Journal of Applied Microbiology 1997, 82, 537-551

A REVIEW

Detection of Escherichia coli 0 157 : H7 and other

verocytotoxin-producing E. coli (VTEC) in food
C. Vernozy-Rozand
Unite de Microbiologie, Epidemiologie moleculaire, Ecole Nationale Veterinaire de Lyon, Marcy I'Etoile, France
5959110/96: received 23 October 1996, revised 23 January 1997 and accepted 27 January 1997

1. Introduction, 537
2. Isolation and identification of E. coli 0157 : H7
2.1 Use of biochemical characteristics, 538
2.2 Immunoblotting with antibodies to 0157 antigen, 541
2.3 Use of DNA probe specific for serotype 0157 : H7, 543
2.4 Confirmatory tests for E. roli 0157 identification, 543
3. Methods for detection of verocytotoxin production and V T
genes
3.1 Immunoblotting with antibodies to verocytotoxins, 544
3.2 Use of DNA probe specific for V T genes, 544
4. Tests in the reference laboratory
4.1 Phenotypic tests, 546

1. INTRODUCTION
Enterohaemorrhagic Esrherirhiu roli (EHEC) are recognized
as the primary cause of haemorrhagic diarrhoea and haemolytic uraemic syndrome (HUS). The pathogenicity of
EHEC appears to be associated with a number of several
cytotoxins referred to as shiga-like toxins (SLT) or verotoxins
(VT) (Karmali 1989). Several serotypes of E. roli have been
shown to produce one or both of these toxins, but bloody
diarrhoea1 disease caused by serotypes of VT-producing E.
roli (VTEC) other than 0 1 5 7 : H 7 is uncommon, and no
outbreaks due to these other serotypes have been reported in
the US, Canada or the UK.
Most outbreaks caused by E. roli 0157 : H7 have been
food or water related. Likely vehicles of infection have been
undercooked ground beef according to a report by the Centres
for Disease Control in 1993. Additionally raw milk, cold
sandwiches, vegetables and water have been implicated as
sources of some outbreaks (Karmali 1989 ; McGowan et a/.
1989 ; Griffin and Tauxe 1991 ; Swerdlow et al. 1992). Cattle
Correspondence to :Dr C. 1 >rnoz,pRozand, Uniti de .2licrohrologir,
Epidimiolo,pe mol6culuirr, Ecole Nutionule I 2tPrinuirc dr L;yun, I
arrnue BourXelut, BP 83, F-hY-780.2lurry I'Etuile, Frunce.

0 1997 The Society for Applied Bacteriology

4.1.1 Serotyping, 546

4.1.2 Biotyping, 546
4.1.3 Phage typing, 546
4.2 Genotypic tests, 546
4.2.1 Plasmid analysis, 546
4.2.2 V T gene analysis, -546
4.2.3 Multilocus enzyme electrophoresis, 546
4.2.4 Pulse-field electrophoresis, 546
4.2.5 Phage j. probe analysis, 547
5. Conclusions, 547
6. Acknowledgement, 547
7. References, 547

are found consistently to be a reservoir for this organism in

the environment (Borczyk et al. 1987 ; Chapman et al. 1993).
However, isolation from ground beef is uncommon (Okrend
et al. 1990a). These organisms may be present in low numbers
in implicated foods containing high levels of competing microflora (Willshaw et al. 1993). Two recent outbreaks were
unusual in that they were both linked to consumption of lowp H foods, which have traditionally been considered safe : an
outbreak in Massachusetts was associated with drinking one
brand of apple cider (Besser et ul. 1993); and the other
outbreak with ingestion of mayonnaise-containing food from
an Oregon restaurant chain (Keene et ul. 1993). In both cases
laboratory experiments showed that E. roli 0157 :H7, while
dying rapidly in these acid foods a t room temperature, survived for weeks at refrigeration temperature (Besser et d.
1993 ; Weagant et ul. 1994).
Many approaches have been taken in developing isolation
and detection procedures for this organism, and these can
be generally divided into three categories: (i) the use of
biochemical characteristics somewhat specific to strains of E.
roli 0157 : H 7 (e.g. the inability to ferment sorbitol and the
lack of fi-glucuronidase activity) ; (ii) the use of DNA probes
for verotoxins or markers associated with verotoxin-pro-

538 C.V E R N O Z Y - R O Z A N D

ducing E. coli; and (iii) immunoblotting with antibodies to

verotoxin (VT) or 0157 antigen in conjunction with hydrophobic grid membrane filters.
The purpose of this review is to describe techniques for
isolation and identification of E. coli 0 1 5 7 : H 7 and other
verocytotoxin-producing strains of E. roli in food.
Throughout this report, the following convention has been
adopted; when the term VTEC is used, it includes verocytotoxin-producing E. coli of all serogroups ; the term E. coli
0157 is used to refer to E. coli of that serogroup where the
precise H antigen type is unknown or not specified ; the term
E. coli 0157 : H7 is used to refer to bacteria of that serotype
only, which is frequently VT-producing.
The predominating view held by Washington State epidemiologists, USDA and FDA officials is that the only verocytotoxin-producing E. coli of importance to human disease
is E. coli 0 1 5 7 : H7. This is contrary to the view held in
Canada and the UK. In the USA, it is accepted that verocytotoxin-producing E. coli other than 0157 : H7 can cause
human disease. However, present detection technology poses
many obstacles to efficient and economical diagnosis of non0157 serogroups and efforts are concentrated solely on 0157.
2. ISOLATION AND IDENTIFICATION OF 15.
COLl 0 1 57 : H7
2.1 Use of biochemical characteristics

Most biochemical reactions of E. coli 0157 : H7 are typical of

E. roli, with the exception of sorbitol fermentation and pglucuronidase activity (Wells rt al. 1983 ; Doyle and Schoeni
1987). About 930.0 of E. coli isolates of human origin ferment
sorbitol within 24 h ; however, E. coli 0157 :H7 was reported
as not fermenting sorbitol (Honish 1986). But, in recent
reports some strains of 0157 VTEC strains fermented sorbitol within 24 h (Gunzer et ul. 1992; Pearce et al. 1994;
Hayes et a/. 1995). The prevalence of such strains is unknown
but they would be discarded using the standard screening
method described here. Additionally, 93910 of E. roli possess
the enzyme 8-glucuronidase but the vast majority of 0157
VTEC do not produce P-glucuronidase (Okrend et al. 1990b).
Growth studies in trypticase soy broth (TSB) indicated
that the organism grows rapidly between 30 and 42"C, with
generation times ranging from 0 4 9 h at 37C to 0.64 h at
42C (Doyle and Schoeni 1994). T h e organism grows poorly
at 4.C15"C and does not grow within 48 h at 10 or 45.5"C
(Raghubeer and Matches 1990). Many procedures to detect
faecal coliforms and subsequently E. coli in food use incubation temperatures in the range of 4445C. Hence, E. coli
0157 : H7 would not likely be detected in normal screening
for faecal coliforms by standard procedures with incubation
at 44.5"C.
The potential low infective dose of this serotype of E. roli

means that it is necessary to be able to detect low numbers

in foods and the lack of sensitivity of direct plating has led
to the development of enrichment culture media, to allow
numbers of contaminating cells to multiply to detectable
levels (Doyle and Schoeni 1987 ; Okrend et al. 1990a ;Padhye
and Doyle 1991b). Several liquid media for the enrichment
of 0157 VTEC have been reported and some are listed in
Table 1. Modified trypticase soy broth (mTSB) is supplemented with either novobiocin or acriflavin to reduce the
growth of Gram-negative organisms. Another enrichment
medium is buffered peptone water (BPW) with vancomycin,
cefsulodin and cefixime to suppress the growth of ..leromonas
spp. and Proteus spp. respectively. Optimal recovery of 0 1 57
VTEC was obtained with growth for 6 h (Chapman et a/.
1991).
After growth in enrichment media a number of methods
can be used to detect E. coli 0 1 57 strains.
In an attempt to reduce the length of routine microbiological analysis of foods and to negate problems associated with a rapid detection system (interference from food
debris and background micro-organisms ; lack of sensitivity),
there has been much interest in the development of separation/concentration techniques. Many techniques have
been studied for this purpose, including centrifugation (Bassel rt al. 1983), filtration (Sharpe and Peterkin 1988; Bobbitt
et nl. 1993), lectin-based biosorbents (Payne et al. 1992),
aqueous biphasic systems (Bennett e t a / . 1994)and ultrasound
(Miles et ul. 1995). Perhaps the most successful of approaches,
however, has been the use of immunomagnetic separation.
T h e use of immunomagnetic separation (IMS) has been suggested as a method of reducing total analysis time improving
sensitivity of detection. Para-magnetic particles coated with
antibodies specific to a target organism are added to a food
system. The target organism is captured onto the magnetic
particles and the whole complex removed from the system
by application of a magnetic field. Target organisms are thus
removed from food debris and background micro-organisms
which potentially will interfere with the detection systems,
and by resuspending isolated cells in a reduced volume. The
concentration of cells can be rapidly increased, thus increasing
the sensitivity of the detection system. One problem experienced by numerous researchers was non-target carryover.
Meadows (1971) reported that the non-target organism carryover effect was possibly due to bacteria adhering to the walls
of the glass test tubes. T h e basic protein salmine (protamine),
which is positively charged at neutral pH, reduced attachment
because it adsorbed to the bacteria and to the glass and hence
reduced their net negative charge. More precisely, Meadows
demonstrated that protamine reduced the number of .leronzonas liyuefuriens, E. roli and Psrullomonasftuorescrns adhering
to glass. Okrend et al. (1992) solved the problem of nontarget organism carryover by adding 1 ml of aqueous protamine solution (0.05 mg ml-') to 10 ml ofenrichment culture

0 1997 The Society for Applied Bacteriology, Journal of Appried Microbiology82, 537-551

DETECTION OF E. COLl 0 1 57 : H7 I N FOOD 539

Table 1 Enrichment liquid media for

Escherirhzu solz 0157 : H7

Designation

Composition (l-')

Reference

mTSB

Trypticase sop broth, 30 g

Bile salts 3, 1.5 g
Dipotassium phosphate (K2HP0.,), 1.5 g
Novobiocin, 20 mg

Doyle and Schoeni 1987

dm TSB-CA

Trypticase soy broth, 30 g

Bile salts 3, 1.5 g
K2HP04,1.5 g
Casamino acids, 10 g
Acriflavine-HC1, 10 mg

Padhye and Doyle 1991b

BPW-VCC

Buffered peptone water (Oxoid)

Vancomycin, 8 mg
Cefixime, 045 mg
Cefsulodin, 10 mg

Chapman et t2l. 1994

mECn

Tryptone, 20 g
Bile salts 3, 1.12 g
Lactose, 5 g
K2HP0,, 1 g
KH,PO1, 1.5 g
NaCI, 5 g
Novobiocin, 20 mg

Organon Teknica 1993

(mEC novobiocin) before adding the magnetic beads (Dynal"'' DynabeadsThl M-280, Dynal, Inc., Great Neck, NY).
T h e beads were washed three times in sterile physiological
saline, and changing the test tubes with each wash. These
modifications reduced the non-target colony counts obtained
from uninoculated meat samples. This procedure enabled
consistent recovery of E. roli 0157 : H7 from inoculated meat
samples. The percentage of E. roli 0 1 57 : H7 cells captured,
compared to the total number of cells captured, ranged from
48 to 10O0/o.
Wright et a/. (1994) considered that IMS is rapid, technically simple and is a specific method for isolation of E.
coli 0157 and to be useful in epidemiological studies. They
reported a 100-fold increase in sensitivity of detection of E.
co/z 0 157 from inoculated minced beef using IMS compared
to direct subculture from BPW supplemented with vancomycin, cefsulodin and cefixime (BPW-VCC) to cefisime tellurite sorbitol MacConkey (CT-SMAC) following incubation
for 6 h at 37C.
More recently, Bennett et a/. (1995, 1996) demonstrated
the ability of the commercially available Dynabeads IMS
procedure using anti-E. roli 0157, to detect a few cells of E.
roli 0157 in 25 g of inoculated minced beef. This gave results
1 d earlier than a cultural analysis of similar sensitivity. The
use of IMS can increase the rate of isolation of E. coli 0157
depending upon the choice of enrichment broth. At levels of

less than 10 cells per 25 g, the direct plating method (24 h

enrichment) and the use of Dynabeads following enrichment
in BPW-VCC isolated 0157 from 47% of inoculated samples
whilst Dynabeads separation following enrichment in
mEC n isolated 0 1 57 from 64% of inoculated samples. The
use of BPW-VCC has been suggested (Chapman et a/. 1991)
as it does not contain lactose. It is believed that other organisms present in food might metabolize lactose to compounds
that inhibit E. coli 0157. The inhibitory effect of microbial
lactose metabolism on E. roll 0157 was reported by Hinton
et a/. (1991). However, in the study of Bennett et ul. (1996),
any growth of competitor organism in m E C + n did not
appear to result in metabolites that were significantly inhibitory to growth of E. coli 0157. Thus with mEC+n, IMS
may increase isolation rate of E. coli 0157 compared to that
obtained using conventional cultural methods.
Total analysis time is increased by a working day when
using the direct plating method and thus if speed of analysis
is an important criterion in method selection, the use of
Dynabeads would be recommended (Bennett et ul. 1996).
After enrichment the beads are usually cultured on one of
the selective media listed in Table 2 .
Escherirhia roll 0157 :H7 do not ferment sorbitol whereas
most other E. roli do and sorbitol MacConkey agar (SMAC)
has been widely used for their isolation. However, SMAC
medium relies entirely on differential sugar fermentation and

0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82, 537-551

540 C . V E R N O Z Y - R O Z A N D

Designation

Composition

Table 2 Solid media for isolation of

Esrherirhiu roli 0157 : H7

Reference
-

SMAC

MacConkey agar
D-Sorbitol, 1%

Farmer and Davis 1985

CR-SMAC

MacConkey Sorbitol agar (Oxoid)

Rhamnose, 0.5%
Cefixime, 0.05 mg I-'

Chapman et ul. 1991

CT-SMAC

MacConkey Sorbitol agar (Oxoid)

Cefixime, 0.05 mg 1 - l
Potassium tellurite, 1 mg I-'

Zadik et nl. 1993

MSA-hIUG

MacConkey Sorbitol agar (Difco)

MUG, 0.01",0

Padhye and Doyle 1991b

PRS-hlUG

Phenol red base + 29.6 agar

ri-Sorbitol, O.Sno

Okrend et ul. 1990c

4-Meth y lumbellifer yl
B-D-glucuronide, 0.005%

MSA-BCIG

MacConkey Sorbitol agar (Difco)

Okrend et ul. 1990b

5-Bromo-4-chloro-3-indoxyl-

fi-Ii-glucuronic acid
c>-clobexylammoniumsalt, 0.01nh
HC

Trvptone, 20 g 1-'
Bile salts 3, 1.12 g 1-'
Sodium chloride (NaCI), 5 g I-'
Sorbitol, 20 g 1-'
MUG, 0.01%
Bromocresol purple, 0,015 g 1-'

Szabo et d. 1986

does not select V T E C E. roli 0 1 5 7 : H7 from other E. roli or

sorbitol non-fermenting genera and therefore lacks sensitivity. Modifications of S M A C have been described with the
aim of improving the selectivity for 0 1 5 7 VTEC. Okrend et
al. (1990b) reported that the addition of 5 b r o m o - k h l o r o indoxy-P-l>-glucuronide (BCIG) at the 0.1 g l-' level to
S M A C plates aided in the isolation of E. roli 0 1 5 7 : H7 from
raw ground beef samples differentiating P-glucuronidasepositive from P-glucuronidase-negative colonies. Escherirhia
roli 0 1 5 7 : H7 colonies being sorbitol-negative, P-glucuronidase-negative, remained white, while sorbitol-negative, B-glucuronidase-positive turned green to blue. T h e
addition of B C I G to the S M A C agar reduced the number of
false suspect colonies picked from the primary plating medium by 3ho/o when compared to SMAC. Esrherirhiu roli
0157:H7 was isolated from 11 out of 12 inoculated meat
samples using SMAC-BCIG as compared to eight out of 12
samples using S M A C without BCIG.
Thompson et al. (1990) developed a rapid fluorogenic assay
for E. coli 0157. T h i s assay used .l-methylumbelliferyl-~-D-

glucuronide ( M U G ) as an indicator which is hydrolysed to a

fluorogenic product by the enzyme P-glucuronidase (Rippey
et al. 1987). However, recently McCleery and Rowe (1995)
reported that S M A C supplemented with MUG (SMACM U G ) performed poorly when stressed cells of the pathogen
are present. T h e incorporation of a resuscitation period (2 h
at 25C) on trypticase soy agar (TSA) before overlay with
S M A C - M U G was found to significantly (P < 0.01) improve
recovery of heat-stressed (52"C/60 min) E. roli 0 1 5 7 : H7
cells. Maximal recovery was, however, obtained by adding
catalase (1000 U) to the T S A before overlaying with SMACMUG.
Rocelle et al. (1995) studied the suitability of selective
plating media for recovering heat- or freeze-stressed E. roli
0157 : H7 from T S B and ground beef. A mixture of five
strains of E. roli 0 1 5 7 : H7 and five non-0157 strains of E.
roli was heated in T S B at 52", 54" or 56C for 10, 20 and 30
min, or frozen at - 20C. Recovery of E. rolz 0 1 57 : H7 was
significantly higher on modified eosin methylene blue agar
than on SMAC.

0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82, 537-551

DETECTION OF E . C O L 1 0 1 5 7 : H7 I N FOOD 541

Zadik et al. (1993) described another modification in which

the SMAC contained tellurite (2.5 mg 1-') and cefixime (0.05
mg 1- '), because minimal inhibitory concentrations (MIC)
were higher for 0157 VTEC than for other E. roli and for
non-sorbitol fermenting enterics including .-ieromonas ssp.,
Plesiomonas ssp., Morganellu morganii, Prodencia ssp. and
Hafnia ulvei (Weagant et al. 1995).
Bolton et al. (1996) have evaluated an enrichment and
four subculture procedures for detection of E. rolz 0157.
Compared to traditional subculture using 10 pl and 100 pl
inocula and culture of centrifuged deposits, the combination
of enrichment in modified tryptone broth (MTSB) incubated
at 42C for 6 h, followed by immunomagnetic separation and
subculture onto SMAC with tellurite and cefixime (CTSMAC), was the most sensitive and selective procedure. This
method utilized incubation of the enrichment broth (MTSB)
at 42C which is more selective than incubation at 37C
according to Bolton et al. (1995). The main advantage is the
inhibition of other non-sorbitol fermenting organisms such
as Hafnia ahei which can be a problem when testing raw
meats (Bennett et al. 1995).
2.2 lmmunoblotting with antibodies to 0157 antigen

Colony immunoblotting has also been employed to detect E.

roli 0157. After enrichment, cultures are spread on agar plates
and colonies are transferred to nitrocellulose membranes. An
alkaline phosphatase-conjugated 0157 antiserum is used for
the detection of positive colonies. This immunoblot procedure which is called the Hydrophobic Grid Membrane
Filter-immunoblot procedure ( H G M F ) was developed by
Doyle and Schoeni (1987). Food samples were selectively
enriched in an enrichment medium composed of TSB, bile
salts No. 3, dipotassium phosphate and novobiocin. Enrichment cultures were filtered through hydrophobic grid membranes and the membranes were incubated on nitrocellulose
paper on selective agar. An immunoblot of each nitrocellulose
paper was prepared using antiserum raised against E. rolz
0157 : H7. Culture filtrates and positive colonies were confirmed as E. coli 0157 : H7 with antisera to E. coli 0157 : H7,
vero cell cytotoxicity tests and biochemical tests. T h e procedure claimed to isolate as few as 1.5 cfu E. roli 0157 : H7
per g of food, but the procedure was not amenable to routine
testing because of its complexity and extensive need for personnel time. An additional drawback of the procedure was
the lack of specificity. T h e polyclonalOl57 antiserum crossreacted with Brucellu abortus, Brurella melitensis, 1-eninin
enterocolitica serogroup 0 : 9, Salmonella group N and Pseudomonas multophilia 555 (Caroff et al. 1984; Bundle et ul. 1987 ;
Di Fabio et al. 1987). T h e common epitope responsible for
these cross-reactions was the rare sugar 4-amino-4,6 dideoxyD-mannose present in the cell wall lipopolysaccharide (Perry
et al. 1986).

Another method based on the hydrophobic grid membrane

and an 0-antigen-specific monoclonal antibody to E. roli
0157 was developed by Todd et ul. (1988). Compared to the
procedure developed by Doyle and Schoeni (1987), there was
no enrichment of the food sample. T h e latter was mixed with
peptone water, macerated by stomaching, filtered to remove
food particles, and then filtered through a hydrophobic grid
membrane. The method yielded presumptive identification
within 24 h and recovered on average 9590 of E. roli 0157 : H7
artificially inoculated into meat. This method could detect 10
cfu E. rolz 0157 : H 7 per g of food ; however, because of the
potential of false-positive identification of group N salmonellae, further testing was necessary to confirm that the isolates
were E. roli 0157 : H7.
A rapid latex agglutination assay (E. roli latex test, Oxoid
Ltd, Hampshire, UK) is commercially available for rapid
presumptive detection of E. roli belonging to serogroup 0157.
March and Ratnam (1989) evaluated the latex agglutination
test using clinical isolates of E. rolz 0 1 5 7 : H 7 and faecal
specimens obtained during an outbreak of haemorrhagic
colitis. The latex test showed a complete and rapid agglutination with all 200 E. rolz Ol57:H7 strains and a clear-cut
negative reaction with the 50 non-E. coli 0157 strains tested.
Esrherirhiu coli 0157 : H7 was not detected in any of the 400
outbreak-related food samples studied, although about 130
food samples yielded presumptive colonies on cellobiose
MacConkey agar. None of the colonies were identified as E.
coli 0157 : H 7 by the latex agglutination assay, and all were
later identified as Enterobarter, Flai1omonus or Hafnia spp.
Borczyk et ul. (1990) demonstrated with the latex agglutination test that non-specific agglutination occurred among
several sorbitol-negative Esrherirhia bacteria, including E.
hermanii, E. roli 0148 : N M and E. coli 0 1 17 : H27.
An improved screening procedure was described by
This method was devised incorporating
Okrend et al. (1990~).
a commercially available reactive disc blot ELISA for E.
coli 0157 antigen into a cultural screening program for the
isolation of E. roli 0157 : H7 from meat and poultry products.
The method included the inoculation of a raw or cooked meat
sample into an enrichment broth (consisting of EC broth with
1.12 g bile salts No. 3 and 10 mg novobiocin l-'), incubation
with shaking at 37C for 6-8 h, followed by inoculation of
3M PetrifilmT" E. roli count (3M Company, St Paul, MN)
plates with dilutions of the enrichment culture. The Petrifilm
plates were incubated at 42C for 18 h. After incubation, the
top plastic film of the Petrifilm plate was carefully raised and
colonies were replica plated onto reactive discs by contacting
the discs with the Petrifilm for 2 min. Colonies reacting with
antiserum to E. roli 0157 were isolated by either USDA
cultural methods from enrichment media (Okrend et a/.
1990a) or by direct picking from the Petrifilm and streaking
on SMAC with BCIG media. This screening procedure
identified negative and presumptive positive samples in 26-

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542 C . V E R N O Z Y - R O Z A N D

28 h, and its minimum level of sensitivity was 0.6 cfu E. coli

0157 : H 7 per g of food with 0910 false-negative and 290 falsepositive results. Since the procedure used 0157 polyclonal
antisera, all presumptive positive samples that could be falsepositive results must be confirmed for E. rolz 0157 : H 7 by
isolating the organism. T h e isolation and identification of the
bacteria required an additional 3 4 d.
T o detect E. roli 0 1 5 7 : H 7 in retail ground beef, Kim
and Doyle (1992) developed a dipstick immunoassay using a
sandwich type assay (with a polyclonal antibody to E. rolz
0157 as the capture antibody and a monoclonal antibody to
E. ruli 0157 : H7 as the detection antibody) on a hydrophobic
polyvinylidine difluoride-based membrane. Escherzrhia roli
0157 : H 7 could be detected in ground beef containing as few
as 0.1 cfu of E. colz 0 1 5 7 : H 7 per g after enrichment in
mTSB. Two false-positive results were observed during the
incubation ; they may have resulted because of denaturation
or degradation of the capture antibody. Detection antibody
or enzyme-conjugated antibody may non-specifically bind to
denatured capture antibody. Kim and Doyle (1992) observed
that rehydrated E. roli 0157 capture antibody held frozen
(- 8OoC) for more than 6 months or refrigerated (5-10C)
for more than a week occasionally resulted in false-positive
reactions.
Padhye and Doyle (1991a, 1992) described a rapid sandwich enzyme-linked immunosorbent assay (EHEC-Tek ;
Organon Teknica, Durham, NC) for the detection of 0157
VTEC in foods. In this test polyclonal 0157 antibody was
used as the capture antibody and a horseradish peroxidaselabelled monoclonal antibody (the MAb 4E8C12), claimed to
be specific to two outer proteins unique to serogroups 0157
and 0 2 6 - t h e two serotypes associated most frequently with
H U S (Padhye and Doyle 1991a)-was used as the detection
antibody. T h e sensitivity of the procedure was determined
by using ground beef and dairy products inoculated with E.
roli 0157:H7. It could detect as few as 0.2 cfu E. coli
0157 : H7 per g of food after overnight enrichment. According to Padhye and Doyle (1991a), this procedure was highly
specific, sensitive, rapid, easy to perform and amenable to use
by laboratories performing routine microbiological testing.
More recently, the specificity of this test was investigated
by Johnson et al. (1995). They reported that the target antigens of the detection reagent, MAb 4E8C12, were present in
numerous serotypes of E. rolz (022 : H8,026H11,046 : H38,
088:H49, 091:H21, 0 1 0 3 : H 2 and 0 l l l : H l l ) and that
their ELISA reactivity was influenced by bile salts, acriflavine
and heat. Acriflavine enhanced ELISA reactivity, principally
that of 0157 strains, whereas bile salts plus heating induced
reactivity in all non-0157 strains. On the basis of these
results, a modified protocol was devised to eliminate falsepositive reactions while retaining the enhancing effects of
acriflavine and heat. T h e above ELISA-positive strains were
grown in TSB for 18 h at 37C. One ml aliquots were trans-

ferred to microcentrifuge tubes and mixed gently for 10 min

with 0.2 ml of magnetic beads (M-280; Dynal) coated with
antibodies to E. coli 0157 : H7 strains. The beads were captured on the side of the tube with a magnetic particle concentrator, washed and resuspended in 0.5 ml of mTSBacriflavine for overnight cultivation at 37C. When heated
aliquots of these cultures were tested in the ELISA, E. cdi
0 1 5 7 : H7 remained strongly positive, while 10 of the 12
previously ELISA-positive strains tested negative. Thus, the
modified protocol, incorporating immunocapture, greatly
improved the specificity of the EHEC-Tek ELISA while
maintaining the assays low limits of detection. Chapman and
Siddons (1996) compared the EHEC-Tek with IMS followed
by culture to cefixime-tellurite-sorbitol MacConkey
(IMS/C) for detecting E. rolz 0157 in artificially inoculated
samples and naturally contaminated beefburgers. When the
EHEC-Tek and IMS/C were repeated using BPW/VCC
and mECn in parallel as the primary enrichment, the results
were much more encouraging with BPW/VCC enrichment,
following which the EHEC-Tek detected E. roli 0157 in four
of five naturally contaminated samples and IMS/C detected
the organism in all five samples. Both EHEC-Tek and
IMS/C failed to detect E. roli 0157 in all five naturally
contaminated samples enriched in mECn, confirming the
unsuitability of this type of medium for enrichment of E. rolz
0157. T h e EHEC-Tek immunoassay was improved by use
of BPW/VCC in place of mECn as the primary enrichment
medium.
Other rapid sandwich enzyme-linked immunosorbent
assays are now available. For example, the VIDAS E. colz
0157 (ECO) assay is intended for use with the Vitek Immuno
Diagnostic Assay System (VIDAS) as an automated qualitative enzyme-linked fluorescent immunoassay (ELFA) for
the detection of E. coli 0157 in food, food ingredients and
environmental samples. All of the assay steps are performed
automatically by the VIDAS instrument. The solid phase
receptacle (SPR), a pipette tip-like disposable unit serves as
the solid phase as well as the pipetter during the process. The
SPR is coated with polyclonal anti-E. roli 0157 antibodies.
Reagents for the assay are sealed in reagent strips. An aliquot
of the enrichment broth is placed into the reagent strip and
the sample is cycled in and out of the SPR for a specific
length of time. This test was evaluated by Kerdahi and Cohen
(1996), for use in the detection of E. rolz 0157 in a variety of
artificially contaminated soft, semi-soft and hard cheeses.
Sixty-five cheese samples were artificially contaminated at
low (2-1 cfu per 25 g) and high (7-10 cfu per 25 g) levels of
contamination with one of two strains of enterohaemorrhagic
E. rolz 0157 : H7. All cheeses artificially contaminated with
high levels of inoculum were detected by the VIDAShl,
whereas five cheeses (7.7%) inoculated with low levels were
negative. In 15 additional cheeses inoculated with coldstressed cells, both V1DASTh1and the Bacteriological Ana-

0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology82, 537-551

DETECTION OF E. COL/0157:H7IN FOOD 543

lytical Manual cultural assay (consisting of spreading 0.1 ml

of overnight growth from mTSB enrichment broth on H C
agar plates) detected all high and low levels of contamination.
No false positives or interference from product background
fluorescence were encountered in any of the cheeses tested
by VIDAS. Results of negative cheeses can be obtained in 24
h, after overnight incubation in selective broth and the 45
min assay.
All of these protocols use specific antibodies in screening
techniques to identify a broth culture or an area of a filter or
semisolid medium which would be promising for focusing
further cultural isolation efforts in order to obtain an isolated
culture for additional tests.
2.3 Use of DNA probe specific for serotype 0 157 : H7

Esrherii.hia roli 0157 : H i was shown to contain zid A gene

sequences that encode for the fl-glucuronidase enzyme (Feng
rt d. 1991 ; Feng and Lampel 1992). Analyses demonstrated
that the nucleotide sequence of the 5 terminus of the rid A
structural gene of 0157 : H7 was identical to that of MUGpositive E. roli, except for a substitution 90 bases downstream
from the initiation codon. In another experiment, Feng (1993)
used an oligonucleotide probe, PF-27, directed to this region,
containing a unique base substitution in the allele of the rid
A gene, to identify isolates of E. roli 0 1 5 7 : Hi. Colony
hybridization analysis of 239 bacteria, including E. roli and
other enteric isolates showed that the probe reacted only with
the 17 isolates of 0 1 57 : H7 serotype. Interestingly, the probe
did not hybridize with the 73 MUG-positive E. roli, the 13
MUG-positive Shigella or eight MUG-positive Salmonrllu
isolates analysed. Except for the single nucleotide base difference, the PF-27 sequence is identical to that rid A gene,
which was present in almost all E. roli regardless of M U G
phenotype. Hence, the absence of probe hybridization with
E. cdi indicated that PF-27 could discriminate the single
nucleotide difference between the 5 region of the zlid A gene
of E. coli and its allele in the 0 157 : H7 serotype. T o further
verify probe specificity, DNA from E. roli and 0 1 5 7 : H 7
were digested with Hznfl enzyme and examined by Southern
blotting. Southern analyses showed that the PF-27 was specific for the base substitution region of the allele. T h e PF-27
probe appeared to be specific solely for serotype 0157 : H i
because it did not hybridize with isolates from the other
VT-producing EHEC serotypes ( 0 2 6 : H11 and 0 1 11 : NM)
studied. The stringent specificity of the PF-27 probe may be
valuable for clinical diagnosis and for the identification of
0157 : H i isolates in foods. The stringency of PF-27 probe
would also eliminate the need for serological confirmation,
and thus, incidences of false-positive identification caused by
antibody cross-reactivity with other organisms.
More recently, Feng (1995) showed that the probe also
detected phenotypic variants of 0157 serotype that were

non-motile, MUG-negative and fermented sorbitol. These

atypical pathogenic 0 157 strains were isolated from haemolytic uraemic syndrome patients in Germany and obtained
from Gunzer et al. (1992). Unlike biochemical differentiations
such as sorbitol fermentation or fl-glucuronidase activity,
probe reactions do not rely on enzymatic activities and are
therefore unaffected by media interference or the presence of
bacteria, such as E. hermunii, which has similar phenotypes.
An isolate of E. hermanii examined by Feng (1993) did not
hybridize with PF-27.
2.4 Confirmatory tests for E. cofi0157 identification

Colonies that appear to be E. roli 0157 must be confirmed as

E. roli using biochemical tests. These would exclude any nonE. roli that give false-positive agglutination tests with the
0 1 57 antiserum. Esrherirhia hermunii is biochemically and
serologically similar to E. roli and cross reacts with polyclonal
antisera to E. rnli (Lior and Borczyk 1987). However, E. colz,
unlike E. hernzanii, does not ferment cellobiose and does not
grow in the presence of potassium cyanide. In addition,
strains of E. hermanii ferment rhamnose and are sensitive to
tellurite and therefore would not be detected on CR-SMAC
or CT-SMAC.
Strains that appear to be E. roli 0157 should be confirmed
serologically with antisera against 0 and H antigen. 0157
VTEC usually have the flagellar antigen H i although some
strains are non-motile. All confirmed E. rolz 0157 strains
should be tested for production for verocytotoxin (VT) or
the presence of V T genes.
Selective screening of food samples with sorbitol MacConkey agar will miss a proportion of 0157 : H7 strains which
are sorbitol + / V T . There is increasing evidence suggesting that phenotypic variations exist among the isolates
within E. roli 0157 : H7. In Germany, Gunzer et al. (1992)
found that 41 VT2-producing E. roli 0157 strains isolated
from patients with diarrhoea or haemolytic uraemic syndrome
(HUS), fermented sorbitol and were M U G positive. Phenotypic variants of E. roli 0157 have also been isolated in other
parts of central Europe and in the United States (Feng 1995 ;
Hayes et ul. 1995). Food microbiologists should be aware of
the emergence of these phenotypic variants and recognize
that these strains may not be identified by routine culture
methods or by biochemical tests used to characterize serotype
0157: H7. Goldwater and Bettelheim (1994) showed that
in Australia, 0157 : H i was uncommon but other less well
recognized serotypes (e.g. 0 1 11 : H-, 0 6 :H31, 0 9 8 : H-,
0 4 8 : H21) were responsible for H U S and bloody diarrhoea
in Australia. These findings suggested the possibility that
transmission of phage-encoded V T genes to native strains of
E. roli could occur and current focus on E. coli 0157 :H7
could be broadened to include methods that would detect all
VTEC. VTEC of serogroups other than 0157 have no reliable

0 1997 The Society for Applied Bacteriology,Journal of Applied Microbiology82, 537-551

544 C . V E R N O Z Y - R O Z A N D

biochemical, serological or morphological characteristics

(other than V T production itself) to distinguish them from
commensal non-VTEC E. roli. T o detect VTEC other than
0157 and the phenotypic variants of E. coli 0157 in food, we
have to use methods for detection of verocytotoxin production and V T genes.
3. METHODS FOR DETECTION OF
VEROCYTOTOXIN PRODUCTION AND
VT GENES
3.1 lmmunoblottingwith antibodies to
verocytotoxins

Verocytotoxin is detected by its cytotoxic effect on vero cells

(Konowalchuk et al. 1977). Faecal suspensions, cultures filtrates or live cultures can be tested (Scotland et al. 1980;
Lior and Borczyk 1987). Strains are grown in T S B and
culture filtrates are added to monolayers of Vero cells. Cells
round up and become detached in the presence of VT. The
monolayers can be examined after 1 d for early cytotoxic
effects using an inverted microscope. Final readings are usually made after incubation fGr 3 4 d when the cells are fixed
and stained (Smith and Scotland 1993). In some early studies
of V T production bacteria were grown in iron-restricted
media and such growth conditions may increase production
of VT1, but not VT2. For routine testing, concentrations of
V T are adequate in ordinary broth media as described below.
A considerable amount of V T is not liberated into the medium
but remains cell bound. It can be released by sonication, with
the use of a French press or by polymyxin treatment, and
these techniques have been used for preparing large quantities
of VT (Karmali et a/. 1985). It has been shown that VTEC
are not isolated in the absence of polymyxin-releasable VT,
whereas V T is frequently present where the organism itself
cannot be isolated (Karmali et ul. 1985 ; Clarke et al. 1988).
T o confirm that cytotoxic effects on Vero cells are due to
the presence of VT, neutralization tests using antisera against
VT1 or VT2 should be performed (Scotland et al. 1988). The
heat lability of the toxin should be confirmed by showing that
V T tests on some samples heated at 100C for 15 min are
negative. Several ELISAs have been described for the detection of V T (Basta et ul. 1989 ; Downes et a/. 1989 ; Acheson
et a/. 1990). Some bind V T to glycolipids containing a terminal a D-Gal-( 1 4 I)-rl-Gal and purified globotriosyl ceramide (GbJ, lyso-Gb, and hydatid cyst fluid isolated from
sheep infected with El-hinororrusgr~mdosus,have been used.
In other studies ELISA monoclonal antibodies against V T
were used to bind toxin. In general, these test have not proved
to be as sensitive as the Vero cell test. In addition, as the
toxins show considerable variation in their antigenicity and
binding properties (even within the VT2 class toxins), care
must be taken in the choice of reagents if the aim is to detect

all VT-producing strains from a clinical specimen (Smith and

Scotland 1993).
Milley and Sekla (1993) developed a colony enzyme-linked
immunosorbent assay using a hydrophobic grid membrane
filter for the isolation of VTEC from human and food
samples. T h e method utilized monoclonal antibodies directed
against the verotoxins and is sensitive to all verotoxin 1
and/or 2 producing serotypes. When applied to meat, 11 of
20 samples positive for verotoxin by polymyxin extraction
yielded verotoxigenic E. roli of a variety of serotypes including
0157 : H7. According to Milley and Sekla (1993), reasons for
why not all VT-positive samples yield a VTEC isolate might
include a low number of VTEC present or a low proportion
of VTEC relative to other Gram-negative organisms. Intrinsic to this problem were the facts that the VTs are a family
of highly potent biological toxins that will exhibit a profound
effect on Vero cell cultures at extremely low concentration
and there are no bacteriological media or temperatures that
will select for VTEC over other Gram-negative organisms
(including non-toxigenic E. roli). Thus, the sensitivity of the
cell culture assay for V T is superior to the sensitivity of
isolation procedures, and non-VTEC coliforms can compete
with VTEC for space on the isogrid membrane.
3.2 Use of DNA probe specific for VT genes

In addition to immunoblotting with antibodies to verotoxin,

polynucleotide probes for V T l and VT2 derived from cloned
genes (Willshaw et a / . 1985, 1987), and synthetic oligonucleotide probes for the detection of different V T genes
have also been developed (Levine et a/. 1987 ; Newland et
al. 1988; Scotland et al. 1988; Karch and Meyer 1989a;
Samadpour et al. 1990; Thomas et a / . 1991; Smith and
Scotland 1993).
Karch and Meyer (1989a) examined four oligonucleotide
probes of various lengths (20 and 40 bases) representing
different regions of the VT1 structural genes and one oligonucleotide (11 bases) derived from the VT2 gene of E. rolz
0157 : H7 strain for the identification of E. roli that produced
cytotoxins for Vero or Hela cells. The 20-base probe appeared
to be as valid as the Il-base probes with regard to specificity
and sensitivity of the hybridization reaction. Fifty isolates of
five different serotypes of producing strains of E. coli were
detected using a colony blot hybridization assay, whereas
none of 416 non-verotoxinogenic E. roli strains was detected.
Escherirhia roli strains that synthesized VT1 alone or E. cola
0 1 5 7 : H 7 isolates that co-expressed V T l and VT2 were
hybridized with all four probes that were complementary to
the V T I genes, suggesting that they had toxin genes with
great homologj7 in all the regions examined. The colony blot
hybridization with the oligonucleotide probes described by
Karch and Meyer (1989) could serve as a specific and sensitive
test with potential diagnostic value.

0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82, 537-551

DETECTION OF E . C O L l 0 1 5 7 : H 7 IN FOOD 545

Levine et al. (1987) prepared a DNA probe, CVD419,

developed from a 3 4 kb Hind111 fragment of the large ca 60MDa plasmid that encodes for an intimate form of adhesion
and is typically carried by E. coli 0157 : H7 and other VTEC
(Framatico et al. 1991 ; Toth et ul. 1991 ; Ashkenazi et nl.
1992). The probe hybridized with 99Yo of all E. roli 0157 : H7
and 77% of E. roli 0 2 6 : H l l , both of which belong to the
VTEC group. The probe hybridized with 21 out of 26 VTEC
(8l0/o) and with only one of the non-VTEC E. coli, indicating
99.8% specificity. Even though the probe proved to be very
specific for EHEC and VTEC, because plasmids of E. colz
may be lost during isolation, this probe would not detect
EHEC and VTEC isolates that no longer carry the 60-MDa
plasmid (Ratnam et al. 1988).
Huck e t a / . (1995) developed a probe for detection of 0157
serogroup E. roli. For that, plasmid DNA extracts from 16
E. coli strains that hybridized with CVD419 probe were
screened for restriction fragments present in plasmids of
0157 serogroup E. coli strains. A 2.0 kb SmuI fragment probe
(VPM1) was the most specific for serogroup 0157 EHEC.
However, this probe hybridized with five of 50 non-0157 E.
coli strains which were verotoxin or CVD419 probe-positive.
With another stringent condition of overnight hybridization
at 45C, two of the five strains that tested false-positive
gave negative results and the other three showed only trace
responses which were easily distinguishable from positive
responses.
Recently, the eue A (for E. roli attaching and effacing) from
E. coli 0157 : H7, necessary for attachment to and effacement
of the microvilli of enterocytes during infection, was cloned
into a multicopy plasmid and sequenced (Jerse et ul. 1990;
Beebakhee et al. 1992; Yu and Kaper 1992). T h e gene from
the 0 157 strain was 97% homologous to enteropathogenic E.
coli (EPEC) rue A. T h e region corresponding to the ear probe
was in the central part of the gene within the highly conserved
region (Jerse and Kaper 1991).
Jerse and Kaper (1990) showed that 29 of 30 VTEC strains
belonging to serotype 0157 :H7 or 0 2 6 : H11 hybridized
with the eae probe. In a study of other VTEC serogroups,
however, a much smaller proportion of strains (35O.
/ o) wasear
positive (Willshaw et ul. 1992). According to Willshaw et a/.
(1994a), hybridization with a probe such as ear 0157 (from
the 3 end of the eae A gene homologue of an 0157 VTEC)
is valuable in the differentiation of 0157 strains. Probing of
colonies from food or faecal specimens with V T and ear
0157 sequences in combination targets 0 1 57 VTEC, whereas
techniques based on immunological detection of the 0 157
antigen identify all strains of this serogroup and some crossreacting organisms.
Amplification of part of the V T gene, using the polymerase
chain reaction (PCR), has also been used to test for the
presence of VTEC. With this procedure, DNA is amplified
to increase the level of target DNA when VTEC are present

in very low numbers. The system first developed used

degenerated primers so that defined sequences of both V T l
and VT2 were amplified (Karch and Meyer 1989b). PCR
products were identified by hybridization using specific oligonucleotide probes complementary to part of the amplified
sequence. It was possible to identify VT1 or VT2 sequences
but variants of VT2 could not be distinguished from VT2.
In order to detect all types of VTEC isolated from animal
and food sources, Read et d . (1992) developed a PCR using
a pair of oligonucleotide primers, targeting conserved
sequences found in VTI, VT2 and VTE genes. Supernatant
fluids of boiled broth cultures of VTEC (233 strains) isolated
from ground beef, ground pork, raw milk, bovine faeces
and porcine faeces, non-VTEC E. roli (72 strains) and other
enteric and food bacteria (76 strains) were tested by PCR.
T h e verocytotoxigenicity of these strains was verified by Vero
cell assay. All 223 VTEC isolates, comprising over 50 different serotypes, were detected by- the PCR procedure. Shzgellu uysentrriue type 1 was the only other bacterium that was
positive in this assay. As little as 1 pg of VTEC DNA and as
few as 17 cfu of VTEC could be detected with this method.
The results suggested that these primers detect VTEC over
a wide range of serotypes. This method might be applicable
as a screening procedure for the detection of VTEC in samples of foods and faeces (Gannon r t ul. 1992).
More recently, Begum and Jackson (1995) adapted a PCE
technique to make it suitable for the identification of VTEC
directly from contaminated ground beef without isolation
of the bacterium or purification of its DNA. Ground beef
hornogenates were diluted 1000-fold to reduce the concentration of components which inhibit the thermostable
polymerase. As few as 30 VTEC per ml of a ground beef
homogenate were detected using the PCR technique,
although it was necessary to enrich six of the samples for
positive detection. Assessment of four different ground beef
samples using the PCR detection technique revealed that fat
content was the major inhibitory component. Masters et (11.
(1994) examined the relationship between viability assessed
by plate counts and detectability by PCR techniques with cells
of E. rolz previously exposed to a range of stress treatment. In
all cases the organisms were detectable by PCR after plate
counts had declined to zero. Treatment with acid or hydrogen
peroxide caused loss of PCR soon after viability was lost, but
strong PCR signals were obtained from starved or desiccated
cells long after cells became non-viable. Exposure to temperatures up to 100C had little effect on detection by PCR
and even autoclaving cells at 121C for 15 min failed to
abolish PCR detection completely. There is thus no simple
relationship between viability and detectability by PCR.
Detection of pathogens by PCR in environmental monitoring
requires additional evidence of viability before risk can be
properly assessed.
In order to maximize epidemiological information or to

0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology82,537-551

546 C. VERNOZY-ROZAND

determine if E. rolz 0157 : H7 human outbreak isolates are

the same strain or a mixture of strains, a variety of techniques
are used. This identification and sub-typing are performed
in a reference laboratory.
4. TESTS IN THE REFERENCE
LABORATORY
4.1 Phenotypic tests
4.1.1 Serotyping. Full serotyping of VTEC requires the
facilities of a reference centre as investigation with antisera
against 173 0 antigens and 56 H antigens is needed. V T
production has been reported in strains belonging to many
different serotypes (Gross et ul. 1985 ; Karmali 1989). Strains
of 0157 are most common, usually with the flagellar antigen
H7 ; non-motile strains have also been reported and this may
be a useful distinguishing feature. Some VTEC belong to
classic enteropathogenic serogroups such as 026, 055, 0 1 11
and 0128. Therefore, strains of these serogroups isolated
from cases of haemorrhagic colitis or HUS or outbreaks of
diarrhoea should be sent to a reference laboratory for further
tests.
4.1.2 Siotyping. The biochemical characteristics of VTEC
are generally the same as E. roli. As described above, most
0157 VTEC are unusual in their ability to ferment sorbitol
or to produce P-glucuronidase. These tests have been suggested as aids in the confirmation of 0157 VTEC. A few
0 157 VTEC and other unusual properties have been reported
including indole-negative or urease-positive strains and
strains able to use citrate (Aleksic r t al. 1991). These characteristics could prove useful markers and care must be taken
not to eliminate such atvpical strains of E. rolz in the early
investigation of food samples. Although different biotypes of
0157 VTEC with respect to fermentation patterns have been
reported, most workers have considered the tests too irreproducible to be used in the differentiation of this group.
4.1.3 Phage typing. A phage typing scheme for VT-producing strains of E. coli 0157 was developed in Canada
for epidemiological investigations (Ahmed et al. 1987). T h e
scheme which uses 16 phages now recognizes over 80 types
(Khahria rt al. 1990; Frost et ul. 1989, 1993). Twenty-four
phage types have been identified in the UK although most
strains belong to phage types 1, 2, 4 and 49. This phage
typing is not used for VTEC of serogroups other than 0157.
4.2 Genotypic tests

4.2.1 Plasmidanalysis. Plasmid analysis of 0157 VTEC can

be used to identify strains in outbreaks and sporadic cases of

infection (Scotland et ul. 1987; Frost et al. 1993). Virtually

all 0157 VTEC isolates carry a plasmid with a molecular
weight of about 60 x 10" but certain strains carry additional
plasmids. Identification of phage type, V T type and plasmids
provides a very useful combination for the characterization
of 0157 VTEC.
4.2.2 VTgene analysis. Two major types of VT, VT1 and
VT2 have been defined but several variants of VT2 have now
been identified (Scotland et al. 1983; O'Brien et al. 1984;
Scotland et (11. 1985 ; Weinstein et al. 1988 ; Gannon et al.
1990 ;Ito rt al. 1990 ;Schmitt et ul. 1991).T h e genes encoding
these variants have been sequenced and this has led to the
development of specific oligonucleotides that can be used
as probes in DNA hybridization tests or primers in PCR
amplification (Tyler etul. 1991 ;Thomas et &I. 1993). Further
differentiation of VT2 genes can be achieved by restriction
fragment length polymorphism (RFLP) analysis of the amplification products. Alternatively, RFLP analysis with DNA
probes can be performed using genomic DNA (Thomas et
al. 1993).
4.2.3 Multilocus enzyme electrophoresis. Strains of 0 157
VTEC have been examined by multilocus enzyme electrophoresis. In a study by Whittam et u1. (1993), genetic
relatedness was estimated from allelic \-ariation among 20
enzyme-encoding genes. Little variation was found in the
0157 : H7 strains and 9596 of 369 strains belonged to a single
electrophoretic type, ET11. T h e profiles of the closely related
variant 0 1 57 : H7 strains are most closely related to those of
0 5 5 : H7 (Whittam et ul. 1993).
Grimm et ul. (1995) studied the molecular epidemiology
of the recent fast-food restaurant chain-associated E. roli
0 1 5 7 : H 7 outbreak in Washington State. Genomic DNAs
prepared from strains isolated from 433 patients were probed
with radiolabelled V T l and VT2 genes and bacteriophage 2
DNA and were subsequently analysed for their RFLP
patterns. VT RFLP and 1. RFLP analyses appeared to be very
sensitive and specific methods for the interstrain differentiation of E. roli 0157 : H7 and could assist in the epidemiological investigation of outbreaks caused by this
organism.
4.2.4 Pulse-field electrophoresis. Pulse-field
electrophoresis (PFGE) of genomic DNA has been performed
on 0157 VTEC strains from different origins (Bohm and
Karch 1992; Harsono r t al. 1993). In general, among the E.
r o l i 0157 : H7 strains the restriction patterns were either
identical or differed only by a few fragment bands. The
enzymes found to be the most useful in these studies were
-YbaI and Sfl. These rare cutting endonucleases generated

0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82, 537-551

DETECTION OF E. C O L / 0 1 5 7 :H7 I N FOOD 547

six and 10 distinct genomic profiles, respectively, for the 22

strains analysed (Harsono et al. 1993). It was concluded that
PFGE should be used together with other typing methods in
epidemiological studies of 0 1 57 VTEC infections.

the currently available methods for testing food should be

fully evaluated so that there is consistency of approach.

6. ACKNOWLEDGEMENT
4.2.5 Phage 2 probe analysis. Recently a phage A probe has
been used in the analysis of genomic DNA from 0157 VTEC
(Paros et ul. 1993; Samadpour et al. 1993). T h e RFLPs
obtained with the A phage probe differentiated the 72 strains
into 23 groups. The use of 1-RFLPs together with toxin
types and plasmid profiles provided further differentiation of
0157 VTEC of human and bovine origin. A V T phage probe
has also been used to examine 0157 VTEC (Rietra et uI.
1989), and this probe can subdivide strains within some of
the common phage types such as PT2 and PT49 (Willshaw et
ul. 1994b). It is recommended that a combination of methods
should be used to allow maximal differentiation of 0157
VTEC.

5. CONCLUSIONS
Because of the potential low infection dose, laboratory diagnosis of 0157 VTEC in food samples has developed over
recent years with the use of liquid enrichment and the development of methods such as immunomagnetic separation.
Solid media (sorbitol MacConkey agar) with improved selectivity for the isolation of 0157 VTEC have been described.
However, sorbitol-fermenting 0 1 57 VTEC strains such as
those reported in Germany would not be detected.
VTEC of serogroups other than 0157 have no reliable
biochemical, serological or morphological characteristics
(other than V T production itself) to distinguish them from
commensal E. colz. Thus to detect VTEC other than 0157
and phenotypic variants of E. roli 0157 in food, the use of
methods for detection of verocytotoxin production and V T
genes is recommended. Such a technique has been shown to
be extremely sensitive and useful as a 'broad brush' to find
potential disease-producing VTEC and would reduce the
chances of missing VTEC present in low numbers. But most
laboratories only test for 0157 VTEC as tests able to detect
all VTEC are not yet suitable for clinical laboratories and the
clinical and epidemiological importance of non-0157 VTEC
cannot be fully assessed at present.
VTEC isolates should be sent to a reference laboratory
for species identification using biochemical testing of probepositive colonies followed by 0 and H antigen determination.
In some cases further epidemiological typing, e.g. phage and
plasmid typing, as well as DNA fingerprinting may be necessary. There is a need for research into improved isolation
media for 0157 VTEC, rapid methods to detect VTEC of all
serogroups and verocytotoxin in food. Improved sub-typing
methods for VTEC are also needed, especially for 0157 and

The author would like to thank Sylvie Ray-Gueniot for her

secretarial assistance in the preparation of this manuscript.

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0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82, 537-551