Associations between Virulence Factors of Shiga Toxin-Producing Escherichia coli and Disease in Humans

Associations between known or putative virulence factors of Shiga toxin-producing Escherichia coli and disease in humans were investigated. Univariate analysis and multivariate logistic regression analysis of a set of 237 isolates from 118 serotypes showed significant associations between the presence of genes for intimin (eae) and Shiga toxin 2 (stx₂) and isolates from serotypes reported in humans. Similar associations were found with isolates from serotypes reported in hemorrhagic colitis and hemolytic-uremic syndrome. The enterohemorrhagic E. coli (EHEC) hemolysin gene was significantly associated with isolates from serotypes found in severe diseases in univariate analysis but not in multivariate logistic regression models. A strong association between the intimin and EHEC-hemolysin genes may explain the lack of statistical significance of EHEC hemolysin in these multivariate models, but a true lack of biological significance of the hemolysin in humans or in disease cannot be excluded. This result warrants further investigations of this topic. Multivariate analysis revealed an interaction between the eae and stx₂ genes, thus supporting the hypothesis of the synergism between the adhesin intimin and Shiga toxin 2. A strong statistical association was observed between the stx₂ gene and severity of disease for a set of 112 human isolates from eight major serotypes. A comparison of 77 isolates of bovine origin and 91 human isolates belonging to six major serotypes showed significant associations of the genes for Shiga toxin 1 and EspP protease with bovine isolates and an increased adherence on HEp-2 cell cultures for human isolates, particularly from diarrheic patients and healthy persons.     

Pathogenesis and Diagnosis of Shiga Toxin-Producing Escherichia coli Infections

Since their initial recognition 20 years ago, Shiga toxin-producing Escherichia coli (STEC) strains have emerged as an important cause of serious human gastrointestinal disease, which may result in life-threatening complications such as hemolytic-uremic syndrome. Food-borne outbreaks of STEC disease appear to be increasing and, when mass-produced and mass-distributed foods are concerned, can involve large numbers of people. Development of therapeutic and preventative strategies to combat STEC disease requires a thorough understanding of the mechanisms by which STEC organisms colonize the human intestinal tract and cause local and systemic pathological changes. While our knowledge remains incomplete, recent studies have improved our understanding of these processes, particularly the complex interaction between Shiga toxins and host cells, which is central to the pathogenesis of STEC disease. In addition, several putative accessory virulence factors have been identified and partly characterized. The capacity to limit the scale and severity of STEC disease is also dependent upon rapid and sensitive diagnostic procedures for analysis of human samples and suspect vehicles. The increased application of advanced molecular technologies in clinical laboratories has significantly improved our capacity to diagnose STEC infection early in the course of disease and to detect low levels of environmental contamination. This, in turn, has created a potential window of opportunity for future therapeutic intervention.     

Characterization of Urinary Tract Infection-Associated Shiga Toxin-Producing Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC), a subgroup of Shiga toxin (Stx)-producing E. coli (STEC), is a leading cause of diarrhea and hemolytic-uremic syndrome (HUS) in humans. However, urinary tract infections (UTIs) caused by this microorganism but not associated with diarrhea have occasionally been reported. We geno- and phenotypically characterized three EHEC isolates obtained from the urine of hospitalized patients suffering from UTIs. These isolates carried typical EHEC virulence markers and belonged to HUS-associated E. coli (HUSEC) clones, but they lacked virulence markers typical of uropathogenic E. coli. One isolate exhibited a localized adherence (LA)-like pattern on T24 urinary bladder epithelial cells. Since the glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) are well-known receptors for Stx but also for P fimbriae, a major virulence factor of extraintestinal pathogenic E. coli (ExPEC), the expression of Gb3Cer and Gb4Cer by T24 cells and in murine urinary bladder tissue was examined by thin-layer chromatography and mass spectrometry. We provide data indicating that Stxs released by the EHEC isolates bind to Gb3Cer and Gb4Cer isolated from T24 cells, which were susceptible to Stx. All three EHEC isolates expressed stx genes upon growth in urine. Two strains were able to cause UTI in a murine infection model and could not be outcompeted in urine in vitro by typical uropathogenic E. coli isolates. Our results indicate that despite the lack of ExPEC virulence markers, EHEC variants may exhibit in certain suitable hosts, e.g., in hospital patients, a uropathogenic potential. The contribution of EHEC virulence factors to uropathogenesis remains to be further investigated.     

Recent Progress of Shiga Toxin Neutralizer for Treatment of Infections by Shiga Toxin-Producing Escherichia coli

Infection with Shiga toxin (Stx)-producing Escherichia coli (STEC), including O157:H7, causes bloody diarrhea and hemorrhagic colitis in humans, occasionally resulting in fatal systemic complications, such as neurological damage and hemolytic-uremic syndrome. Because Stx is a major virulence factor of the infectious disease, a series of Shiga toxin neutralizers with various structural characteristics has been developed as promising therapeutic agents. Most of these agents function to bind to the toxin directly and inhibit the binding to its receptor present on the target cells. Other neutralizers do not inhibit receptor binding but induce aberrant intracellular transport of the toxin, resulting in effective detoxification. Such a novel type of Stx neutralizer provides a new therapeutic strategy against STEC infections. Here, recent progress of the development of Stx neutralizers is reviewed.     

Identification and Characterization of a Newly Isolated Shiga Toxin 2-Converting Phage from Shiga Toxin-Producing Escherichia coli

Shiga toxins 1 (Stx1) and 2 (Stx2) are encoded by toxin-converting bacteriophages of Stx-producing Escherichia coli (STEC), and so far two Stx1- and one Stx2-converting phages have been isolated from two STEC strains (A. D. O’Brien, J. W. Newlands, S. F. Miller, R. K. Holmes, H. W. Smith, and S. B. Formal, Science 226:694–696, 1984). In this study, we isolated two Stx2-converting phages, designated Stx2Φ-I and Stx2Φ-II, from two clinical strains of STEC associated with the outbreaks in Japan in 1996 and found that Stx2Φ-I resembled 933W, the previously reported Stx2-converting phage, in its infective properties for E. coli K-12 strain C600 while Stx2Φ-II was distinct from them. The sizes of the plaques of Stx2Φ-I and Stx2Φ-II in C600 were different; the former was larger than the latter. The restriction maps of Stx2Φ-I and Stx2Φ-II were not identical; rather, Stx2Φ-II DNA was approximately 3 kb larger than Stx2Φ-I DNA. Furthermore, Stx2Φ-I and Stx2Φ-II showed different phage immunity, with Stx2Φ-I and 933W belonging to the same group. Infection of C600 by Stx2Φ-I or 933W was affected by environmental osmolarity differently from that by Stx2Φ-II. When C600 was grown under conditions of high osmolarity, the infectivity of Stx2Φ-I and 933W was greatly decreased compared with that of Stx2Φ-II. Examination of the plating efficiency of the three phages for the defined mutations in C600 revealed that the efficiency of Stx2Φ-I and 933W for the fadL mutant decreased to less than 10⁻⁷ compared with that for C600 whereas the efficiency of Stx2Φ-II decreased to 0.1% of that for C600. In contrast, while the plating efficiency of Stx2Φ-II for the lamB mutant decreased to a low level (0.05% of that for C600), the efficiencies of Stx2Φ-I and 933W were not changed. This was confirmed by the phage neutralization experiments with isolated outer membrane fractions from C600, fadL mutant, or lamB mutant or the purified His₆-tagged FadL and LamB proteins. Based on the data, we concluded that FadL acts as the receptor for Stx2Φ-I and Stx2Φ-II whereas LamB acts as the receptor only for Stx2Φ-II.     

Four-Year Experience with Simultaneous Culture and Shiga Toxin Testing for Detection of Shiga Toxin-Producing Escherichia coli in Stool Samples

We report our experience with universal Shiga toxin-producing Escherichia coli (STEC) screening using culture and Shiga toxin antigen testing over 4 years. Twelve cases were detected—8 detected by both culture and Shiga toxin immunochromatographic assay (IA), 3 by culture, and 1 by IA only. The addition of Shiga toxin testing is of questionable benefit over culture alone for detection of STEC in areas of low prevalence.     

Real-Time PCR Assay for Detection and Differentiation of Shiga Toxin-Producing Escherichia coli from Clinical Samples

Timely accurate diagnosis of Shiga toxin-producing Escherichia coli (STEC) infections is important. We evaluated a laboratory-developed real-time PCR (LD-PCR) assay targeting stx₁, stx₂, and rfbE_O157 with 2,386 qualifying stool samples submitted to the microbiology laboratory of a tertiary care pediatric center between July 2011 and December 2013. Broth cultures of PCR-positive samples were tested for Shiga toxins by enzyme immunoassay (EIA) (ImmunoCard STAT! enterohemorrhagic E. coli [EHEC]; Meridian Bioscience) and cultured in attempts to recover both O157 and non-O157 STEC. E. coli O157 and non-O157 STEC were detected in 35 and 18 cases, respectively. Hemolytic uremic syndrome (HUS) occurred in 12 patients (10 infected with STEC O157, one infected with STEC O125ac, and one with PCR evidence of STEC but no resulting isolate). Among the 59 PCR-positive STEC specimens from 53 patients, only 29 (54.7%) of the associated specimens were toxin positive by EIA. LD-PCR differentiated STEC O157 from non-O157 using rfbE_O157, and LD-PCR results prompted successful recovery of E. coli O157 (n = 25) and non-O157 STEC (n = 8) isolates, although the primary cultures and toxin assays were frequently negative. A rapid “mega”-multiplex PCR (FilmArray gastrointestinal panel; BioFire Diagnostics) was used retrospectively, and results correlated with LD-PCR findings in 25 (89%) of the 28 sorbitol-MacConkey agar culture-negative STEC cases. These findings demonstrate that PCR is more sensitive than EIA and/or culture and distinguishes between O157 and non-O157 STEC in clinical samples and that E. coli O157:H7 remains the predominant cause of HUS in our institution. PCR is highly recommended for rapid diagnosis of pediatric STEC infections.     

Subtilase Cytotoxin-Encoding subAB Operon Found Exclusively among Shiga Toxin-Producing Escherichia coli Strains

The presence of subAB was investigated for 3,453 Escherichia coli strains of various pathogenic categories. The occurrence of other virulence genes in subAB-positive strains was investigated. The subAB operon was detected among some Shiga toxin-producing E. coli (STEC) serotypes devoid of eae and carrying ehxA. Most subAB-positive strains also harbored stx₂, iha, saa, and lpfA_O113.Subtilase cytotoxin, a new member of the AB₅ toxin family, was identified for the first time in 2004 in a virulent O113:H21 Shiga toxin-producing Escherichia coli (STEC) strain that caused an outbreak of hemolytic-uremic syndrome in South Australia (16, 18). The presence of subAB genes was further detected in other STEC strains belonging to different serotypes (19). Subsequently, subAB genes were identified among STEC strains isolated in other countries (3, 8, 9, 14, 25).To evaluate how widely distributed the subAB operon is, we studied a large collection of STEC serotypes from nonhuman sources and E. coli strains of different pathogenic categories associated with human infections. The subAB-positive strains were further characterized regarding the presence of other virulence genes.A total of 2,255 E. coli strains isolated from humans and belonging to enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), extraintestinal pathogenic E. coli (ExPEC), and E. coli strains not belonging to the diarrheagenic categories described so far were randomly selected. STEC strains isolated in Brazil from humans have previously been tested for the presence of subAB by our group (3). The 1,198 STEC strains from nonhuman sources were isolated from dairy cattle, beef cattle, buffaloes, and goats. Overall, 109 different STEC serotypes were tested. An STEC strain of serotype O113:H21 (3) was used as a reference strain for subAB, cdt-V, and lpfA_O113, and E. coli strain DH5α was used as a negative control.The strains were screened for the presence of the subAB operon (encoding subtilase cytotoxin) using colony hybridization assays (21). The 1,823-bp subAB-specific DNA probe was derived from the STEC serotype O113:H21 (3) strain by PCR as previously described (19). Hybridization assays were performed under stringent conditions, and the probe was labeled with [α-³²P]dCTP (Amersham), using the Ready-To-Go DNA labeling kit (Amersham). All strains which yielded a positive or weak signal in hybridization assays with the subAB probe were retested by PCR (18, 19), and only those confirmed by PCR were considered to be carrying this sequence.The genetic profiles of the subAB-positive strains were determined using our previously reported data for the same strains (6, 7, 12, 13, 20, 24) regarding the presence of the ehxA, eae, stx₁, stx₂, and adhesin-encoding genes (1, 10, 11, 15, 17, 22, 23).A total of 130 STEC strains carrying the subAB operon, representative of each serotype and isolated from different animals, were analyzed by PCR for the presence of genes encoding Lpf_O113 and cytolethal distending toxin (Cdt-V) (2, 5).Expression of the SubAB and Cdt-V toxins was investigated using Chinese hamster ovary cells according to the methods of Paton et al. (18) and Bielaszewska et al. (2), respectively. Cells were exposed to filter-sterilized bacterial culture supernatants and observed daily for a period of 7 days. To confirm the loss of viability or morphological changes, trypan blue, violet crystal, and/or 4′,6-diamidino-2-phenylindole (DAPI) staining were performed. Control strains were included in all assays.As shown in Table ​Table1,1, the subAB operon was detected exclusively among STEC strains and corresponded to 25.5% (306/1,198) of the STEC collection. The presence of subAB was identified in 44.2% (141/319), 27.1% (29/107), and 23.8% (129/542) of STEC strains isolated from dairy cattle, buffaloes, and beef cattle, respectively. Only 3% (7/230) of STEC strains isolated from goat carried subAB. Among the 109 different STEC serotypes tested, 21 carried the subAB operon. The presence of the subAB operon probably is associated with some STEC serotypes. In the present study, the rate of carriage of this sequence within each serotype ranged from 5.5 to 100% (Table ​(Table2).2). Among caprine STEC strains, only those belonging to O113:H21 carried the subAB operon. A total of 306 (306/1,198) STEC strains carrying subAB were detected. We have previously reported that among 49 human STEC strains isolated in Brazil, none carried the subAB sequence (3).

TABLE 1.

E. coli strains of different pathogenic categories searched for the subAB operon

Immunoproteomic Analysis To Identify Shiga Toxin-Producing Escherichia coli Outer Membrane Proteins Expressed during Human Infection

Shiga-toxin producing Escherichia coli (STEC) is the etiologic agent of acute diarrhea, dysentery, and hemolytic-uremic syndrome (HUS). There is no approved vaccine for STEC infection in humans, and antibiotic use is contraindicated, as it promotes Shiga toxin production. In order to identify STEC-associated antigens and immunogenic proteins, outer membrane proteins (OMPs) were extracted from STEC O26:H11, O103, O113:H21, and O157:H7 strains, and commensal E. coli strain HS was used as a control. SDS-PAGE, two-dimensional-PAGE analysis, Western blot assays using sera from pediatric HUS patients and controls, and matrix-assisted laser desorption ionization–tandem time of flight analyses were used to identify 12 immunogenic OMPs, some of which were not reactive with control sera. Importantly, seven of these proteins have not been previously reported to be immunogenic in STEC strains. Among these seven proteins, OmpT and Cah displayed IgG and IgA reactivity with sera from HUS patients. Genes encoding these two proteins were present in a majority of STEC strains. Knowledge of the antigens produced during infection of the host and the immune response to those antigens will be important for future vaccine development.     

Cytotoxic and Apoptotic Effects of Recombinant Subtilase Cytotoxin Variants of Shiga Toxin-Producing Escherichia coli

In this study, the cytotoxicity of the recently described subtilase variant SubAB_2-2 of Shiga toxin-producing Escherichia coli was determined and compared to the plasmid-encoded SubAB₁ and the chromosome-encoded SubAB_2-1 variant. The genes for the respective enzymatic active (A) subunits and binding (B) subunits of the subtilase toxins were amplified and cloned. The recombinant toxin subunits were expressed and purified. Their cytotoxicity on Vero cells was measured for the single A and B subunits, as well as for mixtures of both, to analyze whether hybrids with toxic activity can be identified. The results demonstrated that all three SubAB variants are toxic for Vero cells. However, the values for the 50% cytotoxic dose (CD₅₀) differ for the individual variants. Highest cytotoxicity was shown for SubAB₁. Moreover, hybrids of subunits from different subtilase toxins can be obtained which cause substantial cytotoxicity to Vero cells after mixing the A and B subunits prior to application to the cells, which is characteristic for binary toxins. Furthermore, higher concentrations of the enzymatic subunit SubA₁ exhibited cytotoxic effects in the absence of the respective B₁ subunit. A more detailed investigation in the human HeLa cell line revealed that SubA₁ alone induced apoptosis, while the B₁ subunit alone did not induce cell death.     

Evolution of Enterohemorrhagic Escherichia coli Hemolysin Plasmids and the Locus for Enterocyte Effacement in Shiga Toxin-Producing E. coli

This study assessed the diversity of the enterohemorrhagic Escherichia coli (EHEC) hemolysin gene (ehxA) in a variety of Shiga toxin-producing E. coli (STEC) serotypes and the relationship between ehxA types and virulence markers on the locus for enterocyte effacement (LEE). Restriction fragment length polymorphism of the ehxA gene and flanking sequences and of the E. coli attaching and effacing (eae) gene was determined for 79 EHEC hemolysin-positive STEC isolates of 37 serotypes. Two main groups of EHEC hemolysin sequences and associated plasmids, which corresponded to the eae-positive and the eae-negative isolates, were delineated. Comparisons of the ehxA gene sequences of representative isolates of each group showed that this gene and the rest of the EHEC hemolysin operon are highly conserved. Digestion of an ehxA PCR product with the restriction endonuclease TaqI showed a unique restriction pattern for eae-negative isolates and another one for isolates of serotypes O157:H7 and O157:NM. A conserved fragment of 5.6 kb with four potential open reading frames was identified on the EHEC hemolysin plasmid of eae-positive STEC. Phylogenetic analysis of a subset of 27 STEC isolates, one enteropathogenic E. coli isolate, and a K-12 reference isolate showed that eae-positive STEC isolates all belong to a single evolutionary lineage and that the EHEC hemolysin plasmid and the ehxA gene evolved within this lineage without recent horizontal transfer. However, the eae gene and the LEE appear to have been transferred horizontally within this STEC lineage on several occasions. The reasons for the lack of transfer or maintenance of the LEE in other STEC lineages are not clear and require further study.     

Usability and Performance of CHROMagar STEC Medium in Detection of Shiga Toxin-Producing Escherichia coli Strains

The performance and usability of CHROMagar STEC medium (CHROMagar Microbiology, Paris, France) for routine detection of Shiga toxin-producing Escherichia coli (STEC) strains were examined. The ability of the medium to selectively propagate STEC strains differing by their serotypes and virulence genes was studied with a collection of diarrheagenic E. coli isolates (n = 365) consisting of 49 different serotypes and with non-STEC and other bacterial isolates (n = 264). A total of 272 diarrheagenic E. coli (75.0%) isolates covering 24 different serotypes grew on CHROMagar STEC. The highest detection sensitivities were observed within the STEC serogroups O26 (90.0%), O111 (100.0%), O121 (100.0%), O145 (100.0%), and O157 (84.9%), and growth on CHROMagar STEC was highly associated with the presence of the tellurite resistance gene (terD). The specificity of the medium was 98.9%. In addition, CHROMagar STEC was used in parallel with a Shiga toxin-detecting immunoassay (Ridaquick Verotoxin/O157 Combi; R-biopharm, Darmstadt, Germany) to screen fecal specimens (n = 47) collected from patients suffering from hemorrhagic diarrhea. Positive growth on CHROMagar STEC was confirmed by the Premier EHEC enzyme immunoassay (Meridian Bioscience, Inc., Cincinnati, OH), and discrepant results between the two screening methods were confirmed by stx gene-detecting PCR. All 16 of the 47 stool samples that showed positive growth on CHROMagar STEC were also positive in the confirmatory tests. CHROMagar STEC proved to be an interesting option for STEC screening, allowing good detection sensitivity and specificity and permitting strain isolation for further outbreak investigations when required.     

Multiplex PCR for Enterotoxigenic, Attaching and Effacing, and Shiga Toxin-Producing Escherichia coli Strains from Calves

A multiplex PCR was developed to identify enterotoxigenic, attaching and effacing, and Shiga toxin-producing Escherichia coli strains by amplifying genes encoding K99 and F41 fimbriae, heat-stable enterotoxin a, intimin, and Shiga toxins 1 and 2. This multiplex PCR was specific and sensitive. It will be useful for identification of E. coli strains which cause diarrhea in calves.     

Shiga Toxin-Producing Escherichia coli in Finland from 1990 through 1997: Prevalence and Characteristics of Isolates

During the past 10 years Shiga toxin-producing Escherichia coli (STEC) has emerged as one of the most important causes of food-borne infections in industrialized countries. In Finland, with a population of 5.1 million, however, only four STEC O157:H7 infections were identified from 1990 through 1995; the occurrence of non-O157 STEC infections was unknown. In 1996, we established a national prospective study to determine the prevalence of STEC serotypes in feces of Finns with bloody diarrhea. During this enhanced 1-year study period eight sporadic cases of STEC infection were found; of them, only two were indigenously acquired O157:H7 infections. In 1997, O157 infections increased dramatically, with O157 strains causing 51 of all 61 STEC infections. Altogether 14 non-O157:H7 STEC strains were found in Finland in the 1990s: O26:H11 (four strains), O26:HNM (HNM indicates nonmotile), O2:H29, O91:H21, O91:H40, O101:HNM, O107:H27, O157:HNM, O165:H25, OX3:H21, and Rough:H49. All O157:H7 and O26:H11 isolates produced enterohemolysin, but seven of the other STEC strains did not. Most (n = 63) of the 71 STEC strains isolated carried the stx₂ gene only, five carried the stx₁ gene only, and three carried both genes. The eaeA gene was detected in all other isolates except five non-O157 strains. There were seven distinct pulsed-field gel electrophoresis (PFGE) genotypes among 57 O157 strains and three distinct PFGE types among four O26:H11 strains. The main PFGE type was found among 65% of all O157 isolates.     

Emergence of a Multidrug-Resistant Shiga Toxin-Producing Enterotoxigenic Escherichia coli Lineage in Diseased Swine in Japan

Enterotoxigenic Escherichia coli (ETEC) and Shiga toxin-producing E. coli (STEC) are important causes of diarrhea and edema disease in swine. The majority of swine-pathogenic E. coli strains belong to a limited range of O serogroups, including O8, O138, O139, O141, O147, O149, and O157, which are the most frequently reported strains worldwide. However, the circumstances of ETEC and STEC infections in Japan remain unknown; there have been few reports on the prevalence or characterization of swine-pathogenic E. coli. In the present study, we determined the O serogroups of 967 E. coli isolates collected between 1991 and 2014 from diseased swine in Japan, and we found that O139, O149, O116, and OSB9 (O serogroup of Shigella boydii type 9) were the predominant serogroups. We further analyzed these four O serogroups using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing, and virulence factor profiling. Most of the O139 and O149 strains formed serogroup-specific PFGE clusters (clusters I and II, respectively), whereas the O116 and OSB9 strains were grouped together in the same cluster (cluster III). All of the cluster III strains belonged to a single sequence type (ST88) and carried genes encoding both enterotoxin and Shiga toxin. This PFGE cluster III/ST88 lineage exhibited a high level of multidrug resistance (to a median of 10 antimicrobials). Notably, these bacteria were resistant to fluoroquinolones. Thus, this lineage should be considered a significant risk to animal production due to the toxigenicity and antimicrobial resistance of these bacteria.     

Virulence Properties of Shiga Toxin-Producing Escherichia coli (STEC) Strains of Serogroup O118, a Major Group of STEC Pathogens in Calves

Shiga toxin-producing Escherichia coli (STEC) strains of serogroup O118 are the most prevalent group among STEC strains in diarrheic calves in Germany (L. H. Wieler, Ph.D. thesis, University of Giessen, 1997). To define their virulence properties, 42 O118 (O118:H16 [n = 38] and O118:H− [n = 4]) strains were characterized. The strains displayed three different Stx combinations (Stx1 [36 of 42], Stx1 and Stx2 [2 of 42], and Stx2 [4 of 42]). A total of 41 strains (97.6%) harbored a large virulence-associated plasmid containing hly_EHEC (hly from enterohemorrhagic E. coli). The strains’ adhesive properties varied in relation to the eukaryotic cells tested. Only 28 of 42 strains (66.7%) showed localized adhesion (LA) in the human HEp-2 cell line. In contrast, in bovine fetal calf lung (FCL) cells, the number of LA-positive strains was much higher (37 of 42 [88.1%]). The locus of enterocyte effacement (LEE) was detected in 41 strains (97.6%). However, not all LEE-positive strains reacted positively in the fluorescence actin-staining (FAS) test, which indicated the attaching and effacing (AE) lesion. In HEp-2 cells, only 22 strains (52.4%) were FAS positive, while in FCL cells, the number of FAS-positive strains was significantly higher (38 of 42 [90.5%; P < 0.001]). In conclusion, the vast majority of the O118 STEC strains from calves (41 of 42 [97.6%]) have a high virulence potential (stx, hly_EHEC, and LEE). This virulence potential and the high prevalence of STEC O118 strains in calves suggest that these strains could be a major health threat for humans in the future. In addition, the poor association between results of the geno- and phenotypical tests to screen for the AE ability of STEC strains calls the diagnostic value of the FAS test into question.     

Evaluation of Enzyme Immunoassays and Real-Time PCR for Detecting Shiga Toxin-Producing Escherichia coli in Southern Alberta,Canada

Two immunoassays (Shiga Toxin Chek and Shiga Toxin Quik Chek) and real-time PCR were used to detect Shiga toxin-producing Escherichia coli. For enriched culture, the sensitivity and specificity of the three methods ranged from 80.0% to 98.2% and 98.0% to 100.0%, respectively. STEC isolates were identified in 2.6% of the 784 samples.