Frédéric Auvray, Emmanuel Jamet, Jordan Madic, Hubert Brugère, Noémie Vingadassalon, Carine Peytavin de Garam, Flemming Scheutz, Muriel Marault, Institut Technique du Lait et des Produits Laitiers, École nationale vétérinaire d'Alfort (ENVA), Statens seruminstitut, Interactions hôtes-agents pathogènes [Toulouse] (IHAP), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Ministere de l'Alimentation, de l'Agriculture et de la Peche, National Interprofessional Center for the Dairy Economy (CNIEL), Paris, France, ACTILAIT [241/2007], Association Nationale de la Recherche Technique (ANRT), and Transversalite AFSSA-INRA grant
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.