Veterinary Microbiology 140 (2010) 360–370

Contents lists available at ScienceDirect

Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic

Review

Verocytotoxin-producing Escherichia coli (VTEC)

Mohamed A. Karmali a,*, Victor Gannon a, Jan M. Sargeant b
a
Laboratory for Foodborne Zoonoses, Public Health Agency of Canada, Canada
b
Centre for Public Health and Zoonoses and Department of Population Medicine, University of Guelph, Canada

A R T I C L E I N F O A B S T R A C T

Article history: Escherichia coli O157:H7 and other Verocytotoxin-producing E. coli (VTEC) are zoonotic
Received 17 February 2009
pathogens associated with food and waterborne illness around the world. E. coli O157:H7
Received in revised form 27 March 2009
has been implicated in large outbreaks as well as in sporadic cases of haemorrhagic colitis
Accepted 3 April 2009
and the sometimes fatal haemolytic uremic syndrome. VTs produced by these bacteria are
thought to damage host endothelial cells in small vessels of the intestine, kidney and brain
Keywords:
VTEC resulting in thrombotic microangiopathy. All VTs have the same subunit structure,
Verocytotoxin-producing Escherichia coli glycolipid cell receptor and inhibit protein synthesis. During VTEC infection, it is thought
Shiga toxin-producing Escherichia coli one or more bacterial adhesins initiates colonization and establishes intimate attachment
STEC and is responsible for the translocation of a variety of effectors which alter the structure
Zoonosis and function of host cells. VTEC are widespread in animals but ruminants are thought to be
their natural reservoir. E. coli O157:H7 colonizes the terminal colon of cattle and can be
shed in very large numbers by specific herdmates known as ‘‘supershedders’’. Faeces
containing these organisms act as a source of contamination for a variety of foods and the
environment. Many VTEC control efforts have been investigated along the ‘‘farm to fork’’
continuum including, vaccination of cattle with colonization factors, and the use of novel
antimicrobials, such as bacteriocins, chloral hydrate, bacteriophage and substances which
disrupt quorum sensing. In addition, many barriers have been developed for use in the
slaughter and food processing industry such as steam pasteurization and irradiation.
Despite these efforts many scientific, technical and regulatory challenges remain in the
control and prevention of VTEC-associated human illness.
ß 2009 Elsevier B.V. All rights reserved.

Contents

1. Introduction and historical perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361

2. The infectious agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
2.1. Population genetics of VTEC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
2.2. The genome of E. coli O157:H7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
2.3. Disease mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
2.4. The verocytotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
2.5. Antimicrobial resistance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
3. Clinical illness and epidemiology in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
3.1. Reservoirs and sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364

* Corresponding author at: Laboratory for Foodborne Zoonoses, Public Health Agency of Canada, 110 Stone Road West, Guelph, Ontario N1G 3W4, Canada.
Tel.: +1 519 822 3300; fax: +1 519 822 2280.
E-mail address: [email protected] (M.A. Karmali).

0378-1135/$ – see front matter ß 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetmic.2009.04.011
 M.A. Karmali et al. / Veterinary Microbiology 140 (2010) 360–370 361

4. VTEC in cattle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364

4.1. Non-O157 VTEC in cattle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
5. Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
5.1. Laboratory diagnosis in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
5.1.1. Rapid screening methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
5.1.2. Laboratory culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
5.1.3. Typing methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
5.1.4. Serological methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
5.2. Isolation of VTEC from animals, foods and the environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
6. Therapy and prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
6.1. Therapy in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
6.2. Immunity, vaccines and toxin blocking compounds in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
7. Prevention and control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
7.1. On-farm strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
7.2. Abattoir. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
7.3. Retail and consumer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
8. Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367

1. Introduction and historical perspective serotype, O157:H7 which was later shown to produce VT.
Also in 1983, Karmali et al. (1983) established a link
Verocytotoxin (VT)-producing Escherichia coli (VTEC), between infection by VTEC belonging to several serotypes,
also known as Shiga toxin-producing E. coli (STEC), are including O157:H7, and the haemolytic uraemic syndrome
zoonotic agents which cause a potentially fatal human (HUS), a condition of hitherto unknown cause that was first
illness whose clinical spectrum includes diarrhoea, hae- described in 1955 (Gasser et al., 1955). Karmali et al. (1983,
morrhagic colitis, and the haemolytic uraemic syndrome 1985) suggested that VT was of direct significance in the
(HUS). VTEC are of serious public health concern because of genesis of HUS because VT was present in faecal filtrates of
their association with large outbreaks and with HUS, which HUS patients, it was a common denominator among
is the leading cause of acute renal failure in children. strains of different E. coli serotypes that were associated
Although many VTEC serotype have been associated with with HUS, and significant antibody responses to VT were
human illness, the vast majority of reported outbreaks and observed in the sera of several patients. They postulated
sporadic cases of VTEC-infection in humans have been that VT was responsible for the damage to capillaries in the
associated with serotype O157:H7. Ruminants, especially glomeruli and other organs, probably through a direct
cattle, are the main reservoirs of VTEC. Infection is typically cytotoxic action on endothelial cells, to produce the
acquired through the ingestion of contaminated food or characteristic microangiopathic features of HUS.
water, through direct contact with animals, or via person-
to-person transmission. 2. The infectious agent
VTEC were first recognized in the late 1970s by
Konowalchuk and colleagues in Canada (Konowalchuk Most members of the species E. coli are commensals in
et al., 1977) who observed that culture filtrates from some the gastrointestinal tracts of animals and humans. How-
E. coli strains produced a profound irreversible cytopathic ever there are pathogenic groups that cause enteric disease
effect in Vero cells. Given that most of the strains in animals and/or humans that include VTEC, enterotoxi-
demonstrating the irreversible verocytotoxic (VT) effect genic E. coli, enteroinvasive E. coli, enteropathogenic E. coli
were from infants with diarrhoea, Konowalchuk and serotypes, and enteroaggregative E. coli (Nataro and Kaper,
colleagues speculated that VTs had a role in the genesis 1998).
of diarrhoea. In 1983, O’Brien and LaVeck, 1983 purified a Over 380 different VTEC OH serotypes have now been
HeLa-cell lethal toxin from Konowalchuk’s prototype VTEC isolated from humans with gastrointestinal disease and
strain, H.30, and showed that it was a polypeptide subunit many of these serotypes as well as others have been
toxin that was structurally and antigenically similar to recovered from animals (Beutin et al., 1998; World Health
Shiga toxin produced by Shigella dysenteriae type 1 and Organization, 1999; Karmali et al., 2003b). However,
described it as a ‘‘Shiga-like’’ toxin. among these VTEC serotypes only a limited number
The clinical significance of VTEC in human disease appear to be associated with the majority of human
remained uncertain until the early 1980s when Riley et al. disease. While these serotypes vary in frequency with the
(1983) and Karmali et al. (1983) linked these bacteria with country and year, the list commonly include members of O
two diseases of hitherto unknown etiology, haemorrhagic seogroups 26, 91, 103, 111, 113, 121, 145 and 157 (Karmali
colitis and haemolytic uraemic syndrome (HUS), respec- et al., 2003b). Among these VTEC serotypes, O157:H7 is the
tively. In a report in 1983 of two outbreaks of ‘haemor- most frequently associated with both outbreaks and
rhagic colitis’, Riley et al. (1983) found a close link between sporadic cases of severe disease. This quantitative and
disease, consumption of undercooked hamburger patties qualitative difference in disease association among VTEC
from a fast-food outlet, and infection by a ‘‘rare’’ E. coli has given rise to various classification schemes. The
362 M.A. Karmali et al. / Veterinary Microbiology 140 (2010) 360–370

simplest and most open-ended divides VTEC into E. coli 2.2. The genome of E. coli O157:H7
O157 and non-O157 VTEC. The term enterohaemorrhagic
E. coli (EHEC) (Levine et al., 1987) has been applied to VTEC The genome sequences of two epidemic strains of E. coli
serotypes that have the same clinical, epidemiological, and O157:H7, EDL 933 (Perna et al., 2001) and the Sakai strain
pathogenetic features associated with the prototype strain (Hayashi et al., 2001) have been published, and additional
E. coli O157:H7. The VTEC seropathotype (SPT) classifica- O157 genome sequencing projects are under way. The EDL
tion is based on a serotype-specific spectrum of disease 933 and Sakai genomes have a size of 5.5 Mb, compared
frequency and severity with discrete intervals ranging to the 4.6 Mb genome size of E. coli K-12 strain MG1655,
from the most pathogenic serotype E. coli O157:H7 (SPT A), and all three genomes share a common backbone of
to VTEC serotypes either frequently, occasionally or 4.1 Mb. The O157 genomes have about 1.34 million base
infrequently associated with clinical disease (SPTs B–D) pairs more than the K-12 genome while the latter contains
to VTEC that have never been associated with human about half a million bases that are absent in the O157
disease (SPT E) (Karmali et al., 2003b). genomes. In the EDL 933 genome, for example, the 1.34 Mb
of DNA which is absent in the K-12 genome, comprise 1387
2.1. Population genetics of VTEC genes that are organized into 177 gene cassettes greater
than 50 bp in size (known as O-islands) that were probably
Whittam and colleagues investigated the clonal origin acquired by horizontal transfer. Several of the latter, which
of VTEC serotype O157:H7 strains by assaying allelic are of bacteriophage origin, contain virulence genes,
variation at up to 20 different enzyme loci by multilocus including the VT-converting bacteriophages and the locus
enzyme electrophoresis. Strains of E. coli O157:H7 were of enterocyte effacement (LEE) which encodes the
found to constitute a genetically distinct clonal group that structural, accessory, and effector molecules of a type III
is only distantly related to VTEC of other serotypes secretion system (TTSS) whose actions result in character-
(Whittam et al., 1988). The O157:H7 clone was shown istic attaching and effacing lesions on enterocytes.
to be most closely related to a clone of EPEC serotype
O55:H7 that is associated worldwide with outbreaks of 2.3. Disease mechanisms
infantile diarrhoea (Whittam et al., 1993). A model has
been developed (Feng et al., 1998; Wick et al., 2005) for the Current knowledge of VTEC’s disease mechanisms is
stepwise evolution of E. coli O157:H7 from a non-toxigenic based primarily on studies with VTEC serotype O157:H7
EPEC serotype O55:H7 progenitor with the sequential (Nataro and Kaper, 1998). Following passage through the
acquisition of a VT2-encoding bacteriophage, the O157 stomach the two key virulence strategies of VTEC are VT-
antigen, and the large pO157 plasmid. Further lineages production and colonization of the bowel. While bowel
then developed with, in one, the classical E. coli O157:H7 colonization is likely a complex process requiring several
clone, the loss of the properties of sorbitol fermentation mechanisms, the only established process involves colo-
(Sor) and beta-glucuronidase (GUD) and gain of a VT1 nization of the mucosal epithelial cells of, probably, the
phage, and in another, the retention of Sor and GUD, but large bowel with a characteristic ‘‘attaching and effacing’’
the loss of motility. The later Sor+ O157:H-clone has (AE) cytopathology, which is encoded for by genes on the
become increasingly prevalent in Central Europe (Karch LEE pathogenicity island (Nataro and Kaper, 1998). The AE
et al., 1999). lesion of E. coli O157:H7 and other EHEC serotypes
Using Octamer-Based Genome Scanning, (Kim et al., resembles that associated with enteropathogenic E. coli
1999; Kim et al., 2001) showed that GUD- and Sor- E. coli (EPEC) and consists of the destruction of microvilli, an
O157:H7 strains have diverged into two distinct lineages, intimate effacing adherence of the organism to the
I and II, and that lineage I strains are more commonly enterocyte membrane, and changes in the cytoskeletal
associated with human disease than lineage II strains. structure of the enterocyte associated with the accumula-
Comparative genomic studies (Zhang et al., 2007) have tion of polymerized actin and other cytoskeletal proteins
identified significant differences in the genomic content beneath the site of bacterial attachment (Nataro and Kaper,
of the two lineages thus supporting the notion that the 1998). The LEE pathogenicty island consists of genes that
two lineages differ in virulence. Recently, (Manning et al., encode the structural and accessory components of a type
2008) have used single nucleotide polymorphism (SNP) III secretion system, the adhesin intimin and its translo-
analysis at 96 genetic loci to group 500 clinical isolates cated receptor Tir, and several translocated effector
of E. coli O157:H7 into nine clades. The workers found molecules that subvert the host cell signalling processes
that clade 8 included isolates from a 2006 California and the actin cytoskeleton and result in the attaching and
spinach-associated outbreak (Cooley et al., 2007) and effacing cytopathology (Caron et al., 2006; Coburn et al.,
that members of this clade were more frequently 2007). Recent evidence indicates that the pathogenesis of
associated with severe disease (HUS and hospitalization) VTEC/EHEC infection involves many effector molecules
than other E. coli O1577: H7 clades. Recent work (Ziebell encoded on pathogenicity islands outside the LEE (Karmali
et al., 2008) has shown that phage type 2 and a group of et al., 2003b; Deng et al., 2004; Tobe et al., 2006; Coburn
lineage intermediate strains (lineage I/II) identified by et al., 2007; Coombes et al., 2008). These so-called non-
Zhang et al. (2007) also belong to clade 8. Collectively LEE-encoded effectors (Nles) comprise as many as 32
these findings suggest that several discrete genotypes molecules distributed among 20 families encoded on
that differ in virulence exist within E. coli O157:H7 prophages, and include NleC and NleD (OI-36), NleF, NleG,
populations. and NleA (OI-71), and NleB and NleE (OI-122) (Karmali
 M.A. Karmali et al. / Veterinary Microbiology 140 (2010) 360–370 363

et al., 2003b; Deng et al., 2004; Tobe et al., 2006; Coburn 3. Clinical illness and epidemiology in humans
et al., 2007; Coombes et al., 2008).
The publication of genome sequences of E. coli O157:H7 VTEC cause sporadic infection and outbreaks in
(Hayashi et al., 2001; Perna et al., 2001) and of its plasmid humans, with the majority of reported outbreaks of VTEC
pO157 (Burland et al., 1998; Karch et al., 1998; Makino infection associated with VTEC serotype O157:H7, and
et al., 1998) has greatly expanded the list of putative with sporadic cases occurring more frequently than
virulence factors, many of which are under investigation. outbreak cases (Griffin and Tauxe, 1991). The peak age-
related frequency of VTEC-associated diarrhoea and HUS is
2.4. The verocytotoxins in young children (Griffin and Tauxe, 1991), although the
elderly also are at increased risk. The number of outbreak
Human VTEC strains elaborate at least four potent and sporadic cases of VTEC O157 and non-O157 VTEC
bacteriophage-encoded VTs: VT1, VT2, VT2c, and VT2d. typically peaks in the summer months (Griffin and Tauxe,
Each may be present alone, or in a combination of two or 1991). This may be related to factors such as increased
three different VTs (Melton-Celsa and O’Brien, 1998; consumption of barbecued hamburgers, increased shed-
Nataro and Kaper, 1998). VT1 is virtually identical to ding of VTEC by cattle, higher bacterial load in ground beef,
Shiga toxin, but is serologically distinct from members of or increased environmental contamination or survival.
the VT2. The toxins share a common polypeptide subunit In the USA between 1982 and 2002, there were 350
structure consisting of an enzymatically-active A subunit outbreaks (more than 2 cases with a common epidemio-
(32 kDa) linked to a pentamer of B subunits (7.5 kDa) logical exposure) of E. coli O157, representing over 8500
(Karmali, 1989; O’Brien et al., 1992; Paton and Paton, cases with 17% of cases resulting in hospitalization, and
1998). After binding to the glycolipid receptor, globo- 0.5% in death (Rangel et al., 2005). Estimates of the
triaosylceramide (Gb3)(Lingwood et al., 1998) on the incubation period in outbreaks of E. coli O157 have ranged
target endothelial cell, the toxins are internalized by from about 1 to 9 days, with an average of about 3–5 days.
receptor-mediated endocytosis (Sandvig and van Deurs, Attack rates in outbreaks range from 0.1% to 71% (Griffin
1996), an A subunit fragment with N-glycosidae is and Tauxe, 1991).
released into the cytosol and removes an adenine group Clinically, VTEC infection is associated with a wide
from position 2324 from the eukarytotic 28S RNA from spectrum of manifestations that include non-specific
the 60S ribosomal subunit (Endo et al., 1988). This results diarrhoea, haemorrhagic colitis, and HUS (Karmali, 1989;
in the inhibition of protein synthesis (O’Brien et al., 1992) Griffin and Tauxe, 1991). Haemorrhagic colitis (Riley et al.,
and precipitates apoptosis in eukaryotic cells (Monnens 1983) is characterized by the sudden onset of severe
et al., 1998). Cytokines, especially TNF-a and IL1-b, abdominal cramping, followed within hours by watery
which are thought to be produced by VT stimulation of diarrhoea which progresses rapidly to profuse bloody
monocytes, potentiate toxin action on endothelial cells discharge resembling lower gastrointestinal haemorrhage.
through upregulation of the cellular receptor; Gb3 Fever is typically absent or low-grade. The white blood cell
(Monnens et al., 1998). In addition, other cytokines such count may be elevated with a slight left shift. Barium
as IL6, IL8, monocyte chemotractant proeien-1, basic enema examination typically shows a ‘pseudotumour’ or
fibroblast growth factor and platelet aggregating factor ‘thumbprinting’ pattern, suggesting submucosal oedema
are also elevated in the urine (Tarr et al., 2005). The or haemorrhage, usually in the ascending or transverse
eventual outcome of VT action on endothelial cells is colon. Colonoscopy may show erythema, haemorrhage,
thrombotic microangiopathy in the renal glomeruli and and oedema in the ascending and proximal transverse
other organs. colon. An inflammatory exudate may also be seen. Most
patients recover fully. Severe illness may be complicated
2.5. Antimicrobial resistance by HUS, and rarely, by bowel stricture.
Haemolytic uraemic syndrome (HUS) (Karmali et al.,
Although treatment of E. coli O157 infections with 1985; Karmali, 1989; Tarr et al., 2005) is defined by the
antibiotics is generally contra-indicated, numerous studies three features: acute renal failure, thrombocytopenia, and
have been performed to evaluate antimicrobial resistance microangiopathic haemolytic anaemia. HUS associated
patterns. Antimicrobial resistance is common in E. coli with VTEC infection, also referred to as ‘‘classical’’ or D+
O157 and other VTEC serotypes, including multiple drug (diarrhoea associated) HUS, is the most common form of
resistance to streptomycin, sulfisoxazole, and tetracycline HUS. Other uncommon varieties include ‘atypical’ HUS (in
(Kim et al., 1994; Mora et al., 2005), and there is some which the prodrome is typically a respiratory illness),
evidence that resistance may be increasing over time (Kim childhood forms that are inherited, and adult forms that
et al., 1994; White et al., 2002). Although some studies occur in association with pregnancy, oral contraceptive
have shown a higher rate of antimicrobial resistance in E. usage, malignant hypertension, and various chronic ill-
coli O157 bovine strains compared to human strains (Meng nesses. VTEC-associated HUS, which has its highest
et al., 1998; Mora et al., 2005), Schroeder et al. (2002) incidence in children, presents, typically, a few days after
reported similar prevalence of resistance among these host the onset of an acute diarrhoeal ‘prodromal’ illness. The
sources. There is also evidence that antimicrobial resis- incidence of HUS in outbreaks of VTEC serotype O157:H7
tance patterns vary among phage types (Mora et al., 2005; infection has been typically about 7–10% although a much
Ziebell et al., 2008) and between lineages (Ziebell et al., higher incidence of HUS has been reported in some
2008). outbreaks (Carter et al., 1987; Manning et al., 2008). HUS
364 M.A. Karmali et al. / Veterinary Microbiology 140 (2010) 360–370

may also occur as a complication of VTEC-associated et al., 2003). It has been proposed that faecal shedding may
urinary tract infection (Tarr et al., 2005). The severity of be either transient, wherein cattle shed the pathogen
HUS varies from an incomplete and/or clinically mild briefly following exposure, or more long term, resulting
condition to a severe and fulminating illness with multiple from colonization of the gastrointestinal tract (Rice et al.,
organ involvement including the gastrointestinal tract, 2003). There is evidence to suggest that a proportion of
heart, lungs, pancreas, and central nervous system (CNS) positive animals may shed E. coli O157 at much higher
(Upadhyaya et al., 1980; Richardson et al., 1988; Tarr et al., concentrations than others—the so-called super-shedders
2005). The renal histopathology is characterized by (Naylor et al., 2005a). High concentration of E. coli O157 in
glomerular thrombotic microangiopathy with endothelial faeces or prolonged shedding may result from colonization
cell swelling and subendothelial deposits (Upadhyaya in the terminal rectum (Naylor et al., 2005a). Super-
et al., 1980; Richardson et al., 1988; Tarr et al., 2005). shedders are thought to have a substantial impact on the
Similar thrombotic microangiopathy may be seen in the on-farm epidemiology of E. coli O157:H7; while they
capillaries of the brain, gastrointestinal tract, and other constitute a small proportion of cattle, it has been
organs (Upadhyaya et al., 1980; Richardson et al., 1988; estimated that they may be responsible for over 95% of
Tarr et al., 2005). the E. coli O157:H7 bacteria shed by cattle (Naylor et al.,
2005a).
3.1. Reservoirs and sources
4.1. Non-O157 VTEC in cattle
Cattle are the main animal reservoir of VTEC strains
implicated in human disease, and foods of bovine origin, Research has also been conducted to determine the
especially undercooked ground beef patties and unpas- prevalence and epidemiology of non-O157 VTEC in
teurized milk, constitute major sources of human infection ruminants. Non-O157 VTEC strains with the potential to
(Griffin and Tauxe, 1991; Rangel et al., 2005). In studies of cause disease in humans, such as O26, O103, O111, and
retail ground beef in North America, the prevalence of O145, have been isolated from healthy cattle (Jenkins et al.,
VTEC ranged from 9% to 36.4%, with E. coli O157 isolated 2003; Cobbold et al., 2004; Hussein and Sakuma, 2005;
from 0% to 3.7% of the samples tested (Doyle and Schoeni, Renter et al., 2005, 2007; Pearce et al., 2006; Murphy et al.,
1987; Read et al., 1990). 2007).
Other sources linked to sporadic infection and out-
breaks of illness in humans (reviewed in (Rangel et al.,
5. Diagnostics
2005)). Rangel et al. (2005) include lettuce, alfalfa sprouts,
radish sprouts, spinach, and apple cider, consumption or 5.1. Laboratory diagnosis in humans
recreational use of water, direct contact with cattle or
animal excreta, attendance at agricultural fairs, and 5.1.1. Rapid screening methods
recreational use of pastures. Secondary transmission from Several rapid screening methods are now available
infected persons, for example, within families, day-care including the polymerase chain reaction (PCR) (Nataro and
centres, and health-care institutions is well-recognized Kaper, 1998) to detect VTEC-specific DNA sequences and
(Spika et al., 1986; Carter et al., 1987; Rowe et al., 1993; various immunospecific methods to detect VT antigen in
Rangel et al., 2005). faeces, either directly or after broth-culture enrichment, or
in bacterial culture filtrates (Nataro and Kaper, 1998).
4. VTEC in cattle
5.1.2. Laboratory culture
A growing number of non-O157 VTEC serotypes have Laboratory diagnosis of VTEC infection involves the use
been isolated from animals, and many of these serotypes of a selective and differential medium, such as sorbitol
have been associated with human disease (Beutin et al., MacConkey agar (March and Ratnam, 1986), to isolate
1998; World Health Organization, 1999). The majority of VTEC serotype O157:H7, from patient stools. Unlike most
VTEC strains implicated in human disease, including E. coli other E. coli, most strains of serotype O157:H7 do not
O157:H7, do not appear to be associated with clinical ferment sorbitol within 24 h of incubation. However,
disease in cattle (Mohammed et al., 1985; Naylor et al., SMAC medium is limited because: (1) it is not selective
2005a). E. coli O157 is common in cattle; prevalence with respect to other enteric bacteria and therefore small
estimates in North American beef cattle range from 10% to numbers of the organism will be missed, and (2) it cannot
28% and most, if not all, farms have positive animals at be used for most sorbitol-fermenting E. coli which includes
some time (Gannon et al., 2002; Sargeant et al., 2003; most non-O157 VTEC and the E. coli O157:H7:NM clone
Naylor et al., 2005a). Studies of beef and dairy cattle in prevalent in central Europe (Karch et al., 1999).
Europe have reported similar prevalence estimates (Heu-
velink et al., 1998; Blanco et al., 2003; Gunn et al., 2007; 5.1.3. Typing methods
Alonso et al., 2007). Serological evidence from cattle in the Typing and subtyping of strains is a valuable aid to
USA suggests that most range-fed beef calves have been identify outbreaks, detect them at an earlier stage, trace
exposed to E. coli O157 by the time of weaning (Laegreid their sources, and increase the specificity of case defini-
et al., 1999). Within pen prevalence in feedlot cattle shows tion (Nataro and Kaper, 1998). The main methods
temporal peaks with 80% or more of the cattle shedding E. currently used for typing and subtyping include pulsed-
coli O157 at some points in time (Smith et al., 2001; Khaitsa field gel electrophoresis (PFGE) (Gerner-Smidt et al.,
 M.A. Karmali et al. / Veterinary Microbiology 140 (2010) 360–370 365

2006), phage-typing (Ahmed et al., 1987) and MLVA control, and fluid and electrolyte balance. The role of
(Hyytia-Trees et al., 2006). PulseNet, USA, established in antimicrobial agents remains controversial and they
1996, is a network of federal and state public health should probably be avoided (Tarr et al., 2005). Anti-
agencies that use a standardized PFGE protocol to subtype motility drugs, opiates, and non-steroidal anti-inflamma-
E. coli O157 (Gerner-Smidt et al., 2006). The PulseNet tory agents also should not be employed (Cimolai et al.,
network has been implemented in Canada, Europe, the 1994; Tarr et al., 2005).
Asia Pacific region, and Latin America who, along with the
USA, work together to share molecular epidemiological 6.2. Immunity, vaccines and toxin blocking compounds in
information (Gerner-Smidt et al., 2006). humans

5.1.4. Serological methods The peak age-related incidence of HUS is in childhood

When culture or rapid screening methods for VTEC are (Karmali, 1989). The incidence then declines with age, but
negative and a specific diagnosis is considered necessary, appears to rise again during old age (Griffin et al., 1988).
serological diagnosis may be helpful. Antibody responses Seroepidemiological studies show that the age-related
to the O157 lipopolysaccharide correlate well with recent frequencies of immunoglobulin G antibodies to VT1 and
infection by E. coli O157:H7, and are thus useful in VT2 are inversely related to the age-specific incidence of
diagnosing VTEC infection associated with this particular HUS (Karmali et al., 2003a), suggesting strongly that the
serotype (Barrett et al., 1991; Chart et al., 1991; Reymond presence of such antibodies is associated with anti-toxin
et al., 1996). VTEC serotype O157 infections also may be immunity.
diagnosed by assaying saliva for serological response to Despite in vitro (Richardson et al., 1992; Bielaszewska
O157 (Ludwig et al., 2002a). et al., 1997; Ludwig et al., 2002b) and in vivo (Karmali et al.,
2003a) evidence that antibody responses to the VTs are
5.2. Isolation of VTEC from animals, foods and the associated with protective immunity, little progress has
environment been made in developing VT toxoid immunization for
human populations. However, an E. coli O157 O-specific
VTEC serotypes associated with diarrhoea in calves are polysaccharide conjugate vaccine, in which the O-specific
isolated using routine bacteriological techniques used in polysaccharide is conjugated to Pseudomonas aeruginosa
the isolation of other enteropthogenic E. coli. VT produc- exotoxin A, was found to be safe and immunogenic in
tion can be assessed through tissue culture assays or young children (Ahmed et al., 2006).
possession of VT and other virulence genes by PCR. Efforts have also been made to block VT-mediated
Bacteriological testing of faeces for E. coli O157 in cellular damage through the enteric and systemic admin-
animals generally is conducted for research purposes, as E. istration of multivalent toxin receptor analogues (Karmali,
coli O157 is not associated with clinical illness. Culture 2004). While a number of compounds have been devel-
methods to identify the presence or absence of faecal oped that show promise in experimental animals the
shedding typically involve the use of an enrichment broth, blocking agents must be delivered before VT has bound to
immunomagnetic separation, and the use of selective and and been internalized by host cells.
differential media such as sorbitol MacConkey agar
containing cefixime and tellurite, with latex agglutination 7. Prevention and control
used to detect the presence of the O157 antigen (Moxley,
2003). Optimum control of VTEC needs to involve all stages of
Similar procedures are used for the isolation of E. coli food production, from farm to fork. Quantitative risk
O157:H7 water and soils samples. Testing of specific food assessments and simulation models are available which
products, such as ground beef, may be part of a regulatory describe stages in the farm-to-fork continuum that
program (http://www.fsis.usda.gov/PDF/Mlg_5A_01.pdf). contribute to an increased risk of foodborne illness and
allow potential control measures to be assessed (Cassin
et al., 1998; Jordan et al., 1999; Ebel et al., 2004).
6. Therapy and prevention

6.1. Therapy in humans 7.1. On-farm strategies

Most patients with uncomplicated VTEC infection Controlling VTEC at the farm level would be highly
recover fully with general supportive measures (Griffin desirable not only to reduce the risk of contamination of
et al., 1988; Karmali, 1989). Tarr et al. (2005) recommend beef carcasses at slaughter, but also to reduce human
that patients with confirmed VTEC infection and bloody illness associated with direct animal contact, environ-
diarrhoea should be initially admitted to hospital to mental contamination, contamination of crops following
observe for any signs of progression to HUS and also to manure application and contamination of water used for
limit spread of the infection to the community. There are irrigation, recreation and as a drinking water source. The
no specific therapies for HUS and patients should, as far as large number of different VTEC serotypes isolated from the
possible, be managed by specialists with experience in the faeces of cattle and other ruminant species limits hope that
management of renal failure, and of any other complica- a single specific strategy will resolve the issue. Therefore,
tions such as CNS symptoms, with strategies, including most studies on the control of VTEC on farms have been
dialysis, to ensure optimal renal perfusion, blood pressure directed toward serotype E. coli O157:H7.
366 M.A. Karmali et al. / Veterinary Microbiology 140 (2010) 360–370

Identifying and modifying management strategies potential for development of antibiotic resistance and
associated with faecal shedding could reduce animal neomycin has not been approved for use in cattle as an
exposure and transmission. An advantage of this approach intervention for E. coli O157. Inoculation studies and a
is that it tends to target management practices related to randomized control trial evaluating sodium chlorate found
faecal-oral transmission, and thus could have an impact on a significant reduction in E. coli O157 when used in the feed
enteric pathogens in general, rather than specifically or water (reviewed in (Sargeant et al., 2007)). Bacter-
VTECs. However, numerous studies in dairy and beef iophages active against E. coli O157:H7 have been
cattle have investigated associations between manage- identified in ruminants (Kudva et al., 1999). Research
ment practices and E. coli O157 and have not identified has shown that expression of the LEE genes is turned on in
consistent associations with faecal shedding of E. coli O157 response to host epinephrine and norepinephrine and
(Herriott et al., 1998; Garber et al., 1999; Smith et al., 2001; related bacterial molecules. This information is being used
Sargeant et al., 2004). to develop a new class of antimicrobial agents which bind
Another option for controlling E. coli O157 in cattle is to to the receptor, QseC, and inhibit this signalling quorum
increase herd resistance to infection. Examples of inter- sensing pathway and animal colonization (Rasko et al.,
ventions using this approach include probiotics, vaccina- 2008). The effectiveness of bacteriophages in natural
tion, antimicrobials, sodium chlorate, and bacteriophages conditions has been extremely variable and more research
(reviewed in (Callaway et al., 2004; Lonergan and if needed to determine whether this is a viable interven-
Brashears, 2005; Sargeant et al., 2007)). tion strategy in ruminants (Callaway et al., 2004).
Probiotics are commensal bacteria fed to reduce At present, on-farm control programs are voluntary and
pathogenic bacteria in the gastrointestinal tract through tend to consist of general good production practices that
competitive inhibition (Fuller, 1989). Randomized con- are not targeted specifically at VTEC control (Haines, 2004).
trolled trials using these products have produced variable
results (reviewed in (Sargeant et al., 2007)). In small 7.2. Abattoir
groups of feedlot cattle using a combination of L.
acidophilus NP51 and P. freudenreichii significant reduc- Contamination of carcasses with VTEC can occur when
tions in the prevalence of E. coli O157 were reported, gut contents or faecal matter come into contact with meat
although the same combination was not significantly surfaces. There is a significant correlation between the
associated with reduced faecal prevalence of the pathogen prevalence of E. coli O157 in faeces and hides and carcass
in a commercial feedlot. This probiotic combination is contamination (Elder et al., 2000). Cross-contamination
currently licensed in the United States of America as between carcasses may occur during processing (Edwards
Bovamine Culture Complex Probiotic (Nutrition Physiol- and Fung, 2006). The current trend for concentration and
ogy Corp). rapid distribution systems in the food-processing sector
Progress in understanding the biology of VTEC coloni- has resulted in plants that produce vast quantities of
zation in cattle may open up avenues for immunizing product on a daily basis with distribution to large
cattle against VTEC (Stevens et al., 2002). The LEE geographic areas (Altekruse et al., 1997). This can result
pathogenicity island is responsible for the colonization in large outbreaks when coupled with improper cooking
of the terminal rectum of cattle by VTEC serotype O157:H7 and handling procedures at the retail or consumer level.
(Low et al., 2005; Naylor et al., 2005b). Vaccines using LEE- However, such concentration of volume can also facilitate
encoded TSS proteins (Tir, EspA, and EspB) are being the implementation of effective control strategies, result-
evaluated for use in cattle. In a randomized controlled trial, ing in the production of large volumes of high-quality, safe
Potter and colleagues reported a significant reduction in food.
the faecal prevalence of E. coli O157:H7 when the vaccine Food safety in the processing sector is based on a hazard
was administered 3 times (Potter et al., 2004). Similarly, analysis and critical control point (HACCP) approach
Peterson and colleagues have reported that a two-dose (Hulebak and Schlosser, 2002). This approach is not
vaccination regime resulted in feedlot cattle being 98.3% specific to the control of VTEC, but broadly addresses
less likely to have their terminal rectal mucosa colonized biological, chemical, and physical hazards. HACCP pro-
by E. coli O157:H7 (Peterson et al., 2007a,b). In contrast, grams in the processing sector include microbial testing of
VanDonkersgoed and colleagues did not find a significant product for indicator bacteria and/or specifically for E. coli
reduction in E. coli O157:H7 prevalence following vaccina- O157. The specific methods used in the processing sector
tion of feedlot cattle (Van Donkersgoed et al., 2005). to meet HACCP requirements vary, but fall into the
However, the prevalence of E. coli O157:H7 in the later categories of hide interventions, carcass interventions, or
study was low. A further consideration in EHEC vaccine trim interventions (Koohmaraie et al., 2005). Interventions
development is the degree of cross-protection provided by include the use of chemicals or antimicrobial products,
an E. coli O157 vaccine against colonization of cattle by knife trimming, vacuuming, washing, stream pasteuriza-
other EHEC serotypes tion, multiple hurdle interventions, gamma irradiation,
Antimicrobial products may directly affect bacterial low-dose low-penetrating radiation, and good manufac-
populations in the gastrointestinal tract. Neomycin sulfate turing practices in the processing line (Koohmaraie et al.,
has been shown to significantly decrease faecal shedding 2005; Edwards and Fung, 2006). While E. coli O157:H7 is
of E. coli O157:H7 in cattle (Elder et al., 2002; Ransom, highly sensitive to gamma radiation, consumer acceptance
2004; Woerner et al., 2006). However, the use of antibiotics of irradiated products has posed a problem. Recent
in food animals is controversial due to concerns about the marketing of irradiated chicken in the US has shown that
 M.A. Karmali et al. / Veterinary Microbiology 140 (2010) 360–370 367

consumer attitudes are changing and that irradiation may quantitative. Thus, interventions that reduce pathogen
be a viable approach to reduce VTEC contamination of concentration, but do not eliminate shedding, may be
retail foods (Basaran et al., 2004; Oteiza et al., 2005). effective but not observed as such using prevalence or
There is some evidence from temporal analyses that incidence data. Development of valid methods to quantify
intervention strategies may be reducing the occurrence of the concentration of E. coli O157:H7 in cattle faecal
E. coli O157:H7 in beef. Although outbreaks remain samples would aid in evaluating on-farm control strate-
common, the median number of cases per outbreak and gies.
HUS and case-fatality rates declined in the USA between Most of the regulatory focus on the prevention of VTEC
1982 and 2002 (Rangel et al., 2005). The incidence of E. coli has been in the processing sector. HACCP is mandatory in
O157 in the USA decreased 42% between 1996 and 2004 some countries and other countries are moving towards
(Centers for Disease Control, 2005). However, the inci- mandatory implementation. Internationally, food inspec-
dence of VTEC O157 infections in humans increased in tion systems are under increasing trade pressure to comply
2005 and again in 2006 (Anon, 2007). In addition, E. coli with international standards such as the ISO 9000 series.
O157:H7 outbreaks associated with fruits, raw edible Many nations and municipalities have guidelines and
vegetables and drinking and recreational water remain regulations setting standards for internal cooking tem-
causes for concern. peratures and sanitary measures at the retail level, that,
when properly enforced, also serve as important barriers to
7.3. Retail and consumer human infection.
Public health surveillance of VTEC infections can play
In many countries public health regulations dictate an important role in devising and implementing control
minimal cooking temperatures and outline good food measures. Whereas reporting of human cases of VTEC
hygiene practices in restaurants and commercial food infection (or only O157:H7 infection) to public health
processing facilities. In the home, high-risk food-handling, authorities is mandatory in some countries, it is not so in
preparation, and consumption practices are common, others. Consistent reporting by all countries would make a
revealing the need for food safety education of consumers vital contribution to international surveillance and pro-
(Redmond and Griffith, 2003). Programs aimed at con- mote global strategies to control VTEC-associated infec-
sumer food-safety education, such as FightBacTM tions and to evaluate the public health significance of
(www.fightbac.org), have been developed. These programs existing and emerging VTEC serotypes.
emphasise the need for frequent washing of hands and
surface areas in contact with food, separation of foods Conflict of interest
during storage and preparation to avoid cross-contamina-
tion, proper cooking temperatures to kill pathogens that None.
may be present, and prompt refrigeration of purchased and
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