Detection of Escherichia coli O157:H7 from Musca domestica (Diptera: Muscidae) at a cattle farm in Japan

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 was isolated for the first time from Musca domestica L. A total of 310 fly samples was collected from 4 different farms in Obihiro-City, Hokkaido, in the summer and autumn of 1997;5 samples carried E. coli serotype O157:H7. Using ELISA and Vero cell cytotoxicity assay, 3 isolates from 1 cattle farm produced both active Shiga-toxin type 1 (Stx1) and 2 (Stx2). These isolates also carried hemolysin and eaeA genes and harbored the 90-kb virulence plasmid of EHEC O157:H7. Based on plasmid profiles, antibiotic patterns, polymerase chain reaction (PCR)-based DNA finger printing analysis using random amplified polymorphic DNA, pulsed field gel electrophoresis analysis, and DNA sequences of stx1 and stx2, all 3 isolates from fly samples were identical. These results indicate that the house fly is capable of carrying the toxigenic EHEC O157:H7 involved in human disease.     

Multivariate analyses revealed distinctive features differentiating human and cattle isolates of Shiga toxin-producing Escherichia coli O157 in Japan

Genotypes of Shiga toxin-producing Escherichia coli (STEC) O157 isolated from humans and cattle were analyzed by uni- and multivariable logistic regression, and population structure methods, to gain insight into transmission and the nature of human infection. Eleven genotyping assays, including PCR typing of five virulence factors (stx(1), stx(2), stx(2c), eae, and ehxA) and a lineage-specific polymorphism assay using six markers (LSPA6), were considered in the analyses. The prevalence of the stx(1), stx(2), and stx(2c) virulence factors was significantly different between human and cattle isolates. However, multivariable regression revealed that the presence of only the stx(2) gene was significantly associated with human isolates after controlling for confounding effects. LSPA6 typing demonstrated an apparent difference in the distribution of LSPA6 lineages between human and cattle isolates and a strong association between stx genotypes and LSPA6 genotypes. Population genetics tools identified three genetically distinct clusters of STEC O157. Each cluster was characterized by stx genotypes and LSPA6 genotypes. The human isolates typically comprised LSPA6 lineage I with stx(1) stx(2) strains and LSPA6 lineage I/II with stx(2c) or stx(2) stx(2c) strains [corrected]. In contrast, the cattle isolates comprised LSPA6 lineage II strains withstx(2c) or stx(1) stx(2c) strains [corrected] in addition to the clusters identified for the human isolates. Our analyses provide new evidence that the stx(2) gene is the most distinctive feature in human isolates compared to cattle isolates in Japan, and only a subset of the genetically diverse population isolated from cattle is involved in human illnesses. Our results may contribute to international comparisons and risk assessments of STEC O157.     

Isolation of a lysogenic bacteriophage carrying the stx(1(OX3)) gene, which is closely associated with Shiga toxin-producing Escherichia coli strains from sheep and humans.

A specific PCR for the detection of a variant of the gene encoding Shiga toxin 1 (stx(1)) called stx(1(OX3)) (GenBank accession no. Z36901) was developed. The PCR was used to investigate 148 Stx(1)-producing Escherichia coli strains from human patients (n = 72), cattle (n = 27), sheep (n = 48), and a goat (n = 1) for the presence of the stx(1(OX3)) gene. The stx(1(OX3)) gene was present in 38 Shiga toxin-producing E. coli (STEC) strains from sheep belonging to serogroups O5, O125, O128, O146, and OX3 but was absent from Stx(1)-positive ovine STEC O91 strains. The stx(1(OX3)) gene was also detected in 22 STEC strains from humans with nonbloody diarrhea and from asymptomatic excreters. Serotypes O146:H21 and O128:H2 were most frequently associated with stx(1(OX3))-carrying STEC from sheep and humans. In contrast, Stx(1)-producing STEC strains from cattle and goats and 50 STEC strains from humans were all negative for the stx(1(OX3)) gene. The stx(1(OX3))-negative strains belonged to 13 serotypes which were different from those of the stx(1(OX3))-positive STEC strains. Moreover, the stx(1(OX3)) gene was not associated with STEC belonging to enterohemorrhagic E. coli (EHEC) serogroups O26, O103, O111, O118, O145, and O157. A bacteriophage carrying the stx(1(OX3)) gene (phage 6220) was isolated from a human STEC O146:H21 strain. The phage was able to lysogenize laboratory E. coli K-12 strain C600. Phage 6220 shared a similar morphology and a high degree of DNA homology with Stx(2)-encoding phage 933W, which originates from EHEC O157. In contrast, few similarities were found between phage 6220 and Stx(1)-encoding bacteriophage H-19B from EHEC O26.     

stx genotype and molecular epidemiological analyses of Shiga toxin-producing Escherichia coli O157:H7/H? in human and cattle isolates

The relationship between human diseases caused by infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) O157 strains and O157 strains isolated from cattle was investigated in an area where stockbreeding is prolific. For this purpose, the stx genotypes, the molecular epidemiological characteristics of 268 STEC O157 strains including 211 human-origin strains and 57 cattle-origin strains, and clinical manifestations of 210 STEC-infected people were analyzed. Of 211 human-origin strains, 92 strains (44%) were of the stx1/stx2 genotype, and 74 strains (35%) were of the stx2c genotype. Most of the people infected with stx2c genotype strains presented no symptoms or mild symptoms such as slight diarrhea, except for 3 patients with bloody diarrhea. Of the 57 cattle-origin strains, 27 strains (47%) were of the stx2c genotype and 17 strains (30%) were of the stx1/stx2 genotype. Pulsed-field gel electrophoresis (PFGE) and insertion sequence (IS) analysis demonstrated that 11 isolates (41%) of the 27 cattle isolates of the stx2c genotype had high homology (>95% identity) with human isolates. These results suggest that some genetic patterns of the stx2c genotype strains might be preserved in cattle or their surrounding environment for several years, and during these periods, they might have opportunities to infect people through various routes. Because of the mild virulence of the stx2c genotype strains, they seemed to be transmitted asymptomatically from cattle to humans and then spread from person to person. It may be a public health concern. Further, they occasionally cause severe symptoms in humans; therefore, caution is warranted for infections by stx2c genotype O157 strains, in addition to stx2-possessing genotype O157 strains.     

Influence of RecA on in vivo virulence and Shiga toxin 2 production in Escherichia coli pathogens.

The enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains 933 and 86-24 as well as the uropathogenic E. coli (UPEC) strain 536 were compared with their isogenic rec A mutants and rec A trans -complemented strains in intravenous lethality and lung toxicity assays in mice. While the wild-type EHEC strains were fully virulent, the virulence of the rec A mutants was strongly reduced. Complementation of the EHEC rec A mutants with the cloned E. coli recA gene restored their virulence capacity. The stx2EHEC mutant TUV86-2 as well as its isogenic rec A mutant were completely avirulent in both assays. In contrast, RecA had no influence on the virulence of UPEC strain 536. We conclude that the lethality observed with EHEC is presumably mainly due to Shiga toxin, which is severely down-regulated in the rec A mutants as a result of lacking spontaneous phage induction. Therefore, the EHEC rec A+strains 933 and 86-24 were compared for their Shiga toxin 2 (Stx2) production with the respective rec A-counterparts. The rec A mutants of the EHEC strains were significantly reduced in toxin synthesis and were devoid of Stx2 specific phage production. Complementation of the EHEC rec A mutants with the cloned rec A gene enabled the rec A mutants to restore toxin and phage production. These results suggest that the higher level of Stx2 synthesis in the EHEC strains is the result of a higher level of spontaneous Stx2 specific phage induction, which is controlled by RecA.     

Clinical Escherichia coli strains carrying stx genes: stx variants and stx-positive virulence profiles

Altogether, 173 Shiga toxin-producing Escherichia coli (STEC) serotype O157 (n = 111) and non-O157 (n = 62) isolates from 170 subjects were screened by PCR-restriction fragment length polymorphism for eight different stx genes. The results were compiled according to serotypes, phage types of O157, production of Stx toxin and enterohemolysin, and the presence of eae. The stx genes occurred in 11 combinations; the most common were stx(2) with stx(2c) (42%), stx(2) alone (21%), and stx(1) alone (16%). Of the O157 strains, 64% carried stx(2) with stx(2c) versus 2% of the non-O157 strains (P < 0.001). In the non-O157 strains, the prevailing gene was stx(1) (99% versus 1% in O157 strains; P < 0.001). In addition, one strain (O Rough:H4:stx(2c)) which has not previously been described as associated with hemolytic-uremic syndrome (HUS) was found. Ten stx-positive virulence profiles were responsible for 71% of all STEC infections. Of these profiles, five accounted for 71% of the 21 strains isolated from 20 patients with HUS or thrombotic thrombocytopenic purpura (TTP). The strains having the virulence profile that caused mainly HUS or TTP or bloody diarrhea produced Stx with titers of >/=1:128 (90%) more commonly than did other strains (51%; P < 0.001). These strains were also more commonly enterohemolytic (98% versus 68% for other strains; P < 0.001) and possessed the eae gene (100%) more commonly than did other strains (74%; P < 0.001). A particular virulence profile, O157:H7:PT2:stx(2):stx(2c):eae:Ehly, was significantly more frequently associated with HUS and bloody diarrhea than were other profiles (P = 0.02) and also caused the deaths of two children. In this study, the risk factors for severe symptoms were an age of <5 years and infection by the strain of O157:H7:PT2 mentioned above.     

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 and phenotypic profiling of sorbitol-fermenting Escherichia coli O157:H– human isolates from Finland

This study investigated the occurrence of virulence-associated genes, including stx1, stx2, stx2c, stx2d, stx2e, eae and its subtypes (alpha, beta, gamma, epsilon), efa1, cdt-V cluster, enterohaemorrhagic Escherichia coli (EHEC)-hlyA, katP, espP, etpD, sfpA and the flagellar fliC gene, in nine sorbitol-fermenting (SF), beta-glucuronidase-positive E. coli O157:H- (non-motile) isolates obtained from humans in Finland between 1997 and 2001. In addition, the production of Shiga toxin (Stx), cytolethal distending toxin (CDT)-V and EHEC haemolysin (EHEC-Hly) was studied, and the phage type (PT) and pulsed-field gel electrophoresis (PFGE) types were determined. All nine isolates carried eae-gamma, efa1, EHEC-hlyA, etpD, sfpA and fliC; eight also harboured the cdt-V gene cluster and five were positive for stx2. None of the isolates harboured stx1, stx2c, stx2d, stx2e, katP or espP. All isolates harbouring the corresponding genes also produced Stx2 and CDT-V in titres ranging from 1:32 to 1:128 and from 1:2 to 1:4, respectively. None of the isolates expressed EHEC-Hly on enterohaemolysin agar. Seven isolates belonged to PT88 and two had a PT88 variant pattern. Seven isolates showed a close genetic relationship, with a PFGE similarity index (SI) of 92-98%. Two isolates, temporally the first and last, obtained 5 years apart, were the most divergent (SI of 71% and 85%, respectively). The study demonstrated that SF E. coli O157:H- isolates from Finland are closely related and show a close relationship with SF E. coli O157 strains isolated in Germany. This finding suggests a clonality of SF E. coli O157:H- isolates from different geographical regions.     

Enterohemorrhagic Escherichia coli (EHEC) strains of serogroup O118 display three distinctive clonal groups of EHEC pathogens

A recent case report of a child infected with enterohemorrhagic Escherichia coli (EHEC) of serotype O118:H16 in Bavaria, in association with the isolation of a bovine O118 strain on the same farm (A. Weber, H. Klie, H. Richter, P. Gallien, M. Timm, and K. W. Perlberg, Berl. Muench. Tieraerztl. Wochenschr. 110:211-213, 1997), prompted us to investigate the relationship between bovine and human strains of serogroup O118. A total of 29 human O118 E. coli strains from Europe (21), Canada (4), and Peru (4) were compared by virulence typing and macrorestriction analysis with 7 bovine O118 EHEC strains isolated in Bavaria. Twenty-five of the human strains were characterized as EHEC. By serotyping and determination of the virulence-associated factors Shiga toxin (stx1 stx2 stx2 variants), intimin (eae), and EHEC hemolysin (Hly(EHEC)), three distinctive groups of O118 human pathogens were identified. Most of the strains belonged to serotype O118:H16, displaying the virulence traits Stx1, intimin, Hly(EHEC), and EspP/PssA (group 1). In addition, we identified strains of serotype O118:H12 (Stx2d only; group 2) and of serotype O118:H30 (Stx2 and intimin; group 3). Macrorestriction analysis with BlnI and XbaI revealed that all strains with a single O118 serotype profile (O118:H12, O118:H16, and O118:H30) belonged to one clonal cluster, irrespective of their origin. Group 1 strains clustered in the same clonal group as the bovine O118:H16 strains. Moreover, four pairs of strains of different origins and indistinguishable by all other methods applied were identified as group 1 strains. Our data support the direct transmission of an EHEC O118:H16 strain from a calf to a 2-year-old boy in the above-mentioned case report. Since bovine and human O118:H16 strains represent the same clones, they must be considered zoonotic EHEC pathogens. In contrast, EHEC strains of serotypes O118:H12 and O118:H30 have been isolated only from humans, indicating a reservoir for certain human O118 EHEC strains other than bovines.     

Structural and functional differences between disease-associated genes of enterohaemorrhagic Escherichia coli O111

We analysed 72 clinical isolates of enterohaemorrhagic Escherichia coli (EHEC) O111 from patients with diarrhoea or haemolytic uraemic syndrome (HUS) isolated during the period from 1955 to 2005 and identified six motile strains (flagellar antigens 8, 10 and 11); the remaining 66 (92%) were nonmotile (NM) and could not be typed by conventional H serotyping. To improve subtyping methodologies, we determined genotypes of the flagellin-encoding fliC. Three fliC genotypes were found which were identical to those of motile EHEC O111 with H antigens 8, 10 and 11 and designated fliC(H8), fliC(H10) and fliC(H11). The IS629 insertion element was present, identically located, in six epidemiologically unrelated isolates with fliC(H8). The prevalence of the fliC genotypes in the 72 EHEC O111 strains were fliC(H8) (89%), fliC(H10) (7%) and fliC(H11) (4%). Within these fliC genotypes, a high degree of homogeneity for the presence of disease-associated genes was found. The adhesins-encoding genes eae and efa-1 were present in all strains with fliC(H8) and fliC(H11), but absent from strains with fliC(H10). The latter strains have not been reported previously. Strains with fliC(H10) and fliC(H11), but not those with fliC(H8), retained intact cadA and cadC loci and decarboxylated lysine. Three different stx genotypes including stx(1), stx(2) and stx(1)/stx(2) were determined among the 72 EHEC O111. We observed a significant increase over time in the frequency of strains harbouring both stx(1) and stx(2). The presence of stx(2) both alone and in combination with stx(1) was significantly (chi(2)=23.16, P<0.00001, CI(95) [2.29; 9.76]) associated with HUS. Therefore, the emergence of EHEC O111 should be monitored carefully. We conclude that EHEC O111 strains can be differentiated using specific loci required for motility, adherence, Stx production, and lysine decarboxylation. The divergence within EHEC O111 makes it possible to subtype these emerging pathogens in the laboratory thereby providing a basis for further investigations into their ecological niches and survival capabilities.     

志贺毒素阴性的大肠埃希菌O157菌株的毒力因子检测

目的 研究志贺毒素阴性的大肠埃希菌0157血清群菌株可能携带的毒力因子,以确定其致病群类型。方法 对8株分离自杭州的志贺毒素阴性的大肠埃希菌0157血清群菌株及分离自美国和我国江苏的EHEC0157菌株,应用PCR技术检测stx、hly、EPEC和EHEC共有eaeA、EHEC特异的eaeA,ELISA检测ETEC的耐热和不耐热解毒素,HEP－2细胞粘附试验测定细菌与上皮细胞的粘附能力,并进行菌株质粒图谱分析。结果 8株志贺毒素阴性的大肠埃希菌0157菌株均未检出stx、hly、EHEC特异的eaeA以及ETEC的耐热和不耐热肠毒素,仅其中2株菌株携有EPEC和EHEC共有eaeA,另3株菌株对HEP－2细胞有集聚粘附能力。结论 目前我国检获的大肠埃希菌0157菌株其致病类型表现为多样性,部分菌株尚不能明确其毒力因子。EHEC0157的鉴定必须重视毒力因子的检测。     

Serotypes, virulence genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from human patients: prevalence in Lugo, Spain, from 1992 through 1999

We have analyzed the prevalence of Shiga toxin-producing Escherichia coli (STEC) in stool specimens of patients with diarrhea or other gastrointestinal alterations from the Xeral-Calde Hospital of Lugo City (Spain). STEC strains were detected in 126 (2.5%) of 5,054 cases investigated, with a progressive increase in the incidence from 0% in 1992 to 4.4% in 1999. STEC O157:H7 was isolated in 24 cases (0.5%), whereas non-O157 STEC strains were isolated from 87 patients (1.7%). STEC strains were (after Salmonella and Campylobacter strains) the third most frequently recovered enteropathogenic bacteria. A total of 126 human STEC isolates were characterized in this study. PCR showed that 43 (34%) isolates carried stx(1) genes, 45 (36%) possessed stx(2) genes and 38 (30%) carried both stx(1) and stx(2). A total of 88 (70%) isolates carried an ehxA enterohemolysin gene, and 70 (56%) isolates possessed an eae intimin gene (27 isolates with type gamma1, 20 with type beta1, 8 with type zeta, 5 with type gamma2, and 3 with type epsilon). STEC isolates belonged to 41 O serogroups and 66 O:H serotypes, including 21 serotypes associated with hemolytic uremic syndrome and 30 new serotypes not previously reported among human STEC strains in other studies. Although the 126 STEC isolates belonged to 81 different seropathotypes (associations between serotypes and virulence genes), only four accounted for 31% of isolates. Seropathotype O157:H7 stx(1) stx(2) eae-gamma1 ehxA was the most common (13 isolates) followed by O157:H7 stx(2) eae-gamma1 ehxA (11 isolates), O26:H11 stx(1) eae-beta1 ehxA (11 isolates), and O111:H- stx(1) stx(2) eae-gamma2 ehxA (4 isolates). Our results suggest that STEC strains are a significant cause of human infections in Spain and confirm that in continental Europe, infections caused by STEC non-O157 strains are more common than those caused by O157:H7 isolates. The high prevalence of STEC strains (both O157:H7 and non-O157 strains) in human patients, and their association with serious complications, strongly supports the utilization of protocols for detection of all serotypes of STEC in Spanish clinical microbiology laboratories.     

Genetic diversity among Escherichia coli O157:H7 isolates and identification of genes linked to human infections

An Escherichia coli oligonucleotide microarray based on three sequenced genomes was validated for comparative genomic microarray hybridization and used to study the diversity of E. coli O157 isolates from human infections and food and animal sources. Among 26 test strains, 24 (including both Shiga toxin [Stx]-positive and -negative strains) were found to be related to the two sequenced E. coli O157:H7 strains, EDL933 and Sakai. However, these strains showed much greater genetic diversity than those reported previously, and most of them could not be categorized as either lineage I or II. Some genes were found more often in isolates from human than from nonhuman sources; e.g., ECs1202 and ECs2976, associated with stx2AB and stx1AB, were in all isolates from human sources but in only 40% of those from nonhuman sources. Some (but not all) lineage I-specific or -dominant genes were also more frequently associated with isolates from human. The results suggested that it might be more effective to concentrate our efforts on finding markers that are directly related to infection rather than those specific to certain lineages. In addition, two Stx-negative O157 cattle isolates (one confirmed to be H7) were significantly different from other Stx-positive and -negative E. coli O157:H7 strains and were more similar to MG1655 in their gene content. This work demonstrates that not all E. coli O157:H7 strains belong to the same clonal group, and those that were similar to E. coli K-12 might be less virulent.     

Genotypic variations of Shiga toxin-converting phages from enterohaemorrhagic Escherichia coli O157: H7 isolates

Pulsed-field gel electrophoresis (PFGE) analysis revealed that enterohaemorrhagic Escherichia coli (EHEC) O157:H7 strains had considerable variations in their genomes. This study investigated whether or not the molecular profile of Shiga toxin (Stx) 1- and Stx2-converting phages isolated from EHEC O157:H7 strains, derived from various sources in the USA and Japan, corresponded to the variations of host strains' genotypes as determined by PFGE. A total of 51 Stx-converting phages including 12 Stx1-converting phages and 37 Stx2-converting phages was isolated from seven USA isolates and 20 Japanese isolates. The average Dice coefficient values showed 44% similarity between phage DNAs in Stx2-converting phages digested with SmaI and 55% in Stx1-converting phages digested with HindIII, indicating considerable variation among phage DNA. In particular, restriction fragment length polymorphism (RFLP) patterns of Stx2-converting phage DNA varied according to the PFGE type of their host strain, which suggests that the phage genomes have altered their genotypic characteristics with those of host genomes. However, there are several exceptions: the RFLP patterns of some Stx2-converting phages were quite similar irrespective of the different genotypes of the host strains, indicating that horizontal transfer of Stx2-converting phage may also occur under some circumstances.     

Identification, characterization, and distribution of a Shiga toxin 1 gene variant (stx(1c)) in Escherichia coli strains isolated from humans

By using sequence analysis of Shiga toxin 1 (Stx 1) genes from human and ovine Stx-producing Escherichia coli (STEC) strains, we identified an Stx1 variant in STEC of human origin that was identical to the Stx1 variant from ovine STEC, but demonstrated only 97.1 and 96.6% amino acid sequence identity in its A and B subunits, respectively, to the Stx1 encoded by bacteriophage 933J. We designated this variant "Stx1c" and developed stxB(1) restriction fragment length polymorphism and stx(1c)-specific PCR strategies to determine the frequency and distribution of stx(1c) among 212 STEC strains isolated from humans. stx(1c) was identified in 36 (17.0%) of 212 STEC strains, 19 of which originated from asymptomatic subjects and 16 of which were from patients with uncomplicated diarrhea. stx(1c) was most frequently (in 23 STEC strains [63.9%]) associated with stx(2d), but 12 (33.3%) of the 36 STEC strains possessed stx(1c) only. A single STEC strain possessed stx(1c) together with stx(2) and was isolated from a patient with hemolytic-uremic syndrome. All 36 stx(1c)-positive STEC strains were eae negative and belonged to 10 different serogroups, none of which was O157, O26, O103, O111, or O145. Stx1c was produced by all stx(1c)-containing STEC strains, but reacted weakly with a commercial immunoassay. We conclude that STEC strains harboring the stx(1c) variant account for a significant proportion of human STEC isolates. The procedures developed in this study now allow the determination of the frequency of STEC strains harboring stx(1c) among clinical STEC isolates and their association with human disease in prospective studies.     

Serotypes, virulence genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from cattle in Spain and identification of a new intimin variant gene (eae-xi)

A total of 514 Shiga toxin-producing Escherichia coli (STEC) isolates from diarrheic and healthy cattle in Spain were characterized in this study. PCR showed that 101 (20%) isolates carried stx(1) genes, 278 (54%) possessed stx(2) genes, and 135 (26%) possessed both stx(1) and stx(2). Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 326 (63%) and in 151 (29%) of the isolates, respectively. STEC isolates belonged to 66 O serogroups and 113 O:H serotypes (including 23 new serotypes). However, 67% were of one of these 15 serogroups (O2, O4, O8, O20, O22, O26, O77, O91, O105, O113, O116, O157, O171, O174, and OX177) and 52% of the isolates belonged to only 10 serotypes (O4:H4, O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2, and ONT:H19). Although the 514 STEC isolates belonged to 164 different seropathotypes (associations between serotypes and virulence genes), only 12 accounted for 43% of isolates. Seropathotype O157:H7 stx(2) eae-gamma1 ehxA (46 isolates) was the most common, followed by O157:H7 stx(1) stx(2) eae-gamma1 ehxA (34 isolates), O113:H21 stx(2) (25 isolates), O22:H8 stx(1) stx(2) ehxA (15 isolates), O26:H11 stx(1) eae-beta1 ehxA (14 isolates), and O77:H41 stx(2) ehxA (14 isolates). Forty-one (22 of serotype O26:H11) isolates had intimin beta1, 82 O157:H7 isolates possessed intimin gamma1, three O111:H- isolates had intimin type gamma2, one O49:H- strain showed intimin type delta, 13 (six of serotype O103:H2) isolates had intimin type epsilon and eight (four of serotype O156:H-) isolates had intimin zeta. We have identified a new variant of the eae intimin gene designated xi (xi) in two isolates of serotype O80:H-. The majority (85%) of bovine STEC isolates belonged to serotypes previously found for human STEC organisms and 54% to serotypes associated with STEC organisms isolated from patients with hemolytic uremic syndrome. Thus, this study confirms that cattle are a major reservoir of STEC strains pathogenic for humans.     

Shiga toxin 1c-producing Escherichia coli strains: phenotypic and genetic characterization and association with human disease

The distribution of the stx(1c) allele among Shiga toxin (Stx)-producing Escherichia coli (STEC) and the virulence characteristics of stx(1c)-harboring STEC are unknown. In this study, we identified stx(1c) in 76 (54.3%) of 140 eae-negative, but in none of 155 eae-positive, human STEC isolates (P < 0.000001). The 76 stx(1c)-harboring E. coli isolates belonged to 22 serotypes, and each produced Stx1c as demonstrated by latex agglutination. Characterization of putative virulence factors demonstrated the presence of the locus of proteolysis activity (LPA) and the high-pathogenicity island in 65.8 and 21.1%, respectively, of the 76 Stx1c-producing E. coli isolates. Moreover, all but three of these strains contained saa, the gene encoding an STEC autoagglutinating adhesin. The virulence profiles of Stx1c-producing E. coli isolates were mostly serotype independent and heterogeneous. This enabled us to subtype the isolates within the same serotype. The individuals infected with Stx1c-producing E. coli strains were between 3 months and 72 years old (median age, 23.5 years) and usually had uncomplicated diarrhea or were asymptomatic. We conclude that Stx1c-producing E. coli strains represent a significant subset of eae-negative human STEC isolates, which belong to various serotypes and frequently possess LPA and saa as their putative virulence factors. The phenotypic and molecular characteristics determined in this study allow the subtyping of Stx1c-producing STEC in epidemiological and clinical studies.     

Characterization of verotoxigenic Escherichia coli (VTEC) isolates from faeces of small ruminants and environmental samples in southern Jordan

This study was conducted to determine the prevalence rate of VTEC in slaughtered sheep and goats and to evaluate the contamination rate of VTEC in slaughterhouses and butchers' shops in southern Jordan. 201 E. coli isolates from animals' faecal samples and 33 E. coli isolates from slaughterhouse/butcher shop samples were characterized by multiplex PCR (mPCR) reaction for detection of stx1, stx2, eae A and E‐hly A virulent genes. Twenty‐six virulent E. coli isolates were characterized by mPCR to seven different virulent patterns: stx1, stx1+stx2, stx1+eae A, stx1+E‐hly A, stx1+eae A+E‐hly A, eae A and E‐hly A. It was found that VTEC comprised 6.4% and 21% of the total E. coli isolates from slaughtered small ruminants and slaughterhouses/ butchers' shops, respectively. The VTEC comprised 76.2% of the virulent isolates. The proportion of stx1:stx1+stx2 patterns was 19:1. It was found that the characterized complex VTEC (containing eae A and/or E‐hly A) possessed three virulence patterns, including (VTEC) stx1 +eae A, (VTEC/EHEC) stx1 +E‐hly A and (VTEC/EHEC) stx1 +eae A +E‐hly A in percentages of 30%, 25% and 10%, respectively, in relation to the total VTEC isolates. Only two VTEC isolates were characterized as E. coli O157 and O26 serotypes, as highly pathogenic strains. Each of the O157 and O26 VTEC isolates was in a percentage of 0.4% in relation to the total E. coli isolates with virulent patterns stx1, eae A and E‐hly A. The rest of the VTEC isolates were non‐O157 VTEC. The antibiotic sensitivity test showed that the isolated VTEC was highly sensitive to gentamicin and co‐trimoxazole and highly resistant to tetracycline and ampicillin. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Human neutrophils and their products induce Shiga toxin production by enterohemorrhagic Escherichia coli

The Shiga toxins (Stx) are critical virulence factors for Escherichia coli O157:H7 and other serotypes of enterohemorrhagic E. coli (EHEC). These potent toxins are encoded in the genomes of temperate lambdoid bacteriophages. We recently demonstrated that induction of the resident Stx2-encoding prophage in an O157:H7 clinical isolate is required for toxin production by this strain. Since several factors produced by human cells, including hydrogen peroxide (H2O2), are capable of inducing lambdoid prophages, we hypothesized that such molecules might also induce toxin production by EHEC. Here, we studied whether H2O2 and also human neutrophils, an important endogenous source of H2O2, induced Stx2 expression by an EHEC clinical isolate. Both H2O2 and neutrophils were found to augment Stx2 production, raising the possibility that these agents may lead to prophage induction in vivo and thereby contribute to EHEC pathogenesis.     

Identification and characterisation of Escherichia coli strains of O157 and non-O157 serogroups containing three distinct Shiga toxin genes

Three Shiga toxin (Stx)-producing Escherichia coli (STEC) strains from patients with diarrhoea were identified, each of which contained three distinct stx genes (stx1, stx2 and stx2c). The strains belonged to the serotypes O52:H19, O75:H- and O157:H- and harboured eae and EHEC-hly sequences. Colony-blot immunoassay was used to demonstrate that both major types of Stx were expressed. The association of stx genes with either phage or phage DNA was demonstrated in all three strains. Isolated phage DNA from all strains contained stx1 sequences, but stx2 sequences were found only in phage DNA of two of these strains. The presence of three distinct stx genes may enhance the virulence of STEC strains and should be monitored. The observations demonstrate not only the potential of stx genes to spread within different serotypes, but also their capacity to accumulate within a single strain.