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Journal of Applied Microbiology
1997,
82
537-551
A
REVIEW
Detection of Escherichia coli 0 1 7 :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
595911
0/96:
received 2 3 October 1996, revised 23 January 1997 and accepted 27 January 1997
1.
Introduction, 537
2. Isolation and identification of E.
coli
0 1 5 7 :H 7
2.1 Us e of biochemical characteristics, 538
2.2 Imm unoblotting with antibodies to 01 57 antigen, 541
2.3 Use of DN A probe specific for serotype 015 7
:
H7, 543
2.4 Confirmatory tests for E. roli 01 57 identification, 543
3. Me thods for detection
of
verocytotoxin production and V T
genes
3.1 Im mu nob lottin g with antibodies to verocytotoxins, 544
3.2 Use
of
D N A probe specific for V T genes, 544
4.1 Pheno typic tests, 546
4. Te sts in the reference laboratory
4.1.1 Serotyping, 546
4.1.2 Biotyping, 546
4.1.3 Phage typing, 546
4.2 Ge noty pic tests, 546
4.2.1 Plasmid analysis, 546
4.2.2 V T gene analysis, -546
4.2.3 Mu ltilocus 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
1 INTRODUCTION
are found consistently to be a reservoir for this organism in
Enterohaemorrhagic
Esrherirhiu
roli (EHEC) are recognized
as the primary cause of haemorrhagic diarrhoea and hae-
molytic uraemic syndrome (HUS). The pathogenicity of
EHEC appears to be associated with a number of several
cytotoxins referred to as shiga-like toxins (S L T ) or verotoxins
(V T) (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
(VT EC ) other than 0 15 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
0 1 5 7
:
H7 have been
food or water related. Likely vehicles of infection have been
undercooked grou nd beef according to a report by the C entres
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 e t al. 1992). Cattle
the enviro nmen t (Borczyk
et
al. 1987
;
Chapman
et
al. 1993).
However, isolation from gro und beef is uncommon (Okre nd
e t
al .
1990a). The se organisms may be present in low numbers
in implicated foods containing high levels of competing mic-
roflora (Willshaw
et
al. 1993). Two recent outbreaks were
unusua l in that they were both linked to consumption of low-
p 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 w ith ingestion of mayonnaise-containing food from
an Oregon restaurant chain (Keene et ul. 1993). In b oth cases
laboratory experiments showed that
E.
roli 0 1 5 7 :H7, while
dying rapidly in these acid foods a t room temperature, sur-
vived for weeks at refrigeration temperature (Besser
e t
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 int o three catego ries: (i) the use of
biochemical characteristics somewhat specific to strains of E.
roli
0 1 5 7 :H 7 (e.g. the inability to ferm ent sorbitol and the
lack of fi-glucuronidase activity) ; ii) the use of D NA probes
for verotoxins or markers associated with verotoxin-pro-
Correspondence
t o Dr C. >rnoz,pRozand,
Uniti de .2licrohrologir
Epidimiolo,pe mol6culuirr, E c o l e Nutionule 2tPrinuirc dr
L;yun,
I
arrnue
BourXelut,
BP
83 F-hY-780.2lurry I Etuile,Frunce.
1997
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for
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538
C
E R N O Z Y - R O Z A N D
ducing E.
c o l i ;
and (iii) immu noblotting with antibodies to
verotoxin (V T) or 015 7 antigen in conjunction w ith hydro-
phobic grid m emb rane filters.
The purpose of this review
is
to describe techniqu es for
isolation and identification of
E. coli
0 1 5 7 : H 7 a nd o t he r
verocytotoxin-producing strains of
E.
roli in food.
Thro ugho ut this report, the following convention has been
adopted; when the term VTEC is used, it includes vero-
cytotoxin-producing
E.
coli
of all serogroups
;
he 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 ; he term
E. coli
0 1 5 7 :H7 is used to refer to bacteria of that serotype
only, which is frequently VT-producing.
The predominating view held by Washington State epi-
demiologists, USDA and FDA officials
is
that t he only vero-
cytotoxin-producing E.
col i
of importance to human disease
is
E.
col i 0 1 5 7 : H7. T hi s is contrary to the view held in
Canada and the
UK.
In the USA, it is accepted that vero-
cytotoxin-producing
E.
coli
other than 0157
:
H7 can cause
human disease. How ever, present detection technology poses
many obstacles to efficient and economical diagnosis of non-
01 57 serogroups and efforts are concentrated solely on 0 15 7.
2 . ISOLATION AND IDENTIFICATION OF 15
COLl
0 1 7 : H7
2.1 Use of biochemical characteristics
Mo st biochemical reactions of
E. co l i
0 1 5 7
:
H 7 are typical of
E.
r o l i ,
with the exception of sorbitol fermentation and
p-
glucuronidase activity (W ells
r t
al. 1983 ;Doyle and Schoeni
1987). Abou t 930.0 of
E.
coli
isolates of hum an origin ferm ent
sorbitol within 24 h
;
however, E.
coli
0 1 5 7 :H7 was reported
as not ferm enting sorbitol (H onish 1986). But, in recent
reports some strains of 0157 VTEC strains fermented sor-
bitol within
24
h (Gunzer
et
ul. 1992; Pearce
e t al.
1994;
Hayes
et a/.
1995). T h e 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
V TE C do not produce P-glucuronidase (Okrend e t
al.
1990b).
Gro wth stu dies in trypticase soy broth (T SB ) 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 (D oyle 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 M atches 1990). Many procedures to detect
faecal coliforms and subsequently
E. coli
in food use incu-
bation temperatures in the range of 4445C. Hence, E.
coli
0 1 5 7 : 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 (Do yle and Schoen i 1987
;
Okrend
e t al.
1990a adhye
and Doyle 1991b). Several liquid media for the enrichment
of 0157 VTEC have been reported and some are listed in
Tabl e 1. Modified trypticase soy broth (m TS B) is sup-
plemented with either novobiocin or acriflavin to reduce the
growth of Gram-negative organisms. Another enrichment
medium is buffered peptone w ater (BP W ) with vancomycin,
cefsulodin an d cefixime to suppre ss the growth of ..leromonas
spp. and Proteus spp. respectively. O ptimal recovery of 0 1 7
VTEC was obtained with growth for 6 h (Chapman
e t a/.
1991).
After growth in enrichment media a number of methods
can be used to detect E. co l i 0 1 7 strains.
In an attempt to reduce the length of routine mic-
robiological analysis of foods and to negate problems associ-
ated with
a
rapid detection system (interference from
food
debris and background micro-organisms
;
ack of sensitivity),
there has been much interest in the development of sep-
aration/concentration techniques. Many techniques have
been studied for this purpose, including centrifugation (Bas-
sel
r t al.
1983), filtration (Sh arp e and Peterkin 19 88 ; Bobbitt
et nl .
1993), lectin-based biosorbents (P ayn e
et al.
1992),
aqueous biphasic systems (Benne tt
e t a / . 1994)
and ultrasound
(Miles
et ul.
1995). Perh aps the most successful of approaches,
however, has been the use of immunomagnetic separation.
T h e use of immunomagnetic separation (IM S) has been sug-
gested as a met hod of reducing total analysis time improv ing
sensitivity of detection. Para-magnetic particles coated with
antibodies specific to a target organism are added to a food
system. Th e target organism is captured onto th e magnetic
particles and the whole complex removed from the system
by application of a magnetic field. Tar get 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, thu s increasing
the sensitivity of the detection system. One problem experi-
enced by numerous researchers was non-target carryover.
Meadows (1971) reported that the non-target organism car-
ryover effect was possibly due to bacteria adh ering to the walls
of the glass test tubes. T h e basic protein salmine (prota min e),
which
is
positively charged at neutral
pH,
reduced attachmen t
because it adsorbed to th e bacteria and to the glass and hence
reduced their net negative charge. More precisely, Meadows
demonstrated that protamine reduced the number of
.lero-
nzonas
liyuefuriens,
E.
ro l i
and
Psrullomonasftuorescrns
adhering
to glass. Okrend
e t
al. (1992) solved the problem of non-
target organism carryover by adding 1 ml of aqueous pro-
tamine solution
(0.05
mg ml- ') to 10 ml ofenrichment culture
1997
The Society
for
Applied Bacteriology
Journal of Appried Microbiology
82,
537-551
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DETECTION
OF
E .
COLl 0 1
57
: H7
IN
F O O D 539
Table
1
Enrichment liquid media for
Escherirhzu
solz
0157
:
H7 Designation Compo sition
( l - ' )
Reference
m T S B
dm TSB-CA
BPW-VCC
mECn
Trypticase sop broth, 30 g
Bile salts 3, 1.5 g
Dipotassium phosphate (K2HP0.,), 1.5 g
Novobiocin, 20 mg
Doyle and Schoeni 1987
Trypticase soy broth, 30 g
Bile salts 3, 1.5 g
K 2 H P 0 4 ,1.5 g
Casamino acids, 10 g
Acriflavine-HC1, 10 mg
Padhye and Doyle 1991b
Buffered peptone water (Oxoid)
Vancomycin, 8 mg
Cefixime, 0 4 5 mg
Cefsulodin, 10 mg
Chapman
et
t2l. 1994
Tryptone, 20 g
Bile salts
3
1.12 g
Lactose, 5
g
K 2H P0 , , g
KH,PO1, 1.5
g
NaCI, 5
g
Novobiocin, 20 mg
Organon
Teknica 1993
( m E C
+
novobiocin) before ad ding the magnetic beads (Dyn -
al ' DynabeadsT hl M-280, Dy nal, 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 th e non-target colony counts obtained
from uninoculated meat samples. This procedure enabled
consistent recovery of E. ro l i 0 1 5 7 :
H7
from inoculated meat
samples. The percentage of E.
roli
0 1 7
:
H 7 cells captured,
compared to the total num ber of cells captu red, ranged from
48
to 10O0/o.
Wright
et
a / .
(1994) considered that IMS is rapid, tech-
nically simple and is a specific method for isolation
of E .
coli
01 57 and to be useful in epidemiological studies. The y
reported a 100-fold increase in sensitivity of detection of
E.
co/z
157 from inoculated minced beef using IMS compared
to direct subculture from BPW supplemented with vanco-
mycin, cefsulodin and cefixime (BPW -VCC) to cefisime tel-
lurite sorbitol MacConkey (C T-SM AC ) following incubation
for
6
h at 37C.
More recently, Bennett
e t a/.
(1995, 1996) demonstrated
the ability of the commercially available Dynabeads I M S
procedure using anti-E. roli 01 57 , to detect a few cells of E.
roli
0157 in
25
g of inoculated minced beef. Th is gave results
1 d earlier than a cultural analysis of similar sensitivity. The
use of IM S can increase the rate of isolation of E.
coli
0 1 5 7
depending upon the choice of enrichment broth. At levels of
less than 10 cells per 25 g, the direct plating method
(24
h
enrich men t) and the use of Dynabeads following enrichment
in BPW-VCC isolated 01 57 from
47%
of inoculated samples
whilst Dynabeads separation following enrichment in
m E C
+
n isolated
0 1
7 from
64%
of inoculated samples. Th e
use of BPW-VCC has been suggested (Chapman
et a/.
1991)
as it does not contain lactose. It is believed that o ther organ-
isms present in food might metabolize lactose to compounds
that inhibit
E. coli
0157. The inhibitory effect
of
microbial
lactose metabolism on E.
ro l l
0157 was reported by Hinton
et
a/.
(1991). However, in the study of Bennett
et
ul.
(1996),
any growth of competitor organism in mE C + n did not
appear to result in metabolites that were significantly inhibi-
tory to growth of
E. coli
0 1 5 7 . T h u s w ith m E C + n , I M S
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 (B ennett
et
ul. 1996).
After en richment the beads are usually cultured o n one
of
the selective media listed in Table
2
Escherirhia ro l l
0 1 5 7 :H 7 do not ferment sorbitol whereas
most other
E. roli
do and sorbitol MacConkey agar (SMA C)
has
been widely used for their isolation. However, SMAC
med ium relies entirely on d ifferential sugar fermentation and
1997 The Society
for
Applied Bacteriology,
Journal
of
Applied Microbiology
82
537-551
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540 C . V E R N O Z Y - R O Z A N D
Designation Composition
Reference Esrherirhiu roli 0157 :H 7
Table 2 Solid media for isolation of
SMAC
CR-SMAC
CT-SMAC
MSA-hIUG
PRS-hlUG
MSA-BCIG
H C
MacConkey
agar
D-Sorbitol, 1%
MacConkey Sorbitol
agar
(Oxoid)
Rhamnose,
0.5%
Cefixime,
0.05
mg I -
Farmer and Davis
1985
Chapman
et
ul.
1991
MacConkey Sorbitol agar (Oxoid)
Cefixime,
0.05
mg
1 - l
Potassium tellurite, 1 mg I -
MacConkey Sorbitol agar (Difco)
M U G ,
0.01 ,0
Phenol red base+29.6 agar
ri-Sorbitol, O.Sno
4-Meth y lumbellifer yl
B-D-glucuronide,
0.005%
MacConkey Sorbitol agar (Difco)
5-Bromo-4-chloro-3-indoxyl-
fi-Ii-glucuronic acid
c>-clobexylammonium alt, 0.01
nh
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-
Zadik et nl. 1993
Padhye and Doyle 1991b
Okrend et
ul.
1990c
Okrend et ul. 1990b
Szabo et d 1986
does not select V T E C
E. roli
0 1 5 7 :H7 f rom o the r
E.
roli o r
sorbitol non-fermenting genera and therefore lacks sensi-
t ivi ty. Modif ications of SM A C have been described with the
aim of improving the select ivi ty for 01 57 V TE C. O krend e t
al. (1990b) reported that the addition of 5 b r o m o - k h l o r o -
indoxy-P-l>-glucuronide (B CI G) at the 0.1 g l- ' level to
SM A C plates a ided in the isolat ion of E.
r o l i
0 1 5 7 :
H7
f rom
raw ground beef samples differentiating P-glucuronidase-
positive from P-glucuronidase-negative colonies.
Escherirhia
roli 0 1 5 7
:
H7 colonies being sorbitol-negative, P-glu-
curonidase-negative, remained white, while sorbitol-nega-
tive,
B-glucuronidase-positive
t u rned green to b lue . The
addit ion of BCI G to the S MA C agar reduced the number of
false suspect colonies picked fro m th e prim ary plating m ed-
ium by 3ho/o when compared to SMAC.
Esrherirhiu roli
0 1 5 7 : H 7 was isolated from 11 out of 12 inoculated meat
samples us ing SMAC-BCIG as compared to eight out of 12
samples us ing S M A C wi thout BCIG.
T h o m p s o n e t
a l .
(1990) developed a rapid fluorogenic assay
for
E.
coli 01 57 . This assay used . l-methylumbelliferyl-~-D-
glucuronide (M U G ) as an indicator which is hydrolysed to a
f luorogenic produ ct
by
the enzyme P-glucuronidase (Rippey
e t al. 1987). However, recently McCleery and Rowe (1995)
repor t ed tha t S M AC supplemented wi th M U G ( S M A C -
M U G ) performed poorly when s t ressed cel ls of the pathogen
are present. T h e incorporation o f a resuscitation period (2 h
at 25C) on trypticase soy agar (T SA ) before overlay with
SM AC -M UG was found to s ignif icantly (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 SMA C-
M U G .
Rocelle
et al .
(1995) studied the suitability of selective
plating med ia for recovering heat- or freeze-stressed E. roli
0 1 5 7
: H7
from T S B and groun d beef . A mixture of f ive
s t ra ins of
E.
roli 0 1 5 7
:H7
and f ive non-0157 s t ra ins of
E.
ro l i was heated in T S B at 52 , 54 or 6C for 10,
20
and 30
min, or frozen at 0C. Recovery of
E . rolz
0 1
7
:
H7
was
significantly higher on modified eosin methylene blue agar
t h an o n S M A C .
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542 C .
V E R N O Z Y - R O Z A N D
28 h, and its m inim um level of sensitivity was 0.6 cfu E.
coli
0 1 5 7
:
H 7 per g of food with
0910
false-negative an d 290 false-
positive results. Since the procedure used 0157 polyclonal
antisera, all presumptive positive samples that could be false-
positive results must be confirmed for
E.
rolz
0 1 5 7
:
H 7 by
isolating the organism. T h e isolation and identification of the
bacteria required an additional
3 4
.
T o detec t
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
01 57 as the capture antibody and a monoclonal antibody to
E.
ruli
0 1 5 7
:
H 7 as the detection antibo dy) on
a
hydrophobic
polyvinylidine difluoride-based membrane.
Escherzrhia roli
0 1 5 7 :H 7 could be detected in grou nd beef containing as few
as 0.1 cfu of E. colz 0157:H7 per g af ter enr ichment in
mT SB . Tw o false-positive results were observed durin g the
incubation
;
hey 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 an d Doy le (1991a, 1992) described a rapid sand -
wich 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 peroxidase-
labelled monoclonal antibody (th e MA b 4E8C12), claimed to
be specific to two outer proteins uniqu e to serogroups 01 57
and 02 6 -t h e two serotypes associated most frequently with
H U S (Padhye and Doyle 1991a)-was used as the detection
antibody. The 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
0 1 5 7
:
H 7 per g of food after overnight enrich men t. Accord-
ing to Padhye and Doyle (1991a), this procedure was highly
specific, sensitive, rapid, easy to perform and am enable to use
by laboratories performing routi ne microbiological testing.
Mo re recently, the specificity of this test was investigated
by Johnson
et
al. (1995). Th ey rep orted th at the target anti-
gens of the detection reagent, MAb 4E8C12, were present in
numerou s serotypes of
E.
rolz ( 0 2 2
:
H 8 , 0 2 6 H 1 1 , 0 4 6
:
H38,
0 8 8 : H 4 9 , 0 9 1 : H 2 1 , 0 1 0 3 : H 2 a nd
0 l l l : H l l )
and that
their ELISA reactivity was influenced by bile salts, acriflavine
and heat. Acriflavine enhanced EL ISA 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 false-
positive reactions while retaining the enhancing effects of
acriflavine and heat. T h e above ELISA-po sitive strains were
grown in T SB for 18 h at 37C. One m l 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 0 1 5 7 : H 7 strains. T he beads were cap-
tured on the side of the tube with a magnetic particle con-
centrator, washed and resuspended in 0.5 ml of mTSB-
acriflavine for overnight cultivation at 37C. When heated
aliquots of these cultu res were tested in the EL ISA,
E. cdi
0 1 5 7 : H 7 remained strongly positive, while 10 of the 12
previously ELISA -positive strains tested negative. T hu s, the
modified protocol, incorporating immunocapture, greatly
improved the specificity of the EHEC-Tek ELISA while
maintaining the assays low limits of detection. Ch apm an and
Siddons (1996) compared the EHEC -Tek with IM S 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 m ECn in parallel as the primary enrichment, the results
were much more encouraging with BPW/VCC enrichment,
following which the E HEC -Tek detected
E. roli
01 57 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
01 57 . Th e EHEC-T ek 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
01 57 ( EC O) assay is intended for use with th e Vitek Imm uno
Diagnostic Assay System (VIDAS) as an automated quali-
tative enzyme-linked fluorescent immun oassay (EL FA ) 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. Th e
SPR is coated with polyclonal anti-E.
roli
0157 antibodies.
Reagents for the assay are sealed in reagent strip s. An aliquot
of the enrichmen t broth is placed in to the reagent strip and
the sample is cycled in and out of the SPR for a specific
length of time. Thi s test was evaluated by Kerd ahi and Cohen
(1996), for use in the d etection of E.
rolz
01 57 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 on e of two strain s of enterohaemorrhagic
E.
rolz
0 1 5 7
:
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 cold-
stressed cells, both V1DASTh1 nd the Bacteriological Ana-
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N F O O D
543
lytical Manu al cultural assay (consisting of sprea ding 0.1 ml
of overnight growth from mT SB enrichment broth on H C
agar plates) detected all high and low levels of contamination.
N o false positives or interference from product background
fluorescence were encountered in any of the cheeses tested
by VID AS . 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 cultu re or an area of a filter or
semisolid medium which would be promising for focusing
furth er cultural isolation efforts in ord er to obtain an isolated
culture for additional tests.
2.3 Use of
DNA
probe specific for serotype
0 157 :
H7
Esrherii.hia roli
0 1 5 7 :
H i
was shown to contain z i d A gene
sequences that encode for the fl-glucuronidase enzym e (Fen g
r t
d
1991 Feng and Lam pel 1992). Analyses demon strated
that the nucleotide sequence of the 5 terminus of the rid A
structural gene of 01 57 : H7 was identical to that of M UG -
positive
E.
roli, except for a su bstitution 90 bases downstream
from the initiation codon. In a nother experim ent, Feng (1993)
used an oligonucleotide probe, PF-27, directed to this region,
containing a unique base substitution in th e 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
othe r enteric isolates showed tha t the probe reacted only w ith
the 17 isolates of
0 1
7
:
H7 serotype. Interestingly, the probe
did not hybridize with the 73 MUG-posit ive E. roli, the 13
MUG-posit ive
Shigella
or eight MUG-positive
Salmonrllu
isolates analysed. Except for the single nucleotide base dif-
ference, the PF-27 sequence is identical to that
rid
A gene,
which was present in almost all
E. roli
regardless of MU G
phenotype. Hence, the absence of probe hybridization with
E . cdi
indicated that PF-27 could discriminate the single
nucleotide difference between th e
5
region of the
zlid
A gene
of E. coli and its allele in th e
0 1
7 :H 7 serotype. T o fur ther
verify probe specificity, DNA from
E. roli
a n d 0 1 5 7 : H 7
were digested with Hznfl enzyme and examined by Southern
blotting. So uth ern analyses showed th at the PF-27 was spec-
ific for the base subst itutio n 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-produc ing EH EC se ro types (02 6 H11 and 0 1 1
:
N M )
studied. T he stringent specificity of the PF-27 probe may be
valuable for clinical diagnosis and for the identification of
0 1 5 7 :
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 oth er 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 1 7 strains were isolated from hae-
molytic uraemic syndrome patients in Germ any and obtained
from G unzer
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 med ia interference or th e 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 t ests for E. ofi0157 identification
Colonies that appear to be
E . roli
01 57 m ust be confirmed as
E. roli using biochemical tests. The se would exclud e any non-
E.
r o l i
that give false-positive agglutination tests with the
0 1 7 a n t is e rum .
Esrherirhia hermunii
is biochemically and
serologically similar to E.
roli
and cross reacts w ith polyclonal
antisera to
E.
rnli
(Li or and Borczyk 1987). How ever,
E.
colz,
unlike
E. hernzanii,
does not f erme nt 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 01 57 should be confirmed
serologically with antisera against and
H
antigen. 0157
VTEC usually have the flagellar antigen Hi although some
strains are non-motile. All confirmed
E. rolz
0157 stra ins
should be tested for produ ction for verocytotoxin ( V T ) or
the presence of V T genes.
Selective screening of food samples with sorbitol Mac-
Conkey agar will miss a proportion of 0 1 5 7
:
H 7 strains which
are sorbitol + V T + There is increasing evidence sug-
gesting that phenotypic variations exist among the isolates
within E. roli 0 1 5 7 :
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
(HU S), fermented sorbitol and were M U G positive. Pheno-
typic variants of
E. roli
0157 have also been isolated in other
parts of central Europe an d in the Unite d States (Feng 1995
;
Hayes
et ul.
1995). Foo d microbiologists sho uld be aware of
the emergence of these phenotypic variants and recognize
that these strains may not be identified by routine culture
meth ods or by biochemical tests used to characterize serotype
01 57 : H7. Goldw ater and Bettelheim (1994) showed that
in Australia, 0157 :
H i
was uncommon but other less well
recognized serotypes (e.g. 0 1 1
:H-, 0 6
H 3 1 , 0 9 8
:
H-,
0 4 8
:
H21 ) were responsible for H U S and bloody diarrhoea
in Australia. T he se findings suggested th e possibility that
transmission of phage-encoded V T genes to native strains of
E.
roli could occur and current focus on E .
coli
0 1 5 7 :H 7
could be broadened to include methods that would detect all
VT EC . VT EC of serogroups other than 0 15 7 have no reliable
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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 VTE C other than
01 57 and the phenotypic variants of
E. coli
157 in food, we
have to use methods for detection of verocytotoxin pro-
duction and V T genes.
3. METHODS F O R DETECTION OF
VEROCYTOTOXIN PRODUCTION AND
VT GENES
3.1 lmmunoblo tting with antibodies to
verocytotoxins
Verocytotoxin is detected by its cytotoxic effect on vero cells
(Konowalchuk
et
al .
1977). Faecal su spensions, cultu res fil-
trates or live cultures can be tested (Scotland
et
al. 1980;
Lior and Borczyk 1987). Strains are grown in T S B and
cultu re 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 m icroscope. Final readings are usu-
ally made after incubation fGr 3 4 when the cells are fixed
and stained (Sm ith and Scotland 1993). In some early studie s
of V T production bacteria were grown in iron-restricted
media and such growth conditions may increase production
of VT 1, but not V T2. F or routine testing, concentrations of
V T are adequate in ordinary broth media as described below.
A considerable amoun t of VT is not liberated into th e medium
but remains cell bound. I t can be released by sonication, with
the use of a French press or by polymyxin treatment, and
these techniques have been used for pre parin g large quantities
of VT (Karmali
et a/.
1985). It has been shown that V T EC
are not isolated in the absence of polymyxin-releasable VT,
whereas VT is frequently pre sent w here the organism itself
cannot be isolated (Karmali
et
ul.
1985 Clarke
et
al.
1988).
T o confirm t hat cytotoxic effects on Vero cells are due to
the presence of V T, neutralization tests using antisera against
VT 1 or VT 2 should be performed (Scotland
et
al. 1988). Th e
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 ELIS As have been described for th e detec-
tion of V T (Basta
et ul.
1989
;
Downes
e t a / .
1989
;
Acheson
et a/.
1990). Som e bind V T to glycolipids containing a ter-
minal a D-Gal-( 1 )-rl-G al and purified globotriosyl cer-
amide (GbJ, lyso-Gb, and hydatid cyst fluid isolated from
sheep infected with
El-hinororrus r~mdosus ,
ave been used.
In other studies ELIS A monoclonal antibodies against V T
were used to bind toxin. In general, these test have no t 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 strain s from a clinical specimen (Sm ith 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. Th e meth od utilized monoclonal antibodies directed
against the verotoxins and is sensitive to all verotoxin 1
and /or 2 producing serotypes. Whe n applied to meat, 11 of
20 samples positive for verotoxin by polymyxin extraction
yielded verotoxigenic E.
roli
of a variety of serotypes including
0 1 5 7
:
H7.
According to Milley an d Sek la (1993), reasons for
why not all VT-positive samples yield a VT E C isolate might
include a low num ber of VT EC p resent or a low proportion
of VT EC relative to o ther Gram-negative organisms. Intrin-
sic to this problem were the facts that the VTs are a family
of highly pote nt 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
(inclu ding non-toxigenic
E. roli).
Th us, the sensitivity of the
cell cultu re assay for V T is superior to th e sensitivity of
isolation procedures, and non-VTEC coliforms can compete
with V TE C for space on the isogrid mem brane.
3.2 Use of DNA
probe specific for
VT
genes
In addition to im muno blotting with antibodies to verotoxin,
polynucleotide probes for VT l and V T2 derived from cloned
genes (Willshaw
et a/ .
1985, 1987), and synthetic oligo-
nucleotide 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 19 89a;
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 oli-
gonucleotide
(11
bases) derived from the VT2 gene of E.
rolz
0 1 5 7
:
H 7 strain for the identification of
E. roli
that produced
cytotoxins for Vero or Hela cells. Th e 20-base probe appeared
to be as valid as the Il-b ase probes with regard to specificity
and sen sitivity of the hybridization reaction. Fift y 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 V T2 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. T h e colony blot
hybridization with the oligonucleotide probes described by
Karc h and M eyer (198 9) could serve as
a
specific and sensitive
test with po tential diagnostic value.
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Levine
et
al. (1987) prepared a DNA probe, CVD419,
developed from a 3 4 kb Hind111 fragment of th e large ca 60-
MD a plasmid that encodes for an intimate form of adhesion
and is typically carried by
E.
coli
0 1 5 7
:
H7 and o the r VT EC
(Framatico
et
al. 1991 Toth
e t
ul.
1991 Ashkenazi
et
nl.
1992). T h e probe hybridized with 99Yo of all
E.
roli 0 1 5 7
:
H 7
and 77% of
E.
ro l i 0 2 6 :H l l , both of which belong to the
VT EC group. Th e probe hybridized with 21 out of 26 VTE C
(8l0/o) and with only one of the non -VT EC
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
EH EC and V TE C isolates that no longer carry the 60-MDa
plasmid (Ratnam
et
al .
1988).
Huck
e t a / .
(1995) developed a probe for detection of 0 1 5 7
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
01 57 serogroup
E.
coli
strains. A 2.0 kb
SmuI
fragment probe
(VPM 1) was the most specific for serogroup 0 15 7 EH EC .
However, this probe hybridized with five of 50 non-0157
E.
coli
strains which w ere verotoxin or CVD 419 probe-positive.
With another stringent condition of ov ernight hybridization
a t
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
attach ing and effacing) from
E.
coli 0 1 5 7 :H7 , necessary for attach men t 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 K aper 1992). T h e gene from
the 0 1 7 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 Kap er (1990) showed that 29 of 30 VT EC strains
belonging to serotype 0157 :H7 o r 0 2 6 :H11 hybridized
with the
eae
probe. In a study of other VTEC serogroups,
however, a mu ch smaller prop ortio n of strains (35O.
o
wasear
positive (Willshaw
et
ul.
1992). According to W illshaw
et a/.
(1994a), hybridization with a probe such as
ear
015 7 ( f rom
the
3
end of the eae A gene homologue of an 015 7 VT EC )
is valuable in the differentiation of 0157 strains. Probing of
colonies from food or faecal specimens w ith V T and
ear
01 57 sequences in combination targets 0 1 7 VTE C, whereas
techniques based on immunological detection of the 0 1 7
antigen identify all strains of this serog roup and some cross-
reacting organisms.
Amplification of part of the V T gene, using th e polymerase
chain reaction (PC R), has also been used to test for the
presence of VTE C. W ith this procedure, D N A is amplified
to increase the level of target D N A when V T EC 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 oli-
gonucleotide probes complementary to part of the amplified
sequence. It was possible to identify V T1 or VT 2 sequences
but variants of V T2 could not be distinguished from V T2.
In order to detect all types of VTEC isolated from animal
and food sources, Read
e t d
1992) developed a PC R using
a pair of oligonucleotide primers, targeting conserved
sequences found in V TI , VT 2 and V TE genes. Supernatant
fluids of boiled broth c ultu res
of
VT EC (233 strains) isolated
from ground beef, ground pork, raw milk, bovine faeces
and porcine faeces, non-VTEC E.
ro l i
(72 strains) and other
enter ic and food bacteria ( 76 strains) were tested by P CR .
T h e verocytotoxigenicity
of
these stra ins was verified by V ero
cell assay. All 223 VTEC isolates, comprising over
50
dif-
ferent serotypes, were detected by- the P CR procedure.
Shz-
gellu
uysentrriue
type 1 was the only oth er bacterium th at was
positive in this assay. As little as 1 pg of VT EC D N A and as
few as 17 cfu of VTEC could be detected with this method.
T he results suggested that these primers detect VTE C over
a
wide range of serotypes. T hi s method might be applicable
as a screening procedure for the detection of VTEC in sam-
ples of foods and faeces (Ga nn on
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 con-
centration 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 coun ts and detectability by PC R techniques w ith cells
of E. rolz previously exposed to a range of stress treatment. In
all cases the organisms were detectable by PCR after plate
cou nts had declined to zero. Tre atm ent w ith acid or hydrogen
peroxide caused loss of PCR soon after viability was lost, but
stron g PCR signals were obtained from starved or desiccated
cells long after cells became non-viable. Exposure to tem-
peratures u p to 100C had little effect on detection by PCR
and even autoclaving cells at 121C for 15 min failed to
abolish PCR detection completely. The re 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
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of Applied Microbiology82,537-551
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DETECTION O F E. C O L / 0 1 5 7 :H7 N FOOD 5 7
six and 10 distinct gen omic profiles, respectively, for th e 22
strains analysed (Harsono e t al. 1993). It was concluded that
PF G E should be used together with other typing methods in
epidemiological studies of 0 1 7 VT EC infections.
4.2.5
Phage
2
probe analysis.Recently a phage A probe has
been used in the analysis of genomic DN A from
1
57 V T E C
(Paros e t ul. 1993; Samadpour e t al. 1993) . The 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 furt her differentiation of
01 57 V TE C of human and bovine or igin. A V T phage probe
has also been used to examine 0157 VTEC (Rietra
e t uI.
1989), and this probe can s ubdivide strains within some of
the common phage types such as PT 2 and P T4 9 (Willshaw
et
ul. 1994b). It is recommended that a combination of meth ods
should be used to allow maximal differentiation of 0157
VTEC.
5 CONCLUSIONS
Because of the potential low infection dose, laboratory diag-
nosis of 0157 VTEC in food samples has developed over
recent years with the use of liquid enrich men t and the devel-
opment of methods such as immunomagnetic separation.
Solid media (sorbitol MacConkey agar) with improved sel-
ectivity for the isolation of 0 15 7 VT E C have been described.
However, sorbitol-fermenting 0 1 7 VT E C strains such as
those reported in Germany would not be detected.
V T E C
of
serogroups other than 0157 have no reliable
biochemical, serological or morphological characteristics
(other than V T produ ction itself) to distinguish th em from
commensal
E. colz
Th us to de tec t VT EC othe r than 015 7
and phenotypic variants of
E.
r o l i
0157 in food, the use of
methods for detection of verocytotoxin production and V T
genes is recommended. Such a tech nique 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 V T E C present in low numbers. Bu t most
laboratories only test for 0157 VTEC as tests able to detect
all
V T E C are not yet suitable for clinical laboratories and the
clinical and epidemiological im portance of non-015 7 VT EC
cannot be fully assessed at present.
VTEC isolates should be sent to a reference laboratory
for species identification using biochemical testing of probe-
positive colonies followed by
and H antigen determination.
In
some cases furt her epidemiological typing, e.g. phage and
plasmid typing, as well as D N A fing erprin ting may be necess-
ary. There is a need for research into improved isolation
media for 015 7 VT EC, rapid methods to detect VTE C of all
serogroups and verocytotoxin in food. Improved sub-typing
methods for V TE C are also needed, especially for 0 15 7 and
the currently available methods for testing food should be
fully evaluated
so
that t here is consistency of approach.
6.
ACKNOWLEDGEMENT
The author would like to thank Sylvie Ray-Gueniot for her
secretarial assistance in the preparation of this manu script.
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