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 di

Foggia, Via Napoli 25, 71100 Foggia, Italy

Abstract - Escherichia coli O157:H7 is one of the most studied food-borne pathogen bacteria, because of its widespread diffusion, low-dose infectiveness, and severe symptoms associated. It is necessary to focus the attention on the factors (like temperature, pH and aw) that may affect growth and survival of E. coli O157:H7 in food and in the environment since there is a wide range of foods that can be contaminated by this strain, causing haemorrhagic colitis 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 research and diagnostic laboratories by molecular methods. PCR techniques, particularly the latest quantitative real-time-PCR, make possible to reach specificity and sensitivity level of few CFU 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, enteroaggregative E. coli (EAggEC) has also been found to be a diarrhoegenic strain. Among EHEC strains, E. coli O157:H7 is one of the most studied food-borne pathogens, because of its widespread diffusion, peculiar tolerance to some physical and chemical treatments, severity of illness and low dose infectiveness. This paper describes the characteristics of E. coli O157:H7, strain that distinguish it from the harmless commensal type of E. 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 particular emphasis to molecular techniques are reported.

* Corresponding author. Phone: +39-0881589352; Fax: +39-0881740211; E-mail: l.beneduce@unifg.it

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GENERAL PHYSIOLOGICAL CHARACTERISTICS Escherichia coli O157:H7 possess metabolic characteristics distinct from other E. coli. Most isolated strains are unable to ferment sorbitol within 24h and cannot hydrolyze 4-methylumbrelliferyl-D-glucuronide (as lacking -glucuronidase enzyme) (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 other E. 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-producing E. coli O157:H7 and other serogroups can cause haemorrhagic colitis, haemolytic-uraemic syndrome (HUS) and occasionally mild nonbloody diarrhoea in man, although some infections may be asymptomatic. HUS complicates about 10% of cases of E. coli O157:H7 infection and carries a mortality rate of 2-10%. Enterohaemorragic E. coli O157:H7 and related organisms are also referred to as Shiga toxin-producing E. coli (STEC) because of their ability to produce Stxs (formerly called Shiga-like toxins). The infectious dose of E. 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. Several 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 of E. 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 noteworthy 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
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because they are usually not heated before consumption, have been vehicles of outbreaks (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 waterborne transmission have recently been increasing (Meng et al., 2001).

OTHER STEC SEROTYPES Even if E. 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 to the 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).

TABLE 1 Isolation of principal STEC serotypes in Continental Europea Country O157 Austria Belgium Czech Republic Denmark Finland France Germany Greece Italy Spain Switzerland The Netherlands HAF HA H HAF H H HF H HAF HAF HA HA (F). HA HA HA H HA HA STEC serotypesb O26 HA HA H H H H HA H HA O111 HA HA H H

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

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FACTOR INFLUENCING GROWTH AND SURVIVAL IN FOOD The optimum temperature for E. coli O157:H7 is 37 C, the same as that of other E. 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 of E. 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 than E. coli Broth (EC) and Trypticase Soy Broth (TSB) (Doyle and Schoeni, 1984). The resistance to heat of E. 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 Mozzarella cheese should be free of E. coli O157:H7 if temperatures higher than 80 C are used during milk processing (Table 2). Few elements are clear about tolerance of E. coli O157:H7 to freezing temperatures. 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 of E. 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 surface 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 of E. coli O157:H7 has been observed in foods at temperatures of 8 C in non-fermented cider and at 12 C in salads (Abdoul Raouf et al., 1993). Escherichia coli O157:H7 is also able to survive and to grow in raw milk stored at 8C for 17 days (Massa et al., 1999). Refrigeration storage at less than 5 C should be adequate to inhibit growth of E. 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 for Escherichia coli O157:H7 and Salmonella spp. in ground beefa Temp (C) D value (s) Escherichia coli O157:H7 30.5% fat 51.7 57.2 62.8
aFrom Meng et al. (2001). bND, not detected.

Salmonella spp.

17-20% fat NDb 4.5 0.4 54.3 5.43 0.54

115.5 5.3 0.47

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1992). Another study confirmed these results on TSB medium and determined that the population of E. 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 different foods and the interaction between low aw levels and other factors in food processing. The first clear information about the peculiar tolerance to acidity of E. 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 of E. coli O157:H7 plays a key role in food-borne illness linked to this pathogen. The minimum pH for E. 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 contaminated by E. coli O157:H7 (Morgan et al., 1993). Massa et al. (1997) evaluated 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 that E. coli O157:H7 was detectable even after 7 days of storage. The population of E. coli O157:H7 decreased by only 1 to 1.5 log10 CFU/ml during the treatment. This study confirmed 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 other E. coli are inability to ferment sorbitol, lack of glucuronidase enzyme and weak 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; Wilson et al., 1992). The culture isolation techniques used to verify the suspect presence of E. 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 laborintensive. The need for a rapid diagnostic test for recognizing contamination associated with bloody diarrhoea and HUS forced the rapid development of new techniques for isolation and identification of STEC in foods. Immunoassays such as enzyme-linked immunosorbent assay (ELISA) were very useful for rapid screening of E. 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 of E. coli O157:H7 in foods makes it necessary to have a sensitive isolation method, using enrichment and selective procedures. Several studies reported the development of culture media specific for E. coli O157:H7 selective enrichment, (Doyle and Schoeni, 1987; Padhye and Doyle, 1991; Chapman 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 of E. coli but are inhibitory to other species. The best selective media for presumptive identification of E. coli O157:H7 was found to be Sorbitol MacConkey Agar (SMAC), in which E. coli O157:H7 colonies remain uncoloured (lack of sorbitol fermentation) while other E. coli appear red (March and Ratnam, 1986). Okrend et al. (1990), and Tesh et al., (1991) suggested the addition of 5bromo-4-chloro-indoxyl--D-glucuronide (BCIG) on SMAC, to differentiate strains 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 of E. coli O157:H7, by using 4-methylumbelliferyl--glucuronide (MUG). This substance can evidence -glucuronidase activity based upon production of a fluorescent hydrolysis compound (Rippey et al., 1987). Positive colonies are fluorescent after ultraviolet light exposure, while E. coli O157:H7 give no fluorescence. Currently SMAC is usually employed with addition of cefixime and tellurite (CT-SMAC), as reported by Zadik et al. (1993). Cefixime inhibits Proteus spp. At a concentration not inhibitory to E. coli, and O157 STEC strains are generally less susceptible to tellurite than are many other non-sorbitol fermenters such as Aeromonas spp., Morganella 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 isolating E. 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 distinctive black colour, whereas typical non-O157 STEC colonies are blue or purple and most non-toxigenic E. coli colonies are reddish (Meng and Doyle, 1998). To confirm presumptive O157 isolates from culture methods several immunological 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 of E. 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 of E. 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 of E. coli O157:H7 in food, an immunomagnetic separation system (IMS, Dynal, Oslo, Norway) was developed. It consists of superparamagnetic polystyrene microspheres coated with specific antibodies for E. 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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FIG. 1 Target genes for DNA detection of VTEC and Escherichia coli 0157:H7 (modified from Vernozy-Rozand, 1999).

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 molecular methods for detecting STEC, particularly E. coli O157:H7. These includes: attaching-and-effacing (eae) gene (Louie et al., 1994; Yu and Kaper, 1992) Shiga toxin (stx genes) (Karch and Meyer, 1989; Newland and Neill, 1988), the -glucuronidase (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 plasmid (Johnson et al., 1995), and the haemolysin (hlyA) gene (Levine et al., 1987; Schmidt et al., 1995) (Fig. 1).
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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 to match 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 marked with 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 on E. coli O157:H7 was realized by Karch and Meyer (1989) with degenerated primers (a mix of oligonucleotides able to amplify DNA fragment without knowing the exact sequences of the annealing 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 identification. Read et al. (1992) produced a PCR with primers developed on the conserved region of stx1, stx2 and stxE genes, in order to detect STEC in food and faeces 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 separately 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 plasmid). This multiplex gives three positive reactions only for E. coli O157:H7 and O157:NM. UidA gene is another important molecular marker for E. coli O157:H7. This gene codifies for -glucuronidase enzyme, present in the genome of this serotype even if the phenotype is not exhibited. Feng (1993) developed a molecular probe on uidA gene, specific for E. coli O157:H7 (called PF-27). The same author demonstrated that this probe is able also to detect E. coli O157:NM (Feng,
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1995). Other authors used the combination of different primers able to detect multiple 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 and E. coli O157:NM. In this case it is possible to distinguish these two serotypes from other E. coli and to show the presence of one or both Shiga toxins. FliC gene codifies for H antigene of E. 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 fliC gene 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) involves fliC, stx1, stx2 and eae and is able to detect all STEC and to identify 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. Desmarchelier et al. (1998) developed a couple of primers able to amplify rfbE gene, 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 reaction 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 pathogenic bacteria in food have been developed. There are two commercial PCR kits able to detect E. coli O157:H7, the BAX system for screening E. 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 hybridization assay. In both cases the commercial kits possess good specificity and sensibility, 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 coupling the pre-enrichment step with the use of IMS for selectively separating the target bacteria (Fratamico et al., 2000; Chapman et al., 2001). To facilitate PCR reaction it may also be useful to filter the broth culture before DNA extraction, because catabolites 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 bacterial 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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TABLE 3 Molecular tools for detection of Escherichia coli O157:H7 and other STEC Technique Target gene(s) Serotype STEC STEC Reference Karch and Meyer, 1989 Read et al., 1992

PCR stx1, stx2 (degenerated primers) PCR PCR PCR DNA probe Multiplex PCR PCR multiplex PCR PCR restriction profile multiplex PCR PCR PCR PCR multiplex PCR multiplex PCR RT-PCR R-PCR (Taqman) multiplex R-PCR (Taqman) multiplex R-PCR (Taqman) R-PCR (molecular beacon) stx1, stx2, stxE eaeGEN, eaeO157 eaeO157/55, eaeO111 uidA eae, stx, hly933 stx1, stx2,uidA fliC

O157:H7/NM, Gannon et al., 1993 O55:H7, O145 :NM O157:H7/NM, O55:H7/NM O157:H7 O157:H7/NM O157:H7/NM O157:H7/NM O157:H7/NM, O55:H7 Louie et al., 1994 Feng, 1993 Fratamico et al., 1995 Fratamico et al., 1995 Cebula et al., 1995 Fields et al., 1997 Gannon et al. 1997 Meng et al. 1997 Desmarchelier et al. 1998 Paton and Paton, 1998 Nagano et al., 1998 Fagan, 1999 McIngvale et al., 2002 Oberst et al., 1998 Sharma, 2002 Ibekwe et al., 2002 Blanger et al., 2002

fliCH7, stx1, stx2,eae O157:H7/NM Seq. upst. eae and stx O157:H7 rfbE rfbO157, rfbO111 rfb, stx stx1, stx2,eae, hlyA stx1, stx2 eaeA stx1, stx2. eaeO157, eaeO111, eaeO26 stx1, stx2, eae stx1, stx2 O157:H7/NM O157, O111 O157:H7/NM STEC STEC O157 :H7 O157, O111, O26 O157:H7 STEC

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
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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 RPCR 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 ethidium bromide. This technique has been recently developed for the detection and quantification of pathogen bacteria with the use of Taqman probes or molecular beacons, oligonucleotides coupled with reporter and quencher dyes at 5 and 3 ends, respectively. In an intact probe or beacon the quencher dye suppresses the fluorescence emission of the reporter dye. The hydrolysis of the Taqman probe by cleavage 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 target DNA and thus the starting number of cells of a bacterial pathogen. In the last 5 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 protocol. Sharma and Carlson (2000) obtained good sensitivity (about 10 CFU/g sample) in a multiplex R-PCR assay able to detect both E. coli O157:H7 and Salmonella 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) with three 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 efficiency 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 of E. 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 the importance 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 heatresistance. 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 widely 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 eneterohaemorrhagic strains with high sensitivity. More studies are needed to avoid timeconsuming enrichment steps and to prevent the action of PCR inhibitor substances.

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