26 dierent phages
were found (Kili et al., 1996; Lu et al., 2003).
It is known that phages are also prevalent in
the bodies of humans and animals and in
their secretions and excre ments. Phages are
particularly abundant in the intestines of
mammals in view of the presence of
abundant bacterial microora. This issue is
discussed in detail by Letarov (Chapter 2,
this volume).
170 N. Olszowska-Zaremba et al.
Safety of phage therapy general
considerations
The omnipresence of bacteriophages in the
environment implies constant exposure of
humans to phages. Thus, not surprisingly,
bacteriophages administered for therapeutic
purposes are well tolerated by patients. Side
eects of phage therapy are observed very
rarely. In a study by lopek et al. (1983), side
eects included the intolerance of phage
preparations, and allergic symptoms were
observed in only three out of 138 patients
(2.2%). Between days 3 and 5 of the treatment,
a transient liver area discomfort was noted
that could have been caused by massive
endotoxin release following phage-mediated
lysis of bacterial cells. As reported by Morello
et al. (2011), however, components of bacterial
cells released from the cells following phage-
induced lysis do not exert pro-inammatory
activity. They showed that the administration
of phages to mice infected by Pseudomonas
aeruginosa is not associated with an increase
in the production of cytokines.
Similar conclusions were drawn by
Midzybrodzki et al. (2009), who examined
the level of selected inammatory markers
C-reactive protein (CRP), white blood cells
(WBCs) and erythrocyte sedimentation rate
(ESR) in a group of 37 patients subjected to
phage therapy. A retrospective analysis of the
results showed no signicant changes in CRP,
WBCs or ESR values between days 5 and 8 of
the treatment compared with the cor-
responding values found in the patients prior
to phage administration. Moreover, between
days 9 and 32 of the treatment, signicant
decreases in the levels of CRP and WBCs
were observed. These results suggest that
lysis of bacteria by phages does not exert any
pro-inammatory eects; in fact, they
indicate that phages could exert anti-
inammatory activity (Midzybrodzki et al.,
2009).
A strong argument for the safety of
phage therapy is the high specicity of the
antibacterial activity of phages. Phages kill
only bacteria from certain strains or
subspecies. Therefore, unlike antibiotics, they
are less likely to disturb the balance of the
bacterial microora. A lack of any deleterious
eects of phages on the microora was found
in a study by Brutin and Brssow (2005).
They reported that oral administration of T4
phage at a titre of 10
3
or 10
5
plaque-forming
units (PFU) ml
1
to healthy individuals did
not result in a decrease in Escherichia coli
counts in faeces. Furthermore, no side eects
atributable to the administration of phage
preparations were observed in any subject.
An important topic relevant to the
problem of the safety of phage therapy is the
possibility of phage-mediated transfer of
detrimental genes (e.g. those encoding
virulence factors and toxins) between
bacterial cells. This problem is discussed by
Christie et al. (Chapter 4, this volume).
An additional potential source of side
eects during phage therapy can be dierent
components of bacterial cells, especially
endotoxins contaminating bacteriophage
preparations, which are released from the
bacteria in which phages are propagated.
In the early stages of the development of
phage therapy (1930s and 1940s), it was
observed that intravenous administration of
preparations containing a large amount of
endotoxin causes a number of side eects,
including an elevation of temperature,
shivers and headache (Chanishvili et al.,
2001). Eective methods have since been
developed to purify phage preparations from
dierent components of bacterial cells,
including endotoxins. However, so far, phage
lysates have been used for most treatment of
humans, as purication of phage preparations
from endotoxins is associated with a loss of
active phage particles. To minimize side
eects, such preparations are administered
orally or topically (Sulakvelidze and Kuter,
2005). Studies on using phage lysates in the
treatment of chronic bacterial infections in
humans have been conducted since 1952 at
the Hirszfeld Institute of Immunology and
Experimental Therapy PAS, Wrocaw, Poland.
No signicant side eects of phage therapy
have been observed so far (Weber-Dbrowska
et al., 2000a; Grski et al., 2009; and
unpublished results).
Preparations of phages specic to Gram-
positive bacteria can also contain some
components of bacterial cells. There are data
in the literature, however, to show that it
is possible to obtain safe preparations of
these phages. For example, an apyrogenic
preparation of a staphylococcal phage
intended for intravenous administration was
developed in Georgia. A study performed on
900 patients, including 494 subjects who were
administered antibiotics with phages or
phages only, and 406 subjects who were
administered antibiotics only, showed that
intravenous administration of this phage
preparation did not cause any signicant side
eects (Chanishvili et al., 2001).
Phages and oxidative stress
An essential component of non-specic
immune responses associated with phago-
cytosis of microbes is the respiratory burst, in
which reactive oxygen species (ROS) are
generated. These species, including super-
oxide, hydrogen peroxide, hydroxyl radicals
and singlet oxygen, exert bactericidal activity.
When produced in excess, however, they can
also induce oxidative stress and exert
cytotoxic activity, resulting in damage of
tissues (Gulam and Ahsan, 2006). Oxidative
stress resulting from excessive production of
ROS is implicated in the pathogenesis of
cancer, sepsis, multiple organ dysfunction
syndrome, atherosclerosis and neuro-
degenerative diseases, especially Alzheimers
disease and Parkinsons disease (Knight,
1995; Sikora, 2002; Gulam and Ahsan, 2006).
Therefore, the evaluation of the eects of
phages on ROS generation is essential.
It was shown that phages can decrease
both E. coli and lipopolysaccharide (LPS)-
induced production of ROS by neutrophils
(Midzybrodzki et al., 2008). Furthermore, the
incubation of E. coli with homologous phages
resulted in a decrease in the intensity of the
respiratory burst in both neutrophils and
monocytes, which could result from the lysis
of bacteria by phages (Przerwa et al., 2006).
This result suggests that lysis of bacteria by
phages is not likely to induce oxidative stress
in cells, arguing for the safety of phage
therapy. Another study revealed that both
lysates and puried preparations of T4 phage
and staphylococcal phage A3/R induced only
a weak respiratory burst in human monocytes
and neutrophils in vitro (Borysowski et al.,
2010). T4 and A3/R phage-induced production
of ROS was so low that it probably would not
induce oxidative stress in vivo (Przerwa et al.,
2006; Midzybrodzki et al., 2008). The capacity
of phages to decrease the production of ROS
by phagocytic cells can explain the e cacy of
phages in the treatment of sepsis (Weber-
Dbrowska et al., 2003; Przerwa et al., 2006).
Our hypothesis to explain the eects of
phages on phagocytic cells is based on the
assumption that LPS is bound by phage
virions, which could prevent the binding of
LPS to its receptor that triggers the respiratory
burst (Przerwa et al., 2006).
Immune Responses to Exogenous
Bacteriophages
Inactivation of phages by mechanisms of
the innate immunity
Studies of the distribution of bacteriophages
in mammals suggest that they can penetrate
into the circulation following their admini-
stration by practically any standard route
(Dbrowska et al., 2005; Sulakvelidze and
Kuter, 2005). When there are no host bacteria
in which phages could replicate, they are
fairly rapidly removed from the blood and
the majority of internal organs. As shown by
Dubos et al. (1943), Shigella dysenteriae phages
in healthy mice are cleared from the
circulation within a few hours of their
intraperitoneal administration, while in
infected mice they are detectable in blood,
even at 18 h post-administration (Sulakvelidze
and Kuter, 2005). Phages are internalized
and eliminated by cells of the retic-
uloendothelial system of the liver and spleen.
Inchley (1969) noted that, in mice, over 99%
of T4 phage virions were phagocytosed by
Kuper cells within 30 min of their
intravenous administration. As well as
Kuper cells, spleen macrophages can also
eliminate phages, but their activity in this
regard is fourfold lower (Dbrowska et al.,
2005). Consequently, phage titre in the liver
decreases rapidly, while in the spleen phages
remain at high titre for up to 57 days afer
their administration (Geier et al., 1973). It has
been suggested that phages entrapped in the
spleen can be a source of antigen necessary
for antibody generation (Geier et al., 1973).
Anti-phage antibodies
General considerations
Exogenous phages can induce not only innate
immune responses but also the production of
neutralizing antibodies. Concentration of
these antibodies depends, among other
things, on the route of phage administration
(topical and oral administration result in only
a slight increase in the generation of
antibodies) and dosage protocol (the level of
antibodies is low following a single phage
administration and when phages are
administered several times with small
intervals between doses; Sulakvelidze and
Kuter, 2005). Phage im muno genicity also
depends on phage strain. For example, T1
and T5 phages are less immunogenic than T4
phage (Adams, 1959).
Neutralizing antibodies can limit the
e cacy of phage therapy. This was shown by
Srivastava et al. (2004), who showed that the
clearance of T7 phage from the circulation
was slower in B cell-decient mice compared
with wild-type mice. Pagava et al. (2011)
reported that 14 out of 31 blood samples from
patients who were administered phages
orally contained neutralizing antibodies that
inactivated 52.597.3% of bacteriophages.
Antibodies against Staphylococcus aureus
phages were detected in 12 out of 57 patients
with staphylococcal infections (Kucharewicz-
Krukowska and lopek, 1987). During phage
therapy, these antibodies were detected in
54% of patients. Further analysis of the results
obtained in 30 patients revealed that phage
therapy was ineective in two out of ve
patients in whom anti-phage antibodies were
detected prior to therapy (Kucharewicz-
Krukowska and lopek, 1987). Interestingly,
the clinical state of the remaining three
patients improved following the treatment,
which suggests that the presence of
neutralizing antibodies does not always
result in a lower therapeutic e cacy.
Immunogenic properties of |X174 phage
The immunogenic properties of phages have
found use in the diagnosis of immune system
diseases and monitoring of humoral immune
responses. For example, |X174 phage has
been used to evaluate immune responses in
patients with primary and secondary
immunodeciencies, bone-marrow recipients
and HIV patients (Bearden et al., 2005).
Pescovitz et al. (2011) used this phage to
evaluate the eects of rituximab, a B cell-
depleting antibody, on the production of
antibodies in patients with type I diabetes. It
was found that this assay is a sensitive
indicator of antibody production that enables
the evaluation of humoral immune responses.
This study shows the utility of measuring
anti-|X174 phage antibodies both when
monitoring the e cacy of immunosuppressive
therapy and when evaluating immuno-
competence in patients.
Ochs et al. (1971) showed that this phage
can also be used in dierential diagnosis of
immunodeciencies. In healthy individuals,
intravenous administration of |X174 results
in the generation of IgM antibodies that
neutralize the phage within 34 days
(Bearden et al., 2005). It was observed that a
long presence of phages in the circulation
(1142 days) along with a lack of humoral
immune response is typical of X-linked
agammaglobulinaemia (in patients in whom
a severe immunodeciency was excluded;
Ochs et al., 1971). Phage |X174 was selected
for diagnostic use because it is a strong
antigen and does not cause any side eects in
humans. Furthermore, the evaluation of its
clearance from blood is relatively simple
(Ochs et al., 1971).
Anti-phage cellular immunity
Apart from non-specic immune responses
and humoral immunity, cellular immunity
also plays an important role in combating
viral infections. Some of the rst authors to
show that phages can induce cellular immune
responses were Langbeheim et al. (1978),
who examined delayed-type hypersensitivity
reactions to MS-2 phage and a synthetic
fragment of one of its capsid proteins in
guinea pigs. They found that subcutaneous
injection of phages resulted in a strong
hypersensitivity reaction in all animals.
Cellular responses to MS-2 phage were also
observed in vitro, as revealed by an intense
proliferation of lymphocytes from phage-
sensitized guinea pigs (Langbeheim et al.,
1978). On the other hand, the results of
Srivastava et al. (2004) suggest that cellular
immune responses play only a minor role in
inactivation of phages. They showed that the
clearance of T7 phage in T cell-decient mice
is similar to that found in T cell-procient
mice. Thus, the data are conicting, and
further studies are needed in this regard.
Immunomodulatory Effects of
Bacteriophage Preparations
Effects of phages on granulocytes
Non-specic immune response e cacies are
so high that they are ofen su cient to clear
microbes before the activation of specic
immune responses. The rst cells to migrate
to the site of infection are macrophages and
granulocytes, especially neutrophils. It has
been shown that, in patients subjected to
phage therapy, clearance of infection was
accompanied by a decrease in the number of
mature neutrophils and an increase in the
number of neutrophil precursors in the
peripheral blood (Weber-Dbrowska et al.,
2002). Dierentiation of neutrophils can be
mediated by both phage virions and some
components of bacterial cells present in
phage lysates. Stimulatory eects of phage
preparations on the generation of neutrophil
precursors appear to be benecial because
they result in the enhancement of non-specic
immune responses.
It was also shown that bacteriophages
can aect the migration of phagocytic cells.
Pre-incubation of human granulocytes with
T4 phage results in a slight stimulation of
migration in vitro (Kurzpa et al., 2009).
Moreover, phages were found to relieve the
inhibitory eect of LPS on granulocyte
migration (Kurzpa et al., 2009). The results of
other studies showed that phages had only a
slight eect on the migration of human
granulocytes and monocytes and had no
eect on the intracellular killing of bacteria
(unpublished results). Furthermore, T4 phage
can decrease the migration of mononuclear
cells, and to some extent of neutrophils, to a
skin graf in mice, resulting in a marked
decrease in cellular inltration of the graf at
days 7 and 8 post-transplantation (Grski et
al., 2003a).
Inuence of phages on phagocytosis
Our studies have shown that phages also can
aect phagocytosis (Przerwa et al., 2006;
Kurzpa et al., 2009). Pre-incubation of T4
phage and P. aeruginosa F8 phage with human
phagocytic cells in vitro resulted in a decrease
in the phagocytosis of E. coli cells. A similar
eect was noted when phagocytic cells were
incubated with E. coli and T4 phage at a high
titre (10
10
PFU ml
1
).
This eect was weaker for F8 phage.
Incubation of E. coli cells with homologous
phage (T4), but not with heterologous phage
(F8), resulted in an increase in the phago-
cytosis of bacteria by both neutrophils and
monocytes (Przerwa et al., 2006). It appears
that homologous phages can coat bacteria
(phage opsonization), thus facilitating their
phagocytosis (Grski et al., 2003a). T4 phage,
however, had no eect on the in vitro
phagocytosis of bacteria by monocytes and
neutrophils isolated from either E. coli-
infected or uninfected mice (Przerwa et al.,
2006).
Effects of phages on T cells and
platelets practical implications
Integrins comprise a family of cell-surface
receptors that, by interacting with extra-
cellular matrix proteins, are involved in
various physiological processes including
immune responses, tissue remodelling,
haemostasis and angiogenesis. Moreover,
they have been implicated in the pathogenesis
of various diseases including infectious and
non-infectious diseases (Perdih and Dolenc,
2010). The interactions of integrins with
extracellular matrix proteins are mediated by
their RGD (Arg-Gly-Asp) sequences. Several
pathogenic viruses, including HIV, adeno-
viruses, picornaviruses and coxsackievirus
A9, also contain this sequence for binding to
target cells (Berinstein et al., 1995; Triantalou
et al., 2001; Lafrenie et al., 2002).
In vitro experiments revealed that phages
T4 and HAP1 (a T4 mutant strain; see below)
can bind both platelets and T cells (Kniotek et
al., 2004a), possibly via integrin binding. In
the case of T4 and HAP1 phages, this is very
likely, because one of their capsid proteins,
gp24, contains the KGD (Lys-Gly-Asp)
sequence, which can be a ligand for |3-
integrins that are present on both platelets
and T cells (Grski et al., 2003a; Wierzbicki et
al., 2006; Varon and Shai, 2009). An additional
argument for |3-integrins mediating the
interactions between phages and immune
cells is the capacity of eptibatide (an
inhibitor of o
IIb
|
3
integrins) to block binding
of phages to T cells and platelets (Kniotek et
al., 2004a). We also showed that phages can
reduce the adhesion of platelets and, to a
lesser extent, of T cells, to brinogen (Kniotek
et al., 2004a).
Our studies have shown that phages
can exert immunosuppressive activity. In a
study on the role of bacteriophages in the
develop ment of transplantation tolerance,
Grski et al. (2006a) observed that bacterio-
phages can inhibit CD3-triggered activation
of T cells. Intraperitoneal administration of
puried preparations of phages T4, HAP1
and F8 at the dose 10
6
PFU per mouse
resulted in signicant inhibition of specic
humoral responses in vitro (Kniotek et al.,
2004b). It was also found that a puried T4
phage preparation signicantly decreased
alloantigen-driven in vitro humoral responses
(Kniotek et al., 2004b). Furthermore, intra-
peritoneal administration of T4 phage
extended allograf survival and reduced the
inammatory inltration within the graf,
suggestive of phage-mediated inhibition of
anti-transplant immune responses (Grski
et al., 2003b, 2006a). According to our
hypothesis, phages can aect graf rejection
by inhibiting the interactions between T cells,
brinogen and platelets (Kniotek et al., 2004a).
Effects of phages on the production of
cytokines
The results of some studies show that phages
can aect the production of dierent
cytokines (Weber-Dbrowska et al., 2000b;
Kumari et al., 2010). One of the mechanisms
that could mediate these eects is the
inhibition of nuclear factor (NF)-kB activity.
NF-kB is a transcription factor that plays an
important role in the expression of genes
encoding cytokines, cellular surface receptors,
acute phase proteins and adhesion molecules
(Hiscot et al., 1997). We have shown that T4
phage inhibited HSV-1-induced activity of
NF-kB in mononuclear cells in vitro (Gorczyca
et al., 2007). The inuence of phages on the
activation of NF-kB may be important in view
of the potential use of phages in the regulation
of immune responses to allografs, infections
and inammation.
It was found that bacteriophages can
decrease the production of cytokines induced
by bacterial infections in animals. For
example, in a murine model of lung infection
caused by P. aeruginosa, Morello et al. (2011)
determined the concentration of pro-
inammatory cytokines in bronchoalveolar
lavage samples collected at 20 h afer
intranasal administration of bacteria. The
authors noted a signicant reduction in the
concentration of interleukin (IL)-6 and
keratinocyte chemoatractant in the bron-
choalveolar lavage samples following the
administration of a single dose of P3-CHA
phage both 4 days before and 1 day afer
bacterial inoculation. Moreover, histopatho-
logical examination revealed that damage of
pulmonary tissue was more pronounced in
untreated mice compared with phage-treated
animals. As suggested by Kumari et al. (2010),
it is likely that phages, by decreasing the
production of cytokines, limit the inam-
matory reaction, thus reducing damage of
tissues. It was also shown that treating burn
wound infections caused by Klebsiella
pneumoniae with Kpn5 phage resulted in a
signicant decrease in the level of both pro-
inammatory (IL-1| and tumour necrosis
factor (TNF)-o) and anti-inammatory (IL-
10) cytokines in sera and lungs of the treated
mice. It was suggested that a decrease in the
production of cytokines during phage
therapy can result from a reduction in the
number of pathogenic bacteria that are
eliminated by phages (Kumari et al., 2010).
Our observations of patients subjected to
experimental phage therapy has revealed that
phages can substantially aect the production
of IL-6 and TNF-o, two important pro-
inammatory cytokines (Weber-Dbrowska et
al., 2000b). These results suggest that a
positive outcome of phage therapy might, to
some extent, be mediated by the regulation of
TNF-o production. Interestingly, the eect of
phages on the production of TNF-o depends
on the level of this cytokine before the
administration of phage preparations. In
patients with a low or moderate serum level
of TNF-o, phages increased the production of
this cytokine, whereas in patients with a high
level of TNF-o, phages decreased its
production. In a similar (regulatory) way,
phage therapy can aect spontaneous
production of IL-6 and TNF-o by cultured
mononuclear cells, as well as the LPS-induced
production of TNF-o by mononuclear cells. A
decrease in the LPS-induced production of
TNF-o by mononuclear cells was observed in
patients in whom therapeutic eects were
partial or who did not respond to therapy.
The regulatory eects of phage therapy on
the production of cytokines can be atributed
to one (or a combination) of three factors: (i)
immunomodulatory activity of phages; (ii)
natural recovery of the patient; and (iii) a
combination of the eects of phages and
bacterial components (Weber-Dbrowska et
al., 2000b).
Bacteriophages can also aect the
production of other cytokines. Puried T4
phage preparation signicantly inhibited the
production of IL-2 and, to a lesser extent,
gamma interferon (IFN-) by phyto-
haemagglutinin-activated human leukocytes
(Przerwa et al., 2005; Kurzpa et al., 2009). On
the other hand, Kleinschmidt et al. (1970)
showed that the administration of a puried
T4 phage preparation can result in an increase
in the concentration of IFN- in the serum of
mice. Moreover, DNA from T4 phages can
induce the production of IFN in mice.
Immunomodulatory activity of
staphage lysate
It appears that the lysate of a S. aureus phage,
known as staphage lysate (SPL), could be
used for immunodeciency diagnosis, as well
as in treating staphylococcal infections. It was
reported that SPL induced proliferation in
over 95% of lymphocytes isolated from
healthy individuals (Dean et al., 1975).
Maximal lymphoproliferative responses were
observed on day 6 of culture. Furthermore,
unlike phytohaemagglutinin, SPL activates
both T and B cells (Dean et al., 1975). The
authors suggested that SPL could be used as
an adjunct to mitogens and antigens routinely
used to evaluate cellular immune responses.
In line with these data are results showing
that SPL-induced immune responses are
weaker or undetectable in cancer patients
and in those with immunodeciencies (Dean
et al., 1975).
SPL was shown to increase the
production of immunoglobulins in human
lymphocyte culture and to induce the
production of polyclonal immunoglobulins
by murine splenocytes (Sulakvelidze and
Kuter, 2005). One dose of SPL administered
to mice induced the production of IgG and, to
a lesser extent, IgA (Esber et al., 1985).
According to Esber et al. (1985), SPL can also
stimulate a specic humoral response in
mice. Thus, an additional benecial eect of
SPL in the treatment of chronic staphylococcal
infections may be the enhancement of
antibacterial immune responses. Further-
more, SPL, as a preparation stimulating both
humoral and cellular immunity, could be
particularly useful in the treatment of
infections in immunocompromised patients,
including those receiving cytostatic drugs
and individuals with immunodeciencies
(Esber et al., 1985). The immunostimulatory
eects of phage preparations are probably
mediated by both phage virions and some
components of bacterial cells present in
preparations (Sulakvelidze and Kuter, 2005).
In view of the mitogenic properties of
SPL and its capacity to induce IFN production,
studies were conducted to evaluate its
potential anticancer eects (Mathur et al.,
1988). In a rat model of breast cancer, the
administration of SPL preceded by
immunization with dead S. aureus cells
resulted in a decrease in tumour diameter
(Mathur et al., 1988). A study performed on a
murine sarcoma model, however, did not
conrm the anticancer eects of SPL (Esber et
al., 1981). It was also shown that SPL reduces
the migration of murine B16 melanoma cells
in vitro. However, this eect may be mediated
by some components of staphylococcal cells
that are present in the lysate, as bacterial
lysates without any phage particles also
signicantly decreased the migration.
Further more, neither SPL nor control bacterial
preparations inhibited the proliferation of
cancer cells and did not stimulated their
migration. This is an important result in
regard to the potential therapeutic use of SPL
(Dbrowska et al., 2010).
Phage capsid elements inducing
immunomodulatory effects in mammals
Mammals represent an environment for
phages parasitizing mammalian symbiotic or
pathogenic bacteria (see Letarov, Chapter 2,
this volume). In this environment, phages are
aected by a potent pressure of mammalian
immunity that presumably drives selection
and evolutionary adaptation. Thus, immuno-
modulatory eects of bacteriophages could
result from adaptations to persistence in the
mammalian environment. In general, both
nucleic acids and phage capsid proteins may
contribute to phage eects on the immuno-
logical system. The eects can be anticipated
and unspecic, such as the general eect of
large antigenic objects introduced into a
system, or specic, such as directed inter-
actions of active elements. Although data on
specic means of interactions are poor that
is, which genes, polypeptides or proteins are
involved some possible mediators have
been proposed.
In T4 phage, two proteins were proposed
as active in interactions with mammalian
immunity: gp24 and gpHoc (Grski et al.,
2003a; Dbrowska et al., 2006). These are both
located on the phage head. Gp24 is an
essential protein that builds head corners,
while gpHoc is a dispensable external
element of the capsid (Ishii and Yanagida,
1977; Fokine et al., 2004). The full name of Hoc
protein, highly antigenic outer capsid
protein (Ishii and Yanagida, 1975), indicates
its eects on mammalian immunity. Studies
of Hoc structure furthermore have revealed
the presence of immunoglobulin-like folds in
this protein (Bateman et al., 1997; and
unpublished results). These folds characterize
cell-atachment molecules, ofen regulating
immune systems, and are quite ofen present
in phage genomes (Fraser et al., 2006).
Similarities of the phage protein to mam-
malian proteins engaged in interactions and
regulation of immunity make Hoc a probable
modulator of immune functions. Surprisingly,
in light of its originally identied highly
antigenic nature, data support the hypothesis
that Hoc protein could help the phage to
evade the immune system by causing
Hoc-mediated suppression of immunity
Hoc was also shown to regulate the
adhesion properties of the T4 phage particle
(Dbrowska et al., 2004; Sathaliyawala et al.,
2010), which may contribute additionally to
phage interactions with immune cells.
Importantly, a nonsense mutant of hoc that
lacks gpHoc in its capsid (HAP1 phage (high-
a nity phage), selected for its a nity in
binding to B16 murine melanoma cells)
presented unique and unexpected properties
toward mammalian cells. Although the
mutation did not result in changes in the
mutants general morphology (as determined
by electron microscopy), it resulted in sub-
stantial changes in bacteriophage metabolism
and clearance, with clearance of HAP1 being
signicantly more rapid. The comparison of
concentrations of active wild phages to
concentrations of the Hoc
mutant in murine
blood and liver revealed a preponderance of
the wild phage, by approximately one to
three orders of magnitude (Dbrowska et al.,
2007). It is probable that the degradation of
HAP1 is more e cient than that of T4 phage,
as gpHoc (the external head protein) covers
other capsid elements that are more visible
and stimulatory to the degradation factors.
Hoc protein seems to be a kind of protection
for T4 phage, and this protection prevents
rapid degradation of T4 in mammalian
organisms (Dbrowska et al., 2007).
Phage clearance by reticuloendothelial
system ltration is usually proposed as the
main path of phage removal from non-
immunized mammals (Merril, 1974). Thus,
the innate immunity response to a phage can
be modied according to its virion protein
composition. Probably the very rst virion
protein whose properties inuenced elements
of innate immunity was identied by Merril
et al. (1996) in phage . A substitution of the
amino acid lysine for glutamic acid in the
major head protein E caused a long-
circulating phenotype, that is, -derived
mutants that circulated for longer in murine
blood in comparison with the wild-type
phages. The mutated phages were also more
eective in combating lethal bacterial
infections in mice (Merril et al., 1996).
Additional phage effects
T4 and HAP1 were investigated in in vivo
cancer models with regard to their ability to
interfere with cancer processes. This work
was inspired by the work of Bloch (1940), who
showed that phages can accumulate in cancer
tissue and inhibit the growth of tumours.
Next, it was demonstrated that phages bind
cancer cells in vitro and in vivo and atach to
the plasma membrane of lymphocytes
(Katoch, 1958; Katoch and Mordarski, 1958;
Wenger et al., 1978). As described above, SPL
can interfere with melanoma cell migration.
T4 phage and HAP1 phage (Hoc
) were
investigated and compared in a murine model
of intravenously injected melanoma. The
HAP1 preparation was signicantly more
active in diminution of melanoma colonies in
murine lungs (Dbrowska et al., 2004, 2007).
This eect was correlated with the elevated
adhesive potential of the Hoc
phage on
murine melanoma cells. Its adhesive potential
towards mouse melanoma cells was estimated
to be about eight times higher than that of T4.
Elevated a nity to human melanoma was
also observed. Adhesion was decreased by
typical |3-integrin inhibitors such as
eptibatide or antibodies (Dbrowska et al.,
2004).
Increased activity of phage lacking
gpHoc supports the hypothesis that other
capsid elements can interact and aect
mammalian cells, as Hoc protein is located in
the phage head. These dierences in T4 and
HAP1 inspired a hypothesis (Grski et al.,
2003a) on the potential activity of the KGD
motif (a homologue of RGD) that is present in
gp24 (head vertex protein) of T4. The motif is
well exposed at the phage head, as revealed
by the gp24 structure data (Protein Data Bank
ID: 1YUE). The head vertex protein was
postulated to interact with mammalian-
specic receptors via its KGD motif and thus
to aect cells or cancer processes through |3-
integrins. |3-Integrins are a class of adhesion
receptors that mediate cellcell and cell
extracellular matrix interactions. Thus, they
play a role in, for example, tissue integrity,
cellular migration, cell survival, adhesion
and dierentiation. Disintegrins able to bind
|3-integrins were reported to interfere with
tumour growth, progression and metastasis
(Giancoti and Ruoslahti, 1999; Adair and
Yeager, 2002). Thus, gp24 was proposed as
the protein active in phage adhesion to
mammalian cells and in cancer processes
As adhesion processes as well as
metastasis are related to cell migration, T4
and HAP1 phages were also investigated
regarding their ability to interfere with
cancer-cell migration in vitro. Migration
assays were carried out on Matrigel
TM
(an
extracellular matrix substitution) or bro-
nectin. In both cases, phages were able to
diminish cell migration, but no dierences
between T4 and HAP1 were observed
(Dbrowska et al., 2009). HAP1 and T4 were
also shown to decrease immunoglobulin
production by human lymphocytes in vitro in
response to allogenic cells, as well as anti-
sheep red blood cells antibody production in
mice. Both phages induced comparable
eects, with no signicant dierences in their
intensity (Kniotek et al., 2004b).
Mammalian immunity can be also
inuenced by phage DNA, especially using
phage DNA vaccines. This line of research is
discussed in detail by Clark et al. (Chapter 7,
this volume).
Bacteriophage Translocation
According to the hypothesis of Grski et al.
(2006b), bacteriophages or their products,
like bacteria, can undergo translocation
migration from the gut lumen to mesenteric
lymph nodes and subsequently to other
extra-intestinal organs and sites (OBoyle et
al., 1998; Wiest and Garcia-Tsao, 2005).
Translocation of a small amount of bacteria
can occur in 510% of healthy individuals
and is considered a normal physiological
phenomenon (Balzan et al., 2007). If the
function of the immune system is normal,
then bacteria are rapidly cleared from the
extra-intestinal space and are not deleterious.
Intensive penetration of bacteria from within
the gut, however, has been implicated in the
pathogenesis of many severe diseases, includ-
ing acute pancreatitis, sepsis and multiple
organ dysfunction syndrome (OBoyle et al.,
1998; Balzan et al., 2007).
Reports on the abundance of phages in
the gut microbiota raise the question of the
possibility of phages undergoing trans-
location similarly to bacteria and some
pathogenic human viruses. For example,
coxsackievirus B3 could be detected in the
hearts of mice as early as 24 h afer its
intragastric administration, which suggests a
high e ciency of translocation (Harrath et al.,
2004). Viral translocation is mediated by M
cells and dendritic cells, which bind viruses
and transport them across the epithelium to
Peyers patches (Morin and Warner, 1994;
Mossel and Ramig, 2003; Smith et al., 2003).
From Peyers patches, viruses relocate to
mesenteric lymph nodes and then to
peripheral tissues, blood and internal organs
(the spleen, liver and lungs) (Morin and
Warner, 1994; Mossel and Ramig, 2003). Thus,
if phages can undergo translocation, their
presence can be expected in the peripheral
blood of humans and animals. The presence
of phages in the blood of healthy individuals,
however, was reported in one paper only
(Parent and Wilson, 1971). This study
revealed the presence of mycobacteriophages
not only in the sera of patients with Crohns
disease but also in four out of 18 blood
samples taken from healthy individuals from
the control group (Parent and Wilson, 1971).
A larger body of data has been derived from
animals. For example, Merril et al. (1972)
detected phages in the blood of calves, sheep
and hens. Orr et al. (1975) reported the
presence of phages specic to E. coli in as
many as 90% of blood samples from calves. It
needs to be stressed, however, that in the
majority of studies performed in the 1970s,
only E. coli was used to detect the presence of
phages in the sera of animals. It is impossible,
therefore, to draw any general conclusions
regarding the real scale of the presence of
phages in the blood of animals.
Phage translocation was also demon-
strated in studies conducted to evaluate the
distribution of phages following their oral
administration to humans and animals. For
example, Keller and Engley (1958) reported
that Bacillus megaterium phages could be
detected in blood 5 min afer intragastric
administration to mice. Likewise, Bradley et
al. (1963) showed that phages can translocate
fairly rapidly across the gut wall following
oral administration to mice. The pace of
penetration of phages from the gut to the
blood suggests that it can occur by diusion
(Dbrowska et al., 2005). Duerr et al. (2004),
however, suggested that translocation of
phages across the intestinal barrier can be a
more complex process involving M cells,
dendritic cells and enterocytes. By using the
phage-display technique, it was shown that
translocation of phages requires certain
amino acid sequences in capsid proteins.
These are probable ligands for receptors
present on cells that mediate transport across
the intestinal barrier and M13 phage lacking
such sequences did not undergo translocation.
Litle is known about phage translocation
in humans. Weber-Dbrowska et al. (1987)
evaluated the distribution of phages in
patients who were administered phages
orally to treat bacterial infections. The
presence of phages was detected in 47 out of
56 blood samples taken from patients on
day 10 of treatment. A recent study by Pagava
et al. (2011) performed on 102 children with
bacterial infections conrmed that phages
can be absorbed from the digestive tract.
Therapeutic phages were detected in six out
of seven blood samples and in 48 out of 55
urine samples collected from patients
between days 3 and 5 of the treatment. The
translocation of orally administered phages
through the gut wall enables phage therapy
to be used also in the treatment of systemic
infections and infections of the urinary tract.
In conict with these results are those of
Brutin and Brssow (2005), who evaluated
the safety of oral administration of T4 phage
in 15 healthy volunteers. Phages were not
detected in blood samples taken from patients
either at the beginning or at the end of the
study. There are two potential reasons for the
lack of phage translocation in this study. First,
these investigators did not neutralize the
gastric juice prior to the administration of
bacteriophages, which could result in the
inactivation of virions due to a strongly acidic
pH in the digestive tract. Secondly, the titre of
phage preparations used by these authors
was about 1000 times lower than the titre
of phage lysates employed by Weber-
Dbrowska et al. (1987) for therapeutic
purposes. Moreover, it needs to be taken into
account that, in patients with chronic bacterial
infections, the gut wall can be penetrated by
microorganisms more easily than in healthy
individuals (Grski et al., 2006b).
A growing body of data shows that the
bacterial gut microora is very important for
the proper function of the human organism
(Blaser, 2011; see also Letarov, Chapter 2, this
volume). Consistent with this, it is likely that
bacteriophages present in the intestines, like
bacteria, play an important role in main-
taining the physiological balance of the host
organism. According to the hypothesis of
Grski et al. (2006b), endogenous phages can
aect the function of the immune system in
both the gut and the whole body (as a result
of their translocation). Bacteriophages
present in the gut can also limit bacterial
translocation, thus reducing the inammatory
reaction caused by the migration of bacteria
through the wall of the digestive tract (Grski
et al., 2006b). Another important function of
phages present in the gut could be inhibition
of the activity of dendritic cells, which are
essential for inducing immune responses in
the digestive tract. In this way, phages could
control inammatory reactions in the gut
(Grski and Weber-Dbrowska, 2005; Grski
et al., 2006b).
Concluding Remarks
Theoretically, side eects of phage therapy
might be caused by phage virions themselves,
some components of bacterial cells present in
phage preparations and/or the products of
phage-mediated lysis of bacteria. Both
experimental and clinical studies, however,
show that the administration of phage
preparations does not result in any serious
side eects. Moreover, in view of the high
specicity of antibacterial activity, phages are
less likely to disturb the balance of the normal
microora compared with traditional anti-
biotics. In general, then, bacteriophages
appear to be safe antibacterial agents.
The interactions between phages and the
immune system are quite complex. First,
phage antigens can induce specic humoral
and cellular immune responses. From the
point of view of phage therapy, the former
seem to be more important because the
production of neutralizing antibodies is one
of the main factors potentially reducing the
therapeutic eectiveness of phages. More-
over, a number of studies have shown that
phages can aect dierent functions of major
populations of immune cells including
phagocytosis and the respiratory burst of
phagocytic cells and the production of
cytokines. It appears that at least some of the
interactions between phages and immune
cells are mediated by capsid proteins.
An interesting question is whether
phages, being a signicant component of the
gut microbiota, can undergo translocation
across the wall of the digestive tract. Although
the translocation of bacteria is a relatively
well-documented phenomenon and one that
occurs both in normal physiological setings
and in dierent diseases, the knowledge
about the translocation of phages is scanty.
There are some data in the literature, however,
to suggest that bacteriophages, like bacteria,
could translocate across the wall of the gut. It
is possible that phage translocation could
play a role in the development and/or
function of the immune system in both the
gut mucosa and the whole body.
Acknowledgements
This work was supported by funds from the
Operational Program Innovative Economy,
20072013 (Priority axis 1. Research and
Development of Modern Technologies,
Measure 1.3 Support for R&D projects for
entrepreneurs carried out by scientic
entities, Submeasure 1.3.1, Development
project no. POIG 01.03.01-02-003/08 entitled
Optimization of the production character-
ization of bacteriophage preparations for
therapeutic use) and an intramural grant
from Warsaw Medical University (1MG/
W1/09). We are grateful to R. Ashcrof for
correcting the English version of the manu-
script.
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13 Phage Therapy of Wounds and
Related Purulent Infections
Catherine Loc-Carrillo
1
, Sijia Wu
1
and James Peter Beck
1
1
Department of Orthopaedics, The University of Utah.
Every convenience brings its own inconvenience. Roman proverb.
The widespread use of antibiotics for over
half a century has contributed to the
emergence of antibiotic-resistant bacteria.
The ever-increasing problem of treating
infections caused by these superbugs is
pushing the healthcare system and research
scientists to look at alternative antimicrobial
agents that can be harnessed to aid in the
ght against pathogens while leaving our
normal microbial ora untouched. It is well
established that for every prokaryotic cell in
the human body there are approximately ten
bacterial cells, although we are only able to
culture 1050% of them (Teibelbaum and
Walker, 2002; Gill et al., 2006; Morowitz et al.,
2011). Bacteria inhabit the human body as
symbionts, contributing to the structure and
function of tissues around them and playing
an important part in the balance between
health and disease by promoting the
development and function of our adaptive
immunity (Lai et al., 2009; Lee and Maz-
manian, 2010). However, once a physiological
barrier has been breached that is, the
occurrence of a wound there is an increased
risk of colonization of the deep tissues by
opportunistic pathogens, including those
with antibiotic-resistant properties, and
subsequent infection (Hermans and Tread-
well, 2010; Morowitz et al., 2011). Phage
therapy represents an important strategy by
which otherwise untreatable antibiotic-resist-
ant infections may be brought under control
or cured.
This chapter provides a general
denition of what wounds are and describes
specic wound infections that have been
investigated for their receptiveness to phage
therapy. We highlight some of the early
clinical work, from the pre-antibiotic era,
when phage therapy was rst used to prevent
and treat wound infections, as well as other
localized infections. We also provide insights
into some of the reasons for failure of these
approaches as judged by todays standards.
Finally, we discuss the strengths and
weaknesses in experimental design of various
animal models used to determine the e cacy
of bacteriophages as antimicrobials, as it is
only by following a methodical scientic
approach that the potential use of this
unconventional treatment of wound in-
fections will be accepted by 21st-century
scientists and clinicians alike. For more
on other aspects of phage therapy, see
Olszowska-Zaremba et al., Burrowes and
Harper, and Abedon, Chapters 12, 14 and 17,
this volume, as well as reviews by Abedon et
al. (2011), Maura and Debarbieux (2011) and
Kutateladze and Adamia (2010).
186 C. Loc-Carrillo et al.
Sharing our Bodies with Microbes
The surface of the skin is colonized by
microorganisms, the ecology of which is
dependent on the topographical location,
endogenous host factors and exogenous
environmental factors (Grice and Segre, 2011).
In humans, the acidic surface of the skin (~pH
5) provides favourable conditions for bacteria
such as coagulase-negative staphylococci and
corynebacteria. Certain skin locations, where
large quantities of triglyceride-containing
sebum are found, encourage the presence of
Propionibacterium acnes, while other regions
with conditions of higher temperature and
humidity support the growth of Gram-
negative bacilli and Staphylococcus aureus. Host
factors such as age and sex can also have a
great eect on the colonizing microbiota. A
disinfected skin surface will be recolonized
within hours by microorganisms that reside
in deeper undisinfected locations such as hair
follicles, glands, the oral cavity and urogenital
openings.
Although the skin is estimated to have
~10
12
bacterial inhabitants, the gut holds the
vast majority of microorganisms, containing
an impressive 10
14
bacteria, the majority of
which are composed of just four phyla:
Actinobacteria (Gram-positive bacteria that
include the genera Corynebacterium, Myco-
bacterium and Bidobacterium), Firmicutes
(Gram-positive bacteria that include clostridia
and bacilli), Bacteroidetes (Gram-negative
bacteria including members of the genus
Bacteroides) and Proteobacteria (Gram-
negative bacteria including the genera
Escherichia, Salmonella and Helicobacter),
although at vastly dierent frequencies.
Unlike the skin, however, the gut is permeable,
allowing the e cient absorption of nutrients
while possessing innate defences that can
help prevent bacteria from also crossing the
mucosal barrier (Hopper and Gordon, 2001;
Grice and Segre, 2011).
Dening Wounds and Related
Purulent Infections
For our purposes, we dene the term wound
as any penetration of the protective in-
tegumentary organ of the body that we
commonly call the skin. This penetration
could be from external mechanical forces
such as surgery or trauma, or from infectious
breakdown of the skin from an underlying
abscess. The outcome of such penetration is
open communication between the exterior
and the underlying deep tissues of the body.
All of these tissues, under healthy, normal,
uninjured, homeostatic circumstances, are in
a sterile environment that is kept free of
bacteria by the innate and adaptive immune
systems. Swab samples taken from an incision
during elective surgery, following proper
surgical prepping and draping procedures,
thus should always be sterile.
The main types of wounds encountered
clinically include: surgical wounds, thermal
wounds (involving partial- or full-thickness
burns), chemical injuries, wounds due to
trauma and chronic wounds (such as venous,
diabetic and pressure ulcers) (Hermans and
Treadwell, 2010). During the treatment of any
type of wound or wound infection, proper
wound care is a race to obtain infection-free
closure before nosocomial infection occurs
or superinfection with antibiotic-resistant
bacteria takes over the infection process. Table
13.1 summarizes historical studies investigat-
ing the e cacy of phage therapy of wounds.
Acute wounds
Uninfected surgical wounds or incisions tend
to heal within 3 weeks. However, the rate of
wound healing depends on a healthy blood
supply and varies according to the tissues
blood supply, which is dependent on its
distance from the heart. The highly vascular
tissues of the face thus allow suture removal
within days of clean (or sterile) incision,
while sutures in the lower extremities remain
for 23 weeks until primary uncomplicated
wound healing is achieved.
Burn wounds
Partial supercial burns, also known as rst-
degree burns, tend to blister but heal without
scarring. Infections are uncommon and
healing occurs within 2 weeks (Hermans,

P
h
a
g
e

T
h
e
r
a
p
y

o
f

W
o
u
n
d
s

a
n
d

R
e
l
a
t
e
d

P
u
r
u
l
e
n
t

I
n
f
e
c
t
i
o
n
s

1
8
7
Table 13.1. Summary of historical studies investigating the efcacy of phage therapy of wounds.
Reference Afliation Bacteria Wound type (n) Route of administration Outcome Details
McKinley,
1923
Baylor University
College of
Medicine
Staphylococcus
spp.
Open wounds (4) Injected directly into wound
or subcutaneous, or both
100% success rate Resulted in decreased discharge and
eventual improvement of patient
condition
Larkum,
1929
Michigan
Department of
Health
Staphylococcus
spp.
Furunculosis
(208)
Osteomyelitis (6)
Subcutaneous injection in
combination with local
application
78% successfully treated;
19% had recurrence;
3% showed no
improvement
50% had rapid and
A 2-year study: phage preparations
were provided to participating
physicians in return for clinical
outcome of patients;
149 patients had records of whether a
reaction to the inoculation was
seen: 42% showed no reaction;
47% had mild, local erythema and
soreness; 10% had a rise in
temperature and malaise; and 1%
had severe reactions
complete recovery;
50% had marked
improvement
Rice, 1930 Indiana University
School of
Medicine
Staphylococcus
spp. and E. coli
Boils and
carbuncles
(66)
Applied locally as a wet
dressing; injected into
deep lesions; some
abscesses were injected
into the area as a 1%
agar jelly preparation
83% had excellent results;
8% had intermediate
results; 8% failure rate
Most of the failures for boils and
carbuncles were due to the isolate
being resistant to the phage strains
available for treatment
Abscesses
(27)
4% had intermediate
results; 4% had no
improvement
Excellent results were obtained in
cases where the abscess had not
been opened prior to treatment
Infected wounds
(44)
7% had good
outcomes; 2% had no
effect seen
An increase in pus production of
infected wounds was commonly
seen after the rst application for
the rst 2448 h, but wounds
rapidly healed after cleaning out
the pus
Osteomyelitis (11) 36% had excellent results;
27% had fair outcomes;
36% had no effect seen
If there was dead bone in the lesion,
the phage treatment had no value;
however, if all necrotic bone was
removed, good results were
obtained
Continued
1
8
8

C
.

L
o
c
-
C
a
r
r
i
l
l
o

e
t

a
l
.
Reference Afliation Bacteria Wound type (n) Route of administration Outcome Details
Walker, 1931 Research
Laboratories of
E.R. Squibb &
Sons
Staphylococcus
spp.
Abscess
(15 animals; 40
experiments)
0.5 ml subcutaneous
injection into abscess or
5 ml intravenous
injections
100% failure Rabbits were infected experimentally;
treatment was administered either
2 min after the bacterial inoculum
or once lesions had developed;
each route of administration was
set up as a separate study
Shultz, 1932 Department of
Bacteriology &
Experimental
Pathology,
Stanford
University
Staphylococcus
spp.
Furunculosis (63) Subcutaneous injections;
used to irrigate wounds
70% success rate A 2-year study: appropriate phage
stocks were supplied based on
bacterial cultures isolated from
infected patients
Carbuncles (16) 88% success rate
Albee and
Patterson,
1930
Department of
Orthopaedic
Surgery, New
York Medical
School, NY
Staphylococcus
spp.
Osteomyelitis Gauzes used to dress the
wounds were soaked in
phage ltrate and
Vaseline
Not determined Clinical isolates were tested for their

susceptibility to phage ltrate prior
to treatment; seven cases that
resulted in successful outcome
were showcases in this study; in
three of these cases, native
(temperate) phage were induced
from the bacteria found infecting
the wound
Table 13.1. Continued
Phage Therapy of Wounds and Related Purulent Infections 189
2010). Major burn wounds, involving >15% of
the total body surface area, may look
supercial but tend to develop into deeper,
full-thickness wounds, where excision of
necrotic tissue and closure of the wound by
grafing skin over the area is necessary.
Factors that increase the risk of infection in
these wounds include the prior use of
irradiation and some medications such as
corticosteroids and other immunosuppressive
drugs, stress-induced hyperglycaemia, low
serum cortisol levels, anaemia and the length
of hospital stay. Individuals with burns
covering >30% of the total body surface area
are at risk of rapid bacterial colonization due
to the immunosuppressive eect of the injury
(Rowley-Conwy, 2010). The prolonged
presence of an open wound or delayed initial
burn wound care leads to high incidence
rates of morbidity and mortality.
Typically, within the rst week, endo-
genous Gram-positive bacteria initially
colonize burns but are then rapidly replaced
by antibiotic-susceptible Gram-negative
bacteria. The use of broad-spectrum anti-
biotics for treating these infections tends to
favour subsequent colonization by exogenous
nosocomial organisms, which are generally
antibiotic-resistant bacteria such as methicillin-
resistant S. aureus (MRSA), vancomycin-
resistant enterococci and extended-spectrum
|-lactamase (ESBL)-producing Gram-negative
rods (Church et al., 2006; Raa and Tredget,
2011; see Kuhl et al., Chapter 3, this volume, for
additional discussion of some of these
organisms).
Chronic wounds
Any impediment to the wound-healing
process aects the timely physiological repair
needed for anatomical and functional
integrity. The resulting chronic wounds are
generally dened as taking longer than
3 weeks to heal (Hermans and Treadwell,
2010). The chronic wound state may occur as
part of disorders such as diabetes or pressure
regions (e.g. bed sores) and is not always
initiated by infections.
Once infection occurs, it is imperative to
intervene by undertaking an eective wound-
care strategy that encourages deep tissue
homeostasis, allows the recolonization of
commensals and ultimately achieves wound
healing. In all cases, repeated irrigation of the
wound that removes foreign material and
infectious exudate, and thorough debride-
ment of all potentially devitalized and
necrotic tissue, is necessary. For supercial
local infections, the appropriate topical
antimicrobial(s) and wound dressings may
be all that is required, but for deep wound
and systemic infections, systemic anti-
microbials are necessary (Percival and Dowd,
2010). Co-morbidities and complications that
modify the strategy needed to treat a
particular wound infection include advanced
age, obesity, poor nutritional status, dys-
vascular diseases such as those seen with
smokers and diabetic patients, or circum-
stances where poor blood circulation occurs
around infection sites such as in osteomyelitis
and amputations.
Diabetic wounds
Acute as well as chronic wounds are common
complications of diabetes mellitus, which is
one of the most common causes of chronic
wound states, with or without infection.
Most diabetic wounds are on the feet (far
away from the heart and with the poorest
blood supply). Acute infections tend to be
mono microbial and due to aerobic Gram-
positive cocci, such as S. aureus and
|-haemolytic streptococci (i.e. groups A, C
and G but especially B), while chronic
infections tend towards more complex mix-
tures of organisms (polymicrobial), includ-
ing enterococci, various Entero bacteriaceae,
obligate anaerobes, Pseudo monas aeruginosa
and occasionally other non-fermentative
Gram-negative rods (Lipsky et al., 2004; see
Kuhl et al., Chapter 3, this volume).
Osteomyelitis
Osteomyelitis is an acute or chronic infection
of bone. It is most commonly caused by
bacteria but may also be due to fungi. It can
occur as a secondary complication of trauma,
surgery or the insertion of a foreign device
such as a joint prosthesis. It may also occur by
contiguous spread in regions of poor vascular
function, such as diabetic foot infections or
from a haematogenous dissemination from
infected skin sores or insect bites, urinary
tract infections or dental manipulation.
Haematogenous spread is commonly seen in
children (Lew and Waldvogel, 1997). Various
types of osteomyelitis require dierent
medical and surgical strategies, ranging from
short-term antibiotic therapy and limited
surgical incision and drainage for acute cases
to more extensive surgical debridement of
dead bone and long-term antibiotic treatment
(e.g. 68 weeks of intravenous administration)
in chronic cases. The infecting organism may
vary with age and underlying disease.
Children suering from acute haematogenous
osteomyelitis may tend to be infected by
Streptococcus pyogenes, whereas adults suf-
fering from chronic open wounds to the bone
and sof tissue may have P. aeruginosa.
Overall, 80% of osteomyelitis cases are due to
S. aureus (Howell and Goulston, 2011; see also
Kuhl et al., Chapter 3, this volume).
Early Phage Therapy of Wounds
This section highlights an early phage-
therapy study by MacNeal et al. (1942); we
direct the reader to Table 13.1 for an overview
of earlier studies. We also consider the Soviet
Union phage-therapy experience.
A 500-case study treating
staphylococcaemia
The last large phage-therapy study carried
out in the West, following the phage-therapy-
critical Eaton and Bayne-Jones reviews
(1934a,b,c), was completed by MacNeal et al.
(1942). They reported on the use of phage
therapy in 500 patients suering from
staphylococcaemia in the years 19311940,
collected from 169 hospitals in various parts
of the USA. The systemic infection in most of
these cases had resulted from local infection
such as abscesses, carbuncles and osteo-
myelitis. Patient inclusion in this series of
clinical trials required a request from the
atending physician to use phage therapy on
their patient, and a conrmation of staphy lo-
cocci being present in their bloodstream. It
was noted that a larger number of patients
were male, possibly due to a greater exposure
to external wounds and having less
meticulous care of the skin. In vitro testing of
416 cases that produced staphylococci isolates
from patients found that 89% of these
isolates were susceptible to the phage stock
preparation employed in this study.
Thirty patients did not receive the phage
therapy either due to death occurring prior to
commencing treatment or not wanting
treatment despite being initially included in
the trial these were counted as the negative-
control group. Twenty-ve patients received
phage therapy despite their blood cultures
becoming negative prior to commencing
treatment, and seven of them died. This later
group was considered the positive-control
group (see Abedon, Chapter 17, this volume,
for discussion of the use of controls during
phage therapy). Table 13.2 provides a sum-
mary of the results.
From their ndings, MacNeal et al. (1942)
concluded that early recognition of symp-
toms, a willingness to execute the phage-
therapy programme as prescribed and the
good judgement to use complementary
therapeutic measures would all improve the
rate of recovery. Unfortunately, the com-
bination of other measures such as using
sulfathiazole and fractional transfusions with
phage therapy, which they stated oered a
more favourable outlook in the severe forms
of infection, make it di cult to determine
whether bacteriophages played a part in
treating the infections.
This study, as well as those outlined in
Table 13.1, used phage ltrate preparations
that were only void of bacterial cells through
ltration. Although some researchers
speculated on the presence of bacterial
artefacts in these preparations, they were
unaware of bacterial toxins (i.e. endotoxins)
that we now recognize as responsible for
some of the side eects observed from phage
treatments (see Olszowska-Zaremba et al.,
Chapter 12, this volume, for additional
discussion of these issues).
The Soviet-era experience
Chanishvili et al. (2009) recently collected and
collated Soviet reports conducted during the
1930s to 1970s and translated them into
English. The results are described in this
section. When we consider that this therapy
was all that was available in the pre-antibiotic
era and later that the economics of medical
care in the Soviet Union only allowed limited
access to the variety of antibiotics available in
the West, it can be understood that the need
to care for patients, by whatever means
available, took precedence over rigorous
scientic methodology. Viewed in this
context, it is possible to glean some useful
clinical information from their data.
Early phage-therapy trials established
methods for the intramuscular and intra-
venous use of phage preparations. Phages
were most commonly applied topically at the
infection site and also mixed with local
anaesthetics and injected around the wound.
The results of these early trials indicated a
success rate of 80% in 114 surgical cases
infected with staphylococcus and a 72%
positive outcome in 1888 cases, in which the
28% of failed patient treatments was
atributed to mixed bacterial infections. These
reports prompted the Soviet Military to
institute the universal use of phage wound
therapy during the Finnish Campaign and
the Second World War. At the end of the
Soviet Era, the Eliava Institute, in the Republic
of Georgia, was producing 5 t of phage
products each week primarily for the military.
See Burrowes and Harper (Chapter 14, this
volume) for additional discussion of the
Eliava Institute.
Phage therapy in the
Soviet military
In the Finland Campaign, between 1938 and
1939, phage cocktails containing bacterio-
phages infecting strict anaerobes, staphylo-
cocci and streptococci were used to treat
patients with gas gangrene. In one study, the
mixture was applied to 767 patients with only
a 19% death rate compared with the control
group, which was treated with other
methods and resulted in 42% deaths. Another
series, using the same phage mixture
produced at the Eliava Institute, had 19%
deaths compared with 54% using other
medications. Surgical debridement and
amputation presumably were carried out as
required. The death/survival rates appeared
to be the end points, with no mention of limb
salvage. These studies have been reviewed by
Chanishvili et al. (2009).
Subsequent to the initial therapeutic use
of phage therapy, its prophylactic capacity
against gas gangrene was tested experi-
mentally in the eld. Three mobile
brigades, each composed of six persons
(three surgeons and three bacteriologists),
were to test bacteriophage cocktails specic
for staphylococci, streptococci and Clostridium
perfringens. The rst brigade treated 2500
wounded soldiers, with the result of only
1.4% displaying symptoms of gas gangrene.
In comparison, 4.3% of soldiers in the control
group (n = 7918) became infected. Data from
the other two brigades showed that, of 941
phage-treated soldiers, only 1.4% developed
gas gangrene compared with 6.8% of the
untreated controls (n is unknown). In another
set of 2584 phage-treated soldiers, only 0.7%
Table 13.2. A summary showing staphylococcaemia patients included in the phage therapy study by
MacNeal et al. (1942)
Patients
No phage therapy
positive blood
culture
Phage therapy
despite negative
blood culture
Phage therapy following positive
blood culture
Died Survived Died Survived
Died within
3 days
Died after
3 days Survived
Number of patients 25 5 7 18 137 166 142
Percentage of patients 83.3 16.7 28.0 72.0 30.8 37.3 31.9
developed gas gangrene compared with 2.3%
of the untreated controls (n is unknown). The
combined data indicated a 30% decrease in
the incidence of gas gangrene as a direct
consequence of prophylactic phage therapy.
Time-dependent results of prophylactic
therapy were revealed and it was concluded
that in those that were treated earlier, the
wounds were clear of infection sooner and
granulation appeared rapidly, temperature
was normalized in a shorter period of time
and unpleasant odours did not develop or
were insignicant (Chanishvili et al., 2009).
In 1940, one mobile brigade was moved
into more permanent hospital facilities. This
move allowed a more controlled scientic
environment and more standardized tech-
niques of blood analysis prior to phage
therapy. The times and descriptions of
surgical wound manipulation and phage
application were systematically recorded and
patients were grouped according to wound
infection type and anatomical location. This
work was published in 1941 in a book writen
by Professor Tsulukidze entitled Experience of
the Use of Bacteriophages in Conditions of War
Trauma. Chanishvili et al. (2009) described
Tsulukidzes work. In summary, he found
that, on arrival from the front, mixed bacterial
infections were the general rule and were
unlike infections seen afer long hospital
stays and prolonged manipulations, which
produced more complicated mixed infections
and therapeutic di culties including
nosocomial infection with Proteus species
(against which bacterium they had no phage
preparations). To avoid the in-hospital
evolution of wound infections to those with
complex polymicrobial communities and
Proteus superinfection (which seemed to
dominate other infections), bandaging
protocols were modied to prevent cross-
contamination between patients. In an
atempt to prevent the development of anti-
phage antibodies, subcutaneous injection of
phages was done three to four times each
second day rather than once daily. Apparently,
their assumption was that daily phage
injection was more immunogenic than their
regimen (see Olszowska-Zaremba et al.,
Chapter 12, this volume, for further discussion
of phageimmune system interactions).
Phages were also sprayed on to the wound
with each dressing change.
Sof-tissue wounds treated with ordinary
therapy had treatment with chloramines,
rivanol and Vishnevskys salve and were
dressed with tampons. Many such wounds
had abundant pus and complicated infections
with surrounding inammation as well as
necrotic foci, and retained bullet and wood
fragments. To control serious intoxication due
to sepsis and toxaemia, as well as high fever
and gangrenous inammation, these wounds
required extensive debridement and wound
purication with iodine and alcohol followed
by wound irrigation with a 2% sodium
chloride solution. The wounds were then
sprayed with phages and dressed with
phage-soaked gauze but not packed with
tampons. A volume of 510 ml of phage was
injected simultaneously into the abdominal
wall, shoulder or hip. Afer the rst one or
two phage applications the temperature was
normalized. Only three or four such
treatments were normally required to achieve
a complete cure and the blood composition
was also improved. The wounds were
stitched afer phage therapy on the 6th-8th
day of treatment, so that further infection was
unlikely. (Chanishvilli et al., 2009). Treatment
with phage therapy took a number of days
whereas ordinary therapy took several
weeks. No phage-therapy cases required
additional incisions or any other surgical
intervention.
Bone injuries were treated with the same
phage protocol including debridement,
phage wound dressing and phage injections
remote from the regions of the wounds.
Fractures were immobilized in casts or
traction. Phage-treated open-bone injuries
did not smell under a closed cast and the cast
could remain unchanged for 60 days allowing
fracture healing. In such wounds, without
phage therapy, it would be anticipated that
the casts would rapidly become saturated
with purulent wound drainage and have a
putrid smell, requiring frequent cast changes
and disturbing fracture xation and healing.
In cases that were less successfully treated
with phages, the reasons given included: the
presence of Proteus as the cause of the
infection (which the phages were not active
against), the occurrence of frostbite injuries,
long-term hospitalization (2140 days) and
accompanying diseases such as pneumonia,
inuenza and throat infections.
Topical phage applications were found to
cause few or no side eects. Injecting near the
wound was found to be painful for the patient,
while injecting remotely from the wound was
less painful but apparently similarly eective.
Sometimes redness occurred at the injection
site caused by the meat bullion used to
prepare the phages. A slight rise in
temperature (0.51.0C) was also noted. No
reaction was seen following the second or
third injections, however (see Olszowska-
Zaremba et al., Chapter 12, this volume, for
additional discussion). Tsulukidze had noted
that phage therapy shortened hospital stays
and that, by avoiding large surgical
interventions, he could decrease the number
of new infections.
Phage therapy trials on civilians
The compiled data of Chanishvili et al. (2009)
in general reported comparable responses
using similar techniques when applied in
civilian wound care and infections. Several
additional noteworthy observations are high-
lighted here. First, when treating furunculosis,
subcutaneous phage injections up to three
times in increasing doses (e.g. 1 ml, 2 ml and
3 ml over 3 days), with lancing and drainage
of the abscess and additional phage appli-
cation into the abscess along with phage-
soaked dressings, resulted in more successful
outcomes than extensive incision and
drainage while otherwise following the same
protocol. In summary, Chanishvili et al. (2009)
concluded that phage therapy could shorten
the duration of treatments, although with an
increase in duration if combined with in-
vasive methods such as lancing, incisions or
surgery, the later resulting in the longest
increase.
Secondly, persistent sof-tissue infections
(i.e. lasting 13 months), chronic osteomyelitis
and chronic amputation stump wounds
could be treated with topical liquid phage
cocktails using phage-soaked tampons or
bathing in phages. Such means of phage
administration were less traumatic to the
patient than subcutaneous or intramuscular
injections. Mixtures of phages active against
aerobes and anaerobes were used to soak
bandages. Dressings were changed every
23 days. In one group of 15 patients treated
in this way, 13 patients were completely cured
in 23 weeks, with the remaining two having
improved outcomes.
Lastly, to avoid disturbing open surgical
wounds following debridement of osteo-
myelitis, phages were mixed with 0.7% agar
and used to ll the wound. This allowed
phages to be released slowly and to linger in
the wound, promoting phage persistence for
78 days.
In 1974, Krasnoshekova and Soboleva
reported having used a standard pio-
bacteriophage cocktail produced by the
Eliava Institute (containing phages against
staphylococci and haemolytic Streptococcus
species, E. coli, P. aeruginosa and Proteus
vulgaris, also translated as pyo bacterio-
phage) and adapted further to strains
endemic to the Kazan region for treating 85
patients with chronic wound infections. They
found the pathogens to consist of Staphylo-
coccus species in 93% of the wounds and
selected specic phages against those patho-
gens. They reported 98% eective outcomes
when phage therapy was based on the in vitro
activity of the phages previously adapted to
the bacterial host infecting these patients
(Chanishvili et al., 2009).
Also in 1974, Matusis and co-workers
reported treating 94 patients with non-unions
(where a broken bone is not able to heal),
pseudoarthroses (false joint or established
non-union) of the tibia and amputation
stumps complicated by osteomyelitis. In 93%
of cases, osteomyelitis was caused by
staphylococci and 72% of these isolated
strains were susceptible to the corresponding
phage preparation (Chanishvili et al., 2009).
They had three treatment groups. For group
1, antibiotic therapy alone was employed
(n = 24). Healing of stumps took 112 months
(13 cases). Union (i.e. healing) of fractures,
took 613 months and of these, four occurred
within a 4-month period. Six osteomyelitic
stumps failed to heal, as did three fracture
non-unions. With group 2, a combination of
topical phages along with topical antibiotics
were used (n = 12). Healing of stumps took
0.53 months (12 cases). Fracture healing
occurred in 49 months. Two cases relapsed.
In group 3, treatments consisted solely of
topical phage therapy and/or intramuscular
phage injections (n = 53). Forty-seven of the
stump-wound infections healed in 0.32
months, with six cases taking 79 months.
There were no relapses. In this series, phage
therapy proved superior to both combined
phage and antibiotic therapy as well as
antibiotic therapy alone.
Human Wound Studies in the Late
20th Century
Human treatment of wounds using phages
has been ongoing in Eastern Europe, most
notably in Poland and the former Soviet
Union republic of Georgia.
Polish studies
In addition to the studies highlighted by
Chanishvili et al. (2009), there have been more
recent phage-therapy studies from Eastern
Europe that have been published in English
language journals. Slopek et al. (1987), for
example, reported on 550 hospitalized
patients treated with phage therapy between
1981 and 1986, with most treated within the
surgery departments, in Wrocaw, Poland. Of
the 40 cases of osteomyelitis seen, 29 cases
were Staphylococcus infections, three cases
were Pseudomonas infections and one was a
Klebsiella infection. Polymicrobial infections
were found in 11 cases, and 38 cases had been
resistant to antibiotics. Also summarized was
the treatment of cases of furunculosis,
decubitus ulcers (bed sores) and open
wounds (including burns). Table 13.3 sum-
marizes the outcomes of this study. See also
Table 13.3. Summary of the various types of wound infections treated with phage therapy by Slopek et
al. (1987)
Wound Infection type Result of treatment
a
Total
number
of cases
Number of
cases with
positive
outcome
0 +1 +2 +3 +4
Osteomyelitis Single-microbe
infection
0 2 3 19 5 40 36
(90%)
Mixed-microbial
infection
0 0 0 10 1
Furunculosis Single-microbe
infection
0 0 2 19 29 55 55
(100%)
Mixed-microbial
infection
0 0 0 3 2
Decubitus
ulcer
Single-microbe
infection
0 0 0 2 0 16 13
(81%)
Mixed-microbial
infection
0 3 0 9 2
Open wound
(including
burns)
Single-microbe
infection
1 1 0 13 7 49 42
(86%)
Mixed-microbial
infection
0 5 4 12 6
a
Scores represent the following results: 0, no effect; +1, transient improvement; +2, marked improvement with tendency
to healing; +3, elimination of pus and healing of wounds; +4, complete recovery. Positive outcomes were classed as
wounds that displayed scores of +2 or higher.
Olszowska-Zaremba et al. (Chapter 12, this
volume) for additional consideration of the
Poland experience.
Cislo et al. (1987) applied phage therapy
to 31 selected patients. Patients suering
from suppurative skin infections were chosen
based on no improvements seen afer
treatment with routine methods including
antibiotics, or the bacteria isolated from the
lesions displaying resistance to antibiotics.
The same phage administration strategy as
used by Slopek et al. (1987) was followed. No
other antimicrobial treatment (including local
disinfectants) was used at the same time as
phage therapy was administered. They
reported 16 cases resulting in outstanding
therapeutic eects, seven cases with marked
improvement and negative cultures, and two
cases with transient improvement of the
wound. Treatment was abandoned in six
cases due to side eects, although what
these side eects were was not specied in
the study. One case did not result in any
improvement.
PhagoBioDerm
TM
PhagoBioDerm
TM
is a wound-dressing pro-
duct co-developed by Georgian scientists
Zemphira Alavidze and Ramaz Katsarava,
and supported by an American company
(Intralytix Inc.) along with the University of
Maryland, MD, USA. It is a slow-releasing
biodegradable polymer lm impregnated
with the antibiotic ciprooxacin (0.6 mg cm
2
)
and a mixture of bacteriophage (PyoPhage,
1 10
6
plaque-forming units (PFU) cm
2
)
active against S. aureus, P. aeruginosa, E. coli,
Streptococcus and Proteus. To date, the clinical
experience of PhagoBioDerm has been
published in two uncontrolled studies.
Markoishvili et al. (2002) reported on a
series of clinical cases over a 1-year period,
presenting wounds that were healing poorly
due to inadequate vascularization, including
in patients with diabetes, that were refractory
to standard infection treatments. The wound
dressing was used on 96 patients aged
between 31 and 101 years. Treatment involved
irrigating wounds, applying PhagoBioDerm
lm and dressing with sterile bandages to
immobilize the lm. The wounds were
examined daily for the rst 5 days and once
every 24 days thereafer. Films were replaced
any time they became fragmented or had
completely degraded (i.e. every 37 days).
They were lef untouched if the lms had
tightly atached to the wound or the wound
had healed. Completely healed wounds were
seen in 67 patients (70%) and the time
required for infection-free treated sites
ranged from 6 days to 15 months. The
limitations to their studies were discussed
and included the lack of a placebo group,
which they had avoided because of concerns
that continued application of conventional
treatments would remain ineective.
Jikia et al. (2005) reported on treating
three patients who were exposed to
strontium-90 and had developed radiation
burns, which had subsequently become
infected with MRSA. Afer being treated with
a variety of antibiotics and topical ointments,
their infections persisted. Admission to the
hospital and failure to cure the infection by
standard hospital procedures prompted the
topical application of PhagoBioDerm. Only
the two patients who had persistent infections
were treated. Two days afer the lms were
placed on the skin ulcers, both patients
showed a dramatic decrease in purulent
drainage as well as wound pain. On day 7,
the ulcers were culture negative for S. aureus.
The authors concluded that the elimination of
the infecting bacteria and possibly the
marked improvement in wound healing
observed were due to PhagoBioDerm.
Animal Models
Western interest in phage therapy was
renewed by a series of animal studies in the
1980s investigated by Smith and colleagues in
the UK (Smith and Huggins, 1982, 1983;
Smith et al., 1987a,b; Sulakvelidze et al., 2001;
Abedon et al., 2011). They found that phage
therapy could be used to treat mice infected
with E. coli, and that it was even more eective
than multiple doses of an array of antibiotics
(Smith and Huggins, 1982). A decade later,
Soothill (1994) published one of the rst in
vivo animal experiments related to phage
therapy of wounds. A more detailed review
of several of these studies is given by
Burrowes and Harper (Chapter 14, this
volume). In this section, we review animal
models that have been used to investigate the
e cacy of phage therapy in specic types of
wounds and discuss some of the noteworthy
experimental designs and potential draw-
backs.
Burn wound models
Most of the published literature investigating
the e cacy of phage therapy on wounds has
described infected burn wounds. Many
studies have shown phage therapy to be
suc cessful at protecting animals from suc-
cumbing to infections that would otherwise
lead to death if untreated. Not all phage
products/treatments have been shown to be
equally potent, however.
Soothill (1994) demonstrated that phages
could be used prophylactically to prevent
destruction of skin grafs. To represent an
excised burn, he removed a full-thickness
skin section from the back of guinea pigs. The
wound was then inoculated with 100 l of
bacterial suspension, along with phage or
control suspension. The 0.2 mm thick graf
area that was initially removed was placed
back on to the defect area and covered by a
compression pack followed by a large
dressing. Skin grafs were blindly assessed
afer 5 days. Grafs were considered to be
successfully atached if the skin was pink and
blanched when pressure was applied. Six of
seven animals with grafs infected with P.
aeruginosa (6.0 10
5
colony-forming units
(CFU)) and treated with a puried single
phage, BS24 (1.2 10
7
PFU), had successful
outcomes. This compared with all seven
negative phage-treatment control animals
that had infected grafs but remained
untreated, resulting in grafs that failed.
McVay et al. (2007) conducted a study to
determine the protective ability of a phage
cocktail containing three phages on thermally
injured mice infected with a P. aeruginosa
strain (~3 10
2
CFU) that was directly injected
under the anterior end of the burn. The
cocktail inoculums (~3 10
8
PFU) were
administered intraperitoneally (IP), intra-
muscularly (IM) or subcutaneously (SC)
immediately afer the bacterial challenge.
Mice treated with IP injection of puried
phage showed a mortality rate of 12%
compared with 72% for IM, 78% for SC and
94% for untreated mice. The investigators
suggested that the reason the IP route was
most eective was due to an increased rate of
phage dissemination and therefore a higher
dose of phages delivery to their targets,
which they demonstrate from the detectable
levels of phage concentrated in the liver,
spleen and blood of the treated mice over a
48 h period.
Kumari et al. (2009a) evaluated ve
puried and well-characterized bacterio-
phages individually and as a cocktail.
Klebsiella pneumoniae (100% lethal dose (LD
100
)
= 10
6
CFU) was injected subcutaneously
directly under the anterior end of the burn.
Treated mice (n = 12) were immediately
administered phages (multiplicity of infection
(MOI) of 1.0) via the IP route to test their
protective capacity. Afer 72 h, there was only
an ~6% survival rate for untreated mice versus
8197% with the dierent individual phages
and 94% with the ve-phage cocktail
treatment. Full recovery and complete
regeneration of skin layers was seen on day 20
afer phage treatment. Continuing on from
this study, Kumari et al. (2010a) extended their
experiments using one of the phages in the
cocktail, Kpn5, to determine whether the
protective property of this phage was dose
dependent. Following SC injection of the
same previously used lethal dose of K.
pneumoniae, mice (n = 10) were injected IP with
100 l of the phage preparation at various
doses. Survival rates of 96% were seen with
higher phage doses. At 10-, 100- and 1000-fold
lower doses, survival rates were 53, 13 and
0%, respectively. Delayed phage treat ment
appeared to rescue 73, 47, 27 and 7% of the
animals, if administered at higher doses with
delays of 6, 12, 18 and 24 h, respectively.
Similar results were reported by Malik
and Chhibber (2009) for a mouse third-degree
burn wound model for which a subcutaneous
challenge with ten times the LD
100
of K.
pneumoniae suspension (i.e. 10
7
CFU) was
treated with IP or SC injection of pure
bacteriophage K1 (10
8
PFU) at 30 min and
6 h post-infection. While phage therapy
appeared to have protective eects against
mice with experimental burn wounds
infected with K. pneumoniae, similar burn
wound infections caused by P. aeruginosa
(10
7
CFU ml
1
) could not be treated and
resulted in 100% mortality (Kumari et al.,
2009b). Kumari et al. (2010b) compared the
use of other alternative antimicrobial agents
(i.e. honey and aloe vera gel) against phage
therapy. Topical phage treatment using a
single phage isolate suspended in 3%
hydrogel provided a high level of protection
(67% survival) at day 7 compared with 27
33% with non-phage treatment and 0%
survival without treatment. In a similar study,
Kumari et al. (2011) compared the daily
topical application of silver nitrate (5%) or
gentamicin (1000 mg l
1
) with the same burn
wound model and found these to have
survival rates of 57 and 53%, respectively, on
day 7, whereas a single phage treatment in
3% hydrogel resulted in 63% survivors. These
dierences were not found to be statistically
signicant.
Some concerns regarding the studies
carried out by Kumari and colleagues include
their only stating the concentration and not
the volume of bacteria used to inoculate the
wound. In addition, one must assume that
the phage treatment was administered
immediately afer the bacterial application, as
with their previous studies, although this is
not stated in some of their papers. In addition,
although the MOIs are stated in their articles,
it is di cult to determine the actual dose of
phage used for their therapeutic work. Their
explanations of the phage therapy failing to
treat their P. aeruginosa infected mice include
the possibility of biolm formation inhibiting
phage action, although this bacterial process
would seem unlikely to have occurred by the
time the phage were administered, that is, if
phage had been used as a protective treat-
ment. The development of phage-resistant
bacteria was another reason given as to why
this therapy did not work. However, they had
tested bacteria isolated from dierent organs
of the dead mice and found them to remain
sensitive to the phages, which they actually
state would indicate absence of such
mutants. See Abedon (Chapter 17, this
volume) for a more general discussion of
phage-therapy experimental design including
the various issues applicable commendably
as well as critically to the studies of Kumari
and co-workers. Despite not specifying some
of the technical parameters used in their
studies, Chhibbers group have still carried
out phage-therapy research using methodo-
logical scientic standards and are producing
very promising data.
Abscess models
While most modern in vivo phage-therapy
investigations of animal wound models have
been of infected burn wounds, there have
been a few papers considering animal models
of S. aureus abscesses. Wills et al. (2005), for
example, tested the e cacy of treatment
using a single Staphylococcus phage (LS2a) in
three rabbit studies. In the rst study, rabbits
(n = 8) were inoculated with SC injection of S.
aureus at 8 10
7
CFU and immediately treated
with SC administration of phage at 2 10
9

PFU (i.e. an MOI of 25) at the same site. Afer
4 days, only one rabbit developed an abscess
(area = 64 mm
2
). This abscess was smaller
than those found in the untreated rabbits
(median area = 106 mm
2
). In the second study,
infected rabbits (n = 4 per treated group)
received various doses of phage treatment of
6 10
7
, 6 10
6
or 6 10
5
PFU. On day 4, the
results indicated that the phages had some
dose-dependent prophylactic eect. The
median area of the abscesses in particular
decreased as the dose of phage administered
increased. The third study tested the
implications of delayed treatment. Rabbits
(n = 4) were infected with 5 10
7
CFU of S.
aureus, and 3 10
9
PFU of a Staphylococcus
phage at 6, 12 or 24 h afer bacterial injection.
There was no signicant decrease found in
the median area of the abscesses between the
treated and untreated groups (n = 5). Overall,
Wills and colleagues concluded that, although
some protection was achieved against
S. aureus infection, the results were not as
notable as those seen previously with Gram-
negative bacteria.
Capparelli et al. (2007) published a
thorough investigation on the e cacy of
phage therapy to treat S. aureus-infected mice.
Part of their investigation involved testing the
ability of their Staphylococcus phage (M
Sa
, a
long-circulating mutant) to be used as a
prophylactic agent to inhibit formation of
abscesses. Abscesses were induced in mice
(n = 5) through SC injections to the abdomen
using 10
8
CFU of a S. aureus strain. Phage
treatment (10
9
PFU) was administered
immediately afer, via the same route as the
bacteria. Additional experiments were also
used to determine whether their phages
could help infected mice recover from
established abscesses. A single phage dose,
administered 4 days afer the bacterial
inoculum, was compared with four daily
doses of phage treatment. They found that
the formation of abscesses could be inhibited
if the single phage treatment had been given
concurrently with the bacteria. If phage
treatment as single or multiple doses was
provided 4 days afer infection, however,
abscesses were not inhibited from forming,
although the bacterial load present in the
abscesses was signicantly reduced.
Interestingly, the multiple-dose treat-
ment was more eective at reducing the
overall weight of the abscesses than that of
the single dose. This observation was
interpreted as being due to the need for the
bacterial density to pass a required threshold
in order for active therapy to occur with the
single dose (as seen with their systemic
infection model), whereas the multiple-dose
treatment of the abscesses mimicked passive
therapy, allowing the phages to kill bacterial
cells through contact with large loads of
phage particles but without phage multi-
plication. This speculation seems unlikely for
two reasons: (i) the original bacterial dosage
was su cient to support active treatment;
and (ii) the original phage dosage, at least
potentially, was su cient to supply a good
approximation of a passive treatment. To
date, only a few published animal studies
have used multiple phage dosages in their
treatment, and not enough eort has been
made to understand the kinetics of the phage
and target bacteria. We may now be at a good
point, with our increasing understanding of
phage biology, to stop just thinking of using
phage therapy as a one-time shot and
instead start studying how phages can be
utilized in their best-case scenario such as
with multiple dosing (see Abedon, Chapter
17, this volume, for additional consideration
of this point).
Experimental design limitations
It is worth mentioning that some investigators,
including McVay and Kumari, used blood
collection tubes containing EDTA. EDTA is a
metal chelator and from our experience it has
been shown to aect phage atachment to
their bacterial hosts, resulting in a reduction
in the number of infective phage detected in a
sample. When 1 mM EDTA was present in the
growth medium, phage propagation was
inhibited and resulted in a 4 log dierence in
phage amplication when compared with the
control ask, over a 2 h period (unpublished
data). Citrate, another chelating agent, has
also been used in certain phage propagation
media to inhibit adsorption by those phages
that employ divalent cations as adsorption
cofactors (Hyman and Abedon, 2009).
Therefore, when collecting samples for
enumerating infective phage present, it may
be wise to take this into consideration and
use collection tubes without EDTA or other
chelating agents such as citrate ion present.
When designing the infected-animal
models needed to test the e cacy of phage
therapy, one should also consider the
pathogenicity of the bacterial strain to the
animal species chosen to study a particular
infection. In Walkers study (1931), the
bacterial strain used to infect his rabbits was
found to be avirulent, and therefore large
doses were required to produce any sort of
lesion. One must bear in mind, however,
that, by using large doses for bacterial
challenge, the conditions produced may not
be an accurate representation of infection as it
occurs in humans. Thus, the general practice
of using an inoculum to ensure death as an
end point (if the infection remains untreated)
might induce a vastly dierent host response
than if the infection had originated from a
low inoculum, as with most naturally
occurring infections (Zak and OReilly, 1991).
That said, investigators must also understand
the bacteria and infections they intend to
treat. Using S. aureus as an example, it is
known that this organisms virulence in
humans is in part a consequence of its ability
to extract iron from human red blood cells.
One virulent strain of the human pathogen
was found to be less eective at extracting
iron from mouse red blood cells and this
impedes infection in mice (Lowry, 2011). It is
therefore important to determine which
animal species and bacterial strains are
suitable to represent the infection that the
bacteriophages are being tested to treat. See
Zak and OReilly (1991) for a review on using
animal models in the evaluation of
antimicrobial agents.
Modern-day Clinical Phage-therapy
Trials
It is only through evidence-based science that
the appropriate application of phage therapy
can be determined. Phages are not truly
magic bullets capable of curing all bacterial
infections but they do have the potential,
teamed with the larger arsenal of anti-
bacterials and other techniques, to combat the
increasing problem of antibiotic-resistant
bacteria. Below are some examples of clinical
studies that have tried to follow the modern
standards required by the regulatory agencies
to support the development of therapeutics
that can be used as adjuncts or alternatives to
the currently available antibiotic regimens.
In 2006, Marza et al. published a single
case report involving a 27-year-old man with
a 50% total body surface area burn wound.
The burn wound developed an infection with
P. aeruginosa and, despite antibiotic treatment,
grafed areas required regrafing a number of
times due to rapid bacterial enzymatic graf
breakdown. Afer obtaining ethical approval
and informed consent, phage therapy was
atempted based on previous work by Soothill
(1994) showing its potential. The puried
phage chosen for the treatment was shown to
cause lysis of the P. aeruginosa isolated from
the patient and not to be toxic to human
keratinocytes. Phage suspension (10
3
PFU)
was applied to paper discs (25 mm diameter)
and placed on the infected areas. Afer 48 h,
phage counts in the discs had apparently
amplied by up to 1200-fold. Three days afer
phage treatment, no P. aeruginosa was
cultured from swabs. Marza et al. (2006)
carefully remarked that the improvement in
the infection could also have been due to
intravenous cefazidime the patient was
receiving at the same time as the phage
treatment.
Rhoads et al. (2009) reported results from
a phase I clinical safety trial. The safety of an
eight-phage cocktail suspension (WPP-201;
containing 1 10
9
PFU ml
1
of each phage)
against S. aureus, P. aeruginosa and E. coli was
tested through a randomized, controlled,
double-blind study. However, the report did
not specically mention any improvements
in the infected patients. This may be because
the study was biased towards not harming
the patients and there was no mention about
ensuring that the phages used were
appropriate for the pathogens present in the
infected wounds, which may explain the
poor e cacy seen with this phage therapy.
Merabishvili et al. (2009) produced a
phage cocktail that met the regulatory
standards needed for a therapeutic product
to be used in human clinical trials. Their
cocktail (BFC-1 at 3 10
9
PFU ml
1
) consisted
of two Pseudomonas phages (14/1 and PNM)
and one Staphylococcus phage (ISP), all found
to exhibit a large host range specically
against burn-wound bacterial isolates. Eight
patients suering from infected burn wounds
were treated without any adverse eects
being seen. However, no e cacy results were
published in their paper.
Future endeavours
We are currently investigating the use of
bacteriophages as prophylactic and thera-
peutic agents in a rat model of osteomyelitis.
If proved e cacious, we would anticipate
that this therapy could also be used as a post-
operative prophylactic treatment for ortho-
paedic device implantations or as infection
prevention sprays for wounded soldiers on
the batlefront. In relation to orthopaedic
devices, one particular interest in our
laboratory is the use of phages in infection
prevention sprays or washes for patients with
percutaneous osseointegrated devices, which
are implanted through the skin into the bone
of an amputated limb. Bone integrates with
these implants and the protruding stem
provides a robust skeletal docking system for
articial limbs. This atachment is especially
suited to short-stump amputees and patients
with multiple limb loss, in whom conventional
socket atachment technology fails, which is a
population that has grown as a result of the
Iraq and Afghanistan wars. The signicant
advantages of this system have been proven
in human volunteers in Europe (Tillander et
al. 2010), although infection remains a
problem in 1030% of these implants.
With regard to batleeld wounds, despite
current infection intervention strategies
implemented to treat combat-related injuries
in soldiers, including antibiotics, one-third of
such injuries have been complicated by
infections (Murray, 2008; Brown et al. 2010).
Therefore, we must endeavour to nd
alternative treatments to current practices,
which could even mean revisiting the early
practices of using phages in the batleeld,
duplicating the experience of Soviet military
medicine.
Closing Remarks
Animal models seem to indicate that the
application of bacteriophages, as prophylactic
agents, has certain advantages over currently
approved therapies and other alternative
approaches. The use of phage as therapeutic
agents for treating acute or chronic infections,
however, still remains outside the standards
of Western medical practice. Furthermore,
the data from Eastern Europe has not been
felt worthy of scrutiny by mainstream
Western medicine, the impression being that
these studies lack the prospective, double-
blind evidence required to meet current
regulatory standards for the approved use of
a therapeutic agent. It is therefore imperative
that investigators follow good phage-therapy
practices when designing and conducting
their animal studies (see Abedon, Chapter 17,
this volume), and most importantly when
conducting human clinical trials. From the
growing number of phage-therapy studies
published more recently, and with the
particular emphasis on developing phage
preparations that meet the standards of
regulatory agencies, one could speculate that
a small yet momentous wave of clinical
phage-therapy trials will begin to be
performed within the next 5 years (as
declared by Rhoads et al., 2009 and
Merabishvili et al., 2009; see also Burrowes
and Harper, Chapter 14, this volume). The
results should help determine whether phage
therapy can truly become a standard of care
in Western medicine.
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14 Phage Therapy of Non-wound
Infections
Ben Burrowes
1
and David R. Harper
1
1
Ampliphi Biosciences Corporation.
Antibiotic resistance had been observed
both clinically and experimentally among
bacterial isolates even before the widespread
clinical use of antibiotics (Waksman et al.,
1945; Wainwright and Swan, 1986). At rst,
such resistance was of litle concern, as new
antibiotics were routinely becoming available.
Bacteria, however, have continued to become
ever more drug resistant and, with regard to
the treatment of certain resistant pathogens,
entire classes of antibiotics have become
ineective (Boucher et al., 2009). The relentless
evolutionary pressure imposed by decades of
both antibiotic use and close proximity of
infected or susceptible patients (e.g. in
hospitals) has diminished the clinical value of
most if not all antibiotics. Pathogens and
opportunists such as vancomycin-resistant
Enterococcus (VRE), methicillin-resistant and
vancomycin-resistant Staphylococcus aureus
(MRSA and VRSA, respectively), Pseudomonas
aeruginosa and Acinetobacter baumannii are
extremely di cult to treat using conventional
approaches and have required concerted
eorts to prevent colonization of new patients
principally by following strict barrier
nursing procedures (Boyce et al., 1994; Jarvis,
2010).
Phage therapy is an old idea but one that
is gaining renewed interest in the face of the
clinical challenges being brought by bacterial
drug resistance. As such, phage therapy and
biology have been reviewed extensively in
journals (Sulakvelidze et al., 2001; Summers,
2001; Thiel, 2004; Abedon, 2010; Kuter et al.,
2010) and monographs (Summers, 1999;
Kuter and Sulakvelidze, 2005; Husler, 2006;
Clokie and Kropinski, 2009; Abedon, 2011b),
all of which oer valuable insights into
various aspects of phage therapy and biology.
For the purposes of this book, bacterial
infections have been divided into two
arbitrary groups: wound infections (see Loc-
Carrillo et al., Chapter 13, this volume), in
which the bacteria infect damaged or broken
tissues, and non-wound infections, in which
bacteria infect previously undamaged tissue.
It is possible, however, that one type of
infection can lead to the other, for example,
bacteraemias. In this chapter, we briey
consider the history of phage therapy as it
pertains to non-wound infections, before
detailing more up-to-date experimental
models and results.
Discovery and history
Very soon afer their discovery (dHrelle,
2007), phages were put to use in treating
bacterial diseases (dHrelle, 1922).
Diarrhoeal diseases were among the rst to
be treated, and the positive results led to
work on other human and animal diseases.
B. Burrowes and D.R. Harper
204 B. Burrowes and D.R. Harper
Numerous scientists and clinicians were
su ciently convinced by the results to devote
time and resources to studying or
administering phage therapy (dHrelle,
1921, 1929; Smith, 1924; dHrelle et al., 1928;
Asheshov et al., 1930, 1931; Morison, 1932). Of
particular note was the so-called
Bacteriophage Enquiry in which phages
were used experimentally in India to treat or
prevent cholera, with apparent success
(dHrelle, 1929; Morison, 1932; Summers,
1993). Subsequent mixed results and the rise
of antibiotic use, however, led to substantial
reductions in the use of phage therapy
worldwide (Eaton and Bayne-Jones, 1934;
Krueger and Scribner, 1941). Broader
consideration of the history of phage therapy
can be found elsewhere (Sulakvelidze et al.,
2001; Summers, 1999, 2001; Thiel, 2004;
Husler, 2006; Chanishvili and Sharp, 2009).
For the remainder of this section, we briey
provide an overview of work that went on,
particularly in the former-Soviet republic of
Georgia and Poland and which continues to
the present. Details of work elsewhere, such
as that in France and Switzerland, are
discussed by Abedon et al. (2011).
Georgia
Following positive outcomes of early phage-
therapy trials conducted in various countries,
the Soviet Union commissioned the creation
of a bacteriophage research institution in
Tbilisi, Georgia now called the Eliava
Institute of Bacteriophage, Microbiology and
Virology (IBMV), and this institute is actively
isolating, characterizing and producing
therapeutic phages to this day. Phage therapy
is in fact widely used in Georgia, so much so
that it is essentially considered a standard of
care among local practitioners (Kutateladze
and Adamia, 2008; Chanishvili and Sharp,
2009).
In appreciating the issues of phage host
range and bacterial resistance, the IBMV has
essentially always employed therapeutic
phage cocktails, which are mixtures of phages
that can be active against a wide range of
bacterial strains and species associated with
dierent disease types (Chan and Abedon,
2012). Cocktails were generated and main-
tained for pyogenic, intestinal, urological and
gynaecological diseases, as well as developing
monophage preparations for individual
patients or bacterial strains, and generation of
bespoke cocktails as required. Two phage
preparations in particular are produced in
large quantities for general use and are even
available over the counter at Georgian
pharmacies. Both are derivations of cocktails
originally developed by dHrelle and are
known, variously, as Intestiphage and
Pyophage.
Intestiphage is one of the longest
standing phage preparations (Chanishvili
and Sharp, 2009). The preparation is made
empirically using both environmentally
isolated and laboratory-developed phages
that are only tested for their plaque
morphology and host range. The phages are
then propagated in bulk by growing multiple
phages on a small number of hosts (Dr Z.
Alavidze, IBMV, personal communication).
Intestiphage there fore consists of an
essentially unknown num ber of poorly
characterized phages active against common
gut pathogens. The preparation is still
available over the counter at pharmacies
across the former Soviet Union. Phages active
against Shigella spp., Staphylo coccus, Salmon-
ella spp., Escherichia coli, Proteus spp. and P.
aeruginosa isolates were pooled and sold as a
lter-sterilized crude lysate to be taken orally,
either as a treatment or prophylactically.
Human studies of this preparation, in its
myriad forms, were carried out and usually
reported to be successful. Many of these
studies have been thoroughly reviewed by
Chanishvili and Sharp (2009). In one study,
more than 15,000 children received anti-
dysentery phages prophylactically with
signicant success compared with a no-phage
control group of nearly 3000 children
(Babalova et al., 1968).
Another study, reviewed by Kutateladze
and Adamia (2008), investigated phage
treatment of children using an Intestiphage
preparation. The study divided the children
into three treatment groups: phage treatment
alone, phages plus antibiotics, and antibiotics
alone. The result was a reported superiority
of phage treatment alone compared with
Phage Therapy of Non-wound Infections 205
treatment with antibiotics, with or without
co-treatment with phage. Recovery time fell
from 29 days with antibiotics alone to 9 days
in the group treated only with phage. In yet
another study, discussed in the same review,
more than 18,000 children were enrolled in a
study to assess prophylactic use of anti-
cholera phages. Nearly 16,000 of these
children were administered phages (each
dose equating to approximately 25 ml of
phage culture perhaps 10
8
10
10
plaque
forming units (PFU)) resulting in as much as
a vefold reduction in the number of cholera
cases (Kutateladze and Adamia, 2008).
The Pyophage preparation, commonly
used to treat wound infections in particular,
includes phages active against the common
pathogens E. coli, S. aureus, P. aeruginosa,
Streptococcus and Proteus strains. These
organisms overlap with common vaginal and
urinary tract infections, and Pyophage has
therefore been used to treat non-wound
infections as well. Where Pyophage was
compared with antibiotic treatment, it was
found that combined treatment (phages plus
antibiotics) was preferable (Kutateladze and
Adamia, 2008). Other reports from the
Institute suggest that Pyophage is also
benecial for treating mastitis (Chanishvili
and Sharp, 2009). See Loc-Carrillo et al.
(Chapter 13, this volume) for a more extensive
consideration of the phage therapy of wound
infections.
Poland
In 1952, the Institute of Immunology and
Experimental Therapy was founded in
Wrocaw, Poland, by the Polish Academy of
Sciences. The institute is now named the
Hirszfeld Institute afer its rst director and
founder, Ludwig Hirszfeld. The Hirszfeld
Institute has carried out many studies of
phage therapy (as summarized by Slopek et
al., 1987; see also Olszowska-Zaremba et al.
and Loc-Carrillo et al., Chapters 12 and 13,
this volume), typically with phage treatment
atempted only afer conventional therapies
have failed. Recently, the Institute has also
become involved in phage therapy unrelated
to infection control, such as the potential of
phages, as immunologically active agents, to
treat cancer. For a more in-depth discussion
of this work, see Olszowska-Zaremba et al.
(Chapter 12, this volume).
The 1980s rediscovery and 1990s
renaissance
Western research into phage therapy was
rekindled when Smith and Huggins began to
carry out well-designed, thorough and
scientically rigorous animal trials of phage
therapy (Smith and Huggins, 1982, 1983;
Smith et al., 1987a,b). Central to their design
was the use of either naturally occurring
infections or animal models that closely
paralleled human infections, much the same
approach that dHrelle had practised
(Summers, 1999).
Of mice and ruminants
Smith and Huggins began by using a human
E. coli isolate that was known to be virulent in
both humans and mice (Smith and Huggins,
1982). They then isolated a phage that infected
these cells via the bacterial K1 antigen, an E.
coli outer-membrane antigen that is important
in the virulence of this organism. Mice were
infected intramuscularly (IM) with 3 10
7

colony forming units (CFU; equivalent to
around 100 times the 50% lethal dose (LD
50
))
or intracerebrally with 500 CFU (around ten
times the LD
50
). A single dose of phages
containing 3 10
8
PFU was then inoculated
IM into the opposite ank for those mice that
received an IM bacterial inoculation and
compared with mice who received antibiotics.
Both single and repeat doses of streptomycin,
tetracycline, ampicillin, chloramphenicol and
trimethoprim/sulfafurazole were used as
comparison groups. The only antibiotic
regimen that rescued as many mice as a single
phage dose was eight treatments with strepto-
mycin (two and three mice died, respectively,
from groups of 30), leading the authors to
conclude that phages were generally superior
to currently available treatments (Smith and
Huggins, 1982).
Smith and Huggins then treated juvenile
pigs, sheep and cows with phages as therapy
against experimental infections with entero-
pathogenic E. coli strains of demonstrated
pathogenicity in these animals (Smith and
Huggins, 1983). Experimental animals were
infected orally with su cient bacterial cells
to induce diarrhoea and were then
administered orally with between 10
9
and
10
11
PFU of a mixture of two phages,
depending on the exact protocol. Phages
were given either at 1 or 8 h post-infection, or
at the onset of diarrhoea. Although earlier
phage treatment led to higher survival rates
and faster recovery, even application of
phages at the onset of diarrhoea resulted
in clearly demonstrated phage-mediated con-
trol of diarrhoeal disease. They also sug-
gested that phages in the faeces of the treated
animals could have a protective eect for
other animals sharing the same pen. No
direct comparison was made with antibiotic
treatment, but the use of a single dose of
phage and the protective eect of phage
treatment to other animals were convincing
arguments for the e cacy of phage therapy.
Prevention of E. coli infection was later
demonstrated in calf pens by spraying phages
in pens, or even just by housing calves in
pens previously inhabited by phage-shedding
animals (Smith et al., 1987a). In these
experiments, K1-tropic phages were also
used and the resultant K1
E. coli mutants (i.e.

those bacterial mutants that had lost their K1
capsule and were therefore selected for in the
presence of the K1-tropic phage) were less
virulent than the K1
+
parent again high-
lighting advantageous properties of phage
use over antibiotic treatment (this issue is
discussed at length by Levin and Bull, 1996).
Perhaps the most important aspect of Smith
and Huggins work was that their experi-
ments were rigorously designed, using
appropriate controls and large enough
groups of animals to conrm their central
conclusions. Signicantly, their work also
was conducted in the West (the UK, in
particular) and was published in English
language journals.
At the same time as Smith and Huggins
work was being published, the Hirszfeld
Institute in Poland also began publishing in
English (see discussion of Polish work above;
see also Olszowska-Zaremba et al. (Chapter
12, this volume). Their trials were not as
rigorous as those of Smith and co-workers
but did involve human subjects, and they
reported no serious side eects and provided
general evidence of e cacy.
Going beyond Smith et al.
During the 1980s and into the 1990s, antibiotic
resistance was starting to become a signicant
clinical concern. Alternatives were needed
and both researchers and clinicians were
more open to ideas that previously had been
rejected out of hand. Phage therapy, as a
consequence, gained momentum once more.
This time, however, both the scientic and
regulatory frameworks were far more
stringent than they had been during
dHrelles day, meaning that much of the
work of the Eliava and Hirszfeld Institutes
had to be built on to provide clinical data
acceptable to the regulators.
Addressing this need, the British clinician
James Soothill developed multiple experi-
mental approaches to demonstrate the e cacy
of phage therapy for pathogens other than E.
coli (Soothill et al., 1988; Soothill, 1992, 1994).
Phages were used successfully, in vitro, to
prevent destruction of pig skin samples by P.
aeruginosa (Soothill et al., 1988). Importantly,
Soothill developed a simple mouse model to
assess phage treatment of experimental A.
baumannii, P. aeruginosa and S. aureus in-
fections (Soothill, 1992), thereby enabling
phage therapy of multiple diseases to be
assessed in a single animal model. The results
showed e cacy of phages in treating
Pseudomonas and Acinetobacter infections,
although they did not show e cacy in treating
S. aureus. This later result may have been
associated with the poor in vitro activity of the
phages they used, as generally, and as seen
similarly with antibiotics, poor antibacterial
performance in the test tube is typically
expected to be predictive of poor performance
in vivo (see Abedon, Chapter 17, this volume,
for additional discussion along these lines).
As will be discussed below, however, this
rule of thumb may not always hold.
Recent animal models
Current regulatory frameworks demand
stringent testing of disease treatment
protocols in animals before testing in humans
is considered appropriate. In spite of its
history, this applies as much to phage therapy
as to other medicines. Many in vitro models of
phage activity and therapy exist, but they are
limited in their usefulness for studying the
complex pharmacology of the large, self-
replicating nucleoprotein complexes that are
phages (Payne and Jansen, 2003; Levin and
Bull, 2004; Abedon and Thomas-Abedon,
2010; see also Abedon, Chapter 17, this
volume, for additional discussion of phage-
therapy pharmacology). In order to under-
stand the pharmacology of phage therapy, we
must determine the pharmacokinetics (the
fate of the administered agent in vivo) and
pharmacodynamics (the physiological and
therapeutic activity of the administered
agent) of phage therapeutics, which, due to
both the complexity of phages and their
ability to self-replicate, are fundamentally
dierent in phage therapy compared with
chemotherapeutic approaches, a theme that
has been explored at length by Curtright and
Abedon (2011). It is therefore extremely
valuable to develop relevant, high-quality
animal disease models to study the
application and outcomes of phage therapy.
We will now briey discuss recent animal
models of particular relevance to clinical
applications.
Septicaemia
The ease with which blood-borne bacterial
infections can be induced in mouse models
has been exploited for many bacterial
pathogens, and phage treatment is usually
successful in these models. One of the most
common models involves intraperitoneal (IP)
injection of bacteria to induce bacteraemia in
mice, which is challenged with phages either
prior to infection, simultaneously with
infection or post-infection. Such models have
been used for the treatment of bacterial
infections including E. coli (Merril et al., 1996),
Salmonella typhimurium (Merril et al., 1996), S.
aureus (Matsuzaki et al., 2003), VRE (Biswas et
al., 2002; Wang et al., 2006b), Klebsiella
pneumoniae (Vinodkumar et al., 2005) and P.
aeruginosa (Wang et al., 2006a; Vinodkumar et
al., 2008; Heo et al., 2009). These animal
models typically report e cacy of phage
treatment, although with varying degrees of
therapeutic success. In general, it appears
that factors such as the half-life of phages in
vivo (a pharmacokinetic property), virulence
of the phage(s) chosen both in vitro and in vivo
(pharmacodynamic properties) and other
components of the animal/phage/host system
inuence the success of phage treatment or
prophylaxis. The numbers of phages required
to eectively rescue mice from infection
depends on the ability of the phages to kill
their target cells in situ: phages that are less
e cient at killing their target cells will require
larger numbers to su ciently lyse host
bacteria than would be needed when using
more e cient phages. Dosing parameters are
particularly signicant when considering
active versus passive phage therapy. Active
phage therapy requires phage propagation in
situ to bring about a therapeutic eect,
whereas passive phage therapy uses higher
doses of phages and further phage propa-
gation is not required to generate therapeutic
e cacy. This is discussed further by Abedon
Phage pharmacology is in its infancy but
has been discussed in several studies and
theoretical papers (Geier et al., 1973; Payne
and Jansen, 2003; Levin and Bull, 2004;
Dabrowska et al., 2005; Gill et al., 2006; McVay
et al., 2007; Abedon and Thomas-Abedon,
2010; Abedon, 2011a). Perhaps most
importantly, no correlation between antibiotic
resistance and phage resistance has been
reported so far, suggesting that phage therapy
could prove especially useful in treating
drug-resistant bacteria.
Lung infections
Chronic bacterial colonization of the lung,
especially in immunocompromised patients,
represents a signicant challenge clinically
because it can be di cult to maintain suf-
cient chemotherapeutic concentrations at
the site of infection. Phage treatment of such
infections may be desirable, as phage
propagation may lead to su cient phage
numbers in situ to control infections.
Conversely, it may also be problematic to
deliver su cient phage numbers into the
deep lung tissue to bring about clearing of the
bacteria. Animal models therefore are highly
useful to ascertain such limitations, as well as
the potential of phage treatment of lung
infections.
Intranasal (IN) administration of lung
pathogens has been used by several groups to
assess phage treatment of murine pulmonary
infections, and phages can then be applied by
any route to determine treatment or
prophylactic e cacy. This model has been
used to study phage treatment of K.
pneumoniae (Chhibber et al., 2008), which
causes bacterial pneumonia and is associated
with signicant mortality, especially resulting
from nosocomial spread (Podschun and
Ullmann, 1998). A similar model of
Burkholderia cenocepacia infection also
demonstrated e cacy of phage treatment
(Carmody et al., 2010). In both the K.
pneumoniae and B. cenocepacia models,
however, IP administration of phages proved
e cacious whereas IN administration did
not. This could be problematic, as IP
administration is a time-consuming, invasive
and potentially risky procedure that is rarely
used clinically. When essentially the same
model was used for P. aeruginosa infection, IN
administration was shown to be eective
(Debarbieux et al., 2010; Morello et al., 2011),
raising the question of why IN administration
is eective in this model compared with
others. More work is required to ascertain the
factors involved in phage treatment success
or failure.
Gastrointestinal disease
Despite the long history of phage treatment
of alimentary diseases, few animal studies of
phage therapy of this class of disease are
found in the modern literature. Such models
do not appear to be di cult to work with, as
the bacteria can be applied orally, as well as
the phage, although intragastric adminis-
tration of phages is also used. Studies with E.
coli (Chibani-Chennou et al., 2004; Denou et
al., 2009) and P. aeruginosa (Watanabe et al.,
2007) have all shown phage therapy to be
eective in reducing pathogen load and
rescuing mice from infection, albeit to varying
extents. A rabbit model has also been used to
assess phage treatment of cholera, again with
apparent success (Bhowmick et al., 2009). It is
important to note that prophylactic use of
phages will require phage numbers to be
maintained at su cient levels at the site
of future infection to prevent bacterial
colonization. In some cases, therefore, non-
pathogenic phage hosts present in the normal
ora could sustain phage numbers prior to
pathogen challenge (see Letarov, Chapter 2,
this volume). E. coli, for example, is a major
bacterial component of the normal gut ora
and as such may potentially maintain phage
numbers in the gut prior to infection, whereas
Vibrio cholerae is not present in the gut of
healthy animals, implying that prophylaxis
may be less useful for this type of infection.
While temperate phages are usually
avoided for use in phage therapy, temperate
phages were used for treatment of Clostridium
di cile ileocecitis in a hamster model system
(Ramesh et al., 1999). Currently, no published
reports exist of the isolation of virulent C.
di cile phages, so a temperate phage was the
only option in this case. Phages were
administered orally following neutralization
of gastric acids to minimize phage
degradation in the stomach. Both phages and
bacterial cells were administered intra-
gastrically under anaesthesia, and phage-
treated animals were signicantly rescued
from death compared with antibiotic-treated
or untreated animals. Temperate phages of C.
di cile are known to encode or regulate toxin
production in this species (Govind et al.,
2009), so it would be of great value (see
Abedon, Chapter 17, this volume) to ascertain
whether phage therapy using such phages is
potentially harmful in this model by assessing
toxin production and lysogeny in faecal C.
di cile isolates afer phage treatment. It is
also interesting to note that gastric
neutralization may not always be necessary,
as some phages are able to survive the gastric
pH and reach the bowel in signicant
numbers without this step (Chibani-
Chennou et al., 2004; Brutin and Brussow,
2005), although this characteristic would still
need to be demonstrated on a phage-by-
phage basis.
Although temperate phages are usually
considered unt agents for phage therapy, it
appears that they may nevertheless reduce
bacterial load and symptoms in various
bacterial diseases. Merril et al. (1996) used a
derivative of the temperate bacteriophage ,
which had been serially passaged to increase
its half-life in the murine bloodstream, to
treat bacteraemic mice. This work demon-
strated that even temperate phages are
capable of mitigating bacteraemia symptoms
in their model, especially when the phage
had been evolved to perform beter in vivo by
serial passage. While temperate phages are
generally proscribed for therapy due to their
potential to augment the pathogenicity of
their bacterial hosts, nevertheless they
possibly may still be used where no
alternatives exist, although with due
consideration to their enhanced potential for
genetic transfer (see Christie et al. and
Hendrickson, Chapters 4 and 5, this volume).
Canine otitis
While not all animal diseases are relevant to
human disease, veterinary trials can still be
used to pave the way for human clinical trials
where there is overlap between the animal
and human disease, such as in diseases of
similar bacterial aetiology. Natural occur-
rences of canine otitis were used for a eld
trial as a veterinary precursor to regulatory-
approved human clinical trials in the UK
(Wright et al., 2009; Hawkins et al., 2010).
Pet dogs were selected that demonstrated
chronic, refractory otitis caused by P.
aeruginosa and were treated with a cocktail of
phages by placing the puried phage
preparation into the ear canal. Ten dogs were
treated once with 0.2 ml of a cocktail
containing six phage strains (1 10
5
PFU per
phage strain, equivalent to a total of 2.4 ng of
therapeutic agent). In all treated animals, the
mean bacterial count fell by 67% at 48 h afer
phage inoculation, while phage numbers had
increased approximately 100-fold. Clinical
observations showed that otitis symptoms
had been reduced in all treated dogs. The
positive results of this work were su cient to
allow this phage preparation to progress to
UK Medicines and Healthcare Products
Regulatory Agency-regulated phase I/II
clinical trials, which will be discussed below
(see also Abedon, Chapter 17, this volume).
Modern clinical trials
A brief list of human uses of phage therapy is
given in Table 14.1. Ultimately, phage therapy
is unlikely to be accepted in the West until it
satises the regulatory authorities by
demonstrating e cacy and safety in fully
regulated trials. While it has been suggested
that phage therapy warrants a loosening of
the regulatory standards (Verbeken et al.,
2007), it must none the less prove itself to the
same standards as all modern drugs, no
mater how distinct it is from current
therapies. Meanwhile, the resurgence of
phage therapy in the West has led to the
creation of several small companies. In
addition, some large, multinational com-
panies have expressed an interest in develop-
ing and, ultimately, commercializing phage
products for human use. In some cases, the
clinical research has focused on wound-care
applications, which are discussed by Loc-
Carrillo et al. (Chapter 13, this volume).
The rst modern Western trial of phage
therapy was carried out by the Port
Washington, New York-based company
Exponential Biotherapies Inc. (Thiel, 2004).
The trial was an approved phase I assessment
of toxicity of phages active against strains of
VRE but which unfortunately has yet to be
reported in the literature. Exponential
Biotherapies Inc. is currently trying to
develop small-molecule therapeutics (see
http://www.expobio.com/company-profile/
index.php), and the phage-therapy arm of the
company was spun o as EBI Food Safety
(now called Micreos Food Safety), which is
active in control of food-borne bacterial
pathogens (see htp://www.micreosfood
safety.com) (see also Niu et al., Chapter 16,
this volume).
A recent report described small-scale
production of a puried phage preparation
for use in an as yet unreported clinical trial to
be carried out with the Belgian military
(Merabishvili et al., 2009). While the trial was
for burn patients (a wound), the techniques
and regulatory processes are of great
importance in paving the way for production
of phage preparations for potentially any
application, although more stringent
requirements may be imposed for parenteral
phage use. Conversely, a relatively crude
preparation was used by Nestl for oral
administration in a test of phage safety
(Brutin and Brussow, 2005). T4 phage was
prepared simply by amplication, ltration
and density-gradient ultracentrifugation
before being given to volunteers orally. No
adverse eects were reported and the
company is now moving ahead with a large
trial of phage treatment of E. coli diarrhoea in
children in Bangladesh using an in-house
cocktail comprised of T4-like phages as well
as a commercially supplied phage cocktail
and standard-of-care treatments (htp://
clinicaltrials.gov/ct2/show/NCT00937274).
Another phase I clinical trial of phage
therapy has been carried out, using a cocktail
of phages active against several bacterial
species, in this case of chronically infected
wounds (Rhoads et al., 2009). No adverse
eects due to phage treatment were reported
afer topical administration of the cocktail
(see also Loc-Carrillo et al., Chapter 13, this
volume).
As mentioned above, a canine otitis trial
in the UK (Hawkins et al., 2010) was carried
out as a precursor to clinical trials regulated
by the Medicines and Healthcare Products
Regulatory Agency in the UK (Wright et al.,
2009). This is the rst and currently the only
phase II trial of phage therapy to have been
carried out under full Western regulation.
As a placebo-controlled, double-blinded,
randomized trial, this is the rst work to truly
bring phage therapy to the levels of scrutiny
Table 14.1. A brief list of human uses of potential applications for phage therapy.
Infection site Target organism(s) References
Blood (septicemia) S. aureus, Klebsiella spp., E. coli, P.
aeruginosa, Proteus spp.
Slopek et al., 1987; Weber-Dabrowska
et al., 2003; Husler, 2006;
Kutateladze and Adamia, 2008;
Chanishvili and Sharp, 2009
Eye Staphylococcus spp., Klebsiella spp., P.
aeruginosa, Proteus spp., Neisseria
spp., Streptococcus pyogenes
Slopek et al., 1987; Chanishvili and
Sharp, 2009
Ear P. aeruginosa Weber-Dabrowska et al., 2000;
Wright et al., 2009
Respiratory S. aureus, E. coli, Klebsiella spp.,
Proteus spp., P. aeruginosa,
Streptococcus viridans
Slopek et al., 1987; Kutateladze and
Adamia, 2008
Alimentary Salmonella spp., E. coli, Shigella spp.,
Enterococcus spp., Staphylococcus
spp., P. aeruginosa, Proteus spp.,
Klebsiella spp.
Knouf et al., 1946; Slopek et al., 1987;
Bruttin and Brussow, 2005;
Chanishvili and Sharp, 2009
Genitourinary P. aeruginosa, Staphylococcus spp., E.
coli, Proteus spp.
Slopek et al., 1987; Chanishvili and
Sharp, 2009
Skeletal Staphylococcus spp., P. aeruginosa,
Proteus spp., Klebsiella spp., E. coli
Slopek et al., 1987; Weber-Dabrowska
et al., 2000
The dearth of Western human trials to date means that the work referenced here is primarily from the Eliava
(Kutateladze and Adamia, 2008; Chanishvili and Sharp, 2009) and Hirszfeld (Slopek et al., 1987; Weber-Dabrowska et
al., 2000) Institutes. Western trial references are shown in bold. Further details of Hirszfeld Institute human trials can be
found in Slopek et al. (1983a,b, 1984, 1985a,b,c). Some details of other human applications, research and clinical trials
and other studies are given by Husler (2006) and Abedon (2011a,b). This table, however, illustrates some of the wide
range of potential applications for phage therapy.
required for clinical acceptance in the West. A
total of 24 patients were recruited with
chronic otitis of Pseudomonas aetiology. The
infecting bacteria were demonstrated to be
susceptible to the cocktail prior to the
beginning of the trial. As in the canine otitis
trial, a single dose of 600,000 phages was
administered into the ear canal, and clinical
assessments made on days 7, 21 and 42,
including microbiological analyses and
physician and patient assessments. Both the
test and control groups received ear cleaning
at every visit and the results were the rst
human clinical data to demonstrate to
Western regulators that phage therapy could
be superior to existing therapies in reducing
symptoms and bacterial load. In two patients,
phage application led to bacterial titres falling
below the detection limit within 7 days and
remained undetectable up to 42 days post-
treatment. In three patients, including the
two whose bacterial infection had resolved,
phage treatment led to complete resolution of
the infection. Physician- and patient-assessed
scores, as well as microbiological analysis,
demonstrated the e cacy of phage treatment
compared with the placebo. The results of
this trial were su cient to allow an extension
of this work into phase III trials in Europe. If
successful, then this trial should pave the way
for full regulatory approval.
Additional issues
Should phage therapy successfully complete
clinical trials and become a regular treatment
for bacterial disease, then the issue of phage
resistance and maintenance of phage
preparations will come to the fore. It is likely
that bacteria will eventually become resistant
to any phage preparation, as they have to
antibiotics. Do we therefore atempt to create
phage preparations that will be broadly
eective both geographically and temporally,
or instead should the focus be on development
of bespoke approaches that tailor phages for
individuals or small groups of patients?
These issues are of importance and are
already being discussed by phage-therapy
researchers and companies (Pirnay et al.,
2011). However, within the existing Western
regulatory framework, it is unlikely that
bespoke approaches will be acceptable, at
least in the near term. As mentioned above, it
has been suggested that in vitro phage
activities may be poorly predictive of in vivo
activity (D.R. Harper, AmpliPhi Biosciences,
UK, J.A. Fralick, Texas Tech University Health
Sciences Center, USA and L. Debarbieux,
Institut Pasteur, France, personal com-
munication) and some experimental work is
emerging to corroborate these observations
(Bull et al., 2010; see also Abedon, 2012). If this
is the case, we will need superior tools to
predict the pharmacodynamic properties of
therapeutic phages.
The route of administration is also
important to consider. The applications
discussed above used multiple ways to apply
phages, and phages are partitioned dierently
depending on how they are applied (McVay
et al., 2007). Debarbieux reported the e cacy
of phage treatment of P. aeruginosa lung
infections in mice, as discussed above
(Debarbieux et al., 2010; Morello et al., 2011).
This model, however, is probably not su cient
to truly mimic phage treatment of the cystic
brosis lung, which is severely colonized by
mature, polymicrobial biolm (Harrison, 2007;
Hogardt and Heesemann, 2010; Hiby et al.,
2010; see also Shen et al., Chapter 15, this
volume), as opposed to recently applied
bacterial cells of a single species. In response to
this potential need, research has been carried
out to nd mechanisms to aerosolize phages
as dried particulates for inhalational delivery
into the deep lung space (Golshahi et al., 2008).
Furthermore, what degree of phage puri-
cation will be required for a given application?
Some authors have puried their phages by
the gold standard method of caesium chloride
density-gradient ultra centrifugation (Biswas et
al., 2002), while others have used alternative
methods of purication (e.g. Merabishvili et
al., 2009; Wright et al., 2009). The degree of
purication may depend on the route of
administration, presumably with parenteral
administration requiring far more highly
puried phages than oral applications, for
example (see also Gill and Hyman, 2010).
Is it possible to use temperate phages for
therapy? If no virulent phages can be found
for a given bacterial pathogen, as is currently
the case for C. di cile, then it could be possible
to use temperate phages that have been
modied to block their ability to lysogenize
their hosts. One company, the UK-based
Novolytics, has already applied for a patent
for such an approach (Rapson et al., 2008),
although trials have not been reported to date.
Considering that so many temperate phages
augment the pathogenicity of their hosts (see
Christie et al., Chapter 4, this volume), it is
unlikely that this approach would be used for
all but the most extreme circumstances (see
Abedon, Chapter 17, this volume).
One of the most valued traits of phages is
the ease with which they can be manipulated
in vitro. As mentioned above, directed
evolution has been used successfully to alter
the pharmacokinetic properties of
bacteriophage and P22 such that they
remain longer in the bloodstream of mice
(Merril et al., 1996). The huge global diversity
of phages, combined with phage-display
technology (see Siegel, Chapter 8, this
volume) and other molecular approaches
raises the possibility that phages that target
specic tissues or body compartments could
be selected for. Molecular techniques have
also been used to augment phage antibiolm
properties (Lu and Collins, 2007) or alter
host-cell antibiotic sensitivity (Lu and Collins,
2009), which could extend the e cacy of
phage therapy as either a stand-alone or a
combined therapy, albeit at the cost of
complicating the regulatory approval
pathway (see also Goodridge, 2010, and Shen
et al., Chapter 15, this volume).
Phages can have unpredicted eects on
antibiotic sensitivity (reviewed briey by
Heinemann et al., 2000) and antibiotics can
cause changes in how phages propagate
(Comeau et al., 2007), so it is too early to
predict exactly how phage therapy will work
in combination with antibiotics. It is hoped
that reducing antibiotic use, and thereby
reducing the selective pressure on bacteria to
maintain antibiotic resistance, will reinstate
a certain degree of antibiotic susceptibility,
and there may be evidence to support
this (Kutateladze and Adamia, 2008).
Alternatively, one group has developed a
combinatorial approach to greatly expand the
host range of a single phage by creating a
highly diverse library of phages with
modied host ranges (Pouillot et al., 2010).
Currently, Western regulators require that
any phage formulation should be trialled as it
is intended to be used, so maintenance of the
phage preparation will require dialogue with
the regulators and perhaps using regulatory
pathways such as those used for vaccines that
permit some modication of the formulation
without the need for clinical trials. Otherwise,
addition of new phages to a formula to
maintain e cacy if bacteria become resistant
could mean that the new formulation will
have to be tested again through clinical trials.
If a small number of phages that have already
been shown to be safe for use can be modied
combinatorially to expand their host range,
then such requirements might be avoided.
Conclusions
Phage therapy still has a long way to go
before it becomes a common or preferred
treatment modality for a large range of
bacterial diseases. It is likely that, even afer
Western regulatory approval, phage therapy
will be used initially for refractory diseases.
Antibiotic resistance is still on the rise,
however, and the emerging paradigm of
bacterial biolms also represents a major
issue for antibiotics. Ultimately, this may lead
clinicians to prefer phages to current
conventional treatments, even for some
antibiotic-susceptible bacterial isolates (e.g.
where it is di cult to maintain adequate
antibiotic concentrations, as is the case with
many eye infections). The long history of
phage therapy may nally result, given
regulatory approval, in a treatment regimen
of worldwide appeal, with proven e cacy
and the ability to combat bacterial diseases
for at least another century.
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Use of bacteriophage in the treatment of
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15 Phage-based Enzybiotics
Yang Shen
1
, Michael S. Mitchell
1
, David M. Donovan
2
and
Daniel C. Nelson
1
1
Institute for Bioscience and Biotechnology Research, University of Maryland;
2
Animal Biosciences and Biotechnology Lab, ANRI, ARS, USDA.
The term enzybiotic was coined by Vincent
Fischetis group in 2001 to describe both the
enzymatic and antibiotic properties of
bacteriophage-encoded endolysins (Nelson et
al., 2001). Endolysins are peptidoglycan
hydrolases that function to lyse the bacterial
cell wall for release of progeny virions during
the phage lytic cycle. Timing of lysis is
initiated by holins, which permeabilize the
bacterial membrane and allow the cyto-
plasmically accumulated endolysins access
to the peptidoglycan (Young, 1992; Wang et
al., 2000). Signicantly, in the absence of
holins or parental bacteriophage, these
enzymes can be used exogenously to lyse the
peptidoglycan of susceptible organisms.
Once the structural peptidoglycan is com-
promised, internal turgor pressure, measured
at 2050 atmos pheres for Gram-positive
organisms (Whatmore and Reed, 1990; Doyle
and Marquis, 1994; Arnoldi et al., 2000),
causes a rapid osmotic lysis of the bacterial
membrane resulting in cell death. Non-
endolysin enzybiotics, such as virion-
associated enzymes, have also been identied
(Takac and Blasi, 2005; Rodriguez et al., 2011).
With few exceptions, this enzyme-mediated
lysis from without phenomenon a term
that has been used to describe a variety of
phenomena in which an extracellular agent
destroys a bacterial cell envelope (Abedon,
2011a) is restricted to Gram-positive species,
as the Gram-negative peptidoglycan is
covered by a protective outer membrane that
is not permeable to an exogenous enzyme
under normal conditions. None the less,
enzybiotics encoded by Gram-positive phage
represent an atractive therapeutic option
(Loessner, 2005; Borysowski et al., 2006;
Donovan, 2007; Hermoso et al., 2007; Fischeti,
2010).
In recent years, the enzybiotic moniker
has been extended by some authors to
describe any enzyme that displays general
antibacterial or antifungal properties,
whether phage derived or not (Veiga-Crespo
et al., 2007). These include a wide range of
enzymes such as autolysins and exolysins.
Autolysins are peptidoglycan hydrolases
encoded by bacteria for growth, division and
repair of the bacterial peptidoglycan. Like
their phage-encoded endolysin counterparts,
some autolysins can produce lysis from
without when administered in su cient
quantities. For example, the major pneumo-
coccal autolysin, LytA, was shown to eect a
5 log drop in bacterial counts 4 h afer
intraperitoneal injection in a mouse model of
pneumococcal septicaemia (Rodriguez-
Cerrato et al., 2007). Likewise, fungal glucan-
ases and chitinases could be considered
autolysins with a potential for therapeutic
use (Veiga-Crespo and Villa, 2010). In contrast
to endolysins and autolysins, an exolysin is
218 Y. Shen et al.
an enzyme secreted by a bacterial cell to lyse
the peptidoglycan of a dierent strain or
species. One of the most-studied bacterial
exolysins is lysostaphin, a peptidoglycan
hydrolase secreted by Staphylococcus simulans
that cleaves the Staphylococcus aureus cell wall
but does not hydrolyse the S. simulans cell
wall (Schindler and Schuhardt, 1964). This
enzyme has been investigated for therapeutic
e cacy against S. aureus in such diverse
animal models as burn infections (Cui et al.,
2011), ocular infections (Dajcs et al., 2001,
2002), systemic infections (Kokai-Kun et al.,
2007), keratitis models (Dajcs et al., 2000),
nasal colonization (Kokai-Kun et al., 2003)
and aortic valve endocarditis (Climo et al.,
1998; Patron et al., 1999). Similarly, zoocin A is
an exolysin secreted by Streptococcus equi
subs. zooepidemicus that hydrolyses the
peptidoglycan of Streptococcus pyogenes
(Akesson et al., 2007). In addition to bacterial
exolysins, eukaryotic cells can secrete their
own exolysins. For example, lysozymes
found in human tears and saliva are
eukaryotic exolysins that are part of the
innate immune system providing protection
against bacterial invasion.
While the most comprehensive denition
of enzybiotics would include all endolysins,
autolysins, exolysins and perhaps other
enzymes, we will focus in this chapter on the
phage-encoded enzymes, primarily the
endolysins. Current knowledge about the
mechanisms of action for these enzymes, in
vitro and in vivo activity, synergy with other
enzymes or antibiotics, immune responses
and resistance to these enzymes will be
discussed. In addition, at the end of the
chapter, we will briey review bacteriocins
and depolymerases, two other classes of
phage-encoded enzymes that may also be
considered enzybiotics.
Endolysins
History
The history of endolysins dates back to the
discovery of bacteriophages themselves. It has
been well documented in the literature that
Frederick Twort in 1915 and Felix dHrelle
in 1917 are credited with independently
discovering bacteriophages. What is rarely
mentioned in historical reviews, however, is
that Tworts seminal manuscript also
contained perhaps the rst evidence for phage
endolysins. Specically, he noted that, in
addition to the transmissible properties of the
ultramicroscopic agent (i.e. a staphylococcal
phage), there also appeared to be a non-
transmissible, heat-labile property responsible
for producing transparent zones of lysis
(Twort, 1915). In hindsight, it is likely that
Twort was observing lysis from without,
atributable to either the endolysin or a virion-
associated enzyme of the staphylo coccal
phage. By 1925, he had theorized that, by
denition, a transmissible virus will only act
on live bacteria whereas a non-trans missible
bacterial lysin secreted by the virus should
also act on dead bacteria (Twort, 1925). He
then demonstrated that dead staphylococci
could not be lysed by phage unless a small
amount of live staphylococci was added,
which liberated some nascent agent.
In 1926, Reynals not only conrmed
Tworts lytic ndings on dead staphylococci
but also showed that this phenomenon is
restricted to Gram-positive organisms. When
identical experiments were performed on
Escherichia or Shigella species with their
corresponding phage, no lysis of dead cells
was shown, even in the presence of
susceptible live cells (Reynals, 1926). By 1933,
Rakieten reported that staphylococcal strains
resistant to infection by a staphylococcal
phage were e ciently lysed when a small
amount of sensitive staphylococci was added
(Rakieten, 1933). He concluded that this was
due to a non-transmissible, bacteriophage
lysin, which was presumably produced
during the process of bacteriophagy. By the
end of the 1930s, Alice Evans had shown that
lysis from without was not unique to
staphylococci. In several elegant papers, she
showed that phage B only infected group C
streptococci but its potent principle could
lyse group A, C and E streptococci (Evans,
1934, 1936, 1940). More than 30 years later,
Vincent Fischeti would purify Evans potent
principle (i.e. an endolysin) (Fischeti et al.,
1971) and, eventually, his laboratory would
use it in the rst in vivo therapeutic studies of
a bacteriophage endolysin, henceforth known
as an enzybiotic (Nelson et al., 2001).
Phage-based Enzybiotics 219
Basic cell-wall architecture and types of
endolysin activities
The bacterial peptidoglycan is an essential
scaold found on the outside of the
cytoplasmic membrane of most bacteria. It
functions to maintain a rigid structure and
preserve cell integrity. As its name implies, the
main structural features of peptidoglycan are
linear glycan strands cross-linked by short
peptide stems, which in turn are cross-linked
either directly together or through a peptide
bridge (Fig. 15.1). The glycan strand is
a polymer of alternating N-acetylmuramic
acid (MurNAc) and N-acetylglucosamine
(GlcNAc) residues coupled by |(14)
linkages. The peptide stem is covalently
atached to the glycan polymer by an amide
bond between each MurNAc and an l-alanine
residue. The remainder of the peptide stem
contains alternating l- and d-form amino
acids. For many Gram-positive organisms, the
third residue of the stem peptide is l-lysine,
which is cross-linked to an opposing stem
peptide on a separate glycan polymer through
an interpeptide bridge, the composition of
which varies among species. For example, the
interpeptide bridge of S. aureus is pentaglycine
whereas di-alanine is present in S. pyogenes. In
Gram-negative organisms and some genera of
Gram-positive bacteria (i.e. Bacillus and
Listeria), a meso-diaminopimelic acid (mDAP)
residue is present at position number three of
the stem peptide instead of l-lysine. In these
organisms, mDAP directly cross-links to the
terminal d-alanine of the opposite stem
peptide (i.e. there is no interpeptide bridge).
Whether an interpeptide bridge is present or
not, the joining of opposing stem peptides
gives rise to the three-dimensional latice that
is the dening characteristic of the bacterial
peptidoglycan.
There are in fact only a limited number
of covalent bonds that are available for
D-Ala
D-Ala
D-iso-Glu
L-Lys
D-iso-Glu
L-Lys
L-Ala
MurNAc
L-Ala
MurNAc
A
B
C
D
Fig. 15.1. Structure of the peptidoglycan and cleavage sites by generic endolysins. Glycosidases cleave
the carbohydrate backbone. (A) N-Acetylglucosaminidase cleaves on the reducing side of
N-acetylglucosamine (GlcNAc). (B) Lysozymes, also called muramidases, are N-acetylmuramidases that
cleave on the reducing side of N-acetylmuramic acid (MurNAc). Another glycosidase-like enzyme that
cleaves this same bond but does not require hydrolysis of water is the lytic transglycosylase. (C) The
N-acetylmuramoyl-L-alanine amidase cleaves the rst amide bond between the peptide moiety (L
-alanine) and the glycan moiety (MurNAc) (D). Numerous endopeptidase cleavage sites are present on
the peptidoglycan. Note, X is the transpeptide bridge, the length and composition of which varies
depending on species.
220 Y. Shen et al.
cleavage by endolysins due to the reasonably
conserved overall structure of the bacterial
peptidoglycan. In general, endolysins (as
well as autolysins and exolysins) can be
classied into three groups depending on
their cleavage specicity: those that cleave
between two sugar residues (i.e. glycosidases
and lytic transglycosylases), those that cleave
between a sugar residue and an amino acid
(i.e. N-acetylmuramoyl-l-alanine amidases)
and those that cleave between two amino
acids (i.e. endopeptidases), each of which is
summarized in Fig. 15.1 and select examples
of which are presented in Table 15.1.
Sugar-cleaving enzymes include glyco-
sidases (i.e. N-acetylglucosaminidases and
Table 15.1. Examples of in vitro studies with Gram-positive phage endolysins.
Endolysin Target Catalytic activities In vitro activity Reference(s)
Sa2 Streptococcus
agalactiae
Glycosidase +
endopeptidase
Activity on puried
peptidoglycan as
analysed by mass
spectrometry
Pritchard et al.
(2007)
Cpl-1 Streptococcus
pneumoniae
N-acetylmuramidase 5 log decrease in CFU in
30 s with 100 g
Garcia et al. (1987)
PAL Streptococcus
pneumoniae
N-acetylmuramoyl-L-
alanine amidase
4 log decrease in CFU in
30 s with 100 U ml
1
Loefer et al.
(2001)
PlyGBS
(aka B30)
Streptococcus
agalactiae
N-acetylmuramidase
+ endopeptidase
60 min with 40 U
Cheng et al. (2005);
Pritchard et al.
(2004)
PlyC Streptococcus
pyogenes
N-acetylmuramoyl-L-
alanine amidase
5 s with 10 ng
Nelson et al. (2001)
Ply700 Streptococcus
uberis
N-acetylmuramoyl-L-
alanine amidase
0.5 log decrease in CFU
in milk in 15 min with
50 g ml
1
Celia et al. (2008)
PlyG Bacillus anthracis/
cereus
N-acetylmuramoyl-L-
alanine amidase
15 min with 20 U
Schuch et al.
(2002)
PlyB Bacillus cereus N-acetylmuramidase OD
600
decrease from
0.45 to 0.05 with
2.5 M; PlyG also had
similar results
Porter et al. (2007)
u11 Staphylococcus
aureus
N-acetylmuramoyl-L-
alanine amidase +
endopeptidase
OD
600
decrease from 0.3
to 0.15 in 20 min with
20 g ml
1
Navarre et al.
(1999)
LysK Staphylococcus
aureus
N-acetylmuramoyl-L-
alanine amidases +
endopeptidase
1 h with crude lysate
OFlaherty et al.
(2005)
ClyS Staphylococcus
aureus
N-acetylmuramoyl-L-
alanine amidase
30 min with 250 g
Daniel et al. (2010)
Ply511 Listeria
monocytogenes
N-acetylmuramoyl-L-
alanine amidase
OD
600
decrease from 1.6
180 U ml
1
Gaeng et al. (2000)
PlyPSA Listeria
monocytogenes
N-acetylmuramoyl-L-
alanine amidase
OD
600
decrease from 1.0
2.8 nmol
Korndorfer et al.
(2006)
Ply500 Listeria
monocytogenes
L-alanyl-D-glutamate
endopeptidase
OD
600
decrease from 1.0
to 0.2 in 1 min at
4.8 g/ml or 3 min at
1.6 g ml
1
Schmelcher et al.
(2011)
Ply118 Listeria
monocytogenes
L-alanyl-D-glutamate
endopeptidase
OD
600
decrease from 1.6
60 U ml
1
Gaeng et al. (2000)
N-acetylmuramidases) and lytic trans glyco-
sylases. N-Acetylglucosaminidases cleave the
glycan component of the peptidoglycan on
the reducing side of the GlcNAc residue (A
in Fig. 15.1). This type of activity, which is
commonly found in autolysins, does not
appear to be prevalent in endolysins. None
the less, an N-acetylglucosaminidase has
been described as one of two catalytic
activities associated with the streptococcal
Sa2 endolysin (Pritchard et al., 2007). In
contrast, an N-acetylmuramidase, more
commonly known as a lysozyme or mura-
midase, cleaves the glycan on the reducing
side of the MurNac residue (B in Fig. 15.1)
and is commonly associated with endolysins.
Examples include the pneumococcal Cpl-1
endolysin (Garcia et al., 1987) and the
streptococcal B30 endolysin (Pritchard et al.,
2004). Lytic transglycosylases are very similar
to N-acetylmuramidases in that they also
cleave the |(14) linkages between MurNAc
and GlcNAc. However, lytic transglycosylases
do not require water to catalyse the reaction
whereas N-acetylmuramidases are hydrolases
(i.e. they require water for bond lysis). The
endolysin (Taylor and Gorazdowska, 1974)
and the gp144 endolysin from the uKZ
bacteriophage (Paradis-Bleau et al., 2007) are
two examples of known phage lytic trans-
glycosylases.
The second mechanistic class includes
the N-acetylmuramoyl-l-alanine amidase.
This enzyme is ofen referred to simply as an
amidase, which has become confusing in the
literature because the term amidase suggests
any type of generalized protease activity.
In reality, this enzyme is an amidohydrolase
that only cleaves a specic amide bond
between the glycan component (MurNac)
and the peptide component (l-alanine) of
the peptidoglycan (C in Fig. 15.1). The
N-acetylmuramoyl-l-alanine amidase activity
has been demonstrated for the staphylococcal
endolysins u11 (Navarre et al., 1999) and LysK
(Becker et al., 2009a; Donovan et al., 2009), the
Listeria endolysins Ply511 (Loessner et al.,
1995) and PlyPSA (Korndorfer et al., 2006) and
the streptococcal endolysin PlyC (Nelson et
al., 2006).
The third class is dened by a protease,
or endopeptidase, activity (D in Fig. 15.1).
Endopeptidases could also be described
as amidases, but in contrast to the
N-acetylmuramoyl-l-alanine amidase above,
endopeptidases hydrolyse an amide bond
between two amino acids, which denes a
peptide bond. These cleavages ofen occur in
the stem peptide, such as the Listeria Ply500
and Ply118 l-alanyl-d-glutamate endolysins
(Loessner et al., 1995), or in the interpeptide
bridge, such as the staphylococcal u11
endolysin that cleaves an alanineglycine
bond (Navarre et al., 1999).
Endolysin Modular Structure
Gram-negative endolysin structure
The Gram-negative peptidoglycan is con-
tained within the periplasmic space between
the inner and outer bacterial membranes. It is
relatively thin (ve to ten layers) compared
with Gram-positive peptidoglycan (up to 40
layers) and lysis from within is via a holin
and endolysin during the phage lytic cycle.
Accordingly, endolysins from phages that
infect Gram-negative hosts are typically
comprised of a single globular catalytic
domain, which in most cases supplies one of
the two glycosidase activities. The two
notable exceptions include the Pseudomonas
phage endolysins KZ144 and EL188, which
have a distinct cell-wall-binding domain in
addition to a catalytic domain (Briers et al.,
2007). These binding domains alone were
shown to be su cient to direct high-a nity
binding to puried Pseudomonas aeruginosa
cell walls (Briers et al., 2009). Otherwise, the
catalytic domains do not appear to require
any specic binding domain to recognize and
digest the Gram-negative peptidoglycan.
Gram-positive endolysin structure
The Gram-positive endolysins have a modular
design with one or more catalytic domains
and a cell-wall-binding domain (Garcia et al.,
1988, 1990). One of the best-characterized
endolysin catalytic domains is the cysteine,
histidine-dependent amidohydrolase/pept-
idase (CHAP) domain (Bateman and
222 Y. Shen et al.
Rawlings, 2003; Rigden et al., 2003) with active
site residues that are conserved across many
species and have been shown to be essential
for activity by site-directed mutagenesis
(Pritchard et al., 2004; Nelson et al., 2006).
Other catalytic domains exist and have been
described more fully by Nelson et al. (2012).
In contrast to Gram-negative bacteria,
Gram-positive organisms contain no outer
membrane and their considerable pepti-
doglycan layer is highly cross-linked with
surface carbohydrates and proteins. Con-
sequently, endolysins from Gram-positive-
infecting phages also possess a cell-wall-
binding domain (CBD) that recognizes
epitopes on the peptidoglycan surface. These
epitopes can consist of carbohydrates, teichoic
acids or peptide moieties of the peptidoglycan
and can impart a species- or even strain-
specic binding of the endolysin (Schmelcher
et al., 2011), although the necessity of the CBD
for lysis is still debated. Frequently, the CBD
has been found to be an absolute requirement
for lysis-from-without activity of the endo-
lysin, as the catalytic domain alone has highly
diminished activity against the host organism,
presumably as a consequence of catalysis
occurring afer only random encounters
between the catalytic domain and the sessile
bond of the peptidoglycan, or the enzyme is
completely inactive (Porter et al., 2007). By
contrast, however, there are other reports
where deleting the CBD does not aect the
lytic activity (Low et al., 2005) and even results
in increased lysis from without (Cheng and
Fischeti, 2007; Horgan et al., 2009; Gerova et
al. 2011).
In addition to a catalytic domain and a
CBD, Gram-positive phage endolysins ofen
have a exible linker sequence of 1020 amino
acids that connect the globular domains. This
inherent exibility has led to considerable
trouble crystallizing full-length endolysins.
Many atempts have yielded only the
structures of individual catalytic domains or
isolated CBDs (Low et al., 2005; Porter et al.,
2007; Korndorfer et al., 2008; Silva-Martin et
al., 2010). None the less, recent eorts to
produce full-length structures of PlyPSA, a
listerial N-acetylmuramoyl-l-alanine amidase
(Korndorfer et al., 2006), and Cpl-1, a pneu-
mococcal N-acetylmuramidase (Hermoso et
al., 2003), have been successful. These
structures reveal extreme compartment-
alization of the globular domains, which
suggests that domains can be added, deleted
or swapped to tailor activity (Hermoso et al.,
2007). Indeed, the specicity of clostridial,
lactococcal, pneumococcal and listerial
endolysins has already been modied or
changed by insertion or deletion of CBDs
(Croux et al., 1993; Lopez et al., 1997;
Schmelcher et al., 2011). Although the
modular nature of phage endolysins was
initially described in the early 1990s (Diaz et
al., 1990, 1991; Garcia et al., 1990; Croux et al.,
1993), it was not until recently that exploiting
the modular nature of endolysins has become
a major biotechnological focus for the
development of enzybiotics.
Multiple-domain endolysins
While endolysins from phages infecting
Gram-positive bacteria usually contain both a
catalytic domain and a CBD, as mentioned
above, some endolysins contain multiple
catalytic domains. This is common in those
derived from staphylococcal or streptococcal
phage. The presence of two catalytic domains,
however, does not necessarily indicate that
each contributes equally to the lytic activity.
For example, the staphylococcal endolysin
u11 has both N-acetylmuramoyl-l-alanine
amidase and d-alanyl-glycyl endopeptidase
catalytic domains (Navarre et al., 1999). While
the endopeptidase domain was active by
itself in deletion constructs (Donovan et al.,
2006a; Sass and Bierbaum, 2007), the amidase
domain was shown to be silent by deletion
analysis (Sass and Bierbaum, 2007). Similarly,
LysK, the endolysin from the staphylococcal
phage K, has an active N-terminal endo-
peptidase domain and a virtually inactive
amidase domain (Becker et al., 2009a; Horgan
et al., 2009).
The same phenomenon is also prevalent
in streptococcal endolysins. The streptococcal
SA2 endolysin, for instance, contains a
d-glutaminyl-l-lysine endopeptidase activity
and an N-acetylglucosaminidase activity
(Pritchard et al., 2007). Further analysis
revealed, however, that the endopeptidase
domain possessed nearly all of the catalytic
activity and that the glucosaminidase was
almost devoid of activity (Donovan and
Foster-Frey, 2008). At present, it is not clear if
these silent domains are an artefact of the
assay or substrate used for the assay, were
once catalytically active but lost their activity
through evolution, or function as a secondary
binding domain by positioning the substrate
for optimal cleavage by the non-silent
catalytic domain.
Most of these ndings rely on gene
deletion analysis that yields a truncated
protein product. These strategies raise the
potential for aberrant tertiary structure in the
truncated protein that could be misinterpreted
as diminished domain activity. To address
this concern, the streptococcal B30 endolysin
(also known as PlyGBS; Cheng et al., 2005)
was subjected to site-directed mutagenesis of
key amino acid residues that resulted in
eradication of a single domains enzymatic
activity in full-length protein constructs.
Although this does not fully rule out protein
misfolding, it certainly is much less likely.
The result was that the N-acetylmuramidase
domain was nearly silent, with virtually all
lysis from without due to the d-alanyl-l-
alanyl endopeptidase catalytic domain
(Donovan et al., 2006b). This is despite the fact
that each lytic domain showed enzymatic
activity in the native protein on puried
streptococcal peptidoglycan (Pritchard et al.,
2004).
At present, it is not clear why an
endolysin would evolve two disparate
catalytic domains, particularly when one of
the two has litle to no activity. One of us
(D.M.D.) has speculated that the observed
dierences in activity may be atributable to
inherent variations in cleavage when done
from within versus from without (Donovan
and Foster-Frey, 2008). Alternatively, the
silent domain might aid in binding to the
peptidoglycan and position the active domain
in the proper orientation for optimal cleavage,
or cleavage with the highly active domain
might create a substrate that is more amenable
to the second domain activity.
Although we can only speculate as to the
advantages conferred by the dual domains,
one putative mechanism for the modular
nature and accumulation of two domains is
likely to be due to horizontal gene transfer
and a consequence of recombination. Indeed,
group I introns are ofen found within
endolysin genes from phage that infect
Streptococcus (Foley et al., 2000) and
Staphylococcus species (OFlaherty et al., 2004;
Kasparek et al., 2007; Becker et al., 2009b).
Specically, Foley and colleagues found
group I introns interrupting the endolysin
genes of half of the 62 Streptococcus
thermophilus phage genomes studied (Foley et
al., 2000). Additionally, in at least one pair of
more than 90% identical staphylococcal
phage endolysin genes, the intron is
positioned between the two lytic domains
and thus would allow exonic shu ing of the
lytic domains (Becker et al., 2009b). Clearly,
identication of additional intron-containing
multi-domain enzymes and further experi-
ment ation on the current enzymes is needed
to understand more fully the role of introns in
the evolution of these important enzymes.
Nearly all endolysins, whether derived
from Gram-negative or Gram-positive phages,
and whether they are single- or multi-
domained, are products of individual genes.
The only known exception is PlyC, an
endolysin from the streptococcal C1
bacteriophage. Originally, this enzyme was
believed to be a single protein like all other
endolysins (Fischeti et al., 1971; Nelson et al.,
2001). Later, the gene for this endolysin was
thought to be interrupted when whole-
genome sequencing of the C1 phage
uncovered a putative group I intron between
the two coding sequences known to be present
in the endolysin (Nelson et al., 2003). However,
a subsequent investigation aimed at cloning
the gene(s) and systematically investigating
each open reading frame revealed that PlyC is
composed of two dissimilar gene products:
PlyCA, which contains the catalytically active
l-alanine amidase domain, and PlyCB, which
functions as the CBD (Nelson et al., 2006).
Moreover, PlyCB self-assembles into an
octamer, whether in the presence of PlyCA or
expressed alone. Thus, the mature PlyC
holoenzyme is a nonamer composed of one
PlyCA and eight PlyCB subunits. No other
multi-subunit endolysins have been described
and the implications for such a complex are
224 Y. Shen et al.
not clear. None the less, nanogram quantities
of PlyC can achieve approximately 7 log
killing of streptococcal cells within seconds,
which is several orders of magnitude more
active than any other endolysin (Nelson et al.,
2001). Future structure/function studies may
provide additional insight into this novel
endolysin.
Endolysin In Vitro Activity
Gram-negative organisms
The use of endolysins as enzybiotics against
Gram-negative pathogens has been very
limited. The endolysin-susceptible pepti-
doglycan layer resides between an inner and
outer membrane in Gram-negative organisms
and as such is not directly exposed to the
extracellular environment (i.e. lysis from
without via endolysin digestion of pepti-
doglycan has not been reported in the
literature for untreated Gram-negative
organisms). In order to overcome the hurdle
of the outer membrane, the use of peptides,
detergents and chelators in combination with
endolysins was rst suggested by Vaara
(1992). For example, 50 g ml
1
of the EL188
endolysin in the presence of 10 mM EDTA
(i.e. a metal chelator) can reduce P. aeruginosa
titres by 34 logs in 30 min depending on the
strain tested (Briers et al., 2011). Additionally,
there have been studies in which various
chemical moieties conjugated to exolysins or
hydrophobic peptides have been genetically
fused to endolysins to alter the membrane
permeability of these enzymes (Ito et al., 1997;
Masschalck and Michiels, 2003).
Sometimes naturally occurring mem-
brane permeability domains are present on
endolysins. This was shown recently for
LysAB2, the endolysin from the Acinetobacter
baumannii phage uAB2, which was reported
to lyse live Gram-positive and Gram-negative
bacteria (Lai et al., 2011). Notably, this
endolysin contains a C-terminal amphipathic
region that was shown to be necessary for the
observed antibacterial activity. Another
example is the lys1521 endolysin from a
Bacillus amyloliquefaciens phage. This enzyme
possesses two cationic C-terminal regions
that were shown to be able to permeabilize
the outer membrane of P. aeruginosa
(Muyombwe et al., 1999). The wild-type
enzyme, which contained an N-terminal
catalytic domain as well as both C-terminal
cationic domains, displayed antibacterial
activity against live P. aeruginosa (Orito et al.,
2004). However, caution should be exercised
when considering any of the approaches
presented in this section for human thera-
peutics, as agents known to destabilize the
Gram-negative outer membrane are also
ofen toxic to eukaryotic cell membranes. To
date, no therapeutic use of an enzybiotic
against a Gram-negative organism has been
demonstrated successfully in an animal
model.
Gram-positive organisms
The vast majority of the literature on
endolysins/enzybiotics is devoted to
describing in vitro activity (i.e. lysis from
without) of endolysins against Gram-positive
pathogens. The substrate for these enzymes
is complex (ofen the three-dimensional
superstructure of the peptidoglycan as
well as secondary binding sites for the
CBDs). Thus, synthetic small-molecular-
weight chromo genic or uorogenic substrates
are rarely adequate as substrates for these
enzymes. Instead, isolated cell walls or even
whole cells remain the substrates of choice.
As such, the most common assay involves the
use of a spectrophotometer to measure the
loss of turbidity of a suspension of live cells
due to lysis from without (i.e. drop in optical
density at 600 nm (OD
600
) per unit time). This
method has been used to describe a unit of
enzyme activity based on the dilution of an
endolysin solution needed to reduce the
optical density by half in a dened amount of
time (Fischeti et al., 1971; Loe er et al., 2001).
While approaches that rely on turbido-
metric analysis are simplistic and easy to use,
they are an indirect measure of lysis (and cell
death) and are altered by changes in
numerous biological factors. First, the phase
of growth aects the thickness of the
peptidoglycan, which in turn aects the
apparent rate of lysis. An enzyme tested on
mid-exponential-phase cells may have
several orders of magnitude greater activity
than the same enzyme assayed on stationary-
phase cells. Likewise, the turgor pressure of
the cell is important, as is the osmolarity of
the solution. Similarly, cells in medium lyse
much more slowly than cells resuspended in
distilled water. Also, as cells lyse, they release
DNA and other cellular components, causing
the viscosity of the reaction tube to rise,
making further lysis less e cient. Finally,
because endolysins are ofen species specic,
it is di cult to compare the activity of one
enzyme against another when the substrate is
a dierent bacterial species. Despite these
caveats, Mitchell and colleagues have
developed a mathematical approach to
estimate kinetic constants based on a turbi-
dometric reduction assay (Mitchell et al.,
2010). In addition, autoclaved S. aureus cells
have been used to reduce the day-to-day
variability of cultured cells for the determin-
ation of kinetics for the LysK endolysin
(Filatova et al., 2010).
Another common type of in vitro
endolysin assay is based on colony counts
whereby a known amount of bacteria is
exposed to an endolysin for a given amount
of time, serially diluted and then plated.
Activity is then reported as the log-fold
decrease in colony-forming units (CFU) over
a dened interval. While this method is ofen
used to quantify cell death, the high a nity
of the CBDs for the bacterial surface can allow
a situation where the reported activity (i.e.
rate of cell death) is overestimated. For
example, an endolysin may bind the bacterial
surface via the CBD in seconds, but catalytic
lysis may not occur immediately. If the
endolysin remains bound during the serial-
dilution step, actual lysis and cell death may
take place on the agar plate at a later time
point. As such, reported activity, especially
for short incubation times, can be over-
estimated.
Table 15.1 contains a list of in vitro results
for many endolysins that produce lysis from
without on Gram-positive organisms. This
list is not intended to be comprehensive.
Instead, its purpose is to provide activity data
for many of the endolysins mentioned in this
chapter and to also highlight the diversity of
activity displayed by these enzymes in
dierent assays. A thorough inspection of
this table will reveal something already
obvious to those in the eld, namely that not
all endolysins are created equal. Some kill/
lyse very e ciently in seconds at microgram
Table 15.2. Summary of in vivo studies with phage endolysins.
Bacteria Endolysin Reference
Streptococcus pyogenes C1
a
Nelson et al. (2001)
Streptococcus agalactiae PlyGBS Cheng et al. (2005)
Bacillus anthracis PlyG Schuch et al. (2002)
PlyPH Yoong et al. (2006)
Streptococcus pneumoniae Cpl-1 Loefer et al. (2001, 2003); Loefer and Fischetti
(2003); Jado et al. (2003); Entenza et al. (2005);
McCullers et al. (2007); Grandgirard et al. (2008);
Witzenrath et al. (2009)
PAL Jado et al. (2003); Loefer and Fischetti (2003)
Staphylococcus aureus MV-L Rashel et al. (2007)
CHAP
k
Fenton et al. (2010)
LysGH15 Gu et al. (2011)
ClyS
b
Daniel et al. (2010); Pastagia et al. (2011)
a
Renamed PlyC according to Nelson et al. (2006).
b
Chimaeric construct from the bacteriophage Twort and uNM3 endolysins.
226 Y. Shen et al.
or even nanogram quantities, whereas others
need much higher doses and longer in-
cubation times to show any lysis.
Endolysin In Vivo Studies
Although endolysins have been studied for
their role in the bacteriophage replication
cycle for over half a century, it has only been
in the past 10 years that scientists have begun
evaluating the use of endolysins in animal
infection models of human disease. Table
15.2 shows a complete list to date of all
reported in vivo therapeutic trials that utilize
endolysins, which are further summarized
below.
Fischeti and co-workers were the rst to
use a puried endolysin in an in vivo model of
streptococcal infection (Nelson et al., 2001).
It was found that oral administration of
an endolysin from the streptococcal C1
bacteriophage, named PlyC in a subsequent
publication (Nelson et al., 2006), provided
protection from colonization in mice
challenged with 10
7
S. pyogenes (group A
streptococci) (28.5% infected for endolysin
treatment versus 70.5% infected for PBS
treatment). Furthermore, when this enzyme
was administered orally to nine heavily
colonized mice, no detectable streptococci
were observed at 2 h afer endolysin treatment
and only one mouse had any measurable
streptococcal counts 24 and 48 h later (which
possibly were from intracellular streptococci
that had repopulated the pharyngeal surface).
PlyGBS, also known as the (97%
identical) B30 endolysin (Pritchard et al.,
2004), is another phage endolysin that is
active against group A streptococci as well as
against groups B, C, G and L streptococci.
This enzyme was tested in a murine vaginal
model of Streptococcus agalactiae (group B
streptococci) colonization to see whether
PlyGBS could be a potential therapeutic agent
for pregnant women to prevent transmission
of neonatal meningitis-causing streptococci
to newborns (Cheng et al., 2005). A single
vaginal dose was shown to decrease
colonization of pathogenic group B strepto-
cocci by approximately 3 logs. Notably,
PlyGBS was found to have a pH optimum of
around 5.0, which is similar to the range
normally found within the human vaginal
tract. Furthermore, this enzyme did not
possess bacteriolytic activity against common
vaginal microora such as Lactobacillus
acidophilus, suggesting a pathogen-specic
therapeutic that, unlike broad-range anti-
biotics, would probably reduce the concern of
resistance development in exposed com-
mensal bacteria.
Several phage endolysins have also been
used against germinating spores and
vegetative cells of Bacillus species. PlyG, an
endolysin isolated from the Bacillus anthracis
phage, was shown to rescue 13 out of 19
mice in an intraperitoneal mouse model of
septicaemia and extended the life of the
remaining mice several fold over the controls
(Schuch et al., 2002). Signicantly, this enzyme
displayed a favourable temperature prole
and was able to remain fully active afer
heating to 60C for 1 h. A second Bacillus
endolysin, PlyPH, also active against B.
anthracis, is unique in that it has a high
activity over a broad pH range, from pH 4.0
to 10.5. This enzyme also protected 40% of
mice in an intraperitoneal Bacillus infection
model compared with 100% death in control
mice (Yoong et al., 2006).
The most extensively studied endo-
lysins in animal models are Cpl-1, an
N-acetylmuramidase from the Cp-1 pneumo-
coccal phage, and PAL, an N-acetylmuramoyl-
l-alanine amidase from the Dp-1
pneu mo coccal phage. PAL was shown to
need only 30 s to cause an approximate
4 log drop in viability of 15 dierent Strepto-
coccus pneumoniae serotypes represent ing
multidrug-resistant isolates as well as those
that contained a thick polysaccharide capsule
(Loe er et al., 2001). In a mouse model of
nasopharyngeal carriage, PAL was shown to
eliminate all pneumococci in a dose-
responsive manner. In another study, Cpl-1
was shown to be eective in both a mucosal
colonization model and in blood via a
pneumococcal bacteraemia model (Loe er et
al., 2003). These two enzymes were also
shown to be synergistic in a mouse
interperitoneal model (Jado et al., 2003; see
the following section for more information
about synergy). Cpl-1 was also shown to
work on established pneumococcal biolms
in a rat endocarditis model (Entenza et al.,
2005). In an infant rat model of pneumococcal
meningitis, an intracisternal injection of Cpl-1
resulted in a 3 log decrease in pneumococci in
the cerebrospinal uid, and an intraperitoneal
injection led to a decrease of two orders
of magnitude (Grandgirard et al., 2008).
Additionally, Cp-1 was shown to save 100%
of mice from fatal pneumonia when
administered 24 h afer infection and 42% of
mice when administered 48 h afer infection,
a time point at which bacteraemia was fully
established (Witzenrath et al., 2009). Lastly,
Cpl-1 treatment of mice colonized with S.
pneumoniae in an otitis media model was
shown to signicantly reduce co-colonization
following challenge with inuenza virus
(McCullers et al., 2007). Given that pneumo-
cocci are early colonizers to which additional
pathogens and viruses adhere, eliminating
this population could have a multiplier eect
on controlling infectious diseases.
S. aureus, particularly methicillin-
resistant S. aureus (MRSA), is a major public
health concern and a primary source of
nosocomial and community-acquired
infections (see Kuhl et al., Chapter 3, this
volume). There is considerable interest in
identifying and evaluating highly active
staphylococcal endo lysins. The rst
staphylococcal-specic endolysin investi-
gated in vivo was MV-L, which was cloned
from the uMR11 bacterio phage (Rashel et al.,
2007). This enzyme rapidly lysed all tested
staphylococcal strains in vitro, including
MRSA and vancomycin-resistant clones. In
vivo, this enzyme reduced MRSA nasal
colonization by approximately 3 logs and
provided complete protection in an
intraperitoneal model of staphylococcal
infection when administered at 30 min post-
infection. At 60 min post-infection, the same
amount of MV-L provided protection in 60%
of mice compared with controls. More
recently, an endolysin from the GH15 phage,
LysGH15, showed 100% protection in a
mouse intraperitoneal model of septicaemia
(Gu et al., 2011). Likewise, CHAP
k
, a truncated
version of the endolysin LysK, eected a 2 log
drop in nasal colonization of mice when
given 1 h post-challenge (Fenton et al., 2010).
Lastly, ClyS is the rst engineered endolysin
to be tested in an animal model (Daniel et al.,
2010). This enzyme is a chimaera between the
N-terminal catalytic domain of the Twort
phage endolysin (Loessner et al., 1998) and
the C-terminal CBD of the uNM3 phage
endolysin. Like MV-L, ClyS displayed potent
bacteriolytic properties against multidrug-
resistant staphylococci in vitro. In a mouse
MRSA decolonization model, 2 log reductions
in viability were observed 1 h following a
single treatment of ClyS. Similarly, a single
dose of ClyS provided protection when
administered 3 h afer staphylococcal chal-
lenge in an intraperitoneal septicaemia
model. Additionally, ClyS was shown to be
eective at treating topical infections of S.
aureus (Pastagia et al., 2011).
Synergy
Antimicrobial synergy has been demonstrated
for multiple endolysins, either with other
endolysins or with antibiotics. Synergy
studies typically use a checkerboard broth
microdilution method that allows the
concurrent determination of the minimal
inhibitory concentration (MIC) of each agent
(endolysin or antibiotic). The fractional MIC
values of each agent are then put on an x/y
plot, which is called an isobologram. A linear
relationship corresponds to an additive
eect. For example, if 0.5 MIC of agent
A + 0.5 MIC of agent B displays the same
e cacy as 1.0 MIC of either agent A or B, the
two agents are additive. However, if the
relationship has an inverse, non-linear curve,
the eect is said to be synergistic (i.e. if
0.25 MIC each of agents A and B is equal to
1.0 MIC of either agent alone).
Using this method, the pneumococcal
Cpl-1 endolysin, a muramidase, was shown
to be synergistic with PAL, an l-alanine
amidase (Jado et al., 2003; Loe er and
Fischeti, 2003). Because these enzymes
hydrolyse dierent bonds, synergy is
believed to be due to a greater destabilization
of the three-dimensional peptidoglycan
matrix. Cpl-1 was also found to be synergistic
with penicillin as well as with gentamicin,
but not with levooxacin or azithromycin
(Djurkovic et al., 2005). Interestingly, the
228 Y. Shen et al.
synergy with penicillin was greatest on
strains with the highest levels of penicillin
resistance. In a similar fashion, the staphylo-
coccal endolysin LysH5 was found to be
synergistic with nisin, an antimicrobial
peptide (Garcia et al., 2010), and LysK was
shown to be synergistic with lysostaphin, a
staphylococcal exolysin (Becker et al., 2008).
Finally, ClyS, a fusion endolysin described
above, was shown to be synergistic with
oxacillin and vancomycin in vitro and with
oxacillin in vivo in a mouse model of S. aureus
septicaemia (Daniel et al., 2010).
Immune responses
Because endolysins are globular proteins,
they would be expected to elicit antibodies
that may render them inactive and could
hinder their future development as human or
animal therapeutics. Towards this end, two
decisive studies used in vivo models to
address this issue. First, Loe er et al. (2003)
injected Cpl-1, a pneumococcal endolysin,
intravenously into mice three times a week
for 4 weeks resulting in positive IgG
antibodies against the endolysin in ve out of
six mice. Next, these immunized mice and
nave control mice were challenged with
intravenous pneumococci followed by a
200 g dose of Cpl-1 10 h post challenge.
Surprisingly, pneumococcal titres were
reduced to the same level in both groups of
mice at 15 min. Moreover, the authors found
that hyperimmune rabbit serum did not
neutralize the bacteriolytic activity of Cpl-1
when tested against pneumococci in vitro.
Similar in vitro results have been reported for
endolysins active against B. anthracis and S.
pyogenes (Fischeti, 2005).
In the second study, Jado et al. (2003)
challenged mice with pneumococci followed
by treatment with either of the pneumococcal
endolysins, Cpl-1 or PAL. Ten days later they
conrmed that the recovered mice had high
IgG antibody titres to both enzymes, re-
challenged the mice with pneumococci and
retreated them with the enzybiotics.
Signicantly, pneumococcal titres fell by
23 logs upon administration of the enzymes
and all mice survived with no signs of
anaphylaxis or adverse side events. These
studies suggest that, while antibodies can
readily be raised to endolysins due to their
proteinaceous nature, the antibodies do not
eectively neutralize the bacteriolytic actions
of these enzymes in vitro or in vivo.
Resistance Development
Because of the narrow specicity in which
phage endolysins produce lysis from without,
it can be di cult to determine whether a
strain has developed true resistance to an
endolysin or whether it was never susceptible
in the rst place. To date, there are no reports
of strains sensitive to an endolysin developing
resistance to the same endolysin, however. In
fact, there have been a few reports where
researchers have actively tried, but failed, to
develop resistance to endolysins. In one
study, S. pneumonia, S. pyogenes, and B.
anthracis were exposed to sublethal doses of
Cpl-1, PlyC and PlyG, respectively (Fischeti,
2005). Surviving colonies were grown and
once again exposed to sublethal doses of the
corresponding endolysin. In some cases, over
100 rounds of screening took place. At
dierent cycles, surviving colonies were
tested with lethal doses and in no instance
was resistance observed.
In a separate study, resistance to an
endolysin was investigated more formally
(Schuch et al., 2002). Here, Bacillus species
were screened for spontaneous resistance to
PlyG, a Bacillus-specic endolysin, as well as
two antibiotics, streptomycin and novobiocin.
Resistant colonies were readily isolated
for both antibiotics at a frequency of
approximately 1 10
9
, but no resistance was
found for PlyG at the same screening
frequency. Next, bacilli were exposed to a
chemical mutagen known to induce random
mutations and the screening was repeated.
This time, spontaneous resistance to the
antibiotics occurred at frequencies of around
1 10
6
, but no resistance was observed for
PlyG, even at a frequency of more than
5 10
9
. Therefore, even under conditions that
promoted spontaneous resistance to anti-
biotics by approximately 3 logs, no resistance
could be detected for this endolysin.
An explanation for the lack of observed
resistance to endolysins has been put forth by
Fischeti (2005). In brief, he postulated that
phage and their bacterial hosts have co-
evolved over the millennia such that phages
have evolved to target conserved bonds in the
peptidoglycan in order to guarantee survival
of the progeny phage. As such, resistance, if
present, would be a rare event. None the less,
resistance to these enzymes will always be a
real concern. Most famously, resistance has
been well documented for lysostaphin, an
exolysin secreted by S. simulans (Thumm and
Gotz, 1997). While this enzyme is not a phage
endolysin, it is a peptidoglycan hydrolase
with a similar catalytic and binding domain
to many known phage endolysins (Schindler
and Schuhardt, 1964). One must wonder if it
is only a mater of time before endolysin
resistance is identied.
Bacteriocins
Bacteriocins represent another class of enzy-
biotics. Bacteriocins are proteins and peptides
that comprise a large and functionally diverse
family of potent toxins capable of killing
bacteria at nanomolar concentrations. First
discovered in 1925 (Gratia, 1925), bacteriocin
activity is usually directed against a narrow
spectrum of bacteria taxonomically similar to
the bacteriocin producer, although broader
activity against more distantly related
bacteria and even toxicity towards eukaryotic
cells has been recorded (Gratia, 1989). It
has been suggested that some classes of
bacteriocins have a common origin with
bacteriophage or may even be phage rem-
nants. While this is still a hotly debated area,
we provide a brief overview of the bacteriocin
eld below.
Initially, bacteriocins were classied
either as low-molecular-weight peptides that
were usually thermostable, non-sedimentable
by ultracentrifugation and undetectable on
electron micrographs, or as high-molecular-
weight molecules that were easily sedi-
mented, heat-sensitive, protease resistant and
resolvable by electron microscopy (Bradley,
1967). Over time, it became apparent that the
bacteriocins of Gram-negative and Gram-
positive bacteria were distinct from one
another and only shared their proteinaceous
and antimicrobial natures in common (Daw
and Falkiner, 1996).
Gram-positive bacteriocins
The small-peptide bacteriocins of Gram-
positive bacteria are currently classied into
class I lantibiotics and class II unmodied
peptides (Rea et al., 2011), which are each
further divided into several subclasses.
Bacteriolysins represent a third class of Gram-
positive antimicrobial proteins that were
formerly categorized as class III bacteriocins,
with lysostaphin being the best known
member of this class (Kumar, 2008; Bastos et
al., 2009). With the exception of the particulate
bacteriocins discussed below, most Gram-
positive bacteriocins currently lack an
obvious phage connection, although it was
reported recently that production of the
broad-spectrum lantibiotic sublancin 168 by
Bacillus subtilis strain 168, along with its
dedicated immunity system, is determined
by the SP-| prophage (Dubois et al., 2009).
Gram-negative bacteriocins
The bacteriocins of Gram-negative bacteria
primarily belong to two families: the colicin
and colicin-like high-molecular-weight pro-
teins (3080 kDa), and the low-molecular-
weight peptides (<10 kDa) that are also
referred to as microcins. Escherichia coli
colicins were the rst bacteriocins identied
and, along with the pyocins of P. aeruginosa
(Fyfe et al., 1984), have served as a model
system for understanding bacteriocin
structure and function, genetic organization
and the role of bacteriocins in microbial
diversity and evolution. These bacteriocins
are located in gene clusters that include a
closely linked lysis gene involved in toxin
release via lysis or pseudo-lysis (Pugsley and
Schwartz, 1983a,b) and a dedicated immunity
system that protects the producing strain
from the lethal activity of the toxin (Braun et
al., 1994; Smarda and Smajs, 1998). The
majority of colicin and colicin-like bacteriocins
230 Y. Shen et al.
studied to date exhibit a similar three-domain
structure, consisting of an N-terminal trans-
location domain, a central receptor-binding
domain and a C-terminal killing activity
domain (Braun et al., 1994; Lazdunski et al.,
1998). The killing activity can be a nuclease,
an inhibitor of DNA replication, or one that
blocks protein synthesis.
The Gram-negative bacteriocins have
drawn many parallels to bacteriophages. The
bacteriocin receptors on the target cell surface
ofen double as bacteriophage receptors
(Guterman et al., 1975). Additionally, the host
system confers immunity to the producer
against the toxic eects of the bacteriocin,
which has been compared to host immunity
from lysogeny. Lastly, open reading frames
with sequence similarity to phage genes are
ofen found within or in proximity to
bacteriocin gene clusters (Chavan et al., 2005),
implicating bacteriophages in bacteriocin
acquisition and evolution.
Particulate bacteriocins
Defective bacteriophage and phage-like
complexes have frequently been observed in
the supernatants of bacteriocin-producing
Gram-positive and Gram-negative cells
(Bradley, 1967; Reanney and Ackermann,
1982). Some of these particulate bacteriocins
resemble bacteriophages, with small heads
that may contain randomly packaged DNA
(Hirokawa and Kadlubar, 1969). The PBSX-
like defective prophages of Bacillus are one
well-studied example of phage-like particles
that are inhibitory on a Gram-positive host.
The PBSX killer particles appear to be
complete phage-like particles that do not lack
any components but package random pieces
of genomic DNA into heads that are too small
to package a full genome, and do not inject
their contents into sensitive cells upon
adsorbing to the surface (Okamoto et al.,
1968). Similar to temperate bacteriophage,
these non-infectious particles are spon-
taneously produced at low levels and can be
induced by chemical agents or UV light.
Other particulate bacteriocins resemble
phage heads or headless tails. The phage tail-
like complexes are ofen classied according
to their resemblance to the R-type and F-type
pyocins of P. aeruginosa (Fyfe et al., 1984;
Nakayama et al., 2000). R-type pyocins
appear as rigid contractile sheaths that
resemble the tails of members of the
Myoviridae, while the F-type pyocins are
sheathless, exible rod-like structures with
similarity to the tails of members of the
Siphoviridae. The killing activity of the phage
tail-like bacteriocins occurs by forming pores
in the cell membrane, leading to a rapid loss
of ions and membrane potential (Nakayama
et al., 2000; Strauch et al., 2001, 2003). This is
visualized by noticeable shrinkage of the
cytoplasmic membrane and cell contents
(al-Jumaili, 1976). Signicantly, tail-like
bacteriocins have been shown to have
therapeutic potential both in vitro and in vivo.
For example, enterocoliticin, a phage tail-like
bacteriocin produced by pathogenic Yersinia
enterocolitica serotype O3, was shown to be
therapeutic against Y. enterocolitica growth in
challenged BALB/c mice (Damasko et al.,
2005). Similarly, serracin P from Serratia
plymithicum J7, was active against strains of
the re blight pathogen Erwinia amylovora,
suggesting that this bacteriocin would make
a good biopesticide candidate (Jabrane et al.,
2002).
Depolymerases
Depolymerase is a generic term used to
describe any number of glycanase-like
activities associated with the phage particle
that are used to degrade the bacterial capsule
(Yurewicz et al., 1971; Suthereland et al., 2004).
The capsule serves as a secondary receptor
for the phage but as a primary receptor for
the depolymerase (Hughes et al., 1998a). Afer
the depolymerase degrades the polymer of
the capsule, the phage can bind the primary
receptor on the bacterial surface allowing
infection to proceed. Due to the diversity of
bacterial carbohydrates/capsules, depoly-
merase activities could include glucosidases,
endohexosaminidases, alginate lyases, exo-
polygalacturonic acid lyases, hyaluronate
lyases, guluronan lyases, amylases, cellulases,
dextranases, galactosidases and pullulanases,
to name just a few.
There is considerable interest in using
bacteriophage depolymerases therapeutically
to degrade bacterial biolms. Biolms are
formed when planktonic bacteria (i.e. free,
individual cells) adsorb on to a surface (e.g.
wound or medical implant) and form colonies
(reviewed by Stoodley et al., 2002; Hall-
Stoodley et al., 2004; Stewart and Franklin,
2008). Once the colonies have become
established, phenotypic changes cause them
to oversecrete the capsular carbohydrates (i.e.
polysaccharides) that serve as the structural
backbone for the biolm. Non-cellular
components and debris, including additional
carbohydrates, proteins, lipids and nucleic
acids, become entangled in the polysaccharide
backbone and constitute the slime layer of a
biolm, which is technically known as the
extrapolymeric substance (EPS). Appreciably,
the EPS superstructure of the biolm is
known to protect internal bacteria from
antimicrobials, antibodies and circulating
immune cells (Stoodley et al., 2002; Folkesson
et al., 2008).
Several mechanisms are thought to
contribute to the antimicrobial resistance
associated with biolms: (i) delayed or
restricted penetration of antimicrobial agents
through the biolm EPS matrix; (ii) decreased
metabolism and growth rate of biolm
organisms, which resist killing by compounds
that only atack actively growing cells; (iii)
increased accumulation of antimicrobial-
degrading enzymes; (iv) enhanced exchange
rates of drug resistance genes; and (v)
increased antibiotic tolerance (as opposed to
resistance) through expression of stress-
response genes, phase variation and biolm-
specic phenotype development (Lewis,
2001; Fux et al., 2003; Hall-Stoodley et al.,
2004; Keren et al., 2004). Signicantly, several
groups have shown that whole-phage-based
therapy can be used eectively to reduce
biolms (Bartell et al., 1966; Bartell and Orr,
1969a; Yurewicz et al., 1971; Sutherland et al.,
2004; reviewed by Abedon, 2011b). Unlike the
use of whole-phage therapy, application of
depolymerases to a biolm would not directly
kill bacterial pathogens. However, anything
that digests the EPS layer would act to re-
sensitize the underlying cells to antibiotics,
complement, antibodies and immune cells.
As such, we classify depolymerases as a
phage-derived enzybiotic, along with the
endolysins and bacteriocins.
Numerous depolymerase activities have
been described in the literature, although the
depolymerase nomenclature was more
prevalent 3040 years ago. More recently,
these enzymes are referred to by the specic
enzyme activity they possess (i.e. an
endorhamnosidase) rather than the more
generic depolymerase terminology. Due to the
thick alginate layer, the earliest and most well-
studied depolymerases were from phages
that infect Pseudomonas species (Bartell et al.,
1966, 1968; Bartell and Orr, 1969a,b). In
addition, depolymerases have been puried
and characterized for phage that infect E.
amylovora (Vandenbergh et al., 1985), Azoto-
bacter vinelandii (Davidson et al., 1976, 1977)
and Klebsiella pneumoniae (Bessler et al., 1973;
Thurow et al., 1974, 1975). More recently,
Hughes et al. (1998a,b) specically showed
that bacteriophage depolymerases could
penetrate the EPS layers of Enterobacter
agglomerans and Serratia marcescens biolms.
Finally, in vivo studies have shown that a
recombinant depolymerase can reduce
colonization of E. coli in rats, presumably
through increased ingestion of the de-
capsulated bacteria by macrophages (Mushtaq
et al., 2005).
Tailspike proteins, common to phage
belonging to the family Podoviridae, may
represent a well-conserved depolymerase
family that oers untapped potential for
development of therapeutic depolymerases.
Tailspikes have long been known to degrade
bacterial polysaccharides, but until recently,
have only been investigated in the context of
phagehost relationships and not as potential
enzybiotics. Tailspikes are trimers, contain an
N-terminal head-binding domain that
interacts with the base-plate structural
proteins, and a C-terminal receptor binding
and cleaving domain (for an excellent review,
see Casjens and Thuman-Commike, 2011).
Signicantly, the receptor-binding and
cleaving domains can self-assemble into
trimers and can cleave polysaccharides, even
in the absence of the N-terminal head-binding
domains. Much like the cell-wall-binding
domains of the Gram-positive endolysins,
232 Y. Shen et al.
tailspikes can display extreme species
specicity due to the unique polysaccharide
epitope displayed by susceptible organisms.
Tailspike proteins are ofen resistant to
proteolysis, are SDS stable and can tolerate
high temperatures, which makes them well
suited for therapeutic development. Indeed,
due to the excellent thermostability properties
of tailspike proteins, a heating step has been
proposed as a purication tool for a Klebsiella
depolymerase (Kassa and Chhibber, 2012).
Finally, puried tailspike proteins have
been shown to degrade the capsule of
Pseudomonas putida in vitro (Cornelissen et al.,
2011) and decrease the colonization of
Salmonella species in chicken when
administered orally (Waseh et al., 2010).
Concluding Remarks
The spread of antibiotic-resistant pathogens
has forced physicians and biotechnology and
pharmaceutical companies to consider
alternatives. One option is bacteriophage
therapy, which is described in detail by
Burrowes and Harper, and Abedon (Chapters
1214 and 17, this volume). Exploiting phages
for their enzymes (endolysins, bacteriocins or
depolymerises) is another valid approach.
Much like phage therapy, the use of these
enzymes has beneted from billions of years
of co-evolution between phages and their
hosts to develop antimicrobial proteins that
can specically target pathogens with litle
to no expected adverse eects on other
organisms, such as benecial commensal
species.
While endolysins are not self-replicating
like phages, they are enzymatic in their
actions and thus can act repeatedly to
generate lysis from without. Moreover,
because they are proteins and lack nucleic
acids or genetic mechanisms to recombine or
alter the host genome, endolysins should be
safer and thus face fewer regulatory hurdles
than whole phages for therapeutic use.
Endolysins have been shown to be active in
vitro, in vivo and on sessile bacteria such as
those found in biolms (Sass and Bierbaum,
2007). They also can act synergistically with
each other as well as with conventional
antibiotics. Although antigenic, antibodies to
endolysins do not neutralize their activity,
and as proteins they are more readily
degraded and thus more eco-friendly than
conventional antibiotics. Endolysins lyse
their target organism on contact and only
interact with the most external parts of the
cell surface, thereby avoiding many common
resistance mechanisms to antibiotics (e.g.
modication of target, modication of the
therapeutic agent and e ux pumps to
eliminate the therapeutic). Indeed, resistance
has never been reported in organisms that are
originally sensitive to a particular endolysin.
Finally, the modular nature of endolysins
allows molecular biologists and protein
biochemists an opportunity to engineer these
enzymes for optimal activity and/or optimal
host selection. Phage endolysins thus possess
many qualities that make them of high
interest as a novel class of future anti-
microbials.
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16 Role of Phages in the Control of
Bacterial Pathogens in Food
Yan D. Niu
1
, Kim Stanford
2
, Tim A. McAllister
1
and Todd R. Callaway
3
1
Agriculture and Agri-Food Canada;
2
Alberta Agriculture and Rural Development;
3
Food and Feed Safety Research Unit Agricultural Research Service, USDA.
In spite of the numerous hurdles that have
been implemented throughout the food
production chain to ensure food safety,
foodborne illnesses still aect more than 48
million Americans each year (Scallan et al.,
2011) at a total cost of more than US$150
billion (Schar, 2010). Many of the human
foodborne illnesses result from bacterial
contamination of foods, both produce and
meat-based (Scallan et al., 2011). The indirect
and direct cost each year of the ve most
common foodborne pathogenic bacteria in
the US totals more than US$43 billion and
cause more than 1600 deaths (ERS/USDA,
2001; Schar, 2010; see Kuhl et al., Chapter 3,
this volume, for details of some specic
diseases). To combat this immense drain on
both health and nances, researchers in the
US, the EU and around the world have
developed pathogen reduction strategies for
use in the food supply. While these strategies
have typically improved human health and
food safety, they have been far from perfect.
Thus, additional strategies are currently
being developed and evaluated for use to
reduce foodborne pathogens in our food
supply, with one of these strategies being the
use of bacteriophages. Phages have been
examined for use in food products, as well as
on-farm interventions and environmental
cleaning agents. In this chapter, we will
review: (i) the nature of bacterial con-
tamination of the food supply; (ii) the
isolation of zoonotic-bacteria-specic phages;
and (iii) the use of phages against foodborne
bacterial pathogens, with emphasis on
treatment at the farm, in food processing
plants and in ready-to-eat foods. For more
information on phage therapy, please see
Burrowes and Harper, and Abedon (Chapters
12, 13, 14 and 17, this volume) and for more
on bacterial detection and identication see
Williams and LeJeune, Cox, and Goodridge
and Steiner (Chapters 6, 10, and 11, this
volume).
Bacterial Contamination of the
Food Supply
Foods for human consumption naturally
contain bacteria acquired during growth,
harvest, preparation, processing and pro-
duction. These bacteria are ofen reective of
environmental contamination throughout
the food production and preparation chain
(Doane et al., 2007; Arthur et al., 2008).
Typically, this bacterial population is harm-
less to humans and provides a rst immuno-
logical primer to the body to determine self
from non-self, and a lack of exposure to
these bacteria has been linked to the rise in
human allergies (Callaway et al., 2006b).
Role of Phages in the Control of Bacterial Pathogens in Food 241
When animal and vegetable products are not
cooked or pasteurized, however, they can
ofen be sources of human infections by
pathogenic bacteria (e.g. Cody et al., 1999;
LeJeune and Rajala-Schultz, 2009).
Foodborne pathogenic bacteria can
survive in a variety of environments, includ-
ing animals (below), and have been isolated
from soil. Pathogenic bacteria can colonize
produce by being taken up by plant roots
(Natvig et al., 2002) or can be transferred
during handling/processing (Prazak et al.,
2002; Ilic et al., 2008). These bacteria can be
transmited to fruits and vegetables by direct
contact with animals, as well as by contact
with vectors such as insects, mice, birds and
other mammals, as well as irrigation water
(Manshadi et al., 2001; Jay et al., 2007; Talley et
al., 2009).
Foodborne pathogens are found on all
types of animal production farms and in all
stages of animal growth and production
(Oliver et al., 2005; Alali et al., 2010; LeJeune
and Kersting, 2010) and can be transmited to
meat products during processing (Mackey
and Derrick, 1979). Foodborne pathogenic
bacteria can also live asymptomatically in the
gut or on the skin and hide of food animals
(Porter et al., 1997). Enterohaemorrhagic
Escherichia coli (EHEC; including E. coli
O157:H7), Salmonella, Campylobacter and
Listeria are some of the most common food-
borne pathogenic bacteria isolated from
human outbreaks, and have all been isolated
from catle, swine and poultry (Borland, 1975;
Oliver et al., 2005).
The food production industry has
focused for many years on the development
of strategies that reduce foodborne patho-
genic bacteria in the food supply (Koohmaraie
et al., 2005; Ilic et al., 2008). While many of
these have been focused on strategies within
the processing plant, increasingly research
has focused on reducing populations of
patho gens in live animals to enhance the
eective ness of processing plant-level inter-
ventions (Sargeant et al., 2007; Vandeplas
et al., 2010). Perhaps more signicantly from a
public health perspective, human foodborne
illness outbreaks have been linked to indirect
human contact with faeces via water supplies
(both drinking and irrigation) as well as
direct faecal contact (see Goodridge and
Steiner, Chapter 11, this volume). Increasing
incidences of human illnesses linked to direct
animal contact via peting zoos and fairs/
open farms emphasizes the need for some
strategies to focus on reduction of pathogens
on the farm (Chapman et al., 2000; Durso et
al., 2005). While the point source behind
many foodborne illness outbreaks ofen
remains unknown, it is critical for public
health nevertheless to reduce the prevalence
of these pathogens generally on the farm
(Steinmuller et al., 2006). One of the environ-
mental factors that has been suggested to
play an important role in reducing bacterial
distribution in various food-related environ-
ments is bacteriophages (Wetzel and LeJeune,
2007; Niu et al., 2009b).
Natural Phages in Food-producing
Environments
Phages are found naturally throughout the
food chain (Jorgensen et al., 2002; Aterbury et
al., 2003a; Tsuei et al., 2007) and throughout
animals in general (see Letarov, Chapter 2,
this volume). As interest in using phage as
anti-pathogen interventions in the food supply
has grown, questions about their role in the
environment and food supply have grown as
well. The widespread nature of phages
associated with crops as well as live animal
(and human) intestinal environments
strongly suggests that humans have and do
consume phages regularly. Other studies
have demonstrated the common presence of
phages in many traditional fermented foods
eaten around the world, such as yogurt and
cheeses (Goodridge, 2008). Thus, the addition
of phages to foods to reduce foodborne
pathogenic bacteria is not signicantly
dierent from the phage intake that occurs
with normal food consumption.
Phages against foodborne (zoonotic)
pathogenic bacteria have been isolated from a
wide variety of environmental sources
(Ronner and Cliver, 1990; Aterbury et al.,
2003a; Kim et al., 2008). In animals, researchers
have found that 2355% of feedlot catle pens,
48% of swine faecal samples and 130% of
chicken faecal samples contained phages
(Connerton et al., 2004; Callaway et al., 2006a,
2010; Niu et al., 2009b). Coliphages were
242 Y.D. Niu et al.
isolated from 6979% of grasses and silages
fed to catle, as well as from 5% of processed
feeds (Hutchison et al., 2006). Anti-Salmonella
phage were isolated from swine manure
slurries and lagoon e uents, indicating that
they are common components of the swine
faecal population (McLaughlin et al., 2006;
McLaughlin and King, 2008). Phages
specically active against Listeria species
have been isolated from turkey processing
plants (Kim et al., 2008), and naturally
occurring anti-Campylobacter phages were
also widely isolated from retail poultry
products in the UK (Aterbury et al., 2003a).
Collectively, these data illustrate the fact that
humans already consume a great many
phages via the food chain, and have probably
done so for millennia.
Phages against Foodborne Bacterial
Pathogens
In the West, the pursuit of phages and anti-
bacterial therapeutics remained relatively
dormant until the 1980s when Williams Smith
and co-workers demonstrated superior
e cacy with phage therapy as compared
with antibiotics in a series of experiments
with mice, pigs, calves and lambs (see Loc-
Carrillo et al. and Burrowes and Harper,
Chapters 13 and 14, this volume). In these
early studies, phages were used against
enteropathogenic E. coli (EPEC) induced
diarrhoea and splenic enterotoxigenic E. coli
(ETEC) colonization in calves (Barrow, 2001).
Other research groups concluded that phages
had both prophylactic and therapeutic eects
on the reduction of experimentally induced
diarrhoea in weaned pigs experimentally
infected with E. coli (Jamalludeen et al., 2009).
Further studies have shown that phage
treatment is capable of reducing necrotic
enteritis caused by Clostridium perfringens in
broiler chickens (Miller et al., 2010).
Phages have also been used in atempts
to treat other non-gastrointestinal veterinary
diseases, such as bovine mastitis caused by
Staphylococcus aureus (Gill et al., 2006) and E.
coli (Bicalho et al., 2010). Other in vitro studies
have demonstrated that eld isolates of
phages isolated from the uteri of postpartum
Holstein dairy cows have activity against
pathogenic E. coli (Bicalho et al., 2010; Santos
et al., 2010). Researchers have isolated phages
that are active against avian pathogenic E. coli
(APEC) (Hu et al., 2002, 2005; Oliveira et al.,
2010) that cause air sacculitis (a production
disease) in chickens. Oliveira et al. (2010)
found that a cocktail of three phages were
able to protect contaminated birds and
naturally infected ocks from severe coli-
bacillosis APEC infections. These eorts are
in addition to a large literature considering
phage application to animals (and humans)
more generally (see Olszowska-Zaremba et
al., Loc-Carrillo et al., Burrowes and Harper,
and Abedon, Chapters 1214 and 17, this
volume).
On-farm Use of Phages Against
Zoonotic Bacteria
Of the 1415 species of infectious organisms
recorded as pathogenic for humans, 868 (61%)
are characterized as zoonotic (Cleave land et
al., 2001; Taylor et al., 2001). Of 28 pathogens
causing clinically signicant bacterial
zoonoses (Christou, 2011), at least ten includ-
ing Helicobacter sp., Campylobacter sp., Listeria
sp., Salmonella sp., Shigella sp., S. aureus, E. coli
O157:H7, Streptococcus sp., Vibrio sp. and
Yersinia sp. have been demonstrated to be
sensitive to phages in both in vitro and in vivo
clinical trials (OFlaherty et al., 2005). One of
the most eective ways to protect humans
from these infections is to reduce the
prevalence and incidence of the pathogens on
farm, although total elimination is unlikely
(Hynes and Wachsmuth, 2000). Thus, in recent
years, eorts have focused on developing a
continuum of non-antibiotic pathogen re-
duction strategies from farm to fork, which
has included the development of pre-harvest
pathogen interventions for use in and on live
animals (Callaway et al., 2004; Loneragan and
Brashears, 2005; LeJeune and Wetzel, 2007;
Sargeant et al., 2007; Vandeplas et al., 2010). In
North America, while the use of phages in the
treatment of bacterial diseases in humans and
animals has received the most atention, the
use of phages to combat pathogenic bacterial
contamination of food as a public health
measure is far more developed from both a
regulatory and a commercial perspective. In
this section we review the literature
concerning phage use to control zoonotic
bacterial pathogens in food. Additional,
recent reviews of this subject include Greer
(2005), Johnson et al. (2008), Goodridge (2010)
and Hagens and Loessner (2010).
On-farm use of phages against foodborne
pathogenic bacteria
Phage use is being explored as a control
measure for a variety of infectious diseases in
livestock, but is most advanced in poultry
(Connerton et al., 2004; Higgins et al., 2005;
Loc Carrillo et al., 2005; Sillankorva et al.,
2010). The use of phage in broilers is most
eective against Salmonella and Campylobacter
in growing ocks (Sklar and Joerger, 2001;
Loc Carrillo et al., 2005; Sillankorva et al.,
2010). Poultry are ofen found to be positive
for Salmonella and Campylobacter in studies
(Jorgensen et al., 2002), and poultry account
for most of the food-associated salmonellosis
in humans (Guo et al., 2011). While natural
anti-Salmonella phage populations in poultry
occur at a low incidence (approximately 1%;
T.R. Callaway, unpublished data), they have
been isolated from a variety of sources and
can be used to reduce populations of
Campylobacter and Salmonella in the crop and
intestine of chickens (Sklar and Joerger, 2001;
Loc Carrillo et al., 2005; Toro et al., 2005;
Wagenaar et al., 2005; Sillankorva et al., 2010).
While these experiments have shown promise
as proof of concept, to date phages have not
been commercially introduced into the
poultry industry because of expense and the
need to apply treatments to entire poultry
ocks or broiler houses simultaneously.
Swine are ofen associated with carriage
of Salmonella spp. (Davies, 2011; Rostagno
and Callaway, 2012), with prevalence in
faeces ranging from 7 to 20% (Davies, 2011).
Reports suggest that as many as 25% of US
human cases of salmonellosis can be
atributed to consumption of Salmonella-
contaminated pork products (Guo et al., 2011;
Rostagno and Callaway, 2012). Naturally
occurring phages that are active against
Salmonella spp. have been isolated from swine
faeces (Callaway et al., 2010), as well as from
swine wastewater lagoons (McLaughlin et al.,
2006; McLaughlin and King, 2008), which can
harbour faecal Salmonella populations (Hill
and Sobsey, 2003). Researchers found that,
while 48% of the swine faecal samples
contained phages, the occurrence of phages
that specically infected Salmonella Typhi-
murium was approximately 1% (Callaway et
al., 2010). They suggested that, while phages
may be widespread in the national swine
herd, nding a phage active against a specic
Salmonella serotype is possibly linked to the
presence of that specic serotype in the
animal environment. When these researchers
amplied the anti-Salmonella phages from
commecial swine, and inoculated them into
swine experimentally infected with Salmonella
Typhimurium, populations in the cecum and
rectum were reduced (Callaway et al., 2011).
When other researchers used Salmonella
phages isolated from municipal wastewater,
they found that phage treatment reduced
experimentally inoculated Salmonella popu-
lations in nished swine (Wall et al., 2010).
These researchers found that a phage cocktail
reduced Salmonella populations in the ileum
and caecum under conditions similar to those
encountered in commercial production
systems (e.g. in pens in an open barn) (Wall et
al., 2010; Zhang et al., 2010). Results from a
follow-up study from this research group
also appeared to be promising in the
development of a viable phage cocktail
product for use in the swine industry (Zhang
et al., 2010). In both of these proof-of-concept
studies, the number of pigs that were positive
for Salmonella was reduced by phage treatment,
but the pathogen was never completely
eliminated from all phage-treated swine (Wall
et al., 2010; Zhang et al., 2010; Callaway et al.,
2011). Despite the lack of elimination of
Salmonella in these animals, some individual
pigs did stop faecal Salmonella shedding
(reduction in prevalence). Additionally, the
degree of reduction of Salmonella populations
(concentrations reduced from 1 to 3 log
10
colony-forming units (CFU) g
1
digesta)
associated with phage treatment was
signicant enough to cause a reduction in the
total Salmonella pathogen load entering the
244 Y.D. Niu et al.
food chain and should thus result in a
signicant reduction in human foodborne
illnesses (Hynes and Wachsmuth, 2000). In an
intriguing study, a cocktail of phages
signicantly reduced the severity of diarrhoea
in pigs experimentally infected with ETEC
O149 but did not aect total intestinal E. coli
populations (Jamalludeen et al., 2009). Phages
were demonstrated to replicate in ETEC-
infected pigs within 2 days (Jamalludeen et
al., 2009).
Unfortunately for swine and poultry
producers, Salmonella intervention strategies
present unique challenges. There are more
than 2500 serotypes of Salmonella currently
known, and thousands of these serotypes
have been isolated from food animals in
recent years (Popo et al., 2004; USDA/FSIS,
2006). Studies using phages to control
Salmonella in live animals have been
hampered by this broad diversity of sero-
types, because typically phages have been
selected against one or two common sero-
types (Callaway et al., 2010, 2011). The
development of phage cocktails that are
active against multiple serotypes is likely to
alleviate this hurdle, but much research into
the ecology and specicity of serotypes and
their atendant phages is needed to be able to
use phage eectively to control Salmonella in
live animals on a large scale.
Phages in ruminant animals
Because of their unique physiology,
ruminants pose more challenges than single-
stomached animals with regard to the use of
any intervention strategies against foodborne
pathogenic bacteria (Loneragan and
Sargeant et al., 2007; Oliver et al., 2008).
Ruminants also have relatively large caecum
where a distinct microbial population
performs a secondary fermentation. The
relatively recent discovery of E. coli O157:H7
and other EHEC (also known as Shiga
toxigenic E. coli or STEC) as natural residents
of the ruminant gastrointestinal tract,
especially at the recto-anal junction, has
brought new focus to research into ways to
reduce pathogens, especially EHEC, in the
intestinal tract of live catle (Loneragan and
Sargeant et al., 2007). Because E. coli O157:H7
is classied as an adulterant in ground beef,
there is zero tolerance for this organism,
meaning that any positive lots of ground beef
must be destroyed, which represents a
signicant cost to the catle industry. Over the
past 18 years, the beef industry has spent in
excess of US$1 billion in combating E. coli
O157:H7 at both the processing plant and live
animal levels (Kay, 2003). A wide variety of
potential E. coli O157:H7 intervention
strategies have been proposed for use in catle
and are reviewed elsewhere (Loneragan and
Sargeant et al., 2007). We will focus on the use
of phages in this role, because the highly
impactful nature of this pathogen has resulted
in some elegant proofs of the ability of phage
to target specic foodborne pathogenic
bacteria in the gastrointestinal tract.
Phages have been isolated from ruminant
animals for many years (Adams et al., 1966;
Callaway et al., 2006a; Viazis et al., 2011), and
have been used in atempts to modify ruminal
fermentation with variable success (Orpin
and Munn, 1973; Klieve et al., 1991). Phages
that target E. coli O157:H7 were isolated from
10% of bovine and ovine faecal samples
(Kudva et al., 1999). In other studies, anti-E.
coli O157:H7 phages were found in 15% of the
individual faecal samples and in 55% of US
commercial feedlot pens (Callaway et al.,
2006a) and from approximately 23% of the
faecal samples in Canadian feedlot pens (Niu
et al., 2009b). Phages from these and other
sources (Jamalludeen et al., 2007; Viazis et al.,
2011) have been used in a variety of studies in
ruminants to reduce E. coli O157:H7 popu-
lations in the gastrointestinal tract, albeit with
variable success (Kudva et al., 1999; Bach et
al., 2002; Bach et al., 2003; Sheng et al., 2006;
Bach et al., 2009; Rozema et al., 2009). Phages
have exhibited high virulence in killing
STEC/EHEC O157:H7 and non-O157 STEC/
EHEC in broth culture (Niu et al., 2008, 2009a,
2010; Stanford et al., 2010; Viazis et al., 2011)
as well as experimentally infected catle and
sheep (Callaway et al., 2006a; Raya et al., 2006,
2011; Sheng et al., 2006). Other studies have
had less denitive results, although this was
dependent on the method used to treat the
catle with phage (i.e. oral versus rectal
administration, or feed versus waterborne
dosing) or on the phages selected for use
(Bach et al., 2003, 2009; Rozema et al., 2009).
Early studies examining the eect of
phages on E. coli O157:H7 in vitro involved
isolation of phages from ovine and bovine
sources. These studies found that the phages
were only highly active against E. coli
O157:H7 when aerated, which mitigated their
utility in the anaerobic intestinal tract (Kudva
et al., 1999). In one of the rst studies applying
phages orally to ruminants, a single phage
type that had worked well against E. coli
O157:H7 in vitro (Bach et al., 2002) did not
reduce experimentally inoculated E. coli
O157:H7 populations in sheep, but this was
hypothesized to be related to adsorption or
inactivation of the phage (Bach et al., 2003). In
the rst successful proof-of-concept study,
oral addition of a single phage to sheep that
were experimentally inoculated with E. coli
O157:H7 resulted in a decrease in intestinal
populations within 2 days (Raya et al., 2006).
Following up on this study, oral addition of
a phage cocktail to sheep reduced
experimentally inoculated E. coli O157:H7
populations numerically in the rumen and
signicantly in the caecum and rectum
(Callaway et al., 2008). Interestingly, in this
study, the eect of dierent phage:target
ratios was examined (see Abedon, Chapter
17, this volume, for additional discussion of
such ratios), and a ratio of 1:1 (phage:target)
reduced experimentally inoculated E. coli
O157:H7 populations to a greater extent than
a 10:1 or 100:1 ratio (Callaway et al., 2008). In
later studies, some of the early phages
isolated from ruminants (Kudva et al., 1999)
were eective in reducing E. coli O157:H7
populations when added at a multiplicity of
infection of 100 directly to the recto-anal
junction (Sheng et al., 2006). This reduction
was signicant, but again pathogen shedding
was not eliminated (Sheng et al., 2006).
Further studies by the Lethbridge
Research Group led by T.A. McAllister and K.
Stanford indicated that phage treatment
reduced experimentally inoculated E. coli
O157:H7, but total E. coli populations were
not aected. The need for phages to bypass
the abomasum, however, was determined to
be important by these researchers (Bach et al.,
2009). In a follow-up study, these researchers
found that orally phage-treated steers shed
less E. coli O157:H7 cells than rectally or oral
and rectally phage-treated steers, but there
was no elimination of shedding by any
treatment. The benecial eect of phages on
shedding was possibly due to the ability of
phages to propagate extensively in the rumen
and constantly be washed to the lower
intestinal tract where they can aect EHEC
populations (Rozema et al., 2009).
Interestingly, in this study the spread of
phages from treated to non-treated animals
was noted, indicating that the concept of
spreading a biocontrol agent among catle
may be simply accomplished through the use
of a few seeder animals rather than treating
a herd or pen as a whole (Rozema et al., 2009).
When a polymer-encapsulated bacteriophage
was used in experimentally inoculated E. coli
O157:H7 animals, pathogen shedding was
decreased, again suggesting that protection
of phages from hydrolysis may improve
phage treatment e cacy (Stanford et al.,
2010).
In another study, researchers found that
sheep naturally colonized with an O157:H7
infecting phage were more resistant to
experimentally inoculated E. coli O157:H7
colonization than non-phage-colonized or
experimentally phage-colonized sheep, sug-
gesting that natural phage populations may
play a role in the transient nature of E. coli
O157:H7 colonization in catle (Raya et al.,
2011). In this study, phage addition was also
found to reduce experimentally inoculated E.
coli O157:H7 populations (Raya et al., 2011).
Furthermore, several studies have demon-
strated that the use of a phage cocktail is
preferable to the use of a single phage in
controlling E. coli O157:H7 populations (Raya
et al., 2011; Viazis et al., 2011).
Collectively, these results indicate that
real-world use of phage in food animals
requires further development and optimi-
zation. Elucidating factors such as appropriate
phage selection and preparation (including
from which environment), and protection of
phages from acidic and enzymatic hydrolysis,
as well as administration strategy (i.e. dose,
246 Y.D. Niu et al.
frequency and delivery system) is critical for
successful utilization of phage as a method to
reduce foodborne pathogenic bacteria within
the gut of all food animals (Johnson et al.,
2008; Rozema et al., 2009; Stanford et al., 2010).
For additional discussion of E. coli O157:H7,
see Kuhl et al., Christie et al., Williams and
LeJeune, and Goodridge and Steiner
(Chapters 3, 4, 6 and 11 this volume)
Phages on hides
While E. coli O157:H7 is a member of the
microbial ecosystem of catle, it, along with
Salmonella spp., can also be found on the hide
of catle as they enter the abatoir. The hide-
based pathogens can then be transferred to
the carcass during processing; as a result,
several interventions have been proposed to
reduce this pathogen transfer including acid
and chemical rinses (Bosilevac et al., 2005;
Arthur et al., 2007; Loretz et al., 2011).
Interestingly, in spite of what was thought to
be a high regulatory barrier to the use of
bacteriophages, a hide spray has been
approved for use on catle in the US (FDA,
2006). Finalyse
TM
is a hide spray product that
has begun use in eld studies to specically
reduce E. coli O157 on catle prior to proces-
sing and thus far has exhibited promising
results in eld trials. Other potential hide
reduction sprays are in development around
the world and have shown promise in initial
studies (Coey et al., 2011).
Phages and environmental contamination
Other issues that involve live animals as a
source of human foodborne illnesses involve
direct faecal contact (Chapman et al., 2000;
Pritchard et al., 2000) as well as runo from
farms that can penetrate water supplies used
for drinking or crop irrigation (Anon., 2000;
Manshadi et al., 2001; Ferens and Hovde,
2011). Further vectors of pathogens from the
farm to other crops and humans include ies
(Talley et al., 2009) and other animal vectors
(e.g. wild pigs) that have been responsible for
large-scale human outbreaks (Jay et al., 2007).
In an eort to combat this potential source of
foodborne illness, phages isolated from
sewage e uent have been used as a cocktail
to reduce Salmonella Typhimurium more than
100-fold within 4 h within composted
manure, a potential source of pathogens
(Heringa et al., 2010). This phage cocktail
oers a potential on-farm treatment to reduce
the environmental impact and spread of
pathogen shedding.
Phages in foods and in processing
plants
One of the most direct and logical ways to
utilize phages to improve food safety comes
in the realm of use on foods and surfaces in
processing plants (Greer, 2005; Hudson et al.,
2005; Karmali et al., 2010). Phages have been
approved by the FDA for use in food
processing plants for cleaning of surfaces,
and amendments to the rules have been
applied for, in order to allow use in ready-to-
eat products as of mid-2011 by Intralytix
(FDA, 2006). Thus, we have entered a new era
for the development of phage products for
use in food processing.
Research studies have shown that phages
can reduce populations of Salmonella and
Vero toxin-producing E. coli (VTEC)/STEC in
fruits and vegetables (Pao et al., 2004;
Abuladze et al., 2008; Patel et al., 2011).
Sprouts have been a source of human food-
borne illnesses several times, and these have
been linked to contaminated seeds. Among
many strategies aimed at reducing this
contamination (Nandiwada et al., 2004; Singla
et al., 2011), phages have been examined and
were found to eectively reduce Salmonella
populations (Pao et al., 2004). Spinach and
other green leafy vegetables have also been
sources of several outbreaks (Ilic et al., 2008).
Phages have been sprayed on to spinach to
reduce E. coli O157:H7 populations (Abuladze
et al., 2008), and recent studies have found
that phage treatment of harvester blades
could reduce E. coli O157:H7 populations
(Patel et al., 2011). Other researchers have
found that phage treatment at high densities
could reduce experimentally inoculated E.
coli O157:H7 populations on broccoli, spinach
and tomatoes (Abuladze et al., 2008).
Phages have also been used experi-
mentally to reduce pathogens on retail and
ground beef (both raw and cooked) products
(Abuladze et al., 2008; Bigwood et al., 2008).
Other research has indicated that the
inclusion of phages on chicken carcasses or
retail cuts can eectively reduce Salmonella
and Campylobacter contamination (Aterbury
et al., 2003b; Goode et al., 2003; Higgins et al.,
2005). In one of the rst real-world type of
proof-of-concept studies, I.F. Connertons
research group demonstrated that phage
application to chicken skin inoculated with
Campylobacter jejuni resulted in a 1 log
10
CFU
reduction within 2 days (Aterbury et al.,
2003b). Other researchers found that the
addition of a low ratio (1:1) of phage to
experimentally inoculated Campylobacter and
Salmonella resulted in a modest decrease in
pathogen populations (Goode et al., 2003).
When the phage:target ratio was increased,
the populations decreased by more than
2 log
10
CFU, and when sprayed on carcasses
with a very low pathogen presence with a
multiplicity of infection of 1000, Salmonella
was eliminated entirely (Goode et al., 2003;
see also Abedon (Chapter 17, this volume) for
additional discussion of phage:bacterial
ratios). Raw and cooked beef inoculated with
Campylobacter and Salmonella had reduced
pathogen counts in the order of 25.9 log
10

CFU cm
2
dependent on incubation
temperature and food type (Bigwood et al.,
2008). Other studies have found that the use
of sprays and carcass rinses using a cocktail
of up to 72 phage varieties could reduce
carcass populations of experimentally
inoculated Salmonella Enteritidis (Higgins et
al., 2005). Finally, the addition of a relatively
high dose of phage (10
8
10
10
plaque-forming
units) reduced experimentally inoculated E.
coli O157:H7 populations from 1 to 4 log
10

CFU in ground beef (Abuladze et al., 2008).
Dairy products have long been known to
contain phages due to the common use of
Lactococcus and Streptococcus in cheese and
yogurt production (Coetzee et al., 1960; Zhang
et al., 2006). Raw milk cheeses are known to
harbour pathogenic bacteria and have caused
outbreaks of human illness, and studies have
shown that phage addition can reduce
Salmonella populations in raw milk and
pasteurized cheeses (Modi et al., 2001).
Further exciting studies have demonstrated
that Listeria populations can be signicantly
reduced in raw cheeses by the addition of
phage (Carlton et al., 2005; Schellekens et al.,
2007; Guenther et al., 2009). Collectively, these
results indicate that phages have denite
potential in reducing foodborne pathogen
carriage in dairy products.
Cross-contamination of foods within the
plant is a signicant route of human food-
borne illnesses. The use of phages as cleaning
agents within processing plants therefore
oers some signicant opportunities to
reduce the pathogen load entering the food
chain, especially with regard to the creation
of biolms (see below). From use as a method
to clean machinery used in harvesting/
processing (Patel et al., 2011) to the cleaning
of food preparation surfaces (Abuladze et al.,
2008; Brooks, 2009), phage cleaning products
have a signicant role to play in the
disinfection of processing facilities. The use
of anti-listerial phage sprays is especially
pertinent to facilities that prepare ready-to-
eat foods, which are not further cooked by
consumers and thus are a direct route of
contamination (Guenther et al., 2009).
Role of Biolms in Food
Contamination and Prospects of
Phage Treatment
Biolms are a concern in food production
(Brooks, 2009; Abedon, 2011; Myszka and
Czaczyk, 2011), and have been addressed
primarily in the postharvest or processing
type of environment as a method to install
another hurdle to pathogen entry to the
continuum of food production (e.g. acid
washes, UV light and salt treatments) (Sofos
and Smith, 1998; Koohmaraie et al., 2005; Fox
et al., 2008). One of the most signicant
pathogens associated with biolms is Listeria,
which can form or take part in other bacterial
biolms on food-processing surfaces and
equipment (Bernbom et al., 2011). Other
common foodborne pathogenic bacteria such
as Salmonella, Campylobacter and E. coli
O157:H7 can be found participating in
biolms in various environments (Dourou et
248 Y.D. Niu et al.
al., 2011; Hasegawa et al., 2011). Contact
between foods or equipment and these
biolms can transfer the bacteria to the
foodstu, thus ensuring the presence of
spoilage organisms or, more signicantly,
foodborne pathogenic bacteria. Furthermore,
the presence of a biolm can isolate bacteria
from these kinds of physical treatments,
preventing pH or osmotic shocks or even UV
light penetration to targeted pathogens
(Bernbom et al., 2011; Dourou et al., 2011),
thus ensuring their survival and passage into
the food supply on raw, processed or ready-
to-eat products.
Phages are an alternative treatment that
has the potential to obviate some of the
protective benets of biolms (Abedon,
2011). Of note, phages can produce poly-
saccharide depolymerases to degrade the
extracellular polysaccharide matrix of the
biolm (Hughes et al., 1998; see also Shen et
al., Chapter 15, this volume). Accordingly,
phages were used to eliminate Pseudomonas
uorescens biolms in the rst 5 days of
development (up to 80% of biolm removal)
in a model system (Sillankorva et al., 2004).
An in vitro study showed that an engineered
T7 phage, with the capability of producing a
biolm-degrading enzyme, completely
reduced E. coli counts in biolms and
improved biolm removal compared with a
wild-type phage that lacked this activity (Lu
and Collins, 2007). Further studies have
indicated that phage could disperse biolms
made by C. jejuni (Siringan et al., 2011). Thus,
it appears that phage sprays can be used
against pathogens that would otherwise be
impervious to conventional anti-pathogen
treatments due to the presence of a biolm on
the work surface or processing machinery.
Further research is under way into this
exciting area of phage treatment.
Concluding Remarks
Because foodborne pathogenic bacteria can
penetrate the food supply chain at many
points from the farm to the fork (known as
the hazard analysis critical control point, or
HACCP, approach), the ability to counteract
these pathogens directly is crucial. Because of
the nature of phage activity, they have been
proposed for a variety of uses to improve
human public health through a variety of
mechanisms. Phage therapy oers a natural,
safe and eective means to treat and in some
cases prevent bacterial disease, as pathogens
can be specically targeted without aecting
commensal ora in the environment. As the
problem of antibiotic resistance has grown,
researchers have striven to develop phage-
based strategies to treat or prevent bacterial
diseases, which may be the only means of
controlling pathogens that exhibit resistance
to multiple antibiotics. By using phage as
treatments in the eld, on animals and crops
as they enter the processing facility, as a
cleaning spray and as a preparation on
ready-to-eat foods, we can use phage as an
adjunct to existing pathogen-reduction
strategies. By erecting multiple hurdles to
prevent patho gens reaching human con-
sumers, we can signicantly reduce human
illnesses. As can be seen by the wide variety
of approaches of phage treatment and usage
in the food industry, we are well on the way
to imple ment ing these predators of patho-
gens to further ensure the safety of our food
supply.
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17 Phage-therapy Best Practices
Stephen T. Abedon
1
1
Department of Microbiology, The Ohio State University.
S.T. Abedon
Phage therapy, or phage-based bacterial
biocontrol (Abedon, 2009c), is the use of
bacteriophages to combat nuisance or patho-
genic bacteria. This is done to augment the
use of chemical antibacterials or, especially, to
serve as alternatives to anti biotics. Phage
therapy is useful, particularly: (i) in light of
bacterial evolution to antibiotic resistance; (ii)
given the limitations to chemical antibacterial
e cacy such as against bacterial biolms; (iii)
in terms of the potential for certain antibiotics
to cause relatively severe side eects; and (iv)
due to concerns over the negative impact of
chemical antibacterials on environments. The
later can be a consequence, for example, of
antibiotic release into municipal sewage
systems following disposal or elimination
down the drain, or due to the use of chemical
antibacterials outdoors to control bacterial
pathogens of plants (Balogh et al., 2010).
Phage therapy can be impressively safe,
highly e cacious and even relatively
economical; see Olszowska-Zaremba et al.
(Chapter 12, this volume) for a discussion of
phage safety and Olszowska-Zaremba et al.,
Loc-Carrillo et al. and Burrowes and Harper
(Chapters 1214, this volume), as well as the
review by Abedon et al. (2011), for a discussion
of phage-therapy e cacy. It is also important,
nevertheless, for phage formulations and
protocols to be developed knowledgeably.
Specically, there exist a number of
approaches to phage choice (Gill and Hyman,
2010), application and experimentation that
should only be deviated from for explicit
reasons rather than avoided due solely to a
lack of appreciation of their importance or
utility. Here, I provide a guide to such phage-
therapy best practices, principles that have
been formulated in the course of reviewing
the modern phage-therapy and phage-based
bacterial biocontrol literature (Abedon and
Thomas-Abedon, 2010; Kuter et al., 2010;
Abedon, 2010a,b, 2011b; Abedon et al., 2011)
in combination with extensive consideration
of related issues of phage ecology (Abedon,
2006, 2008a,b, 2009a,d, 2011b,c; Abedon et al.,
2009; Chan and Abedon, 2012a). These ideas I
present as answers to a series of questions,
twelve in all, which I believe should be
generally considered over the course of
phage-therapy research and development.
Four more issues are also presented at the
end of the chapter those of host range,
unusual results, the dependence of phage-
mediated bacterial killing on phage titres,
and a discussion of the meaning or lack
thereof of positive results. To avoid mis-
interpretation of the third point, which seems
to occur all too ofen that is, dependence of
phage-mediated bacterial killing on phage
titres I will be redundant in noting that it is
phage densities in particular that determine
rates of bacterial killing during phage therapy
and not bacterial densities or ratios of added
phages to bacteria.
Phage-therapy Best Practices 257
In addition to helping guide the design
of more eective phage-therapy experi-
mentation, the ideas presented can also be
used towards rational debugging of phage-
therapy protocols that have proven to be
insu ciently e cacious. The hope is to
improve on the overall rigour as well as the
eectiveness of phage therapy and to do so
particularly by providing consideration of
how phage therapy should not be practised.
This undertaking can also be viewed as an
extension of ongoing eorts to tackle issues
of phage-therapy pharmacology (Abedon,
2009b,c, 2010a 2011a,b,d, 2012; Abedon
and Thomas-Abedon, 2010; Curtright and
Abedon, 2011; Ryan et al., 2011; Chan and
Abedon, 2012b).
Phage-therapy Pharmacology
In phage therapy, phages are applied to
bodies specically to impact on the con-
stitution of a bacterial community. As bodies
exist, pharma cologically, as a com bination of
both body cells and associated ora (see
Letarov, Chapter 2, this volume), this use of
phages as antibacterial agents can be viewed
as a form of body modication intentionally
undertaken for the sake of combating disease.
In other words, phages can be productively
considered as variations on the concept of an
antibacterial drug (Abedon, 2012). As the
primary goal of pharmacology is to enhance
drug utility, so too should the perspective of
this chapter be viewed as basically
pharmacological. Accordingly, in this section,
I provide an introduction to the ideas of
pharmacology in general and phage-therapy
pharmacology in particular.
Pharmacology is the study of drugbody
interactions. Relevant to phage-therapy
pharmacology, symbionthost interactions
and organismenvironment interactions can
be studied from ecological perspectives. Just
also as ecological interactions can be
considered as occurring between environ-
ments and organisms (Abedon, 2011c), so too
can drugs be viewed in terms of impacting on
bodies or, instead, in terms of bodies
impacting on drugs. In pharmacology, these
distinct views are described respectively in
terms of pharmacodynamics and pharma-
cokinetics. Pharmacodynamics in particular
can be dierentiated into positive versus
negative consequences of drug action on the
body, such as control of bacterial infections
versus causing adverse side eects.
Pharmacodynamic eects occur as a
function of drug densities as found in specic,
target tissues within the body (with the body
dened as a combination of both eukaryotic,
i.e. human cells and the associated micro-
biome). Thus, for example, we can consider
the impact of antibiotic densities found in the
vicinity of bacteria-infected tissues on those
bacteria (anti-bacterial eects) as well as side
eects aecting human cells and tissues
(which have typically negative consequences).
Drug density in turn is a consequence of a
combination of dosing and pharmacokinetic
considerations. Specically, most drugs fol-
lowing dosing will access the systemic cir-
culation of the blood (absorption), penetrate
into non-blood tissues (distribution), be
subject to inactivation (metabolism) and may
also be eliminated from the body (excretion).
For certain drugs, including phages as well as
pro-drugs, metabolism that is, chemical
reactions as they occur in for example human
cells, human tissues or associated micro-
organisms can also lead to increases in
active pharmaceutical densities in situ.
As with most antibacterials, successful
bacterial control can require substantially
more phages than there are bacteria present.
The generation of these phage densities via
dosing alone has been described as a passive
treatment, whereas the harnessing of phage
population growth to achieve phage densities
that are su cient to result in bacterial control
instead has been termed active treatment
(Payne et al., 2000; Payne and Jansen, 2001,
2003; Abedon and Thomas-Abedon, 2010;
Abedon, 2012). In either case, antibacterial
activity with phage therapy is accomplished
via a bactericidal action and, other than in
terms of phage properties, is entirely
dependent on the establishment of su cient
phage densities within the vicinity of the
target bacteria, such as the achievement of
densities of approximately 10
8
free phages
ml
1
.
258 S.T. Abedon
Phage-therapy Best Practices I: Basic
Considerations
It is my premise that phage-therapy experi-
mentation could benet from a more rigorous
pharmacological approach. Although in part
increases in pharmacological rigour may be
addressed in terms of beter modelling of
phage impact or greater consideration of
phage pharmacokinetics, in fact I am referring
to much simpler as well as more achievable
practices, such as improved consideration of
basic microbiology, controls and experimental
design. In this section, I consider phage-
therapy best practice numbers 14, which
address the issues of phage isolation, phage
choice, phage characterization and disease
models. I then provide a discussion within
this section before presenting, in subsequent
sections, phage-therapy best practice num-
bers 58 (controls) and then 912 (dosing and
enumeration).
1. Has a good strategy been used to
isolate highly efcacious phages?
As with chemical antibiotics, the rst step in
phage therapy development is the isolation of
appropriate phages (Balogh et al., 2010; Gill
and Hyman, 2010). Appropriate phages are
those that are capable of killing bacteria
under the expected in situ conditions. This
criterion should be viewed as basic to phage
choice for phage-therapy use, with phage-
isolation protocols designed with this goal in
mind. Methods for such isolation ideally will
involve enrichment-culture techniques that
use bacterial hosts and conditions that are
well matched to the expected hosts and
environ ments during treatment, perhaps
especially the former (Abedon, in press).
Isolation without enrichment, such as via
direct plating of environmental or tissue
samples, will ofen provide numerous phages
that can kill bacteria but not necessarily those
phages that are the most eective at doing so.
A red ag is the use of phages for phage-
therapy studies that are obtained using
mechanisms biased primarily towards con-
venience, such as through the American Type
Culture Collection, unless the goal is to
characterize specic phage isolates.
Phage-therapy publications should
provide su cient detail on approaches
employed for phage isolation including
strengths, shortcomings and rationales
justifying their use; enriching for phages at
37C for use at 15C, for example, should
represent an obvious error in experimental
design. Alternatively, it is important for
researchers to keep in mind that in situ phage
growth characteristics are relevant to phage
choice only if phage-therapy protocols are to
be reliant on active rather than passive
treatment. This is because passive treatment
is dependent solely on phage bactericidal
eects rather than also depending on phage
in situ amplication. Of course, to a large
degree, a demonstration of e cacy (positive
results) in conjunction with phage-therapy
protocols will negate phage isolation
concerns. As will be discussed subsequently,
however, not all phage therapy positive
results are equivalently e cacious. An
additional issue concerning phage choice and
isolation is that of host range, which I address
towards the end of the chapter.
2. Have the phages been adequately
characterized ex situ?
From a pharmacodynamic perspective,
phages should be chosen for phage therapy
based not only on their ability to kill bacteria
but also in terms of their relative safety
(Abedon and Thomas-Abedon, 2010; Abedon,
2012; see also Olszowska-Zaremba et al.,
Chapter 12, this volume). With both of these
criteria in mind, there are generally three
characteristics that should be avoided in
phage therapeutics, the rst two perhaps
more importantly avoided than the third.
These include an inability to display lysogenic
cycles (i.e. non-temperate phages), a failure to
encode bacterial virulence factors (see
Christie et al., Chapter 4, this volume) and an
inability to display generalized transduction
of bacterial genes (see Abedon, Chapter 1,
this volume).
Fortunately, lack of these three properties
can positively correlate with phage ability to
kill bacteria. A phages potential to display
lysogenic cycles, however, may not always be
obvious or necessarily easy to establish.
Notwithstanding the later caveat, (i) an
inability to display lysogenic cycles means
that no bacteria will become phage immune
in the course of successful phage infection
(Fogg et al., 2010); (ii) phage encoding of
bacterial virulence factors seems to be
associated particularly with temperate
phages or their immediate descendants
(Hyman and Abedon, 2008); and (iii) non-
temperate phages ofen disrupt bacterial
chromosomes in a manner that makes
generalized trans duction less likely (Wilson
et al., 1979; Kuter et al., 1994).
All phage-therapy studies if at all
possible should employ phages that have
been shown, as explicitly indicated in
publications, to form clear plaques when
plated on potential target bacteria (and, for a
higher level of stringency, demonstration that
phage-resistant bacteria isolated from the
plaques in fact are not lysogens). This is
rather than forming plaques with turbid
centres, which serves as a marker for phage
temperance, although not necessarily with
100% accuracy (Stent, 1963). Furthermore, not
all temperate phages form lysogens on all
bacteria they are capable of infecting
productively. None the less, at a minimum, a
failure to produce clear plaques should be
taken as a red ag with regard to the use of a
given phage isolate for phage-therapy
purposes.
Under certain circumstances, the use of
temperate phages may be necessary, such as
when there is a lack of availability of non-
temperate phages. The choice of using
temperate phages, however, must be justied
in publications, and even defended. Given
the association of bacterial toxin genes in
particular with temperate phages, it is also
reasonable to expect greater levels of phage
characterization by investigators who are
using either temperate phages or phages that
are derived from temperate phages, that is,
when employing phages that are not
professionally lytic (Curtright and Abedon,
2011). This is additional characterization,
particularly in terms of virulence factor
encoding.
It is my view that this more stringent
characterization should not be an absolute
requirement for publication, at least in terms
of the early-stage development of phage-
therapy protocols. For later-stage develop-
ment of phage-therapy protocols, especially
ones using human subjects, phage encoding
of bacterial toxins and virulence factors
should be ruled out prior to phage use, even
for professionally lytic phages. By contrast,
study of the phage potential to inadvertently
transduce bacterial virulence factor genes via
generalized or specialized transduction has
not been a priority among phage-therapy
researchers. Presumably, the development of
standardized as well as relatively inexpensive
protocols for such characterization would be
useful, however. In addition, where possible,
the propagation of phages in vitro for
subsequent use in vivo should be conducted
using bacterial hosts that lack virulence factor
genes.
3. Has reasonable ex situ phage anti-
bacterial virulence been observed?
In vivo or in situ experimentation can be
expensive. In addition, in vivo studies can be
subject to ethical considerations, and this is so
even if such experiments have a reasonable
likelihood of successful outcome. For human
testing, of course, these later considerations
are explicit, but issues of expense and
researcher time also suggest obvious benets
to developing techniques using simplied
experimental or theoretical models prior to
more involved testing.
Although various authors have pre-
sented in silico approaches to understanding
phage therapy processes, these methods are
neither well developed nor well tested for
predictive accuracy (Gill, 2008). Less
complicated approaches exist that can allow
researchers to calculate the minimum number
of supplied phages necessary to achieve
phage-therapy success (Abedon and Thomas-
Abedon, 2010; Abedon, 2011a,b). These too,
however, are not always adequately robust
means of phage characterization with regard
to bacterial killing ability, as they may be
performed prior to ramping up to more
260 S.T. Abedon
complex procedures such as animal testing.
What then constitutes reasonably robust in
vitro characterization of phage-mediated
bacterial killing?
First, if at all possible, in vitro (i.e. ex situ)
characterization should at least atempt to
simulate in situ conditions (Nasser et al., 2002;
Gill et al., 2006b; Bull et al., 2010), perhaps
particularly in terms of bacterial physiology
and density. Thus, if bacteria in situ are
presumed to be in stationary phase and
present at densities of 10
4
CFU ml
1
, then in
vitro testing should employ stationary-phase
bacteria and bacterial densities of 10
4
CFU
ml
1
. Similarly, if biolms are explicitly
serving as antibacterial targets, then a phages
anti-biolm capabilities might also be
explored in vitro before moving on to animal
testing (Abedon, 2010a; 2011b).
Secondly, measuring bacterial killing in
terms of bacterial viable counts can provide
more information than optical density or total
bacterial cell count determinations. In part,
this is because the later readily determines
only a few log reduction in bacterial density.
Instead, a good rule of thumb is that one
should be able to atain at least 4 log
reductions, in vitro, over reasonable spans of
time (Kasman et al., 2002; Abedon and
Thomas-Abedon, 2010; Abedon, 2011b, 2012)
when using an approximation of those phage
densities that are actually obtainable in vivo or
in situ. Clearly, it is hard to defend moving up
to in vivo testing using phages for which this
substantial amount of bacterial killing has not
been or cannot rst be demonstrated in vitro
using presumed in situ phage densities.
Thirdly, during in vitro testing, phage
bacterial community interactions should be
followed over relatively short time intervals
so that bacterial growth, giving rise to
substantial increases in bacterial densities,
does not occur over the course of experi-
mentation. This means, in particular, that
overnight incubations of phages with bacteria
do not represent an optimal means of
characterizing phage impact on bacteria
(Abedon, 2011a). Kinetic analyses also are
typically preferable to end point determin-
ations when studying phagebacterial
interactions, whether in terms of phage
adsorption, phage growth character istics or
phage potential to clear cultures (Hyman and
Abedon, 2009).
A standard assay for phage anti-bacterial
virulence consists of adding phages at
dierent initial titres to a standardized
bacterial culture. Those phages that can clear
and otherwise reduce culture viable counts
from the lowest starting titre are deemed the
most virulent. From Smith and Huggins
(1983, p. 2660): Ten ml amounts of nutrient
broth were inoculated with 3 l0
8
viable E.
coli organisms and three-fold falling numbers
of phage particles and incubated for 5 h. The
lowest inoculum of phage particles that
produced complete clearing of the bacterial
cultures was then recorded. Such assays are a
measure of multiple phage parameters that
can contribute to phage population growth
rates as well as anti-bacterial activity,
although not all of these parameters are
necessarily relevant to all phage-therapy
protocols (Abedon, 2009b).
Alternative approaches to measuring a
phages bacterial killing ability have been
described by Abedon and Thomas-Abedon
(2010) and Abedon (2011a,b, 2012). Further-
more, unless actively designed to do so, in
vitro assays are not necessarily a measure of a
phages ability to penetrate to target bacteria
in vivo, such as into biolms or in terms of
phage movement between body com-
partments. Still, before moving on to animal
or otherwise more costly in situ experi-
mentation, phages should at least display in
vitro therapeutic abilities, such as 4 log
reductions in bacterial viable counts at phage
densities that are readily atainable in situ. In
any case, it is not so much that robust and
eective in vitro phage characterization is
crucial to successful phage therapy develop-
ment but instead that such eorts are cheaper,
less time-consuming and frankly more
humane than relying entirely on animal
testing to characterize phage isolates for
bacterial killing ability.
4. Has bacterial colonization been
established prior to phage application?
While ex situ testing is important for practical
as well as economic reasons, it is crucial to
make sure that subsequent testing employs
animal or other models that reasonably
represent the actual circumstances under
which phage therapy is envisaged. What
constitutes a good model for in vivo or in situ
phage therapy e cacy? At a minimum, eort
should be made to make sure that bacterial
colonization along with tissue invasiveness
and other infection details occur to an extent
that is similar to that seen with typical
infection presentation (see, for example, Loc-
Carrillo et al., Chapter 13, this volume). For
instance, if biolms are normally present, and
have developed for days, weeks or months
prior to the onset of antibacterial treatment,
then such biolms should also be present in
the disease model employed to explore the
potential for phage-therapy e cacy (Ramage
et al., 2010; Abedon, 2010a; 2011b).
The ultimate indication of poor technique
in terms of failing to allow establishment of
bacterial colonization prior to phage
application is the mixing of phages with
bacteria prior to bacterial challenge, although
simultaneous or even just short delays
between bacteria and phage addition should
also be viewed as suspect. Indeed, ofen the
result of such practices is what appear to be
bacterial reductions only to below minimal
lethal densities rather than a curing of
existing disease. At a minimum, therefore,
eort should be made during phage-therapy
experimentation to avoid mixing bacterial
challenges with phages in a manner that
mimics phagebacterial interactions as they
can occur in broth cultures. Usually such
avoidance will be the case given substantial
delays between bacterial challenge and phage
addition.
Researchers should consider treatment
di culties that may be encountered because
of such delays as an indication of a need for
further development of phage-therapy
protocols and/or a need for identication of
more eective phages. Such considerations
would contrast with observation of reductions
in e cacy given substantial delays between
bacterial challenge and phage application
serving instead as study end points, as too
ofen is the case in the phage-therapy
literature. Such a reduction in e cacy rather
should serve as the starting point in terms of
in situ protocol development. In particular,
unless protocols have been designed
specically to test for the prevention of
initiation of bacterial infections (prophylaxis),
then at a minimum multiple hours should
separate bacterial challenge and subsequent
phage addition (and if testing of prophylaxis
is envisioned, then phage addition should
precede bacterial challenge by a substantial
length of time rather than being simultaneous
or near simultaneous to bacterial application).
An alternative approach involves applying
phages and bacteria to dierent body com-
partments, although determining whether
such approaches are truly good models for
determining phage-therapy e cacy should
be the subject of rigorous pharmacokinetic
testing rather than simply assumed.
Discussion
Given the above considerations, I envisage a
logical four-step process of initial phage-
therapy development: (i) rational phage
isolation including in terms of avoidance of
temperate phages as well as the carriage of
virulence-factor genes; (ii) in vitro phage
characterization for anti-bacterial virulence;
(iii) in vivo or in situ proof-of-principle eorts
using models in which e cacy is highly
expected while at the same time limitations
of technique may be identied (particularly
in terms of delays between bacterial challenge
and phage application); and then (iv) use of
more realistic disease models to develop
clinically useful therapeutic techniques, that
is, that improve on e cacy at or beyond the
limits of proof-of-principle success (again,
particularly following substantial delays
between bacterial challenge and the initiation
of phage treatment). In short, the eld of
phage therapy, even in Western literature,
has moved beyond the point of proof of
principle (e.g. see Abedon et al., 2011). Thus,
if animal experimentation is indicated, then
that experimentation really ought to be
properly done. This means, perhaps more
than anything else, that eort should be
made to improve the accuracy of disease
models (see Loc-Carrillo et al., Chapter 13,
this volume).
262 S.T. Abedon
Ultimately phage-therapy protocols
need to be tested against naturally occurring
infections, although in my opinion this
represents a later step in development. Thus,
in terms of publication, I envisage possibly a
four-step process: Step (iv), as described
above, might be the rst publication in the
series. Next, a step (v) could seek to expand
the step (iv) type eorts to a greater range of
bacterial target strains or treatment
conditions. Step (vi) might then incorporate
treatment of a small number of naturally
occurring infections, representing essentially
a limited trial. Step (vii) would then entail a
fairly substantial trial, perhaps under more
real-world conditions. For human application,
there will additionally be phase I safety
testing, prior to limited e cacy testing (phase
II), and then full-blown clinical trial (phase
III), all of which will follow what testing can
be accomplished using animal models.
Animal testing of the treatment of human
diseases, however, may not always be
extendable beyond step (v).
By way of example, consider the develop-
ment of phage-therapy treatment against
Pseudomonas ear infections (see Burrowes and
Harper, Chapter 14, this volume, for ref-
erences). Here, the rst publication addressed
phage treatment of a naturally acquired
infection in a single dog, the second publi-
cation considered the treatment of naturally
acquired infections in ten additional dogs, the
third publication was a safety and limited
e cacy trial in humans, and currently a
phase III clinical trial is either planned or
under way (note that the quotes are because
the third publication came out prior to the
second publication).
Phage-therapy Best Practices II:
Controls
The subject of controls during phage-therapy
experimentation is complicated by the issue
of double blinding of well-designed clinical
trials. Thus, in addition to researcher
blinding, it is important not to overlook the
more basic experimental controls negative
controls, positive controls and controlled
variables. In this section, I consider best
practice numbers 58, all of which involve
questions of how to properly control as well
as reduce biases during phage-therapy
experimentation.
5. Has a positive phage-killing control
been used?
In this section, I will concentrate on positive
controls; that is, can a phage-therapy protocol,
under some approximation of the best of all
possible circumstances, achieve phage-
therapy e cacy even if it fails to do so under
other, perhaps more clinically realistic,
conditions? Generally, the best of all possible
circumstances will involve procedures that
assure that su cient phage densities are
present to allow substantial killing of target
bacteria such as may be equivalent to that
observed during in vitro testing (above). This
would particularly be the use of an
approximation of passive treatment, meaning
supplying via dosing enough phages to
achieve bacterial clearing, including by
means of repeated phage dosing (below). A
good rule-of-thumb denition of enough,
whether provided by active or passive means,
may be the achievement of a density of at
least 10
9
phages ml
1
to the actual physical
location of target bacteria, or at least 10
8
given
somewhat ideal conditions (Abedon and
Thomas-Abedon, 2010; Abedon, 2011a,b,
2012; Curtright and Abedon, 2011). Keep in
mind, furthermore, that the dosing of animals
may need to be higher than that of broth
cultures to account for phage loss (excretion),
dilution (associated with absorption, dis-
tribution and ine ciencies in movement
between body compartments), decay (as due
to metabolism) or ine ciencies of phage
penetration to bacteria (ine ciencies in
distribution as described above but also
ine ciencies in penetration into biolms).
It should be strongly stressed that
positive controls are most relevant, and
arguably perhaps only relevant, if phage-
therapy protocols fail to otherwise achieve
reasonable levels of e cacy (i.e. su ciently
positive results). In other words, negative
experimental results generally are more
meaningful to the extent that positive
experimental results phage-mediated
bacterial clearing positive controls are
demonstrably possible, such as might be
achieved by adding more phages, employing
more eective phages, preparing infections
beter for phage treatment and/or dosing
more frequently (Abedon and Thomas-
Abedon, 2010; Abedon, 2010a, 2011a,b). To
improve on experimental phage therapy
outcomes, it therefore may be necessary for
issues of convenience, or of economy of use of
materials, to not be allowed to interfere with
the achievement of greater e cacy, par-
ticularly should results otherwise prove
disappointing. Studies should not be
published if only poor or negative results are
obtained unless a robust eort has been made
to improve on these results.
Another way of making the later point is
that publication of negative results is
unquestionably important but only if those
negative results are assuredly not simply a
consequence of poor or otherwise insu cient
experimental technique. Before publishing
negative results, there thus should always be
concern that su cient eort has been made
to achieve desired outcomes. Above all, one
should always be worried that the phages
employed were in some manner inadequate,
that the doses used were in some manner
insu cient or that the rates or duration of
dosing were to some degree in need of further
increase. Thus, try to establish especially
whether supplying more phages will result in
greater overall levels of bacterial killing.
Note that a phage-killing positive control
is not the only kind of positive control that is
possible in phage experiments. For example,
if one is comparing phage performance with
a normal standard of care, then an antibiotic-
positive control may be employed. The use of
such a control, however, does not substitute
for the use of a phage-killing positive control.
This is because the question that is being
addressed with phage-killing positive
controls is not that of phage therapy e cacy
in comparison with other treatment options
but instead whether, in the absence of desired
levels of bacterial-treatment success, in-
su ciencies in dosing might be to blame. In
other words, there is litle information that
may be obtained should phage application
result in poor outcomes under circumstances
in which antibiotics nevertheless are eective,
except that more eort may need to be put
into phage-therapy protocol development.
6. Has a reasonable negative-treatment
control been used?
The best negative control to employ during
phage-therapy protocols is not necessarily
either obvious or straightforward. None the
less, the use of a reasonable negative control is
crucial to determining phage-therapy e cacy.
To simplify the discussion, it can be easier to
view negative controls not in terms of negative
e cacy but rather in terms of positive
infection outcomes, such as continued subject
morbidity or resulting subject death. Thus,
the negative control is an approximation of
the normal phage-therapy protocol but one
that allows a normal infection outcome, as
seen without phage treatment. Clearly, the
easiest means of ataining this outcome is to
avoid applying phage formulations altogether.
This approach, however, is complicated by
the need to dierentiate phage-associated
therapy e cacy from e cacy that is associated
instead with phage carriage material or other
aspects of treatment protocols that might not
be employed in the absence of phage
application. Particularly, non-phage compon-
ents of phage formulations can contain
complex bacterial-lysis products (Gill and
Hyman, 2010). Therefore, as phage-therapy
negative controls, it is beter practice to use
some kind of mock treatment that is a good
approximation of the phage formulation, only
lacking in active phages.
Phages can be removed from formu-
lations via ltration or destroyed via heating.
Formulations otherwise can be created in a
phage-free manner that approximates the
phage-generated material. Alternatively,
phages can be su ciently puried prior to
use such that a simple buer, as equivalent to
that in which the phages have been sus-
pended, may be employed as the negative
control. Unfortunately, not much eort has
been put into determining whether these
various substitutions provide similar or even
264 S.T. Abedon
reasonable performance as true phage-
negative controls. None the less, it is crucial
to employ negative controls during phage-
therapy testing that allow a denitive
distinction between phage-mediated e cacy
and e cacy that is due instead to non-phage
components of the treatment.
7. Have experiments been blinded?
It is the gold standard of clinical trials to
employ double blinding, such that nobody
knows who has received the treatment versus
the placebo (negative control) until the data
has been collected from the trials. These
methods are important for the sake of
removing biases from what can be subjective
observations, such as how sick the subject is.
In addition, blinding is also important to
remove biases in the treatments themselves,
particularly if the treatments are relatively
complicated or, instead, if there is any potential
that subjects may be chosen or taken care of
dierently depending on the treatment type.
It is my opinion that if clinical trials
should be expected to display double
blinding, then at a minimum some eort
should be made to remove biases from in vivo
or in situ testing as well, such as by blinding
researchers who are directly involved in a
protocol from the treatment being employed.
This approach can be seen, for example, in
the mastitis study of Gill et al. (2006a). More
generally, subtle dierences between treat-
ment results, even if shown to be statistically
signicantly dierent, should not be con-
sidered to be denitive indicators of e cacy
unless, at a minimum, some form of bias
reduction such as blinding has been
employed.
8. Has testing for phage formulation
toxicity been explicitly performed?
An ongoing concern with regard to phage
therapy is the potential for introduction of
bacterial toxins into patients in the course of
phage application. This occurs because
bacterial lysis is involved in phage stock
preparation, and lysis has the potential to
release a number of bacterial toxins, in
particular endotoxin from Gram-negative
bacteria (Gill and Hyman, 2010). To guard
against the introduction of these toxins into
patients, phage preparations can be puried.
Even given such purication, in the course of
experimentation an important control is to
show that phage application in and of itself
will not lead to toxicity. Indeed, it can be
important to ascertain what level of phage
purication is actually necessary to protect
subjects from side eects.
Fortunately, toxicity testing is easily
accomplished through the application of
phage doses to animals. The importance of
such controls is not just to show that
formulations as well as the phages themselves
that are employed in a given study are non-
toxic but also to build up in the literature a
record of phage toxicity testing. Should
formulation toxicity in fact turn out to be the
case, then production or delivery approaches
may be modied. For example, greater lysate
purication or less invasive application the
later being, for example, topical use rather
than systemic use may be employed. Note
that in vitro testing of phage formulations,
especially for endotoxin presence, is also
routinely available (Boratynski et al., 2004).
See Olszowska-Zaremba et al. (Chapter 12,
this volume) for further discussion on issues
of the potential for phages to display toxicity.
Phage Therapy Best Practices III:
Dosing and Enumeration
Certain additional details can be relevant to
successful phage therapy experimentation. In
this section, I consider issues of dosing
number, dosing level and dosing description,
as well as bacterial enumeration. These
constitute best practice numbers 912.
9. Has multiple or continuous dosing
been attempted?
A conceit too ofen found in the phage-therapy
literature is the notion that phages, as
potentially self-amplifying antibacterials,
ought to be e cacious even if applied only
once. By contrast, it is atypical among pharma-
ceuticals to demand e cacy following only a
single application, although certainly that can
be desirable from a treatment perspective.
Notwithstanding such issues, it seems
unreasonable to expect a priori that phages
should consistently work despite such in-
tentional hobbling in terms of dosing. For that
reason, phage-therapy protocols, particularly
where human treat ment is the goal, should
not be intentionally designed to employ only
a single dosing unless benets gained from
such an approach are substantial such as in
terms of economics, convenience or reduced
invasiveness while eectiveness is only
minimally lost.
The alternative perspective is that, to the
extent that a phage-therapy protocol has
failed to provide su ciently e cacious
results, strategies ought to be explored that
are aimed at improving outcomes. Such
strategies might include providing phages
multiple times over the course of treatment,
providing phages with increasing frequency
(e.g. such as multiple times per day rather
than only once per day) or even providing
phages continuously (Abedon, 2011a, 2012).
Of course, if single dosing can be shown to be
e cacious and to provide su cient benets
in terms of convenience and/or economy,
then it would not be unreasonable to limit
phage application to just one dose per
treatment. It is dangerous, however, to
assume that single dosing represents a default
means by which phage therapy is imple-
mented. In particular, where phage therapy is
actually practised clinically, such as in
Georgia and Poland, single dosing does not
represent the standard of care (Kuter et al.,
2010; Abedon et al., 2011). See Loc-Carrillo et
al. (Chapter 13, this volume) for additional
discussion of this issue.
10. Has there been inappropriate reliance
on active treatment?
Similar to the idea of a utility associated with
multiple rather than single dosing, so too
there exists a usefulness to supplying
adequate phage numbers per dose. Supplying
su cient phage numbers is an obvious
dosing criterion given consideration of phage
therapy from a pharmacological perspective.
Even so, the prospect of phages multiplying
while in the presence of target bacteria,
potentially resulting in active treatment, can
lead to assumptions that such multiplication
might always occur, plus occur to a degree
that phages will achieve su cient densities to
eradicate bacteria (Abedon and Thomas-
Abedon, 2010; Abedon, 2010a, 2011a,b, 2012).
Notwithstanding this potential, and
unless there is strong reason to believe
otherwise, in the course of debugging phage-
therapy protocols that is, experimentally
addressing the question of why results may
not be as e cacious as anticipated or desired
one should always question whether
su cient phages were applied per dose. In
addition, one should question whether a
su cient frequency or number of doses was
applied, as well as whether the phages
themselves have su cient antibacterial
activity. Thus, for example, if one observes a
single log killing with an application of 10
7

phages per dose, where such minimal killing
should not be a desired end point of any
phage-therapy research and development
programme, then it would not be unrealistic
to consider whether adding 10
8
phages per
dose might improve outcomes or supplying,
for instance, ten rather than a single phage
dose to increase levels of bacterial eradication.
Further considerations include the
following: (i) when employing phages that
have been engineered to not reproduce, such
as to avoid in situ bacterial lysis, which will
release endotoxin (Bull and Regoes, 2006;
Goodridge, 2010; Abedon and Thomas-
Abedon, 2010), then only those phages
supplied will be present and passive treat-
ment occurs by default; (ii) the rate at which
phages nd, adsorb and then kill bacteria is a
function of phage density that is, e cient
bacterial adsorption by phages generally is
not a function of either bacterial densities or
ratios of phages added to bacteria but instead
is almost entirely dependent on the phage
titres that are present within an environment.
Consequently, even if an excess of phages
over bacteria is employed, this does not mean
that phages will rapidly adsorb and then kill
266 S.T. Abedon
the bacteria. Indeed, at low phage and low
bacterial densities it can take hours, days,
weeks or even longer for a reasonable fraction
of bacteria to become phage adsorbed
(Goodridge, 2008; Hagens and Loessner,
2010; Abedon, 2011a,d, 2012).
To reiterate these rst two points, if
insu cient phage densities are present
within the environment, then insu cient
bacterial killing will occur and such
insu ciencies in bacterial killing are possible
even given a substantial excess of phages
over bacteria. The later is the case because
generally the only absolute determinant of
the likelihood or rate at which a given
bacterium becomes phage adsorbed, other
than phage properties, is the density of phage
particles that are present in the environment
of the bacterium. Furthermore, this statement
is dierent from the question of the overall
rate of phage-to-bacterium adsorption that
will occur within an environment, which will
increase based on phage densities, bacterial
densities or indeed ratios of phages present to
bacteria. Thus, if more phages and bacteria
are present within an environment, then of
course there will be more phage-to-bacterium
encounters. The important issue in phage
therapy, however, is the per capita rate at
which bacteria become adsorbed by phages,
and that rate is entirely a function of phage
densities, phage adsorption properties and
the ability of phages to reach target bacteria,
that is, the basic pharmacological parameters
of drug dosing, drug functionality given
access to targets and drug ability to reach
targets. Similarly, we expect rates at which
bactericidal antibiotics kill bacteria to be a
function particularly of antibiotic densities.
Alternatively, (iii) interference between
phages making up phage cocktails may be
greater when phages are supplied at higher
versus lower densities, particularly given co-
infection of individual bacteria by
heterogeneous phages (Abedon, 1994). The
result could be a utility associated with
supplying phages at lower densities per dose,
perhaps in combination with repeated or
continuous dosing. See Chan and Abedon
(2012b) for further discussion of this
admitedly somewhat hypothetical issue, as
well as Abedon (2011e) for a related issue
that of lysis from without. Lastly, (iv) if
contamination of formulations with bacterial
lysis products such as endotoxin is a problem,
then using more phages per treatment will
result in patient exposure to more of these
toxins. Thus, while using more phages per
dose can be an important consideration in
optimizing phage-therapy protocols, there
are potential tradeos associated with this
strategy, although these are all tradeos that
may be readily explored experimentally.
11. Has the concept of multiplicity of
infection been used correctly?
To achieve bacterial killing, phages must be
present in su cient densities such that at best
only a small fraction of susceptible bacteria
remain uninfected. These phage densities can
be supplied as a direct consequence of dosing
(passive treatment) or can be generated in situ
as a result of phage multiplication on target
or other bacteria (active treatment). In either
case, it is the titre of phages that are present in
the vicinity of target bacteria that determines
the degree to which bacterial densities are
reduced, rather than necessarily the ratio of
phages supplied to bacteria present (Abedon
and Thomas-Abedon, 2010). The later has
been described as an input multiplicity of
infection (Kasman et al., 2002) and is a awed
approach to dosing considerations (Abedon,
2011a,b).
The reason to avoid framing phage
dosing in terms of input multiplicities is that
what maters in terms of bacterial killing is
the multiplicity of phages that actually
succeed in adsorbing (actual multiplicity of
infection). This multiplicity, however, will
vary not only with phage densities, phage
properties and in situ conditions but also with
bacterial densities. Thus, if bacterial densities
happen to be low, then the input multiplicity
of a given dosage of phages (titre) will be
high, but if bacterial densities happen to be
high, then the input multiplicity associated
with that same phage dose will be low.
Furthermore, in a treatment situation, actual
bacterial densities ofen will not be known
and therefore dosing expressed as an input
multiplicity of infection will not be possible.
Phage dosing thus should be described in
terms of phage titres along with formulation
volumes if phages are added directly to
bacteria. Alternatively, absolute virion num-
bers supplied may be more relevant if phage
application to bacteria is less direct, such as
oral dosing. The botom line is that not only is
describing phage dosing solely in terms of
multiplicities of infection usually not useful,
it can also be misleading, and in most or all
cases should be avoided.
12. Have the bacteria been adequately
protected during enumeration?
There are a number of means of assessing
phage impact during treatment of bacterial
infections. These include reductions in subject
mortality, improvement in subject health and
various measures of infection severity. In
terms of infection severity, the most objective
measure can be of in situ bacterial density,
and this is particularly so since the phage
impact on bacteria during phage therapy is to
directly reduce bacterial viable counts.
Although it ofen is desirable to assess
phage-therapy e cacy in terms of deter min-
ing bacterial viable counts, actually deter-
mining those counts, unfortunately, is
complicated by the potential for phages to
infect bacteria during the enumeration
process itself. The result of such infection can
be perception of a reduction in bacterial
densities that exceeds actual in situ reductions.
Thus, enumeration procedures if nothing else
should be designed as well as assessed in
such a manner that litle doubt exists that the
observed bacterial killing occurred in situ
rather than ex situ during the enumeration
process. Note that the primary reason that
this is a concern is when the enumeration
step involves disruption of spatial structure
associated with bacterial infections such that
phage penetration to bacteria is more e cient
during the enumeration procedure than it
had been during the actual treatment process.
Such disruption might occur following tissue
or biolm suspension and/or homogenization
into liquid solutions.
There are three ways to address this
concern of phage ex situ killing of bacteria: (i)
inactivate phages prior to the enumeration
step (particularly prior to any bacterial
resuspension in buer step); (ii) dramatically
dilute phage densities in the course of
enumeration (e.g. 100-fold or more depending
on phage concentrations in situ since, as
discussed above as well as in the following
section, the phage killing potential is a
function of phage densities rather than of
ratios of phages to bacteria); and (iii) employ
enumeration controls that assure the integrity
of enumeration results. Such controls involve
determining phage densities during enumer-
ation and/or the degree to which bacterial
densities decline in the course of prolonged
ex situ but pre-plating incubation. In short, a
carefully designed phage therapy experiment
will include vigilant consideration of the
shortcomings associated with various means
of outcome determination, and an obvious
concern is the potential for phages to adsorb
bacteria following homogenization of phage-
and bacteria-containing samples within
buer. In some cases, protection of phages
during their enumeration can also be an
issue, as discussed by Loc-Carrillo et al.
Additional Issues
In this section, I discuss additional issues that
can be relevant to phage-therapy ex-
perimentation. These are the relevance of
phage host range, particularly to laboratory
phage-therapy experimentation as typically
practised, and the need to follow up
unexpected results. In addition, I provide a
quick overview of the logic of phage
adsorption theory, which results in phage
titres being more relevant to rates of bacterial
decline due to phage adsorption than
bacterial densities, and, nally, a reiteration
about the relevance, or lack thereof, of
positive results.
Host range and laboratory
experimentation
In the presented discussions, I have mostly
ignored the topic of phage host range (Hyman
268 S.T. Abedon
and Abedon, 2010; Abedon, 2011a; Chan and
Abedon, 2012a). Host range is typically
measured in terms of phage impact on
bacteria in solid or semi-solid media, that is,
in terms of plaque or spot formation.
Measured in this way, host range is ofen of
primary concern within phage-therapy
publications, and sometimes is seemingly
emphasized more than issues of actual
phage therapy e cacy. Notwithstanding this
emphasis, host range is relevant mostly in
terms of modifying the spectrum of activity
of phage therapeutics, including in terms of
the use of fewer versus greater numbers of
phage types per formulation (Chan and
Abedon, 2012b).
By contrast, the range of bacterial strains
that phages can spot on should not directly
impact the approach that is taken to most
phage-therapy experiments, particularly as
practised in the laboratory (versus in the
clinic or on the farm). This is because such
experiments typically involve the application
of specic phage isolates to infections caused
by specic target bacterial strains, that is,
bacteria against which phages have already
been characterized. In any case, it is phage
anti-bacterial virulence, in situ phage-
infection performance and the various details
of phage-formulation application that are
salient to phage-therapy success. Thus, while
phage host range is relevant to phage choice
for subsequent experimentation, the range of
bacteria a phage can plaque or spot on itself
does not directly impact that experimentation,
at least as usually practised in the laboratory,
unless treatments specically are of infections
by bacteria that a priori display unknown
phage susceptibilities. See Williams and
LeJeune as well as Cox (Chapters 6 and 10,
this volume) for further consideration of
phage spoting and plaque formation.
Follow-up of unexpected results
Experimentation can be both expensive and
time-consuming. Good scientic method
none the less involves not only implementing
all necessary controls but also employing a
valid theoretical framework within which
experiments are conducted. This framework
includes knowledgeable hypothesis making
and testing, as well as prediction of experi-
mental outcomes. More than occasionally,
results will be unexpected in some manner.
To the extent that such results are germane to
the study being conducted, it is then
imperative that additional experiments and/
or controls be designed and conducted to
achieve a greater understanding of ones
experimental system.
If adding phages to bacteria does not
result in the degree of bacterial killing that
one expects (see Abedon (2011a,b, 2012) for
tips on how to make such predictions), then
clearly factors not taken into account may be
operating within the system under study.
More importantly, if one observes un-
expectedly poor e cacy, then it can be
important for these results to be followed up
with additional experimentation, if possible
with these additional experiments published
at the same time as the original unexpected
observation. An important example of such a
result occurs when dose-response curves
(phage density added relative to treatment
outcome) are non-linear, and particularly so
where adding more phages results in
substantial reductions in e cacy (Abedon
and Thomas-Abedon, 2010; Abedon, 2012) or,
hypothetically, should multiple dosing prove
inferior to single-dosing protocols. In such
circumstances it is necessary for both the
development of the eld as a whole and the
development of the specic system under
study to show that such results can be
replicated ex situ, explained mechanistically
and/or overcome via beter treatment design.
For example, in vitro exploration might be
atempted when lysis from without is
suspected (Abedon, 2011e). In short, publi-
cation of unexpected results can be confusing
to the eld and particularly so when such
results are not subject to reason able, sub-
sequent exploration.
Importance of phage titres to phage-
therapy efcacy
A basic premise in pharmacology, only rarely
violated, is that the administration of more
drug results in greater physiological impact
(although not always generating beter
results). The same is true for phage appli-
cation, at least in terms of rates of phage
adsorption by bacteria. In particular, basic
phage adsorption theory dictates that the
rates of phage-bacterial encounters will be a
function of both phage and bacterial
densities. Crucially, however, the rate at
which a given bacterium becomes phage
adsorbed is a function of phage density and
not of bacterial density, except to the extent
that bacterial densities can impact on phage
densities.
Specically, the rate of phagebacterial
adsorptions in a given environment is equal
to PNk where P is phage density, N is bacterial
density and k is the likelihood of encounter,
and subsequent adsorption of one phage to
one bacterium. The number of phage
adsorptions to a specic bacterium, per unit
time, thus is equal to Pk, as N has been
dened, in this scenario, as equal to 1 (that is,
a specic bacterium). Indeed, no mater how
many bacteria are present, the likelihood of
phage adsorption to any one bacterium
remains Pk because this view is from the
bacteriums perspective rather than that of
the phage. Key to this calculation, however, is
that P is being held constant, meaning in
eect that phages are present in excess.
By analogy, in war the more ammunition
(phage equivalent) that one throws at ones
enemy (bacterial culture or infection
equivalent), then the greater the likelihood
that any given enemy soldier (bacterium
equivalent) will die. The more enemy soldiers
that are present, the more enemy soldiers that
will die, but unless soldiers are shielding
other soldiers, the per capita rate of enemy
soldier loss will be a function of how much
ammunition you throw at them rather than
how many enemy soldiers happen to be
present. Keep in mind, however, that greater
impact in the short term (e.g. winning batles)
does not always translate into greater impact
in the longer term (e.g. winning wars), just as
using more phages per dose does not always
translate into consistently beter treatment
outcomes (above). For additional con-
sideration of these ideas, see, for example,
Abedon (1990, 1999), Kasman et al. (2002),
Goodridge (2008), Abedon (2009c), Hagens
and Loessner (2010), Abedon and Thomas-
Abedon (2010) and Abedon (2011a,b,d, 2012).
Positive results
The goal of phage-therapy research and
development should be an improvement in
treatment results. Well-documented studies
that achieve positive results therefore can be
inherently more valuable than studies that
fail to demonstrate e cacy, although good
documentation in the later case may provide
clues as to what strategies not to employ, or
indeed what may have gone wrong.
Notwithstanding these considerations, the
concept of a positive treatment result in fact
has some ambiguity. One concern is that there
is a dierence between statistically signicant
positive results and biologically or clinically
meaningful outcomes: very small but none
the less reproducible results may not be
worth exploring further, although they might
hint instead at a potential for greater real-
world signicance, for example were more or
dierent phages employed.
An additional ambiguity concerns the
suitability of experimental models. Again,
although positive results are preferred to
negative experimental results, positive results
are valuable only to the extent that they are
meaningful. Thus, infection models that do
not provide realistic inhibitions on phage
penetration to bacteria, or which otherwise
fail to allow bacterial colonization prior to the
initiation of treatment, may bias results
unrealistically towards e cacy. Authors
should strive to recognize as well as to fully
explain aws in the systems they are using
and, of course, endeavour to improve upon
those systems such that major or substantive
problems are reduced or eliminated.
Although such advice should be obvious
when experimental results are disappointing,
self-criticism is important even given positive
experimental outcomes.
Conclusion
The development of any eld requires
advancements in techniques, not just in terms
270 S.T. Abedon
of technology but also such that poor practices
are discouraged or eliminated. Phage therapy
has beneted from ongoing improvements in
techniques over the last century since the
discovery of phages, especially in terms of
phage molecular characterization. This
increasing technical mastery does not always
translate into an implementation of best
practices, however. In addition, techniques,
no mater how powerful, always have
limitations. The quality of actual scientic
output thus will depend on a substantial
appreciation of which practices are most
relevant towards achieving meaningful
phage-therapy e cacy along with a reason-
able appreciation of the limitations of these
techniques.
The greatest strength of phages as
antibacterial agents is that, in many instances,
they seem to give rise to bacterial-control
e cacy even without a robust pharmacological
understanding (Abedon and Thomas-
Abedon, 2010). A weakness, however, is that
phages are relatively easy to work with,
thereby lowering barriers of entry into the
eld. The result can be a lack of subtle
understanding of phage biology, phage
therapy, experimental design and even of the
phage literature in the course of designing
phage-therapy approaches. In this chapter, I
have endeavoured to point out technical aws
that seem to be commonly associated with
modern phage-therapy studies. It is my hope
that this discussion will help to provide a
roadmap for researchers towards improve-
ment in real-world phage-therapy e cacy.
More generally, phages possess vast potential
in terms of combating bacterial disease and
otherwise improving health, but eective
realization of that potential will require both
innovative and well-considered research and
development.
Acknowledgements
Thank you to Catherine Loc-Carrillo, Jason
Gill and, of course, Paul Hyman, for their
help in editing this chapter.
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273
Acute wounds 186
AFLP see Amplied fragment length
polymorphism (AFLP)
Amplication, phage
infection cycle 137
and LFIs 138141
MALDI-TOF MS 137138
plaque assay 135136
reporter genes 137
virionhost interactions 136
Amplied fragment length polymorphism (AFLP)
80, 134
Antibacterial interactions
bacteriophagehost interaction-based
development 126, 127
cell-cycle arrest 126
host-cell growth 126
synthetic molecule inhibitors 128
Antibody discovery, phage display
applications 108, 110
cell-surface antigens 113
framework region 110
immune libraries 111
indirect agglutination assay 112
nave libraries 111
natural eector function 109110
synthetic libraries 112
Anti-microbial resistance (AMR) genes 81
Anti-phage antibodies
cellular immunity 172173
immunogenic properties, X174 phage 172
neutralizing antibodies 172
APEC see Avian pathogenic E. coli (APEC)
Avian pathogenic E. coli (APEC) 242
Bacillus infection model 226
Bacterial colonization
biolms and broth cultures 261
ex situ testing 260261
prior to phage application and development 261
reductions, e cacy 261
Bacterial contamination, food supply
animal production farms 241
colonization 241
EHEC 241
food-related environments 241
fruits and vegetables 241
natural phages
anti-pathogen interventions 241
Anti-Salmonella 242
coliphages 241242
Listeria 242
yogurt and cheeses 241
pathogens 242
plant-level interventions 241
Bacterial detection and identication
AFLP 134
uorescent phage endolysin cell wall-binding
domain 145146
MALDI-TOF MS 135
PCR 134
phage amplication 135141
phage-encoded reporter genes 142145
phage immobilization 141142
QDs 145
signal amplication method 146
23S rRNA gene sequencing 134
Bacterial epidemiology
antibiotic resistance 81
Index
274 Index
Bacterial epidemiology continued
animal hides 61
bacterial genomes 80
biological and genetic properties 76
epidemiology and ecology 82
Phage type (PT) 7680
Bacterial pathogens
capabilities, pathogenicity 6870
E. coli 6768
environmental contamination 246
evolutionary timescales 71
evolution, bacterial 70
foodborne pathogenic bacteria 243244
genetic elements 71
genomes 67
HGT detection 6267
hides 246
pathogenic lifestyles 71
processing plant and foods
anti-listerial phage sprays 247
chicken skin and clean machinery 247
dairy products and cross-contamination 247
FDA 246
Lactococcus and Streptococcus 247
Listeria populations 247
raw and cooked beef 247
retail cuts/chicken carcasses 247
spinach and sprouts 246
VTEC/STEC 246
Phage role in Pathogen development 7071
ruminant animals
caecum and anti-E. coli O157:H7 244
E. coli O157:H7 classication 244
elucidating factors 245246
isolation and STEC 244
non-O157 STEC/EHEC and STEC/EHEC
O157:H7 broth culture 244245
non-phage-colonized/experimentally phage-
colonized sheep 245
ovine and bovine sources 245
polymer-encapsulated bacteriophage 245
seeder animals 245
zoonotic bacteria 242243
Bacteriocins
classication 229
F and R-type pyocins 230
Gram-negative 229230
Gram-positive 229
narrow spectrum 229
PBSX-like defective prophages 230
tail-like 230
Bacteriophage preparations
granulocytes 173
immunomodulatory activity, staphage lysate
175176
immunomodulatory eects, mammals 176177
phage eects 177178
phagocytosis 173
production of cytokines 174175
T cells and platelets 173174
Bacteriophage Enquiry 204
Biolms 9, 12, 14, 70, 155, 212, 227, 231, 232, 247,
248, 256, 260262
Listeria 247
pH/osmotic shocks/UV light penetration 248
polysaccharide depolymerases 248
Pseudomonas uorescens 248
BoNTXs see Botulinum neurotoxins (BoNTXs)
Botulinum neurotoxins (BoNTXs) 34
Botulism
Botox treatments 24
Clostridium-associated diseases 23
food poisoning 23
paralysis 23
Burn wounds
corticosteroids 189
rst-degree burns 186
models
CFU 196
ve-phage cocktail treatment 196
Klebsiella pneumoniae 196197
Phage-resistant bacteria in mutants 197
phage treatment 197
skin grafs 196
MRSA 189
CAI see Codon adaptation index (CAI)
CBD see cell wall-binding C-terminal domain
(CBD)
CDT see Cytolethal distending toxin (CDT)
Cellular cytochemistry and virulence
Clostridium 3435
Corynebacterium 3334
E. coli 4346
phage conversion 33
Pseudomonas 4748
Salmonella 47
Staphylococcus 3541
Streptococcus 4143
V. cholerae 4647
Cell-wall architecture, endolysins
Gram-positive phage, in vitro studies 220221
lytic transglycosylases 221
N-acetylglucosaminidase 221
N-acetylmuramoyl-l-alanine amidase 221
peptide bridge 219
protease/endopeptidase 221
Cell-wall-binding domains (CBDs) 222
CFU see Colony-forming units (CFU)
Chemotaxis inhibitory protein of S. aureus (CHIPS)
39, 40
CHIPS see Chemotaxis inhibitory protein of S.
aureus (CHIPS)
Cholera
faecal contamination, water 28
phages 28
Index 275
seasonal epidemics 28
treatment 29
Chronic wounds 189
Clinical applications, phage display
allergic/anaphylactic reactions 113
antibody discovery 106113
bacterial infectivity 102
binding a nity 103
biological assays 101
Crohns disease 114
in vitro a nity maturation 114
M13 lamentous genomic DNA 102
peptide and protein libraries 103106
pIII fusion protein 103
selection strategies 107109
Clostridium
BoNTXs 34
lysogenic conversion 34
virion structural proteins 35
Clustered, regularly interspaced, short
palindromic repeats (CRISPR) 66
Codon adaptation index (CAI) 63
Colony-forming units (CFU)
IP/SC injection 197
Klebsiella pneumoniae 196
Corynebacterium
C. diphtheriae 34
lysogenicity and toxinogeny 3334
CRISPR see Clustered, regularly interspaced, short
palindromic repeats (CRISPR)
Crohns disease 114
C-terminal domain (CBD) 145, 146
Cytolethal distending toxin (CDT) 46
Depolymerases
alginate layer 231
biolms 231
capsule 230
EPS 231
tailspike proteins 231232
Diarrhoeal diseases 13, 17, 21, 22, 23, 26, 27, 46,
153, 203, 206, 210, 242, 244
cholera 2829
fatal dehydration 28
shiga toxin-associated diseases 29
toxin eects, kidneys 28
Diphtheria
exotoxin-mediated disease 22
treatment and prevention 23
upper respiratory tract infection 23
Diseases
bacterial diseases 21
botulism 2324
diphtheria 2223
genomic sequencing 21
lysogenic-converting genes 22
Pasteurella infection 28
phage therapy 30
prevention 30
Pseudomonas infections 2930
Staphylococcus infections 2427
Streptococcus infections 2728
toxigenic prophages and diseases 22
treatment 30
DNA vaccines
antibody responses 89
EGFP 8889
intramuscular immunization 89
VLPs 89, 90
Dosing and enumeration, phage therapy
active treatment
debugging 265
low phage and low bacterial densities 266
single log killing 265
multiple/continuous
self-amplifying antibacterials 264265
single dosing 265
multiplicity of infection 266267
protection 267
ECIS see Electrical cell-substrate impedance
(ECIS)
EGFP see Enhanced green uorescent protein
(EGFP)
EHEC see Enterohaemorrhagic Escherichia coli
(EHEC)
Electrical cell-substrate impedance (ECIS) 141
Endolysins
autolysins 217
cell-wall architecture and types 219221
domains in detection 145146
exolysin 217218
Gram-negative structure 221
Gram-positive structure 221222
in vitro activity 224226
in vivo studies 226228
lyse 232
multiple-domain 222224
peptidoglycan hydrolases 217
phage therapy 232
S. aureus 218
transmissible and non-transmissible virus 218
ultramicroscopic agent 218
zoocin A 218
Enhanced green uorescent protein (EGFP)
8889
Enterohaemorrhagic Escherichia coli (EHEC) 43,
241
Enzybiotics
bacteriocins 229230
denition 218
depolymerases 230232
endolysins see Endolysins
resistance development 228229
276 Index
Escherichia coli (E.coli)
cytolethal distending toxin (CDT) 46
characteristics, sequenced Stx phages 45
EHEC 43
genetic map, late gene regulatory region 44
lytic cycle 45
Shiga-like toxigenic phages (SLT-phages) 43
STEC 43
VT phages 43
ETs see Exfoliative toxins (ETs)
Exfoliative toxins (ETs) 37
Exogenous bacteriophages
anti-phage antibodies see Anti-phage antibodies
anti-phage cellular immunity 172173
innate immunity 171172
Exolysin 217218, 220, 224, 228, 229
Ex situ phage antibacterial virulence
biolms and bacterial killing measurement 260
in silico approaches 259260
in situ conditions 260
in vitro testing and assays 260
kinetic analyses and standard assay 260
Extrapolymeric substance (EPS) 231
Faecal contamination, phage detection
assessing water quality, indicator organisms
154155
B. fragilis bacteriophages 162163
cryptosporidiosis outbreak 153
description 153
inadequate water quality 153
indicator and index microorganisms 155156
male-specic coliphages see Male-specic
coliphages
somatic coliphages see Somatic coliphages
water quality 154
Fluorescent phage endolysin cell wall-binding
domains
CBD 145, 146
magnetic separation 146
peptidoglycan hydrolases 145, 146
Fluorescent protein-based reporter phages
phage adsorption 144
YFP 145
Foodborne pathogenic bacteria
Campylobacter 243, 244
CFU 243244
poultry 243
Salmonella 243, 244
sources of 246247
swine 243
Gastrointestinal tract
description 9
gut 1014
oral cavity and pharynx 910
Gene therapy 87, 9293
Gram-negative bacteriocins 229230
Gram-negative endolysin structure 221
Gram-positive bacteriocins 229
Gram-positive endolysin structure
catalytic domains 221222
CBDs 222
Gut
bacterial diversity 11
culture-based analyses 1213
eukaryotic viruses 11
F-specic phages 1314
human intestinal phage 12
limitations, phage replication 1415
metagenomic sequences, human 12
non-cultured viral community 11
Red Queen dynamics 12
temperate phages 13
total viral counts 1011
HACCP see Hazard analysis critical control point
(HACCP)
Hazard analysis critical control point (HACCP)
248
HGT see Horizontal gene transfer (HGT)
Horizontal gene transfer (HGT)
acquisition, functions 21, 30, 35, 41, 49, 6667
bacterial biolms 70
bacterial genomes 5, 67
CAI 63
chromosome sequence architecture 66
CRISPR 66
E. coli 6768
genome fusion 64
genomic uidity 35, 41, 49, 69, 223
lysogeny/integration 65
parametric methods 63
phage involvement, cycling antibiotic
production 68, 69
phylogenetic incongruity 63
prokaryotes 62
spectrum, host interactions 68
transduction, antibiotic resistance genes 25, 69
transformation 64
transmitance, genetic material 2, 5, 21, 30, 41,
49, 65
Host genome replication
DNA replication pathway 123
inhibition mechanisms. 123
lytic phages 123
phagehost interactions 123, 124
Host immune system 40
Hosts and phages
bacterial metabolic processes 120
cell-wall biosynthesis 126
development, antibiotic resistance 119
drug-design eld 120
Index 277
global host metabolism 125
host genome replication 123124
host transcription apparatus 120123
interactions, antibacterials 126128
intracellular phage 120
microbial resistance 128
pharmaceutical companies 119
restriction enzymes and proteases 120
target-molecule selection 120
translation machinery 124125
Host transcription apparatus
bacterial RNA polymerases 120
inhibition 123
phagehost interactions 121, 122
Human microbiome and phages
antibacterial immunity 7
ecotope 7
gastrointestinal tract 9, 1015, 16, 158, 162, 168,
178, 179
macro-host homeostasis 78
monographs 6
non-cultured viral communities 8
physiology 7
respiratory tract 89
skin 8
vagina 1516
virome and host 17
Human wound studies
abscess models 197198
burn wound models see Burn wounds
description 195196
experimental design limitations 198199
phage-therapy trials 199200
PhagoBioDerm
195
Polish studies 194, 195
Innate immunity 171172
In vitro activity, endolysins
Gram-negative organisms 224
Gram-positive organisms
colony counts 225
isolated cell walls 224
phage 225226
three-dimensional superstructure 224
turbidometric analysis 224225
In vivo studies, endolysins
animal infection models 226
ClyS and CHAP
k
227
Cpl-1 226227
immune responses 228
PlyG and PlyGBS 226
S. aureus and MRSA nasal colonization 227
streptococcal infection 226
synergy 227228
Lateral ow immunoassays (LFIs)
detection, analytes 138
human bacterial pathogens 140
microbial identication methods 141
myriad, analytes 140
phage infection reaction 141
Leukotoxins
PVL 37
skin infections 39
LFIs see Lateral ow immunoassays (LFIs)
Luc-based reporter phages
bacterial luciferase genes 143
mycobacterial detection 144
phage-based bioluminescence 142143
Lysogenic conversion
Phage conversion
toxigenic conversion 4, 5, 2223, 3334, 4748,
81
Lysogeny
denitions 45
description 34
lysogen 4
prophage 4
MALDI-TOF MS see Matrix-assisted laser
desorption ionization time-of-ight mass
spectrometry (MALDI-TOF MS)
Male-specic coliphages
description 157158
DNA 158
faecal contamination, river water 162
genotype identication 161
PCR methods 162
primer/probe sets 161162
RNA
desired host bacterium 161
FRNA 158
individual serogroups 158
morphology and survival characteristics 158
origin, faecal pollution 159
seasonal uctuations in FRNA 160
serotyping 160
sewage pollution 160
taxonomy, FRNA phages 159
RT-PCR assays 161
somatic coliphages 161
Matrix-assisted laser desorption ionization time-
of-ight mass spectrometry (MALDI-TOF MS)
analysis and identication 137
bacterial cultures 137
biological analysis 137
CFU 138
mass spectrum 138
phage protein proling 138, 139
species-specic bacterial infection 137
Maximum contaminant levels (MCL)
drinking water 154155
recreational waters 155
total coliforms 154
278 Index
MCL see Maximum contaminant levels (MCL)
Methicillin-resistant S. aureus (MRSA) 185, 227
Mouse intraperitoneal model 227
MRSA see Methicillin-resistant S. aureus (MRSA)
Mucosal colonization model 226
Multiple-domain, endolysins
endopeptidase and LysK 222
exonic shu ing and putative mechanism
223
N-acetylmuramidase and gene deletion
analysis 223
PlyC 223224
silent and non-silent 223
staphylococcal/streptococcal phage 222
streptococcal SA2 222223
Murine vaginal model 226
Negative-treatment control 263264
NMR analysis see Nuclear magnetic resonance
(NMR) analysis
Non-wound infections
animal models
canine otitis 209
gastrointestinal disease 208209
lung infections 207208
septicaemia 207
clinical trials
applications, phage therapy 209, 210
microbiological analyses 211
phase I assessment 209
phase II 210
phase III 211
puried phage preparation 210
description 203
diarrhoeal diseases 203
in Georgia
cocktails 204
Eliava Institute of Bacteriophage Microbiology
and Virology (IBMV) 191, 204
Intestiphage 204
Pyophage preparation 205
treatment 204205
P. aeruginosa lung infections 211
pharmacokinetic properties 212
in Poland 205
rediscovery and renaissance
mice and ruminants 205206
pig skin samples 206
tailor(ed) phages 211
temperate phages 211212
Nuclear magnetic resonance (NMR) analysis
48
Oral cavity and pharynx
oropharyngeal swabs 9
saliva microbiota 10
sequencing, virome metagenomes 910
Veillonella and Enterococcus faecalis bacteriophage
10
VLPs 9
Osteomyelitis 189190
PantonValentine leukocidin (PVL) 37
PCR see Polymerase chain reaction (PCR)
Peptide and protein phage-display libraries
antibody fragments 104
construction, phagemid-based antibody
fragment 104, 105
discovery approach 103
inhibition, pilus formation 106
mAbs 94, 103
Phage formulation toxicity
endotoxin, Gram-negative bacteria 264
testing 264
Phage immobilization, bacterial detection
assay sensitivity 142
ECIS 141
real-time PCR 142
Phage therapy
antibacterial activity 170
antibacterial virulence see Ex situ phage
antibacterial virulence
antibiotics 256
bacterial colonization 260261
blinding 264
dosing and enumeration 264267
ex situ, characterization
early-stage and later-stage development
protocols 259
employ and temperate 259
potential, display lysogeny 259
therapeutics 258
follow-up, unexpected results 268
formulation toxicity 264
host range and laboratory experimentation
267268
initial development 261
isolation 258
municipal sewage systems 256
negative-treatment control 263264
PFU 170
phages and oxidative stress 171
pharmacology 257
positive phage-killing control 262263
positive results 269
protocols 262
rational debugging 257
real-world e cacy 270
safe, highly e cacious and economical 256
staphylococcal phage 171
titres, e cacy 268269
treatment vs. Pseudomonas 262
Index 279
Phage-therapy trials
combat-related injuries 200
orthopaedic devices 200
prophylactic treatment 199200
regulatory agencies 199
Phage typing (PT)
bacteria, bacteriophages 77
CRISPR 79
discriminatory power 78
environmental factors 79
epidemiological concordance 78
Fourier transform infrared spectroscopy 80
host susceptibility 77
lysogenization 78
PCR 80
phage-based typing method 7677
phage-mediated transfer, DNA 78
phages lytic 77
phenotypic assessment tool 77
phenotypic methods 80
reproducibility 79
restriction enzyme systems 77
typeability 78
PhagoBioDerm
TM
195
Phage vaccines 87, 8892, 178
Plaque assay
bacterial identication 135
Petri dish 135
species-specic phage 135, 136
Pneumococcal bacteraemia model 226
Polymerase chain reaction (PCR) 31, 36, 40, 79, 80,
104, 105, 110, 111, 112, 134, 141, 156, 159, 160,
161, 162, 163
Positive phage-killing control
description 262
phage experiments 263
Protein vaccines
Cytos Biotechnology 92
lamentous phage display 90, 91
genetic engineering 91
humoral immune response 90
orphan diseases 92
tissue tropism 92
Pseudomonas
lysogeny and polylysogeny 47
NMR analysis 48
O-antigenic conversion 4748
PT see Phage typing (PT)
PVL see PantonValentine leukocidin (PVL)
QDs see Quantum dots (QDs)
Quantum dots (QDs) 145
SCIN see Staphylococcal complement inhibitor
(SCIN)
Selection strategies, phage display
antibody phagemid library 106
biotinylated antigen approach 107
cell-surface panning 108, 109
peptide/antibody 106
solid-phase panning 107
western blot/immunohistochemistry 108
Shiga-like toxigenic phages (SLT-phages) 43, 81
Shiga-toxigenic E.coli (STEC) 22, 29, 43, 78, 81, 244
Skin and lung infections
phage-encoded toxins 26
S. aureus 25
SLT-phages see Shiga-like toxigenic phages
(SLT-phages)
Somatic coliphages
colonize biolms 157
degradation 156
description 156
E. coli strains WG5 and CN13 157
IAWPRC 157
in sewage-contaminated waters 157
SSSS see Staphylococcal scalded-skin syndrome
(SSSS)
Staphylococcaemia
patients, phage therapy 190, 191
systemic infection 190
Staphylococcal complement inhibitor (SCIN)
39
Staphylococcal scalded-skin syndrome (SSSS)
Riters disease 25
toxin production 25
TSS 26
Staphylococcus infection
ear gene 41
Exofoliative toxins (ETs) 37
gastrointestinal intoxication 26
genome analysis 35
high-frequency mobilization, SaPIs 41
horizontal phage transfer 35
immune evasion cluster 3941
leukotoxins 37, 39
location and organization, conversion genes.
36
lysogenic conversion 37
PVL 24
sau42I gene 41
S. aureus-converting phages 3739
skin and lung infections 2526
SSSS 25
TSS 2627
STEC see Shiga-toxigenic E.coli (STEC)
Streptococcus infection
neutrophil extracellular traps (NETs) 27, 42
prophage and prophage-like elements 41, 42
SpyCI and mutator phenotype 4243
stationary-phase bacteria 28
TSS 27
virulence factors 27
virulence genes 43
280 Index
Tailspike proteins 231232
Therapeutic delivery vehicles, phages as
amyloid plaques 94
applications 86
characteristics, material 86
cytotoxic agents 9394
description 92
DNA vaccines 8890
gene therapy 87, 88, 9293
immunogenic molecules 95
ligand density 95
mammalian physiology 88
phage-delivered cargo 87
phage genome 86
protein distribution 94
protein vaccines 9092
transfection 93
Time-resolved uorescence resonance energy
transfer (TR-FRET) 126
Toxic shock syndrome (TSS)
erythematous rash 2627
staphylococcal disease 26
treatment 27
Transduction
genetic exchange 1, 5, 35, 4850, 64, 69, 81,
259
horizontal gene transfer 30, 41, 4850
lytic phages 49
methods, genetic exchange 48
Translocation
antibacterial agents 169
bacterial gut microora 179
description 168, 178
exogenous bacteriophages see Exogenous
bacteriophages
gut microbiota 178
immunomodulatory eects, bacteriophage
preparations see Bacteriophage
preparations
mesenteric lymph nodes 178
oral administration 178
pathogenic viruses 169
proliferation, T and B cells 168
treatment, systemic infections 179
TR-FRET see Time-resolved uorescence
resonance energy transfer (TR-FRET)
TSS see Toxic shock syndrome (TSS)
Verotoxigenic phages (VT phages) 43
Vero toxin-producing E. coli (VTEC) 246
Vibrio cholerae (V. cholerae) see Disease, cholera
genetic structure, CTX prophage 28, 4647
hybrid phage genomes 47
Virus-like particles (VLPs) 3, 912, 15, 16, 89, 90,
9192, 9495
VLPs see Virus-like particles (VLPs)
VTEC see Vero toxin-producing E. coli (VTEC)
Water quality assessment
Clostridium perfringens 154
EPA and MCL 154155
faecal contamination 155
faecal pollution 154
limitations, bacterial indicators 155
Wounds and purulent infections
acute wounds 186
burn wounds 186, 189
chronic wounds 189
description 185
diabetic wounds 189
early phage therapy
bone injuries 192
Clostridium perfringens 191
pre-antibiotic era 191
prophylactic 192
sof -tissue wounds 192
staphylococcaemia see Staphylococcaemia
surgical debridement 191
topical phage applications 193
trials on civilians 193194
wound infection type and anatomical location
192
e cacy, phage therapy 187188
human wound studies see Human wound
studies
microbes 186
osteomyelitis 189190
types 186
unconventional treatment 185
Yellow uorescence protein (YFP) 145
YFP see Yellow uorescence protein (YFP)

Bacteriophages in Health and Disease

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Bacteriophages in Health and Disease

Bacteriophage T4 seen obliquely from the base plate end

), contains killed whole cells of V.

bovine and porcine strains.

), approved by the FDA in 2011

Not determined Clinical isolates were tested for their

E. coli mutants (i.e.

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