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The pks island codes for the enzymes necessary for synthesis of the genotoxin colibactin, which contributes to the virulence of Escherichia coli strains and is suspected of promoting colorectal cancer. From a collection of 785 human and bovine E. coli isolates, we identified 109 strains carrying a highly conserved pks island, mostly from phylogroup B2, but also from phylogroups A, B1 and D. Different scenarios of pks acquisition were deduced from whole genome sequence and phylogenetic analysis. In the main scenario, pks was introduced and stabilized into certain sequence types (STs) of the B2 phylogroup, such as ST73 and ST95, at the asnW tRNA locus located in the vicinity of the yersiniabactin-encoding High Pathogenicity Island (HPI). In a few B2 strains, pks inserted at the asnU or asnV tRNA loci close to the HPI and occasionally was located next to the remnant of an integrative and conjugative element. In a last scenario specific to B1/A strains, pks was acquired, independently of the HPI, at a non-tRNA locus. All the pks-positive strains except 18 produced colibactin. Sixteen strains contained mutations in clbB or clbD, or a fusion of clbJ and clbK and were no longer genotoxic but most of them still produced low amounts of potentially active metabolites associated with the pks island. One strain was fully metabolically inactive without pks alteration, but colibactin production was restored by overexpressing the ClbR regulator. In conclusion, the pks island is not restricted to human pathogenic B2 strains and is more widely distributed in the E. coli population, while preserving its functionality.

The pks island codes for the enzymes necessary for synthesis of the genotoxin colibactin, which contributes to the virulence of Escherichia coli strains and is suspected of promoting colorectal cancer. From a collection of 785 human and bovine E. coli isolates, we identified 109 strains carrying a highly conserved pks island, mostly from the phylogroup B2, but also from phylogroups A, B1 and D. Different scenarios of pks acquisition were deduced from whole genome sequence and phylogenetic analysis. In the main scenario, pks was introduced and stabilized into certain sequence types (ST) of the B2 phylogroup, such as ST73 and ST95, at the asnW tRNA locus located in the vicinity of the yersiniabactin-encoding High Pathogenicity Island (HPI). In a few B2 strains, pks inserted at the asnU or asnV tRNA loci close to the HPI and occasionally was located next to the remnant of an integrative and conjugative element. In a last scenario specific to B1/A strains, pks was acquired, independently of the HPI, at a non-tRNA locus. All the pks-positive strains except 18 produced colibactin. Sixteen strains contained mutations in clbB or clbD, or a fusion of clbJ and clbK and were no longer genotoxic but most of them still produced low amount of potentially active metabolites associated with the pks island. One strain was fully metabolically inactive without pks alteration, but colibactin production was restored by overexpressing the ClbR regulator. In conclusion, the pks island is not restricted to human pathogenic B2 strains and is more widely distributed in the E. coli population, while preserving its functionality.IMPACT STATEMENTColibactin, a genotoxin associated with the carcinogenicity of certain strains of E. coli, is encoded by a pathogenicity island called pks. We took advantage of a large collection of non-clinical E. coli strains originating from human and bovine hosts to explore the distribution, conservation and functionality of the pks island. We found that the pks island was not only present in the phylogroup B2 (and more specifically to certain B2 sublineages), but also in other genetic phylogroups, highlighting its capacity to disseminate though horizontal gene transfer. We identified various genetic pks configurations indicative of an introduction of the pks island into E. coli on multiple independent occasions. Despite the existence of various acquisition scenarios, we found that the pks sequences were highly conserved and pks-carrying strains were overwhelmingly capable of producing colibactin, suggesting that the pks island is under selective pressure, through the production of colibactin or other secondary metabolites. Future implications include the identification of such metabolites and their biological activities that could be advantageous to E. coli and enable its adaptation to various ecological niches.DATA SUMMARYAll sequence data of the 785 E. coli used in this study are freely available from the NCBI BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/) under the accession number PRJDB5579. This database was updated to include the sequence data obtained using ONT MinION for the E. coli reference strain SP15 and for E. coli strains ECSC054, JML285, KS-NP019, NS-NP030 and SI-NP020. The sequence data of E. coli strain UPEC129 obtained using PacBio instrument were deposited in the NCBI BioProject database and are available at https://www.ncbi.nlm.nih.gov/Traces/study/ under the accession number PRJNA669570. Hybrid MinION-Illumina and PacBio-Illumina assemblies are available at the NCBI nucleotide database. The genome sequences of 36 other E. coli reference strains and 7 non-E. coli strains were retrieved from NCBI.

Wastewater treatment plants (WWTP) receiving effluents from food-producing animals and humans may contribute to the spread of extended-spectrum β-lactamases (ESBL)-carrying plasmids. This study was designed to investigate extended-spectrum cephalosporin resistant Escherichia coli strains, CTX-M distributions and the genetic lineage of blaCTX-M -carrying plasmids from urban and slaughterhouse wastewaters. The level of extended-spectrum cephalosporin-resistant E. coli in slaughterhouse wastewater entering the WWTP was negligible compared with that of urban wastewater. The blaCTX-M-1 gene was predominant in slaughterhouse wastewater whereas diverse blaCTX-M genes were encountered in urban wastewater and WWTP outlet. Characterization of the main CTX-M-producing E. coli isolates by antibiotic resistance phenotyping, genotyping and typing of plasmids carrying blaCTX-M genes revealed that blaCTX-M-1 and blaCTX-M-15 genes were harboured by the predominant blaCTX-M-1 IncI1/ST3 and blaCTX-M-15 F31:A4:B1 plasmids, which were recovered from unrelated E. coli genotypes in both slaughterhouse and urban wastewaters. This study highlighted the spread of predominant blaCTX-M-1 and blaCTX-M-15 plasmid lineages in diverse E. coli genotypes from humans and food-producing animals, their mixing in WWTP and final release into the aquatic environment. This could have a serious negative impact on public health and requires further evaluation.

The aim of this study was to compare the antimicrobial resistance profiles of top five enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) to E. coli isolated from the fecal flora of the same adult cattle. Previous prevalence studies had led to the isolation by immunomagnetic separation (IMS) of 39 EHEC and 80 EPEC. Seven EHEC were resistant (17.9%), and six were multidrug resistant (MDR) (15.4%). None of the top five EHEC was resistant to azithromycin. Nine EPEC O26:H11 (11.3%) were resistant. They were all resistant to tetracycline, and four were MDR (5.0%). An E. coli strain was isolated from the feces (without preselection by IMS) of 97 bovine carriers of top 5 strains. All these strains were susceptible to antibiotics. Comparative analyses did not reveal any differences between the cytotoxic activities of resistant EHEC and their susceptible counterparts or in the production of attachment and effacement lesions. These results highlighted the higher percentage of resistance of EHEC and EPEC strains compared to other E. coli. They also showed that resistance traits did not have any impact on the expression of virulence phenotypes in EHEC strains.

International audience; Wastewater of human and animal may contain Shiga toxin-producing (STEC) and enteropathogenic (EPEC) Escherichia coli. We evaluated the prevalence of such strains in a wastewater treatment plant (WWTP) receiving both city and slaughterhouse wastewater. PCR screenings were performed on 12,248 E. coli isolates. The prevalence of STEC in city wastewater, slaughterhouse wastewater and treated effluent was 0.22%, 0.07% and 0.22%, respectively. The prevalence of EPEC at the same sampling sites was 0.63%, 0.90% and 0.55%. No significant difference was observed between the sampling points. Treatment had no impact on these prevalences. Enterohemorrhagic E. coli (EHEC) O157:H7 and O111:H8 were isolated from the treated effluent rejected into the river. The characteristics of STEC and EPEC differed according to their origin. City wastewater contained STEC with various stx subtypes associated with serious human disease, whereas slaughterhouse wastewater contained exclusively STEC with stx2e subtype. All the EPEC strains were classified as atypical and were screened for the ε, γ1 and β1 subtypes, known to be associated with the EHEC mainly involved in human infections in France. In city wastewater, eae subtypes remained largely unidentified; whereas eae-β1 was the most frequent subtype in slaughterhouse wastewater. Moreover, the EPEC isolated from slaughterhouse wastewater were positive for other EHEC-associated virulence markers, including top five serotypes, the ehxA gene, putative adherence genes and OI-122 associated genes. The possibility that city wastewater could contain a pool of stx genes associated with human disease and that slaughterhouse wastewater could contain a pool of EPEC sharing similar virulence genes with EHEC, was highlighted. Mixing of such strains in WWTP could lead to the emergence of EHEC by horizontal gene transfer.

Enterohemorrhagic Escherichia coli (EHEC) with serotype O157:H7 is a major foodborne pathogen. Here, we report the draft genome sequence of EHEC O157:H7 strain MC2 isolated from cattle in France. The assembly contains 5,400,376 bp that encoded 5,914 predicted genes (5,805 protein-encoding genes and 109 RNA genes).

Enterohemorrhagic Escherichia coli (EHEC) with serotype O157:H7 is a major foodborne pathogen. Here, we report the draft genome sequence of EHEC O157:H7 strain MC2 isolated from cattle in France. The assembly contains 5,400,376 bp that encoded 5,914 predicted genes (5,805 protein-encoding genes and 109 RNA genes).

International audience; Enteroaggregative Shiga-toxin-producing Escherichia coli strains were responsible for a massive outbreak in Europe in 2011, and had been previously isolated from French patients. The objective of this study was to investigate the presence of enteroaggregative E. coil (EAEC) in slaughterhouse effluents (wastewater, slurry, sludge and effluents), and in river waters near these slaughterhouses. A total of 10,618 E. coli isolates were screened by PCR for the presence of EAEC-associated genetic markers (aggR, aap and aatA). None of these markers was detected in E. coli isolated from slaughterhouse samples. A unique enteroaggregative E. coli (EAEC) O126:H8 was detected in river water sampled upstream from slaughterhouse effluent discharge. These results confirmed that animals might not be reservoirs of EAEC, and that further studies are required to evaluate the role of the environment in the transmission of EAEC to humans.

International audience; We compared the prevalence of pathogenic and extended-spectrum beta-lactamase (ESBL) producing Escherichia coli in effluents of a municipal wastewater treatment plant (WWTP) receiving wastewater from a slaughterhouse. A total of 1248 isolates were screened for the presence of virulence genes associated with enterohemorrhagic E. calf (EHEC) (stx(1), stx(2), and eae) and extraintestinal pathogenic E. coli (ExPEC) (sfa/focDE, kpsMT K1, hlyA, papEF, afa/draBC, clbN, f17A and cnf). The prevalence of atypical enteropathogenic E. coli (EPEq was 0.7%, 0.2% and 0.5% in city wastewater, slaughterhouse wastewater and in the treated effluent, respectively. One stx(1a) and stx(2b)-positive E. coli isolate was detected in city wastewater. The prevalence of ExPEC was significantly higher in city wastewater (8.4%), compared to slaughterhouse wastewater (1.2%). Treatment in the WWTP did not significantly impact the prevalence of ExPEC in the outlet effluent (5.0%) compared to city wastewater. Moreover, the most potentially pathogenic ExPEC were isolated from city wastewater and from the treated effluent. ESBL-producing E. coli was also mainly detected in city wastewater (1.7%), compared to slaughterhouse wastewater (0.2%), and treated effluent (0.2%). One ESBL-producing E. coli, isolated from city wastewater, was eae-beta 1 positive. These results showed that pathogenic and/or ESBL-producing E. coli were mainly detected in human wastewater, and at a lesser extend in animal wastewater. Treatment failed to eliminate these strains which were discharged into the river, and then these strains could be transmitted to animals and humans via the environment. (C) 2013 Elsevier Ltd. All rights reserved.

Wastewater treatment plants (WWTP) receiving effluents from food-producing animals and humans may contribute to the spread of extended-spectrum β-lactamases (ESBL)-carrying plasmids. This study was designed to investigate extended-spectrum cephalosporin resistant Escherichia coli strains, CTX-M distributions and the genetic lineage of bla

Aims: To develop a duplex real-time PCR assay targeting enterohaemorrhagic Escherichia coli (EHEC) type III effector TccP/TccP2-encoding genes which are pivotal to EHEC-mediated actin cytoskeleton reorganization in human intestinal epithelial cells. Methods and Results: The specificity of the assay was demonstrated with DNA from EHEC reference strains and non-E. coli bacterial species. The detection limit was determined as five tccP or tccP2 copies per reaction. The assay was then evaluated on a large collection of 526 E. coli strains of human, animal, food and environmental origins. The results showed that tccP was restricted to a limited number of serotypes (i.e. O5:H−, O55:H7, O125:H6 and O157:H7). The tccP2 gene was present in a higher number of serotypes including the five most frequent EHEC serotypes (i.e. O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7), and a few other serotypes that caused human infections (i.e. O4:H−, O45:H2 and O55:H7). A minority of O26:H11 and O103:H2 strains however tested negative for tccP2, though it is not known whether the lack of tccP2 affected their pathogenic potential. Real-time PCR analysis of 400 raw milk cheeses revealed the presence of tccP and/or tccP2 genes in 19·75% of the cheese enrichment suspensions. Conclusions: A highly specific and sensitive duplex real-time PCR method was developed for rapid and simultaneous detection of tccP and tccP2. Unpasteurized dairy products may be contaminated with E. coli strains carrying tccP and/or tccP2. Significance and Impact of the Study: The developed real-time PCR assay represents a valuable alternative to conventional PCR tests and should be useful for characterization of the virulome of pathogenic E. coli strains.

EnteropathogenicEscherichia coli(EPEC) and enterohemorrhagicE. coli(EHEC) inject effector proteins into host cells via a type III secretion system encoded by the locus of enterocyte effacement (LEE). One of these effectors is Cif, encoded outside the LEE by a lambdoid prophage. In this study, we demonstrated that the Cif-encoding prophage of EPEC strain E22 is inducible and produces infectious phage particles. We investigated the distribution and functional expression of Cif in 5,049E. colistrains of human, animal, and environmental origins. A total of 115E. coliisolates from diverse origins and geographic locations carriedcif. The presence ofcifwas tightly associated with the LEE, since all thecif-positive isolates were positive for the LEE. These results suggested that the Cif-encoding prophages have been widely disseminated within the natural population ofE. colibut positively selected within the population of LEE-positive strains. Nonetheless, 66% ofcif-positiveE. colistrains did not induce a typical Cif-related phenotype in eukaryotic cells due to frameshift mutations or insertion of an IS element in thecifgene. The passenger region of the prophages carryingcifwas highly variable and showed various combinations of IS elements and genes coding for other effectors such asnleB,nleC,nleH,nleG,espJ, andnleA/espI(some of which were also truncated). This diversity and the presence of nonfunctional effectors should be taken into account to assess EPEC and EHEC pathogenicity and tropism.

International audience; The goal of this study was to investigate the involvement of bovine slaughterhouse effluents and bio-solids in the risk of environmental dissemination of pathogenic and antibiotic-resistant Escherichia coli. Several samples were collected from one adult cattle and one veal calf slaughterhouse wastewater treatment plant (WWTP). The treatment process had no impact on the percentage of Shiga toxin-producing E. coli (STEC) and on the percentage of atypical enteropathogenic E. coli (aEPEC). A STEC O157:H7 was isolated from the thickened sludge of the adult cattle slaughterhouse. As thickened sludge is intended to be spread on agricultural lands, the detection of this pathogenic strain is a public health issue. The percentage of antibiotic-resistant E. coli was 5.0% and 87.5% in wastewater from the adult cattle and the veal calf slaughterhouse, respectively. These percentages were not significantly different after treatment. Integron-bearing E. coli isolates were only detected in the veal calf slaughterhouse WWTP with percentages above 50.0% for all sampling points whatever the step of the treatment process. Taken together, these findings highlighted the fact that different public health risks might be associated with adult cattle or veal calf slaughterhouses regarding the dissemination of pathogenic and antibioticresistant E. coli isolates into the environment.

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.

International audience; In May-June 2011, a massive outbreak of haemolytic uraemic syndrome caused by enteroaggregative Shiga toxin (Stx)-producing Escherichia coli (STEC) O104:H4 occurred in Europe, which was linked to the consumption of sprouted seeds. As ruminants are known reservoirs of STEC, this study investigated whether cattle could be a reservoir of enteroaggregative STEC O104:H4 and a potential source of transmission to humans. A total of 1468 French cattle were analysed for faecal carriage of the outbreak strain by PCR assays targeting stx2, wzx(O104), fliC(H4) and aggR genetic markers. None of the faecal samples contained the four markers simultaneously, indicating that cattle is not a reservoir of this recently emerged E. coli pathotype. (C) 2012 Elsevier B.V. All rights reserved.

The consumption of raw milk cheese can expose populations to Shiga toxin-producing Escherichia coli (STEC). We report here the genome sequence of an E. coli O26:H11 strain isolated from humans during the first raw milk cheese outbreak described in France (2005).

The main pathogenic enterohemorrhagic Escherichia coli (EHEC) strains are defined as Shiga toxin (Stx)-producing E. coli (STEC) belonging to one of the following serotypes: O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28. Each of these five serotypes is known to be associated with a specific subtype of the intimin-encoding gene ( eae ). The objective of this study was to evaluate the prevalence of bovine carriers of these “top five” STEC in the four adult cattle categories slaughtered in France. Fecal samples were collected from 1,318 cattle, including 291 young dairy bulls, 296 young beef bulls, 337 dairy cows, and 394 beef cows. A total of 96 E. coli isolates, including 33 top five STEC and 63 atypical enteropathogenic E. coli (aEPEC) isolates, with the same genetic characteristics as the top five STEC strains except that they lacked an stx gene, were recovered from these samples. O157:H7 was the most frequently isolated STEC serotype. The prevalence of top five STEC (all serotypes included) was 4.5% in young dairy bulls, 2.4% in young beef bulls, 1.8% in dairy cows, and 1.0% in beef cows. It was significantly higher in young dairy bulls ( P < 0.05) than in the other 3 categories. The basis for these differences between categories remains to be elucidated. Moreover, simultaneous carriage of STEC O26:H11 and STEC O103:H2 was detected in one young dairy bull. Lastly, the prevalence of bovine carriers of the top five STEC, evaluated through a weighted arithmetic mean of the prevalence by categories, was estimated to 1.8% in slaughtered adult cattle in France.

Shiga toxin-producing Escherichia coli (STEC) strains belonging to serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 are known to be associated with particular subtypes of the intimin gene ( eae ), namely, γ1, β1, ε, θ, and γ1, respectively. This study aimed at evaluating the usefulness of their detection for the specific detection of these five main pathogenic STEC serotypes in cattle feces. Using real-time PCR assays, 58.7% of 150 fecal samples were found positive for at least one of the four targeted eae subtypes. The simultaneous presence of stx , eae , and one of the five O group markers was found in 58.0% of the samples, and the five targeted stx plus eae plus O genetic combinations were detected 143 times. However, taking into consideration the association between eae subtypes and O group markers, the resulting stx plus eae subtype plus O combinations were detected only 46 times. The 46 isolation assays performed allowed recovery of 22 E. coli strains belonging to one of the five targeted STEC serogroups. In contrast, only 2 of 39 isolation assays performed on samples that were positive for stx , eae and an O group marker, but that were negative for the corresponding eae subtype, were successful. Characterization of the 24 E. coli isolates showed that 6 were STEC, including 1 O157:H7, 3 O26:H11, and 2 O145:H28. The remaining 18 strains corresponded to atypical enteropathogenic E. coli (aEPEC). Finally, the more discriminating eae subtype-based PCR strategy described here may be helpful for the specific screening of the five major STEC in cattle feces.

International audience; F17 fimbriae are produced by pathogenic Escherichia coli involved in diarrhea and septicemia outbreaks in calves and lambs. These proteins result from the expression of four different clustered genes, namely f17A, f17D, f17C and f17G, encoding a pilin protein, a periplasmic protein, an anchor protein and an adhesin protein, respectively. Several variants of f17A and f17G genes have been reported and found genetically associated with typical virulence factors of bovine pathogenic E. coli strains. In this study, a new F17e-A variant, closely related to F17b-A, was identified from a collection of 58 E. coli isolates from diarrheic calves in Iran. While highly prevalent in Iranian F17-producing clinical isolates from calves, this variant was rare among E. coli from a French healthy adult bovine population, suggesting a possible association with virulence. The f17Ae gene was also found in the genome of the Shiga-like toxin variant Stx1d–producing bovine E. coli strain MHI813, and belonged to a gene cluster also encoding a new F17-G3 variant, which greatly differed from F17-G1 and F17-G2. This gene cluster was located on a pathogenicity island integrated in the tRNA pheV gene. The gene coding for a third new F17f-A variant corresponding to a combination of F17c-A and F17d-A was also identified on the pVir68 plasmid in the bovine pathogenic E. coli strain 6.0900. In conclusion, we identified three new F17-A and F17-G variants in cattle E. coli, which may also have significant impact on the development of new diagnostics and vaccination tools.

Enterohemorrhagic Escherichia coli (EHEC) strains are a subset of Shiga toxin (Stx)-producing E. coli (STEC) strains that are isolated from human patients and are responsible for severe clinical symptoms, such as hemorrhagic colitis (HC) and the potentially lethal hemolytic uremic syndrome (HUS) (28, 29). Although most outbreaks of HC and HUS have been attributed to serotype O157:H7/H−, an increasing number of human infections are caused by other serotypes, such as O26:H11/H−, O103:H2, O111:H8/H−, and O145:H28/H− (13, 20, 28). O157:H7/H− and these four serotypes were classified into seropathotypes A and B, respectively, which occur most frequently in human diseases and outbreaks and in patients with severe symptoms (30). Shiga toxins, the main virulence factors contributing to pathogenicity, consist of two major types, Stx1 and Stx2, each including several variants (47). In addition to Stx, typical EHEC strains carry on their chromosomes the locus for enterocyte effacement (LEE), a large pathogenicity island that is shared with enteropathogenic E. coli (EPEC). The LEE is responsible for attaching and effacing (A/E) lesions on enterocytes (34); it encodes several virulence factors, including the outer membrane adhesin intimin and its translocated receptor Tir, as well as components of a type III secretion machinery and its effector proteins. Intimin is involved in the tight attachment of bacteria to the enterocytes. It is encoded by the eae gene, 18 types and 9 subtypes of which have been described on the basis of its variable 3′ region (25). The EHEC serotypes O157:H7 and O145:H28 are known to be associated with the eae-γ1 subtype, whereas EHEC O26:H11, O103:H2, and O111:H8 harbor the eae-β1, eae-ɛ, and eae-γ2/θ subtypes, respectively (7, 11, 37, 51). Additional candidate pathogenicity islands, or “O islands” (OI), that may contribute to virulence have been identified. They include OI-71 and OI-122, which contain a number of type III non-LEE-encoded effector (nle) genes whose presence correlates with outbreak and HUS potential (17). Finally, EHEC O157:H7 contains a large plasmid with putative virulence genes encoding the EHEC-hemolysin (EHEC-hlyA), a serine protease (espP), and a catalase peroxidase (katP). However, strains lacking one or more of these genes have been involved in HUS, and high variability in the gene composition of this large plasmid has also been reported in various STEC serotypes (14). Domestic ruminants have long been identified as a major reservoir for STEC (29). Although STEC transmission to humans is frequently associated with consumption of raw or undercooked meat, raw milk and dairy products have also been implicated in human disease (4). Detection and isolation of STEC in foodstuffs by traditional culture methods is rather laborious and time-consuming and is complicated by the lack of common biochemical characteristics distinguishing most STEC from other E. coli strains. Development of rapid methods for the detection of the most pathogenic STEC strains is essential to ensure the safety of food products. A sequential approach based on real-time PCR assays specific for stx, wzxO26, wzxO103, wbdIO111, ihp1O145, and rfbEO157 genetic markers has been described for the detection of STEC O26, O103, O111, O145, and O157 in foods (3, 40). A similar strategy that includes an additional eae amplification step has been proposed by working group 6 of the European Committee for Standardization (CEN) TC275 for a technical specification (currently under International Organization for Standardization [ISO] evaluation) regarding the detection of these five STEC serogroups in foods (21). Recently, we developed real-time PCR assays for detection of the eae variants and fliC alleles associated with the five most common EHEC serotypes (32). The aim of the present study was to investigate whether these newly developed assays could further refine the diagnostic result obtained from PCR analysis of foods. To this end, the prevalence of stx, O group genetic markers, eae variants, and fliC alleles typical of EHEC O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7 was investigated in 400 raw-milk cheeses. STEC and E. coli strains belonging to the five targeted serotypes were then isolated for confirmation of the PCR results, and their virulence traits were characterized. As the term EHEC includes a clinical connotation (28, 29), the stx-positive E. coli isolates recovered from food are designated STEC (not EHEC) here, irrespective of the serotype.

Consumers' high expectations in terms of meat quality and the way in which the animals for slaughter are bred have turned breeding into a key component within the beef production industry. A clearer understanding of consumers' expectations should not only allow the industry to meet them, but also anticipate them. As for “consumers-citizens”, the expectations for beef quality greatly exceed the meat's organoleptic and nutritional qualities alone, and also concern the fields of food safety, animal welfare and environmental impacts. To meet these expectations, quality-driven approaches are initiated by the different sector players, particularly the breeders. At the same time, major and increasingly stringent regulations govern the production and processing of meat to comply with safety, environmental and ethical requirements. The breeding sector, the first link in the industry's chain, is fully committed to the quality approaches and the programmes for controlling the health quality of the meat. The meat industry needs to listen to the consumers' demands and fears to interpret them and try to formulate a response, as well as sometimes adjust them by providing information that is unknown to the public but required to promote a good understanding of trade practices.; Les fortes attentes des consommateurs en matière de qualité des viandes et sur le mode d’élevage des animaux dont elles sont issues font de l’élevage un composant majeur de la filière de production de viande bovine. Mieux connaître les attentes des consommateurs doit permettre non seulement d’y répondre mais aussi de les anticiper. Pour les consommateurs-citoyens, les attentes de qualité de la viande bovine dépassent largement les seules qualités organoleptiques et nutritionnelles de la viande, mais concernent aussi les domaines de la sécurité sanitaire des aliments, du bien-être des animaux et des impacts environnementaux. Pour répondre à ces attentes des démarches qualité voient le jour, initiées par les différents acteurs des filières et en particulier par les éleveurs. Parallèlement, une réglementation importante et de plus en plus exigeante encadre la production et la transformation des viandes pour répondre aux attentes d’ordre sanitaire, environnemental et éthique. Premier maillon des filières, le secteur de l’élevage est totalement impliqué dans les démarches qualité et dans les programmes de maîtrise de la qualité sanitaire des viandes. Il faut entendre les demandes et les craintes des consommateurs pour pouvoir les interpréter et tenter d’y répondre, mais aussi parfois pouvoir les moduler par l’apport des informations méconnues du public mais nécessaires à une bonne compréhension des pratiques professionnelles.

International audience; The microbial community of a pig slurry was monitored on a farm for 6 months using both molecular and cultural approaches. Sampling was carried out at all the different stages of effluent handling, from the rearing building up to slurry spreading. Total DNA of each sample was extracted and analyzed by PCR-SSCP using primers targeting the 16S ribosomal RNA genes from the archaeal and bacterial domains but also the Eubacterium and Clostridium, the Bacillus-Streptococcus-Lactobacillus and the Bacteroides-Prevotella groups. Comparison of the SSCP profiles showed a rapid modification of the dominant bacterial community during the first 2 weeks of anaerobic storage and its relative stability thereafter. Several bacterial populations, identified as closely related to uncultured Clostridium and Porphyromonas and to Lactobacillus and Streptococcus cultured species commonly isolated from pig feces, remained present and dominant from the rearing building up to the time of spreading. Numeration of fecal indicators (Enterococci and Escherichia coli) performed in parallel using cultural methods revealed the same trends. On the other hand, the archaeal community adapted slowly during pig slurry storage and increased its diversity. A shift was observed between two hydrogenotrophic methanogenic Methanobrevibacter populations from the storage pit up into the pond. Microorganisms present in pig slurry at the time of spreading could no longer be detected in soil thereafter, either by molecular or cultural technique, probably because of the detection limit inherent in both techniques.; La communauté microbienne d'un liser de porc a été suivi à la ferme pendant 6 mois en utilisant des approches moléculaires et culturales. Des prélèvements ont été réalisés à toutes les étapes de la gestion de l'effluent, du bâtiment d'élevage jusqu'a l'épandage. L'ADN total de chaque échantillon a été extrait et analysée par PCR-SSCP en utilisant des amorces ciblant les gènes ribosomaux de l'ARN 16S pour les domaines archaea et bactéries mais également pour les groupes bactériens Eubacterium-Clostridium, Bacillus-Streptococcus-Lactobacillus et pour le groupe Bacteroides-Prevotella. La comparaison des profils SSCP a montré une modification rapide de la communauté bactérienne dominante pendant les 2 premières semaines du stockage anaérobie ensuite cette communauté se stabilise. Plusieurs populations bactériennes, identifiées comme étant proches des Clostridium non cultivés, des Porphyromonas, des Lactobacillus et des Streptococcus couramment isolées des fèces de porcs, ont été misent en évidence dans le lisier et dominent l'écosystème bactérien du bâtiment d'élevage jusqu'a l'épandage. La numération des indicateurs fécaux (les entérocoques et Escherichia coli) réalisée en parallèle avec les techniques de mise culture montre les mêmes tendances. Par contre, le suivi de la communauté archaea montre une lente adaptation pendant la période de stockage du lisier de porc ainsi qu'une augmentation de la diversité. On a observé un décalage entre deux populations methanogenic hydrogenotrophic de Methanobrevibacter entra la fosse de stockage et la lagune. Les micro-organismes présents dans le lisier de porc après épandage ne sont plus détectés dans le sol ensuite, par technique moléculaire ou culturale, probablement en raison de la limite de détection inhérente aux deux techniques.

International audience; Enterohaemorrhagic Escherichia coli (EHEC) constitute a subset of serotypes (E. coli O157 and some other serogroups) of Shiga toxin (Stx)-producing E. coli (STEC) firmly associated with severe human illnesses like bloody diarrhoea and haemolytic uraemic syndrome. Stx production is essential but not sufficient for EHEC virulence. Most strains are capable of colonising the intestinal mucosa of the host with the "attaching and effacing" mechanism, genetically governed by a large pathogenicity island (PAI) defined as the Locus of Enterocyte Effacement. Other virulence factors carried by mobile genetic elements like PAI and plasmids have been recently described, and their role in the pathogenic process has not been fully elucidated. EHEC are zoonotic pathogens. They rarely cause disease in animals, and ruminants are recognised as their main natural reservoir. Cattle are considered to be the most important source of human infections with EHEC O157, and the ecology of the organism in cattle farming has been extensively studied. The organism has also been reported in sheep, goats, water buffalos, and deer. Pigs and poultry are not considered to be a source of EHEC and the sporadic reports may derive from accidental exposure to ruminant dejections. The epidemiology of EHEC infections has remarkably changed during the past ten years and an increasing number of unusual food vehicles have been associated with human infections. New routes of transmission have emerged, like contact with animals during farm visits and a wide variety of environment-related exposures. As for other zoonotic agents, having animals and raw products that are free from EHEC is not possible in practice. However, their occurrence can be minimised by applying high standards of hygiene in all the steps of the food production chain.

Cattle are known to be reservoir of enterohemorrhagic Escherichia coli (EHEC), pathogenic for humans and antibiotic resistant E. coli as well. In a first stage, we assessed frequencies of these strains in two bovine slaughterhouse wastewater treatment plants, one slaughtered only adult cattle and the other only veal calves. Percentages of resistant and class 1 integron-bearing E. coli were significantly higher in veal calves effluents and thickened sludges (87.5%, 56.2%) compared to those of adult cattle (5.0%, 0%). These percentages were not impacted by treatment process. The treatment had no impact on percentages of Shiga toxin-producing E. coli (STEC) either. A STEC O157:H7 highly pathogenic for humans was isolated from the thickened sludge of the adult cattle slaughterhouse, intended to be spread on agricultural lands. These results confirmed that bovine slaughterhouse effluents might contribute to the environmental dissemination of antibiotic resistant and/or pathogenic E. coli and underlined that the risks of dissemination differ according to slaughtered bovine category. In a second stage, we assessed the antibiotic resistance of top 5 STEC (O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7) isolated from adult cattle fecal samples. Seven of the 39 top 5 STEC were resistant, of which 6 resistant to at least 3 classes of tested antibiotics. Non-top 5 STEC and aEPEC E. coli strains from fecal flora of the same bovine carriers were all susceptible to the tested antibiotics, indicating a possible link between EHEC-associated virulence genes and antibiotic resistance genes. We showed that ehxA gene, which is a reliable marker of the EHEC virulence plasmid, and antibiotic-resistance genes blaTEM, strA-strB, tet(A), sulII were located on a same large plasmid in 4 antibiotic-resistant top 5 STEC strains (1 O26:H11, 1 O103:H2 and 2 O111:H8). This genetic association raises the concern about the clonal selection of such pathogenic strains by antibiotic use in bovine carriers., Les bovins sont porteurs de souches d'Escherichia coli entérohémorragiques (EHEC), pathogènes pour l'homme et également de souches d'E. coli résistantes aux antibiotiques. Dans un premier temps, nous avons évalué la fréquence de ces souches dans les effluents de station d'épuration des eaux usées de deux abattoirs, l'un de bovins adultes et l'autre de veaux de boucherie. Les pourcentages d'E. coli antibiorésistantes et porteuses d'intégrons de résistance de classe 1 étaient significativement plus élevés dans les effluents et boues d'abattoirs de veaux (87,5%, 56,2%) par rapport aux bovins adultes (5,0%, 0%). Ces pourcentages n'étaient pas modifiés par le traitement épuratoire. Le traitement épuratoire n'a également eu aucun impact sur les pourcentages de souches d'E. coli productrices de shigatoxines (STEC). Une souche STEC O157:H7 hautement pathogène a été isolée des boues destinées à l'épandage agricole dans la station d'épuration d'abattoir de bovins adultes. Ces résultats ont confirmé qu'il existe des risques de dissémination environnementale d'E. coli antibiorésistantes et/ou pathogènes via les effluents d'abattoir de bovins et ont mis en évidence que ce risque était différent selon la catégorie de bovins abattus. Dans un deuxième temps, nous avons évalué la résistance de souches STEC du top 5 (O26:H11, O103:H2, O111:H8, O145:H28 et O157:H7) isolées de fèces de bovins adultes. Sept des 39 souches STEC testées étaient résistantes, dont 6 à au moins 3 classes d'antibiotiques. Les souches non-STEC et aEPEC du top 5 d'E. coli de la flore fécale de ces mêmes bovins étaient toutes sensibles, indiquant un possible lien génétique entre gènes de résistance et virulence. Nous avons mis en évidence que le gène ehxA, marqueur fiable du plasmide de virulence des EHEC, et les gènes de résistance blaTEM, strA-strB, tet(A), sulII étaient localisés sur un même plasmide de grande taille pour 4 souches STEC (1 O26:H11, 1 O103:H2 et 2 O111:H8). Cette association génétique soulève la problématique de la sélection clonale de ces souches pathogènes lors du traitement des bovins porteurs avec des antibiotiques.