Bacterial Biofilms With Emphasis On Coagulase-Negative

Received: May 27, 2008 J. Venom. Anim. Toxins incl. Trop. Dis.
Accepted: August 19, 2008 V.14, n.4, p.572-596, 2008.

Abstract published online: September 1, 2008 Review article.
Full paper published online: November 30, 2008 ISSN 1678-9199.
BACTERIAL BIOFILMS WITH EMPHASIS ON COAGULASE-NEGATIVE

STAPHYLOCOCCI
OLIVEIRA A (1), CUNHA MLRS (1)
(1) Department of Microbiology and Immunology, Botucatu Biosciences Institute, São

Paulo State University, UNESP, Botucatu, São Paulo State, Brazil.
ABSTRACT: In addition to their capacity to attach to surfaces, various groups of

microorganisms also produce an extracellular polymeric substance known as “slime”.
This slime forms a thin layer around cells known as biofilm. Thus, biofilm structure
comprises bacterial cells and an extracellular polymeric substance. It also presents a
defined architecture, providing the microorganisms with an excellent protective
environment and favoring the exchange of genetic material between cells as well as
intercellular communication. The ability to produce biofilm is observed in a large
group of bacteria, including coagulase-negative staphylococci (CNS) which are the
predominant microorganisms of normal skin flora and have been implicated as the
causative agents of hospital infections. Bacteremia caused by these agents is
common in immunodepressed persons, in patients with cancer, in adult and neonatal
intensive care units (ICU) and in patients using catheters or other prosthetic devices.
The pathogenicity of CNS infections is probably related to the production of slime,
which adheres preferentially to plastic and smooth surfaces, forming a biofilm that
protects against attacks from the immune system and against antibiotic treatment, a
fact hindering the eradication of these infections. The main objective of the present
review was to describe basic and genetic aspects of biofilm formation and methods
for its detection, with emphasis on biofilm creation by CNS and its relationship with
diseases caused by these microorganisms which are becoming increasingly more
frequent in the hospital environment.
KEY WORDS: coagulase-negative staphylococci, biofilm, slime, infection,

microbiology.
CONFLICTS OF INTEREST: There is no conflict.
FINANCIAL SOURCE: Fapesp.
CORRESPONDENCE TO:
MARIA DE LOURDES RIBEIRO DE SOUZA DA CUNHA, Departamento de
Microbiologia e Imunologia, Instituto de Biociências, UNESP, Caixa Postal 510,
Botucatu, SP, 18618-000, Brasil. Fax: +55 14 3815 3744. Email:
[email protected].
A. Oliveira and M. L. R. S. Cunha BACTERIAL BIOFILMS WITH EMPHASIS ON COAGULASE-NEGATIVE
STAPHYLOCOCCI. J. Venom. Anim. Toxins incl. Trop. Dis., 2008, 14, 4, p. 573
INTRODUCTION
Biofilms are not simple organism-containing viscous layers; they represent highly
organized systems in which bacteria establish functional structured and coordinated
communities (27). Some groups of microorganisms are able to adhere to a surface
and to develop biofilms. These biofilm-associated cells can be distinguished from
suspended planktonic cells by the production of an extracellular polymeric substance
(EPS), by the reduced growth rates of the colonies and by the regulation of specific
genes. The attachment process is highly complex and regulated by different
characteristics of the growth medium, substrate and cell surface. The biofilm
structure comprises microbial cells and EPS, has a defined architecture and provides
an excellent environment for the exchange of genetic material among cells (29).
Coagulase-negative staphylococci (CNS) have recently been identified as important
causes of hospital infections. An important step in the development of catheter- or
implant-associated infections caused by CNS is the adhesion and attachment of
these bacteria to biomaterial surfaces (5-7, 9, 15, 18, 25, 35, 39, 48, 56, 68).
Among the various mechanisms involved in bacterial adhesion, the production of an
extracellular polysaccharide substance called slime plays a relevant role. This
substance strengthens in surface adhesion like cement, permitting the agglomeration
of bacterial cells into biofilms or multilayers. Once formed, these biofilms render the
cells less accessible to the defense system of the organism, thus impairing the action
of antibiotics and, in turn, represents basic survival strategies of these
microorganisms, a fact that explains why biofilms are considered to be of great
importance for public health. Therefore, studies and diagnostic methods identifying
virulent bacterial strains, i.e., strains with a capacity for slime production and
consequent biofilm formation, are necessary to develop effective strategies for biofilm
control and improvement of patient care (3, 5, 6, 9, 13, 22, 25, 27, 56, 68).
Since biofilm-producing CNS are potentially involved in the occurrence of catheter-
associated infections, transforming microorganisms from the normal flora to important
opportunistic pathogens in immunocompromised patients – such as preterm
newborns, neutropenic oncology patients, seniors with severe underlying diseases,
hospitalized patients submitted to invasive procedures and those with permanent
plastic devices –, the objective of the present review was to provide further data
regarding biofilms that may contribute to future studies on this subject.
A HISTORY OF BIOFILMS
Donlan and Costerton (30) cite Van Leeuwenhoek, who observed “animalcules” in
the plaque of his own teeth using a simple microscope, as the pioneer in the
discovery of microbial biofilms. Heukelekian and Heller (42), studying marine
microorganisms inside a bottle, observed that bacterial activity and growth were
markedly increased in this container due to the incorporation of a surface to which
these organisms could adhere. Analyzing seawater, Zobell (69) noticed that the
number of bacteria attached to surfaces was higher than in the surrounding medium.
However, a detailed study of biofilms only became possible with the advent of the
electron microscope, which permits high-resolution photomicroscopy at
magnifications hundreds of times higher than those obtained with the light
microscope. Jones et al. (46), employing scanning electron microscopy, investigated
biofilms in a water treatment plant and, based on cell morphology, demonstrated that
biofilms consist of a variety of organisms. In addition, the authors were able to show
that the matrix material surrounding and enclosing cells in these biofilms consisted of
polysaccharides. Characklis (11) investigated microbial slimes in industrial water
systems and showed that they were not only persistent but also resistant to
disinfectants like chlorine. However, the theory of biofilm predominance was only
promulgated in 1978 by Costerton et al. (19) based on observations of dental plaque
and sessile communities in mountain streams. This comparison of organisms in their
natural environment and in dental plaque permitted the understanding of the
mechanisms whereby these microorganisms adhere to living and nonliving materials
and the benefits of this type of ecological niche. According to Donlan (29) and
Costerton and Lashen (20), since 1978 studies of biofilms related to public health,
industry and ecology have been carried out in parallel. Scanning electron microscopy
and standard microbiological culture techniques have been important tools for the
characterization of biofilms in the last few decades, during which two main tools have
revolutionized the understanding of biofilms: laser scanning microscopy and the
investigation of the genes involved in cell adhesion and biofilm formation.
Numerous studies on biofilms published over the most recent decades have
contributed to the understanding of numerous infectious diseases associated with
biofilm growth. Many infections that occur in hospitalized patients who use medical
devices, such as prostheses or catheters, are related to biofilms. Bacteria from
normal skin flora of patients or hospital personnel come in contact with objects on
which biofilms develop, facilitating the entry of these microorganisms into the
organism and rendering the infection persistent due to the several advantages
encountered by bacterial cells when enclosed in these biofilms (29).
Staphylococcus epidermidis, the most frequently detected CNS species and one of
the main inhabitants of normal skin and mucosal flora, has been identified as the
major nosocomial pathogen associated with infections due to implanted medical
devices as a result of its great biofilm production capacity (55).
Since biofilms play a role in numerous human activities, researchers from various
areas have conducted a large number of studies that aimed to prevent infections
related to biofilms or to their accumulation in water or industrial equipment where
they cause damage and, consequently, great financial losses. In view of the large
number of scientific fields involved, an arsenal of techniques is available for the
biofilm study, ranging from molecular microbiological methods applied laboratory-
cultured monospecies biofilms to the use of microsensors for the study of algal
carpets (51).
DEFINITION OF BIOFILMS
According to Donlan and Costerton (30), the definition of biofilms has evolved over
the last 25 years. In 1976, Marshall described the biofilm as the envelopment of “very
fine extracellular polymeric fibrils” attached to bacteria. Many authors define biofilms
as associations of microorganisms and their extracellular products adhering to biotic
or abiotic surfaces (27).
IMPORTANCE OF BIOFILMS FOR BACTERIA

According to Donlan (29), biofilms are a survival strategy used by bacteria in natural
and industrial systems and on medical devices such as prostheses and catheters as
a protection against chemical antibacterial substances (including natural antibiotics),
environmental bacteriophages and phagocytic cells. In bacterial infections associated
with biofilm formation, microorganisms are resistant to antibiotic therapy as well as to
host defense mechanisms such as antibodies and phagocytes.
BIOFILM STRUCTURE
Studies on the structure of biofilms have shown that they primarily consist of
microbial cells and EPS that accounts for 50 to 90% of their total organic carbon and
can be considered their main formation material. Although EPS varies in its physical
and chemical properties, it is primarily composed of polysaccharides. Biofilm
polysaccharides can be neutral or polyanionic, as in gram-negative bacteria. One
important characteristic is the anionic property of EPS conferred by the presence of
uronic acids (D-glucuronic, D-galacturonic and mannuronic acids). This property
favors the association of bivalent cations, such as calcium and magnesium, that have
been shown to bind to the polymers and contribute to the binding strengh in a
developed biofilm. Some bacteria like Staphylococcus present a cationic chemical
composition of EPS (30). Hussain et al. (43) demonstrated that CNS slime consists of
a teichoic acid mixed with small amounts of proteins. EPS can incorporate large
amounts of water into its structure due to is hydrogen bonding ability and are
therefore highly hydrated.
According to Sutherland (66), the composition and structure of polysaccharides
determine their primary configuration, as is the case of many bacterial EPS, which
possess backbone structures that contain 1,3-β or 1,4-β-hexose residues and tend to
be more rigid, less malleable and, in certain cases, poorly soluble or insoluble,
whereas others are readily soluble in water. In addition, EPS is not uniform but may
vary spatially and temporally. Leriche et al. (53), employing the binding specificity of
lectins for simple sugars to evaluate the development of bacterial biofilms in different
organisms, concluded that different organisms produce variable amounts of EPS and
that the quantity of EPS increases with the age of the biofilm. EPS can also associate
with metal ions and macromolecules (proteins, DNA, lipids and organic substances).
Some culture medium conditions – excessive carbon availability and limited amounts
of nitrogen, potassium or phosphate – affect EPS production. Slow bacterial growth
also results in an increase of EPS production. Since EPS is a highly hydrated
compound, it prevents desiccation in natural biofilms.
Tolker Nielsen and Molin (67) showed that biofilms contain microcolonies of bacterial
cells enclosed in an EPS matrix and that these structures are separated by interstitial
voids (water channels). The water channels are important for fluid circulation, thus
permitting the flow of nutrients, oxygen and microorganisms from one site to another.
Even pure culture biofilms such as those found on instrument surfaces or associated
with medical devices are heterogenous. According to Stoodley et al. (63), biofilms
generally comprise a thin base film that ranges from a monolayer of cells to a film of
several layers that contain water channels. The structure of biofilms is also
influenced by the organisms that comprise the biofilm.
According to James et al. (45), biofilm thickness may be affected by the number of
microorganisms that compose it. Pure cultures of Klebsiella pneumoniae or
Pseudomonas aeruginosa grown in a laboratory reactor presented thinner biofilms
(15 and 30 µm, respectively), whereas a biofilm containing both species was thicker.
Jones et al. (46) suggested that this finding may be due to the fact that one species
increases the stability of another. On the basis of the aforementioned studies, we
conclude that biofilm architecture is heterogenous in space and time. Donlan (29)
reported that biofilm structure and architecture may also be influenced by the
interaction of particles of nonmicrobial components from the host or environment.
One example is the accumulation of erythrocytes and fibrin during biofilm formation.
Another classic example of this interaction is observed on natural heart valves, where
microcolonies develop a biofilm – from a matrix of platelets, fibrin and EPS – while
the organisms are protected inside the forming fibrin capsule from host leukocytes,
causing infective endocarditis.
BIOFILM ECOLOGY
Biofilms present in natural and industrial water systems or on medical devices
provide the ideal environment for the exchange of energy and material among
microorganisms, including gene exchange and nutrient circulation. This environment
is favorable due to the proximity of cells within microcolonies or between
microcolonies. Bacteria in biofilms are subject to predation by protozoans and to
competition with other bacteria, although some species are able to coexist
harmoniously (28). Murga et al. (58) demonstrated the colonization and consequent
predation in heterotrophic biofilms by Hartmannella vermiformis, a free-living
protozoan. Predation has also been observed by Acanthamoeba spp. in biofilms of
storage cases for contact lens.
James et al. (45) reported that competition also occurs inside biofilms and
demonstrated that invasion of a Hyphomicrobium sp. biofilm by Pseudomonas putida
resulted in the dominance of the latter, although the numbers of biofilm-associated
Hyphomicrobium remained relatively constant. Donlan (29) cited the study of Stewart
who investigated biofilms containing K. pneumoniae and P. aeruginosa and observed
that both species are able to coexist in a stable community.
Several decades ago, it was proposed that bacteria represent interactive organisms
that are able to act collectively, facilitating their adaptation to environmental changes.
The establishment of a multicellular behavior is necessary for the formation of a
biofilm consisting of one or several species, which is reflected in coordinated
activities of interaction and communication among various organisms (27) (Figure 1).
One of the mechanisms of interbacterial communication that has been proven
extremely important for the formation and development of biofilms is “quorum
sensing” (27), a system by which bacteria communicate with one another through
signaling molecules, called autoinducers, that regulate bacterial behavior according
to population density (64).
Figure 1. Example of microbial community ecology. The four central microcolonies

correspond to organisms that generate and consume hydrogen. The fermenters use
sugars and produce organic acids that are used by hydrogen producers. In addition
to metabolic interactions, intra- and intercellular communication may be mediated
by signaling molecules. Adapted from Davey and O’Toole (27).
Gene Transfer
The proximity of cells inside microcolonies or among microcolonies also provides an
excellent environment for the exchange of genetic material. The mechanism of
conjugation, i.e., the transfer of plasmids between bacteria, occurs at a higher
proportion in biofilm bacterial cells than among planktonic cells (29). Ghigo (37)
suggested that clinically relevant strains of bacteria containing conjugative plasmids
develop biofilms more readly. The author showed that the F conjugative pilus
(encoded by the TRA operon of the F plasmid) acts as an adhesion factor in cell-to-
surface and cell-to-cell interactions, resulting in a three-dimensional biofilm of
Escherichia coli. Many organisms that did not carry plasmids produced only
microcolonies, but when these microorganisms received the plasmid from donor
bacteria they also started to produce biofilms. In addition to cell-to-cell contact, the
biofilm environment provides adherence while plasmids may also encode resistance
to multiple antimicrobial agents. This association with the biofilm also provides a
selection mechanism and promotes the dissemination of bacterial resistance to
antimicrobial agents.
Biofilm Formation Mechanisms, Attachment and Substrate Effects

The ideal environment for the attachment and growth of microorganisms is a solid-
liquid interface between a surface and an aqueous medium (e.g. water or blood).
Various factors influence the biofilm formation mechanisms such as substrate effects,
the types of conditioning film forming on the substrate, characteristics of the medium
and properties of the cell surface. As a surprise to the medical community, bacteria
preferentially form biofilms on very smooth surfaces. This fact was confirmed in
studies investigating the adhesion of laboratory and wild-type bacterial strains to
smooth and rough surfaces. The results showed that smooth surfaces are colonized
as easily as rough surfaces. Once a biofilm has formed and the exopolysaccharide
matrix has been secreted, the resultant structure resembles rubber, i.e., it is viscous
and elastic (12, 30).
Biofilms formed on rougher surfaces have been shown to possess a low tensile
strength and to break easily, whereas biofilms formed on much smoother surfaces
are stronger and more resistant to rupture, although the solid surface seems to
present many characteristics important to the attachment process (21, 30).
Characklis (11) showed that the extent of microbial colonization is heightened with
increasing surface roughness, because shear forces are reduced and the surface
area of rougher surfaces is larger. Another factor that exerts a strong influence on the
rate and extent of attachment is represented by the physicochemical properties of the
surface (2, 21).
Vuong and Otto (68) described biofilm formation as follows: in the first stage, bacteria
may become attached to the free surface of the polymer; in the second, they become
attached to the surface of the protein layer, which will subsequentially cover the
polymer. Once attached, bacteria start to multiply and form numerous layers
(accumulative phase). The biofilms of S. epidermidis do not contain other bacterial
species probably because of the action of bacteriocins produced by this species. A
capsular polysaccharide molecule, called polysaccharide adhesin, seems to
influence attachment to the uncoated surface. After the surface is coated with a
protein layer, S. epidermidis interacts with it through different surface molecules.
Fibrinogen-binding protein of S. epidermidis mediates bacterial adherence to
fibrinogen. Bacterial layers are formed by the binding of cells to one another through
polysaccharide intercellular adhesin (PIA), a polysaccharide molecule that favors
adhesion between cells, and through an extracellular protein called accumulation-
associated protein.
The study by Vuong and Otto (68) showed a significant increase in PIA during
tricarboxylic acid (TCA) cycle stress. The authors reported abundant PIA production
in a nutrient-replete medium even when iron was limited or under conditions of low
oxygen availability. However, a stress-inducing stimulus such as the addition of sugar
motivated fermentation, with a consequent production of ethanol and an increase in
salt concentration, thus elevating the PIA production. This condition of the medium
was also found to repress TCA cycle activity and raised the hypothesis that changes
in TCA cycle activity affect PIA production. Culturing S. epidermidis with a low
concentration of the TCA cycle inhibitor fluorocitrate dramatically increased PIA
production without affecting glucose catabolism. These data led the authors to
speculate that alterations in the growth medium and/or in the TCA cycle cause
changes in the intracellular levels of ATP biosynthesis or even in the redox status of
the cell. These changes in bacterial metabolic status may result in the attenuation of
PIA production.
PIA is a linear homopolymer of up to 130 β-1,6-linked N-acetylglucosamine residues
which consists of two polysaccharide fractions: polysaccharide I (> 80%) which
contains 15 to 20% diacetylated residues and is therefore positively charged, and

polysaccharide II (< 20%) which is structurally related to polysaccharide I but
contains a smaller number of non-N-acetylated D-glucosamine residues and
phosphate and ester-linked succinate, and is therefore anionic (4, 33, 39, 57).
Genes Controlling Biofilm Formation in Staphylococcus aureus and

Staphylococcus epidermidis
Many saprophytic organisms, including Staphylococcus epidermidis, are part of the
normal microflora of mucosa and skin. Whereas S. aureus has always been a
causative agent of infections in humans, under certain conditions S. epidermidis may
also cause infections; therefore, it is considered an opportunistic agent. Over the
most recent decades, S. epidermidis has emerged together with S. aureus as a
frequent etiological agent of infections associated with catheters, prostheses and
other medical devices. One of the main mechanisms responsible for the development
of severe device-associated infections is the formation of biofilms that facilitate the
bacterial adhesion and colonization of these materials. Recent studies have shown
that both S. aureus and S. epidermidis are able to produce biofilms. According to
Arciola et al. (5), the genetic control of slime production has recently begun to be
elucidated, first in S. epidermidis and then in S. aureus.
The genes encoding the most important substances and proteins, especially a
polysaccharide adhesin, that participate in biofilm formation belong to the ica operon.
This operon contains the icaADBC genes and the regulatory icaR gene which is
transcribed in the direction opposite to the ica operon. Briefly, the synthesis of the
polysaccharide capsule is mediated by the ica operon. When this operon is activated,
PIA and poly-succinyl-glucosamine are synthesized and support cell-to-cell contacts
by means of a multilayer biofilm. PIA is formed from UDP-N-acetylglucosamine by
the enzyme N-acetylglucosamine transferase, which is encoded by the intercellular
adhesion (ica) locus, particularly the icaA gene. The expression of icaA alone in
isolation induces only low enzymatic activity, but coexpression of icaA and icaD leads
to a significant increase in the activity of N-acetylglucosamine transferase, which
catalyzes the formation of oligomers with approximately 20 residues. Expression of
the icaC gene leads to the synthesis of longer oligomers (130 residues) that react
with PIA-specific antisera. The function of the icaB gene has not been established (4,
6, 9, 16, 23, 32, 35, 38, 48, 56, 57, 59, 60) (Figure 2).
Figure 2. Model of PIA synthesis. IcaA and IcaD synthesize oligomers, derived from
UDP-N-acetylglucosamine, consisting of 10 to 20 residues. In the presence of IcaC,
longer oligomers containing 130 residues are formed by reacting with two PIA-
specific antisera. Due to IcaB’s sequence similarity with diacetylases, it may catalyze
diacetylation.
CM: cytoplasmic membrane.
Adapted from Heilmann et al. (41), Gerke et al. (36) and O. Schweitzer (unpublished results).
Some studies have suggested that icaR gene product is a transcription repressor that
plays an adaptive role in the regulation of the ica operon expression depending on
environmental conditions. Some factors such as anaerobic growth, the presence of
antibiotics like tetracycline at subinhibitory concentrations, and environmental stress
including high osmolarity, may increase the expression of the ica operon. The genes
encoding virulence factors in S. epidermidis, as well as the ica operon, seem to be
controlled by the global agr and sar regulatory systems, similar to what is observed in
S. aureus (5).
Li et al. (54) confirmed that biofilm formation in S. epidermidis depends on PIA,
whose biosynthesis is mediated by the ica operon. The authors transferred a plasmid
containing the ica operon to three ica-negative bacterial strains. Using rats as
experimental models, the authors infected these animals with bacteria harboring the
ica operon. These bacteria, then, produced a biofilm and were pathogenic to rats,
thus confirming the importance of the ica operon for biofilm production and for the
pathogenesis of S. epidermidis.
Arciola et al. (4) investigated the role of the icaA and icaD genes in slime production
in clinical staphylococcal samples. The coexpression of icaA and icaD was analyzed
by the polymerase chain reaction (PCR) in a collection consisting of: 91
Staphylococcus strains (68 S. epidermidis and 23 S. aureus strains) isolated from
catheter-associated infections, ten strains obtained from skin and mucosa of healthy
volunteers, and two reference strains. The phenotypic slime-forming ability was
tested by the Congo red agar method. Among the analyzed isolates, 49% of S.
epidermidis strains were from catheters and 61% of S. aureus strains produced slime
and were positive for icaA and icaD. None of the saprophytic strains produced slime
and all were negative for icaA and icaD. Two S. aureus strains and one S.
epidermidis strain isolated from catheter tips, were positive for icaA and icaD by
PCR, and were slime producers by the Congo red method, in which they exhibited
small red dots in the center of black colonies after 48 hours. The presence of these
variants could not be attributed to a mutagenic potential of Congo red which, in the
Ames test, did not present mutagenicity. PCR showed that these red variants were
negative for icaA and icaD and were even lacking the icaADBC operon. These data
indicate an important role of the ica genes as a virulence factor in intravenous
catheter-associated staphylococcal infections. Regarding the genetic factors studied,
the authors concluded that the recent advances in genome research will have a
crucial influence on future investigations concerning the pathogenesis of
Staphylococcus.
Slime Production by Coagulase-negative Staphylococci and Its Association

with Infections and Diseases
Various CNS species including S. epidermidis have become the main causes of
nosocomial infections, especially those affecting newborns, immunodepressed
individuals and patients hospitalized in the ICU using catheters or other prosthetic
devices. These infections can be endogenous or exogenous, arising from the
hospital environment or from medical staff hands.
In addition to the conditions of the patient, the pathogenicity of CNS is favored by the
ability of these microorganisms to attach to smooth surfaces and to produce large
amounts of extracellular substances which form a biofilm that confers protection
against the defense mechanisms of the host and the action of antibiotics. As an
important virulence factor, this biofilm-producing capacity is one of the mechanisms
that explains the colonization of medical devices and possible infections with
staphylococci (1, 3, 7, 18, 22, 35, 39, 47, 50, 56, 57).
Controversial results have been reported by some investigators regarding the
association between slime production and clinically significant infections caused by
CNS or even concerning differences in the pathogenic potential between slime-
producing and non-producing strains. No evidence is available about the role of host
protein receptors expressed by CNS. In this respect, some studies have clearly
demonstrated the ability of S. epidermidis to adhere to immobilized plasma proteins,
whereas others reported that these proteins reduce or block the staphylococcal
adhesion. These contrasting results were attributed to a “masking action” of slime-
covering protein receptors in in vitro assays (3).
In view of the findings regarding CNS, Alcaraz et al. (1) conducted a study on CNS
species identification in terms of slime production and oxacillin susceptibility of strains
isolated from nosocomial infections. Most slime-producing CNS strains
(environmental and clinical) were resistant to oxacillin. The authors concluded that
precise detection and effective control of these microorganisms are necessary in
order to prevent their dissemination in hospitals, particularly among
immunocompromised patients.
In contrast to S. aureus, S. epidermidis is unable to produce large amounts of
enzymes and toxins, so that infections caused by the latter are subacute or chronic.
S. epidermidis has become an important agent of hospital infections because of its
ability to adhere to polymer surfaces and to form a biofilm, the latter being considered
its main virulence factor (55).
Cerca et al. (10) demonstrated the importance of biofilm production in the
development of infections and diseases which confers resistance against antibiotics,
by quantitatively comparing the antibiotic susceptibility of biofilm-producing S.
epidermidis and S. haemolyticus with the susceptibility of planktonic bacteria. The
results showed that biofilm-producing bacteria were resistant to antibiotics which
inhibited cell wall synthesis, but were susceptible to inhibitors of RNA and protein
synthesis, whereas planktonic cultures were susceptible to both.
Ishak et al. (44), investigating slime production by CNS, reported that the presence of
slime seems to adversely influence opsonization and phagocytosis, inhibiting
polymorphonuclear leukocyte (PMN) chemotaxis, which induces PMN degranulation,

inhibiting the oxygen-dependent metabolic activities of PMN which normally occur
during phagocytosis, and subsequent intracellular death, influencing the interactions
of surface opsonins, which are a prerequisite for the processing of phagocytic.
One of the frequent causes of death in patients treated for hydrocephalus has been
ventriculoperitoneal shunt infection. Antimicrobial therapy has been successful in
some of these cases, but otherwise removal of the shunt and concomitant
appropriate antimicrobial therapy are recommended. Diaz-Mitoma et al. (28) reported
that CNS are responsible for 36 to 80% of these infections. Slime-producing strains
were the most common contaminants of the foreign bodies used. In view of
speculations by researchers about whether slime production is involved in the
pathogenesis of these infections, Diaz-Mitoma et al. (28) determined slime
production in CNS isolated from ventriculoperitoneal shunt infections. Eleven cases
of infection were caused by slime-producing CNS and eight cases were caused by
non-producing strains. The shunt obstruction and abdominal pain were more frequent
when infectious cases were due to slime-producing CNS than when they were
caused by non-producing CNS. Despite appropriate antimicrobial therapy, the mean
duration of fever was longer and failure to eradicate the infectious organisms was
more frequent when the infectious cases were due to slime-producing CNS.
Several investigators have indicated exopolysaccharide or slime production as an
epidemiological marker of infection in pediatric patients (31, 40). Similar studies
mainly conducted on adult patients have reported a higher frequency of slime-
producing CNS strains to be involved in the etiology of infections compared to non-
producing strains (8, 14). Kotilainen (50), evaluating the importance of slime
production as a virulence factor in strains isolated from septicemia patients in two
Finish hospitals attended by the same microbiology laboratory, reported conflicting
results, suggesting that slime production was certainly an important virulence factor.
However, this relationship between slime production and clinically significant
infections may not be a universal phenomenon. Probably, virulence factors other
than slime production also contribute to the pathogenesis of these microorganisms,
with these factors varying in different environments.
Cunha et al. (26) have shown that only a small proportion of strains producing this
exopolysaccharide (22.2%) is associated with infectious processes in newborns.
Other authors also reported lack of evidence that slime is a virulence factor (13, 51).
On the other hand, slime-producing CNS strains have been more frequently isolated
from patients with sepsis than from patients without invasive disease, indicating the
risk of invasive disease in patients harboring slime-producing CNS (40). Similar
results have been observed by Cunha et al. (26) who found a significantly higher
frequency of slime-producing S. epidermidis strains in blood cultures (20.4%) and
foreign bodies (21.9%) than in secretions (0%). Cunha et al. (24) also conducted
studies in order to identify CNS species and to determine the influence of slime
production during the course of peritonitis in patients submitted to continuous
ambulatory peritoneal dialysis (CAPD). Slime production was independently
associated with the non-resolution of peritonitis cases caused by CNS during CAPD,
with peritonitis caused by non-slime-producing CNS presenting a 27 times higher
chance of cure than episodes caused by slime-positive CNS.
Slime production may also vary from species to species. According to Christensen et
al. (15), slime production is more frequent in strains of S. capitis, S. epidermidis, S.
hominis and S. saprophyticus. Among CNS species isolated in various studies (24-
26, 33), slime production was observed in S. epidermidis, S. lugdunensis and S.
hominis.
Methods for the Detection of Slime Production in Coagulase-negative

Staphylococci
Different methods are currently employed to detect biofilms, including visual
techniques including scanning electron, transmission electron and epifluorescence
contrast microscopy, and non-visual methods such as the measurement of
impedance and bioluminescence. Qualitative analyses – like the tube method by
Christensen et al. (13) and the Congo red agar method by Freeman et al. (34) – and
quantitative assays – such as tissue culture plate (TCP) method by Christensen et al.
(15) – have also been described. In addition, molecular methods (PCR) are used to
provide direct evidence of the genetic basis of slime production. Electron microscopy
has been used since the first study on biofilms for their examination and
measurement. This technique requires gradual dehydration of the samples since the
water is not compatible with vacuum employed by the electron beam. Graded
solvents such as alcohol, acetone and xylene are used for this purpose. The
technique disadvantage is that it can cause distortion of samples since the EPS
consists of approximately 95% water and the liquid loss led them to appear more like
fibers surrounding the cells than like a gelatinous matrix (30). The advent of the
transmission electron microscope, which uses specific polysaccharide stains such as
ruthenium red, allowed researchers to identify the nature of the extracellular fibers in
biofilms and lead to a better understand of their association with cells. Despite some
limitations, the electron microscope is being employed until today for the examination
and characterization of biofilms on medical devices and in human infections.
Because of its excellent resolution properties, the electron microscope represents an
important tool for biofilm investigation. Although at lower magnifications, another type
of microscope used is the confocal laser scanning which permits in situ examination
of biofilms without the limitations of the electron microscope, namely, the intact
biofilm matrix state (30).
In a study regarding to microbial biofilm detection on venous catheters removed from
ICU patients, Stort et al. (65) used scanning electron microscopy and reported that
the extracellular biofilm matrix appears as an amorphous material on the catheter
surface, similar to the observation of Donlan (29).
Confocal microscopy is the method of choice for the structural analysis of biofilms
(52) since it permits the optical sectioning in hydrated state without destroying the
samples, providing three-dimensional data of the structure. In addition, confocal
microscopy permits the analysis of multiple-channel images and thus provides spatial
information about the various biofilm functions. The noninvasive characteristics of the
confocal microscope also favor the investigation of biofilms cultured on flow cells,
which are small chambers whose top and bottom consist of glass lamellae and which
can be coupled to a microscope for the observation of biofilms growing on the inner
walls (61).
Contrast microscopy is recommended for the real-time monitoring of biofilms
developing on a transparent surface. Epifluorescence microscopy is an excellent
alternative for the quantification of surface-adhered cells. For the visualization of
bacterial adhesion, fluorescent dyes such as acridine orange are used for direct
staining of the cells, or fluorescent antibodies that bind to the cells (17).
Electron microscopy is more indicated for the evaluation of interactions that occur in
the biofilm matrix. The samples are fixed with chemical agents, such as
glutaraldehyde, paraformaldehyde and osmium, or are cryofixed, a process in which
the sample is quickly frozen to prevent cell damage caused by the ice crystals (17).
Non-visual methods used for the evaluation of bacterial adherence and biofilm
formation are impedance and bioluminescence measurement. Impedance measuring
is based on the principle that when metabolizing components are present in the
culture medium, microorganisms transform large molecules into small ones that are
electrically charged, an event leading to a change in the resistance or impedance of
the medium. The change in conductivity can be measured and the number of
microorganisms adhered to the surface is related to the conductivity obtained (62).
The bioluminescence assay is based on the content of adenosine triphosphate
(ATP), the universal energy currency in biological systems, whose generation in the
bacterial cell is similar to that observed for all life forms. ATP is generated by the
oxidation of nutrient molecules such as glucose, fatty and amino acids. The amount
of ATP in a sample can be measured by the bioluminescence reaction between
luciferin and the enzyme luciferase (39).
The most commonly used qualitative method for the analysis of biofilm production is
the tube adherence method described by Christensen et al. (13). This method has
been employed by several investigators with reliable results and has been shown to
be efficient, presenting excellent sensitivity and adequate specificity, providing a
consistent and adequate diagnosis in routine applications (24, 27, 28, 38, 44, 50). In
a study comparing the reliability of three biofilm detection methods in S. aureus, a
TCP assay, a tube test and Congo red agar, Knobloch et al. (49) reported good
correlation between the tube test (13) and TCP assay for strongly biofilm-producing
strains, whereas weak producers were not safely discriminated from non-producing
strains. In contrast, screening on Congo red agar showed a low correlation with the
tube test and TCP assay (15) and was therefore not recommend for the investigation
of biofilm production by S. aureus. Similar results have been reported by Mathur et
al. (56) who evaluated three different selection methods, including the tube
adherence test, Congo red agar method and TCP assay, for the detection of biofilm
formation in clinical staphylococcal isolates. The TCP assay was highly satisfactory in
terms of biofilm-positive phenotype detection, with easy discrimination between
strong, moderate or weak producers and non-producers. The tube adherence test
presented high correlation with the TCP method, but weak producers were difficult to
discriminate from biofilm-negative isolates. In contrast, the Congo red agar method
did not presented a good correlation with either of the two other methods. The
authors concluded that the TCP method was the most sensitive and accurate
selection method showing good reproducibility for the detection of biofilm formation in
Staphylococcus and had the advantage of being a quantitative method for the study
of the adherence of Staphylococcus to medical devices.
The advances in DNA technology additionally offer the possibility of detecting genes
encoding this polysaccharide. Arciola et al. (5), investigating the presence of the icaA
and icaD genes in slime-producing staphylococcal strains from catheter-association
infections, used PCR because they considered the method simple, fast, reliable and
reported that it required minimal amounts of DNA for the detection of these genes in
S. epidermidis and S. aureus strains. Lopes et al. (55) obtained excellent results with
PCR, with the method permitting the differential detection of the ica locus between S.
aureus and S. epidermidis.
CONCLUSIONS
Bacterial infections have been one of the main causes of implant failure, especially
those caused by bacteria of normal flora which show an excellent performance in the
colonization of prosthetic and other medical devices, rendering these organisms
pathogenic to the host. This success in the colonization and development of infection
has been related to the capacity of these microorganisms to produce slime, which
also interferes with the treatment of infections, impairing the action of host immune
cells and compromising antibiotic efficacy.
Studies on biofilms have shown that S. epidermidis is the most frequently isolated
slime-producing CNS and is also the most common cause of nosocomial infections in
patients with catheters, medical implants or other invasive devices. There is still a
wide field of research on this subject; for example, what makes S. epidermidis a
successful colonizer of the human skin and mucosal membranes; how does bacterial
competition works; how bacterium-host interaction can be explained; and how
saprophytic microorganisms become pathogenic? Regarding to slime production and
the consequent biofilm formation, many aspects still need to be explored since
divergences exist in the literature. Some studies suggest that CNS infections are
mainly caused by adherent slime producers, whereas others do not confirm this
finding. Therefore, further studies about the formation of biofilms by these
microorganisms are necessary, as well as the improvement of detection methods and
treatments to prevent their production.
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Bacterial Biofilms With Emphasis On Coagulase-Negative

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Received: May 27, 2008 J. Venom. Anim. Toxins incl. Trop. Dis.

Accepted: August 19, 2008 V.14, n.4, p.572-596, 2008.

BACTERIAL BIOFILMS WITH EMPHASIS ON COAGULASE-NEGATIVE

OLIVEIRA A (1), CUNHA MLRS (1)

(1) Department of Microbiology and Immunology, Botucatu Biosciences Institute, São

ABSTRACT: In addition to their capacity to attach to surfaces, various groups of

KEY WORDS: coagulase-negative staphylococci, biofilm, slime, infection,

CONFLICTS OF INTEREST: There is no conflict.

FINANCIAL SOURCE: Fapesp.

IMPORTANCE OF BIOFILMS FOR BACTERIA

Figure 1. Example of microbial community ecology. The four central microcolonies

Biofilm Formation Mechanisms, Attachment and Substrate Effects

contains 15 to 20% diacetylated residues and is therefore positively charged, and

Genes Controlling Biofilm Formation in Staphylococcus aureus and

Slime Production by Coagulase-negative Staphylococci and Its Association

polymorphonuclear leukocyte (PMN) chemotaxis, which induces PMN degranulation,

Methods for the Detection of Slime Production in Coagulase-negative

1 ALCARAZ LE., SATORRES SE., LUCERO RM., CENTORBI ONP. Species

45 JAMES GA., BEAUDETTE L., COSTERTON JW. Interspecies bacterial

68 VUONG C., OTTO M. Staphylococcus epidermidis infections. Microbes Infect.,

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