Pathogenic Escherichia coli

N Fegan and KS Gobius, CSIRO Animal, Food and Health Sciences, Werribee, VIC, Australia
GA Dykes, Monash University, Bandar Sunway, Malaysia
r 2014 Elsevier Ltd. All rights reserved.

Glossary Fimbriae A thin surface appendage produced by many

Bacteremia The presence of bacteria in the
blood.
Bacteriophage Any virus that can infect bacteria.
Epithelium A tissue that lines surfaces and cavities in
the body.
bacteria.
Pathogenicity island A horizontally transferred group of
genes that confer virulence to a bacterium.
Plasmid A deoxyribonucleic acid molecule that is not part
of the chromosome and can replicate independently of it.

Introduction organisms to the intestines and, in some cases, the production

Most Escherichia coli live as harmless commensals in the in-
testines of warm-blooded animals, but certain types can cause
disease in their hosts; these latter organisms are referred to as
pathogenic E. coli (PEC). The diseases that might be caused by
PEC are varied and depend on the site of infection.
Extraintestinal PEC (ExPEC) cause diseases outside the gas-
trointestinal tract, the most common of which are urinary tract
infections resulting from infection with uropathogenic E. coli.
ExPEC have also been associated with sepsis, meningitis, pneu-
monia, and wound infections. The sources of ExPEC are believed
to be the intestines of humans, although evidence is emerging
that animals might be a source of at least some ExPEC.
Of greater current concern with respect to food are E. coli
that cause diseases of the gastrointestinal tract. Gastroenteritis
can be caused by a variety of different types of PEC. The major
types are described in Table 1. Symptoms range from diarrhea,
which can be either acute or chronic, to more severe compli-
cations that can result in death. These pathogenic types are
grouped on the basis of the diseases they cause and on specific
virulence factors, which are involved in the disease process.
Those factors that are known to be important in gastrointest-
inal diseases tend to be associated with attachment of the
of toxins.
Enteroinvasive E. coli are closely related to Shigella spp.
They cause a disease similar to shigellosis, with invasion of the
intestinal epithelium and inflammation of the intestines. The
genes that confer invasiveness are carried on a plasmid referred
to as pInv. Enterotoxigenic E. coli (ETEC) are the major cause
of traveler's diarrhea. Infection is initiated through attachment
to and subsequent colonization of the intestines. Attachment
involves the production of fimbriae called colonization fac-
tors. Once the intestines have been colonized, ETEC produce
heat-stable (ST) and heat-labile toxins, which cause a watery
diarrhea that might be mild, short lived, and self-limiting or
severe and possibly fatal, as in patients with cholera.
Enteroaggregative E. coli (EAEC) have been associated with
persistent diarrhea (i.e., lasting more than 14 days) in infants
in developing countries, but EAEC can also cause acute diar-
rhea in adults and infants and are the second greatest cause of
traveler's diarrhea. This group of E. coli is diverse with respect
to virulence factors but generally display aggregative adherence
(AA), i.e., the cells attach to the gut wall and form layers with
one another in a stacked-brick appearance. Most EAEC pro-
duce AA fimbriae, which are involved in AA and can also be
important in attachment to the host tissues. EAEC can also

Table 1 The diseases and major virulence factors of pathogenic Escherichia coli associated with intestinal illnesses

Type Time to illness Disease Major virulence factors

Attachment Toxins

Enteroinvasive E. coli 8–24 h Watery diarrhea and dysentery Invasion-related plasmid

Enterotoxigenic E. coli
(ETEC)
Enteroaggregative E. coli
(EAEC)
Enteropathogenic E. coli
Enterohemorrhagic
E. coli and Shiga
toxin-producing E. coli
with blood and mucus
8–44 h Watery diarrhea and traveler's
diarrhea
8–48 h Watery diarrhea, acute and
persistent diarrhea, and the
second greatest cause of
traveler's diarrhea after ETEC
12–36 h Watery diarrhea with mucus
and infant diarrhea
3–4 days Diarrhea, bloody diarrhea,
hemolytic uremic syndrome,
and death
(pInv)
Colonization factors Heat labile and heat stable
Aggregative adherence EAEC heat-stable enterotoxin
fimbriae
Locus of enterocyte
effacement (LEE) and
bundle-forming pilus
LEE and others Shiga toxins (Stx1 and Stx2)

Encyclopedia of Meat Sciences, Volume 2 doi:10.1016/B978-0-12-384731-7.00035-0 357

358 Microbiological Safety of Meat | Pathogenic Escherichia coli
produce the enteroaggregative ST toxin, which is similar to ST
found in ETEC, and can possess genes for a variety of other
possible virulence factors.
Enteropathogenic E. coli (EPEC) are a leading cause of
diarrhea in infants in developing countries. EPEC cause at-
taching and effacing (A/E) lesions in the intestines as a result
of the intimate attachment that occurs between the bacteria
and the host cells. The cell components required for this in-
timate attachment are encoded by genes located on a patho-
genicity island termed the locus of enterocyte effacement
(LEE). Most EPEC can also produce fimbriae called bundle-
forming pili, which are responsible for bacterium–bacterium
attachment and can be involved in the initial attachment of
EPEC to intestinal cells.
Enterohemorrhagic E. coli (EHEC) can cause bloody diar-
rhea (hemorrhagic colitis) or more severe disease, such as
hemolytic uremic syndrome (HUS) that can lead to death.
EHEC are a subset of the broader group of Shiga toxin-pro-
ducing E. coli (STEC) that can produce Shiga toxins (Stx).
EHEC is the term given to STEC that have caused clinical
disease, although STEC is often used more broadly when re-
ferring to EHEC. The term verotoxin-producing E. coli is an
earlier synonym of STEC that is still sometimes used in the
literature and refers to the toxicity of these strains against Vero
monkey kidney cells in vitro. In addition to Stx, most EHEC
also carry LEE or other adherence factors that assist in colon-
ization of the host's intestines. EHEC of serotype O157:H7
have been responsible for large foodborne outbreaks, many of
which have been linked to the consumption of meat and meat
products. For this reason, in 1994, E. coli O157:H7 was de-
clared an adulterant of ground beef by the Food Safety and
Inspection Service (FSIS) of the United States Department of
Agriculture (USDA). The term STEC has been used to refer to
this group of strains in its broader sense throughout most of
this article as opposed to the more specific EHEC.
Other E. coli that are able to cause diarrhea include diffusely
adherent E. coli, cell-detaching E. coli, and cytotoxic necrotizing
E. coli, but little is known about the significance and impact of
these E. coli as human pathogens or their relationships with
animals used for meat production. Many of the virulence
factors of PEC are mobile and can move between strains of
E. coli to produce new pathogenic types. Thus, the E. coli O104:
H4 outbreak in Germany and other parts of Europe during
2011, which affected more than 4000 people, with more than
900 cases of HUS and at least 50 deaths, was caused by a strain
of serotype O104:H4 that was essentially an EAEC which had
acquired Stx and multiple antibiotic resistances. New com-
binations of virulence factors in E. coli are most likely to lead
to new pathogenic types arising in the future.
All gastrointestinal PEC can be spread from human to
human, either directly or through food and water that has
been contaminated by humans. In addition to human sources,
STEC have animal reservoirs and can be transmitted to
humans through consumption of foods made from con-
taminated meat or by direct contact with contaminated ani-
mals. Ruminants, particularly cattle, are the major reservoir of
STEC, and milk and meat have been implicated in outbreaks.
For this reason the focus of the remainder of this article
is on STEC as they are the most significant PEC associated
with meat.
Clinical Significance
Human infection with STEC can cause a spectrum of diseases
ranging from asymptomatic carriage to death. Worldwide, E.
coli O157:H7 is the most common cause of HUS. However, in
several outbreaks, the E. coli O157:H7 outbreak strain was
isolated from stools of asymptomatic persons as well. The
normal signs of E. coli O157:H7 gastroenteritis include ab-
dominal pain, nonbloody diarrhea followed by bloody diar-
rhea after 1–4 days, absence of fever, and five or more bowel
movements per day. HUS develops 5–13 days after initial
diarrhea in 10–15% of patients, most commonly children who
are less than 5 years of age. HUS is characterized by an acute
onset of kidney damage. Additional severe extrarenal compli-
cations, including neurological impairment, increased pan-
creatic enzymes, edema, necrosis of the colon wall, and
myocardial and central nervous system damage, can result
from HUS and often lead to death. STEC infections generally
do not result in bacteremia, indicating that the systemic
complications of HUS are attributable to circulating Stx. The
cell surface receptor glycosphingolipid globotriaosylceramide
(Gb3) is present on cells in the human kidney. Stx binds to
Gb3 and is internalized by endocytosis, resulting in protein
synthesis inhibition through disruption of the 28S ribosomal
subunit.
Molecular Aspects of Pathogenicity
Production of Stx is essential for the pathogenesis of bloody
diarrhea and HUS caused by both E. coli O157:H7 and non-
O157 STEC. Two main Stx types are encoded by bacterio-
phages integrated into the chromosomes of STEC/EHEC
strains. Stx1 is closely related to the Stx of Shigella dysenteriae,
but Stx2 shows only 55–60% deoxyribonucleic acid (DNA)
and amino acid similarity to Stx1. Although a variety of genetic
subtypes of stx1 and stx2 genes have been described, expression
of the stx2 gene is most strongly associated with significant
clinical manifestation of bloody diarrhea and HUS. In add-
ition to Stx production, all E. coli O157:H7 and most non-
O157 STEC (e.g., serotypes O26, O45, O103, O111, O121,
and O145) also encode the LEE pathogenicity island. How-
ever, HUS is also caused by some LEE-negative serotypes (e.g.,
O91, O104, and O113), indicating that LEE is not essential for
HUS causation and that additional virulence factors might
contribute to the severe illnesses caused by these serotypes.
There are phylogenetic and geographic variations among E.
coli O157:H7 strains in the potential for disease causation and
the severity of the illness they cause. Three lineages of E. coli
O157:H7, LI, LII, and LI/II, have been identified. Although all
lineages are found in the bovine O157 reservoir, LI and LI/II
are overrepresented among human O157:H7 isolates. LI pre-
dominates in North America and Japan, whereas LI/II is most
prevalent in the Netherlands. Stx bacteriophage association
with the different lineages are also frequently observed: LI
isolates carry stx1 and stx2 bacteriophages; LII isolates carry stx2c
bacteriophage; and LI/II isolates carry stx2 alone or stx2 and
stx2c bacteriophages. Eight virulence clades were identified
among North American human isolates and these clades were
correlated with historical E. coli O157:H7 outbreaks occurring
 Microbiological Safety of Meat | Pathogenic Escherichia coli
in the United States. Infections caused by clades 1, 2, and 3
(also typed as LI) isolates from 1982 and 1993 hamburger
outbreaks were less severe than clade 8 (also typed as LI/II)
isolates from the 2006 spinach and lettuce outbreaks.
Ecology
STEC shed in animal feces can remain viable in the environ-
ment for up to several months. Animal manure and wastes
that carry STEC can enter water sources. Contaminated water
used for irrigation contaminates fresh produce. Swimming in
or drinking contaminated water can lead to human illness, as
can direct contact with animals on farms or at petting zoos.
Most of what is known about the ecology of STEC comes
from the study of E. coli O157:H7. Much less is known about
STEC of other serotypes. The major reservoir of E. coli O157:
H7 are ruminant animals, particularly cattle. Escherichia coli
O157:H7 has also been isolated from sheep, deer, and goats.
Nonruminant animals, such as pigs, horses, rabbits, birds, and
flies, have also been found to carry E. coli O157:H7 on occa-
sion. Escherichia coli O157:H7 does not cause disease in cattle.
It is found in the intestines of healthy animals and, if so, is
shed in feces, which commonly contaminate animal hides.
The organism is also found in the mouths of animals. The
prevalence of E. coli O157:H7 in herds of cattle can vary over
time. In the Northern Hemisphere, the highest prevalence is
observed in the warmer summer months. Not all animals
within a herd may shed E. coli O157:H7 at the same time, and
those animals which do, mostly shed only very low numbers.
Occasionally, an animal may shed high numbers (4 10 000
cfu g 1) of E. coli O157:H7. These animals, which have been
termed super shedders, are thought to pose the greatest risk for
meat contamination during slaughter and processing. The
reasons why some animals on occasion shed high numbers of
E. coli O157:H7 are unknown. Various means of preventing
shedding of E. coli O157:H7 have been proposed with a view
to reducing contamination of meat and meat products.
However, no method has as yet been shown to have consistent
and substantial effects in practice.
STEC generally are commonly found in ruminant and
other animals. STEC other than E. coli O157:H7 can account
for more than half of STEC illnesses in humans. Non-O157
STEC are transmitted to humans in the same manners as are
E. coli O157:H7.
Presence and Survival on Meat
Most published data on STEC in meat and meat products are
for the prevalence of E. coli O157:H7. The prevalence of E. coli
O157:H7 on carcasses and on retail product is generally less
than 0.3%. However, some studies have reported prevalence
on carcasses and retail product 430% and 43%, respectively.
To cause foodborne disease, STEC need to survive and
possibly grow on meat products. There is very little evidence to
suggest that PEC on meat products behave much differently
from E. coli generally. On raw chilled or frozen meats, E. coli
O157:H7 and other STEC can survive for extended periods but
do not grow. Although some decrease in numbers of the
359
pathogen might occur during storage under refrigeration, this
is not usually sufficient to eliminate them. Their presence on
refrigerated products is of concern because of their relatively
low infectious doses and because cross-contamination of other
food products may occur. STEC is eliminated in properly
cooked products. The survival and growth of STEC in pro-
cessed meats is dependent on factors such as the pH, water
activity, and the presence of preservatives in the products.
Control of these qualities in some products, such as certain
types of salami, is critical for preventing the growth of these
pathogens. Although it has been suggested that E. coli O157:
H7 are more resistant to acidic conditions than other E. coli,
the available evidence indicates that this is not the case.
Control of PEC growth can generally be achieved by the same
measures that control the growth of generic E. coli (see Section
‘Control’ below).
Detection
There are many challenges associated with the detection and
isolation of PEC in meat and meat products. These include low
prevalence, low numbers if present, and the presence of other
bacteria, including non-PEC in meat and meat products.
Sampling plans and test methods have been progressively
developed to counter these challenges. An example of this is
the microbiological testing program for E. coli O157:H7 in
beef developed by the USDA FSIS. This was introduced in
1994 when US legislation categorized E. coli O157:H7 as an
adulterant of ground beef. There have been several changes
made since then to improve the sensitivity of the methods, for
example, adoption of improved detection methods and in-
creasing the amount of meat tested from 25 to 375 g. The
general approach for detection of PEC in meat involves en-
richment, screening to determine whether the target E. coli is
present, and then isolation and confirmative identification of
the pathogen. Enrichment is used to increase the numbers of
PEC; however, this also increases the numbers of other bac-
teria. Selective agents, such as antimicrobials or other chem-
icals, may be added to inhibit competing bacteria. After
enrichment, samples may be screened, using molecular
methods (such as polymerase chain reaction) or immuno-
logical methods (such as enzyme immunoassays), for the
presence of virulence factors, such as Stx and the E. coli at-
taching and effacing gene (eae) component of LEE, and specific
serotypes, such as O157, O26, and O111.
If a molecular screening test is positive, the specific E. coli
of interest must be isolated and confirmed. Within a single
sample, the target serotype and individual virulence factors
might each reside in a different E. coli. For example, a sample
might test positive for Stx (or stx), eae, or LEE and serotype
O111 on screening, implying that non-O157 STEC of ser-
otype O111 is present in the sample. However, the stx gene
might be carried in a nonpathogenic STEC, the eae gene in a
different E. coli, and the O111 strain might be in yet another
E. coli. In this case, the sample is negative for non-O157 STEC
of serotype O111. The isolation of PEC from enriched sam-
ples can be difficult because of the presence of large numbers
of other bacteria. Immunocapture can assist isolation of
specific serotypes of E. coli through concentration of the target
360 Microbiological Safety of Meat | Pathogenic Escherichia coli
organisms. For this, antibodies specific to a serotype are
coated on magnetic beads or other particles. These antibodies
bind cells of the target serotype by forming antibody–bacteria
complexes. The coated particles can then be removed from
the sample. In the case of magnetic particles, a magnet is used
to remove the particles. This process is termed immuno-
magnetic separation. The antibody–bacteria complexes may
then be tested further for the presence of the target serotype
using selective and differential plating media or molecular
detection methods.
Differentiating target E. coli from other E. coli on selective
and differential media presents challenges as most E. coli
share common phenotypic properties. A range of selective
and differential media have been tested for the isolation of E.
coli O157:H7 and non-O157 STEC. Most E. coli O157:H7 are
phenotypically different from other E. coli in that they are
unable to ferment sorbitol. Consequently, most E. coli O157:
H7 produce colorless colonies on Sorbitol MacConkey Agar
(SMAC), whereas other E. coli produce pink colonies. Cefix-
ime and tellurite are commonly added to SMAC to improve
its selectivity by inhibiting the growth of competing bacteria.
Escherichia coli O157:H7 are also unable to produce the en-
zyme glucuronidase, a trait that differentiates them from
most other E. coli. Such variations in biochemical abilities of
different E. coli strains have been exploited to produce a range
of chromogenic media for detection of different strains of E.
coli. These media contain substrates that change color when
cleaved by enzymes produced by bacteria. Unlike E. coli
O157:H7, the non-O157 STEC are not easily differentiated
from other E. coli on the basis of phenotypic differences.
Once suspect colonies have been obtained, confirmation is
necessary to ensure that the organism is an E. coli that carries
the specific virulence factors and belongs to the serotype of
interest. A range of biochemical, molecular, or immuno-
logically methods can be used for confirmation.
There are many commercial test kits (including molecular
and immunological) for detection of E. coli O157:H7. Meth-
ods for the non-O157 STEC are becoming commercially
available in response to six of the non-O157 STEC serotypes
being declared adulterants of ground beef by the USDA FSIS.
Ongoing research into the pathogenic mechanisms of E. coli
can identify more specific targets and lead to improvements of
detection and isolation methods in the future.
Control
Numerous methods have been developed and applied in an
attempt to control PEC on meat in general and beef in par-
ticular. These methods are applied at points in the production
and supply chain from on the farm through primary process-
ing to preservation of retail products. Some of the methods
used and found to be effective not only control PEC but also
affect generic E. coli and other bacteria as well. General ap-
proaches to hygiene and preservation fall into this category.
More specific approaches to the control of PEC in meat have
tended to focus on the control of E. coli O157:H7.
Results obtained with on-farm methods for the control of
E. coli O157:H7 generally have been inconclusive, with many
being found effective under some conditions but not under
others. Given the diversity of agricultural practices associated
with the production of meat, this is not surprising. Manipu-
lation of diet has been one approach with, for example, high-
fiber/low-energy and low-fiber/high-energy diets reportedly
resulting in different prevalence and shedding rates of E. coli
O157:H7 in various meat animal species. In particular, it has
been postulated that the ratios of volatile fatty acids in and
the pH of the rumen, which change with different diets,
contribute to this effect. The gastrointestinal tracts of ru-
minants are, however, very complex and many factors might
influence their functioning. Thus, no clear recommendations
on the best diet to use to reduce E. coli O157:H7 carriage and
shedding can be made. Vaccines represent another type of
intervention that has been investigated for the control of E.
coli O157:H7 in cattle. Although showing some promise,
none of the vaccines developed so far can eliminate or con-
sistently reduce carriage of this pathogen. Other methods that
have been investigated to control E. coli O157:H7 on farm
have been the supplementation of the feed or water of live-
stock with probiotics (live microbes with beneficial effects),
bacteriophages (viruses that infect and kill specific bacteria),
and chemicals, such as sodium chlorate. Although these
methods have proved effective in trials, their practical value
has generally been shown to be not as great as initially
thought. On-farm interventions have included attempts to
control PEC in the farm environment in order to restrict
cross-contamination and reinfection of animals. Increased
sanitation of equipment, such as water troughs, is an example
of this form of control. Although such interventions are
commendable and may reduce spread of a range of bacteria,
they are unlikely to greatly reduce the risks posed by these
bacteria.
A very wide range of methods have been applied for con-
trol of PEC (particularly E. coli O157:H7) during primary
processing and in retail ready products. Many of these are
general antimicrobial techniques that have either been tested
on, or refined for effect on, PEC. Interventions that have been
applied include steam pasteurization and treatment of car-
casses, cuts trimmings, and other forms of meat with ionizing
radiation, ozone, ultraviolet light, and chemical anti-
microbials, such as chlorine and organic acids. Many of the
methods have been shown to be effective in reducing both
prevalence and numbers of, in particular, E. coli O157:H7 on
carcasses and in retail products. None of the techniques have
been shown to be completely effective for eliminating this
pathogen under all conditions. In addition, many of the
methods used have drawbacks associated with them, including
the presence of potential harmful residues or a reduction in
the quality of the meat product to which they have been ap-
plied. In most cases, a series or combination of rigorously
tested and approved (by regulation) treatments used in com-
bination have been shown to reduce the risks associated with
these pathogens but not eliminate them.
The control of PEC throughout the food system is an area
of active and ongoing research. Progress has been made in
controlling PEC, particularly E. coli O157:H7, but the recent
emergence of new serotypes and strains of concern to public
health has complicated this endeavor. The search for effective
means of controlling an ever-increasing array of pathogenic
strains of E. coli will, undoubtedly, continue.
 Microbiological Safety of Meat | Pathogenic Escherichia coli
See also: Foodborne Zoonoses. Meat-Borne Hazards,
Concepts and Methods for Mitigating Risks Related to.
Microbial Contamination: Decontamination of Fresh Meat;
Decontamination of Processed Meat; Microbial Contamination of
Fresh Meat; Microbial Contamination of Processed Meat.
Microbiological Analysis: Indicator Organisms in Meat. Parasites
Present in Meat and Viscera of Land Farmed Animals.
Spoilage, Factors Affecting: Microbiological
Further Reading
Bach, S.J., McAllister, T.A., Veira, D.M., Gannon, V.P.J., Holley, R.A., 2002.
Transmission and control of Escherchia coli O157:H7 − A review. Canadian
Journal of Animal Science 82, 475–490.
Ferens, W.A., Hovde, C.J., 2011. Escherichia coli O157:H7: Animal reservoir and
sources of human infection. Foodborne Pathogens and Disease 8, 465–487.
361
Holck, A.L., Axelsson, L., Rode, T.M., et al., 2011. Reduction of verotoxigenic
Escherichia coli in production of fermented sausages. Meat Science 89,
286–295.
Karama, M., Gyles, C.L., 2010. Methods for genotyping verotoxin-producing
Escherichia coli. Zoonoses and Public Health 57, 447–462.
Nataro, J.P., Kaper, J.B., 1998. Diarrheagenic Escherichia coli. Clinical Microbiology
Review 11, 142–201.
Rhoades, J.R., Duffy, G., Koutsoumanis, K., 2009. Prevalence and concentration of
verocytotoxigenic Escherichia coli, Salmonella enterica and Listeria
monocytogenes in the beef production chain: A review. Food Microbiology 26,
357–376.
Schmidt, M.A., 2010. LEEways: Tales of EPEC, ATEC and EHEC. Cellular
Microbiology 12, 1544–1552.
Relevant Website
http://www.fsis.usda.gov
Food Safety and Inspection Service − United States Department of Agriculture.