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Applied and Environmental Microbiology, May 2006, p. 3245-3251, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3245-3251.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Characterization of Shiga Toxin Gene (stx)-Positive and Intimin Gene (eae)-Positive Escherichia coli Isolates from Wastewater of Slaughterhouses in France

Estelle Loukiadis,1 Monique Kérourédan,1 Lothar Beutin,2 Eric Oswald,1 and Hubert Brugère1*

UMR 1225 INRA-ENVT Interactions Hôtes-Agents Pathogènes, Equipe Pathogénie moléculaire et cellulaire des Escherichia coli, Ecole Nationale Vétérinaire de Toulouse, 31076 Toulouse Cedex 3, France,1 National Reference Laboratory for Escherichia coli, Federal Institute for Risk Assessment, 12277 Berlin, Germany2

Received 22 November 2005/ Accepted 21 February 2006


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ABSTRACT
 
Wastewater samples from 12 slaughterhouses located in different regions in France were tested for the presence of stx-positive and eae-positive Escherichia coli isolates, and characteristics of the isolates obtained were determined. A total of 224 wastewater samples were collected in wastewater treatment plants at different stages of wastewater processing. Altogether, 5,001 E. coli isolates were obtained by colony counting and screened for the presence of stx and eae genes by multiplex PCR. stx-positive and eae-positive E. coli isolates were detected in 25% of the samples collected; they were found in 13% and 3% of the samples obtained from treated effluent and sludge, respectively, suggesting that they could be spread into the environment. Screening of the samples collected by immunomagnetic separation allowed us to isolate 31 additional E. coli serogroup O157 isolates. Four of these isolates harbored stx and eae genes. All stx-positive and eae-positive E. coli isolates were analyzed for eae and stx genetic variants, as well as for additional virulence factors and serotypes. Our results suggest that the majority of the stx- and eae-positive E. coli isolates from wastewater have low virulence for humans. However, the diversity of the enterohemorrhagic E. coli-associated virulence factors in the strains indicates that the environment may play an important role in the emergence of new pathogenic enterohemorrhagic E. coli strains.


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INTRODUCTION
 
Enterohemorrhagic Escherichia coli (EHEC) is a recent emerging group of food-borne pathogens. Since 1982, EHEC strains have caused worldwide outbreaks of hemorrhagic colitis which have led in 10% of the cases to life-threatening hemolytic-uremic syndrome (HUS) (7).

E. coli of serotype O157:H7 is the leading cause of EHEC infections in humans, but other EHEC serotypes, such as O26:H11 or O26:H-, O103:H2, O111:H-, O113:H21, and O145:H28 or O145:H-, have been increasingly associated with outbreaks and sporadic cases of EHEC-associated disease in humans (3, 7). The reason certain serotypes are associated with disease in humans has not been fully elucidated. The inventory and precise functions of EHEC virulence factors are still not known, but epidemiological studies have suggested that associations between virulence factors could increase the ability of some serotypes to cause disease in humans (5). Certain E. coli strains (Shiga toxin [Stx]-producing E. coli [STEC]) produce Shiga toxins that are encoded by stx genes and damage intestinal, vascular, and renal cells (20). stx genes are carried by lysogenic bacteriophages and can be acquired by horizontal gene transfer (25, 26). Therefore, it has been suggested that the emergence of EHEC strains has resulted from successive horizontal transfers of virulence factors between STEC and enteropathogenic E. coli (EPEC) (7). Stx production is essential but not sufficient for EHEC virulence. The majority of EHEC strains produce a characteristic histopathological feature at the microvillus brush border of enterocytes of humans, known as an "attaching and effacing" (A/E) lesion, by subverting the intestinal epithelial cell function. The formation of A/E lesions is governed by a pathogenicity island known as the locus of enterocyte effacement (LEE), which contains the eae (E. coli attachment effacement) gene encoding the intimin protein (7). The presence of the eae gene correlates highly with the presence of other genes of the LEE (1) in EHEC strains, as well as in EPEC strains. The majority of EHEC strains harbor both stx and eae genes, but some eae-negative STEC strains, such as O91:H21 and O113:H21 strains, which cause bloody diarrhea and HUS in humans, have been described previously (7).

A wide range of animal species are known to carry STEC and EHEC strains, but ruminants are the most important natural reservoir and excrete these bacteria with their feces (7). Although the main infection routes are person-to-person transmission, as well as consumption of contaminated meat and milk, ingestion of contaminated vegetables or water and direct contact with animals or soil have also been associated with EHEC-associated outbreaks. It has been reported that human infections can result from ingestion of fewer than 100 viable EHEC cells (7). Moreover, EHEC and STEC can persist and remain infectious for several weeks in slurries, farmyard manure, and sewage sludge, as well as on pasture land (10, 17, 23). Land application of sludge and effluent discharge in surface water contribute to the spread of STEC in the environment. Contamination of the environment, followed by uptake of STEC by farm animals on pasture, maintains the epidemiological cycle of STEC and is a public health concern. In addition, data on the presence and characteristics of STEC in urban and slaughterhouse wastewater are limited and difficult to compare (11, 12, 14, 18, 22, 37).

The aim of this study was to determine the presence and characteristics of stx-positive and eae-positive E. coli strains in slurry, wastewater, and river water collected in and near slaughterhouses located in different parts of France. Detection and characterization of virulence genes, together with serotyping of stx- and eae-positive E. coli strains and analysis of the function of LEE-encoded genes, allowed us to estimate the significance of these organisms for public health. The combination of the different virulence factors found mainly on mobile genetic elements and the different phenotypic characteristics allowed us to determine the diversity of E. coli strains better. Altogether, our results helped us to determine the role of the environment in the emergence of new pathogenic STEC and EHEC strains.


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MATERIALS AND METHODS
 
Sample description.
A total of 224 samples of slurry, wastewater, and river water were collected in and near 12 slaughterhouses in France. Samples were obtained from each slaughterhouse seasonally between February 2002 and September 2003. Six slaughterhouses located in the west of France (Bretagne and Normandie regions) each had a complete wastewater treatment plant and released their effluents into rivers. Six slaughterhouses located in the southwest of France (Aquitaine and Midi-Pyrénées regions) did not have a complete wastewater treatment plant and passed their effluents to an urban wastewater treatment plant. Wastewater samples were collected at different points during wastewater treatment. Two liters of raw wastewater (40 samples), 2 liters of screened wastewater (40 samples), and 2 liters of primary treated wastewater (34 samples) were collected from each slaughterhouse. Samples of slurry (35 2-liter samples) and sludge (29 2-liter samples) intended to be spread on land were also collected. Samples of treated wastewater intended to be released into rivers were obtained only from slaughterhouses with complete wastewater treatment plants (16 2-liter samples). River water samples were collected upstream (15 5-liter samples) and downstream (15 5-liter samples) of the effluent discharges of slaughterhouses that released their effluents into rivers.

Isolation of E. coli.
Briefly, decimal dilutions of the 224 homogenized samples collected were plated on selective medium for E. coli (Petrifilm Select Coli; BioMérieux, Marcy l'Etoile, France) and cultured at 44°C for 24 h. For each sample, 20 to 24 CFU were selected at random, biochemically confirmed to be E. coli with an API 20E test (BioMérieux), and grown separately with agitation overnight at 37°C in Luria-Bertani broth (Invitrogen, Paisley, Scotland). Broth cultures of each isolate were stored at –80°C in 30% sterile glycerol.

To improve the sensitivity of detection of E. coli O157, a complementary isolation procedure was performed. Aliquots of the 224 homogenized samples were enriched in modified Trypticase soy broth at 42°C for 16 h as described previously (9). Immunomagnetic separation of E. coli O157 was performed according to the manufacturer's instructions (anti-E. coli O157 Dynabeads; Dynal Biotech, Bromborough, United Kingdom). The beads were plated on sorbitol MacConkey agar (Oxoid, Hampshire, United Kingdom) and cultured at 37°C for 24 h. Presumed E. coli O157 strains were confirmed by biotyping with the API 20E test (BioMérieux) and by latex agglutination (E. coli O157 test kit; Oxoid) with non-sorbitol-fermenting isolates from the plates.

Reference strains.
Broth cultures of all reference strains listed in Table 1 were stored at –80°C in 30% sterile glycerol. Each strain was grown with agitation overnight at 37°C in Luria-Bertani broth.


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  		TABLE 1. Pathotypes, serotypes, and virulence factors of reference strains used for genotypic and phenotypic analysis of E. coli strains isolated from slurry, wastewater, and river water collected in and near French slaughterhouses

Laboratory nonpathogenic E. coli strain MG1655 was used as a negative control for all virulence factors investigated. EHEC O157:H7 strain RIMD 050992 (Sakaï) was used as a positive control for detection of the stx1, stx2 (conserved region), and eae (conserved region) genes by multiplex PCR. EHEC, EPEC, and STEC strains listed in Table 1 were used as positive controls for detection of other virulence factors and for typing and subtyping of the eae and stx2 genes. E. coli strains CL37, 95NR1, 4795/95, 6044/95, and 7476/97 were kindly provided by H. Schmidt, Stuttgart, Germany. E. coli strain EF73 was kindly provided by S. Morabito, Rome, Italy.

Laboratory nonpathogenic E. coli strain MG1655 was used as a negative control for the fluorescence actin staining (FAS) test. EPEC O127:H6 strain E2348/69 and EHEC O103:H2 strain PMK5 were used as positive controls for the FAS test.

Detection of virulence genes by PCR.
DNA from the isolates of E. coli was subjected to multiplex PCR for detection of the virulence genes eae, stx1, and stx2 by using combinations of the eaeA B52, eaeA B53, sltI B54, sltI B55, sltII B56, and sltII B57 specific primers as described previously (8). The amplification products were analyzed by electrophoresis on a 2% agarose gel.

Plasmid-borne virulence genes were screened by PCR. The presence of the enterohemorrhagic E. coli hemolysin (EHEC-HlyA) encoded by the ehxA gene was investigated as described previously (33). The genes encoding the bundle-forming pili (bfpA), the STEC autoagglutinating adhesin (saa), and the ToxB adhesin (toxB) were investigated as previously described (15, 30, 34). PCR amplification products were analyzed by electrophoresis on 1.5% agarose gels.

Variants of the B subunit of stx2 genes.
Subtypes of the B subunit of stx2 genes were determined by PCR, followed by analysis of restriction endonuclease-digested PCR products (restriction fragment length polymorphism [RFLP]) as described previously (19, 32). stx2-EDL 933, stx2vh-a, stx2vh-b, stx2d-Ount, and stx2d-OX3a variants were differentiated by PCR using the VT2-e and VT2-f primers, followed by digestion with HaeIII and PvuII of the PCR amplification products (32). The presence of variant stx2e was investigated by PCR using primers Vte-a and Vte-b as described previously (19). However, these methods failed to detect some of the stx2 variants that are present in patient isolates, and Nakao et al. (27) have described a multiplex PCR to detect the most prevalent stx2 variants in EHEC strains classified as variants stx2-EDL 933, stx2ent, and stx2-O48 and group stx2-G1 corresponding to the variants stx2c, stx2cf, stx2-OX3b, stx2vh-a, stx2vh-b, stx2vh-c, stx2vh-c(Lin), and stx2vh-d, and an unnamed stx2 variant (GenBank accession no. AB017524). Variants stx2-EDL 933, stx2ent, and stx2-O48 were investigated by using combinations of primers mStx2, G1-F, and mG1-R primers, and group stx2-G1 was investigated by using combinations of primers mStx2, G1-F, and mStx2-R, as described previously (27). The amplification products and restriction analysis products were examined by electrophoresis on 1% agarose gels.

Types and subtypes of intimin genes.
Eight intimin types ({alpha}, ß, {gamma}, {varepsilon}, {zeta}, {eta}, {iota}, and {kappa}) were investigated by PCR using combinations of forward primer SK1 and reverse primers LP2 to LP10, as described previously (38). To further differentiate the different types of intimin genes, PstI restriction analysis of the PCR amplification products was performed to differentiate {alpha}1, {alpha}2, ß1, ß2/{delta}, {gamma}1, and {gamma}2/{theta} variants, as described previously (29). The amplification products and restriction analysis products were examined by electrophoresis on 1% agarose gels.

FAS test with HeLa cells.
Expression of LEE-encoded genes leading to A/E lesions was tested by visualization of filamentous actin accumulation at sites of bacterial adhesion on cultured human epithelial cells. The FAS test was performed by using a protocol described previously (29).

Serotype identification.
The O antigens of all isolates that had eae, stx1, and stx2 genes were determined with O antisera specific for the 11 most prevalent O antigens detected in EHEC strains (O26, O48, O91, O103, O111, O113, O118, O128, O145, O146, and O157) according to information found at the Laboratorio de Referencia de E. coli website (http://www.lugo.usc.es/ecoli/SEROTIPOSHUM.htm). The H types of motile isolates that belonged to one of the serogroups screened were analyzed by serotyping using antisera specific for 53 different H antigens (H1 to H56). Nonmotile isolates were investigated to determine their H-type-specific (fliC) genes by PCR, followed by HhaI digestion of fliC PCR products and evaluation of RFLP patterns, as previously described (4).

All antisera were obtained and absorbed with the corresponding cross-reacting antigens to remove nonspecific agglutinins. The O antisera were provided by the Laboratorio de Referencia de E. coli (Lugo, Spain), and the H antisera were obtained from the Statens Serum Institute (Copenhagen, Denmark). Additional O and H typing of E. coli strains was performed at the National Reference Laboratory for Escherichia coli at the Federal Institute for Risk Assessment, using O and H antisera produced at the Federal Institute for Risk Assessment.


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RESULTS
 
Presence of stx-positive and eae-positive E. coli isolates.
A total of 224 samples of slurry, wastewater, and river water were collected in and near 12 slaughterhouses in France in different seasons and at different points in connected wastewater treatment plants. Altogether, 5,001 isolates of E. coli were obtained and screened for the presence of eae, stx1 and stx2 genes by multiplex PCR.

Twenty-five percent (55/224) of all samples contained at least one E. coli isolate carrying eae, stx1, and stx2 genes. The positive samples were found to be equally distributed in all of the slaughterhouses investigated. However, one slaughterhouse was more contaminated with stx-positive and eae-positive E. coli (80% of the samples were contaminated). The high contamination rate was later explained by malfunctioning and shutting down of the wastewater treatment plant. Characteristics of samples containing stx-positive and eae-positive E. coli are shown in Table 2. The total numbers of E. coli organisms were determined for all samples and were found to be similar for all sampling points independent of the season and slaughterhouse. In general, samples from early stages of wastewater treatment (raw, screened, and primary treated wastewater samples) were more contaminated with E. coli and with stx-positive and eae-positive types than samples from the final stages (effluent thrown out and effluent sludge) were. Nevertheless, stx-positive and eae-positive E. coli isolates were detected in all kinds of effluent that were released into the environment (treated effluents flowing into rivers, sludge and slurry intended to be spread on land, and primary treated wastewater flowing into urban wastewater treatment plants). In general, river water collected upstream from the slaughterhouses was less contaminated than river water collected downstream.


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  		TABLE 2. Characteristics of wastewater samples containing stx- and eae-positive E. coli isolates, as determined by colony counting

Characterization of stx-positive E. coli isolates.
Thirty independent isolates of stx-positive E. coli were detected among the 5,001 E. coli isolates by colony counting. These 30 STEC isolates were considered to be independent since they were obtained from different samples or (when they were isolated from the same sample) had different virulence factors or biotypes, as determined by the API biochemical code.

The Shiga toxin types and variants of the stx2 and eae genes of the 30 STEC isolates were determined by PCR and PCR-RFLP analysis and are shown in Table 3.


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  		TABLE 3. Variants of stx and eae genes for the most frequent EHEC serogroups detected for 30 independent STEC isolates by colony counting in samples of slurry, wastewater, and river water collected in and near French slaughterhousesa

Except for O128, most of the STEC isolates did not belong to the 11 serogroups frequently associated with EHEC strains.

The STEC strains were heterogeneous for the stx genes; 14 strains harbored only stx1, and 15 strains harbored only stx2. One STEC strain contained both stx1 and stx2-vhb. Three stx2-positive strains harbored more than one stx2 gene. The most frequent stx2 variant was stx2e (detected in six STEC strains), followed by stx2vh-a or stx2vh-b (detected in three STEC strains) and stx2-EDL933 (detected in two STEC strains). One stx2-positive STEC strain had an stx2 variant that belonged to group 1 as defined by Nakao et al. (27), which is the most important stx2 variant group in terms of human pathogenicity. Five stx2-positive STEC strains contained stx2 genes which were nontypeable with the methods described above.

Most of the STEC isolates were eae negative; only two eae-positive STEC strains were detected, and they were associated with intimin ß1.

Characterization of eae-positive E. coli isolates.
It is known that Shiga toxin gene-negative (stx-negative) and intimin gene-positive (eae-positive) strains of E. coli can acquire stx genes by horizontal transfer, increasing their pathogenicity for humans (25, 26). The present work provided new information because we studied not only STEC isolates but also stx-negative, eae-positive E. coli isolates. On the other hand, stx-negative, eae-positive strains of E. coli could be derivatives of STEC strains that have lost their phage-encoded stx genes (24).

Fifty-four independent stx-negative, eae-positive E. coli isolates were detected among the 5,001 E. coli isolates by colony counting. The intimin types and intimin variants of these isolates were determined by PCR and PstI RFLP analysis (Table 4).


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  		TABLE 4. Intimin types and variants for the most frequent EHEC serogroups detected for 54 independent stx-negative, eae-positive E. coli isolates by colony counting in samples of slurry, wastewater, and river water collected in and near French slaughterhouses

Most of the stx-negative, eae-positive E. coli strains did not belong to the 11 serogroups frequently associated with EHEC. The most frequent EHEC-related serogroups detected were O26 and O103, followed by O145 and O157.

Neither intimin {alpha}, intimin {varepsilon}, nor intimin {iota} was detected in any isolate. The most frequently found intimin was intimin {gamma}2/{theta} (detected in 18 isolates), followed by intimin ß1 (detected in 13 isolates) and intimin {gamma}1 (detected in 12 isolates). Six isolates harbored intimin ß2/{delta}, {kappa}, {eta}, or {zeta}. Five isolates carried intimin variants different from the 11 variants screened.

All stx-negative, eae-positive E. coli isolates were bfp negative and could therefore be classified as "atypical EPEC" (28).

Detection of E. coli O157 isolates.
E. coli O157 STEC strains were not present among the 5,001 E. coli strains isolated from wastewater samples. This collection of E. coli isolates was probably representative of the dominant populations of E. coli and did not take into account nondominant populations of E. coli present in slaughterhouse wastewater. In order to screen specifically for E. coli O157, O157 immunomagnetic separation enrichment followed by a latex agglutination assay with sorbitol-negative E. coli isolates was performed for all samples. In this analysis, 31 E. coli O157 strains were isolated and screened for the presence of eae, stx1, and stx2 genes by multiplex PCR. Variants of the eae and stx2 genes were screened by PCR and PCR-RFLP analysis. Four E. coli O157 isolates were positive for intimin {gamma}1, and two of these harbored both stx2vh-a and stx2vh-b variants. These four isolates belonged to the O157:H7 serotype.

Characterization of the putatively most virulent E. coli isolates.
All stx-positive E. coli strains and stx-negative, eae-positive E. coli strains isolated from slaughterhouse wastewater were characterized further in order to assess their significance for public health.

E. coli strains belonging to serotypes O26:H11 or O26:H-, O103:H2, O111:H-, O113:H21, O145:H28 or O145:H-, and O157:H7 are recognized as classical EHEC types (3). In addition, apart from their capacity to produce Shiga toxins and intimins, most EHEC strains have accessory virulence factors, such as enterohemolysin (EHEC-HlyA), which seems to be associated with severe clinical disease (5, 33). Furthermore, intestinal adherence factors distinct from intimin, such as Saa and ToxB, play a role in the virulence of some E. coli strains by increasing the capacity for adhesion (31, 35).

Detection of these additional virulence factors, together with determination of the complete serotype, allowed us to select 13 isolates which belonged to serotypes associated with illness in humans (Table 5). Most of these isolates were detected in the early stages of wastewater treatment, but four isolates (two serotype O128:H8 STEC isolates and two stx-negative, eae-positive serotype O26:H11 or O157:H7 E. coli isolates) were detected in wastewater released into the environment. Three eae-positive STEC isolates, including one O128:H8 isolate and two O157:H7 isolates, harbored more than three different virulence factors that are particularly associated with EHEC strains. The last two STEC isolates belonged to serotype O128:H8 and were negative for eae, but they harbored a potential adhesion factor encoded by the saa gene. Seven of the eight stx-negative, eae-positive isolates of E. coli that belonged to a serotype associated with human illness contained an intimin variant frequently associated with HUS, four isolates that belonged to serotype O26:H11, O26:H- or O103:H2 were positive for ß1 intimin, and three serotype O145:[H28] or O157:H7 isolates harbored {gamma}1 intimin. The last stx-negative, eae-positive isolate, which belonged to serotype O157:H7, harbored a nontypeable variant of the eae gene.


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  		TABLE 5. Phenotypes, genotypes, and origins of the 13 putatively most virulent E. coli isolates from slurry, wastewater, and river water collected in and near French slaughterhouses

The ability of eae-positive isolates of E. coli to produce A/E lesions on cultured HeLa cells was determined by the FAS test. Eight of 11 eae-positive strains which belonged to serotypes associated with illness in humans induced accumulation of filamentous actin at sites of adhesion.


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DISCUSSION
 
One aim of the present study was to determine the presence of stx-positive and eae-positive E. coli isolates in slurry, wastewater, and river water collected in and near 12 French slaughterhouses.

Twenty-five percent of the samples collected were contaminated with stx-positive and eae-positive E. coli isolates, and positive samples were found to be equally distributed in all of the slaughterhouses studied. These results show that potentially pathogenic E. coli isolates could be found ubiquitously in French slaughterhouses and are in agreement with the results of a previous study of contamination of the environment with STEC in France (37).

Higher numbers of stx- and eae-positive E. coli isolates were detected in samples from early stages of wastewater treatment than in samples from the final stages of wastewater treatment, confirming the results of a previous report on reduction of STEC in wastewater after treatment (12). Thus, wastewater treatment could be appropriate for reducing the survival of STEC and eae-positive E. coli, as it is for reducing E. coli viability. Nevertheless, the total numbers of E. coli organisms in samples could not be considered a reliable indicator of the presence of STEC and eae-positive E. coli. Indeed, some stx-positive and eae-positive E. coli isolates were detected in sludge samples that contained relatively low numbers of E. coli organisms, whereas stx-positive and eae-positive E. coli isolates were not detected in some samples of raw wastewater that contained high numbers of E. coli. Some stx-positive and eae-positive E. coli isolates were detected in slaughterhouse wastewater that was ready to be released into the environment. Moreover, river water collected upstream from the slaughterhouses was less contaminated than river water collected downstream from the slaughterhouses. These results suggest that STEC and eae-positive E. coli can persist during all stages of wastewater treatment and are able to adapt to environmental stress in aquatic systems and survive, thus confirming results of a previous study of the resistance of E. coli O157:H7 to routine water treatment procedures (21). Persistence of STEC in wastewater during treatment has been observed previously in France (37), Germany (18), and Tasmania (22). However, it was difficult to compare the numbers of STEC isolates since the isolation procedures were different. Nevertheless, the results indicate that contamination of natural water sources can occur directly or indirectly from the regular disposal of sewage, although this possibility was once rejected (14). As a consequence, our results indicate that the environment is a reservoir for human-pathogenic E. coli isolates, which has consequences for public health.

All STEC and stx-negative, eae-positive E. coli isolates originating from wastewater released by slaughterhouses were characterized further in order to assess their potential as possible human pathogens.

Although some stx1-positive STEC isolates have previously been associated with severe disease (6), epidemiological studies have demonstrated that Stx2 is the most important virulence factor associated with severe human disease (5, 20) and is significantly associated with an increased risk of HUS in persons infected with O157 or non-O157 STEC (6). In the present study, one-half of the STEC isolates (15/30) harbored stx2 genes and can thus be considered to be more closely associated with an increased risk of HUS than the stx1-positive STEC isolates.

Twenty stx2 variants have been identified on the basis of sequence homology, and at least 12 stx2 variants produced by EHEC strains from patients have been described previously (2, 27). Some other variants, which are associated with STEC strains isolated from specific hosts, such as sheep (stx2d) and pigs (stx2e), are considered to be less pathogenic for humans (7). The type of stx variant could thus reflect not only the origins and relationships but also the virulence of the different STEC strains. In our collection of environmental STEC isolates, the most frequent stx2 variants were stx2e and nontypeable variants of stx2, which are probably less pathogenic for humans. The stx2e variants were isolated from samples collected in slaughterhouses in which pigs are slaughtered, a finding which is in agreement with previously published data (7). However, stx2 variants produced by EHEC strains, such as stx2vh-a, stx2vh-b, stx2-EDL933, and variants of the G1 group, which was defined by Nakao et al. (27) as the most important stx2 variant group in terms of human pathogenicity, were detected in STEC isolates. Our results indicate that very diverse stx2 variants are associated with STEC isolates, confirming the results of previous findings for STEC strains isolated from the environment in France (36). The majority of STEC strains probably originated from livestock, but lysogenization of E. coli strains that harbor stx2 variants produced by EHEC strains may lead to conversion of new strains of E. coli and to the emergence of new human pathogens (25).

The presence of the eae gene in EHEC strains is significantly associated with an increased risk of bloody diarrhea in humans (6), and at present 11 variants of intimin have been described previously (38). The distribution of these variants among EHEC and EPEC strains isolated from different species suggests that the host and/or the tissue tropism of the different A/E bacteria may be influenced by the variants of intimin that they express. In our collection of E. coli isolates, the majority of eae-positive isolates can be considered "atypical EPEC" and are probably characteristic of animal strains, which could be explained by the nature of the samples collected. Indeed, intimin {alpha}, which seems to be specifically expressed by human classical EPEC strains (29), was not found in any eae-positive E. coli isolate. Furthermore, even if the majority of eae-positive E. coli isolates harbored intimins {gamma}2/{theta}, ß1, and {gamma}1, which are expressed by human strains (29, 38), most of them did not belong to serogroups frequently associated with EHEC strains and were probably less pathogenic for humans. In addition, intimin variants that are probably rare among strains associated with severe human disease, such as intimins ß2/{delta}, {kappa}, {eta}, and {zeta}, or intimin variants that are different from those screened (38) were detected in eae-positive isolates. The presence of these nontypeable intimins could be explained by the frequency of recombination events inside the LEE and especially inside the eae gene (38). Thus, the great diversity of intimin variants, including those associated with EHEC strains, detected in our environmental collection of eae-positive E. coli isolates indicated that the environment could play a role as a reservoir for pathogenic strains and for recombination of intimin genes (38).

Based on the intimin and Stx variants and serotypes, most of the stx-positive and eae-positive E. coli isolates were probably low-virulence strains, which confirmed the results of a previous characterization of STEC strains isolated from Spanish urban sewage and animal wastewater (11). The majority of E. coli isolates in our study had serotypes and combinations of virulence genes that are not frequently associated with hemorrhagic colitis or HUS, and they were probably more specific E. coli strains present in animal digestive microfloras. These isolates may nonetheless play an important role in the assortment of genes between E. coli isolates, which could lead to the emergence of new pathogenic EHEC strains. In addition, 13 strains putatively virulent for humans were also detected. The presence of such virulent isolates could have been underestimated since decimal dilution of the samples collected allowed isolation of strains that were primarily representative of the dominant E. coli populations. Indeed, STEC serotype O157:H7 isolates were present in the nondominant E. coli populations, as demonstrated by the immunomagnetic separation procedure.

In conclusion, this study demonstrated that slurry, wastewater, and river water present in and near French slaughterhouses are contaminated with STEC and with eae-positive E. coli and highlights the importance of appropriate handling and use of slurry and sewage sludge to prevent contamination of the environment and food by EHEC. In addition, the great diversity of the stx- and eae-positive E. coli isolates from wastewater of slaughterhouses suggests that monitoring the flux of EHEC virulence factors in the bacterial population in the environment is necessary to prevent epidemiological risks for public health.


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ACKNOWLEDGMENTS
 
This study was supported by the research program "Environnement et santé 2000 INSERM-contrat no. EN00C04 RD: AC006G" from the Ministère Français de l'aménagement du territoire et de l'environnement.

We thank E. Sandrin-Gabriel-Robez from the Biogram Society for her technical assistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: UMR 1225 INRA-ENVT Interactions Hôtes-Agents Pathogènes, Equipe Pathogénie moléculaire et cellulaire des Escherichia coli, Ecole Nationale Vétérinaire de Toulouse, BP 87 614, 31076 Toulouse Cedex 3, France. Phone: 33 (0)5 61 19 39 05. Fax: 33 (0)5 61 49 12 63. E-mail: h.brugere{at}envt.fr. Back


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Applied and Environmental Microbiology, May 2006, p. 3245-3251, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3245-3251.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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