Clonal diversity of Shiga toxin-producing Escherichia coli O157:H7/H- in Germany--a ten-year study

Two hundred and ten E. coli O157:H7/H- strains isolated from single cases and outbreaks of diarrhoea and haemolytic uraemic syndrome (HUS) in Germany between 1988 and 1998 were characterised by a range of molecular subtyping methods and phage typing in order to analyse their clonal nature. A high clonal heterogeneity, together with a considerable clonal stability, has been identified among the bacterial isolates and no single clonal type appeared to be geographically dominant. It is recommended to apply pulsed-field gel electrophoresis (PFGE) together with P gene profile determination (number and genomic positions of lambdoid bacteriophages) as laboratory tools for an extended epidemiological surveillance of E. coli OOFF phage typing will remain helpful as a first line of analysis, particularly in outbreak situations.     

Restriction endonuclease analysis of RAPD-PCR amplicons derived from Shiga-like toxin-producing Escherichia coli O157 isolates

Shiga-like toxin-producing Escherichia coli O157 isolates were characterised by random amplification of polymorphic DNA by PCR (RAPD-PCR) analysis developed to allow robust epidemiological typing of E. coli. Amplification with primer 1247 or 1290 generated a reproducible profile, but was not capable of distinguishing sufficiently between epidemiologically unrelated strains. Subsequent digestion of the amplicons with selected restriction endonucleases improved the discriminatory ability of this method for strains showing limited differentiation following RAPD-PCR analysis alone. Restriction endonuclease analysis of RAPD-PCR fragments generated from closely related strains has the potential to provide additional discriminatory information without loss of specificity.     

Pathogenic potential of emergent sorbitol-fermenting Escherichia coli O157:NM

Non-sorbitol-fermenting (NSF) Escherichia coli O157:H7 is the primary Shiga toxin-producing E. coli (STEC) serotype associated with human infection. Since 1988, sorbitol-fermenting (SF) STEC O157:NM strains have emerged and have been associated with a higher incidence of progression to hemolytic-uremic syndrome (HUS) than NSF STEC O157:H7. This study investigated bacterial factors that may account for the increased pathogenic potential of SF STEC O157:NM. While no evidence of toxin or toxin expression differences between the two O157 groups was found, the SF STEC O157:NM strains adhered at significantly higher levels to a human colonic cell line. Under the conditions tested, curli were shown to be the main factor responsible for the increased adherence to Caco-2 cells. Notably, 52 of 66 (79%) European SF STEC O157:NM strains tested bound Congo red at 37^οC and this correlated with curli expression. In a subset of strains, curli expression was due to increased expression from the csgBAC promoter that was not always a consequence of increased csgD expression. The capacity of SF STEC O157:NM strains to express curli at 37^οC may have relevance to the epidemiology of human infections as curliated strains could promote higher levels of colonization and inflammation in the human intestine. In turn, this could lead to increased toxin exposure and an increased likelihood of progression to HUS.     

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.     

Use of the duplex TaqMan PCR system for detection of Shiga-like toxin-producing Escherichia coli O157

Real-time PCR assays have been applied for the detection and quantification of pathogens in recent years. In this study two combinations of primers and fluorescent probes were designed according to the sequences of the rfb(Escherichia coli O157) and stx2 genes. Analysis of 217 bacterial strains demonstrated that the duplex real-time PCR assay successfully distinguished the Escherichia coli O157 serotype from non-E. coli O157 serotypes and that it provided an accurate means of profiling the genes encoding O antigen and Shiga-like toxin 2. On the other hand, bacterial strains that lacked these genes were not detected by this assay. The quantitative ranges of the real-time PCR assay for these two genes were linear for DNA concentrations ranging from 10(3) to 10(9) CFU/ml of E. coli O157:H7 in pure culture and milk samples. The real-time PCR allowed the construction of standard curves that facilitated the quantification of E. coli O157:H7 in feces and apple juice samples. The detection sensitivity of the real-time PCR assay ranged from 10(4) to 10(9) CFU/g (or 10(4) to 10(9) CFU/ml) for feces and apple juice and 10(5) to 10(9) CFU/g for the beef sample without enrichment. After enrichment of the food samples in a modified tryptic soy broth, the detection range was from 10(0) to 10(3) CFU/ml. The real-time PCR assays for rfb(E. coli) (O157) and stx2 proved to be rapid tests for the detection of E. coli O157 in food matrices and could also be used for the quantification of E. coli O157 in foods or fecal samples.     

Identification and distribution of the enterohemorrhagic Escherichia coli factor for adherence (efa1) gene in sorbitol-fermenting Escherichia coli O157: H-

Sorbitol-fermenting (SF) Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H- strains are emerging as causes of hemorrhagic colitis and the hemolytic-uremic syndrome in Europe. Using subtractive hybridization between SF STEC O157:H- strain 493/89 and STEC O157:H7 strain EDL933, three different fragments, of approximately 700 bp in length, were identified. Each demonstrated > 99% homology to genes encoding the enterohemorrhagic E. coli factor for adherence (efa1) and lymphostatin (lifA). Therefore, a cosmid library was constructed from SF STEC O157:H- strain 493/89, and one clone containing these fragments was sequenced. This sequencing demonstrated a 9669-bp open reading frame (ORF) that had 99.9% sequence homology to efa1 of STEC O111:H- strain E45035 and to lifA of an enteropathogenic E. coli O127:H6 strain E2348/69. In STEC O157:H7 strain EDL933, only small (ca. 3 kb) initial and terminal fragments of this ORF are present. PCR analysis with primers complementary to the efa1/lifA sequence of strain 493/89 indicated that the complete sequence is present in each of 10 SF STEC O157:H- isolates but in none of 10 STEC O157:H7 strains investigated. The presence of the complete efa1/lifA also in both tested E. coli O55:H7 strains supports the hypothesis that SF STEC O157:H- are phylogenetically closer to the proposed E. coli O55:H7 ancestor than STEC O157:H7. Our data demonstrate the presence of a potential virulence gene in SF STEC O157:H- that is only rudimentarily present in STEC O157:H7.     

Complete sequence of the large virulence plasmid pSFO157 of the sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H- strain 3072/96

The large virulence plasmid pSFO157 of sorbitol-fermenting E. coli O157:H(-) strain 3072/96 has a size of 121,239bp and contains 96 open reading frames >50bp. It is therefore 29,162bp larger (ca. 32%) than plasmid pO157 of E. coli O157:H7 strain EDL933. Major differences between the plasmids are the absence of katP, espP, and toxB in pSFO157 and, instead of these, the presence of the sfp fimbriae gene cluster and a large part of an F-plasmid transfer region, the latter accounting for most of the additional DNA. The differences in the order of the genes and their composition, as well as the presence of a number of replication-associated genes and mobile genetic elements suggests that the large E. coli O157 virulence plasmids have a complex evolutionary origin.     

Molecular detection of sorbitol-fermenting Escherichia coli O157 in patients with hemolytic-uremic syndrome.

Shiga-like toxin-producing Escherichia coli strains of serogroup O157 were identified in 26 of 104 patients with hemolytic-uremic syndrome and in 18 of 668 patients with diarrhea. All strains were identified by colony hybridization with DNA probes complementary to Shiga-like toxin I and Shiga-like toxin II gene sequences and characterized by biochemical tests and serotyping. Seventeen of these 44 patients had E. coli O157 strains which were unusual because they fermented sorbitol within 24 h of incubation and were positive for beta-glucuronidase activity. Culture filtrates of these sorbitol-fermenting strains were highly toxic to Vero cells in culture. Serological tests and DNA analysis performed by restriction endonuclease digestion of B-subunit toxin genes revealed that all 17 isolates produced Shiga-like toxin II. Although by using molecular probes we established a high frequency of sorbitol-fermenting E. coli O157 strains in the patients we examined, further studies on the prevalence of such isolates in other areas of endemic disease are clearly warranted.     

Phylogeny, clinical associations, and diagnostic utility of the pilin subunit gene (sfpA) of sorbitol-fermenting, enterohemorrhagic Escherichia coli O157:H-

The plasmid-borne sfpA gene encodes the pilin subunit in sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H-. We investigated the distribution of sfpA among 600 E. coli isolates comprising the complete E. coli standard reference (ECOR) and diarrheagenic E. coli (DEC) strain collections and clinical isolates associated with enteric disease. sfpA was detected in DEC3F SF EHEC O157:H- strain 493/89, each of 107 SF EHEC O157:H- clinical isolates, and 14 Shiga toxin-negative SF E. coli O157:H- strains which contained eae, which encodes gamma-intimin, and fliC, which encodes the H7 antigen. sfpA was absent from all other strains, including the ECOR strain collection, all non-SF EHEC O157:H7 strains, and all E. coli O55:H7 strains (E. coli O55:H7 is the postulated ancestor of Shiga toxin-producing E. coli [STEC] O157). These results suggest that there was a single acquisition of the sfpA gene in the nonmotile SF E. coli O157 branch, presumably after the eae-encoding pathogenicity island (the locus of enterocyte effacement) was acquired and motility was lost. We then applied the sfpA PCR in combination with rfbO157, stx, and eae PCRs to screen 636 stool samples from patients with diarrhea or hemolytic-uremic syndrome for SF STEC O157:H-. In 27 cases, the simultaneous presence of the sfpA, eae, and rfbO157 amplicons indicated the presence of SF E. coli O157:H- strains, and the result was subsequently confirmed by isolation. All but two of these strains possessed stx2. None of the other stool samples was positive by the sfpA PCR; 59 of these stool samples contained EHEC O157:H7. The sfpA gene can be recommended as a target for screening for SF E. coli O157:H-.     

Cattle can be a reservoir of sorbitol-fermenting shiga toxin-producing Escherichia coli O157:H(-) strains and a source of human diseases

Using the immunomagnetic separation procedure, we isolated sorbitol-fermenting (SF) Shiga toxin-producing Escherichia coli (STEC) O157:H(-) strains from two patients, one with hemolytic-uremic syndrome and the other with diarrhea, and from a dairy cow epidemiologically associated with the patients. The phenotypic and genotypic characteristics of all isolates were identical or closely related. Moreover, the bovine isolate showed a clonal relatedness to SF STEC O157:H(-) strains isolated from patients in Germany and the Czech Republic from 1988 to 1998. This is the first evidence that cattle can be a reservoir of SF STEC O157:H(-) and a source of human diseases.     

Phenotypic and molecular analysis of tellurite resistance among enterohemorrhagic Escherichia coli O157:H7 and sorbitol-fermenting O157:NM clinical isolates

A total of 66 (98.5%) of 67 enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains had increased potassium tellurite (Te) MICs (32 to 1,024 microg/ml), grew on Te-containing media, and possessed Te resistance (ter) genes, whereas 83 (96.5%) of 86 sorbitol-fermenting (SF) EHEC O157:NM strains had Te MICs of 相似文献   

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.     

Urease genes in non-O157 Shiga toxin-producing Escherichia coli: mostly silent but valuable markers for pathogenicity

The distribution of ureC was investigated among 294 Escherichia coli isolates, comprising 72 strains from the E. coli standard reference collection (ECOR), 62 strains from the diarrhoeagenic E. coli (DEC) collection, and 160 clinical isolates of Shiga toxin-producing E. coli (STEC). The ureC gene was more frequent among STEC isolates harbouring eae than among those lacking eae (p < 0.0001). All clinical STEC isolates of serogroups O111 and O145 contained ureC, but only two of 294 isolates expressed urease activity. The silencing of urease expression could not be linked to a stop codon in ureD. The frequent occurrence of ure genes in eae-positive STEC isolates makes them valuable markers for virulence.     

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.     

Characterization of Shiga toxin-producing Escherichia coli O157:H7 isolated in Italy and in France

Twenty-one Escherichia coli O157:H7 strains isolated in northern Italy from sporadic cases of hemolytic-uremic syndrome and from cattle and food were characterized by virulence gene analysis, pulsed-field gel electrophoresis (PFGE) of XbaI-digested DNA, enterobacterial repetitive intergenic consensus (ERIC) sequence-based PCR (ERIC-PCR), and antibiotic resistance patterns and compared to 18 strains isolated in France from human cases of diarrhea, cattle, and the environment. Strains isolated in Sicily (southern Italy) from a local farm (one strain) and from calves just imported from France (11 strains) and Spain (six strains) were also typed. Whereas the eae and hlyA genes were always detected, Shiga toxin gene (stx) analysis showed some differences related to geographic areas. Isolates from northern Italy showed a high frequency of stx(1) and stx(2), while strains isolated in France and from French and Spanish calves imported to Sicily more frequently possessed the stx(2c) gene. The majority of the strains isolated in northern Italy were also resistant to one or more antibiotics, while most of the strains isolated in France and Sicily were fully susceptible. ERIC-PCR analysis was not able to differentiate the strains. PFGE typing after XbaI DNA digestion produced a total of 54 distinct restriction endonuclease digestion profiles (REDPs) among the 57 strains. Phylogenetic analysis was unable to cluster REDPs according to geographic origin. All epidemiologically related isolates showed either identical or >/=91% similar REDPs. Our findings suggest a peculiar circulation of antibiotic-resistant, genetically unrelated strains in northern Italy.     

Molecular epidemiology of Escherichia coli O157:H7 by restriction fragment length polymorphism using Shiga-like toxin genes.

The purpose of this study was to assess a simplified method for interstrain differentiation of Escherichia coli O157:H7 and other Shiga-like toxin-producing E. coli (SLTEC) strains. A method based on the use of nucleic acid probes from Shiga-like toxin (SLT) I and II structural genes was used to generate restriction fragment length polymorphism (RFLP) patterns of SLTEC strains, (SLT-RFLP patterns) resulting from digestion of isolated genomic DNA with four different restriction enzymes (BamHI, EcoRI, HindIII, and PvuII) used separately. A total of 165 SLTEC strains from clinical, food, and environmental sources, including O157:H7 isolates from four food-borne outbreaks in Canada and the United States, were analyzed in the study. SLT-RFLP demonstrated that E. coli O157:H7 strains from each food-borne outbreak had the same unique SLT-RFLP pattern. Fifty-two SLT-RFLP types were found among 96 E. coli O157:H7 isolates from sporadic cases of hemorrhagic colitis and hemolytic uremic syndrome in Washington state. The use of the SLT probes proved to be a very powerful method for interstrain differentiation of SLTEC strains. Although the use of each of the enzymes alone did not give enough differentiative power to be used in epidemiological studies, the combination of patterns generated by two restriction enzymes (EcoRI and PvuII, used separately) provided the desired sensitivity for such studies. The results clearly demonstrate the usefulness of the method for studying the molecular epidemiology of E. coli O157:H7. The method is also suitable for establishing an epidemiological database, in terms of both sensitivity and ease of compilation and interpretation of results.     

Use of ramification amplification assay for detection of Escherichia coli O157:H7 and other E. coli Shiga toxin-producing strains

Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) strains are important human pathogens that are mainly transmitted through the food chain. These pathogens have a low infectious dose and may cause life-threatening illnesses. However, detection of this microorganism in contaminated food or a patient's stool specimens presents a diagnostic challenge because of the low copy number in the sample. Often, a more sensitive nucleic acid amplification method, such as PCR, is required for rapid detection of this microorganism. Ramification amplification (RAM) is a recently introduced isothermal DNA amplification technique that utilizes a circular probe for target detection and achieves exponential amplification through the mechanism of primer extension, strand displacement, and ramification. In this study, we synthesized a circular probe specific for the Shiga toxin 2 gene (stx(2)). Our results showed that as few as 10 copies of stx(2) could be detected, indicating that the RAM assay was as sensitive as conventional PCR. We further tested 33 isolates of E coli O157:H7, STEC, Shigella dysenteriae, and nonpathogenic E. coli by RAM assay. Results showed that all 27 STEC isolates containing the stx(2) gene were identified by RAM assay, while S. dysenteriae and nonpathogenic E. coli isolates were undetected. The RAM results were 100% in concordance with those of PCR. Because of its simplicity and isothermal amplification, the RAM assay could be a useful method for detecting STEC in food and human specimens.     

Cytolethal distending toxin gene cluster in enterohemorrhagic Escherichia coli O157:H- and O157:H7: characterization and evolutionary considerations

We identified a cytolethal distending toxin (cdt) gene cluster in 87, 6, and 0% of sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H(-), EHEC O157:H7, and E. coli O55:H7/H(-) strains, respectively. The toxin was expressed by the wild-type EHEC O157 strains and by a cdt-containing cosmid from a library of SF EHEC O157:H(-) strain 493/89. The cdt flanks in strain 493/89 were homologous to bacteriophages P2 and lambda. Our data demonstrate that cdt, encoding a potential virulence factor, is present in the EHEC O157 complex and suggest that cdt may have been acquired by phage transduction.