07-Detection of E. Coli O157H7 and Other Verocytotoxin-producing E. Coli

8/9/2019 07-Detection of E. Coli O157H7 and Other Verocytotoxin-producing E. Coli

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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

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Journal of Appried Microbiology

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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

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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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DETECTION O F

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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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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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DETECTION O F

E . COLl0157 :

H 7

IN FOOD

545

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

1997

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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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Documents

07-Detection of E. Coli O157H7 and Other Verocytotoxin-producing E. Coli