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Annals of Microbiology, 53 (4), 511-527 (2003)

Escherichia coli O157:H7 general characteristics,isolation and identification techniques

L. BENEDUCE1*, G. SPANO1, S. MASSA1

1Dipartimento di Scienze degli Alimenti (DISA), Facolt di Agraria, Universit degli Studi diFoggia, Via Napoli 25, 71100 Foggia, Italy

Abstract -Escherichia coli O157:H7 is one of the most studied food-borne pathogen bacte-ria, because of its widespread diffusion, low-dose infectiveness, and severe symptoms asso-ciated. It is necessary to focus the attention on the factors (like temperature, pH and aw) thatmay affect growth and survival ofE. coli O157:H7 in food and in the environment sincethere is a wide range of foods that can be contaminated by this strain, causing haemorrhagiccolitis outbreaks. Culture methods and phenotypical assays for isolation and identification of

E. coli O157:H7 are more and more integrated (and often completely replaced) in researchand diagnostic laboratories by molecular methods. PCR techniques, particularly the latestquantitative real-time-PCR, make possible to reach specificity and sensitivity level of fewCFU detection even in complex samples like food and faeces.

Key words:Escherichia coli O157:H7, epidemiology, identification, molecular methods.

INTRODUCTION

Escherichia coli strains that cause diarrhoea include enterotoxigenic (ETEC),

enterophatogenic (EPEC), enteroinvasive (EIEC) and enterohaemorrhagic (EHEC)

strains. Recently, enteroaggregativeE. coli (EAggEC) has also been found to be adiarrhoegenic strain. Among EHEC strains, E. coli O157:H7 is one of the most

studied food-borne pathogens, because of its widespread diffusion, peculiar toler-

ance to some physical and chemical treatments, severity of illness and low dose

infectiveness. This paper describes the characteristics ofE. coli O157:H7, strain

that distinguish it from the harmless commensal type ofE. coli in the human gut.

Summary of the pathogenesis, foodborne outbreaks, resistance and sensitivity to

factors such as temperature, pH, aw and methods of its identification with particu-

lar emphasis to molecular techniques are reported.

511

* Corresponding author. Phone: +39-0881589352; Fax: +39-0881740211; E-mail:[email protected]

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GENERAL PHYSIOLOGICAL CHARACTERISTICS

Escherichia coli O157:H7 possess metabolic characteristics distinct from otherE.

coli. Most isolated strains are unable to ferment sorbitol within 24h and cannot

hydrolyze 4-methylumbrelliferyl-D-glucuronide (as lacking -glucuronidaseenzyme) (Okrend et al., 1990; Strockbine et al., 1998). These two characteristics,together with the inability to grow well at temperatures > 44.5 C (see below), are

very important in discriminating between O157:H7 and otherE. coli. The antigenic

composition of outer cell membrane is the most important characteristic that leads

to identification of this serotype. E. coli O157:H7 have a cell envelope structure

typical of Gram-negative cells, and thus, posses an outer membrane with a

lipopolysaccharide component that is distinct from the cytoplasmic membrane.

The O157 antigen is defined by the carbohydrate composition and structure within

the lipopolysaccharide. The H7 antigen is determined by the unique polypeptide

composition of the flagella.

PATHOGENESIS

Shiga toxin-producingE. coli O157:H7 and other serogroups can cause haemor-

rhagic colitis, haemolytic-uraemic syndrome (HUS) and occasionally mild non-

bloody diarrhoea in man, although some infections may be asymptomatic. HUS

complicates about 10% of cases ofE. coli O157:H7 infection and carries a mortal-

ity rate of 2-10%. EnterohaemorragicE. coli O157:H7 and related organisms arealso referred to as Shiga toxin-producingE. coli (STEC) because of their ability to

produce Stxs (formerly called Shiga-like toxins). The infectious dose ofE. coli

O157:H7 is very low: for example, between 0.3 to 15 CFU/g was enumerated in

lots of frozen ground beef patties associated with an outbreak in USA in 1993. Sev-

eral other cases in which the infectious dose was very low (from 0.2 to 50 CFU/g)

were reported by different authors (Willshaw et al., 1993; Tilden et al., 1996).

FOODBORNE OUTBREAKS

Haemorrhagic colitis that characterised the first registered outbreak ofE. coli

O157:H7 in Oregon, was described with severe abdominal cramps, little or no

fever, severe bloody diarrhoea and colonic mucosal oedema (Riley et al., 1983;

Riley, 1987). This first recognized outbreak was linked to contaminated ground

beef, but it is now claimed that numerous foods have been implicated in outbreaks

and sporadical cases: ground beef (Doyle and Padhye, 1989), roast beef (Rodrigue,

1995), venison jerky (Keene et al., 1997), salami (Centers for Disease Control and

Prevention, 1995), raw milk (Meng et al., 2001), yoghurt (Morgan et al., 1993),

lettuce (Mermin et al., 1997), unpasteurized apple cider or juice (Besser et al.,1993; Centers for Disease Control and Prevention, 1997a), cantaloupe (Beuchat,

1996), potatoes (Morgan et al., 1988), radish sprouts (Izumiya et al., 1997) and

alfalfa sprouts (Centers for Disease Control and Prevention, 1997b). It is notewor-

thy that certain foods such as apple cider and dry-cured salami that previously

were considered safe and ready to consume because of their high acidity and

512 L. BENEDUCE et al.

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because they are usually not heated before consumption, have been vehicles of out-

breaks (Meng and Doyle, 1998). The largest outbreak to date occurred in Japan in

1996, affecting over 9000 people, with contaminated radish sprouts as the possible

source of infection (Michino et al., 1998). Reports of person-to-person and water-

borne transmission have recently been increasing (Menget al.

, 2001).

OTHER STEC SEROTYPES

Even ifE. coli O157:H7 is the major cause of haemorrhagic colitis and HUS

worldwide, several other STEC serotypes have been isolated during past outbreaks.

Among over 200 serotypes of STEC isolated from humans, only few serotypes are

EHEC (able to cause enterohaemorrhagic colitis): the most important serotypes are

O157:NM (non-motile), O111:NM, O26:H11, O111:H8. For these non-O157:H7

strains the food-borne pathogenesis has not yet been completely established, due tothe difficulty for most laboratories to investigate the serotype and the atypical

behaviour of these EHEC (e.g. O157:NM does ferment sorbitol rapidly) (Karch

and Bielaszewska, 2001). In table 1 are listed the 16 countries in Continental

Europe in which the above mentioned STEC serotypes were isolated (Caprioli and

Tozzi, 1998) (Table 1).

Ann. Microbiol., 53 (4), 511-527 (2003) 513

TABLE 1 Isolation of principal STEC serotypes in Continental Europea

Country STEC serotypesb

O157 O26 O111

Austria HAF HA HA

Belgium HA HA HA

Czech Republic H H H

Denmark HAF H H

Finland H H

France H H H

Germany HF HA HA

Greece H

Italy HAF HA HA

Spain HAF HA HA

Switzerland HA HA

The Netherlands HA H

aAdapted from Caprioli and Tozzi (1998).bSTEC from human (H), animal (A), and food (F).

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FACTOR INFLUENCING GROWTH AND SURVIVAL IN FOOD

The optimum temperature forE. coli O157:H7 is 37 C, the same as that of otherE.

coli. The maximum for cell development is 45 C. This detail is very important

because other faecal coliforms are able to grow well at 44 C while growth ofE.

coli O157:H7 may be slowed or inhibited. Culturing conditions may affect the

growth: Brain Heart Infusion Broth (BHI) is more permissive at 42-45 C thanE.

coli Broth (EC) and Trypticase Soy Broth (TSB) (Doyle and Schoeni, 1984). The

resistance to heat ofE. coli O157:H7 is not noteworthy. D values for that pathogen

are 270, 45, 24 and 9.6 s at 57.2, 60.0, 62.8, and 64.3 C, respectively (Doyle and

Schoeni, 1984). Usual pasteurization temperatures are sufficient to kill more than

104 cells per ml (Daoust et al., 1988). Spano et al. (2003) observed that Moz-

zarella cheese should be free ofE. coli O157:H7 if temperatures higher than 80 C

are used during milk processing (Table 2).

Few elements are clear about tolerance ofE. coli O157:H7 to freezing temper-atures. It seems that cell injury and death is very limited after stocking at -20 C for

9 months (Doyle and Schoeni, 1984). Chou et al. (1999) studied the survival ofE.

coli O157:H7 after low temperature treatments of -28, -18 and -5 C and found that

the lowest surviving population was found when stored at -18 C followed by those

stored at -28 C and -5 C. Moreover,E. coli O157:H7 has been isolated from sur-

face water and can survive for many weeks in this kind of environment, especially

at cold temperatures (Rice et al., 1992; Wang and Doyle, 1996).

All STEC are able to grow on laboratory media in a range of temperatures

between 6.5 and 7.2 C (Davies et al., 1992). Growth ofE. coli O157:H7 has beenobserved in foods at temperatures of 8 C in non-fermented cider and at 12 C in

salads (Abdoul Raoufet al., 1993).Escherichia coli O157:H7 is also able to sur-

vive and to grow in raw milk stored at 8C for 17 days (Massa et al., 1999). Refrig-

eration storage at less than 5 C should be adequate to inhibit growth ofE. coli

O157:H7, but is insufficient when other conditions (pH, NaCl, concentration) are

not unfavourable (Zhao et al., 1993).

Escherichia coli O157:H7 seems to have no particular tolerance to salt and low

aw levels. Bacterial growth has been documented with NaCl concentration ranging

from 2.5 to 6.5%, when other growth-affecting factors were favourable (Conner,


TABLE 2 Comparison of D values forEscherichia coli O157:H7 and Salmonella spp. inground beefa

Temp (C) D value (s)

Escherichia coli O157:H7 Salmonella spp.

30.5% fat 17-20% fat

51.7 115.5 NDb 54.3

57.2 5.3 4.5 5.43

62.8 0.47 0.4 0.54

aFrom Meng et al. (2001).bND, not detected.

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1992). Another study confirmed these results on TSB medium and determined that

the population ofE. coli O157:H7 decreased by 1 to 2 log10 CFU/g in fermented

dry sausage during fermentation, drying and storage at 4 C for two months (Glass

et al., 1992). More studies are needed to determine the aw limit for growth in dif-

ferent foods and the interaction between low aw levels and other factors in foodprocessing.

The first clear information about the peculiar tolerance to acidity ofE. coli

O157:H7 emerged during occurrence of an infection linked to consumption of

yoghurt produced from raw milk in 1991 (Morgan et al., 1993). It is now believed

that acidic resistance ofE. coli O157:H7 plays a key role in food-borne illness

linked to this pathogen. The minimum pH forE. coli O157:H7 growth is 4.0-4.5 on

laboratory media, but this limit is dependent upon the interaction of pH with other

growth factors. When temperature and aw are lower than optimum for E. coli

O157:H7 the minimum pH rises to 4.5 (Glass et al., 1992). It has been stated that

yoghurt, despite its low pH and the presence of elevated microflora, can be con-taminated byE. coli O157:H7 (Morgan et al., 1993). Massa et al. (1997) evaluat-

ed the effect of fermentation, refrigeration and storage at 4 C in yoghurt produced

with two different lactic starters (final pH 4.5-4.6) and stated thatE. coli O157:H7

was detectable even after 7 days of storage. The population ofE. coli O157:H7

decreased by only 1 to 1.5 log10 CFU/ml during the treatment. This study con-

firmed that acidic protection in fermented milk is not adequate for E. coli

O157:H7.

ISOLATION AND IDENTIFICATION TECNIQUES

The three most important phenotypic differences between E. coli O157:H7 and

otherE. coli are inability to ferment sorbitol, lack ofglucuronidase enzyme andweak or no growth at temperatures above 44 C. (Meng et al. 1997). However, in

some cases atypical O157 strains may ferment sorbitol (Morgan et al., 1993; Wil-

son et al., 1992). The culture isolation techniques used to verify the suspect pres-

ence ofE. coli O157:H7 in food are based upon these characteristics, and can help

to distinguish between non-pathogenic E. coli and pathogenic E. coli O157:H7

strains. On the other hand, culture isolation requires a lot of time and it is labor-

intensive. The need for a rapid diagnostic test for recognizing contamination asso-

ciated with bloody diarrhoea and HUS forced the rapid development of new tech-

niques for isolation and identification of STEC in foods. Immunoassays such as

enzyme-linked immunosorbent assay (ELISA) were very useful for rapid screening

ofE. coli O157:H7 and non O157 in food. Actually the impressive development of

molecular techniques has made it possible to have real-time identification with

high sensitivity and specificity.

The low infectious dose ofE. coli O157:H7 in foods makes it necessary to

have a sensitive isolation method, using enrichment and selective procedures. Sev-eral studies reported the development of culture media specific forE. coli O157:H7

selective enrichment, (Doyle and Schoeni, 1987; Padhye and Doyle, 1991; Chap-

man et al., 1991) containing bile salts, novobiocin, cefsulodine and cefixime as

selective agents. The US Department of Agriculture (USDA) recommends 24 h

enrichment on selective media at 35 C, in order to provide conditions that promote

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growth ofE. coli but are inhibitory to other species. The best selective media for

presumptive identification ofE. coli O157:H7 was found to be Sorbitol Mac-

Conkey Agar (SMAC), in which E. coli O157:H7 colonies remain uncoloured

(lack of sorbitol fermentation) while otherE. coli appear red (March and Ratnam,

1986). Okrend et al. (1990), and Tesh et al., (1991) suggested the addition of 5-bromo-4-chloro-indoxyl--D-glucuronide (BCIG) on SMAC, to differentiatestrains that lack-glucuronidase (like E. coli O157:H7), which appears white,while the colonies which possess -glucuronidase activity turn green or blue.Thompson et al. (1990) developed a rapid fluorescent test for detection ofE. coli

O157:H7, by using 4-methylumbelliferyl--glucuronide (MUG). This substancecan evidence -glucuronidase activity based upon production of a fluorescenthydrolysis compound (Rippey et al., 1987). Positive colonies are fluorescent after

ultraviolet light exposure, whileE. coli O157:H7 give no fluorescence. Currently

SMAC is usually employed with addition of cefixime and tellurite (CT-SMAC), as

reported by Zadiket al. (1993). Cefixime inhibits Proteus spp. At a concentrationnot inhibitory toE. coli, and O157 STEC strains are generally less susceptible to

tellurite than are many other non-sorbitol fermenters such asAeromonas spp.,Mor-

ganella spp., Providencia spp., and most other E. coli strains. Recently a new

selective media was developed by Biolog inc., (Hayward, CA.), called Rainbow

agar O157. This media is more specific than SMAC for isolatingE. coli O157:H7

and it is also useful for isolating and differentiating other STEC serotypes from

nontoxigenic E. coli because it has both selective and chromogenic properties.

Most bacteria, other than O157 and non O157 STEC, are inhibited or grow as

white or cream coloured colonies.E. coli O157:H7 colonies are unique, with a dis-tinctive black colour, whereas typical non-O157 STEC colonies are blue or purple

and most non-toxigenicE. coli colonies are reddish (Meng and Doyle, 1998).

To confirm presumptive O157 isolates from culture methods several immuno-

logical methods have been developed to detect O and H antigens. These methods

are rapid (from 15 min. to 2 h) and are employed after a pre-enrichment step (24 h).

Immunological one step methods are those most used by industries because of

their rapidity and the easy performing kit provided. Reaction kits are constituted by

a membrane to which a specific antibody to O157 and/or H7 antigens adheres. The

membrane is filled with fresh cell culture and after 10 - 20 min the agglutination to

antibodies is detectable. ELISA assays are based upon the same reaction, with

monoclonal antibody (MAb) reactive with low-molecular-weight outer membrane

antigens ofE. coli O157:H7, but are performed in microplates and the antibody is

coupled with an enzyme that allows colorimetric screening. It has been revealed

that the target antigens of the MAb are present in other serotypes ofE. coli and that

their expression and detection are influenced by culture conditions and sample

preparation. A modified protocol which provided high specificity for E. coli

O157:H7 was developed (Johnson et al., 1995). In order to improve the sensitivity

of detection ofE. coli O157:H7 in food, an immunomagnetic separation system

(IMS, Dynal, Oslo, Norway) was developed. It consists of superparamagneticpolystyrene microspheres coated with specific antibodies forE. coli O157:H7. In

this way it is possible to separate target bacterial cells from other substances and

background microflora present in a complex sample like food, and to concentrate

pathogen cells for further identification studies. Because numerous outbreaks

linked to non O157 STEC have been reported in recent years, a new ELISA kit has


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been developed, able to detect both Shiga toxins stx1 and stx2. Nevertheless this

assay cannot differentiate between the two toxins. After an enrichment step this

new ELISA kit is able to detect one STEC cell per g of ground beef (Acheson et al.,

1996).

Molecular methods

Molecular methods offer extremely sensitive and focused techniques that are

potentially able to detect and to quantify pathogenic bacteria with a sensitivity and

specificity not achievable by culture techniques and biochemical or serological

tests. Genotypic identification of bacteria avoids all inconvenience of phenotypic

assays, like variation in enzymatic activity when bacteria are cultured in different

media, emergence of biochemical mutants and presence of strain of different

species that are very closely related and possess the same phenotype but different

genotype. Several genes and DNA sequences have been targeted to develop molec-

ular methods for detecting STEC, particularly E. coli O157:H7. These includes:

attaching-and-effacing (eae) gene (Louie et al., 1994; Yu and Kaper, 1992) Shigatoxin (stx genes) (Karch and Meyer, 1989; Newland and Neill, 1988), the -glu-curonidase (uidA) gene (Cebula et al., 1995; Feng, 1993) the DNA sequence

upstream of the eae gene (Zhao et al., 1995; Meng et al., 1996) the 60-MDa plas-

mid (Johnson et al., 1995), and the haemolysin (hlyA) gene (Levine et al., 1987;

Schmidt et al., 1995) (Fig. 1).

Ann. Microbiol., 53 (4), 511-527 (2003) 517

FIG. 1 Target genes for DNA detection of VTEC andEscherichia coli 0157:H7 (modi-

fied from Vernozy-Rozand, 1999).

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DNA probes

The hybridization technique consists in developing specific DNA oligonucleotides

(probes) labelled by radioactive isotopes, or enzymatic markers. These probes can

hybridize with single stranded DNA from target bacteria, fixed onto nitrocellulose

or nylon membranes. The presence of homologous sequences allows the probe tomatch to the target DNA section, allowing the detection of the gene of interest.

DNA probes able to detect stx1 and stx2 STEC genes were developed (Karch and

Meyer, 1989; Newland and Neill, 1988; Willshaw et al., 1987). Samadpour et al.

(1994) used probes targeted to stx genes to detect STEC in faecal samples and

foods, by using colony hybridization and dot blotting. These assays guaranteed

good sensitivity (about 1.0 CFU/g) but inadequate specificity. Levine et al. (1987)

developed a DNA probe based on the 60-MDa plasmid common among EHEC and

found high specificity with strains isolated from patients with haemorrhagic colitis

and HUS (about 99%). In order to avoid problems linked to manipulation of

radioactive-labelled probes Thomas et al. (1991) developed DNA probes markedwith dioxigenine or biotine detectable after enzymatic coloured reaction, specific

for stx1, stx2 and stx2 variant genes. The use of DNA hybridization techniques is

certainly the best choice for its sensitivity but the high cost and the labor-intensive

procedures makes them unsuitable for routine assays in laboratory.

The suitability, time-saving and relatively low-cost of PCR techniques makes

possible to develop sensible and specific assays to detect E. coli O157:H7 and

other STEC, amenable to the requirements of most health surveillance and food

control laboratories. Some PCR assays are now available commercially as gene

detection diagnostics. The first PCR experiment onE. coli O157:H7 was realizedby Karch and Meyer (1989) with degenerated primers (a mix of oligonucleotides

able to amplify DNA fragment without knowing the exact sequences of the anneal-

ing sites) built up to detect stx1 and stx2 gene. DNA hybridization with specific

probes was assayed on the obtained amplicons, in order to confirm the genes iden-

tification. Read et al. (1992) produced a PCR with primers developed on the con-

served region ofstx1, stx2 and stxEgenes, in order to detect STEC in food and fae-

ces samples. By this method 50 different STEC serotypes were detected, with a

sensibility of about 20 UFC. The eae gene codifies for intimin, a protein that plays

an important role in attaching and effacing bacterial cell to intestinal epithelium.

Gannon et al. (1993) showed that is possible to use eaeGEN primers for amplifying

a portion of the gene common to all EHEC and eaeO157 primers for detecting

specifically E. coli O157:H7 (together with E. coli O157:NM, O55:H7 and

O145:NM). Louie et al. (1994) developed an eae based PCR able to identify sepa-

rately serotypes O157:H7/NM, O55:H7/NM (that possess the same eae gene

sequence) and O111:H7/NM. Meng et al. (1997) used primers that amplify a DNA

sequence upstream of the eae and stx genes. This multiplex assay revealed a better

specificity than the ones based only on eae gene. Fratamico et al. (1995) developed

a multiplex PCR based on three genes (eae, stx and a portion of a 60 MDa plas-

mid). This multiplex gives three positive reactions only for E. coli O157:H7 andO157:NM. UidA gene is another important molecular marker forE. coli O157:H7.

This gene codifies for -glucuronidase enzyme, present in the genome of thisserotype even if the phenotype is not exhibited. Feng (1993) developed a molecu-

lar probe on uidA gene, specific for E. coli O157:H7 (called PF-27). The same

author demonstrated that this probe is able also to detectE. coli O157:NM (Feng,


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1995). Other authors used the combination of different primers able to detect mul-

tiple gene sequences, in a multiplex PCR reaction: Cebula et al. (1995) developed

a multiplex PCR for stx1, stx2 and uidA genes capable of detecting both E. coli

O157:H7 andE. coli O157:NM. In this case it is possible to distinguish these two

serotypes from otherE. coli and to show the presence of one or both Shiga toxins.FliCgene codifies for H antigene ofE. coli and is involved in the production of

flagelline, a flagellar protein with high variability among EHEC species. Fields et

al. (1997) developed a PCR coupled with restriction profile on fliCgene able to

detect and distinguish between E. coli O157:H7, O157:NM and O55:H7 with a

detection limit due to some differences in the restriction profile of some strains

belonging to the same serogroup. A multiplex PCR produced by Gannon et al.

(1997) involvesfliC, stx1, stx2 and eae and is able to detect all STEC and to identi-

fy specifically E. coli O157:H7 and O157:NM. Rfb is another important marker

gene that codifies for an enzyme linked to biosynthesis of O157 antigen. Des-

marchelier et al. (1998) developed a couple of primers able to amplify rfbEgene,capable of detecting serotypes O157:H7 and O157:NM. Paton and Paton (1998)

developed another assay with two couples of primers able to detect rfbO157 and

rfbO111 thus making it possible to differentiate between E. coli O157 and O111

(another important toxigenic serotype). Nagano et al. (1998) coupled a PCR reac-

tion with primers for rfb gene and for stx genes in order to detect and distinguish

between O157:H7 able to produce Shiga toxins and non toxigenic strains. Other

multiplex PCR reactions were realized by Paton and Paton (1998) and Fagan et al.

(1999), with primers built on stx1, stx2, eae and hlyA (a gene that codifies for

enterohaemolysin) (Table 3).In spite of the recent interest in genetic analysis for detecting pathogen

microorganisms in food industries, a large number of PCR protocols for most path-

ogenic bacteria in food have been developed. There are two commercial PCR kits

able to detectE. coli O157:H7, the BAX system for screeningE. coli O157:H7

(Qualicon, Inc. U.S.A.), in which primers, DNA polymerase, and nucleotides are

combined in a single ready-to-use tube, and Probelia PCR System-E. coli

O157:H7 (BioControl Systems, Inc., U.S.A.) based on PCR plus DNA hybridiza-

tion assay. In both cases the commercial kits possess good specificity and sensibil-

ity, are easy to use and require only 24 h to be performed. All PCR protocols

applied on complex samples like food, faeces, etc. may often be subjected to some

inconvenience, like presence of DNA polymerase inhibitors (like humic acids),

substances that bind to magnesium, or denature DNA (nucleases). In order to avoid

this problem it is necessary to isolate bacteria (or their DNA) from the sample

debris. There are different ways to obtain the reduction of polymerase inhibitors:

the most widely used is a pre-enrichment step of 6-18 h capable of increasing the

target bacterial cells. It is also possible to minimize PCR inhibitor effects by cou-

pling the pre-enrichment step with the use of IMS for selectively separating the tar-

get bacteria (Fratamico et al., 2000; Chapman et al., 2001). To facilitate PCR reac-

tion it may also be useful to filter the broth culture before DNA extraction, becausecatabolites produced during bacterial growth may inhibit the polymerase

(Venkateswaran et al., 1997). It is important to remember that DNA is a relatively

stable molecule, able to maintain its molecular structure even after death of bacte-

rial cells. For this reason PCR may occur even if bacterial cells are dead or in a

viable-non culturable (VNC) state, inducing an overestimation of potential

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pathogen charge of a given sample. A recent paper (McIngvale et al., 2002)

focused on the detection of viable STEC cells based on reverse-transcriptase (RT)

PCR. This technique is based on the retro-transcription of mRNA into a copy of the

original DNA (DNA copy , cDNA). By using a retro-transcriptase enzyme, mRNA

targets were detected in 12-h cooked ground meat enrichments with a an initial


TABLE 3 Molecular tools for detection ofEscherichia coli O157:H7 and other STEC

Technique Target gene(s) Serotype Reference

PCR stx1, stx2 STEC Karch and Meyer,

(degenerated primers) 1989PCR stx

1, stx

2, stx

ESTEC Read et al., 1992

PCR eaeGEN, eaeO157 O157:H7/NM, Gannon et al., 1993O55:H7, O145 :NM

PCR eaeO157/55, eaeO111 O157:H7/NM, Louie et al., 1994O55:H7/NM

DNA probe uidA O157:H7 Feng, 1993

Multiplex PCR eae, stx, O157:H7/NM Fratamico et al.,

1995

PCR hly933 O157:H7/NM Fratamico et al.,1995

multiplex PCR stx1, stx

2,uidA O157:H7/NM Cebula et al., 1995

PCR fliC O157:H7/NM, Fields et al., 1997restriction profile O55:H7

multiplex PCR fliCH7, stx1, stx2,eae O157:H7/NM Gannon et al. 1997

PCR Seq. upst. eae and stx O157:H7 Meng et al. 1997

PCR rfbE O157:H7/NM Desmarchelier et al.1998

PCR rfbO157

, rfbO111

O157, O111 Paton and Paton,1998

multiplex PCR rfb, stx O157:H7/NM Nagano et al., 1998

multiplex PCR stx1, stx2,eae, hlyA STEC Fagan, 1999

RT-PCR stx1, stx2 STEC McIngvale et al.,2002

R-PCR (Taqman) eaeA O157 :H7 Oberst et al., 1998

multiplex R-PCR stx1, stx2. eaeO157

, O157, O111, O26 Sharma, 2002(Taqman) eaeO111, eaeO26

multiplex R-PCR stx1, stx2, eae O157:H7 Ibekwe et al., 2002(Taqman)

R-PCR stx1, stx2 STEC Blanger et al., 2002(molecular beacon)

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inoculum of 1 CFU/g. A new PCR-based quantitative and qualitative technique,

with high specificity and sensitivity potential is Real Time PCR (R-PCR). With R-

PCR it is possible to have immediate quantitative detection of target-gene specific

amplified products, with the advantage of avoiding manipulation of the sample by

gel electrophoresis and the use of carcinogenic intercalating dyes like ethidiumbromide. This technique has been recently developed for the detection and quan-

tification of pathogen bacteria with the use of Taqman probes or molecular bea-

cons, oligonucleotides coupled with reporter and quencher dyes at 5 and 3 ends,

respectively. In an intact probe or beacon the quencher dye suppresses the fluores-

cence emission of the reporter dye. The hydrolysis of the Taqman probe by cleav-

age of Taq polymerase or a conformational change in a molecular beacon during

the annealing and extension phases of the PCR process results in an increase in the

reporter dyes fluorescence intensity. The measurement of increasing fluorescence

for each PCR cycle makes it possible to estimate the starting concentration of tar-

get DNA and thus the starting number of cells of a bacterial pathogen. In the last5 years different authors have developed real time PCR methods for detection of

E. coli O157:H7 and other STEC in food and faecal samples. Oberst et al. (1998)

developed a R-PCR Taqman assay for E. coli O157:H7 based on eaeA gene,

allowing sensitivity of about 103 CFU/ml in enrichment broth, improved to 102

CFU/ml when an enrichment step and DNA purification were added to the proto-

col. Sharma and Carlson (2000) obtained good sensitivity (about 10 CFU/g sam-

ple) in a multiplex R-PCR assay able to detect both E. coli O157:H7 and Salmo-

nella strains in a single reaction, but a 6-18 h pre-enrichment step was needed.

Another multiplex real time PCR assay was developed by Sharma (2002) withthree sets of primers and Taqman probes, specific for serotype O157, O111 and

O26, together with two sets of primers and probes for stx1 and stx2 genes. The effi-

ciency of R-PCR technique has been tested also in different samples like soil and

waste water (Ibekwe et al., 2002). In this case a multiplex R-PCR based on stx and

eae genes gave high specificity even in the presence of etherotropic microflora and

with complex samples in which the presence of inhibitors constitutes a serious

problem for PCR reaction. The need for a rapid, specific and sensitive assay for

detecting E. coli O157:H7 and other STEC, useful for clinical diagnostic, lead

(Blanger et al., 2002) to the development of a multiplex molecular beacon R-PCR

assay able to detect and quantify STEC in about 1 h.

CONCLUDING REMARKS

The role ofE. coli O157:H7 and other STEC as food-borne pathogens has been

extensively investigated. Since the first outbreak in 1982, a wide variety of foods

have been involved in STEC contamination, some of them, like apple cider and

mayonnaise, previously thought to be safe because of their acidity. The increasing

of the international nature of STEC outbreaks (Duffel et al., 2003) emphasises theimportance of close collaboration between organisations in the management of

outbreaks, of ensuring international standards in food safety, and of agreeing a

common standard in STEC typing. The survival of STEC in food and tolerance to

physical and chemical stresses has been principally focused on acidity- and heat-

resistance. Further studies are needed to investigate the effect of aw on the survival

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and growth of enterohaemorrhagic E. coli. Molecular methods greatly improved

the specificity and sensibility of detection techniques for E. coli O157:H7 and

other STEC serotypes. DNA probes, and multiplex PCR are more and more wide-

ly used in diagnostic laboratories. Detection of live cell by RT-PCR and rapid

quantisation by real-time PCR will allow rapid (about 1h) detection of enetero-haemorrhagic strains with high sensitivity. More studies are needed to avoid time-

consuming enrichment steps and to prevent the action of PCR inhibitor substances.

REFERENCES

Abdoul-Raouf U.M., Beuchat L.R., Ammar M. S. (1993). Survival and growth ofEscherichia coli O157:H7 on salad vegetables. Appl. Environ. Micriobiol., 59:1999-2006.

Acheson D., Lincicome L., Breucker S., Keusch, G. (1996). Detection of Shiga-like toxin-producing Escherichia coli in ground beef and milk by commercial enzymeimmunoassay. J. Food Prot., 59: 344-349.

Blanger S.D., Boissinot M., Mnard C., Picard F.J., Bergeron M.G. (2002). Rapid detec-tion of shiga toxin-producing bacteria in feces by multiple PCR with molecular bea-cons on the smart cycler. J. Clin. Microb., 40: 1436-1440.

Besser R., Lett S., Weber J., Doyle M., Barrett T., Wells J., Griffin, P. (1993). An outbreakof diarrhea and hemolytic uremic syndrome fromEscherichia coli O157:H7 in freshpressed apple cider. JAMA., 269: 2217-2220.

Beuchat L.R. (1996). Pathogenic microorganisms associated with fresh produce. J. FoodProt., 59: 203-216.

Caprioli A., Tozzi E. (1998). Epidemiology of Shiga toxin-producing Escherichia coliinfections in Continental Europe. In:. Kaper J.B, OBrien A., Eds, Escherichia coliO157:H7 and Other Shiga Toxin-ProducingE. coli Strains. ASM Press, Washington,D.C., pp. 38-48.

Cebula T.A., Payne W.L., Feng P. (1995). Simultaneous identification of strains ofEscherichia coli serotype O157:H7 and their Shiga-like toxin type by mismatchamplification mutation assay-multiplex PCR. J. Clin. Microbiol., 33: 248-250.

Centers for Disease Control and Prevention. (1995).Escherichia coli O157:H7 outbreaklinked to commercially distributed dry-cured salami- Washington and California,1994. Morbid. Mortal. Weekly Rep., 44: 157-160.

Centers for Disease Control and Prevention. (1997a). Outbreaks ofEscherichia coliO157:H7 infection and cryptosporidiosis associated with drinking unpasteurizedapple cider- Connecticut and New York, October 1996. Morbid. Mortal. Weekly Rep.,46: 4-8.

Centers for Disease Control and Prevention. (1997b). Outbreaks ofEscherichia coliO157:H7 infection associated with eating alfalfa sprouts- Michigan and Virginia,June-July 1997. Morbid. Mortal. Weekly Rep., 46: 741-744.

Chapman P., Siddons C., Zadik P., Jewes L. (1991). An improved selective medium for theisolation ofEscherichia coli O157. J. Med. Microbiol., 35: 107-110.

Chapman P.A., Ellin M., Ashton R., Shafique W. (2001). Comparison of culture, PCR andimmunoassays for detectingEscherichia coli O157 following enrichment culture andimmunomagnetic separation performed on naturally contaminated raw meat prod-ucts. Int. J. Food Microbiol., 68: 11-20.

Chou C.C., Cheng S.J., Wang Y.C., Chung K.T. (1999). Behavior ofEscherichia coliO157:H7 andListeria monocytogenes in Tryptic soy broth subjected to various lowtemperature treatments. Food Res. Int., 32: 1-6.


7/27/2019 beneduce_53_511

13/17

Conner D.E. (1992). Temperatures and NaCl affects growth and survival ofEscherichiacoli O157:H7 in poultry-based and laboratory media. J. Food Sci., 57: 532-533.

Daoust J., Park C., Szabo R., Todd E., Emmons B., Mckellar R. (1988). thermal inactiva-tion ofCampilobacterspecies, Yersinia enterocolitica, and hemorrhagicEscherichiacoli O157:H7 in fluid milk. J. Dairy Sci., 71: 3230-3236.

Davies A.R., Slade A., Blood R.M., Gibbs P.A. (1992). Effect of temperature and pH valueon the growth of verotoxigenic E. coli. Leatherhead Food Research Association.,Research Report n 691.

Desmarchelier P.M., Bilge S.S., Fegan N., Mills L., Vary J.C., Tarr P.I. (1998). A PCR spe-cific forEscherichia coli O157 based on the rfb locus encoding O157 lypopolysac-charide. J. Clin. Microbiol., 36: 1801-1804.

Doyle M.P., Schoeni J.L. (1984). Survival and growth characteristics ofEscherichia coliassociated with haemorrhagic colitis. Appl. Environ. Microbiol., 48: 855-856.

Doyle M.P., Schoeni, J.L. (1987). Isolation ofEscherichia coli O157:H7 from retail fresh

meats and poultry. Appl. Environ. Microbiol., 53: 2394-2396.Doyle M., Padhye V. (1989). Escherichia coli. In: Doyle M., Ed., Foodborne Bacterial

Pathogens. Marcel Dekker Inc., New York, N.Y., pp. 236-282.

Duffel E., Espi E., Nichols T., Adak G.K., De Valk H., Anderson K., Stuart M. (2003).Investigation of an outbreak ofE. coli O157 infections associated with a trip to Franceof schoolchildren from Somerset, England. Euro Surveillance, 8: 81-86.

Fagan P.K., Hornitzky M.A., Bettelheim K.A., Djordjevic S.P. (1999). Detection of shiga-like toxin (stx1 and stx2), intimin (eaeA), and enterohemorrhagic E. coli (EHEC)hemolysin (EHEC hlyA) genes in animal feces by multiplex PCR. Appl. Environ.Microbiol., 65: 868-872.

Feng P. (1993). Identification ofEscherichia coli serotype O157:H7 by DNA probe spe-cific for an allele ofuidA gene. Mol. Cell. Probes., 7: 151-154.

Feng P. (1995).Escherichia coli serotype O157:H7: novel vehicles of infection and emer-gence of phenotypic variants. Emerg. Infect. Dis., 1: 16-21.

Fields P.I., Blom K., Hugues H.J., Hesel L.O., Feng P., Swaminathan B. (1997). Molecu-lar characterization of the gene encoding H antigen inEscherichia coli and develop-ment of a PCR-Restriction Fragment Length Polymorphism test for identification ofE. coli O157:H7 and O157:NM. J. Clin. Microbiol., 35: 1066-1070.

Fratamico P.N., Sackitey S.K., Wiedmann M., Deng M.Y. (1995). Detection ofEscherichia

coli O157:H7 by multiplex PCR. J. Clin. Microbiol., 33: 2188-2191.Fratamico P.N., Bagi L.K., Pepe T. (2000). A multiplex polymerase chain reaction assay

for rapid detection and identification ofEscherichia coli O157:H7 in foods andbovine faeces. J. Food Prot., 63: 1032-1037.

Gannon V.P.J., Rashed M., King R.K., Golsteyn-Thomas E.J. (1993). Detection and char-acterization of the eae gene of Shiga-like toxin-producing Escherichia coli usingpolymerase chain reaction. J. Clin. Microbiol., 31: 1268-1274.

Gannon V.P., De Souza S., Graham T., King R.K., Rahn K., Read S. (1997). Use of the fla-gellar H7 gene as a target in multiplex assays and improved specificity in identifica-tion of enterohaemorrhagicEscherichia coli strains. J. Clin. Microbiol., 35: 656-662.

Glass K.A., Loeffelholz J.M., Ford J.P., Doyle M.P. (1992). Fate ofEscherichia coliO157:H7 as affected by pH or sodium chloride in fermented, dry sausage. Appl. Env-iron. Microbiol., 58: 2513-2516.

Ibekwe A.M., Watt P.M., Grieve C.M., Sharma V.K., Lyons S.R. (2002). Multiplex fluoro-genic real-time PCR for detection and quantification ofEscherichia coli O157:H7 indairy wastewater wetlands. Appl. Environ. Microbiol., 68: 4853-4862.

Ann. Microbiol., 53 (4), 511-527 (2003) 523

7/27/2019 beneduce_53_511

14/17

Izumiya H., Terajima J., Wada A., Inagaki Y., Itoh K.I., Tamura K., Watanabe H. (1997).Molecular typing of enterohemorrhagicEscherichia coli O157:H7 isolates in Japanby using pulsed-field gel electrophoresis. J. Clin. Microbiol., 35: 1675-1680.

Johnson R.P., Durham R.J., Johnson S.T., MacDonald L.A., Jeffrey S.R., Butman B.T.(1995). Detection ofEscherichia coli O157:H7 in meat by an enzyme-linked

immunosorbent assay, EHEC Tek. Appl. Environ. Microbiol., 61: 386-388.

Karch H., Bielaszewska M. (2001). Sorbitol-fermenting shiga toxin-producingEscherichia coli O157:H- strains: epidemiology, phenotypic and molecular character-istics, and microbiological diagnosis. J. Clin. Microbiol., 39: 2043-2049.

Karch H., Meyer T. (1989). Evaluation of oligonucleotide probes for identification ofShiga-like-toxin-producingEscherichia coli. J. Clin. Microbiol., 27: 1180-1186.

Keene W.E., Sazie E., Kok J., Rice D.H., Hancock D.D., Balan V.K., Zhao T., Doyle M.P.(1997). An Outbreak ofEscherichia coli O157:H7 infections traced to jerky madefrom deer meat. JAMA, 277: 1229-1231.

Levine M., Xu J., Kaper J., Lior H., Prado V., Tall B., Cataro J., Karch H., Wachsmuth,K. (1987). A DNA probe to identify enterohemorrhagicEscherichia coli of O157:H7and other serotypes that cause hemorrhagic colitis and haemolytic uremic syndrome.J. Infect. Dis., 156: 175-182.

Louie M., De Azavedo J., Clarke R., Borczyk A., Lior H., Richter M., Brunton J. (1994).Sequence heterogeneity of the eae gene and detection of verocytotoxin-producingEscherichia coli using serotype-specific primers. Epidemiol. Infect., 112: 449-461.

March S.B., Ratnam S. (1986). Sorbitol MacConkey medium for detection ofEscherichiacoli O157:H7 associated with hemorrhagic colitis. J. Clin. Microbiol., 23: 869-872.

Massa S., Altieri C., Quaranta V., De Pace R. (1997). Survival ofEscherichia coli

O157:H7 in yoghurt during preparation and storage at 4 degrees C. Lett. Appl. Micro-biol., 24: 347-350.

Massa S., Goffredo E., Altieri C., Natola K. (1999). Fate ofEscherichia coli O157:H7 inunpasteurized milk stored at 8 C. Lett. Appl. Microbiol., 28: 89-92.

McIngvale S.C., Elhanafi D., Drake M.A. (2002). Optimization of Reverse TranscriptasePCR to detect viable shiga-toxin-producingEscherichia coli. Appl. Environ. Micro-biol., 68: 799-806.

Meng J., Doyle M.P. (1998). Microbiology of Shiga toxin-producing Escherichia coli infoods. In: Kaper J.B., OBrien A., Eds, Escherichia coli O157:H7 and Other ShigaToxin-ProducingE. coli Strains. ASM Press, Washington, D.C., pp. 92-108.

Meng J., Doyle M.P., Zhao T., Zhao S. (2001). Enterohemorrhagic Escherichia coli.In: Doyle M.P., Beuchat L.R., Montville T.J.,Eds, Food Microbiology: Fundamentalsand Frontiers, 2nd edn., ASM Press, Washington, D.C., pp. 193-213.

Meng J., Zhao S., Doyle D., Mitchell S., Kresovich S. (1996). Polymerase chain reactionfor detectingEscherichia coli O157:H7. Int. J. Food Microbiol., 32: 103-114.

Meng J., Zhao S., Doyle D., Mitchell S., Kresovich S. (1997). A multiplex PCR for iden-tifying Shiga-like toxin-producingEscherichia coli O157:H7. Lett. Appl. Microbiol.,24: 172-176.

Mermin J., Hilborn E., Voetsch A., Swartz M., Lambert-Fair M., Farrar J., Vugia D.,

Hadler J., Slutsker L. (1997). A multistate outbreak ofEscherichia coli

O157:H7infections associated with eating mesclun mix lettuce, In: VTEC 97: 3rd Internation-al Symposium and Workshop on Shiga Toxin (Verocytotoxin)-producingEscherichiacoli Infections. Abstr. V74/I, p. 9.

Michino H., Araki K., Minami S., Nakayama T., Ejima Y., Hiroe K., Tanaka H., Fujita N.,Usami S., Yonekawa M., Sadamoto K., Takaya S., Sakai N. (1998). Recent outbreaksof infections caused byEscherichia coli O157:H7 in Japan. In: Kaper J.B., OBrien


7/27/2019 beneduce_53_511

15/17

A., Eds,Escherichia coli O157:H7 and Other Shiga Toxin-ProducingE. coli Strains.ASM Press, Washington, D.C., pp. 73-81

Morgan D., Newman C., Hutchinson D., Walker A., Rowe B., Majid F. (1993). Verotoxinproducing Escherichia coli O157 infections associated with the consumption ofyoghurt. Epidemiol. Infect., 111: 181-187

Morgan G., Newman C., Palmer S., Allen J., Shepherd W., Rampling A., Warren R., GrossR., Scotland S., Smith H. (1988). First recognized community outbreak of haemor-rhagic colitis due to verotoxin-producingEscherichia coli O157:H7 in the UK. Epi-demiol. Infect., 101: 83-91.

Nagano I., Kunishima M., Itoh Y., Wu Z., Takahashi Y. (1998). Detection of verotoxin-pro-ducingEscherichia coli O157:H7 by multiplex polymerase chain reaction. Microbiol.Immunol., 42: 371-376.

Newland J.W., Neill R.J. (1988). DNA probes for Shiga-like toxins I and II and for toxin-converting bacteriophages. J. Clin. Microbiol., 26: 1292-1297.

Oberst R.D., Hays M.P., Bohra L.K., Phebus R.K., Yamashiro C.T., Paszko-Kolva C.,Flood S.J.A., Sargeant J.M., Gillespie J. R. (1998). PCR-based DNA amplificationand presumptive detection ofEscherichia coli O157:H7 with an internal fluorogenicprobe and the 5 nuclease (Taqman) assay. Appl. Environ. Microbiol., 64: 3389-3396.

Okrend J.G., Rose B.E., Lattuada C.P. (1990). Use of 5-Bromo-4-Chloro-3-Indoxyl-_-D-Glucuronide in MacConkey Sorbitol Agar to aid in the isolation ofEscherichia coliO157:H7 from ground beef. J. Food Prot., 53: 941-943.

Padhye N.V., Doyle M.P. (1991). Rapid procedure for detectingEscherichia coli O157:H7in food. Appl. Environ. Microbiol., 57: 2693-2698.

Paton J.C., Paton A.W. (1998). Detection and characterization of Shiga toxigenic

Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, enterohemor-rhagicE. coli hlyA, rfbO111, and rfbO157. J. Clin. Microbiol., 36: 598-602.

Read S.C., Clarke R.C., Martin A., De Grandis S.A., Hill J., McEwen S., Gyles G.L.(1992). Polymerase chain reaction of verocytotoxigenic Escherichia coli isolatedfrom animal and food sources. Mol. Cell. Probes., 6: 153-161.

Rice E.W., Johnson C.H., Wild D.K. Reasoner, D.J. (1992). Survival ofEscherichia coliO157:H7 in drinking water associated with a waterborne disease outbreak of hemor-rhagic colitis. Lett. Appl. Microb., 15: 38-40.

Riley L.W., Remis R.S., Helgerson S.D., McGee H.B., Wells J.G., Damis B.R., HerbertR.J., Olcott E.S., Johnson L.M., Hargrett N.T., Blake P.A., Cohen M.L. (1983). Hem-

orrhagic colitis associated with a rare Escherichia coli serotype. N. Engl. J. Med.,308: 681-685.

Riley L.W. (1987). The epidemiologic, clinical, and microbiologic features of hemorrhag-ic colitis. Ann Rev. Microbiol., 41: 383-407.

Rippey S.R., Chandler L.A., Watkins W.D. (1987). Fluorimetric method for enumerationofEscherichia coli in molluscan shell-fish. J. Food. Protect., 50 :685-690.

Rodrigue D. (1995). A university outbreak ofEscherichia coli O157:H7 infections associ-ated with roast beef and an unusually benign clinical course. J. Infect. Dis.,172: 1122-1125.

Samadpour M., Ongerth J.E., Liston J., Tran N., Nguyen D., Whittam T.S., Wilson R.A.,Tarr P.I. (1994). Occurrence of Shiga-like toxin-producingEscherichia coli in retailfresh seafood, beef, lamb, pork, and poultry from grocery stores in Seattle, Washing-ton. Appl. Environ. Microbiol., 60: 1038-1040.

Schmidt H., Beutin L., Karch H. (1995). Molecular analysis of the plasmid-encodedhemolysin ofEscherichia coli O157:H7 strain EDL 933. Infect. Immun., 63:1055-1061.

Ann. Microbiol., 53 (4), 511-527 (2003) 525

7/27/2019 beneduce_53_511

16/17

Sharma V.K., Carlson S.A. (2000). Simultaneous detection of Salmonella strains andEscherichia coli O157:H7 with fluorogenic PCR and single-enrichment-broth culture.Appl. Environ. Microbiol., 66: 5472-5476.

Sharma V.K. (2002). Detection and quantitation of enterohemorrhagic Escherichia coliO157, O111, and O26 in beef and bovine feces by real-time polymerase chain reac-

tion. J. Food Prot., 65: 1371-1380.

Spano G., Goffredo E., Beneduce L., Tarantino D., Dupuy A., Massa S. (2003). Fate ofEscherichia coli O157:H7 during the manufacture of Mozzarella cheese. Lett. Appl.Microbiol., 36: 73-76.

Strockbine N.A., Wells J.G., Bopp C.A., Barret T.J. (1998). Overview of detection andsubtyping methods. In: Kaper J.B., OBrien A.D., Eds,Escherichia coli O157:H7andOther Shiga Toxin ProducingEscherichia coli (STEC) Strains. ASM Press. Washing-ton D.C., pp. 331-335.

Tesh V., Samuel J.E., Perera L.P., Sharefkin J.B., OBrien A.D. (1991). Evaluation of therole of Shiga-like toxins in mediating direct damage to human vascular endothelialcells. J. Infect. Dis., 64: 344-352.

Thomas A., Smith H.R., Willshaw G.A., Rowe B. (1991). Non-radioactively labelledpolynucleotide and oligonucleotide DNA probes, for selectively detectingEscherichia coli strains producing Vero cytotoxins VT1, VT2 and VT2 variant. Mol.Cell. Probe., 5: 129-135.

Thompson J.S., Hodge D.S., Borczyk A.A. (1990). Rapid biochemical test to identifyverocytotoxin-producing Escherichia coli serotype O157. J. Clin. Microbiol.,28: 2165-2168.

Tilden J. JR., Young W., McNamara A.M., Custer C., Boesel B., Lambert-Fair M.A.,

Majkowski J., Vugia D., Werner S.B., Hollingsworth J., Morris J.G. JR. (1996).A new route of transmission forEscherichia coli: Infection from dry fermented sala-mi. Am. J. Public Health., 86: 1142-1145.

Venkateswaran K., Kamijoh Y., Ohashi E., Nakanishi H. (1997). A simple filtration tech-nique to detect enterohemorrhagicEscherichia coli O157:H7 and its toxins in beef bymultiplex PCR. Appl. Environ. Microbiol., 63: 4127-4131.

Vernozy-Rozand C. (1999). VerotoxigenicE. coli and Shigella spp. detection by culturalMethods In: Robinson R.K., Batt C.A., Patel P.D., Eds, Encyclopedia of Food Micro-biology, Vol. 1. Academic Press, London, pp. 2232-2236.

Wang G., Doyle M. (1996). Survival ofEscherichia coli O157:H7 in drinking and recre-

ational water. In: Abstracts of the Annual Meeting of the International Association ofMilk, Food and Environmental Sanitarians. Abstr. 184, p. 78.

Willshaw G.A., Smith H.R., Roberts D., Thirlwell J., Cheasty T., Rowe B. (1993). Examina-tion of raw beef products for the presence of Vero cytotoxin-producingEscherichia coli,particularly those of serogroup O157. J. Appl. Bacteriol., 75: 420-426.

Willshaw G.A., Smith H.R., Scotland S.M., Field A.M., Rowe B. (1987). Heterogeneity ofEscherichia coli phages encoding Vero cytotoxins: comparison of cloned sequencesdetermining VT1 and VT2 and development of specific gene probes . J. Gen. Micro-biol., 133: 1309-1317.

Wilson J.B., McEwen S.A., Clarke R.C., Leslie K.E., Wilson R.A., Walter-Toews D.

(1992). Distribution and characteristic of verocytotoxigenicEscherichia coli isolatedfrom Ontario dairy cattle. Epidemiol. Infect., 108: 423-439.

Yu J., Kaper J. (1992). Cloning and characterization of the eae gene of enterohemorrhagicEscherichia coli O157:H7. Mol. Microbiol., 6: 411-417.

Zadik P.M., Chapman P.A., Siddons C.A. (1993). Use of tellurite for the selection of vero-cytotoxigenicEscherichia coli O157. J. Med. Microbiol., 6: 411-417.


7/27/2019 beneduce_53_511

17/17

Zaho T., Doyle M.P., Besser R.E. (1993). Fate of enterohaemorragic Escherichia coliO157:H7 in apple cider with and without preservatives. Appl. Environ. Microbiol.,59: 2526-2530.

Zhao S., Mitchell S., Meng J., Doyle M., Kresovich S. (1995). Cloning and nucleotidesequence of a gene upstream of the eaeA gene of enterohemorrhagicEscherichia coli

O157:H7. FEMS Microbiol. Lett., 133: 35-39.

Ann. Microbiol., 53 (4), 511-527 (2003) 527

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