Evaluation of the premier EHEC assay for detection of Shiga toxin-producing Escherichia coli.

An enzyme-linked immunosorbent assay for the detection of Shiga toxins (Premier EHEC assay; Meridian Diagnostics, Inc.) was compared to conventional sorbitol-MacConkey culture for the recovery of enterohemorrhagic Escherichia coli. A total of 74 enteric pathogens, including 8 E. coli O157:H7 isolates, were recovered from 974 stool specimens. Two of these specimens were not tested by Premier assaying due to insufficient sample and are not considered in the data analysis. The Premier EHEC assay detected the 6 evaluable specimens which were culture positive for E. coli O157:H7 and identified an additional 10 specimens as containing Shiga toxin. Seven isolates were recovered from these 10 specimens by an immunoblot assay and were confirmed as toxin producers by a cytotoxin assay. Of these seven, four isolates were serotype O157:H7, one was O26:NM, one was O6:H-, and one was O untypeable:H untypeable. Three specimens contained Shiga toxin by both EHEC immunoassaying and cytotoxin testing; however, no cytotoxin-producing E. coli could be recovered. The sorbitol-MacConkey method had a sensitivity and a specificity of 60 and 100%, respectively, while the Premier EHEC assay had a sensitivity and a specificity of 100 and 99.7%, respectively, for E. coli O157:H7 only. The Premier EHEC assay also detected an additional 20% Shiga toxin-producing E. coli (STEC) that were non-O157:H7. Thus, the Premier EHEC assay is a sensitive and specific method for the detection of all STEC isolates. Routine use would improve the detection of E. coli O157:H7 and allow for determination of the true incidence of STEC other than O157:H7. The presence of blood in the stool and/or the ages of the patients were poor predictors of the presence of STEC. Criteria need to be determined which would allow for the cost-effective incorporation of this assay into the routine screen for enteric pathogens in high-risk individuals, especially children.     

Evaluation of performance and potential clinical impact of ProSpecT Shiga toxin Escherichia coli microplate assay for detection of Shiga Toxin-producing E. coli in stool samples

Shiga toxin-producing Escherichia coli bacteria (STEC) are emerging pathogens capable of producing sporadic and epidemic diarrhea, hemorrhagic colitis, and potentially life-threatening hemolytic-uremic syndrome. Although the presence of E. coli O157 can be readily detected in stool by sorbitol-MacConkey agar culture (SMAC), STEC non-O157 serotypes cannot. In contrast to culture, testing for the presence of Shiga toxins 1 and 2 in stool detects both O157 and non-O157 STEC serotypes capable of causing disease. Over two consecutive summers, we evaluated the performance of the ProSpecT Shiga toxin E. coli Microplate assay (Alexon-Trend, Ramsey, Minn.), an enzyme immunoassay for the detection of Shiga toxins 1 and 2, on all stools submitted for culture of enteric pathogens, and the potential clinical impact of Shiga toxin detection. Twenty-nine stool specimens were STEC positive by ProSpecT assay. Twenty-seven of 29 STEC-positive isolates were confirmed by SMAC and serotyping or by a second enzyme immunoassay and PCR (positive predictive value, 93%). Thirteen of 27 confirmed Shiga toxin-producing strains were serotype O157. The remaining 14 strains represented 8 other serotypes. The ProSpecT assay was 100% sensitive and specific for detection of E. coli O157 in stool (7 of 7) compared to SMAC. In addition, the ProSpecT assay detected twice as many STEC as SMAC. Fifty-two percent of confirmed STEC-positive stools were nonbloody. Thus, in our population, screening strategies that test only visibly bloody stools for STEC would miss a majority of cases. Eleven (41%) STEC-positive patients were hospitalized, and eight (30%) developed severe disease (two developed hemolytic-uremic syndrome, and six developed hemorrhagic colitis). Prior to detection of STEC infection, seven (26%) and eight patients (30%) underwent unnecessary diagnostic procedures or received potentially deleterious empirical treatment, respectively. We propose that establishing a specific diagnosis of STEC may have prevented these potentially harmful interventions. We conclude that the ProSpecT assay is sensitive and specific for the detection of Shiga toxins 1 and 2 in stool and has potentially significant clinical impact for the individual patient and public health. Shiga toxin assays should be considered for routine use in settings where prevalence of STEC disease warrants testing.     

Characterization of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli serotypes isolated from sheep.

The isolation and characterization of Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli (STEC) strains from sheep are described. One flock was investigated for E. coli O157:H7 over a 16-month period that spanned two summer and two autumn seasons. Variation in the occurrence of E. coli O157:H7-positive sheep was observed, with animals being culture positive only in the summer months but not in the spring, autumn, or winter. E. coli O157:H7 isolates were distinguished by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA and toxin gene restriction fragment length polymorphism (RFLP) analysis. Ten PFGE patterns and five RFLP patterns, identified among the isolates, showed that multiple E. coli O157:H7 strains were isolated from one flock, that a single animal simultaneously shed multiple E. coli O157:H7 strains, and that the strains shed by individuals changed over time. E. coli O157:H7 was isolated only by selective enrichment culture off 10 g of ovine feces. In contrast, strains of eight STEC serotypes other than O157:H7 were cultured from feces of sheep from a separate flock without enrichment. The predominant non-O157 STEC serotype found was O91:NM (NM indicates nonmotile), and others included O128:NM, O88:NM, O6:H49, and O5:NM. Irrespective of serotype, 98% of the ovine STEC isolates possessed various combinations of the virulence-associated genes for Shiga toxin(s) and the attaching-and-effacing lesion (stx1, stx2, and eae), suggesting their potential for human pathogenicity. The most common toxin-eae genotype was positive for stx1, stx2, and eae. A Vero cell cytotoxicity assay demonstrated that 90% of the representative STEC isolates tested expressed the toxin gene. The report demonstrates that sheep transiently shed a variety of STEC strains, including E. coli O157:H7, that have potential as human pathogens.     

Detection in Escherichia coli of the genes encoding the major virulence factors,the genes defining the O157:H7 serotype,and components of the type 2 Shiga toxin family by multiplex PCR

Strains of Shiga toxin-producing Escherichia coli (STEC) have been associated with outbreaks of diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans. Most clinical signs of disease arise as a consequence of the production of Shiga toxin 1 (Stx1), Stx2 or combinations of these toxins. Other major virulence factors include enterohemorrhagic E. coli hemolysin (EHEC hlyA), and intimin, the product of the eaeA gene that is involved in the attaching and effacing adherence phenotype. In this study, a series of multiplex-PCR assays were developed to detect the eight most-important E. coli genes associated with virulence, two that define the serotype and therefore the identity of the organism, and a built-in gene detection control. Those genes detected were stx(1), stx(2), stx(2c), stx(2d), stx(2e), stx(2f), EHEC hlyA, and eaeA, as well as rfbE, which encodes the E. coli O157 serotype; fliC, which encodes the E. coli flagellum H7 serotype; and the E. coli 16S rRNA, which was included as an internal control. A total of 129 E. coli strains, including 81 that were O157:H7, 10 that were O157:non-H7, and 38 that were non-O157 isolates, were investigated. Among the 129 samples, 101 (78.3%) were stx positive, while 28 (21.7%) were lacked stx. Of these 129 isolates, 92 (71.3%) were EHEC hlyA positive and 96 (74.4%) were eaeA positive. All STEC strains were identified by this procedure. In addition, all Stx2 subtypes, which had been initially identified by PCR-restriction fragment length polymorphism, were identified by this method. A particular strength of the assay was the identification of these 11 genes without the need to use restriction enzyme digestion. The proposed method is a simple, reliable, and rapid procedure that can detect the major virulence factors of E. coli while differentiating O157:H7 from non-O157 isolates.     

Molecular Profiling of Escherichia coli O157:H7 and Non-O157 Strains Isolated from Humans and Cattle in Alberta,Canada

Virulence markers in Shiga toxin-producing Escherichia coli (STEC) and their association with diseases remain largely unknown. This study determines the importance of 44 genetic markers for STEC (O157 and non-O157) from human clinical cases and their correlation to disease outcome. STEC isolated from a cattle surveillance program were also included. The virulence genes tested were present in almost all O157:H7 isolates but highly variable in non-O157 STEC isolates. Patient age was a significant determinant of clinical outcome.     

Molecular characterization of a serotype O121:H19 clone,a distinct Shiga toxin-producing clone of pathogenic Escherichia coli

Most illnesses caused by Shiga toxin-producing Escherichia coli (STEC) have been attributed to E. coli serotype O157:H7, but non-O157 STEC infections are now increasingly recognized as public health problems worldwide. The O121:H19 serotype is being isolated more frequently from clinical specimens and has been implicated in one waterborne outbreak. We used multilocus virulence gene profiling, a PCR-based assay, to characterize the virulence gene content of 24 isolates of serotype O121:H19 and nonmotile variants. We also performed multilocus enzyme electrophoresis and multilocus sequencing to establish the clonal relatedness of O121 isolates and to elucidate the relationship of O121 to common STEC clones. The 24 isolates were found to represent a single bacterial clone, as there was no allelic variation across 18 enzyme loci among the isolates. The complete nucleotide sequence of the intimin gene differed by four substitutions from that of the epsilon (Int- epsilon ) allele of O103:H2 strain PMK5. The typical O121 virulence gene profile was similar to the profiles of enterohemorrhagic E. coli (EHEC) clones of E. coli: it included a Shiga toxin 2 gene (stx(2)), two genes on the EHEC plasmid (toxB and ehxA), and the gene encoding intimin (eae). Despite the similarities, putative virulence genes distributed on O islands-large chromosomal DNA segments present in the O157:H7 genome-were useful for discriminating among STEC serotypes and the O121:H19 clone had a composite profile that was distinct from the profiles of the other major EHEC clones of pathogenic E. coli. On the basis of sequencing analysis with 13 housekeeping genes, the O121:H19 clone did not fall into any of the four classical EHEC and enteropathogenic E. coli groups but instead was closely related to two eae-negative STEC strains.     

Inhibition of Water Absorption and Selective Damage to Human Colonic Mucosa Are Induced by Subtilase Cytotoxin Produced by Escherichia coli O113:H21

Shiga toxin-producing Escherichia coli O157:H7 (STEC) is by far the most prevalent serotype associated with hemolytic uremic syndrome (HUS) although many non-O157 STEC strains have been also isolated from patients with HUS. The main virulence factor of STEC is the Shiga toxin type 2 (Stx2) present in O157 and non-O157 strains. Recently, another toxin, named subtilase cytotoxin (SubAB), has been isolated from several non-O157 strains and may contribute to the pathogenesis of HUS. Here, we have demonstrated that an O113:H21 STEC strain expressing SubAB and Stx2 inhibits normal water absorption across human colon and causes damage to the surface epithelium, necrosis, mononuclear inflammatory infiltration, edema, and marked mucin depletion. This damage was less marked, but nevertheless significant, when purified SubAB or E. coli O113:H21 expressing only SubAB was assayed. This is the first study showing that SubAB may directly participate in the mechanisms of diarrhea in children infected with non-O157 STEC strains.     

Antimicrobial resistance of Shiga toxin (verotoxin)-producing Escherichia coli O157:H7 and non-O157 strains isolated from humans, cattle, sheep and food in Spain

A total of 722 Shiga toxin-producing Escherichia coli (STEC) isolates recovered from humans, cattle, ovines and food during the period from 1992 to 1999 in Spain were examined to determine antimicrobial resistance profiles and their association with serotypes, phage types and virulence genes. Fifty-eight (41%) out of 141 STEC O157:H7 strains and 240 (41%) out of 581 non-O157 STEC strains showed resistance to at least one of the 26 antimicrobial agents tested. STEC O157:H7 showed a higher percentage of resistant strains recovered from bovine (53%) and beef meat (57%) than from human (23%) and ovine (20%) sources, whereas the highest prevalence of antimicrobial resistance in non-O157 STEC was found among isolates recovered from beef meat (55%) and human patients (47%). Sulfisoxazole (36%) had the most common antimicrobial resistance, followed by tetracycline (32%), streptomycin (29%), ampicillin (10%), trimethoprim (8%), cotrimoxazole (8%), chloramphenicol (7%), kanamycin (7%), piperacillin (6%), and neomycin (5%). The multiple resistance pattern most often observed was that of streptomycin, sulfisoxazole, and tetracycline. Ten (7%) STEC O157:H7 and 71 (12%) non-O157 strains were resistant to five or more antimicrobial agents. Most strains showing resistance to five or more antimicrobial agents belonged to serotypes O4:H4 (4 strains), O8:H21 (3 strains), O20:H19 (6 strains), O26:H11 (8 strains eae-beta1), O111:H- (3 strains eae-gamma2), O118:H- (2 strains eae-beta1), O118:H16 (5 strains eae-beta1), O128:H- (2 strains), O145:H8 or O145:H- (2 strains eae-gamma1), O157:H7 (10 strains eae-gamma1), O171:H25 (3 strains), O177:H11 (5 strains eae-beta1), ONT:H- (3 strains/1 eae-beta1) and ONT:H21 (2 strains). Interestingly, most of these serotypes, i.e., those indicated in bold) were found among human STEC strains isolated from patients with hemolytic uremic-syndrome (HUS) reported in previous studies. We also detected, among non-O157 strains, an association between a higher level of multiple resistance to antibiotics and the presence of the virulence genes eae and stx(1). Moreover, STEC O157:H7, showed an association between certain phage types, PT21/28 (90%), PT23 (75%), PT34 (75%), and PT2 (54%), with a higher number of resistant strains. We conclude that the high prevalence of antimicrobial resistance detected in our study is a source of concern, and cautious use of antibiotics in animals is highly recommended.     

Shiga toxin-producing Escherichia coli in Central Greece: prevalence and virulence genes of O157:H7 and non-O157 in animal feces, vegetables, and humans

In Greece, Shiga toxin-producing Escherichia coli (STEC) have only been sporadically reported. The objective of this study was to estimate the prevalence of STEC and Escherichia coli O157:H7 in farm animals, vegetables, and humans in Greece. A total number of 1,010 fecal samples were collected from farm animals (sheep, goats, cattle, chickens, pigs), 667 diarrheal samples from humans, and 60 from vegetables, which were cultured in specific media for STEC isolates. Enzyme-linked immunosorbent assay (ELISA) was used to detect toxin-producing colonies, which, subsequently, were subjected to a multiplex polymerase chain reaction (PCR) for stx1, stx2, eae, rfbE _O157, and fliC _h7 genes. Eighty isolates (7.9 %) from animal samples were found to produce Shiga toxin by ELISA, while by PCR, O157 STEC isolates were detected from 8 (0.8 %) samples and non-O157 STEC isolates from 43 (4.2 %) samples. STEC isolates were recovered mainly from sheep and goats, rarely from cattle, and not from pigs and chickens, suggesting that small ruminants constitute a potential risk for human infections. However, only three human specimens (0.4 %) were positive for the detection of Shiga toxins and all were PCR-negative. Similarly, all 60 vegetable samples were negative for toxin production and for toxin genes, but three samples (two roman rockets and one spinach) were positive by PCR for rfbE _O157 and fliC _h7 genes. These findings indicate that sheep, goats, cattle, and leafy vegetables can be a reservoir of STEC and Escherichia coli O157:H7 isolates in Greece, which are still rarely detected among humans.     

Comparative pathogenicity of Escherichia coli O157 and intimin-negative non-O157 Shiga toxin-producing E coli strains in neonatal pigs

We compared the pathogenicity of intimin-negative non-O157:H7 Shiga toxin (Stx)-producing Escherichia coli (STEC) O91:H21 and O104:H21 strains with the pathogenicity of intimin-positive O157:H7 and O157:H(-) strains in neonatal pigs. We also examined the role of Stx2d-activatable genes and the large hemolysin-encoding plasmid of O91:H21 strain B2F1 in the pathogenesis of STEC disease in pigs. We found that all E. coli strains that made wild-type levels of Stx caused systemic illness and histological lesions in the brain and intestinal crypts, whereas none of the control Stx-negative E. coli strains evoked comparable central nervous system signs or intestinal lesions. By contrast, the absence of intimin, hemolysin, or motility had little impact on the overall pathogenesis of systemic disease during STEC infection. The most striking differences between pigs inoculated with non-O157 STEC strains and pigs inoculated with O157 STEC strains were the absence of attaching and effacing intestinal lesions in pigs inoculated with non-O157:H7 strains and the apparent association between the level of Stx2d-activatable toxin produced by an STEC strain and the severity of lesions.     

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.     

Shiga-toxigenic Escherichia coli detection in stool samples screened for viral gastroenteritis in Alberta, Canada

Shiga-toxigenic Escherichia coli (STEC) is an important cause of diarrheal disease. The most notorious STEC serotype is O157:H7, which is associated with hemorrhagic colitis and hemolytic-uremic syndrome (HUS). As a result, this serotype is routinely screened for in clinical microbiology laboratories. With the bias toward the identification of the O157 serogroup in routine diagnostic processes, non-O157 STEC has been largely underrepresented in the epidemiology of STEC infections. This diagnostic bias is further complicated by the fact that many non-O157 STEC infections cause nonspecific gastroenteritis symptoms reminiscent of enteric viral infections. In this study, real-time PCR was used to amplify Shiga toxin genetic determinants (stx(1) and stx(2)) from enriched stool samples that were initially submitted for the testing of enteric viruses in patients with suspected viral gastroenteritis between May and September of 2006, 2007, and 2008 (n = 2,702). Samples were submitted from the province of Alberta, Yukon, the Northwest Territories, and Nunavut, Canada. A total of 38 samples (1.4%) tested positive for Shiga toxin genes, and 15 isolates were cultured for further characterization. Several of the serotypes identified (O157:H7, O26:HNM, O26:H11, O103:H25, O121:H19, and O145:HNM) have been previously associated with outbreaks and HUS. This study outlines the importance of combining molecular methods with classical culture techniques to enhance the detection of emerging non-O157 as well as O157 serotypes in diarrheal stool samples. Furthermore, atypical diarrhea disease caused by non-O157 STEC can be routinely missed due to screening only for viral agents.     

Antimicrobial resistance patterns of Shiga toxin-producing Escherichia coli O157:H7 and O157:H7- from different origins

Shiga toxin-producing Escherichia coli (STEC) serotypes including O157:H7 (n = 129) from dairy cows, cull dairy cow feces, cider, salami, human feces, ground beef, bulk tank milk, bovine feces, and lettuce; and O157:H7- (n = 24) isolated from bovine dairy and bovine feedlot cows were evaluated for antimicrobial resistance against 26 antimicrobials and the presence of antimicrobial resistance genes (tetA, tetB, tetC, tetD, tetE, tetG, floR, cmlA, strA, strB, sulI, sulII, and ampC). All E. coli exhibited resistance to five or more antimicrobial agents, and the majority of isolates carried one or more target antimicrobial resistance gene(s) in different combinations. The majority of E. coli showed resistance to ampicillin, aztreonam, cefaclor, cephalothin, cinoxacin, and nalidixic acid, and all isolates were susceptible to chloramphenicol and florfenicol. Many STEC O157:H7 and O157:H7-isolates were susceptible to amikacin, carbenicillin, ceftriaxone, cefuroxime, ciprofloxacin, fosfomycin, moxalactam, norfloxacin, streptomycin, tobramycin, trimethoprim, and tetracycline. The majority of STEC O157:H7 (79.8%) and O157:H7- (91.7%) carried one or more antimicrobial resistance gene(s) regardless of whether phenotypically resistant or susceptible. Four tetracycline resistant STEC O157:H7 isolates carried both tetA and tetC. Other tetracycline resistance genes (tetB, tetD, tetE, and tetG) were not detected in any of the isolates. Among nine streptomycin resistant STEC O157:H7 isolates, eight carried strA-strB along with aadA, whereas the other isolate carried aadA alone. However, the majority of tetracycline and streptomycin susceptible STEC isolates also carried tetA and aadA genes, respectively. Most ampicillin resistant E. coli of both serotypes carried ampC genes. Among sulfonamide resistance genes, sulII was detected only in STEC O157:H7 (4 of 80 sulfonamide-resistant isolates) and sulI was detected in O157:H7- (1 of 16 sulfonamide resistant isolates). The emergence and dissemination of multidrug resistance in STEC can serve as a reservoir for different antimicrobial resistance genes. Dissemination of antimicrobial resistance genes to commensal and pathogenic bacteria could occur through any one of the horizontal gene transfer mechanisms adopted by the bacteria.     

TccP2 of O157:H7 and non-O157 enterohemorrhagic Escherichia coli (EHEC): challenging the dogma of EHEC-induced actin polymerization

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) trigger actin polymerization at the site of bacterial adhesion by inducing different signaling pathways. Actin assembly by EPEC requires tyrosine phosphorylation of Tir, which subsequently binds the host adaptor protein Nck. In contrast, Tir(EHEC O157) is not tyrosine phosphorylated and instead of Nck utilizes the bacterially encoded Tir-cytoskeleton coupling protein (TccP)/EspF(U), which mimics the function of Nck. tccP is carried on prophage CP-933U/Sp14 (TccP). Typical isolates of EHEC O157:H7 harbor a pseudo-tccP gene that is carried on prophage CP-933 M/Sp4 (tccP2). Here we report that atypical, beta-glucuronidase-positive and sorbitol-fermenting, strains of EHEC O157 harbor intact tccP and tccP2 genes, both of which are secreted by the LEE-encoded type III secretion system. Non-O157 EHEC strains, including O26, O103, O111, and O145, are typically tccP negative and translocate a Tir protein that encompasses an Nck binding site. Unexpectedly, we found that most clinical non-O157 EHEC isolates carry a functional tccP2 gene that encodes a secreted protein that can complement an EHEC O157:H7 DeltatccP mutant. Using discriminatory, allele-specific PCR, we have demonstrated that over 90% of tccP2-positive non-O157 EHEC strains contain a Tir protein that can be tyrosine phosphorylated. These results suggest that the TccP pathway can be used by both O157 and non-O157 EHEC and that non-O157 EHEC can also trigger actin polymerization via the Nck pathway.     

Serum antibodies to Escherichia coli serotype O157:H7 in patients with hemolytic uremic syndrome

Sera from 13 patients with hemolytic uremic syndrome (HUS) and 8 healthy control subjects were examined for antibodies specific for bacterial antigens of Eschericia coli serotype O157:H7. Bacterial components, including outer membrane proteins (OMPs), lipopolysaccharide (LPS), and flagella, were reacted with sera by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting and by enzyme-linked immunosorbent assay. All 13 serum samples from HUS patients contained high-titered antibodies of the immunoglobulin M class against O157 LPS and some OMPs. These same sera reacted weakly with some of the major OMPs, but not the LPS, of non-O157 strains of E. coli. Sera from patients did not contain antibodies to non-O157 LPS or H7 flagella. The possibility of using E. coli serotype O157 LPS in an enzyme-linked immunosorbent assay for the routine diagnostic testing of sera from HUS patients for evidence of O157:H7 infection is discussed.     

Prevalence and clinical manifestations of shiga toxin-producingEscherichia coli infections in Austrian children

The prevalence and clinical manifestations of infections associated with Shiga toxin-producingEscherichia coli (STEC) among Austrian children were assessed. Stool samples from 280 pediatric patients were analyzed by enzyme immunoassay (EIA) for the presence of free fecal Shiga toxin (Stx) 1 and 2, and by culture on sorbitol MacConkey agar. Specimens testing positive by the EIA were subjected to a cytotoxicity assay, polymerase chain reaction analysis, and a colony hybridization test. Direct culture on MacConkey agar demonstrated the presence of threeEscherichia coli O157:H7-positive stools. These were also positive by EIA and by the DNA-based methods. An additional six samples were positive by EIA, and in four of these, non-O157 STEC of serotypes O111H^–, O146: H^–, and O113:H53 could be isolated. Analysis of stools for a variety of enteric pathogens demonstrated that STEC was the third most common bacterial pathogen. The clinical manifestations of STEC infections were difficult to distinguish from those of infections caused by other enteric pathogens, as most patients presented with watery diarrhea. The median age of children with STEC infections was 27.6 months (range, 7 months to 5.75 years); children withSalmonella orCampylobacter infections were younger on average, while those withRotavirus infections were older. This study demonstrated that althoughEscherichia coli O157:H7 could be identified with the same sensitivity by both EIA or agar-based methods, the identification of non-O157 STEC strains was enhanced by the use of EIA followed by colony hybridization. Analysis of overnight cultures from 53 STEC isolates revealed that all strains producing Stx1, Stx2, or Stx2c reacted in the EIA. However, culture supernatants from Stx2e-producingEscherichia coli O101 were negative in the EIA. Despite this disadvantage, the EIA is easy to perform and time efficient and can be recommended as a screening test for non-O157 STEC in children with diarrhea.     

Enterohemorrhagic Escherichia coli O157:H7 Shiga toxins inhibit gamma interferon-mediated cellular activation

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a food-borne pathogen that causes significant morbidity and mortality in developing and industrialized nations. EHEC infection of host epithelial cells is capable of inhibiting the gamma interferon (IFN-γ) proinflammatory pathway through the inhibition of Stat-1 phosphorylation, which is important for host defense against microbial pathogens. The aim of this study was to determine the bacterial factors involved in the inhibition of Stat-1 tyrosine phosphorylation. Human HEp-2 and Caco-2 epithelial cells were challenged directly with either EHEC or bacterial culture supernatants and stimulated with IFN-γ, and then the protein extracts were analyzed by immunoblotting. The data showed that IFN-γ-mediated Stat-1 tyrosine phosphorylation was inhibited by EHEC secreted proteins. Using two-dimensional difference gel electrophoresis, EHEC Shiga toxins were identified as candidate inhibitory factors. EHEC Shiga toxin mutants were then generated and complemented in trans, and mutant culture supernatant was supplemented with purified Stx to confirm their ability to subvert IFN-γ-mediated cell activation. We conclude that while other factors are likely involved in the suppression of IFN-γ-mediated Stat-1 tyrosine phosphorylation, E. coli-derived Shiga toxins represent a novel mechanism by which EHEC evades the host immune system.     

Sorbitol-MacConkey medium for detection of Escherichia coli O157:H7 associated with hemorrhagic colitis.

Escherichia coli serotype O157:H7 is a recently recognized human pathogen associated with hemorrhagic colitis. Unlike most E. coli strains, E. coli O157:H7 does not ferment sorbitol. Therefore, the efficacy of MacConkey agar containing sorbitol (SMAC medium) instead of lactose as a differential medium for the detection of E. coli O157:H7 in stool cultures was determined in comparison with MacConkey agar. The relative frequency of non-sorbitol-fermenting (NSF) organisms other than E. coli O157:H7 in feces was low at 10 to 20% (95% confidence limits), and NSF organisms also occurred mostly in small numbers. In a field trial involving over 1,000 diarrheal stools, E. coli O157:H7 was isolated from 18 stools, all of which were from patients with bloody diarrhea. In every instance, the growth of E. coli O157:H7 on SMAC medium was heavy and occurred in almost pure culture as colorless NSF colonies in contrast to fecal flora, which are mostly sorbitol fermenting and hence appear pink on this medium, whereas on MacConkey agar cultures, the growth of E. coli O157:H7 was indistinguishable from fecal flora. SMAC medium permitted ready recognition of E. coli O157:H7 in stool cultures. Detection of E. coli O157:H7 on SMAC medium had a sensitivity of 100%, a specificity of 85%, and an accuracy of 86%. SMAC medium stool culture is a simple, inexpensive, rapid, and reliable means of detecting E. coli O157:H7, and we recommend routine use of SMAC medium especially for culturing bloody stools.     

Prevalence of EHEC O157:H7 in patients with diarrhoea in Lagos, Nigeria

The prevalence of sorbitol-nonfermenting Escherichia coli O157:H7 (EHEC) was assessed in 100 patients with diarrhoea by stool culture on sorbitol MacConkey agar. The cytotoxicity of the EHEC strains was analysed by Vero cell assay and the antimicrobial susceptibility pattern of the isolates was determined. Detection rate of EHEC O157:H7 was 6%. Five of the six patients were males. Three of the isolates were from children and one was from a teenager. All strains induced cytotoxic effects in the Vero cell assay. All isolates were susceptible to most of the antimicrobials tested. The results showed that diarrhoea caused by EHEC O157:H7, a potentially life-threatening pathogen, has remained common particularly among the child population of Lagos during the past 10 years (5). There must therefore be adequate meat and food inspection to improve the general hygiene of local fast food restaurants, so-called 'bukkas', which are regarded as likely sources of infection.     

Evaluation of CHROMagar STEC and STEC O104 Chromogenic Agar Media for Detection of Shiga Toxin-Producing Escherichia coli in Stool Specimens

The performance of CHROMagar STEC and CHROMagar STEC O104 (CHROMagar Microbiology, Paris, France) media for the detection of Shiga toxin-producing Escherichia coli (STEC) was assessed with 329 stool specimens collected over 14 months from patients with suspected STEC infections (June 2011 to August 2012). The CHROMagar STEC medium, after an enrichment broth step, allowed the recovery of the STEC strain from 32 of the 39 (82.1%) Shiga toxin-positive stool specimens, whereas the standard procedure involving Drigalski agar allowed the recovery of only three additional STEC strains. The isolates that grew on CHROMagar STEC medium belonged to 15 serotypes, including the prevalent non-sorbitol-fermenting (NSF) O157:H7, O26:H11, and O104:H4 serotypes. The sensitivity, specificity, and positive and negative predictive values for the CHROMagar STEC medium were between 89.1% and 91.4%, 83.7% and 86.7%, 40% and 51.3%, and 98% and 98.8%, respectively, depending on whether or not stx-negative eae-positive E. coli was considered atypical enteropathogenic E. coli (EPEC) or STEC that had lost Shiga toxin genes during infection. In conclusion, the good performance of CHROMagar STEC agar medium, in particular, the high negative predictive value, and its capacity to identify NSF O157:H7 as well as common non-O157 STEC may be useful for clinical bacteriology, public health, and reference laboratories; it could be used in addition to a method targeting Shiga toxins (detection of stx genes by PCR, immunodetection of Shiga toxins in stool specimens, or Vero cell cytotoxicity assay) as an alternative to O157 culture medium. This combined approach should allow rapid visualization of both putative O157 and non-O157 STEC colonies for subsequent characterization, essential for real-time surveillance of STEC infections and investigations of outbreaks.