Non-O157 Shiga Toxin–Producing Escherichia coli in Foods

Review

Non-O157 Shiga ToxinndashProducing Escherichia coli in Foods

EMILY C MATHUSA YUHUAN CHEN ELENA ENACHE AND LLOYD HONTZ

Grocery Manufacturers Association 1350 I Street NW Suite 300 Washington DC 20005 USA

MS 10-033 Received 21 January 2010Accepted 8 May 2010

ABSTRACT

Non-O157 Shiga toxinndashproducing Escherichia coli (STEC) strains have been linked to outbreaks and sporadic cases

of illness worldwide Illnesses linked to STEC serotypes other than O157H7 appear to be on the rise in the United States

and worldwide indicating that some of these organisms may be emerging pathogens As more laboratories are testing for

these organisms in clinical samples more cases are uncovered Some cases of non-O157 STEC illness appear to be as

severe as cases associated with O157 although in general cases attributed to non-O157 are less severe There is much

variation in virulence potential within STEC serotypes and many may not be pathogenic Of more than 400 serotypes

isolated fewer than 10 serotypes cause the majority of STEC-related human illnesses Various virulence factors are

involved in non-O157 STEC pathogenicity the combined presence of both eae and stx genes has been associated with

enhanced virulence A scientific definition of a pathogenic STEC has not yet been accepted Several laboratories have

attempted to develop detection and identification methods and although substantial progress has been made a practical

method of STEC detection has yet to be validated Worldwide foods associated with non-O157 STEC illness include

sausage ice cream milk and lettuce among others Results from several studies suggest that control measures for O157

may be effective for non-O157 STEC More research is needed to uncover unique characteristics and resistances of non-

O157 STEC strains if they exist The public health significance of non-O157 STEC and the implications for industry

practices and regulatory actions are discussed

Escherichia coli is one of the most studied microor-

ganisms (11) Part of the Enterobacteriaceae family E coliis a facultatively anaerobic rod-shaped gram-negative

bacterium It is a nonndashspore-former and some strains may

be motile with peritrichous flagella Certain strains of E colihave been recognized as human pathogens since the 1940s

subsequently additional strains have been linked to human

illness

Several serotypes of STEC have been linked to

foodborne illness but not all STEC strains are capable of

causing human disease (38) The different enteropathogenic

groups of E coli are broken down into six pathotypes (46)The pathotype of interest in this review is verocytotoxigenic

E coli (VTEC) also known as Shiga toxinndashproducing Ecoli (STEC) (11) The terms Shiga toxin (Stx) and verotoxin

are interchangeable they refer to the production of cellular

cytotoxins (53 64 79 102) Enterohemorrhagic E coli(EHEC) strains are a subset of STEC that cause bloody

diarrhea (79) All EHEC strains are considered to be

pathogenic because they are correlated to specific clinical

connotations whereas not all STEC strains are pathogens

EHEC strains are defined as possessing a ca 60-MDa

plasmid expressing Stx and having the ability to cause

attaching and effacing (AE) lesions on epithelial cells (78)

In 2003 Karmali et al (67) proposed that STEC be

classified into five seropathotypes A to E Seropathotype A

consists of serotypes considered to be most virulent this

group consists of O157H7 and O157NM (nonmotile)

Seropathotype B consists of serotypes that are able to cause

severe illness and outbreaks but occur less frequently

Examples of serotypes in this group according to the data

reported include O26H11 O103H2 O111NM O121H19

and O145NM Organisms belonging to seropathotype B are

the focus in this review Seropathotype C is composed of

serotypes that are infrequently associated with sporadic

hemolytic uremic syndrome (HUS) but not often with

outbreaks including O91H21 and O113H21 Seropathotype

D consists of serotypes able to cause diarrhea and

seropathotype E includes all the other STEC serotypes that

have not been implicated in human disease (53 67)Scheutz (92) argued that there are problems in using

this classification system for STEC because it associates

serotype with degree of illness instead of the organismrsquos

virulence profile Scheutz suggests a classification of STEC

into three groups The first group would include HUS-

inducing andor epidemic outbreak potential STEC the

second group would consist of human diarrheandashinducing

STEC and the third group would be animal-associated

STEC (92) STEC strains may be placed into each category

based on information on pathogenicity of the specific strain

and the group classification may change as new data

become available Author for correspondence Tel 202-637-4807 Fax 202-639-5993

E-mail emathusagmaonlineorg

1721

Journal of Food Protection Vol 73 No 9 2010 Pages 1721ndash1736Copyright G International Association for Food Protection

ILLNESSES AND OUTBREAKS ATTRIBUTED TONON-O157 STEC

In the United States active surveillance of infections

attributed to non-O157 STEC began in 2001 (29) The

number of non-O157 STEC infections reported in the

United States between 2000 and 2005 increased from 171 to

501 cases suggesting a higher burden of illness than

previously thought (1) An increase in testing for Stx in

diarrheal cases and for STEC serotypes other than O157 is

likely the reason for the increase in incidence of cases and

outbreaks attributed to non-O157 STEC (60) Human

infections with STEC can occur with ingestion of

contaminated food or water or by direct contact with

animals Transmission can also occur through person-to-

person contact (53)Illnesses reported due to STEC serotypes other than

O157 are on the rise worldwide (78) It is estimated that 20

to 50 of all STEC infections can be attributed to non-

O157 strains but the percentages differ greatly from country

to country and among regions within a country (66 78) It is

estimated that less than 10 of HUS cases in North

America are caused by non-O157 STEC strains (52) In

Germany Italy and the United Kingdom it is estimated that

non-O157 STEC strains have caused 10 to 30 of sporadic

cases of HUS (23) Estimating the true percentage of

infections caused by non-O157 STEC strains is difficult

because these strains are not routinely subject to testing

(78) There is currently no convenient method that can

reliably screen for non-O157 STEC strains which compli-

cates the determination of incidence of disease due to these

organisms (78)There have been at least 22 outbreaks attributed to non-

O157 STEC strains in the United States since 1990 (19 3851) The sources for the non-O157 STEC strains were not

determined in some of these outbreaks and the vehicles

included foods and nonfoods (Table 1) Food vehicles

implicated in the outbreaks included milk (26) salad bar

(27) punch (21) apple cider (19) and iceberg lettuce (951) Mead et al (77) estimate that in the United States Ecoli O157H7 causes 73000 illnesses annually and non-

O157 STEC strains cause at least 37000 illnesses annually

Illnesses attributed to non-O157 STEC strains are most

frequently reported in the summer months (21)In 1994 there was an outbreak involving postpasteur-

ization contaminated milk with 11 confirmed and seven

suspected cases of illness Sixteen of the patients developed

bloody stools diarrhea and abdominal cramps Isolates

from three patients were identified as E coli O104H21 and

were positive for Stx2 A confirmed case was defined as

acute infection with E coli O104H21 isolated from patient

stool with serological confirmation Based on a case-control

study one brand of milk was significantly associated with

the illnesses The incident strain could not be isolated from

samples taken from the dairy at which the milk was

produced (26)In 1999 an outbreak of E coli O111H8 associated

with ice and salad from a salad bar occurred at a camp in

Texas Of 521 campers interviewed 58 had symptoms that

met the definition of illness either bloody diarrhea or

nonbloody diarrhea accompanied by abdominal cramps and

occurring within 14 days of the start of camp Two patients

developed HUS The meal served on the first night of camp

was significantly associated with development of illness

Several possible food items were identified but two were

significantly and independently associated with illness ice

and salad from the salad bar E coli O111H8 was isolated

from 2 of the 11 stool specimens submitted by ill patients

PCR was used to determine that both stx1 and stx2 were

present No food samples from the indicated meal were

available for testing (27)According to a newspaper report that was cited in the

review by Eblen (38) E coli O121H19 was linked to an

outbreak of illness associated with iceberg lettuce from a

fast food restaurant in 2006 Of 73 people who became ill

three developed kidney failure Iceberg lettuce was

pinpointed by local health officials because it was the only

common food among sickened patients The iceberg lettuce

was not tested Before the outbreak occurred the implicated

restaurant passed a health inspection and no issues were

discovered when another health inspection was performed

during the outbreak (9 38)To date there are no conclusive epidemiological data

that link meat products to non-O157 STEC illness in the

United States In 2006 there were two individual cases of

illness in the United States involving ground beef that may

have been due to non-O157 STEC but efforts to pinpoint

the source and the pathogen were inconclusive The first

involved a patient who consumed undercooked ground beef

An indistinguishable strain of E coli O103 was detected

using pulsed-field gel electrophoresis (PFGE) from both the

patient and leftover ground beef The original source of Ecoli O103 in this case was left undetermined because of

possible cross-contamination of a meat grinder (38) In the

second case a patient ill with E coli O157 provided ground

beef samples in which Stx was present but from which no

O157 could be recovered When the ground beef sample

was sent to the Centers for Disease Control and Prevention

(CDC) for characterization E coli O6H34 was found but

the ground beef could not be confirmed as the source of

illness (38)Recently in 2008 there was an outbreak attributed to

E coli O111NM in Locust Grove Oklahoma that involved

341 cases of illness 70 hospitalizations and 1 death The

outbreak was linked to a local restaurant but the

contamination route to the restaurant andor food source

remain undetermined Cross-contamination of food through

handling surface contact and storage areas was likely It

was reported that several employees of the restaurant

worked on days they experienced diarrhea but the strain

of E coli was not isolated from employee stool specimens

Clinical specimens were tested for Stx using a Shiga toxin

enzyme immunoassay screened for stx1 and stx2 genes

using real-time PCR and typed by PFGE Six Xbal PFGE

patterns were determined among E coli O111 isolates

These patterns were uploaded to the national PulseNet

database The state investigation of this outbreak found

many asymptomatic infections of E coli O111 in which

1722 MATHUSA ET AL J Food Prot Vol 73 No 9

serum immunoglobulin (Ig) M antibodies to O111 were

found in patients that experienced no clinical illness Of the

135 persons from whom serum or plasma specimens were

tested for E coli O111 IgM antibodies 66 (49) were

asymptomatic 8 (6) had mild illness 12 (9) were

suspected cases 22 (16) were probable cases and 26

(19) were confirmed cases (83) In many outbreaks

involving non-O157 STEC the source of infection remains

unidentified (78) especially for outbreaks that occurred

prior to 2000 (Table 1)

In Australia in 1995 an outbreak of HUS was linked to

semidry uncooked fermented sausage contaminated with

STEC O111NM Sixteen (70) of the patients required

dialysis and one patient died Stool samples were screened

for genes encoding Stx using PCR 87 were positive for

both stx1 and stx2 4 were positive for stx2 only and 9

were negative E coli O111NM was isolated from 16 of the

stool samples and other E coli strains were recovered from

three of the patients Another 62 patients with bloody and

nonbloody diarrhea who had consumed the implicated

sausage were reported but E coli O111NM was isolated

from only 3 of the patients Of 10 sausage samples taken

from patientsrsquo homes 8 were positive for stx and E coliO111NM was isolated from 4 (25)

In 2007 there was an outbreak of E coli O145 and O26

in Belgium associated with ice cream produced on a dairy

farm Twelve people became severely ill with five children

developing HUS E coli O145 was isolated from stool

samples from three HUS patients from one stool sample E

coli O26 was also isolated Stool samples were cultured on

sorbitol-containing MacConkey (SMAC) agar and colonies

were identified as E coli O145 or O26 through biochemical

tests PCR and an agglutination assay For the E coli O145

strains PCR analysis revealed the presence of stx1 stx2 eaeand ehxA (enterohemolysin) PFGE was used to compare

the genetic profiles of all STEC strains isolated Undistin-

guishable strains of E coli O145 and O26 were found in

stool samples ice cream samples and environmental

samples collected on the dairy farm (33)In Denmark in 2007 an outbreak of E coli O26H11

was associated with organic fermented beef sausage

Twenty people were involved in the outbreak with the

majority of cases in children Two unopened samples and

two opened samples of sausage tested positive for the

infection strain of E coli O26H11 Leftover beef used to

make the sausage also tested positive for the strain which

was stx1 positive and eae positive The reported symptoms

of illness were mild but there was one case involving

bloody diarrhea Several samples of stool also tested

positive for other diarrheal pathogens (two Campylobacterspecies two Yersinia enterocolitica one norovirus and two

eae-positive but stx-negative E coli strains) (43 44)In the United States Canada United Kingdom and

Japan E coli O157H7 is currently the STEC serotype most

frequently linked to illness but in other countries other

STEC serotypes have been associated with disease and

outbreaks (21 38 42 99) In Europe Argentina Australia

Chile and South Africa non-O157 STEC infections are just

TABLE 1 Selected outbreaks of non-O157 STEC in the United States and worldwide

Year Serotype(s) present Country (state) No of persons illa HUS Source ID methodb Reference(s)

1986 O111H8 Germany 4 1 Undetermined

1990 O111 USA (OH) 55 (5 conf) Yes Undetermined 41994 O104H21 USA (MT) 18 (conf) Yes Milk 261995 O111NM Australia 158 (26 conf) 23 Sausage PCR 251998 O121 USA (MT) 40 Unknown Undetermined 381999 O121 USA (CT) 11 (conf) Yes Lake water 751999 O111H8 USA (TX) 56 (conf) Yes Salad bar 271999 O145H28 Germany 2 No Undetermined 161999 O26H11 Germany 3 3 Undetermined 162000 O103 USA (WA) 18 (conf) Yes Punch 212000 O26H11 Germany 11 No Day care beef PCR PFGE 1012001 O145H28 Germany 6 1 Undetermined 162001 O111 USA (SD) 3 No Day care 212001 O26 USA (MN) 4 No Lake water 212002 O145H28 Germany 2 No Undetermined 162004 O111c USA (NY) 212 (conf) No Apple cider 19 282005 O45NM O45H2 USA (NY) 52 Food handler PCR PFGE 32006 O45 USA Day care 192006 O121 USA Day care 192006 O121H19 USA (UT) 73 No Iceberg lettuce 92006 O103H25 Norway 17 (conf) 11 Lamb sausage MLVA 932007 O145 O26 Belgium 12 5 Ice cream PFGE 332007 O26 Denmark 20 Beef sausage PCR PFGE 432008 O111 USA (OK) 341 Yes Restaurant PCR PFGE 83

a conf confirmedb PFGE pulsed-field gel electrophoresis MLVA multiple locus variable-number tandem repeat analysisc Cryptosporidium was also isolated

J Food Prot Vol 73 No 9 LITERATURE REVIEW ON NON-O157 SHIGA TOXINndashPRODUCING E COLI IN FOODS 1723

as prevalent if not more prevalent than E coli O157H7

infections according to some assessments (21 38 64 78 99101) In 1999 in Germany two-thirds of the STEC infections

reported were due to non-O157 STEC In Germany the

STEC serotype O26 was the second most frequently reported

after O157 and accounted for 20 of all reported STEC

infections (101) An unpublished study by Acheson in 2001

reported a similar incidence of O157 (54) and non-O157

STEC (46) from clinical stool samples (38) Acheson

concluded that certain strains of non-O157 STEC including

O26 O45 O103 O111 and O145 are just as prevalent and

clinically significant as E coli O157 in the United States (38)Worldwide disease caused by non-O157 STEC is considered

an emerging problem (102)A 2009 study done by Hedican et al (57) and the

Minnesota Department of Health compared the characteris-

tics of infections attributed to O157 versus non-O157

STEC All stool cultures were collected between 2000 and

2006 and were received from two sites in Minnesota a

metropolitan health maintenance organization laboratory

and a hospital laboratory that served a small city and a rural

area They found that O157 STEC infections were more

likely than non-O157 STEC infections to result in bloody

diarrhea (78 versus 54) hospitalization (34 versus 8)

and HUS (7 versus 0) They also noted that when only

isolates that harbored stx2 genes were considered O157

STEC cases were still more likely to result in bloody

diarrhea and hospitalizations than the non-O157 STEC

cases Of the non-O157 STEC cases 74 were represented

by just five serotypes including O26 (27) O103 (21)

O111 (19) O145 (5) and O45 (4) (57) The

incidence of illnesses associated with these serotypes

correlated to Minnesota cases differ slightly from percent-

ages seen for the United States and worldwide indicating

that their prevalence may be unique country to country and

region to region Similar information on comparisons of

O157 and non-O157 STEC infections but on a national

level based on FoodNet data were reported by Gould of the

CDC at a public meeting in Washington DC in late 2009

(51) It was reported that factors such as age gender and

seasonality of O157 and non-O157 STEC infections are

similar Gould noted that non-O157 STEC infections are more

sporadic than infections of O157 and are correlated with fewer

outbreaks E coli O157 has a much higher incidence of HUS

(63 O157 versus 17 non-O157) hospitalizations (42

O157 versus 12 non-O157) and deaths (06 O157 versus

01 non-O157) Another interesting difference was seen

between infections of O157 and non-O157 STEC the

incidence of international travel was five times greater for

patients with non-O157 STEC infection (51)In 2009 McPherson et al (76) collected information on

serogroup-specific risk factors of STEC infections in

Australia from 2003 through 2007 Questionnaires were

used to collect data on clinical illness foods consumed and

exposure to environmental sources from individuals from

six different jurisdictions in Australia Interviewees included

43 case patients infected with O157 STEC 71 case patients

infected with non-O157 STEC and 304 control subjects Of

the non-O157 STECndashinfected patients 14 cases could be

attributed to O111 7 cases to O26 and 1 case each to O103

O113 and O172 Infections due to O157 STEC were

positively associated with eating at a restaurant or catered

event eating hamburgers prior use of antibiotics and

family occupational exposure to red meat There was a

negative association between eating homegrown vegetables

fruits and herbs and O157 STEC infection Infections due

to non-O157 STEC were positively associated with eating at

a catered event eating chicken meat or corned beef from a

delicatessen camping family occupational exposure to

animals and living on or visitation to a farm For non-O157

STEC infections there was a negative association to eating

pork eggs raw and homegrown vegetables fruits and

herbs (76)

PATHOGENESIS OF NON-O157 STEC

There is extensive variation within serotypes of STEC

in the severity of illness caused and more than 120 different

serotypes have been associated with illness (78 92) In the

United States between 1983 and 2002 the six most

commonly occurring serotypes of non-O157 STEC associ-

ated with disease were in descending order O26 O111

O103 O121 O45 and O145 (3 21 53) According to

preliminary data presented by Gould (51) in 2009 these six

serotypes made up 82 (n ~ 803) of FoodNet human

isolates of non-O157 STEC between 2000 and 2007 STEC

infection in humans may result in no illness or mild to

severe symptoms and in some cases may lead to more

severe disease such as hemorrhagic colitis HUS and

thrombotic thrombocytopenic purpura (102)Twardon et al (99) speculate that fewer than 10

bacterial cells of E coli O26 are able to infect humans

however no data were provided by the authors for this

postulation Gyles (53) suggested it to be fewer than 50

cells to a few hundred organisms based on information on

E coli O157 It is estimated that the infectious dose for

non-O157 STEC may be higher than that for E coli O157

which has been shown to be 10 to 100 cells (45) An article

by Paton et al (85) on an outbreak of HUS in dry

fermented sausage that was contaminated with non-O157

STEC found low levels (100 CFUg) of E coli present in

sausages eaten by ill patients In this outbreak E coliO111NM was indicated as the causative agent for illness

STEC O111NM was isolated from both patients and

reserved sausage samples PCR was used to determine that

only 04 to 14 of E coli isolated from the sausage were

STEC Of the STEC strains isolated from the sausage on

MacConkey agar generally less than 10 were identified

as STEC O111NM by colony immunoblotting The

authors suggest that there may have been as little as one

cell of STEC O111NM per 10 g of the sausage which

would indicate a low infectious dose for this organism in

certain foods (85)

Characteristics of disease related to non-O157STEC The incubation period of STEC is usually 3 to

4 days but can be as long as 5 to 8 days or as short as 1 to

2 days Initial symptoms include crampy abdominal pain a

short-lived fever and nonbloody diarrhea Vomiting can


occur during the diarrhea stage of illness but is observed in

only about half of the patients In 1 to 2 days diarrhea may

become bloody with increased abdominal pain and this may

last for up to 10 days Most cases of infection with STEC

will resolve without sequelae but 10 of patients most

commonly young children (younger than 10 years old) and

the elderly may experience the development of HUS (4453 78) Hemorrhagic colitis is characterized by severe

abdominal cramps and watery then grossly bloody diarrhea

with little to no fever HUS was initially described in 1955

and linked to Shiga toxinndashproducing Shigella dysenteriae

HUS is characterized by acute renal failure thrombocyto-

penia and microangiopathic hemolytic anemia Stx is

responsible for damage to both intestinal and renal tissue

(78) Patients suffering from thrombotic thrombocytopenic

purpura experience the same clinical symptoms as HUS

accompanied by fever and formation of thrombi that may

lead to severe neurological disorders (102)Bloody diarrhea is more common with E coli O157H7

than with non-O157 STEC It is estimated that O157 causes

at least 80 of HUS cases associated with STEC infections

while less than 10 of HUS cases can be attributed to non-

O157 STEC (5 52 71) Some Shiga toxigenic non-O157 Ecoli including serotypes O26 and O111 have been

associated with hemorrhagic colitis and HUS (78 79)Some cases of illness from infection with non-O157 STEC

have resulted in symptoms similar to those for E coliO157H7 (53 78) Although in some reported cases the

degree of illness due to non-O157 STEC has been just as

severe as illness due to E coli O157H7 (78) in most of the

reported cases it appears that the overall illness associated

with non-O157 STEC is less severe than illness due to

E coli O157H7 and fewer hospitalizations are reported

(60 79)The disease process for STEC first requires the

organism to overcome host defense mechanisms and

establish itself in the intestine Acid resistance of STEC is

important for its survival in the harsh acidic environment of

the gastrointestinal tract STEC strains that possess the eae(E coli attaching and effacing or intimin) gene can produce

products involved in cell attachment During attachment

eae-positive STEC strains form an AE lesion on intestinal

epithelial cells The AE lesion results in structural changes

in the epithelial cells such as loss of microvilli pedestal

formation and accumulation of cytoskeletal proteins

allowing adherence of the bacteria to the host cell surface

After attachment Stx is absorbed into the host cell through a

transcellular pathway (78) STEC infection appears to be

localized without septicemia but the toxin produced is

absorbed from the intestine and causes the systemic effects

of the disease (53) Translocation of the toxin into the

bloodstream is believed to be aided by damage of the

intestinal epithelium by lipopolysaccharide or the toxin

itself (78)

Virulence factors Over 200 serotypes of E coli can

produce Stx but only about 50 of these serotypes have been

associated with bloody diarrhea or HUS in humans (78)Shiga and Shiga-like toxins can be produced by several

other bacilli including Enterobacter cloacae Citrobacterfreundii and Aeromonas hydrophila (79 99) The ability of

an E coli strain to produce Stx alone does not automatically

confer pathogenicity without other virulence factors (78100)

There are two types of Stx Stx1 and Stx2 Stx1 is

identical to the toxin produced by Shigella dysenteriae type

1 (53) Variants of stx genes have been reported such as

stx1a stx1b stx1c stx1d stx2a stx2b stx2c stx2d stx2e stx2f

and stx2g (13 64) Certain variants including stx2a and stx2c

are more likely to be associated with hemorrhagic colitis and

HUS (13) Several other variants of Stx show no clinical

significance (53 78) A single STEC strain may express

Stx1 Stx2 or both toxins (78 79) Expression of Stx2 has

been associated with a higher risk for developing HUS

especially when the organism is also eae positive (21 5266) It has been suggested that E coli producing Stx2 is

involved in most HUS cases because E coli O157H7

strains that are isolated from patients with HUS usually

produce only Stx2 or both Stx1 and Stx2 E coli producing

only Stx1 has not been isolated from patients with HUS

(90) Stx2 has also been shown to be 1000 times more toxic

for human renal microvascular endothelial cells than Stx1

which may be due to major differences in crystal structure

between the two toxins (53) Boerlin et al (18) found a

strong statistical association between non-O157 STEC

serotypes O26 O103 O111 and O145 expressing stx2

and the severity of human disease They determined that

possession of the stx2 gene makes the organism significantly

more likely to cause serious disease including bloody

diarrhea and development of HUS (18 42)Friedrich et al (47) used PCR to screen 626 STEC

isolates from stool samples collected in Germany from 1996

to 2000 to determine serotype and detect the presence of

stx1 stx2 and stx2 variants and the eae gene Serotypes of

non-O157 STEC were isolated from patients with HUS

including O26 O103 O111 and O145 The most

frequently isolated non-O157 STEC serotype from patients

with HUS was O26 Identical strains of non-O157 STEC

were isolated from both asymptomatic patients and those

with diarrhea STEC strains O26H11NM O145NM

O103H2H18NM and O111NM were isolated from

patients with HUS patients with diarrhea but no HUS

and asymptomatic patients The stx2 variants detected

included stx2c stx2d and stx2e with stx2c as the most

frequent variant found in 148 (236) of the 626 isolates

Variants stx2d and stx2e were eae negative and not detected

in any of the non-O157 STEC serotypes of interest

Of the 626 isolates there were 87 non-O157 STEC

isolates harboring stx2 and nine carrying stx2c Friedrich et

al (47) found that of 87 isolates of non-O157 STEC that did

harbor stx2 which included O26 O103 O121 and O145

83 (954) carried the eae gene Of the non-O157 STEC

isolates harboring the stx2c variant 333 were eae positive

Of the 28 O157 isolates (from the pool of 626 isolates) with

the stx2c variant 100 were eae positive The authors

concluded that STEC stains harboring the stx2c variant are

able to cause HUS but isolates with either the stx2d or stx2e

variant result in milder illness unlikely to produce sequelae


(47) Another study by Beutin et al (15) found that high

production of Stx2e by human-associated STEC strains did

not result in diarrheal disease Strains harboring stx2e genes

were negative for eae and ehxA genes The authors

concluded that Stx2e-producing strains are not good

colonizers of the human intestine probably due to the lack

of receptors on human enterocytes and that strains

producing only Stx2e are not able to cause severe disease

(15)Stx is encoded by phages inserted into the E coli

chromosome (53 78 79) Stx is made up of the basic A-B

subunit structure The B pentamer of the toxin binds to a

specific receptor globotriaosylceramide on the intestinal

cell surface permitting internalization The Stx2e variant

which is associated with disease in swine uses globote-

traosylceramide as its receptor The toxin molecule is taken

up into the cell through receptor-mediated endocytosis The

membrane vesicle containing toxin may fuse with lysosomal

vesicles resulting in destruction of the toxin or may be

transported to the Golgi apparatus and endoplasmic

reticulum The A subunit of the toxin protein possesses

enzymatic activity that cleaves a specific adenine base from

the 28 S rRNA inhibiting protein synthesis (78) This can

result in apoptosis programmed cell death due to

ribocytotoxic stress response (53)Important virulence factors include expression of the

eae gene and the hly (hemolysin) gene (53) Another

hemolysin gene present in some STEC strains ehxA is

correlated with virulence of EHEC (64) The eae gene

expresses intimin also called the eae protein which is

important in the production of AE lesions in the intestine A

pathogenicity island called the locus of enterocyte efface-

ment (LEE) encodes proteins necessary for the formation of

the AE lesion LEE encodes for a type III secretion

apparatus a protein translocation system and an adherence

system that consists of the eae protein which is the outer

membrane protein and its receptor translocated intimin

receptor The translocated intimin receptor protein becomes

inserted into the host cell outer membrane where it acts as

the receptor for the eae protein on the bacterial cell surface

(53) These genes are more common in STEC strains that

are correlated to illness but strains lacking these genes

reportedly have caused clinical illness (79 80) E coliO113H21 does not possess the LEE pathogenicity island

but has been the cause of sporadic illness and outbreaks

The illness cases attributed to E coli O113H21 were

reported to be just as severe as those caused by E coliO157H7 (80)

Fluid secretion associated with diarrhea occurs with

death of absorptive villus tip intestinal epithelial cells by

Stx It is believed that a STEC strainrsquos ability to produce

AE lesions is sufficient to cause nonbloody diarrhea but

Stx production is essential for the development of bloody

diarrhea and hemorrhagic colitis Expression of hemolysin

is widely distributed among non-O157 STEC strains and

causes lysis of red blood cells in vitro Approximately 90

of all STEC strains possess genes encoding hemolysin

(78)

Other toxins produced by STEC may play a role in the

etiology of human disease Cytolethal distending toxin is

produced by a few eae-negative STEC strains that have

been associated with disease (17 53) Subtilase cytotoxin is

also produced by an eae-negative STEC strain O113H21

and the gene is detected in many other STEC strains (5380) Newton et al (80) suggest that subtilase cytotoxin

emerged as a virulence factor in the absence of LEE and

this toxin likely plays a role in the progression of severe

disease Although E coli O113H21 is eae negative it has

been associated with HUS which further complicates the

definition of pathogenicity for these organisms as a whole

(11) Several other gene products have been suggested to

have possible virulence roles for STEC including adhesins

such as the VTEC auto-agglutinating adhesin (saa)

proteases iron acquisition systems lipopolysaccharide

and flagellin (53 64) The virulence of the subtilase

cytotoxin of LEE-negative STEC is partially dependent on

flagellin showing that some of these products may work

with other virulence factors to impart pathogenicity (80)Given that there is no satisfactory animal model that mimics

the disease in humans it is difficult to determine how

significantly these factors contribute to virulence if at all

(53 102)Much of the research on non-O157 STEC has focused

on the serotype O26 A study by Zhang et al (103)examined the molecular characteristics of 55 STEC O26

strains collected in Germany and the Czech Republic

between 1965 and 1999 Virulence genes that were found

in O26 such as hlyA catalase peroxidase (katP) and a

serine protease (espP) that cleaves human coagulation

factor V are also found in STEC O157 They found that

all the STEC O26 strains possessed a high-pathogenicity

island that O157 does not that contains genes encoding

pesticin receptor ( fyuA) and a siderophore called

yersiniabactin An interesting discovery was made regard-

ing the type of stx gene contained by STEC O26 strains

over time Through PCR analysis they found that 16 of 18

strains collected from 1965 to 1996 expressed stx1 alone

with only two additional strains expressing stx1 after 1997

The 37 strains that expressed stx2 alone or in combination

with stx1 were isolated between 1995 and 1999 These

results indicate that there was a shift from stx1 to stx2

expression among STEC O26 Of the 55 STEC O26

isolates 16 clonal subgroups were determined by PFGE

showing the diversity of this serogroup Using PFGE

Zhang et al (103) discovered the emergence of a new

clonal subgroup A with a set of unique virulence genes

including stx2 hlyA and the etp (EHEC type II secretion

pathway) cluster Originally found only in STEC O157

the etp gene cluster which encodes a type II secretion

system which allows for extracellular excretion of

proteins was seen in several O26 strains with identical

plasmid profiles and only after 1995 (94 103) Four

clusters of outbreaks were linked to this subgroup A of

STEC O26 The STEC O26 of subgroup A were shown to

have a high pathogenic potential for humans so any

disease outbreaks correlated to these organisms should be

closely monitored by public health authorities (103)


A shift in the expression of virulence factors and

emergence of virulence strains among STEC strains is also

suggested by evidence for O157 E coli O157H7 was first

reported as a cause of foodborne illness in 1983 by Riley et

al (89) after investigating outbreaks in 1982 involving

undercooked ground beef Before these incidents this

serotype was almost never isolated (10 78 89) After the

link between E coli O157H7 and foodborne illness was

made laboratories around the world reviewed all E colistrains collected between 1973 and 1983 Only one E coliO157H7 was isolated by the CDC laboratories out of 3000

serotyped isolates and the Public Health Laboratory in the

United Kingdom also found just one O157H7 isolate out of

15000 serotyped isolates Only six O157H7 isolates were

found out of 2000 isolates from patients with diarrhea by

Canadarsquos Laboratory Centre for Disease Control Although

illness from O157H7 STEC could have been hidden in the

overall burden of illness from EHEC the limited isolation of

O157H7 prior to 1982 suggests that the presence of this

serotype may have increased since that time instead of

having previously been missed (78)

SOURCES FOR STEC AND DISTRIBUTION

Ruminants especially cattle are an important reservoir

for STEC strains (10 42 53 61) STEC strains have been

recovered from cattle sheep goats pigs cats deer horses

dogs birds and flies (53 78 81) In North America cattle

are the significant reservoir for STEC strains but in other

countries such as Australia sheep are the most important

carrier (53) In the United States beef carcass processing is

the main area targeted for interventions to reduce contam-

ination (53)Generally non-O157 STEC strains are found in cattle

at a much higher prevalence than E coli O157 (10) In a

study by Beutin et al (12) STEC strains were isolated in

632 of feces samples from cattle in one herd (n ~ 19)

over a period of 6 months Of the 33 serotypes of STEC

isolated none were O157 Stx was detected by the Vero cell

test and the presence of stx1 and stx2 was determined by

colony blot hybridization with digoxigenin-11-dUTPndashla-

beled gene probes Almost all of the STEC serotypes

produced Stx2 only one strain produced Stx1 All the

strains but one were negative for the eae gene (12) Most

cattle colonized by STEC are asymptomatic due to the

absence of the globotriaosylceramide receptor in their

intestinal cells that is specific for Stx proteins (99) Rates

of colonization of STEC in cattle have been found to be as

high as 60 but are more typically in the range of 10 to

25 (12 78) In 2007 Hussein estimated that the prev-

alence of non-O157 STEC in dairy cattle may be as high as

74 (61 63) Non-O157 STEC strains isolated from dairy

cattle belonged to 152 different serotypes with an estimated

49 of these being pathogenic when defined as a STEC that

produces one or more of the following virulence factors

Stx1 Stx2 hlyA EHEC-hlyA andor intimin (61) Another

study by Hussein on non-O157 STEC in cattle at slaughter

found prevalence rates of 21 to 701 (62) The rate is

variable and thought to depend on environmental factors

and management practices (62) A 2003 study by Barkocy-

Gallagher et al (6) found the prevalence of non-O157 STEC

in beef cattle at the time of slaughter to be between 139 and

271 depending on the season

Studies have shown that there is a higher frequency of

fecal shedding of STEC by cattle in warmer months than

colder months with a correlating higher incidence of human

illness in summer months (53 78) Age may also play a role

in fecal shedding of STEC in cattle with the lowest

shedding rates in calves before weaning the highest rates in

the postweaning period and intermediate rates in adult

cattle (53) Studies have shown that many bovine isolates of

non-O157 STEC are less likely to carry important virulence

factors other than stx such as eae and hlyA in comparison

to human isolates indicating that these organisms may be

less virulent (2 18 69)Over 435 different serotypes of STEC have been

recovered from cattle and more than 470 STEC serotypes

have been isolated from humans with great overlap Only a

fraction of these STEC serotypes are capable of causing

illness Of human STEC isolates fewer than 10 O groups

are responsible for the majority of illnesses (53 78)

FOODS ASSOCIATED WITH NON-O157 STEC

Foods from which non-O157 STEC strains have been

isolated andor associated with illness include sausage ice

cream postpasteurization contaminated milk punch and

iceberg lettuce (21 38 44 101) Bettelheim (10) suggested

that many of the foods from past outbreaks associated with

illness due to E coli O157 were likely to also contain non-

O157 strains but that only O157 was sought Studies have

screened grocery items such as delicatessen salad raw

milk raw beef minced meat pork lamb poultry fish

shellfish and cheese and were able to detect non-O157

STEC at different frequencies (Table 2) (35 38 86 88 91)A study in the United States by Samadpour et al (91)

sampled raw meat poultry and seafood samples for stxgenes using DNA probes and found them in samples of beef

(23) veal (63) pork (18) chicken (12) turkey

(7) lamb (48) fish (10) and shellfish (5) After

determination of serotypes in the samples they found that

several different non-O157 strains but no O157 strains

were present Comparisons of electrophoretic typing

patterns found that the isolates had a close relationship to

isolates from human and animal disease cases (91) A 2002

study by Arthur et al (2) looked at the prevalence of

non-O157 STEC on beef carcasses in US processing

plants and found that 539 were positive for at least one

strain prior to evisceration This level was reduced to only

83 following processing interventions including steam

vacuum hot water organic acids and steam pasteurization

(2) Studies from around the world have reported differing

postprocessing prevalence of non-O157 STEC on beef

carcasses but this may be due to different STEC isolation

methodologies (69)In 2006 in France Perelle et al (86) screened samples

of raw milk (n ~ 205) and minced meat (n ~ 300) using

PCR-ELISA and found the prevalence of STEC-positive


samples was 174 Of the 205 raw milk samples 43 (21)

were positive for STEC Of the 300 minced meat samples

45 (15) were positive for STEC Of the 88 positive STEC

samples 74 (84) were confirmed positive for stx using a

59-nuclease PCR assay When multiplex real-time PCR was

used to screen for specific serotypes including O26 O103

O111 O145 and O157 they were found in 26 of the raw

milk samples and 48 of the minced meat samples Of the

45 samples of STEC-positive minced meat 7 included

serotype O145 and 2 had serotype O103 Of the 43 samples

of STEC-positive raw milk 9 had serotype O145 2 had

serotype O103 and 1 had serotype O26 Many of the

samples had more than one of the specific STEC serotypes

sought The incidence of E coli O157 in minced meat and

raw milk was 1 which is in line with worldwide values of

incidence but the incidence of E coli O145 was

surprisingly higher 3 of the samples (86) Survey data

were converted to most-probable-number counts following

the previously proposed Halvorson and Ziegler (55)calculation and showed that the contamination was only 1

to 2 most-probable-number STEC cells per kg of sample

Perelle et al (86) determined that the contamination of the

beef and raw milk samples was very low and that the

potential risk of consumer infection by these strains from the

samples is likely very minor

Another French study by Pradel et al (88) looked at the

prevalence of STEC in beef samples and cheese samples At

least one strain of STEC was found in 4 of beef samples

and 1 of cheese samples The investigators screened 220

STEC isolates including isolates of the beef and cheese

samples as well as isolates from stool samples from cattle

and hospitalized patients Of the STEC isolates only 5

carried the eae gene 15 harbored the stx1 gene 53

harbored the stx2 gene and 32 had both genes The

authors concluded that the majority of the STEC isolates

from beef samples and cheese samples were unlikely to be

pathogenic in humans based on the lack of virulence

characteristics associated with clinical isolates (88)In early 2010 results of PCR screening tests for the stx

eae and the O26 O103 O121 O45 O111 and O145 genes

in US Food Safety and Inspection Service (FSIS) archived

lysates of ground beef samples were reported (50) PCR

testing of 224 E coli O157H7 sample enrichments yielded

the following percent positives for each genetic target O26

(31) O103 (36) O121 (18) O45 (201) O111

(04) and O145 (00) (50) These samples had

previously tested negative for E coli O157H7 It was

noted that E coli O111 and O145 did not grow well in the

E coli O157H7 enrichment broth Among the 224 samples

it was found that only 13 of sample enrichments were

positive for all three factors one of the top six serotypes

stx and eae (50) Furthermore these PCR screening tests

yielded presumptive-positive results The archived lysates of

ground beef samples contain lysed cells from sample

enrichment and thus isolates are unavailable for confirma-

tion testing The information presented above suggests that

using the results of serotype screening alone could be

misleading if it is assumed that all positive results represent

pathogenic non-O157 STEC If appropriate virulence

factors are not targeted as part of food sample screenings

it will be difficult to know whether or not identified STEC

strains are pathogenic

DETECTION AND IDENTIFICATION METHODS

Currently there exists no standard cultural method to

identify non-O157 STEC but many laboratories worldwide

are attempting to develop a method (11) The non-O157

STEC serotypes of interest differ from country to country

TABLE 2 Occurrence of STEC in foods

Product tested positive all STECa positive non-O157 STECa Test methods Reference

Beef 23 DNA probes for stx genes 91Veal 63

Pork 18

Chicken 12

Turkey 7

Lamb 48

Fish 10

Shellfish 5

Beef carcasses 719 539 PCR targeting stx genes and colony

hybridization for STEC serotyping

2Treated beef carcasses 101 83

Raw milk 21 48b PCR-ELISA targeting stx genes multiplex

real-time PCR

86Minced meat 15 26b

Beef 4 Not reported PCR targeting stx genes API testing for Ecoli serotyping

88Cheese 1

Lysate from FSIS archived

ground beef samples

Not reported 13c PCR targeting O-antigen stx and eaegenes

50

a Results from PCR screening tests in which an isolate was not obtained for confirmation testing are presumptive positive not confirmed positiveb These values represent the fraction of samples that tested PCR positive for one or more of the serotypes O26 O103 O111 O145 and O157c This value represents the fraction of samples that tested PCR positive for the stx and eae genes as well as positive for one of the six

serotypes (ie O26 O103 O121 O45 O111 or O145)


and there is no widely accepted selective-differential media

available to determine the individual serotypes

Cultural methods with selective and differentialmedia The current cultural method for isolation of E coliO157 is based on the inability of this organism to ferment

sorbitol although a few strains are able to ferment sorbitol

(53) Most E coli strains are capable of fermenting sorbitol

Using SMAC to isolate suspected E coli will result in clear

colonies for E coli O157 Bright pink to mauve colonies

indicate sorbitol-fermenting organisms which include most

non-O157 and other common fecal microflora Gram-

positive microorganisms will be inhibited on this medium

by crystal violet and the bile salts mixture in the

formulation Differentiation of non-O157 STEC colonies

on SMAC is not possible (74)Researchers have been working on developing media to

detect non-O157 STEC In 2008 Posse et al (87) developed

a set of novel differential media for the isolation and

confirmation of non-O157 STEC strains (O26 O103 O111

and O145) from food and feces The first medium is based

on a mixture of carbohydrate sources b-D-galactosidase

activity and selective reagents that result in color-based

differentiation of the four specified non-O157 STEC strains

The composition of this differential medium starts with

MacConkey agar base and is supplemented with sucrose

sorbose bile salts 5-bromo-4-chloro-3-indolyl-b-D-galacto-

pyranoside (X-gal) isopropyl-b-D-thiogalactopyranoside

novobiocin and potassium tellurite The growth of the four

different non-O157 STEC serotypes on this medium

produces different colored colonies STEC O26 colonies

appear bright red to dark purple O103 and O111 colonies

are blue-purple and O145 colonies are green The second

group of media is for confirmation of serotypes based on

specific carbohydrate utilization These agars contain phenol

red broth base supplemented with dulcitol L-rhamnose D-

raffinose or D-arabinose (87)Unpublished studies in our laboratories (40) using the

chromogenic agars described by Posse et al (87) showed

that while some of the serotypes may present the colony

color as indicated in that publication the color and the entire

aspect of the colonies may change as a function of

incubation time how crowded or isolated the colonies are

or the medium or food matrix from which they are isolated

When the colonies are small and crowded they may look

significantly different than when they are well isolated and

larger For example O26 streaked on the chromogenic agar

from a pure culture in tryptic soy broth grew either as small

purple colonies with a darker center or large pink colonies

with a darker center and circled by a blue-grayish edge the

colonies were also shiny and smooth For the same serotype

inoculated in irradiated ground beef and streaked on the

same chromogenic agar resulting colonies were small blue

rough granulated flat and dry or were large pink shiny

and smooth as seen from colonies isolated from tryptic soy

broth When six STEC serotypes were streaked on the

chromogenic agar it was difficult to differentiate between

serotypes (40) Another unpublished study conducted in

2009 used Rainbow agar to detect non-O157 STEC

serotypes (49) Different color reactions on the Rainbow

agar indicate which serotype may be present Serotypes

O26 O103 and O121 may appear pink or magenta on this

agar O45 O111 and O157 may appear gray light blue or

light purple and O145 may appear dark blue (49)Hiramatsu et al (58) have developed a selective

medium specific for E coli O26 using rhamnose called

rhamnose-MacConkey (RMAC) The study showed that all

O26 strains 31 total were able to ferment rhamnose while

108 other STEC strains could not All STEC O26 colonies

were colorless on RMAC while the vast majority of

other STEC (89 of 93 strains which included serotypes

O157 and O111) produced red colonies Most non-STEC

strains (50 of 59 strains) were unable to grow on RMAC

Other studies have also shown that the O26 serotype is

unique in that it is able to ferment rhamnose a characteristic

that could be used in its differentiation from other STEC

serotypes (24 58)Another indicator for STEC is the production of

enterohemolysin and a medium which detects enterohemo-

lysin-producing organisms has been developed (11)Catarame et al (24) reviewed many commercially available

media for their ability to recover STEC serotypes O26 and

O111 from minced beef Different combinations of

enrichment procedures and incubation time and temperature

were tried as well as novel media formulated with a range

of selective antibiotics and carbohydrates Tryptic soy broth

containing cefixime and vancomycin was used as the

enrichment medium for both serotypes with the addition of

potassium tellurite to optimize the enrichment for serotype

O26 A couple of O111 strains were sensitive to potassium

tellurite Catarame et al found that the optimum recovery of

STEC O26 was on MacConkey agar modified by replacing

lactose with rhamnose and supplemented with cefixime and

potassium tellurite Suspect colonies of O26 appear brown

or red on this medium STEC O111 was best recovered on

chromocult agar supplemented with cefixime cefsulodin

and vancomycin colonies indicative of O111 appear purple

Before plating on selective agars O26 and O111 cells were

concentrated using immunomagnetic separation (IMS) The

authors concluded that the serotype-specific enrichment

broth IMS extraction and selective agar with serological

and biochemical confirmation testing are effective methods

for the recovery of these STEC serotypes (24)

Immunological methods IMS and plating is a highly

sensitive method currently used to detect E coli O157H7

and other organisms In this method microscopic iron-

cored beads are coated with specific antibodies to E coliO157H7 allowing for the organismrsquos capture when a

sample is passed over the beads The bead-cell complexes

are then captured using a magnetic concentrator The cells

can be removed from the beads and plated on agar such as

SMAC (100) IMS has also been used to detect STEC in

fecal samples from animals shedding low numbers of STEC

Currently IMS is being used in the detection of O26 O103

O111 and O145 STEC (11 53) Different kits for detection

of STEC have been developed but not all have been

validated (11)


In 2005 in a method comparison study by OrsquoHanlon et

al (82) IMS (Seiken Particles Denka Seiken Tokyo

Japan) was used to extract STEC O26 and O111 from

minced beef In this study PCR was compared with an IMS

culture method The IMS culture method involved IMS

plating on specially formulated media then latex aggluti-

nation using antibodies specific for O26 and O111 They

found that the IMS culture method was not able to detect

O26 when the contaminated minced beef was subjected to

freezing and frozen storage while the PCR method was able

to detect the O26 cells inoculated into the minced beef

samples handled under these conditions (82) This may be

due to reduction in numbers and stress of the organism

during freeze and thaw cycles (37 82) In 2004 Drysdale

et al (37) also worked with an IMS culture method for

detection of STEC O26 and O111 They found that the

recovery of O26 and O111 through IMS was affected by

enrichment protocol high numbers of background micro-

flora and physiological state of the organism (37) The

authors suggested that recovery may be improved by using

media with low nutrients such as buffered peptone water

instead of tryptic soy broth and by using higher enrichment

temperatures (37 39)Several commercial latex kits exist for certain serotypes

of non-O157 STEC of concern including O26 O91 O103

O111 O121 and O145 (3) Several studies have used these

kits to confirm these serotypes after recovery from foods

(24 37 82) There are several methods for determining

whether an E coli strain produces Stx A reversed-passive

latex agglutination test the VTEC-Screen lsquolsquoSeikenrsquorsquo (Denka

Seiken) uses Stx antibodies to detect Stx (31) The Vero

cell assay another reversed-passive latex agglutination test

works in a similar way but was found to be more sensitive

than the VTEC-Screen (68) A new version of these

methods is able to determine if suspect colonies are STEC

strains in less than 3 h (11) Stool samples can be screened

using a Shiga toxin enzyme immunoassay (Meridan

BioScience Cincinnati OH Ridascreen R-Biopharm

Darmstadt Germany) Some enzyme immunoassay meth-

ods may produce false-positive results when Pseudomonasaeruginosa is present (73) Another new method of STEC

detection is the Shiga toxin colony immunoblot (Roche

Diagnostics GmbH Mannheim Germany) used for detec-

tion of Shiga toxin The presence of Shiga toxin on a

primary isolation plate is detected by immunological or

nucleic acid probes The Premier-EHEC ELISA (Meridian

Bioscience Inc Cincinnati OH) detects Stx from samples

immunologically (11)

PCR and subtyping methods There is an enormous

amount of genetic diversity with STEC STEC strains have

been characterized by serotyping based on the O (Ohne)

antigen and the H (Hauch) antigen There are 174 O

antigens which are determined by the polysaccharide

portion of the cell wall lipopolysaccharide and 53 H

antigens determined by flagella protein A limited number

of laboratories are currently capable of E coli serotyping

(53) Phage typing for E coli O157H7 has been developed

and has been useful in conjunction with serotyping and

PFGE Development of phage typing for non-O157 STEC

has been reported (53)Subtyping is important in outbreak investigations and

these methods are extensively developed for E coliO157H7 PFGE is a widely applied method of subtyping

STEC In this method fragments of the bacterial chromo-

some generated by digestion with a restriction enzyme

selected to cut the DNA into about 20 to 25 pieces are

separated by electrophoresis The resulting patterns can be

compared to determine the relationships of isolates to one

another PFGE is believed to be the most discriminatory

identification method when compared with multilocus

sequence typing and repetitive-element PCR (53) PFGE

has made it possible for specific STEC serotypes to be

linked in outbreaks and the information gathered can be

compared across the PulseNet system in the United States

and other countries (53)Another subtyping method is multiple locus variable-

number tandem repeat analysis This method is based on the

occurrence of tandem duplications on stretches of DNA at

specific loci in the chromosome PCR is used to target these

loci (53) Multilocus sequence typing is based on variations

in nucleotide sequences of internal fragments of selected

housekeeping genes and has not been found to be effective

in finding diversity between STEC that was found using

PFGE

The GeneDiscCycler (PallGeneSystems Bruz France)

is a real-time PCR method that is able to identify several

serotypes of STEC (13) This technology offers simulta-

neous detection of stx1 stx2 eae and O-group antigens

O26 O103 O111 O145 and O157 (13 96) The protocol

for use of the GeneDiscCycler for EHEC determination

includes an enrichment step DNA extraction screening for

stx and eae genes and then serotyping (96) Studies have

successfully used the GeneDiscCycler to detect the specific

STEC strains from direct pure colonies on mixed plates

(13 14)Since 1998 in Germany all STEC infections must be

reported and stool samples are sent to the National

Reference Laboratory for subtyping Virulence patterns are

assessed using PCR to determine the presence of genes such

as stx1 and stx2 eae hly and katP Outbreak strains are

distinguished from other strains through PFGE typing and

plasmid profiling A case of STEC infection in Germany is

defined as a person having PCR-confirmed STEC from a

stool sample (101)In the United States in 2000 non-O157 STEC

infections became nationally notifiable and in 2006 the

CDC published recommendations for laboratory identifica-

tion of STEC (3) The CDC released updated recommen-

dations in late 2009 for diagnosis of STEC by clinical

laboratories They recommend that all stools submitted for

testing should be cultured on a selective agar for O157

STEC (eg sorbitol MacConkey agar or cefixime tellurite-

sorbitol MacConkey agar) and simultaneously be assayed

for non-O157 by testing for Shiga toxin using enzyme

immunoassay or PCR Clinical laboratories do not typically

identify non-O157 STEC Clinical samples from which

Shiga toxin or the genes encoding these toxins are found


but from which no O157 STEC was recovered should be

immediately forwarded to a state or local public health

laboratory for identification of the non-O157 STEC The

method used to isolate non-O157 STEC entails streaking

the Shiga toxinndashpositive broth on a relatively less selective

agar such as MacConkey agar SMAC or blood agar

Traditional media used to isolate enteric organisms (eg

xylose lysine desoxycholate agar and Salmonella-Shigellaagar) inhibit many E coli and are not recommended

Colonies suspected to be E coli may be tested using

commercial O-specific antisera for the top STEC serotypes

of importance (O26 O45 O103 O111 O121 and O145)

All non-O157 STEC isolates should be sent to the CDC or

a public health laboratory for confirmation and character-

ization (eg confirmation for Shiga toxin production

serotyping and PFGE subtyping) The CDC believes that

following these recommendations will lead to improved

patient outcome of infection with STEC as prompt

treatment may prevent serious complications (30) The

CDC suggests that there is a need for developing improved

isolation methods for non-O157 STEC and methods for

assessing the potential for the organisms to cause severe

illness (eg detecting the presence of stx2 certain stx2

subtypes and eae)

The importance of cultural confirmation is also

suggested by other researchers A study in which food

samples were screened for non-O157 STEC (O26 O103

O111 and O145) used a method based on enrichment of

samples in nutrient broth (modified EC broth with

novobiocin) then used a duplex 59-nuclease PCR assay

that targeted stx genes followed by a multiplex 59-nuclease

PCR test specific for the different serotypes (86) Pradel

et al (88) suggest that PCR may not be the best tech-

nique for identifying diarrheal illness caused by STEC

because the carriage of genes by an organism does not

guarantee their expression A food or stool sample may

carry multiple strains of STEC with multiple virulence

genes present Unless each organism is culturally

isolated PCR cannot distinguish between organisms to

determine if a single organism carries specific virulence

genes

CONTROL OF NON-O157 STEC IN FOODS(IN COMPARISION TO E COLI O157)

Several factors affect the growth and survival of STEC

in foods These include temperature pH salt content and

water activity (100) Comparisons of studies on recovery of

different STEC serotypes including O157 O26 and O111

suggest that these organisms may behave similarly physi-

ologically when stressed in foods (37)Proper cooking of ground beef and commercial thermal

pasteurization of milk has been shown to be effective in

eliminating E coli O157H7 from these foods (36 100)Currently there is no published data that suggest that non-

O157 STEC have greater heat resistance than O157

Preliminary data on heat resistance of STEC (serotypes

O26 O45 O103 O111 O121 and O145) in our

laboratories substantiate that in general the non-O157 STEC

serotypes of concern have heat resistance similar to or less

than that of O157 (41)Many hide carcass and trim interventions for beef

have been described that are based on elimination of E coliO157H7 in the final product Many of the methods are used

in combination as no intervention is 100 effective (69) In

2000 Cutter and Rivera-Betancourt (32) studied current

interventions used by the meat industry for reduction of

E coli O157H7 and Salmonella on beef surfaces and their

ability to eliminate non-O157 STEC serotypes O26H11 and

O111H8 This study found that E coli O26 and O111 were

reduced on meat surfaces using typical interventions which

included spray washing with water hot water (72uC) 2

acetic acid 2 lactic acid or 10 trisodium phosphate

to the same extent as O157H7 This result was shown

immediately after treatment and after long-term refrigerat-

ed and vacuum-packaged storage The authors concluded

that the interventions currently used by the meat industry to

reduce E coli O157 should be effective against other STEC

serotypes as well (32)A 2009 study by Gilbreth et al (48) tested the

effectiveness of antimicrobial treatments on E coliserotypes O157H7 O145 O111 and O26 on meat

surfaces Antimicrobial solutions were made using acidified

sodium chloride (1000 ppm) octanoic acid (9000 ppm)

and peracetic acid (200 ppm) The antimicrobial solutions

were sprayed for 15 s onto meat surfaces after cells were

applied and allowed 30 min for attachment Samples were

held for 2 h at 2 to 8uC neutralized and then sampled for

survivors by plating on SMAC No significant differences

were found in effectiveness of the different antimicrobials

between strains The authors concluded that the sensitivity

of O157 and non-O157 STEC are equivalent and levels of

antimicrobials used for control would not be different (48)Some studies on acid tolerance of EHEC have shown

that E coli O157H7 was more acid tolerant than other

EHEC strains (8) In 2005 Large et al (70) studied survival

rates of clinical isolates of STEC for the three major acid

resistance mechanisms of E coli the glutamate system the

oxidative system and the arginine system The clinically

isolated serotypes represented three clonal groups of STEC

EHEC clonal group 1 consisted of O157H7 and O157NM

EHEC clonal group 2 consisted of serotypes O26H11 and

O111H8 and the third group was made up of serotype

O121H19 Large et al found that the average survival rate

for the O157H7 clonal group was significantly less than

that of other STEC clones in the acid resistance mecha-

nisms The authors concluded that there was no evidence

that O157H7 has greater acid resistance in any of the single

systems than the other STEC clones They conceded that

there may be other mechanisms of E coli O157H7 that may

contribute to its acid resistance in natural settings (70)In 2004 Baylis et al (7) studied the survival of E coli

O157H7 O111NM and O26H11 in chocolate and

confectionery products during storage at different temper-

atures When chocolate was artificially contaminated with

STEC serotypes at high levels (104 CFUg) they found that

all three serotypes were able to survive storage at 38uC for

up to 43 days but after 90 days only E coli O26 and O111


could be recovered Chocolate with low levels (102 CFUg)

of contamination was stored at 10uC At this temperature Ecoli O157 O26 and O111 were detected at 12 months

After 12 months only E coli O26 and O111 were detected

in the chocolate Survival experiments in chocolate at other

temperatures and levels of contamination showed compara-

ble results for all three serotypes Dissimilar results were

seen in biscuit cream and mallow E coli O157H7 was

recovered after O26 and O111 were no longer detected

Very few strains of STEC were used in the study and the

authors suggest that the prolonged survival of non-O157

STEC strains in comparison to O157 may be strain

dependent instead of associated with serotype This study

found that survival of STEC was longer in products with

lower water activity or stored at lower temperatures than in

products with higher water activity or in storage at higher

temperatures (7)A 2005 study by Hiramatsu et al (59) determined the

desiccation tolerance of STEC on paper disks using 15

strains of O157 15 strains of O26 and 5 strains of O111

All serotypes survived on paper disks after 24 h of drying at

35uC The resistance to desiccation was not dependent on

serotype which indicates that interventions of drying used

for O157 may also work for other STEC serotypes (59)Presently there is no reason to believe that current

interventions in foods for the elimination of Salmonellaand E coli O157 would not be effective against non-O157

STEC (95)

PUBLIC HEALTH SIGNIFICANCE OFNON-O157 STEC

Certain serotypes of non-O157 STEC have been

repeatedly recognized as human pathogens able to cause

serious disease through foodborne contamination The

apparent increase in incidence of non-O157 STEC cases is

likely due to increased laboratory testing for Stx in cases of

diarrhea In 2000 non-O157 STEC infections became

nationally notifiable in the United States (3) As surveillance

for these organisms continues to increase more cases may

be detected (60) This does not necessarily mean that the

illnesses associated with non-O157 STEC are increasing

rather existing cases are being detected more often due to

more frequent testing than in the past (54)These organisms produce Stx which is a major

virulence factor of the established foodborne pathogen Ecoli O157 It has been shown that they are able to produce

other virulence factors as well some are common to E coliO157 Disease-causing serotypes of non-O157 STEC have

been shown to possess multiple combinations of these

virulence factors This lack of a uniform or consistent

pattern of virulence factors makes it extremely difficult to

clearly define pathogenic STEC based solely on serotype

(11 38) Scheutz (92) suggested that the definition of a

pathogenic STEC be based on virulence profile (Stx

production eae presence etc) instead of serotype

Several serotypes of non-O157 STEC dominate

outbreaks worldwide these include O26 O45 O103

O111 O121 and O145 (11) In the United States between

1983 and 2002 the most frequently reported STEC

serotypes of all non-O157 STECndashassociated outbreaks and

sporadic cases were O26 (22) O111 (16) O103 (12)

O121 (8) O45 (7) and O145 (5) (21) In the United

States Asia and Europe strains in serogroup O26 are the

second most frequently isolated outbreak-related STEC after

O157 In Europe another E coli serogroup O91 is ranked

in their top five non-O157 STEC serogroups most

frequently associated with human illness (72) Strains of

E coli O26 are second to E coli O157 as the most frequent

cause of HUS (44) In some reported outbreaks more than

one non-O157 STEC serotype was isolated (38)There have been 22 outbreaks in the United States

involving non-O157 STEC from 1990 to 2007 83 of the

illnesses in these outbreaks were foodborne These 22

outbreaks were attributed to O111 (10) O121 (5) O26 (3)

O45 (2) O104 (1) and O103 (1) (51) Seven of the 22 were

multipathogen outbreaks that involved non-O157 STEC

strains (O111 O121 or O26) and other pathogens including

norovirus Cryptosporidium and Vibrio species In some

reported cases a non-O157 STEC strain was isolated from

patients who had high levels of antibody to O157

lipopolysaccharide in serum This suggests that the patients

may have been coinfected with E coli O157 that was not

isolated but which may have caused the disease symptoms

(97) Many illnesses due to non-O157 STEC are sporadic

infections that occur typically in rural areas (99) This

suggests that in addition to food there appear to be other

vehicles such as contact with animals that may play a

significant role in transmission of non-O157 STEC

Several studies have characterized STEC isolated from

bovine sources and food samples and have determined that

the majority of STEC strains are either not pathogenic to

humans or are less virulent than E coli O157 (2 18 69 88)It has been shown that some strains of non-O157 STEC are

able to cause illness as severe as E coli O157 but the

majority of illnesses associated with non-O157 STEC have

been less severe and have resulted in fewer hospitalizations

(60 78 79 98)The significance of non-O157 STEC strains as

foodborne pathogens is still under debate as more

information is needed to determine their relative importance

(22 84 97) In two separate case-control studies non-O157

STEC strains were recovered at similar rates from patients

with illness and from the healthy controls (22 84) Several

studies have shown that control subjects without diarrhea

and patients with diarrhea had the same frequency of fecal

excretion of non-O157 STEC (22 34 65 84) In one

outbreak investigation involving E coli O111NM it was

determined that a high percentage (46) of people who

tested positive for E coli O111 IgM antibodies in their

serum experienced no clinical symptoms This suggests that

a high percentage of the population may ingest this

organism but experience no illness and may carry these

bacteria in their flora with no symptoms (29) The recovery

of non-O157 STEC from stool samples does not necessarily

mean that an illness occurred in association with that strain

of STEC In cases of illness in which stool samples tested

positive for Shiga toxin but not for E coli O157 non-O157


STEC strains have been assumed to be the cause In two

outbreaks one each in North Carolina and Virginia

illnesses initially were linked to non-O157 STEC and were

later determined to be caused by norovirus (3)

REGULATORY STATUS

In October 2007 FSIS the US Food and Drug

Administration (FDA) and CDC cosponsored a public

meeting to consider the public health significance of non-

O157 STEC In the spring of 2008 FSIS hosted another

public meeting on STEC which included discussion of

Agency plans for addressing non-O157 STEC The policy

strategy being considered at that time involved resolution

of laboratory testing methodology issues assessing the

magnitude of the problem by testing meat samples

determining the circumstances under which non-O157

STEC would be considered an adulterant and informing

stakeholders about that determination before implementa-

tion of the new policy

FSIS continues its collaboration with US Department

of Agriculture Agricultural Research Service on the

development and validation of detection methods for six

non-O157 STEC serotypes There has been no public

indication that an Agency study to determine the prevalence

of the non-O157 STEC of concern has been initiated as of

January 2010 indeed that study awaits finalization of

validated testing methods Researchers have suggested that

the pathogenicity of a non-O157 STEC strain may depend

on the individual organismrsquos virulence profile rather than

simply on its serotype so classification of certain non-O157

STEC strains as adulterants based on serotype alone should

be carefully considered (92)Neither the FSIS nor the FDA has yet established a

regulatory policy specific for non-O157 STEC however it

is clear that non-O157 STEC strains remain a top priority

for FSIS Additional pressure for Agency action is being

applied by consumer advocates as well as by an October

2009 petition to have FSIS administratively declare six non-

O157 STEC serotypes to be adulterants in raw beef

products as was done for E coli O157H7 in 1994 (20 56)It is apparent that some strains of non-O157 STEC

may cause human illness but many questions regarding

their pathogenicity remain Non-O157 STEC isolates

identical to strains associated with illness have been

recovered from asymptomatic patients (10 22 47 84)The industry has programs in place to control E coliO157H7 and based on current research these should be

effective in controlling non-O157 STEC as well In order

to support a practical science-based regulatory policy it is

critical to establish a molecular definition for pathogenic

non-O157 STEC and to further develop and validate a

reference method for pathogenic non-O157 STEC (20)Careful consideration of the relative scope and magnitude

of the public health risk from pathogenic non-O157 STEC

in beef and other products should also be quantified in a

risk assessment to help determine effective risk reduction

strategies and to support risk-based regulation if appro-

priate (20)

ACKNOWLEDGMENTS

The authors acknowledge the contributions from Virginia N Scott

(the Grocery Manufacturers Association [GMA] currently FDA Center for

Food Safety and Applied Nutrition) and from members of the GMA non-

O157 STEC task force in development of the manuscript

REFERENCES

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Safety and Inspection Service notice of public meeting non-

Escherichia coli O157H7 Shiga toxin-producing E coli Fed

Regist 7257285ndash57286

2 Arthur T M G A Barkocy-Gallagher M Riveria-Betancourt and

M Koohmaraie 2002 Prevalence and characterization of non-O157

Shiga toxin-producing Escherichia coli on carcasses in commercial

beef cattle processing plants Appl Environ Microbiol 684847ndash

4852

3 Atkinson R G Johnson T Root T Halse D Wroblewski M

Davies A Byrd L Long L Demma F Angulo C Bopp P

Gerner-Smidt N Strockbine K Greene B Swaminathan P

Griffin J Schaffzin and B Goode 2006 Importance of culture

confirmation of Shiga toxin-producing Escherchia coli infection as

illustrated by outbreaks of gastroenteritis New York and North

Carolina 2005 Morb Mortal Wkly Rep 551042ndash1045

4 Banatvala N M M Debeukelaer P M Griffin T J Barrett K D

Greene J H Green and J G Wells 1996 Shiga-like toxin-

producing Escherichia coli O111 and associated hemolytic-uremic

syndrome a family outbreak Pediatr Infect Dis J 151008ndash1011

5 Banatvala N P M Griffin K D Greene T J Barrett W F Bibb

J H Green and J G Wells 2001 The United States national

prospective hemolytic uremic syndrome study microbiologic

serologic clinical and epidemiologic findings J Infect Dis 183

1062ndash1070

6 Barkocy-Gallagher G A T M Arthur M Rivera-Betancourt X

Nou S D Shackelford T L Wheeler and M Koohmaraie 2003

Seasonal prevalence of Shiga toxin-producing Escherichia coli

including O157H7 and non-O157 serotypes and Salmonella in

commercial beef processing plants J Food Prot 661978ndash1986

7 Baylis C L S MacPhee A J Robinson R Griffiths K Lilley

and R P Betts 2004 Survival of Escherichia coli O157H7

O111H- and O26H11 in artificially contaminated chocolate and

confectionery products J Food Microbiol 9635ndash48

8 Benjamin M M and A R Datta 1995 Acid tolerance of

enterohemorrhagic Escherichia coli Appl Environ Microbiol 61

1669ndash1672

9 Berger S 2006 E coli VTEC non-O157 Lettuce USA (Utah)

Available at httplistuvmeducgi-binwaA3~ind0609aampL~-

SAFETYampE~7bitampP~94506ampB~--ampT~text2Fplain20charset~

US-ASCII Accessed 17 April 2009

10 Bettelheim K A 2001 Enterohaemorrhagic Escherichia coli

O157H7 a red herring J Med Microbiol 50201ndash202

11 Bettelheim K A 2007 The non-O157 Shiga-toxigenic (verocyto-

toxigenic) Escherichia coli under-rated pathogens Crit Rev

Microbiol 3367ndash87

12 Beutin L D Geier S Zimmermann S Aleksic H A Gillespie

and T S Whittam 1997 Epidemiological relatedness and clonal

types of natural populations of Escherichia coli strains producing

Shiga toxins in separate populations of cattle and sheep Appl

Environ Microbiol 632175ndash2180

13 Beutin L S Jahn and P Fach 2008 Direct rapid and reliable

detection of classical enterohaemorrhagic E coli (EHEC) from

single colonies with the lsquolsquoGeneDiscrsquorsquo real-time PCR system

Bundesinstitut fur Risikobewertung (BfR Federal Institute for

Risk Assessment) Berlin

14 Beutin L S Jahn and P Fach 2009 Evaluation of the lsquoGeneDiscrsquo

real-time PCR system for detection of enterohaemorrhagic Esche-

richia coli (EHEC) O26 O103 O111 O145 and O157 strains

according to their virulence markers and their O- and H-antigen-

associated genes J Appl Microbiol 1061122ndash1132


15 Beutin L U Kruger G Krause A Miko A Martin and

E Strauch 2008 Evaluation of major types of Shiga toxin 2e-

producing Escherichia coli bacteria present in food pigs and the

environment as potential pathogens for humans Appl Environ


16 Bielaszewska M 2007 German experience with non-O157 STEC

Presented at the Public Health Significance of Non-O157 Shiga

Toxin-Producing Escherichia coli (STEC) Public Meeting Wash-

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17 Bielaszewska M M F L Greune R Prager A Fruth H Tschape

M A Schmidt and H Karch 2004 Characterization of cytolethal

distending toxin genes and expression in Shiga toxin-producing

Escherichia coli strains of non-O157 serogroups Infect Immun 72

1812ndash1816

18 Boerlin P S A McEwen F Boerlin-Petzold J B Wilson R P

Johnson and C L Gyles 1999 Associations between virulence

factors of Shiga toxin-producing Escherichia coli and disease in

humans J Clin Microbiol 37497ndash503

19 Bopp C 2008 Non-O157 Shiga toxin-producing Escherichia coli

isolation and detection challenges Centers for Disease Control and

Prevention Available at wwwfsisusdagovPPTNon-0157_STEC_

Boppppt Accessed 19 October 2009

20 Brackett R E 2008 Grocery Manufacturers Association comments

to Docket no FSIS-2008-0011 Shiga toxin-producing E coli public

meeting 72 FR 18257 3 April 2008 Available at httpwwwfsis

usdagovOPPDEComments2008-00112008-0011-5pdf Accessed

8 January 2010

21 Brooks J T E G Sowers J G Wells K D Greene P M

Griffin R H Hoekstra and N A Strockbine 2005 Non-O157

Shiga toxin-producing Escherichia coli infections in the United

States 1983ndash2002 J Infect Dis 1921422ndash1429

22 Brown J E P Exheverria D N Taylor J Seriwatana V

Vanapruks U Lexomboon R N Neill and J W Newland 1989

Determination by DNA hybridization of Shiga-like-toxin-producing

Escherichia coli in children with diarrhea in Thailand J Clin


23 Caprioli A A E Tozzi G Rizzoni and H Karch 1997 Non-

O157 Shiga toxin-producing Escherichia coli infections in Europe

Emerg Infect Dis 3578ndash579 (Letter)

24 Catarame T M G K A OrsquoHanlon G Duffy J J Sheridan I S

Blair and D A McDowell 2003 Optimization of enrichment and

plating procedures for the recovery of Escherichia coli O111 and

O26 from minced beef J Appl Microbiol 95949ndash957

25 Centers of Disease Control and Prevention 1995 Community

outbreak of hemolytic uremic syndrome attributable to Escherichia

coli O111NM South Australia 1995 Morb Mortal Wkly Rep 44

550ndash551 557ndash558

26 Centers of Disease Control and Prevention 1995 Outbreak of acute

gastroenteritis attributable to Escherichia coli serotype O104H21

Helena Montana 1994 Morb Mortal Wkly Rep 44501ndash503

27 Centers of Disease Control and Prevention 2000 Escherichia coli

O111H8 outbreak among teenage campers Texas 1999 Morb

Mortal Wkly Rep 49321ndash324

28 Centers of Disease Control and Prevention 2006 Summary of

notifiable disease United States 2004 Morb Mortal Wkly Rep

531ndash79

29 Centers of Disease Control and Prevention 2008 Preliminary

FoodNet data on the incidence of infection with pathogens

transmitted commonly through food 10 states 2007 Morb Mortal

Wkly Rep 57366ndash370

30 Centers of Disease Control and Prevention 2009 Recommenda-

tions for diagnosis of Shiga toxin-producing Escherichia coli

infections by clinical laboratories Morb Mortal Wkly Rep 58

1ndash14

31 Chart H G A Willshaw and T Cheasty 2001 Evaluation of a

reversed passive latex agglutination test for the detection of

verocytotoxin (VT) expressed by strains of VT-producing Esche-

richia coli Lett Appl Microbiol 32370ndash374

32 Cutter C N and M Rivera-Betancourt 2000 Interventions for the

reduction of Salmonella Typhimurium DT 104 and non-O157H7

enterohemorrhagic Escherichia coli on beef surfaces J Food Prot

631326ndash1332

33 De Schrijver K G Buvens B Posse D Van den Branden C

Oosterlynck L De Zutter K Eilers D Peirard K Dierick R Van

Damme-Lombaerts C Lauwers and R Jacobs 2008 Outbreak of

verocytotoxin-producing E coli O145 and O26 ingections associ-

ated with the consumption of ice cream produced at a farm

Belgium 2007 Available at httpwwweurosurveillanceorg

editionv13n07080214_5asp Accessed 15 February 2008

34 de Wit M S M P G Koopmans L M Kortbeek N J van

Leeuwen A I M Bartelds and Y T H P van Duynhoven 2001

Gastroenteritis in sentinel general practices the Netherlands Emerg

Infect Dis 782ndash91

35 Doyle M P and J L Schoeni 1987 Isolation of Escherichia coli

O157H7 from retail fresh meats and poultry Appl Environ


36 Doyle M P T Zhao J Meng and S Zhao 1997 Escherichia coli

O157H7 p 171ndash191 In M P Doyle L R Beuchat and T J

Montville (ed) Food microbiology fundamentals and frontiers

ASM Press Washington DC

37 Drysdale M M MacRae N J C Strachan T M S Reid and I

D Ogden 2004 The detection of non-O157 E coli in food by

immunomagnetic separation J Appl Microbiol 97220ndash224

38 Eblen D R 2007 Public health importance of non-O157 Shiga

toxin-producing Escherichia coli (non-O157 STEC) in the US food

supply US Department of Agriculture Food Safety and Inspec-

tion Service Available at httpwwwfsisusdagovPDFSTEC_

101207pdf Accessed 15 December 2008

39 Edel W and E H Kampelmacher 1973 Comparative studies on

the isolation of lsquolsquosub-lethally injuredrsquorsquo salmonellae in nine European

laboratories Bull WHO 48167ndash174

40 Enache E and E C Mathusa 2009 Personal communication

41 Enache E E C Mathusa P Elliott and G Black 2009 Thermal

resistance parameters for Shiga toxin-producing Escherichia coli in

apple juice p 88 P1-72 Poster abstract presented at the 96th Annu

Meet IAFP Grapevine TX 12 to 15 July 2009

42 Erickson M C and M P Doyle 2007 Food as a vehicle for

transmission of Shiga toxin-producing Escherichia coli J Food

Prot 702426ndash2449

43 Ethelberg S B Smith M Torpdahl M Lisby J Boel T Jensen

and K Molbak 2007 An outbreak of verocytotoxin-producing

Escherichia coli O26H11 caused by beef sausage Denmark 2007

Euro Surveill 12 Available at httpwwweurosurveillanceorg

ViewArticleaspxArticleId~3208 Accessed 26 March 2009


E M Nielsen and K Molbak 2009 Outbreak of non-O157 Shiga

toxin-producing Escherichia coli infection from consumption of

beef sausage Clin Infect Dis 48e78ndashe81

45 Feng P 2009 Analytical methods the needs the reality and the

perspectives Presented at the 96th Annu Meet IAFP Grapevine

TX 12 to 15 July 2009

46 Feng P and S D Weagant 2002 Diarrheagenic Escherichia coli

chap 4a In FDA bacteriological analytical manual online 8th ed

US Food and Drug Administration Washington DC

47 Friedrich A W M Bielaszewska W Zhang M Pulz T Kuczuis

A Ammon and H Karch 2002 Escherichia coli harboring Shiga

toxin 2 gene variants frequency and association with clinical

symptoms J Infect Dis 18574ndash84

48 Gilbreth S E T C Podtburg and P W Bodnaruk 2009 Survival

of non-O157H7 STEC on beef tissue surface following spray

treatment with different antimicrobials p 2ndash31 Presented at the

96th Annu Meet IAFP Grapevine TX 12 to 15 July 2009

49 Glass K C Kasper A Milkowski J Sindelar B Lotz J Kang N

Faith E Enache and A Kataoka 2009 Validation of pepperoni

process for control of Shiga toxin-producing E coli (STEC)

Personal communication unpublished work

50 Goldman D 2010 Transcript FSIS role in PulseNet FSIS product

tracing related to E coli O157H7 from the lab perspective

Presented at the FSIS Product Tracing Related to E coli O157H7

Improving Product Tracing in Food Public Meeting Washington


DC 10 March 2010 Available at wwwfsisusdagovPDF

Transcript_031010_Traceabilitypdf Accessed 20 April 2010

51 Gould H 2009 Update on the epidemiology of STEC in the United

States Centers for Disease Control and Prevention Presented at the

2009 Annual Capital Area Food Protection Association lsquolsquoNon-O157

STEC Waiting for the other shoe to droprsquorsquo Washington DC 15

September 2009

52 Griffin P 2007 CDC perspective on non-O157 Shiga toxin-

producing E coli (STEC) in the United States Presented at the

Public Health Significance of Non-O157 Shiga toxin-producing

Escherichia coli (STEC) Public Meeting Arlington VA 17

October 2007

53 Gyles C L 2006 Shiga toxin-producing Escherichia coli a review

J Anim Sci 8545ndash62

54 Hagan E 2008 Non-O157 STEC what we know and whatrsquos next

Presented at the Shiga Toxin-Producing E coli Addressing the

Challenges Moving Forward with Solutions Public Meeting

Washington DC 9 to 10 April 2008

55 Halvorson H O and N R Ziegler 1933 Applications of statistics

to problems in biology J Bacteriol 25101ndash121

56 Healy A 2009 Marler petitions USDA to declare more strains of E

coli adulterants Food Chem News p 51

57 Hedican E B C Medus J M Besser B A Juni B Koziol C

Taylor and K E Smith 2009 Characteristics of O157 versus non-

O157 Shiga toxin-producing Escherichia coli infections in Minne-

sota 2000ndash2006 Clin Infect Dis 49358ndash364

58 Hiramatsu R M Matsumoto Y Miwa Y Suzuki M Saito and

Y Miyazaki 2002 Characterization of Shiga toxin-producing

Escherichia coli O26 strains and establishment of selective isolation

media for these strains J Clin Microbiol 40922ndash925

59 Hiramatsu R M Matsumoto K Sakae and Y Miyazaki 2005

Ability of Shiga toxin-producing Escherichia coli and Salmonella

spp to survive in a desiccation model system and in dry foods Appl


60 Hurd S 2007 The Connecticut experience with non-O157 STEC

lsquolsquoSeek and ye shall findrsquorsquo Presented at the Public Health

Significance of Non-O157 Shiga toxin-producing Escherichia coli

(STEC) Public Meeting Arlington VA 17 October 2007

61 Hussein H S 2007 Ongoing research and outreach efforts targeted

at non-O157 STEC Presented at the Public Health Significance of

Non-O157 Shiga toxin-producing Escherichia coli (STEC) Public

Meeting Arlington VA 17 October 2007

62 Hussein H S 2007 Prevalence and pathogenicity of Shiga toxin-

producing Escherichia coli in beef cattle and their products J Anim

Sci 85E63ndashE72

63 Hussein H S and T Sakuma 2005 Invited review prevalence of

Shiga toxin-producing Escherchia coli in dairy cattle and their

products J Dairy Sci 88450ndash465

64 International Commission on Microbiological Specifications for

Food 2005 Meat and meat products p 1ndash106 In Microorganisms

in foods 6 microbial ecology of food commodities ICMSF New

York

65 Jelacic J K T Damrow G S Chen S Jelacic M Bielaszewski M

Ciol H M Carvalho A R Melton-Celsa A D OrsquoBrien and P I

Tarr 2003 Shiga toxin-producing Escherichia coli in Montana

bacterial genotypes and clinical profiles J Infect Dis 188719ndash729

66 Johnson K E C M Thorpe and C L Sears 2006 The emerging

clinical importance of non-O157 Shiga toxin-producing Escherichia

coli Clin Infect Dis 431587ndash1595

67 Karmali M A M Mascarenhas S Shen K Ziebell S Johnson

R Reid-Smith J Issac-Renton C Clark K Rahn and J B Kaper

2003 Association of genomic O island 122 Escherichia coli EDL

933 with verocytotoxin-producing Escherichia coli seropathotypes

that are linked to epidemic andor serious disease J Clin Microbiol

414930ndash4940

68 Konowalchuk J J I Speirs and S Starvric 1977 Vero response

to a cytotoxin of Escherichia coli Infect Immun 18775ndash779

69 Koohmaraie M T M Arthur J M Bosilevac M Guerini S D

Shackelford and T L Wheeler 2005 Post-harvest interventions to

reduceeliminate pathogens in beef Meat Sci 7179ndash91

70 Large T M S T Walk and T S Whittam 2005 Variation in acid

resistance among Shiga toxin-producing clones of pathogenic

Escherichia coli Appl Environ Microbiol 712493ndash2500

71 Lynn R M S J OrsquoBrien C M Taylor G K Adak H Chart

T Cheasty J E Coia I A Gillespie M E Locking W J Reilly

H R Smith A Waters and G A Willshaw 2005 Childhood

hemolytic uremic syndrome United Kingdom and Ireland Emerg


72 Madic J C Lecureuil F Dilasser S Derzelle E Jamet P Fach

and F Auvray 2009 Screening of food raw materials for the

presence of Shiga toxin-producing Escherichia coli O91H21 Lett

Appl Microbiol 48447ndash451

73 Manning S D R T Madera W Schneider S E Dietrich W

Khalife W Brown T S Whittam P Somsel and J T Rudrik

2007 Surveillance for Shiga toxin-producing Escherichia coli

Michigan 2001ndash2005 Emerg Infect Dis Available at httpwww

cdcgovEIDcontent132318htm Accessed 4 December 2008

74 March S B and S Ratnam 1986 Sorbitol-MacConkey medium

for detection of Escherichia coli O157H7 associated with

hemorrhagic colitis J Clin Microbiol 23869ndash872

75 McCarthy T A N L Barrett J L Hadler B Salsbury R T

Howard D W Dingman C D Brinkman W F Bibb and M L

Cartter 2001 Hemolytic-uremic syndrome and Escherichia coli

O121 at a lake in Connecticut 1999 Pediatrics 10859ndash65

76 McPherson M K Lalor B Combs J Raupach R Stafford and

M D Kirk 2009 Serogroup-specific risk factors for Shiga toxin-

producing Escherichia coli infection in Australia Clin Infect Dis

49249ndash256

77 Mead P S L Slutsker V Dietz L F McCaig J S Bresee C

Shapiro P M Griffin and R V Tauxe 1999 Food-related illness

and death in the United States Emerg Infect Dis 5607ndash625

78 Nataro J P and J B Kaper 1998 Diarrheagenic E coli Clin

Microbiol Rev 11142ndash201

79 Neill M A 1997 Overview of verotoxigenic Escherichia coli J

Food Prot 601444ndash1446

80 Newton H J J Sloan D M Bulach T Seemann C C Allison

M Tauschek R M Robins-Browne J C Paton T S Whittam A

W Paton and E L Hartland 2009 Shiga toxin-producing

Escherichia coli strains negative for locus of enterocyte effacement

Emerg Infect Dis 15372ndash380

81 Noris M and G Remuzzi 2005 Hemolytic uremic syndrome J

Am Soc Nephrol 161035ndash1050

82 OrsquoHanlon K A T M G Catarame I S Blair D A McDowell

and G Duffy 2005 Comparison of a real-time PCR and an IMS

culture method to detect Escherichia coli O26 and O111 in minced

beef in the Republic of Ireland Food Microbiol 22553ndash560

83 Oklahoma State Department of Health 2009 Epidemiological

investigation of restaurant-associated Escherichia coli O111NM

outbreakmdashMayes County Oklahoma 2008 final report Available at

httpwwwcrossroadsodlstateokuscgi-binshowfileexeCISOROOT~

stgovpubampCISOPTR~8805ampfilename~9063pdf Accessed 13 April

2009

84 Paton A W J C Paton P N Goldwater and P A Manning

1993 Direct detection of Escherichia coli Shiga-like toxin genes in

primary fecal cultures by polymerase chain reaction J Clin


85 Paton A W R M Ratcliff R M Doyle J Seymour-Murray D

Davos J A Lanser and J C Paton 1996 Molecular microbio-

logical investigation of an outbreak of hemolytic-uremic syndrome

caused by dry fermented sausage contaminated with Shiga-like

toxin-producing Escherichia coli J Clin Microbiol 341622ndash1627

86 Perelle S F Dilasser J Grout and P Fach 2006 Screening food

raw materials for the presence of the worldrsquos most frequent clinical

cases of Shiga toxin-encoding Escherichia coli O26 O103 O111

O145 and O157 Int J Food Microbiol 113284ndash288

87 Posse B L De Zutter M Heyndrickx and L Herman 2008

Novel differential and confirmation plating media for Shiga toxin-

producing Escherichia coli serotypes O26 O103 O111 O145 and

sorbitol-positive and -negative O157 FEMS Microbiol Lett 282

124ndash131


88 Pradel N V Livrelli C de Champs J B Palcoux A Reynaud F

Scheutz J Sirot B Joly and C Forestier 2000 Prevalence and

characterization of Shiga toxin-producing Escherichia coli isolated

from cattle food and children during a one-year prospective study

in France J Clin Microbiol 381023ndash1031

89 Riley L W R S Remis S D Helgerson H B McGee J G

Wells B R Davis R J Herbert E S Olcott L M Johnson N G

Hargrett P A Blake and M L Cohen 1983 Hemorrhagic colitis

associated with a rare Escherichia coli serotype N Engl J Med

308681ndash685

90 Ruggenenti P M Noris and G Remuzzi 2001 Thrombotic

microangiopathy hemolytic uremic syndrome and thrombic

thrombocytopenic purpura Kidney Int 60831ndash846

91 Samadpour M J E Ongerth J Liston N Tran D Nguyen T S

Whittam R A Wilson and P I Tarr 1994 Occurrence of Shiga-

like toxin-producing Escherichia coli in retail fresh seafood beef

lamb pork and poultry from grocery stores in Seattle Washington

Appl Environ Microbiol 601038ndash1040

92 Scheutz F 2007 Experiences with non-O157 STEC and implica-

tions on public health programs Presented at the Public Health

Significance of Non-O157 Shiga Toxin-Producing Escherichia coli


93 Schimmer B K Nygard H M Eriksin J Lassen B A Lindstedt

L T Brandal G Kapperud and P Aavitsland 2008 Outbreak of

haemolytic uraemic syndrome in Norway caused by stx2-positive

Escherichia coli O103H25 traced to cured mutton sausages BMCInfect Dis 841 Available at httpwwwbiomedcentralcom1471-

2334841 Accessed 18 April 2009

94 Schmidt H B Henkel and H Karch 1997 A gene cluster closely

related to type II secretion pathway operons of gram-negative

bacteria is located on the large plasmid of enterohemorrhagic

Escherichia coli O157 strains FEMS Microbiol Lett 148265ndash

272

95 Scott V N 2007 Food industry perspective on non-O157 STEC


Toxin-Producing Escherichia coli (STEC) Public Meeting Arling-

ton VA 17 October 2007

96 Spurling D T Orchard and P Arbault 2008 PCR by Pall

GeneSystems one step further to the holy grail conquest Presented

to Grocery Manufacturers Association by PallGeneSystems

97 Tarr P I and M A Neill 1996 Perspective the problem of non-

O157H7 Shiga toxin (verocytotoxin)-producing Escherichia coli JInfect Dis 1741136ndash1139

98 Tauxe R V 2008 E coli O157H7 illness trends and recent data

from outbreak investigations United States Presented at the Shiga

Toxin-Producing E coli Addressing the Challenges Moving

Forward with Solutions Public Meeting Washington DC 9 to 10

April 2008

99 Twardon J B Sobieszczanska A Gonet and M Blaszkoska

2005 Epidemiology of Shiga-like toxin-producing Escherichia colistrains (STEC) Electron J Pol Agric Univ 8 Available at http

wwwejpaumediaplvolume8issue4art-03html Accessed 28 Sep-

tember 2007

100 US Department of Agriculture Food Safety and Inspection

Service 2001 Risk assessment of E coli O157H7 in ground beef

Available at httpwwwfsisusdagovScienceRisk_Assessments

indexaspecoli Accessed 1 April 2008

101 Werber D A Fruth A Liesegang M Littmann U Buchholz R

Prager H Karch T Breuer H Tschape and A Ammon 2002 A

multistate outbreak of Shiga toxin-producing Escherchia coli

O26H11 infections in Germany detected by molecular subtyping

surveillance J Infect Dis 186419ndash422

102 Willshaw G A T Cheasty and H R Smith 2000 Escherichia

coli p 1136ndash1177 In B M Lund T C Baird-Parker and G W

Gould (ed) The microbiological safety and quality of food vol 2

Aspen Publishers Gaithersburg MD

103 Zhang W M Bielaszewska A Liesegang H Tschape H

Schmidt M Bitzan and H Karch 2000 Molecular characteristics

and epidemilogical significance of Shiga toxin-producing Esche-

richia coli O26 strains J Clin Microbiol 382134ndash2140


ILLNESSES AND OUTBREAKS ATTRIBUTED TONON-O157 STEC

In the United States active surveillance of infections

attributed to non-O157 STEC began in 2001 (29) The

number of non-O157 STEC infections reported in the

United States between 2000 and 2005 increased from 171 to

501 cases suggesting a higher burden of illness than

previously thought (1) An increase in testing for Stx in

diarrheal cases and for STEC serotypes other than O157 is

likely the reason for the increase in incidence of cases and

outbreaks attributed to non-O157 STEC (60) Human

infections with STEC can occur with ingestion of

contaminated food or water or by direct contact with

animals Transmission can also occur through person-to-

person contact (53)Illnesses reported due to STEC serotypes other than

O157 are on the rise worldwide (78) It is estimated that 20

to 50 of all STEC infections can be attributed to non-

O157 strains but the percentages differ greatly from country

to country and among regions within a country (66 78) It is

estimated that less than 10 of HUS cases in North

America are caused by non-O157 STEC strains (52) In

Germany Italy and the United Kingdom it is estimated that

non-O157 STEC strains have caused 10 to 30 of sporadic

cases of HUS (23) Estimating the true percentage of

infections caused by non-O157 STEC strains is difficult

because these strains are not routinely subject to testing

(78) There is currently no convenient method that can

reliably screen for non-O157 STEC strains which compli-

cates the determination of incidence of disease due to these

organisms (78)There have been at least 22 outbreaks attributed to non-

O157 STEC strains in the United States since 1990 (19 3851) The sources for the non-O157 STEC strains were not

determined in some of these outbreaks and the vehicles

included foods and nonfoods (Table 1) Food vehicles

implicated in the outbreaks included milk (26) salad bar

(27) punch (21) apple cider (19) and iceberg lettuce (951) Mead et al (77) estimate that in the United States Ecoli O157H7 causes 73000 illnesses annually and non-

O157 STEC strains cause at least 37000 illnesses annually

Illnesses attributed to non-O157 STEC strains are most

frequently reported in the summer months (21)In 1994 there was an outbreak involving postpasteur-

ization contaminated milk with 11 confirmed and seven

suspected cases of illness Sixteen of the patients developed

bloody stools diarrhea and abdominal cramps Isolates

from three patients were identified as E coli O104H21 and

were positive for Stx2 A confirmed case was defined as

acute infection with E coli O104H21 isolated from patient

stool with serological confirmation Based on a case-control

study one brand of milk was significantly associated with

the illnesses The incident strain could not be isolated from

samples taken from the dairy at which the milk was

produced (26)In 1999 an outbreak of E coli O111H8 associated

with ice and salad from a salad bar occurred at a camp in

Texas Of 521 campers interviewed 58 had symptoms that

met the definition of illness either bloody diarrhea or

nonbloody diarrhea accompanied by abdominal cramps and

occurring within 14 days of the start of camp Two patients

developed HUS The meal served on the first night of camp

was significantly associated with development of illness

Several possible food items were identified but two were

significantly and independently associated with illness ice

and salad from the salad bar E coli O111H8 was isolated

from 2 of the 11 stool specimens submitted by ill patients

PCR was used to determine that both stx1 and stx2 were

present No food samples from the indicated meal were

available for testing (27)According to a newspaper report that was cited in the

review by Eblen (38) E coli O121H19 was linked to an

outbreak of illness associated with iceberg lettuce from a

fast food restaurant in 2006 Of 73 people who became ill

three developed kidney failure Iceberg lettuce was

pinpointed by local health officials because it was the only

common food among sickened patients The iceberg lettuce

was not tested Before the outbreak occurred the implicated

restaurant passed a health inspection and no issues were

discovered when another health inspection was performed

during the outbreak (9 38)To date there are no conclusive epidemiological data

that link meat products to non-O157 STEC illness in the

United States In 2006 there were two individual cases of

illness in the United States involving ground beef that may

have been due to non-O157 STEC but efforts to pinpoint

the source and the pathogen were inconclusive The first

involved a patient who consumed undercooked ground beef

An indistinguishable strain of E coli O103 was detected

using pulsed-field gel electrophoresis (PFGE) from both the

patient and leftover ground beef The original source of Ecoli O103 in this case was left undetermined because of

possible cross-contamination of a meat grinder (38) In the

second case a patient ill with E coli O157 provided ground

beef samples in which Stx was present but from which no

O157 could be recovered When the ground beef sample

was sent to the Centers for Disease Control and Prevention

(CDC) for characterization E coli O6H34 was found but

the ground beef could not be confirmed as the source of

illness (38)Recently in 2008 there was an outbreak attributed to

E coli O111NM in Locust Grove Oklahoma that involved

341 cases of illness 70 hospitalizations and 1 death The

outbreak was linked to a local restaurant but the

contamination route to the restaurant andor food source

remain undetermined Cross-contamination of food through

handling surface contact and storage areas was likely It

was reported that several employees of the restaurant

worked on days they experienced diarrhea but the strain

of E coli was not isolated from employee stool specimens

Clinical specimens were tested for Stx using a Shiga toxin

enzyme immunoassay screened for stx1 and stx2 genes

using real-time PCR and typed by PFGE Six Xbal PFGE

patterns were determined among E coli O111 isolates

These patterns were uploaded to the national PulseNet

database The state investigation of this outbreak found

many asymptomatic infections of E coli O111 in which
























































































O111 (19) O145 (5) and O45 (4) (57) The









































herbs (76)




































certain foods (85)




















































itself (78)

















































































































(78)















































































































































































































(31) O103 (36) O121 (18) O45 (201) O111


























Pork 18

Chicken 12

Turkey 7

Lamb 48

Fish 10

Shellfish 5





real-time PCR



88Cheese 1


ground beef samples


50















































































































validated (11)
















































































PFGE























































subtypes and eae)

















genes












O26 O45 O103 O111 O121 and O145) in our















































































STEC (95)























































































REGULATORY STATUS















































priate (20)

ACKNOWLEDGMENTS





REFERENCES









4852
















1062ndash1070












1669ndash1672







































1812ndash1816













8 January 2010





























531ndash79








1ndash14








631326ndash1332

















































TX 12 to 15 July 2009



























September 2009





October 2007

































Sci 85E63ndashE72







York













414930ndash4940

































49249ndash256
























2009


















124ndash131











308681ndash685






















272














April 2008




tember 2007









































































































O111 (19) O145 (5) and O45 (4) (57) The









































herbs (76)




































certain foods (85)




















































itself (78)

















































































































(78)















































































































































































































(31) O103 (36) O121 (18) O45 (201) O111


























Pork 18

Chicken 12

Turkey 7

Lamb 48

Fish 10

Shellfish 5





real-time PCR



88Cheese 1


ground beef samples


50















































































































validated (11)
















































































PFGE























































subtypes and eae)

















genes












O26 O45 O103 O111 O121 and O145) in our















































































STEC (95)























































































REGULATORY STATUS















































priate (20)

ACKNOWLEDGMENTS





REFERENCES









4852
















1062ndash1070












1669ndash1672







































1812ndash1816













8 January 2010





























531ndash79








1ndash14








631326ndash1332

















































TX 12 to 15 July 2009



























September 2009





October 2007

































Sci 85E63ndashE72







York













414930ndash4940

































49249ndash256
























2009


















124ndash131











308681ndash685






















272














April 2008




tember 2007














































O111 (19) O145 (5) and O45 (4) (57) The









































herbs (76)




































certain foods (85)




















































itself (78)

















































































































(78)















































































































































































































(31) O103 (36) O121 (18) O45 (201) O111


























Pork 18

Chicken 12

Turkey 7

Lamb 48

Fish 10

Shellfish 5





real-time PCR



88Cheese 1


ground beef samples


50















































































































validated (11)
















































































PFGE























































subtypes and eae)

















genes












O26 O45 O103 O111 O121 and O145) in our















































































STEC (95)























































































REGULATORY STATUS















































priate (20)

ACKNOWLEDGMENTS





REFERENCES









4852
















1062ndash1070












1669ndash1672







































1812ndash1816













8 January 2010





























531ndash79








1ndash14








631326ndash1332

















































TX 12 to 15 July 2009



























September 2009





October 2007

































Sci 85E63ndashE72







York













414930ndash4940

































49249ndash256
























2009


















124ndash131











308681ndash685






















272














April 2008




tember 2007

































































itself (78)

















































































































(78)















































































































































































































(31) O103 (36) O121 (18) O45 (201) O111


























Pork 18

Chicken 12

Turkey 7

Lamb 48

Fish 10

Shellfish 5





real-time PCR



88Cheese 1


ground beef samples


50















































































































validated (11)
















































































PFGE























































subtypes and eae)

















genes












O26 O45 O103 O111 O121 and O145) in our















































































STEC (95)























































































REGULATORY STATUS















































priate (20)

ACKNOWLEDGMENTS





REFERENCES









4852
















1062ndash1070












1669ndash1672







































1812ndash1816













8 January 2010





























531ndash79








1ndash14








631326ndash1332

















































TX 12 to 15 July 2009



























September 2009





October 2007

































Sci 85E63ndashE72







York













414930ndash4940

































49249ndash256
























2009


















124ndash131











308681ndash685






















272














April 2008




tember 2007








































































(78)















































































































































































































(31) O103 (36) O121 (18) O45 (201) O111


























Pork 18

Chicken 12

Turkey 7

Lamb 48

Fish 10

Shellfish 5





real-time PCR



88Cheese 1


ground beef samples


50















































































































validated (11)
















































































PFGE























































subtypes and eae)

















genes












O26 O45 O103 O111 O121 and O145) in our















































































STEC (95)























































































REGULATORY STATUS















































priate (20)

ACKNOWLEDGMENTS





REFERENCES









4852
















1062ndash1070












1669ndash1672







































1812ndash1816













8 January 2010





























531ndash79








1ndash14








631326ndash1332

















































TX 12 to 15 July 2009



























September 2009





October 2007

































Sci 85E63ndashE72







York













414930ndash4940

































49249ndash256
























2009


















124ndash131











308681ndash685






















272














April 2008




tember 2007









































































































































































(31) O103 (36) O121 (18) O45 (201) O111


























Pork 18

Chicken 12

Turkey 7

Lamb 48

Fish 10

Shellfish 5





real-time PCR



88Cheese 1


ground beef samples


50















































































































validated (11)
















































































PFGE























































subtypes and eae)

















genes












O26 O45 O103 O111 O121 and O145) in our















































































STEC (95)























































































REGULATORY STATUS















































priate (20)

ACKNOWLEDGMENTS





REFERENCES









4852
















1062ndash1070












1669ndash1672







































1812ndash1816













8 January 2010





























531ndash79








1ndash14








631326ndash1332

















































TX 12 to 15 July 2009



























September 2009





October 2007

































Sci 85E63ndashE72







York













414930ndash4940

































49249ndash256
























2009


















124ndash131











308681ndash685






















272














April 2008




tember 2007





























































(31) O103 (36) O121 (18) O45 (201) O111


























Pork 18

Chicken 12

Turkey 7

Lamb 48

Fish 10

Shellfish 5





real-time PCR



88Cheese 1


ground beef samples


50















































































































validated (11)
















































































PFGE























































subtypes and eae)

















genes












O26 O45 O103 O111 O121 and O145) in our















































































STEC (95)























































































REGULATORY STATUS















































priate (20)

ACKNOWLEDGMENTS





REFERENCES









4852
















1062ndash1070












1669ndash1672







































1812ndash1816













8 January 2010





























531ndash79








1ndash14








631326ndash1332

















































TX 12 to 15 July 2009



























September 2009





October 2007

































Sci 85E63ndashE72







York













414930ndash4940

































49249ndash256
























2009


















124ndash131











308681ndash685






















272














April 2008




tember 2007






























































































































validated (11)
















































































PFGE























































subtypes and eae)

















genes












O26 O45 O103 O111 O121 and O145) in our















































































STEC (95)























































































REGULATORY STATUS















































priate (20)

ACKNOWLEDGMENTS





REFERENCES









4852
















1062ndash1070












1669ndash1672







































1812ndash1816













8 January 2010





























531ndash79








1ndash14








631326ndash1332

















































TX 12 to 15 July 2009



























September 2009





October 2007

































Sci 85E63ndashE72







York













414930ndash4940

































49249ndash256
























2009


















124ndash131











308681ndash685






















272














April 2008




tember 2007

































































































PFGE























































subtypes and eae)

















genes












O26 O45 O103 O111 O121 and O145) in our















































































STEC (95)























































































REGULATORY STATUS















































priate (20)

ACKNOWLEDGMENTS





REFERENCES









4852
















1062ndash1070












1669ndash1672







































1812ndash1816













8 January 2010





























531ndash79








1ndash14








631326ndash1332

















































TX 12 to 15 July 2009



























September 2009





October 2007

































Sci 85E63ndashE72







York













414930ndash4940

































49249ndash256
























2009


















124ndash131











308681ndash685






















272














April 2008




tember 2007









































subtypes and eae)

















genes












O26 O45 O103 O111 O121 and O145) in our















































































STEC (95)























































































REGULATORY STATUS















































priate (20)

ACKNOWLEDGMENTS





REFERENCES









4852
















1062ndash1070












1669ndash1672







































1812ndash1816













8 January 2010





























531ndash79








1ndash14








631326ndash1332

















































TX 12 to 15 July 2009



























September 2009





October 2007

































Sci 85E63ndashE72







York













414930ndash4940

































49249ndash256
























2009


















124ndash131











308681ndash685






















272














April 2008




tember 2007










































STEC (95)























































































REGULATORY STATUS















































priate (20)

ACKNOWLEDGMENTS





REFERENCES









4852
















1062ndash1070












1669ndash1672







































1812ndash1816













8 January 2010





























531ndash79








1ndash14








631326ndash1332

















































TX 12 to 15 July 2009



























September 2009





October 2007

































Sci 85E63ndashE72







York













414930ndash4940

































49249ndash256
























2009


















124ndash131











308681ndash685






















272














April 2008




tember 2007























REGULATORY STATUS















































priate (20)

ACKNOWLEDGMENTS





REFERENCES









4852
















1062ndash1070












1669ndash1672







































1812ndash1816













8 January 2010





























531ndash79








1ndash14








631326ndash1332

















































TX 12 to 15 July 2009



























September 2009





October 2007

































Sci 85E63ndashE72







York













414930ndash4940

































49249ndash256
























2009


















124ndash131











308681ndash685






















272














April 2008




tember 2007
































1812ndash1816













8 January 2010





























531ndash79








1ndash14








631326ndash1332

















































TX 12 to 15 July 2009



























September 2009





October 2007

































Sci 85E63ndashE72







York













414930ndash4940

































49249ndash256
























2009


















124ndash131











308681ndash685






















272














April 2008




tember 2007

























September 2009





October 2007

































Sci 85E63ndashE72







York













414930ndash4940

































49249ndash256
























2009


















124ndash131











308681ndash685






















272














April 2008




tember 2007




























308681ndash685






















272














April 2008




tember 2007



















Non-O157 Shiga Toxin–Producing Escherichia coli in Foods

Documents