Impact of microbial diversity on rapid detection of enterohemorrhagic Escherichia coli in surface waters

Enterohemorrhagic Escherichia coli (EHEC) are a physiologically, immunologically and genetically diverse collection of strains that pose a serious water-borne threat to human health. Consequently, immunological and PCR assays have been developed for the rapid, sensitive detection of presumptive EHEC. However, the ability of these assays to consistently detect presumptive EHEC while excluding closely related non-EHEC strains has not been documented. We conducted a 30-month monitoring study of a major metropolitan watershed. Surface water samples were analyzed using an immunological assay for E. coli O157 (the predominant strain worldwide) and a multiplex PCR assay for the virulence genes stx(1), stx(2) and eae. The mean frequency of water samples positive for the presence of E. coli O157, stx(1) or stx(2) genes, or the eae gene was 50%, 26% and 96%, respectively. Quantitative analysis of selected enriched water samples indicated that even in samples positive for E. coli O157 cells, stx(1)/stx(2) genes, and the eae gene, the concentrations were rarely comparable. Seventeen E. coli O157 strains were isolated, however, none were EHEC. These data indicate the presence of multiple strains similar to EHEC but less pathogenic. These findings have important ramifications for the rapid detection of presumptive EHEC; namely, that current immunological or PCR assays cannot reliably identify water-borne EHEC strains.     

Presence of virulence and fitness gene modules of enterohemorrhagic Escherichia coli in atypical enteropathogenic Escherichia coli O26

Enterohemorrhagic Escherichia coli (EHEC) strains of serogroup O26 cause hemolytic-uremic syndrome (HUS) whereas atypical enteropathogenic E. coli (aEPEC) O26 typically cause uncomplicated diarrhea but have been also isolated from HUS patients. To gain insight into the virulence of aEPEC O26, we compared the presence of O island (OI) 122, which is associated with enhanced virulence in EHEC strains, among aEPEC O26 and EHEC O26 clinical isolates. We also tested these strains for the high pathogenicity island (HPI) which is a fitness island. All 20 aEPEC O26 and 20 EHEC O26 investigated contained virulence genes located within OI-122 (efa1/lifA, nleB, nleE, ent). In both aEPEC O26 and EHEC O26, OI-122 was linked to the locus for enterocyte effacement, forming a mosaic island which was integrated in pheU. Moreover, strains of these two pathotypes shared a conserved HPI. These data support a close relatedness between aEPEC O26 and EHEC O26 and have evolutionary implications. The presence of OI-122 in aEPEC O26 might contribute to their pathogenic potential.     

Identification of genetic markers for differentiation of Shiga toxin-producing, enteropathogenic, and avirulent strains of Escherichia coli O26

Shiga toxin-producing Escherichia coli (STEC) O26 is one of the top five enterohemorrhagic E. coli (EHEC) O groups most often associated with hemorrhagic colitis and hemolytic uremic syndrome (HUS) worldwide. STEC O26 is considered to have evolved from enteropathogenic (EPEC) O26 strains through the acquisition of Shiga toxin (Stx)-encoding genes. Our PCR data identified several STEC-like strains expressing all features of STEC except Stx production and carrying remnants of Stx phages that were probably derivatives of EHEC O26. EHEC and EPEC O26 strains phenotypically resemble O26 EHEC-like and apathogenic E. coli O26 strains and are therefore undistinguishable by cultural methods. A clear discrimination between the different O26 groups is required for diagnostics in patients and for control of food safety. To develop an assay for specific detection of EHEC and EHEC-like O26 strains, we used a high-throughput PCR approach for selection of discriminative genetic markers among 33 tested genes mostly encoding type III secretion system effector proteins. The genes ECs1822, nleH1-2, nleA, nleC, nleH1-1, nleG, nleG2, nleG6-1, nleG6-2, espJ, espM2, nleG8-2, espG, ent (or espL2), nleB, nleE, efa1, and espB were detected at different frequencies in O26 EHEC, EHEC-like, and EPEC strains, indicating the possible role of these genes in virulence of human pathogenic O26 strains. The espK and espN genes were detected only in EHEC and EHEC-like O26 strains. espK was present in 99.14% of EHEC and 91.14% of EHEC-like O26 strains and was hence the best candidate as a genetic marker for characterizing these pathogroups. These data were corroborated by a genotyping real-time PCR test based on allelic discrimination of the arcA (aerobic respiratory control protein A) gene. The results indicate that a combination of molecular detection tools for O26 wzx (wzx(O26)), eae-beta, stx, espK, and arcA genotyping is highly discriminative for clear identification of EHEC and EHEC-like E. coli O26 strains. This simple diagnostic test might be applicable in hospital service laboratories or public health laboratories to test strains isolated from stools of patients suffering from diarrhea.     

Enterohemorrhagic Escherichia coli Hemolysin Employs Outer Membrane Vesicles to Target Mitochondria and Cause Endothelial and Epithelial Apoptosis

Enterohemorrhagic Escherichia coli (EHEC) strains cause diarrhea and hemolytic uremic syndrome resulting from toxin-mediated microvascular endothelial injury. EHEC hemolysin (EHEC-Hly), a member of the RTX (repeats-in-toxin) family, is an EHEC virulence factor of increasingly recognized importance. The toxin exists as free EHEC-Hly and as EHEC-Hly associated with outer membrane vesicles (OMVs) released by EHEC during growth. Whereas the free toxin is lytic towards human endothelium, the biological effects of the OMV-associated EHEC-Hly on microvascular endothelial and intestinal epithelial cells, which are the major targets during EHEC infection, are unknown. Using microscopic, biochemical, flow cytometry and functional analyses of human brain microvascular endothelial cells (HBMEC) and Caco-2 cells we demonstrate that OMV-associated EHEC-Hly does not lyse the target cells but triggers their apoptosis. The OMV-associated toxin is internalized by HBMEC and Caco-2 cells via dynamin-dependent endocytosis of OMVs and trafficked with OMVs into endo-lysosomal compartments. Upon endosome acidification and subsequent pH drop, EHEC-Hly is separated from OMVs, escapes from the lysosomes, most probably via its pore-forming activity, and targets mitochondria. This results in decrease of the mitochondrial transmembrane potential and translocation of cytochrome c to the cytosol, indicating EHEC-Hly-mediated permeabilization of the mitochondrial membranes. Subsequent activation of caspase-9 and caspase-3 leads to apoptotic cell death as evidenced by DNA fragmentation and chromatin condensation in the intoxicated cells. The ability of OMV-associated EHEC-Hly to trigger the mitochondrial apoptotic pathway in human microvascular endothelial and intestinal epithelial cells indicates a novel mechanism of EHEC-Hly involvement in the pathogenesis of EHEC diseases. The OMV-mediated intracellular delivery represents a newly recognized mechanism for a bacterial toxin to enter host cells in order to target mitochondria.     

Fenotypic and genotypic characterization of Shiga toxin-producing Escherichia coli O111 strain isolated from patient with hemolytic-uremic syndrome

Shiga toxin-producing E. coli O157 and non-O157 are important emergance pathogens that can cause diarrhea and hemorrhagic colitis with life-threatening complications, such as hemolytic uremic syndrome (HUS). A few cases of EHEC infections are documented per year in Poland. Among them only one patient with EHEC O157 infection developed HUS. We characterized the first VTEC non-O157 strain isolated from child with HUS in Poland. The VTEC O111 strain produced Stx2 which was cytotoxic for Vero cell. Using DNA microarray analysis we have found set of virulence genes in VTEC O111 strain as: stx2A, stx2B, ehly, eae, tir tccP espA, espJ, cif nleA, nleB, lpfA, iha, efa1, cba. The strain was fenotypic resistant to streptomycin, tetracyclin and sulphonamides (strA, tetA, sul2 genes were detected).     

Evaluation of the 'GeneDisc' real-time PCR system for detection of enterohaemorrhagic Escherichia coli (EHEC) O26, O103, O111, O145 and O157 strains according to their virulence markers and their O- and H-antigen-associated genes

Aims:  To evaluate the GeneDisc multiplex real-time PCR assay for detection of enterohaemorrhagic Escherichia coli (EHEC) O26, O103, O111, O145 and O157 strains.
Methods and Results:  GeneDiscs for detection of genes encoding Shiga toxins ( stx ), intimins ( eae ), E. coli O157 ( rfbE _O157) and H7 ( fliC _H7) antigens as well as genes specific for EHEC O26 ( wzx _O26), O103 ( wzx _O103), O111 ( wbd1 _O111), O145 ( ihp1 _O145) and O157 ( ihp1 _O157) were evaluated. The assay was run with native bacteria in 1 h in a GeneDisc Cycler. All genotypes of stx and eae , except stx _2f and eae -rho, were identified. Escherichia coli strains belonging to O-groups O26, O103, O111, O157 as well as EHEC O145:[H28] strains were specifically detected with this assay. The ihp1 _O157 gene was not found specific for EHEC O157. O-rough mutants of EHEC and non-motile EHEC O157 strains were reliably identified with the GeneDisc assay. Two to three colonies of EHEC strains were still detectable in a lawn of 50 000 apathogenic E. coli from agar plates.
Conclusions:  The GeneDisc assay is a specific and reliable assay for detection of major EHEC strains. It is robust enough to detect few EHEC colonies in mixed cultures of bacteria.
Significance and Impact of the Study:  The assay is promising for its use in EHEC diagnostics and for EHEC monitoring with different kinds of samples.     

Identification of human-pathogenic strains of Shiga toxin-producing Escherichia coli from food by a combination of serotyping and molecular typing of Shiga toxin genes

We examined 219 Shiga toxin-producing Escherichia coli (STEC) strains from meat, milk, and cheese samples collected in Germany between 2005 and 2006. All strains were investigated for their serotypes and for genetic variants of Shiga toxins 1 and 2 (Stx1 and Stx2). stx(1) or variant genes were detected in 88 (40.2%) strains and stx(2) and variants in 177 (80.8%) strains. Typing of stx genes was performed by stx-specific PCRs and by analysis of restriction fragment length polymorphisms (RFLP) of PCR products. Major genotypes of the Stx1 (stx(1), stx(1c), and stx(1d)) and the Stx2 (stx(2), stx(2d), stx(2-O118), stx(2e), and stx(2g)) families were detected, and multiple types of stx genes coexisted frequently in STEC strains. Only 1.8% of the STEC strains from food belonged to the classical enterohemorrhagic E. coli (EHEC) types O26:H11, O103:H2, and O157:H7, and only 5.0% of the STEC strains from food were positive for the eae gene, which is a virulence trait of classical EHEC. In contrast, 95 (43.4%) of the food-borne STEC strains carried stx(2) and/or mucus-activatable stx(2d) genes, an indicator for potential high virulence of STEC for humans. Most of these strains belonged to serotypes associated with severe illness in humans, such as O22:H8, O91:H21, O113:H21, O174:H2, and O174:H21. stx(2) and stx(2d) STEC strains were found frequently in milk and beef products. Other stx types were associated more frequently with pork (stx(2e)), lamb, and wildlife meat (stx(1c)). The combination of serotyping and stx genotyping was found useful for identification and for assignment of food-borne STEC to groups with potential lower and higher levels of virulence for humans.     

Enterohaemorrhagic Escherichia coli haemolysin is cleaved and inactivated by serine protease EspPα

The haemolysin from enterohaemorrhagic Escherichia coli (EHEC-Hly) and the serine protease EspPα are putative virulence factors of EHEC. We investigated the interplay between these secreted factors and demonstrate that EspPα cleaves the 107 kDa large EHEC-Hly. Degradation was observed when purified EspPα was added to a growing culture of an EHEC-Hly-expressing strain, with isolated proteins and with coexpressing strains, and was independent of the EHEC serotype. EHEC-Hly breakdown occurred as a multistage process with the formation of characteristic fragments with relative molecular masses of ~82 kDa and/or ~84 kDa and ~34 kDa. The initial cleavage occurred in the N-terminal hydrophobic domain of EHEC-Hly between Leu(235) and Ser(236) and abolished its haemolytic activity. In a cellular infection system, the cytolytic potential of EHEC-Hly-secreting recombinant strains was abolished when EspPα was coexpressed. EHEC in contact with human intestinal epithelial cells simultaneously upregulated their EHEC-Hly and EspP indicating that both molecules might interact under physiological conditions. We propose the concept of bacterial effector molecule interference (BEMI), reflecting the concerted interplay of virulence factors. Interference between effector molecules might be an additional way to regulate virulence functions and increases the complexity of monomolecular phenotypes.     

Prevalence of Escherichia coli strains in the canteens

The prevalence of enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) E. coli strains in stool specimens from asymptomatic human carriers working in the canteens and also in the kitchen and sanitary facilities was evaluated. The E. coli genes coding for the following virulence markers: intimin (eae), enterohaemolysin (hlyA), and verotoxins type I and II (stx1 and stx2) were sought by multiplex PCR assay. E. coli isolates were obtained from 144 stool specimens, 295 swabs taken from kitchen hardware and surrounding facilities, and from 33 meat specimens. Only 66 (8.5%) of total 777 E. coli isolates belonged to O44, O18, O25, O127, O55, O114, O125, and O142 serogroups, the prevalent serogroups in Poland. None of the strains was classified as serogroup O157. The serogroups O44 and O18 were present most often among all typeable strains and their incidence was 51.5% and 25.8% respectively. Among 363 isolates assayed for the presence of the genes encoding virulence markers only 10 isolates (2.8%) carried eae gene. None of the isolates possessing eae gene belonged to the serogroups tested. The hlyA, stx1 and stx2 genes were absent in all E. coli isolates tested.     

Shiga toxin gene loss and transfer in vitro and in vivo during enterohemorrhagic Escherichia coli O26 infection in humans

Escherichia coli serogroup O26 consists of enterohemorrhagic E. coli (EHEC) and atypical enteropathogenic E. coli (aEPEC). The former produces Shiga toxins (Stx), major determinants of EHEC pathogenicity, encoded by bacteriophages; the latter is Stx negative. We have isolated EHEC O26 from patient stools early in illness and aEPEC O26 from stools later in illness, and vice versa. Intrapatient EHEC and aEPEC isolates had quite similar pulsed-field gel electrophoresis (PFGE) patterns, suggesting that they might have arisen by conversion between the EHEC and aEPEC pathotypes during infection. To test this hypothesis, we asked whether EHEC O26 can lose stx genes and whether aEPEC O26 can be lysogenized with Stx-encoding phages from EHEC O26 in vitro. The stx2 loss associated with the loss of Stx2-encoding phages occurred in 10% to 14% of colonies tested. Conversely, Stx2- and, to a lesser extent, Stx1-encoding bacteriophages from EHEC O26 lysogenized aEPEC O26 isolates, converting them to EHEC strains. In the lysogens and EHEC O26 donors, Stx2-converting bacteriophages integrated in yecE or wrbA. The loss and gain of Stx-converting bacteriophages diversifies PFGE patterns; this parallels findings of similar but not identical PFGE patterns in the intrapatient EHEC and aEPEC O26 isolates. EHEC O26 and aEPEC O26 thus exist as a dynamic system whose members undergo ephemeral interconversions via loss and gain of Stx-encoding phages to yield different pathotypes. The suggested occurrence of this process in the human intestine has diagnostic, clinical, epidemiological, and evolutionary implications.     

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.     

Norwegian sheep are an important reservoir for human-pathogenic Escherichia coli O26:H11

A previous national survey of Escherichia coli in Norwegian sheep detected eae-positive (eae(+)) E. coli O26:H11 isolates in 16.3% (80/491) of the flocks. The purpose of the present study was to evaluate the human-pathogenic potential of these ovine isolates by comparing them with E. coli O26 isolates from humans infected in Norway. All human E. coli O26 isolates studied carried the eae gene and shared flagellar type H11. Two-thirds of the sheep flocks and 95.1% of the patients harbored isolates containing arcA allele type 2 and espK and were classified as enterohemorrhagic E. coli (EHEC) (stx positive) or EHEC-like (stx negative). These isolates were further divided into group A (EspK2 positive), associated with stx(2-EDL933) and stcE(O103), and group B (EspK1 positive), associated with stx(1a). Although the stx genes were more frequently present in isolates from patients (46.3%) than in those from sheep flocks (5%), more than half of the ovine isolates in the EHEC/EHEC-like group had multiple-locus variable number of tandem repeat analysis (MLVA) profiles that were identical to those seen in stx-positive human O26:H11 isolates. This indicates that EHEC-like ovine isolates may be able to acquire stx-carrying bacteriophages and thereby have the possibility to cause serious illness in humans. The remaining one-third of the sheep flocks and two of the patients had isolates fulfilling the criteria for atypical enteropathogenic E. coli (aEPEC): arcA allele type 1 and espK negative (group C). The majority of these ovine isolates showed MLVA profiles not previously seen in E. coli O26:H11 isolates from humans. However, according to their virulence gene profile, the aEPEC ovine isolates should be considered potentially pathogenic for humans. In conclusion, sheep are an important reservoir of human-pathogenic E. coli O26:H11 isolates in Norway.     

Prospective genomic characterization of the German enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing technology

An ongoing outbreak of exceptionally virulent Shiga toxin (Stx)-producing Escherichia coli O104:H4 centered in Germany, has caused over 830 cases of hemolytic uremic syndrome (HUS) and 46 deaths since May 2011. Serotype O104:H4, which has not been detected in animals, has rarely been associated with HUS in the past. To prospectively elucidate the unique characteristics of this strain in the early stages of this outbreak, we applied whole genome sequencing on the Life Technologies Ion Torrent PGM? sequencer and Optical Mapping to characterize one outbreak isolate (LB226692) and a historic O104:H4 HUS isolate from 2001 (01-09591). Reference guided draft assemblies of both strains were completed with the newly introduced PGM? within 62 hours. The HUS-associated strains both carried genes typically found in two types of pathogenic E. coli, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). Phylogenetic analyses of 1,144 core E. coli genes indicate that the HUS-causing O104:H4 strains and the previously published sequence of the EAEC strain 55989 show a close relationship but are only distantly related to common EHEC serotypes. Though closely related, the outbreak strain differs from the 2001 strain in plasmid content and fimbrial genes. We propose a model in which EAEC 55989 and EHEC O104:H4 strains evolved from a common EHEC O104:H4 progenitor, and suggest that by stepwise gain and loss of chromosomal and plasmid-encoded virulence factors, a highly pathogenic hybrid of EAEC and EHEC emerged as the current outbreak clone. In conclusion, rapid next-generation technologies facilitated prospective whole genome characterization in the early stages of an outbreak.     

Genetic relatedness of a non-motile variant O157 enteropathogenic Escherichia coli (EPEC) strain and E. coli strains belonging to pathogenic related groups

The study was undertaken to determine the clonal relationship and the genetic diversity among Escherichia coli isolates by comparing a non-motile O157 variant with three O157:H7 EHEC isolates and one O55:H7 enteropathogenic E. coli (EPEC) strain. E. coli strains were characterized by sorbitol phenotype, multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, random amplification polymorphic DNA, and the presence of specific virulence genes (stx, E-hly and LEE genes). Sorbitol fermentation was observed in O157:H- (strain 116I), O55:H7 and O157:H7 (strain GC148) serotypes. stx1 or stx2 and E-hly genes were only detected among O157:H7 isolates. LEE typing revealed specific allele distribution: eaegamma, tirgamma, espAgamma, espBgamma associated with EPEC O55:H7 and EHEC O157:H7 strains (B1/1 and EDL 933), eaealpha, tiralpha, espAalpha, espBalpha related to the 116I O157:H- strain and the GC148 strain presented non-typable LEE sequences. Multilocus enzyme profiles revealed two main clusters associated with specific LEE pathotypes. E. coli strains were discriminated by random amplification of polymorphic DNA-polymerase chain reaction and pulsed-field gel electrophoresis methodologies. The molecular approaches used in this study allowed the determination of the genetic relatedness among E. coli strains as well as the detection of lineage specific group markers.     

Rapid categorization of pathogenic Escherichia coli by multiplex PCR

A one-shot multiplex polymerase chain reaction (PCR) was developed for detecting 12 virulence genes of diarrheagenic Escherichia coli. In order to differentiate between the five categories of diarrheagenic E. coli, we selected the target genes: stx1, stx2, and eaeA for enterohemorrhagic E. coli(EHEC); eaeA, bfpA, and EAF for enteropathogenic E. coli(EPEC); invE for enteroinvasive E. coli(EIEC); elt, estp, and esth for enterotoxigenic E. coli(ETEC); CVD432 and aggR for enteroaggregative E. coli(EAggEC); and astA distributed over the categories of diarrheagenic E. coli. In our multiplex PCR system, all 12 targeted genes (stx1, stx2, eaeA, invE, elt, estp, astA, esth, bfpA, aggR, EAF, and CVD432) were amplified in a single PCR reaction in one tube and detected by electrophoresis. Using our multiplex PCR, the 208 clinically isolated strains of diarrheagenic E. coli in our laboratory were successfully categorized and easily analyzed for the presence of virulence plasmids.     

Prevalence and characteristics of shiga toxin-producing Escherichia coli from healthy cattle in Japan

The prevalence of Shiga toxin-producing Escherichia coli (STEC) in Japan was examined by using stool samples from 87 calves, 88 heifers, and 183 cows on 78 farms. As determined by screening with stx-PCR, the prevalence was 46% in calves, 66% in heifers, and 69% in cows; as determined by nested stx-PCR, the prevalence was 100% in all animal groups. Of the 962 isolates picked by colony stx hybridization, 92 isolates from 54 farms were characterized to determine their O serogroups, virulence factor genes, and antimicrobial resistance. Of these 92 isolates, 74 (80%) could be classified into O serogroups; 50% of these 74 isolates belonged to O serogroups O8, O26, O84, O113, and O116 and 1 isolate belonged to O serogroup O157. Locus of enterocyte effacement genes were detected in 24% of the isolates, and enterohemorrhagic E. coli (EHEC) hlyA genes were detected in 72% of the isolates. Neither the bundle-forming pilus gene nor the enteropathogenic E. coli adherence factor plasmid was found. STEC strains with characteristics typical of isolates from human EHEC infections, which were regarded as potential EHEC strains, were present on 11.5% of the farms.     

The highly virulent 2006 Norwegian EHEC O103:H25 outbreak strain is related to the 2011 German O104:H4 outbreak strain

In 2006, a severe foodborne EHEC outbreak occured in Norway. Seventeen cases were recorded and the HUS frequency was 60%. The causative strain, Esherichia coli O103:H25, is considered to be particularly virulent. Sequencing of the outbreak strain revealed resemblance to the 2011 German outbreak strain E. coli O104:H4, both in genome and Shiga toxin 2-encoding (Stx2) phage sequence. The nucleotide identity between the Stx2 phages from the Norwegian and German outbreak strains was 90%. During the 2006 outbreak, stx(2)-positive O103:H25 E. coli was isolated from two patients. All the other outbreak associated isolates, including all food isolates, were stx-negative, and carried a different phage replacing the Stx2 phage. This phage was of similar size to the Stx2 phage, but had a distinctive early phage region and no stx gene. The sequence of the early region of this phage was not retrieved from the bacterial host genome, and the origin of the phage is unknown. The contaminated food most likely contained a mixture of E. coli O103:H25 cells with either one of the phages.     

Phylogenetically related Argentinean and Australian Escherichia coli O157 isolates are distinguished by virulence clades and alternative Shiga toxin 1 and 2 prophages

Shiga toxigenic Escherichia coli O157 is the leading cause of hemolytic uremic syndrome (HUS) worldwide. The frequencies of stx genotypes and the incidences of O157-related illness and HUS vary significantly between Argentina and Australia. Locus-specific polymorphism analysis revealed that lineage I/II (LI/II) E. coli O157 isolates were most prevalent in Argentina (90%) and Australia (88%). Argentinean LI/II isolates were shown to belong to clades 4 (28%) and 8 (72%), while Australian LI/II isolates were identified as clades 6 (15%), 7 (83%), and 8 (2%). Clade 8 was significantly associated with Shiga toxin bacteriophage insertion (SBI) type stx(2) (locus of insertion, argW) in Argentinean isolates (P < 0.0001). In Argentinean LI/II strains, stx(2) is carried by a prophage inserted at argW, whereas in Australian LI/II strains the argW locus is occupied by the novel stx(1) prophage. In both Argentinean and Australian LI/II strains, stx(2c) is almost exclusively carried by a prophage inserted at sbcB. However, alternative q(933)- or q(21)-related alleles were identified in the Australian stx(2c) prophage. Argentinean LI/II isolates were also distinguished from Australian isolates by the presence of the putative virulence determinant ECSP_3286 and the predominance of motile O157:H7 strains. Characteristics common to both Argentinean and Australian LI/II O157 strains included the presence of putative virulence determinants (ECSP_3620, ECSP_0242, ECSP_2687, ECSP_2870, and ECSP_2872) and the predominance of the tir255T allele. These data support further understanding of O157 phylogeny and may foster greater insight into the differential virulence of O157 lineages.     

Characterization of two major groups of diarrheagenic Escherichia coli O26 strains which are globally spread in human patients and domestic animals of different species

Twenty-three Escherichia coli O26 strains from humans, cattle, sheep, pigs and chicken were investigated for virulence markers and for genetic similarity by pulsed field gel electrophoresis and multi locus sequence typing. Two groups of genetically closely related O26 strains were defined. One group is formed by enteropathogenic (EPEC) and enterohemorrhagic (EHEC) E. coli strains, which do not ferment rhamnose and dulcitol and most of these carry a plasmid encoding enterohemolysin. The other group consists of rhamnose and dulcitol fermenting EPEC strains, which carry plasmids encoding alpha-hemolysin. Multiple species of domestic animals were shown to serve as a reservoir for human pathogenic O26 EPEC and EHEC strains.     

Phylogenetic grouping and virulence potential of extended-spectrum-β-lactamase-producing Escherichia coli strains in cattle

In line with recent reports of extended-spectrum beta-lactamases (ESBLs) in Escherichia coli isolates of highly virulent serotypes, such as O104:H4, we investigated the distribution of phylogroups (A, B1, B2, D) and virulence factor (VF)-encoding genes in 204 ESBL-producing E. coli isolates from diarrheic cattle. ESBL genes, VFs, and phylogroups were identified by PCR and a commercial DNA array (Alere, France). ESBL genes belonged mostly to the CTX-M-1 (65.7%) and CTX-M-9 (27.0%) groups, whereas those of the CTX-M-2 and TEM groups were much less represented (3.9% and 3.4%, respectively). One ESBL isolate was stx(1) and eae positive and belonged to a major enterohemorrhagic E. coli (EHEC) serotype (O111:H8). Two other isolates were eae positive but stx negative; one of these had serotype O26:H11. ESBL isolates belonged mainly to phylogroup A (55.4%) and, to lesser extents, to phylogroups D (25.5%) and B1 (15.6%), whereas B2 strains were quasi-absent (1/204). The number of VFs was significantly higher in phylogroup B1 than in phylogroups A (P = 0.04) and D (P = 0.02). Almost all of the VFs detected were found in CTX-M-1 isolates, whereas only 64.3% and 33.3% of them were found in CTX-M-9 and CTX-M-2 isolates, respectively. These results indicated that the widespread dissemination of the bla(CTX-M) genes within the E. coli population from cattle still spared the subpopulation of EHEC/Shiga-toxigenic E. coli (STEC) isolates. In contrast to other reports on non-ESBL-producing isolates from domestic animals, B1 was not the main phylogroup identified. However, B1 was found to be the most virulent phylogroup, suggesting host-specific distribution of virulence determinants among phylogenetic groups.