Phenotypic and genotypic characteristics of non-O157 Shiga toxin-producing Escherichia coli (STEC) from Swiss cattle

A total of 42 Shiga toxin-producing (STEC) strains from slaughtered healthy cattle in Switzerland were characterized by phenotypic and genotypic traits. The 42 sorbitol-positive, non-O157 STEC strains belonged to 26 O:H serotypes (including eight new serotypes) with four serotypes (O103:H2, O113:H4, O116:H-, ONT:H-) accounting for 38.1% of strains. Out of 16 serotypes previously found in human STEC (71% of strains), nine serotypes (38% of strains) were serotypes that have been associated with hemolytic-uremic syndrome (HUS). Polymerase chain reaction (PCR) analysis showed that 18 (43%) strains carried the stx1 gene, 20 strains (48%) had the stx2 gene, and four (9%) strains had both stx1 and stx2 genes. Of strains encoding for stx2 variants, 63% were positive for stx2 subtype. Enterohemolysin (ehxA), intimin (eae), STEC autoagglutinating adhesin (saa) were detected in 17%, 21%, and 19% of the strains, respectively. Amongst the seven intimin-positive strains, one possessed intimin type beta1 (O5:H-), one intimin gamma1 (O145:H), one intimin gamma2/theta, (O111:H21), and four intimin epsilon (O103:H2). The strains belonged to 29 serovirotypes (association between serotypes and virulence factors). O103:H2 stx1eae-epsilon ehxA, O116:H- stx2, and ONT:H- stx2c were the most common accounting for 29% of the strains. Only one strain (2.4%) of serovirotype O145:H- stx1stx2eae-gamma1ehxA showed a pattern of highly virulent human strains. This is the first study providing characterization data of bovine non-O157 STEC in Switzerland, and underlining the importance of the determination of virulence factors (including intimin types) in addition to serotypes to assess the potential pathogenicity of these strains for humans.     

Serotypes,phage types and virulence genes of shiga-producing Escherichia coli isolated from sheep in Spain

PROBLEM ADDRESSED: Shiga toxin-producing Escherichia coli (STEC), have emerged as food poisoning pathogens which can cause severe diseases in humans. OBJECTIVE: The aim of this study was to determinate the serotypes and virulence genes of STEC strains isolated from sheep in Spain, with the purpose of determining whether sheep represent a potential source of STEC pathogenic for humans. METHODS AND APPROACH: Faecal swabs obtained from 697 healthy lambs on 35 flocks in Spain during the years 2000 and 2001 were examined for STEC using phenotypic (Vero cells) and genotypic (PCR) methods. RESULTS: STEC O157:H7 strains were isolated from seven (1%) animals in six flocks, whereas non-O157 STEC strains were isolated from 246 (35%) lambs in 33 flocks. A total of 253 ovine STEC strains were identified in this study. PCR showed that 110 (43%) strains carried stx(1) genes, 10 (4%) possessed stx(2) genes and 133 (53%) both stx(1) and stx(2). Enterohaemolysin (ehxA) and intimin (eae) virulence genes were detected in 120 (47%) and in 9 (4%) of the STEC strains. STEC strains belonged to 22 O serogroups and 44 O:H serotypes. However, 70% were of one of these six serogroups (O6, O91, O117, O128, O146, O166) and 71% belonged to only nine serotypes (O6:H10, O76:H19, O91:H-, O117:H-, O128:H-, O128:H2, O146:H21, O157:H7, O166:H28). A total of 10 new O:H serotypes not previously reported in STEC strains were found in this study. Seven strains of serotype O157:H7 possessed intimin type gamma1, and two strains of serotype O156:H- had the new intimin zeta. STEC O157:H7 strains were phage types 54 (four strains), 34 (two strains) and 14 (one strain). CONCLUSIONS: This study confirms that healthy sheep are a major reservoir of STEC pathogenic for humans. However, because the eae gene is present only in a very small proportion of ovine non-O157 STEC, most ovine strains may be less pathogenic.     

Serotypes and Shiga toxin genotypes among Escherichia coli isolated from animals and food in Argentina and Brazil

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains isolated from animals and food in Argentina (n=44) and Brazil (n=20) were examined and compared in regard to their phenotypic and genotypic characteristics to evaluate their pathogenic potential. The clonal relatedness of STEC O157 isolates (n=22) was established by phage typing (PT) and pulsed-field gel electrophoresis (PFGE). All O157 strains studied carried eae and enterohemorrhagic E. coli (EHEC)-hly sequences. In Argentina, these strains occurred both in cattle and meat, and 50% of them carried stx2/stx2vh-a genes, whereas in Brazil the O157 strains were isolated from animals, and most harbored the stx2vh-a sequence. At least 13 different O:H serotypes were identified among the non-O157 strains studied, with serotype O113:H21 being found in both countries. All but one non-O157 strains did not carry eae gene, but EHEC-hlyA gene was found in 85.7% of them, and the stx2 genotype was also more prevalent in Argentina than in Brazil (P<0.01), where stx1 alone or in association was most common (68.8%). One STEC strain isolated from a calf in Brazil harbored the new variant referred to as stx2-NV206. PFGE analysis showed that STEC O157 strains were grouped in four clusters. One Brazilian strain was considered possibly related (> or =80%) to Argentinean strains of cluster I. Differences in the pathogenic potential, especially in regard to serotypes and stx genotypes, were observed among the STEC strains recovered from animals and food in both countries.     

High occurrence of Shiga toxin-producing Escherichia coli (STEC) in healthy cattle in Rio de Janeiro State, Brazil

In order to evaluate the prevalence of Shiga toxin-producing Escherichia coli (STEC) strains, 197 fecal samples of healthy cattle from 10 dairy farms, four beef farms and one slaughterhouse at Rio de Janeiro State, Brazil, were examined for Shiga toxin (Stx) gene sequences by polymerase chain reaction (PCR). For presumptive isolation of O157:H7 E. coli, the Cefixime-potassium tellurite-sorbitol MacConkey Agar (CT-SMAC) was used. A high occurrence (71%) of Stx was detected, and was more frequently found among dairy cattle (82% vs. 53% in beef cattle), in which no differences were observed regarding the age of the animals. Dot blot hybridization with stx1 and stx2 probes revealed that the predominant STEC type was one that had the genes for both stx1 and stx2 in dairy cattle and one that had only the stx1 gene for beef cattle. Three (1.5%) O157:H7 E. coli strains were isolated from one beef and two dairy animals by the use of CT-SMAC. To our knowledge, this is the first report of O157:H7 isolation in Brazil. A PCR-based STEC detection protocol led to the isolation of STEC in 12 of 16 randomly selected PCR-positive stool samples. A total of 15 STEC strains belonging to 11 serotypes were isolated, and most of them (60%) had both stx1 and stx2 gene sequences. Cytotoxicity assays with HeLa and Vero cells revealed that all strains except two of serotype O157:H7 expressed Stx. The data point to the high prevalence of STEC in our environment and suggest the need for good control strategies for the prevention of contamination of animal products.     

Serotypes and virulence genes of bovine Shigatoxigenic Escherichia coli (STEC) isolated from a feedlot in Argentina

Grazing-fed cattle were previously demonstrated to be reservoir of non-O157 Shigatoxigenic Escherichia coli (STEC) serotypes in Argentina. The acid-resistance of some STEC strains makes it reasonable to assume the presence in feedlot of particular STEC serotypes. Fifty-nine animals were sampled every 2 weeks during 6 months by rectal swabs. Twenty-seven of 59 animals (45.8%) were shown to be Stx2(+); 3/59 (5.1%) carried Stx1(+) and 7/59 (11.9%) were Stx1(+) Stx2(+). Among 44 STEC isolates, 31 isolates were associated to 10 O serogroups (O2, O15, O25, O103, O145, O146, O157, O171, O174, O175) and 13 were considered non-typable (NT). Six H antigens (H2, H7, H8, H19, H21, H25) were distributed in 21 isolates whereas 23 were non-mobile (H-). Seventeen of 44 strains (38.6%) were eaeA(+) and 14 (31.8%) harbored the 60MDa plasmid. The megaplasmid (Mp) and eaeA gene were simultaneously found in a limited number of serotypes belonging to the enterohaemorrhagic E. coli (EHEC). E. coli O157:H7 strains, isolated from four (6.8%) animals, corresponded to the Stx2(+), eaeA(+), Mp(+) pattern. Three O157:H7 strains belonged to phage type 4 and the other strain was atypical. Many serotypes isolated from grain-fed cattle (O2:H25, O15:H21, O25:H19, O145:H-, O146:H-, O146:H21, O157:H7, O175:H8) also differed from those isolated by us previously from grazing animals. The serotypes O15:H21, O25:H19 and O175:H8 had not been identified at present as belonging to STEC. This work provides new data for the understanding of the ecology of STEC in grain-fed cattle and confirms that cattle are an important reservoir of STEC.     

Dissemination and persistence of Shiga toxin-producing Escherichia coli (STEC) strains on French dairy farms

Some Shiga toxin-producing Escherichia coli strains (STEC), and in particular E. coli O157:H7, are known to cause severe illness in humans. STEC have been responsible for large foodborne outbreaks and some of these have been linked to dairy products. The aim of the present study was to determine the dissemination and persistence of STEC on 13 dairy farms in France, which were selected out of 151 randomized dairy farms. A total of 1309 samples were collected, including 415 faecal samples from cattle and 894 samples from the farm environment. Bacteria from samples were cultured and screened for Shiga toxin (stx) genes by polymerase chain reaction (PCR). STEC isolates were recovered from stx-positive samples after colony blotting, and characterized for their virulence genes, serotypes and XbaI digestion patterns of total DNA separated by pulsed-field gel electrophoresis (PFGE). Stx genes were detected in 145 faecal samples (35%) and 179 (20%) environmental samples, and a total of 118 STEC isolates were recovered. Forty-six percent of the STEC isolates were positive for stx1, 86% for stx2, 29% for intimin (eae-gene) and 92% for enterohemolysin (ehx), of which 16% of the STEC strains carried these four virulence factors in combination. Furthermore, we found that some faecal STEC strains belonged to serotypes involved in human disease (O26:H11 and O157:H7). PFGE profiles indicated genetic diversity of the STEC strains and some of these persisted in the farm environment for up to 12 months. A large range of contaminated samples were collected, in particular from udders and teats. These organs are potential sources for contamination and re-contamination of dairy cattle and constitute an important risk for milk contamination.     

Non-O157 Shiga toxin-producing Escherichia coli isolated from diarrhoeic calves in Argentina

Faecal samples from 76 diarrhoeic calves belonging to 36 farms located in the Pampas plain, Argentina, were examined for Shiga toxin-producing Escherichia coli (STEC). A total of 15 STEC strains were isolated from 12 (15.8%) calves which came from six different farms. All stx positive strains assayed by PCR were also positives in the Vero cell cytotoxicity test. The majority (60.0%) of the STEC strains carried the stx(1) gene. Twelve (80.0%) of the STEC isolates which belonged to serotypes O5:H- (n = 4), O26:H11 (n = 4), O26:H- (n = 1), O111:H- (n = 2), and O123:H38 (n = 1) were also enterohaemolysin (EHly) positive and carried the gene encoding for intimin (eae). All the stx positive strains were negative for the bfpA gene. Localized adherence to HEp-2 cells were observed in 83.3% of the eae+ STEC strains. STEC belonging to serotype O5:H- showed atypical biochemical properties, including urease production. Urease was also produced by two strains belonging to serotypes O153:H? and non-typeable, respectively. Resistance to three or more antibiotics was observed in 12 (80.0%) of the STEC isolates. Most of the serotypes of STEC recovered in this survey carried virulence traits that are associated with increased human and bovine pathogenicity. The present study shows that highly virulent STEC strains are being shed by diarrhoeic calves from farms located in a high incidence area of human STEC infections.     

Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli in healthy cattle, sheep and swine herds in Northern Spain

Three-hundred and forty-five herds (17 swine, 122 dairy sheep, 124 beef and 82 dairy cattle) were investigated for prevalence of Shiga toxin-producing Escherichia coli (STEC). Rectal faecal samples were selectively enriched and then examined by immunodetection techniques (Immunomagnetic Separation with anti-E. coli O157 Dynabeads, ImmunoMagnetic cell Separation (IMS) and automated enzyme-linked fluorescent immunoassay using VIDAS) and polymerase chain reaction (PCR) (rfbE and fliC genes) to assess the prevalence of E. coli O157:H7. Prevalence of non-O157 STEC was estimated by PCR screening for stx genes of 10 lactose-positive colonies grown on MacConkey agar after enrichment. PCR was used on all STEC isolates to detect stx(1), stx(2), eaeA and E-hlyA genes. Both immunodetection methods showed a moderate-good level of agreement (kappa = 0.649) but IMS showed 87.5% complementary sensitivity. Prevalence of positive herds for E. coli O157:H7 was estimated at 8.7% for sheep and 3.8% for cattle, whereas all the porcine herds tested negative. Non-O157 STEC were also absent from swine, but were isolated more frequently from ovine (50.8%) than bovine herds (35.9%). Within-herd prevalences of excretion of E. coli O157:H7 established by individual testing of 279 sheep (six herds) and 30 beef cattle (one herd) were 7.3% and 6.7% respectively. PCR analysis of 49 E. coli O157:H7 and 209 non-O157 isolates showed a different distribution of virulence genes. All E. coli O157:H7 were stx(2) gene-positive, eaeA was detected in 95.9%, and the toxigenic profile stx(2)/eaeA/E-hlyA was present in 75.5% of the isolates. Among the non-O157 STEC, prevalence of eaeA was significantly lower (5.3%) and E-hlyA was present in 50.2% of the isolates but only sporadically associated with eaeA. stx(2) was predominant in non-O157 isolates from cattle, whereas in sheep the combination stx(1)/stx(2) was more prevalent. This study demonstrated the wide distribution of STEC in ruminant herds, which represent an important reservoir for strains that pose a potential risk for human infections.     

Shiga toxin-producing Escherichia coli in the feces of Alberta feedlot cattle

Shiga toxin-producing Escherichia coli (STEC) are a public health concern. Bacterial culture techniques commonly used to detect E. coli O157:H7 will not detect other STEC serotypes. Feces from cattle and other animals are a source of O157:H7 and other pathogenic serotypes of STEC. The objective of this study was to estimate the pen-level prevalence of Shiga toxins and selected STEC serotypes in pre-slaughter feedlot cattle. Composite fecal samples were cultured and a polymerase chain reaction (PCR) was used to detect genes for Shiga toxins (stx1 and stx2) and genes for O157:H7, O111:H8, and O26:H11 serotypes. Evidence of Shiga toxins was found in 23 pens (92%), O157:H7 in 2 (8%), O111:H8 in 5 (20%), and O26:H11 in 20 (80%) of the 25 pens investigated. Although pen-level prevalence estimates for Shiga toxins and non-O157 serotypes seem high relative to O157:H7, further effort is required to determine the human health significance of non-O157 serotypes of STEC in feedlot cattle.     

Serotypes and virulence markers of Shiga toxin-producing Escherichia coli (STEC) isolated from dairy cattle in São Paulo State, Brazil

In order to determine the occurrence, serotypes and virulence markers of Shiga toxin-producing Escherichia coli (STEC) strains, 153 fecal samples of cattle randomly selected from six dairy farms in Sao Paulo State, Brazil, were examined for Shiga toxin (Stx) production by the Vero cell assay. Feces were directly streaked onto MacConkey Sorbitol Agar and incubated at 37 degrees C overnight. Sorbitol-negative colonies (maximum 20) and up to 10 sorbitol-positive colonies from each plate were subcultured onto presumptive diagnostic medium IAL. Sorbitol-negative isolates were screened with O157 antiserum for identification of O157:H7 E. coli. Isolates presenting cytotoxic activity were submitted to colony hybridization assays with specific DNA probes for stx1, stx2, eae, Ehly and astA genes. The isolation rate of STEC ranged from 3.8 to 84.6% depending on the farm analysed. STEC was identified in 25.5% of the animals, and most of them (64.1%) carried a single STEC serotype. A total of 202 STEC isolates were recovered from the animals, and except for the 2 O157:H7 isolates all the others expressed cytotoxic activity. The great majority of the STEC isolates carried both stx1 and stx2 genes (114/202, 56.4%) or stx2 (82/202, 40.6%); and whereas the Ehly sequence occurred in most of them (88%) eae was only observed in O157:H7 and O111:HNM isolates. Serotypes O113:H21, O178:H19 and O79:H14 were the most frequent STEC serotypes identified and widely distributed among animals from different farms, while others such as O77:H18, O88:H25 and O98:H17 occurred only in particular farms. This is the first report on the occurrence of STEC in dairy cattle in Sao Paulo State, and the results point to substantial differences in rate of isolation, serotypes and genetic profile of STEC that has been previously described among beef cattle in our community. Moreover, to our knowledge O79:H14 and O98:H17 represent new STEC serotypes, while O178:H19 has only been recently reported in Spain.     

Subtilase cytotoxin encoding genes are present in human, sheep and deer intimin-negative, Shiga toxin-producing Escherichia coli O128:H2

Shiga toxin-producing Escherichia coli (STEC) O128:H2 is recognised worldwide to be an important non-O157 STEC associated with human illness and in particular with causing haemolytic uraemic syndrome. This serotype is commonly isolated from sheep and is being increasingly isolated from deer. We determined the virulence profile and genetic relationships of one human, six sheep and five deer intimin-negative STEC O128:H2 strains isolated in Spain over a 7-year period. Our goals were to establish the presence of other virulence-associated factors, such as SubAB, in intimin-negative STEC O128:H2 strains involved in human disease and in that case, to determine if sheep and/or deer represent a reservoir of SubAB-positive STEC O128:H2. All the strains lacked the eae gene and carried subtilase cytotoxin (SubAB) encoding genes (subAB) and tia genes, but not saa gene, suggesting the presence of the recently identified new variant of SubAB, encoded on a putative pathogenicity island together with tia. We report for the first time the presence of subtilase cytotoxin encoding genes in intimin-negative STEC O128:H2 strains pathogenic for humans and how this finding might explain their clinical relevance despite neither carrying eae nor stx subtypes associated with severe clinical outcomes, but only stx1c and stx2b. Multilocus sequence typing analysis revealed that STEC O128:H2 strains from sheep and deer belong to the clonal lineage of STEC O128:H2 strains involved in diarrhoeal and haemorrhagic diseases in humans. Our results indicate that sheep and deer represent a reservoir of SubAB-positive STEC O128:H2 strains and thus a potential source of human infection.     

某定点肉牛屠宰场中非O157致病性STEC的分离鉴定

为了了解新疆伊犁地区肉牛屠宰过程中大肠杆菌的污染情况，检测非O157致病性产志贺毒素大肠杆菌（Shiga toxin-producing Escherichia coli，STEC）的感染情况，本试验采集新疆伊犁地区某定点肉牛屠宰场中屠宰肉牛的粪样和屠宰后的胴体表面拭子，并对样品进行了大肠杆菌的分离鉴定、毒力基因（eae、stx1、stx2）的PCR检测、O157鉴定（rfbE）、ERIC-PCR基因分型和小鼠致病性试验。结果显示，在采集的45份样品中分离鉴定出42株大肠杆菌，分离率为93.3%。其中2株菌株同时编码了毒力基因stx1和stx2，检出率为4.8%，毒力基因eae未被检出。PCR鉴定均为非O157 STEC。ERIC-PCR基因分型检测发现，2株菌的基因型非常相似，同源关系密切。对小鼠进行腹腔注射攻毒，攻菌6 h后，小鼠开始出现死亡，立即解剖死亡小鼠发现，其肠道出血，肝脏、脾脏、肾脏明显出血肿大，解剖对照小鼠表现正常，表明菌株具有一定的致病性。综上所述，在肉牛屠宰过程中存在大肠杆菌污染，其中粪便中非O157 STEC菌株对胴体造成了污染，需要加强控制肉牛的屠宰加工关键环节的环境卫生。     

Profile of Shiga toxin-producing Escherichia coli strains isolated from dogs and cats and genetic relationships with isolates from cattle, meat and humans

Pets can be reservoirs of Shiga toxin-producing Escherichia coli (STEC) strains. The aim of this study was to examine nine strains belonging to several serotypes (O91:H21, O91:H16, O178:H19, O8:H19, O22:H8, O22:HNT, ONT:H8), previously recovered from cats or dogs. To this end, we assessed a set of additional virulence genes (stx(2) subtype, subAB, ehxA, eae and saa), cytotoxic activity, and genetic relationships with strains isolated from cattle, meat and humans using pulsed-field gel electrophoresis (PFGE). Most of the isolates carried the stx(2) and/or stx(2vh-b) sequences, while only the O91:H21 isolate presented the mucus-activatable stx(2d) variant, as confirmed by sequencing the genes of subunits A and B. All the strains showed cytotoxic activity in cultured cells. One of the two O178:H19, selected for its high level of cytotoxicity in Vero cells, showed the ability to cause functional alterations in the human colon mucosa in vitro. None of the strains possessed the subAB, eae or saa genes and only the strains belonging to serotype O8:H19 carried the ehxA gene. The isolates shared 90-100% similarity by PFGE to epidemiologically unrelated strains of the corresponding serotypes recovered from cattle, meat or humans. Our results demonstrate that dogs and cats may have a role in the infection of humans by STEC, probably serving as a vehicle for bovine strains in the cycle of human infection, and thus emphasize the health risks for owners and their families.     

Characterisation and clonal relationships of Shiga-toxigenic Escherichia coli (STEC) isolated from Australian dairy cattle

A total of 136 Shiga toxin-producing Escherichia coli (STEC) isolated during a longitudinal survey of three Australian dairy farms were examined to determine their virulence factors, serotype and genomic relationships. This study aimed to assess the potential of these STEC to cause disease in humans and to analyse the on-farm ecology of STEC. Virulence factors (stx, eae, ehxA) were used as determinants of potential to be enterohaemorrhagic E. coli (EHEC) and were examined using polymerase chain reaction (PCR). Among the cattle groups tested, calves, both before and during weaning, shed the most putative EHEC and were the main source of serotypes commonly associated with human disease. E. coli O157:H7 and E. coli O26:H11 represented 9.4 and 7.8% of cattle STEC isolates respectively, with other putative EHEC serotypes reported for the first time from cattle. Based on serotype and virulence factors, 20% of STEC were putative EHEC. Pulsed-field gel electrophoresis (PFGE) was used to compare the genomic profiles of STEC from dairy farms. Isolates common to cattle and the farm environment were identified. Multiple strains of STEC with high clonal turnover were detected in the faeces of cattle, and isolates appeared to be specific to individual farms. To fully assess the pre-slaughter EHEC risk factors on-farm, examination of STEC virulence is as important as determination of STEC prevalence.     

Prevalence and characterization of Shiga toxin-producing Escherichia coli O157 and O26 in beef farms

Rectal content grab samples were collected from 2436 beef cattle reared on 406 beef farms in Japan between November 2007 and March 2008. STEC strains O157 and O26 were isolated from 110 (27.1%) and 7 (1.7%) farms, respectively. Farms that tested positive for STEC O157 were located in 35 out of all 47 Japanese prefectures. This indicates that STEC O157 strains are widespread on beef farms nationwide. Of the 2436 tested beef cattle, 218 (8.9%) and 10 (0.4%) had STEC strains O157 and O26 in the rectal content, respectively. The most common Shiga toxin genes detected in the isolated STEC O157 strains were: stx(2c) alone (32.1%), stx(2)/stx(2c) (27.2%), and stx(1)/stx(2) (21.8%). Almost all of the STEC O157 and STEC O26 strains expressed Shiga toxins (Stx). Most of the STEC O157 and STEC O26 strains possessed eaeA and EHEC-hlyA. These results strongly suggest that STEC strains O157 and O26 from beef cattle would be pathogenic to humans. Therefore, it is important to reduce STEC strains O157 and O26 in beef cattle in order to prevent foodborne disease caused by STEC. The presence of dogs and/or cats on a farm was significantly (P=0.02) associated with the prevalence of STEC O157. More research is needed to clarify the role of dogs and cats.     

Long-term excretion of Shiga toxin-producing Escherichia coli (STEC) and experimental infection of a sheep with O157

To investigate a long-term shedding of Shiga toxin (Stx)-producing Escherichia coli (STEC) from sheep, a fifteen-month study for STEC isolation from a sheep, which had yielded STEC before, was attempted. The sheep continued to shed STEC and 39 STEC were isolated. The number of STEC in the feces was estimated at 1.7 x 10(3) per gram. In addition, although Stx1-negative O157 and stx2-encoding bacteriophage were experimentally infected to the sheep, Stx-positive O157 or Stx2- producing bacterial cells were not detected. The genetical and biochemical characterization of those 39 STEC strains showed that all STEC strains produced Shiga toxin 1 (Stx1) and were divided into three classes (I to III). From phylogenetic analysis of their amino acid sequences, class-I STEC was classified as group 1 comprising mainly human STEC, and classes II/III were as group 2 comprising sheep STEC. Our results suggest that STEC easily colonized in sheep and that the sheep continued to shed STEC, showing that sheep might be an important reservoir for human STEC infection.     

Virulence gene profiles and intimin subtypes of Shiga toxin-producing Escherichia coli isolated from healthy and diarrhoeic calves

The virulence properties of Shiga toxin-producing Escherichia coli (STEC) strains isolated from diarrhoeic and non-diarrhoeic calves were compared. The strains were also tested for O157:H7, O111 and O26 serotypes, using PCR and conventional serotyping methods. E coli strains isolated from 297 faecal samples, from 200 diarrhoeic and 97 non-diarrhoeic calves, were screened by multiplex PCR assay for the stx1, stx2, eae and Ehly virulence genes. STECs were recovered from 8 per cent of diarrhoeic calves and 10.3 per cent of non-diarrhoeic calves. The predominant virulence gene profile was stx1/eae/Ehly (47.3 per cent) among isolates from diarrhoeic calves and eae/Ehly (36.8 per cent) among isolates from non-diarrhoeic calves. Among three tested serogroups, the predominant serogroup was O26 (18.4 per cent), and O157:H7 was not detected. Intimin subtyping by restriction fragment length polymorphism analysis revealed only three intimin subtypes (β, γ and ). A significant difference was observed in the distribution of Int- between two groups. Int- was present in 50 per cent of the isolates from diarrhoeic calves and in 11.1 per cent of the isolates from non-diarrhoeic calves; this difference was statistically significant (P=0.01).     

Serotypes and analysis of distribution of Shiga toxin producing Escherichia coli from cattle and sheep in the lower North Island, New Zealand

AIMS: To serotype a subset of Shiga toxin-producing Escherichia coli (STEC) isolates from cattle and sheep to determine whether any corresponding serotypes have been implicated in human diarrhoeal disease, both in New Zealand and worldwide, and to examine the distribution of STEC and enteropathogenic Escherichia coli (EPEC) amongst cattle (calves, heifers and dairy) and sheep (lambs, rams and ewes), to assess whether carriage of identified bacterial genotypes may be associated with a particular age of animal. METHODS: Recto-anal mucosal swabs (RAMS) were taken from 91 calves, 24 heifers and 72 dairy cattle, and 46 lambs, 50 ewes and 36 rams, from four sites in the Manawatu and Rangitikei regions of New Zealand. Strains of E. coli selected from primary isolation plates were subjected to a multiplex polymerase chain reaction (PCR), to determine the presence of Shiga toxin genes (stx1 and stx2) and the E. coli attaching and effacing gene (eae). RESULTS: Overall, 186/319 (58.3%) animals sampled were positive for stx1, stx2, or eae isolates. More sheep (43.9%) were stx1-positive than cattle (2.7%; p = 0.036), and amongst sheep more lambs and ewes were stx1-positive than rams (p = 0.036). Amongst cattle, more calves and heifers were eae-positive than dairy cows (p = 0.030). Two or more different STEC were isolated from at least 28 (9%) animals (three cattle and 25 sheep), based on their stx/eae genotype. Enterohaemolysin genes were found in 39/51 (76%) isolates serotyped. Twenty-one different serotypes were detected, including O5:H-, O9:H51, O26:H11, O84:H-/H2 and O149:H8 from cattle, and O26:H11, O65:H-, O75:H8, O84:H-, O91:H-, O128:H2 and O174:H8 from sheep; O84:H-, O26:H11, O5:H-, O91:H- and O128:H2 serotypes have been associated with human disease. CONCLUSIONS: If nationally representative, this study confirms that cattle and sheep in New Zealand may be a major reservoir of STEC serotypes that have been recognised as causative agents of diarrhoeal disease in humans. Distribution of STEC and EPEC in cattle and sheep indicates that direct contact with, in particular, calves or their faeces, or exposure to environments cross contaminated with ruminant faeces, may represent an increased risk factor for human disease in New Zealand.     

Comparable stx2a expression and phage production levels between Shiga toxin‐producing Escherichia coli strains from human and bovine origin

Shiga toxin‐producing Escherichia coli (STEC) can cause diarrhoea and severe diseases in humans, such as haemolytic uraemic syndrome. STEC virulence is considered to correlate with the amount of Shiga toxins (Stx) produced, especially Stx2, whose subtype Stx2a is most frequently associated with high virulence. Stx are encoded in prophages, which play an important role in STEC pathogenesis. The aim of this study was to evaluate stx_2a expression levels and Stx2a phage production using qPCR and the double‐agar‐layer method in 29 STEC strains, corresponding to serotypes O26:H11 (6), O91:H21 (1), O145:H‐ (11) and O157:H7 (11), isolated from cattle and humans. Results were then tested for possible associations with serotype, origin or some genetic features. We observed heterogeneous levels of stx_2a expression and Stx2a phage production. However, statistical comparisons identified a higher stx_2a expression in response to mitomycin C in strains isolated from cattle than in those from humans. At the same time, compared to stx_2a/stx_2c strains, stx_2a strains showed a higher increase in phage production under induced conditions. Notably, most of the strains studied, regardless of serotype and origin, carried inducible Stx2a phages and evidenced expression of stx_2a that increased along with phage production levels under induced conditions.     

Prevalence and antimicrobial resistance of porcine O157 and non-O157 Shiga toxin-producing Escherichia coli from India

The aims of this study were to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) strains in pigs as a possible STEC reservoir in India as well as to characterize the STEC strains and to determine the antimicrobial resistance pattern of the strains. A total of 782 E. coli isolates from clinically healthy (n?=?473) and diarrhoeic piglets (309) belonging to major pig-producing states of India were screened by the polymerase chain reaction (PCR) assay for the presence of virulence genes characteristic for STEC, that is, Shiga toxin-producing gene(s) (stx1, stx2), intimin (eae), enterohemolysin (hlyA) and STEC autoagglutinating adhesin (Saa). Overall STEC were detected in 113 (14.4 %) piglets, and the prevalence of E. coli O157 and non-O157 STEC were 4 (0.5 %) and 109 (13.9 %), respectively. None of the O157 STEC isolates carried gene encoding for H7 antigen (fliCh7). The various combinations of virulence genes present in the strains studied were stx1 in 4.6 %, stx1 in combination with stx2 gene in 5.1 %, stx1 in combination with stx2 and ehxA in 0.6 %, stx1 in combination with stx2 and eae in 0.2 % and stx2 alone in 3.7 %. All STEC isolates were found negative for STEC autoagglutinating adhesin (Saa). The number of STEC isolates which showed resistance to antimicrobials such as ampicillin, tetracycline, streptomycin, lincomycin, nalidixic acid, sulfadiazine, penicillin, gentamicin, kanamycin and ceftriaxone were 100, 99, 98, 97, 95, 94, 92, 88, 85 and 85, respectively. Ninety-seven isolates showed resistance to more than 2 antimicrobials, and 8 resistance groups (R1 to R8) were observed. This study demonstrates that pigs in India harbour both O157 and non-O157 STEC, and this may pose serious public health problems in future.