Production of cytolethal distending toxin and other virulence characteristics of Escherichia coli strains of serogroup O86

Genetic and phenotypic virulence markers of different categories of diarrhoeagenic Escherichia coli were investigated in 106 strains of enteropathogenic E. coli (EPEC) serogroup O86. The most frequent serotype found was O86:H34 (86%). Strains of this serotype and the non motile ones behaved as EPEC i.e., carried eae, bfpA and EAF DNA sequences and presented localised adherence to HeLa cells. Serotypes O86:H2, O86:H6, O86:H10, O86:H18, O86:H27 and O86:H non determined, belonged to other categories. The majority of the strains of serotype O86:H34 and non motile strains produced cytolethal-distending toxin (CDT). The ribotyping analysis showed a correlation among ribotypes, virulence markers and serotypes, thus suggesting that CDT production might be a property associated with a universal clone represented by the O86:H34 serotype.     

Virulence of Escherichia coli Serotypes for Mice

A study was undertaken to determine whether virulence in mice could be used to assess the pathogenicity of a variety of Escherichia coli serotypes. Sixty-one E. coli strains isolated from animals, poultry, or humans were serotyped to determine their O, K, and H antigens, and were administered to mice via the intraperitoneal route with and without a mucin adjuvant. The ld(50) dose was then determined for each serotype. The results indicated that the source of the serotype may be associated with virulence for mice. Serotypes isolated from nonenteric, systemic sources showed a greater virulence for mice inoculated intraperitoneally than did the enteric and the nonenteric, nonsystemic (localized) isolates. It was observed that not all serotypes belonging to a specific serogroup were virulent for mice and that the presence or absence of a K antigen had no effect on the virulence of strains of one serotype.     

Virulence markers and serotypes of Shiga toxin-producing Escherichia coli, isolated from cattle in Rio Grande do Sul, Brazil

AIMS: To determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) and serotypes and virulence markers of the STEC isolates from beef and dairy cattle in Rio Grande do Sul, Brazil. METHODS AND RESULTS: Faecal samples from beef cattle were collected at slaughterhouses. The isolates were submitted to colony hybridization assay with specific DNA probes for stx1, stx2 and eae genes, and serotyped for the identification of O and H antigens. Thirty-nine per cent of beef cattle surveyed harboured at least one STEC strain. Among the distinct serotypes identified, 10 were shared by both beef and dairy cattle. Most of the strains isolated harboured stx2. Genotypic and phenotypic profiles allowed the identification of 34 and 31 STEC strains, isolated from beef and dairy cattle, respectively. Serotypes O10:H14, O15:H21, O96:H21, O119:H4, O124:H11, O128:H21, O137:H-, O141:H19, O159:H42, O160:H2 and O177:H11, identified in this study, have not been previously reported as STEC isolated from cattle. CONCLUSIONS: Cattle are an important reservoir of STEC strains associated with human diseases in South America. SIGNIFICANCE AND IMPACT OF THE STUDY: Determining the prevalence, genotypic profile and serotypes of STEC strains isolated from cattle enables the prediction of possible risk for public health.     

Genetic characterization of Escherichia coli O104 isolates from different sources in the United States

Escherichia coli O104 isolates collected from different sources in the United States were examined for virulence genes typical of enterohemorrhagic E. coli and those identified in the O104:H4 isolate associated with the 2011 German outbreak. The unexpected presence of virulence markers in these isolates highlights the importance of screening unusual and potentially pathogenic Shiga toxin-producing E. coli serotypes.     

Verotoxin-producing Escherichia coli in Spain: prevalence,serotypes, and virulence genes of O157:H7 and non-O157 VTEC in ruminants,raw beef products,and humans

In Spain, as in many other countries, verotoxin-producing Escherichia coli (VTEC) strains have been frequently isolated from cattle, sheep, and foods. VTEC strains have caused seven outbreaks in Spain (six caused by E. coli O157:H7 and one by E. coli O111:H- [nonmotile]) in recent years. An analysis of the serotypes indicated serological diversity. Among the strains isolated from humans, serotypes O26:H11, O111:H-, and O157:H7 were found to be more prevalent. The most frequently detected serotypes in cattle were O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2, and OUT (O untypeable):H19. Different VTEC serotypes (e.g., O5:H-, O6:H10, O91:H-, O117:H-, O128:H-, O128:H2, O146:H8, O146:H21, O156:H-, and OUT:H21) were found more frequently in sheep. These observations suggest a host serotype specificity for some VTEC. Numerous bovine and ovine VTEC serotypes detected in Spain were associated with human illnesses, confirming that ruminants are important reservoirs of pathogenic VTEC. VTEC can produce one or two toxins (VT1 and VT2) that cause human illnesses. These toxins are different proteins encoded by different genes. Another virulence factor expressed by VTEC is the protein intimin that is responsible for intimate attachment of VTEC and effacing lesions in the intestinal mucosa. This virulence factor is encoded by the chromosomal gene eae. The eae gene was found at a much less frequency in bovine (17%) and ovine (5%) than in human (45%) non-O157 VTEC strains. This may support the evidence that the eae gene contributes significantly to the virulence of human VTEC strains and that many animal non-O157 VTEC strains are less pathogenic to humans.     

Genomic Diversity and Virulence Profiles of Historical Escherichia coli O157 Strains Isolated from Clinical and Environmental Sources

Escherichia coli O157:H7 is, to date, the major E. coli serotype causing food-borne human disease worldwide. Strains of O157 with other H antigens also have been recovered. We analyzed a collection of historic O157 strains (n = 400) isolated in the late 1980s to early 1990s in the United States. Strains were predominantly serotype O157:H7 (55%), and various O157:non-H7 (41%) serotypes were not previously reported regarding their pathogenic potential. Although lacking Shiga toxin (stx) and eae genes, serotypes O157:H1, O157:H2, O157:H11, O157:H42, and O157:H43 carried several virulence factors (iha, terD, and hlyA) also found in virulent serotype E. coli O157:H7. Pulsed-field gel electrophoresis (PFGE) showed the O157 serogroup was diverse, with strains with the same H type clustering together closely. Among non-H7 isolates, serotype O157:H43 was highly prevalent (65%) and carried important enterohemorrhagic E. coli (EHEC) virulence markers (iha, terD, hlyA, and espP). Isolates from two particular H types, H2 and H11, among the most commonly found non-O157 EHEC serotypes (O26:H11, O111:H11, O103:H2/H11, and O45:H2), unexpectedly clustered more closely with O157:H7 than other H types and carried several virulence genes. This suggests an early divergence of the O157 serogroup to clades with different pathogenic potentials. The appearance of important EHEC virulence markers in closely related H types suggests their virulence potential and suggests further monitoring of those serotypes not implicated in severe illness thus far.     

Genetic characteristics and antimicrobial resistance of Escherichia coli from Japanese macaques (Macaca fuscata) in rural Japan

Escherichia coli was isolated from wild and captive Japanese macaques (Macaca fuscata) to investigate the risk of zoonotic infections and the prevalence of antimicrobial-resistant Escherichia coli in the wild macaque population in Shimokita Peninsula, a rural area of Japan. We collected 265 fresh fecal samples from wild macaques and 20 samples from captive macaques in 2005 and 2006 for E. coli isolation. The predominant isolates were characterized by serotyping, virulence gene profiling, plasmid profiling, pulsed-field gel electrophoresis (PFGE), and microbial sensitivity tests. In total, 248 E. coli strains were isolated from 159 fecal samples from wild macaques, and 42 E. coli were isolated from 17 samples from captive macaques. None of the virulence genes eae, stx, elt, and est were detected in any of the isolates. The relatedness between wild- and captive-derived isolates was low by serotyping, PFGE, and plasmid profiling. Serotypes O8:H6, O8:H34, O8:H42, O8:HUT, O103:H27, O103:HNM, and OUT:H27 were found in wild macaque feces; serotypes O157:H42 and O119:H21 were recovered from captive macaques. O-and H-serotypes of the 26 isolates were not typed by commercial typing antisera and were named OUT and HUT, respectively. Twenty-eight isolates had no flagellar antigen, and their H-serotypes were named HNM. Similarity of PFGE patterns between wild-derived isolates and captive-derived isolates was <70%. No plasmid profile was shared between wild-derived and captive-derived isolates. The prevalence of antimicrobial-resistant E. coli was 6.5% (n=62) in wild macaques, and these isolates were resistant to cephalothin. We conclude that wild Japanese macaques in Shimokita Peninsula were unlikely to act as a reservoir of pathogenic E. coli for humans and that antimicrobial-resistant E. coli in wild macaques may be derived from humans.     

Serotypes and antibiotic resistance of Verotoxigenic (VTEC) and Necrotizing (NTEC) Escherichia coli strains isolated from calves with diarrhoea

Serotypes and antibiotic resistance of 51 Verotoxigenic (VTEC) and 33 Necrotizing (NTEC) bovine Escherichia coli strains were determined and compared with those shown by 205 non-VTEC non-NTEC strains isolated from the same batch of calves. E. coli untypable for O-antigen represented 47% of the VTEC, 12% of the NTEC and 8.8% of the non-VTEC non-NTEC. Typable VTEC belonged to serotypes 02:K?, 0103:K-, 0104:K?, 0128:K?, 0153:K- and O157:K-:H7, whereas typable NTEC were of serotypes 08:K87, 015:K14, 015:K-, 054:K?, 076:K-, 078:K(80), 088:K?, 0123:K-, 0139:K- and 0153:K-. Non-VTEC non-NTEC showed a wide variety of serotypes which were generally unrelated to those found in VTEC and NTEC. VTEC were resistant to antibiotics at higher rates than NTEC and non-VTEC non-NTEC, and showed also the highest multidrug-resistant pattern. Our results show that bovine VTEC strains belonged to O-groups usually found in human VTEC causing sporadic diarrhoea, haemorrhagic colitis and/or haemolytic uraemic syndrome, such as 02, 0103, 0104, 0153 and especially 0128 and O157. In contrast, bovine NTEC strains belonged to serotypes different from those previously found in necrotizing E. coli strains of human origin.     

Differences in virulence among Escherichia coli O157:H7 strains isolated from humans during disease outbreaks and from healthy cattle

Escherichia coli O157:H7 causes life-threatening outbreaks of diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans and significant economic loss in agriculture and could be a potential agent of bioterrorism. Although the prevalence of E. coli O157:H7 in cattle and other species with which humans have frequent contact is high, human infections are relatively uncommon, despite a low infectious dose. A plausible explanation for the low disease incidence is the possibility that not all strains are virulent in humans. If there are substantial differences in virulence among strains in nature, then human disease may select for high virulence. We used a gnotobiotic piglet model to investigate the virulence of isolates from healthy cattle and from humans in disease outbreaks and to determine the correlation between production of Shiga toxin 1 (Stx1) and Stx2 and virulence. Overall, E. coli O157:H7 strains isolated from healthy cattle were less virulent in gnotobiotic piglets than strains isolated from humans during disease outbreaks. The amount of Stx2 produced by E. coli O157:H7 strains correlated with strain virulence as measured by a reduction in piglet survival and signs of central nervous system disease due to brain infarction. The amount of Stx1 produced in culture was not correlated with the length of time of piglet survival or with signs of central nervous system disease. We suggest that disease outbreaks select for producers of high levels of Stx2 among E. coli O157:H7 strains shed by animals and further suggest that Stx1 expression is unlikely to be significant in human outbreaks.     

The use of biochemical markers, serotype and fimbriation in the detection of Escherichia coli clones

Biochemical reactions, O and K serotypes and presence of P-fimbriae were analysed in 116 Escherichia coli strains isolated in blood cultures from patients with bacteraemia and in 99 faecal strains isolated from healthy individuals. By using biochemical typing, the strains could be grouped into six main clusters with similarity index less than 0.8 (Gower, 1971) and altogether 16 subclusters with similarity index 0.82-0.89. The most discriminating tests between the clusters were fermentation of D-tagatose, saccharose, salicin and sorbose. No single biochemical property could differentiate bacteraemic isolates from faecal strains, although strains isolated from blood were significantly more often found in certain subclusters, whereas other subclusters contained mainly control strains. Bacteraemic strains possessed P-fimbriae more often, especially strains isolated from patients with E. coli in the urine concomitantly with bacteraemia. Equally, no single reaction could separate P-fimbriated from non-P-fimbriated strains. D-Tagatose was fermented more often by the P-fimbriated strains; on the other hand, melibiose and lactose fermentation tests were less often positive. Certain O serotypes (O1, O4, O6, O7, O18 and O25) were more common among bacteraemic isolates than controls. K serotypes such as K1, K5 and K52 were also more frequent among blood isolates. We conclude that a combination of biochemical tests, fimbriation and serotyping might be used to identify potentially pathogenic clusters of E. coli.     

Design and evaluation of PCR primers which differentiate Escherichia coli O157:H7 and related serotypes

Aims:  To develop methods to differentiate Escherichia coli O157:H7 and related serotypes by the use of amplicon length polymorphism (ALP) analysis based on identifying DNA sequence deletions within highly homologous regions of three sequenced E. coli strains.
Methods and Results:  Potential primer locations along the ancestral genomic backbone were identified and evaluated against three sequenced genomes and then applied to a reference set of pathogenic E. coli strains. All 16 primer combinations generated the expected diagnostic fragments as predicted for the E. coli K12 MG1655, O157:H7 EDL933, and O157:H7B Sakai genomes.
Conclusions:  This study defines a collection of primers distributed along the length of the E. coli genome that were applied to ALP analysis methods to successfully differentiate between serotypes of E. coli O157:H7 and other E. coli serotypes.
Significance and Impact of the Study:  ALP-PCR analysis method was validated as an independent method of classification when compared with that of rep-PCR. The principles underlying ALP analysis can be readily applied for the detection and differentiation of other closely related microbial species because of the abundance of complete DNA sequence data for a large number of microbial genomes.     

Comparative Genomic Analysis Shows That Avian Pathogenic Escherichia coli Isolate IMT5155 (O2:K1:H5; ST Complex 95, ST140) Shares Close Relationship with ST95 APEC O1:K1 and Human ExPEC O18:K1 Strains

Avian pathogenic E. coli and human extraintestinal pathogenic E. coli serotypes O1, O2 and O18 strains isolated from different hosts are generally located in phylogroup B2 and ST complex 95, and they share similar genetic characteristics and pathogenicity, with no or minimal host specificity. They are popular objects for the study of ExPEC genetic characteristics and pathogenesis in recent years. Here, we investigated the evolution and genetic blueprint of APEC pathotype by performing phylogenetic and comparative genome analysis of avian pathogenic E. coli strain IMT5155 (O2:K1:H5; ST complex 95, ST140) with other E. coli pathotypes. Phylogeny analyses indicated that IMT5155 has closest evolutionary relationship with APEC O1, IHE3034, and UTI89. Comparative genomic analysis showed that IMT5155 and APEC O1 shared significant genetic overlap/similarities with human ExPEC dominant O18:K1 strains (IHE3034 and UTI89). Furthermore, the unique PAI I₅₁₅₅ (GI-12) was identified and found to be conserved in APEC O2 serotype isolates. GI-7 and GI-16 encoding two typical T6SSs in IMT5155 might be useful markers for the identification of ExPEC dominant serotypes (O1, O2, and O18) strains. IMT5155 contained a ColV plasmid p1ColV₅₁₅₅, which defined the APEC pathotype. The distribution analysis of 10 sequenced ExPEC pan-genome virulence factors among 47 sequenced E. coli strains provided meaningful information for B2 APEC/ExPEC-specific virulence factors, including several adhesins, invasins, toxins, iron acquisition systems, and so on. The pathogenicity tests of IMT5155 and other APEC O1:K1 and O2:K1 serotypes strains (isolated in China) through four animal models showed that they were highly virulent for avian colisepticemia and able to cause septicemia and meningitis in neonatal rats, suggesting zoonotic potential of these APEC O1:K1 and O2:K1 isolates.     

Virulence properties and antimicrobial susceptibility of Shiga toxin-producing Escherichia coli strains isolated from healthy cattle from Paraná State, Brazil

The presence of Shiga toxin-producing Escherichia coli (STEC) strains in feces samples of cattle was determined using the cytotoxicity assay on Vero cells and a screening PCR system to detect stx genes. The STEC isolates were serotyped, tested for antimicrobial susceptibility, and analyzed for virulence genes using multiplex PCR. The verocytotoxin-producing E. coli - reverse passive latex agglutination (VTEC-RPLA) assay was also used to detect Shiga toxin production. The frequency of cattle shedding STEC was 36%. The isolates belonged to 33 different serotypes, of which O10:H42, O98:H41, and O159:H21 had not previously been associated with STEC. The most frequent serotypes were ONT:H7 (10%), O22:H8 (7%), O22:H16 (7%), and ONT:H21 (7%). Most of the strains (96%) were susceptible to all antimicrobial agents tested. Shiga toxin was detected by the VTEC-RPLA assay in most (89%) of the STEC strains. The frequency of virulence markers was as follows: stx1, 10%; stx2, 43%; stx1 plus stx2, 47%; ehxA, 44%; eae, 1%; and saa, 38%. Several strains belong to serotypes associated with human disease, and most of them carried a stx2-type gene, suggesting that they represent a risk to human health. The screening PCR assay showed fewer false-negative results for STEC than the Vero-cell assay and is suitable for laboratory routine.     

Studies on Coli-aerogenes and Other Gram-negative Intestinal Bacteria in Various Animals and Birds

S ummary . Some faecal material from native animals and birds (especially) showed either no coli-aerogenes bacteria or contained types other than Escherichia coli I.
Amongst coli-aerogenes isolates, E. coli I was frequent but the high percentage of other biotypes indicated that the animal host may serve as a pool of intermediate and irregular strains. Paracolons and Proteus spp. were abundant, and Salmonella spp. were isolated from dogs and native animals possibly in contact with man.
Animals were only occasionally carriers of enteropathogenic E. coli serotypes and seldom of types locally important in the etiology of infantile diarrhoea, such as E. coli O26:B6 and O55:B5. In this series O86 serotypes other than the diarrhoeal strain isolated from babies were found. The results of the study indicated some degree of host specificity. In the group of 20 O serotypes (referred to as specific E. coli ), O8 was most commonly listed. Most strains were sensitive to chemotherapeutic agents but resistance of some strains especially to sulphonamide was recorded.
The implication of the presence in the animal gut of coli-aerogenes bacteria other than E. coli type I is discussed in relation to bacterial standards for drinking water.     

Escherichia coli Serogroups Isolated from Streams in Pennsylvania, 1965 to 1972

Of 3,200 cultures of Escherichia coli isolated from streams in Pennsylvania over a 7-year period, 82.46% or 2,639 were O serogrouped. The largest number of cultures (33.4%) belonged to O groups 1 to 26, and the second highest number (16.8%) belonged to O groups 60 to 88. The individual E. coli O groups most frequently isolated were ADO3, 18ac, 2a, 3, 7, 73, 139, and OX13. Practically every known standard E. coli O group was found in the streams. It was not possible to identify the K and H antigen of every E. coli isolate. Serotypes of E. coli O2a:K1:H6, O26:K60:H11, O55:K59:H27, O86:K62:H2, 112ab:K68:H2, 125ab:K70:H21, 128ab:K67:H2, and O138:K81:H14 known to be pathogenic for humans and animals were identified. Cultures having the same K antigen but a different H antigen for enteropathogenic E. coli O groups 6, 18ab, 18ac, 111ab, 126, 127a, 139, 141, and 147 were also isolated.     

合肥地区动物源性大肠埃希菌的血清型分布与耐药性分析

目的了解安徽省合肥地区动物源性大肠埃希菌的血清型分布和耐药状况,以期筛选出菌苗株和指导临床合理用药。方法对46份疑似大肠埃希菌病病料进行细菌分离培养、生化编码鉴定和致病性测定。采用玻片凝集试验对分离到的46株致病性大肠埃希菌进行血清型鉴定。同时分别采用K-B纸片琼脂扩散法和双纸片增效法检测致病性大肠埃希菌的耐药性和ESBLs阳性菌株。结果46株致病性大肠埃希菌中,除7株细菌未能定型外,其余39株细菌分布于10个血清型,O127：K63血清型为优势血清型,占定型菌株的33．33％。46株致病性大肠埃希菌对21种抗菌药物均呈现不同程度的耐药性,15个ESBLs阳性菌株表现为多重耐药,对各种抗菌药物的耐药率均高于ESBLs阴性菌株。结论O127：K63血清型为优势血清型,可作为菌苗株。合肥地区动物源性大肠埃希菌耐药性较为严重,尤其是产ESBLs大肠埃希菌多重耐药更为突出。     

Diarrheagenic Escherichia coli categories among the traditional enteropathogenic E. coli O serogroups--a review

The so called enteropathogenic Escherichia coli (EPEC) O serogroups include typical and atypical EPEC, enterohaemorrragic E. coli, enterotoxigenic E. coli, and enteroaggregative E. coli. The aim of this article is to review the composition of each O serogroup and the major serotypes, clones, and additional virulence characteristics of each of these diarrheagenic categories. Their adherence patterns and genetic relationships are also presented. The review is based on the study of 805 strains of serogroups O26, O55, O86, O111, O114, O119, O125, O126, O1127, O128, and O142 most of which isolated in Sao Paulo from children with diarrhea between 1970 and 1990. Since some O serogroups include more than one diarrheagenic category O serogrouping only should be abandoned as a diagnostic method. However serotyping is a reliable method for those serotypes that correspond to clones.     

Genotypic and phenotypic characterization of enterotoxigenic Escherichia coli (ETEC) strains isolated in Rio de Janeiro city, Brazil

Enterotoxigenic Escherichia coli (ETEC) strains have been implicated as important etiological agents of diarrheal disease, especially in developing countries. This group of microorganisms has been associated with a diverse range of genotypic and phenotypic markers. In the present study, 21 ETEC isolates previously defined according to the toxigenic genotypes, were characterized on the basis of O:H typing, cell adherence patterns, and colonization factors (CFs) antigens. Genetic diversity was investigated by random amplification polymorphic DNA (RAPD-PCR), pulsed-field gel electrophoresis (PFGE) and multilocus enzyme electrophoresis (MLEE). LT-I probe-positive isolates belonged to serotypes ONT:HNT, O7:H24, O48:H21, O88:H25, O148:H28, O159:H17 and O159:H21. ST-h probe-positive isolates belonged to serotypes O159:H17, O148:H28 and O6:H-. Serotypes O148:H28, O159:H17 and O6:H- were associated with the CS6, CFA/I and CS1 CS3 antigens, respectively. Most ETEC strains exhibited a diffuse pattern of adherence to cultured epithelial cells. In general, phenotypic and genotypic characteristics correlated well. RAPD-PCR, PFGE and MLEE showed reproducibility and good discriminatory potential. The application of molecular typing systems allowed the detection of significant diversity among the isolates, indicating a non-clonal origin and revealing intra-serotype variation overlooked by classical epidemiological approaches. The phenotypic and genotypic diversity observed lead us to recommend the use of different typing systems in order to elucidate the epidemiology of ETEC infection.     

Molecular evolution and mosaic structure of alpha, beta, and gamma intimins of pathogenic Escherichia coli.

Two types of pathogenic Escherichia coli, enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), cause diarrheal disease by disrupting the intestinal environment through the intimate attachment of the bacteria to the intestinal epithelium. This process is mediated by intimin, an outer membrane protein that is homologous to the invasins of pathogenic Yersinia. The intimin (eae) gene is part of a pathogenicity island, a 35-kb segment of DNA that has been acquired independently in different groups of pathogens. Nucleotide sequences of eae of three EPEC and four EHEC strains representing distinct clonal lineages revealed an exceptionally high level of divergence (15%) in the amino acid sequences of alpha, beta, and gamma intimin molecules, most of which is concentrated in the C-terminal region. The gamma intimin sequences from E. coli strains with serotypes O157:H7, O55:H7, and O157:H- are virtually identical, supporting the hypothesis that these bacteria belong to a single clonal lineage. Sequences of beta intimin of EPEC strains of serotypes O111:H2 and O128:H2 show substantial differences from alpha and gamma intimins, indicating that these strains have evolved independently. Strong nonrandom clustering of polymorphic sites indicates that the intimin genes are mosaics, suggesting that protein divergence has been accelerated by recombination and diversifying selection.     

Detection of Shiga toxin-producing Escherichia coli serotypes O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7 in raw-milk cheeses by using multiplex real-time PCR

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are a diverse group of food-borne pathogens with various levels of virulence for humans. In this study, we describe the use of a combination of multiple real-time PCR assays for the screening of 400 raw-milk cheeses for the five main pathogenic STEC serotypes (O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7). The prevalences of samples positive for stx, intimin-encoding gene (eae), and at least one of the five O group genetic markers were 29.8%, 37.3%, and 55.3%, respectively. The H2, H7, H8, H11, and H28 fliC alleles were highly prevalent and could not be used as reliable targets for screening. Combinations of stx, eae variants, and O genetic markers, which are typical of the five targeted STEC serotypes, were detected by real-time PCR in 6.5% of the cheeses (26 samples) and included stx-wzx(O26)-eae-β1 (4.8%; 19 samples), stx-wzx(O103)-eae-ε (1.3%; five samples), stx-ihp1(O145)-eae-γ1 (0.8%; three samples), and stx-rfbE(O157)-eae-γ1 (0.3%; one sample). Twenty-eight immunomagnetic separation (IMS) assays performed on samples positive for these combinations allowed the recovery of seven eaeβ1-positive STEC O26:H11 isolates, whereas no STEC O103:H2, O145:H28, or O157:H7 strains could be isolated. Three stx-negative and eaeβ1-positive E. coli O26:[H11] strains were also isolated from cheeses by IMS. Colony hybridization allowed us to recover STEC from stx-positive samples for 15 out of 45 assays performed, highlighting the difficulties encountered in STEC isolation from dairy products. The STEC O26:H11 isolates shared the same virulence genetic profile as enterohemorrhagic E. coli (EHEC) O26:H11, i.e., they carried the virulence-associated genes EHEC-hlyA, katP, and espP, as well as genomic O islands 71 and 122. Except for one strain, they all contained the stx1 variant only, which was reported to be less frequently associated with human cases than stx2. Pulsed-field gel electrophoresis (PFGE) analysis showed that they displayed high genetic diversity; none of them had patterns identical to those of human O26:H11 strains investigated here.