Investigation of shiga toxin-producing Escherichia coli in avian species in India

AIMS: To investigate the presence or absence of shiga toxin-producing Escherichia coli (STEC) in avian species in India. METHODS AND RESULTS: Faecal samples originating from 500 chicken and 25 free flying pigeons were screened for the presence of E. coli. A total of 426 (chicken, 401; pigeons, 25) E. coli strains were isolated. Of 426 E. coli strains, 387 were grouped into 77 serogroups, while 70 and 59 strains were untypable and rough, respectively. All isolates were subjected to multiplex polymerase chain reaction (m-PCR) for the detection of stx(1), stx(2), eaeA, hlyA and saa genes. None of the E. coli strains studied showed the presence of stx(1), stx(2) or their variants and saa genes. Overall 11 (2.74%) and seven (1.74%) strains from chickens possessed eaeA and hlyA genes, respectively, while as only six (1.49%) strains from chickens possessed both eaeA and hlyA genes. O9, O8, O60 and O25 serogroups were most predominant of which there were 24 (5.63%), 23 (5.39%), 23 (5.39%) and 20 (4.69%) strains, respectively. None of the isolates from pigeons showed the presence of any of the virulence genes studied. CONCLUSIONS: STEC are absent in chickens and pigeons. However, further studies are required in this direction to confirm or contradict our findings. E. coli strains originating from birds are carrying a low percentage eaeA or hlyA genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study is the first attempt to investigate STEC in chickens and free flying pigeons in India. The chickens and pigeons cannot be considered as important carrier of STEC in India.     

Prevalence and characterization of non-O157 shiga toxin-producing Escherichia coli isolates from commercial ground beef in the United States

Escherichia coli O157:H7 is a Shiga toxin (stx)-producing E. coli (STEC) strain that has been classified as an adulterant in U.S. beef. However, numerous other non-O157 STEC strains are associated with diseases of various severities and have become an increasing concern to the beef industry, regulatory officials, and the public. This study reports on the prevalence and characterization of non-O157 STEC in commercial ground beef samples (n = 4,133) obtained from numerous manufacturers across the United States over a period of 24 months. All samples were screened by DNA amplification for the presence of Shiga toxin genes, which were present in 1,006 (24.3%) of the samples. Then, culture isolation of an STEC isolate from all samples that contained stx(1) and/or stx(2) was attempted. Of the 1,006 positive ground beef samples screened for stx, 300 (7.3% of the total of 4,133) were confirmed to have at least one strain of STEC present by culture isolation. In total, 338 unique STEC isolates were recovered from the 300 samples that yielded an STEC isolate. All unique STEC isolates were serotyped and were characterized for the presence of known virulence factors. These included Shiga toxin subtypes, intimin subtypes, and accessory virulence factors related to adherence (saa, iha, lifA), toxicity (cnf, subA, astA), iron acquisition (chuA), and the presence of the large 60-MDa virulence plasmid (espP, etpD, toxB, katP, toxB). The isolates were also characterized by use of a pathogenicity molecular risk assessment (MRA; based on the presence of various O-island nle genes). Results of this characterization identified 10 STEC isolates (0.24% of the 4,133 total) that may be considered a significant food safety threat, defined by the presence of eae, subA, and nle genes.     

The incidence of Shiga toxin-producing Escherichia coli in cattle with mastitis in Brazil

AIMS: To determine the prevalence and molecular characteristics of Shiga toxin-producing Escherichia coli (STEC) isolates from bovine mastitic milk in Brazil. METHODS AND RESULTS: A total of 2144 milk samples from dairy cattle showing mastitis were screened for the presence of E. coli. A total of 182 E. coli isolates were selected and examined. All were subjected to dot blot analysis using the CVD419 probe for the detection of the enterohaemolysin (hly) gene, and to a multiplex PCR for the detection of stx1, stx2 and eaeA genes. STEC were isolated from 22 (12.08%) milk samples. All the STEC isolates were tested for sensibility to 10 antimicrobials; the resistances most commonly observed were to cephalothin (86.3%), tetracycline (63.6%) and doxycycline (63.6%). CONCLUSION: STEC isolates were found in bovine mastitic milk in Brazil. SIGNIFICANCE AND IMPACT OF THE STUDY: STEC isolates from mastitic milk were potentially pathogenic for human in that they belonged to serogroups associated with diarrhoea and haemolytic-uraemic syndrome, some of them were stx2, eaeA and hly positive.     

Comparison between a PCR-ELISA test and the vero cell assay for detecting Shiga toxin-producing Escherichia coli in dairy products and characterization of virulence traits of the isolated strains

AIMS: This paper provides information on a PCR-ELISA method for detecting Shiga toxin-producing Escherichia coli (STEC), and on their prevalence in dairy products. METHODS AND RESULTS: The sensitivity and specificity of the test was evaluated using pure cultures, spiked and naturally-contaminated samples. A comparative study with vero cytotoxicity testing was conducted, and STEC isolated from naturally-contaminated samples were characterized. The PCR-ELISA test was highly specific and sensitive, and detected 14% more positive samples than the vero cell assay. The prevalence of STEC in raw milk and unpasteurized cheese was 21.5% and 30.5%, respectively, while samples from the 'dairy environment' and from pasteurized cheese were less contaminated. The 34 strains of STEC isolated from natural samples showed that some of them carried virulence genes. CONCLUSION: No conclusion can be drawn at the moment concerning the potential risk to consumers. SIGNIFICANCE AND IMPACT OF THE STUDY: These data show the necessity of valuable screening methods to appreciate the virulence of STEC.     

Shiga toxin-producing Escherichia coli in sheep and pre-slaughter lambs in eastern Australia

Sheep and lambs from 14 farms in southern Queensland and one from central New South Wales were surveyed to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC). STEC, isolated from 45% of 144 sheep faeces collected on the farms and 36% of 72 lamb faeces from abattoir yards, were tested for the presence of genes encoding virulence factors. Most (64%) of the 117 STEC isolates contained Shiga toxin 1 and 2 genes, 22% contained those encoding Shiga toxin 1, and 14% contained genes encoding Shiga toxin 2. The genes encoding the E. coli attaching and effacing factor were present in 2.6% of STEC and 26% contained the enterohaemolysin gene. The isolates that contained the E. coli attaching and effacing gene were serotype O157:H. This study has shown that STEC are widely distributed in eastern Australian sheep and lambs and are shed in their faeces prior to slaughter. Thus, there is potential for contamination of carcasses and entry of STEC into the human food chain.     

产志贺样毒素和肠毒素大肠杆菌分子流行病学

[目的]人和动物腹泻的主要病原菌为大肠杆菌,本文主要研究贵州省致腹泻大肠杆菌毒力因子的分布类型.[方法]采用PCR技术对各毒力因子的基因分布进行研究.[结果]共分离到333株大肠杆菌,其中产肠毒素大肠杆菌(ETEC)在腹泻的人、猪、牛群中占优势,分别为:人群73(n=112),猪群82(n=106),牛群18(n=115).在ETEC菌株中检测到热敏肠毒素(lt)和不耐热肠毒素(st)基因,还存在lt/st并存现象.从人、猪、牛群中还检测到产志贺样毒素大肠杆菌(STEC),其中源自猪的STEC的检出率最高.大部分STEC同时携带lt、st或lt和st同时并存.编码F18菌毛的主亚基由fedA基因编码.对所分离大肠杆菌F18菌毛进行的研究结果表明,fedA基因主要与肠毒素基因共存,与stx基因并存的类型较少,25份猪源STEC菌株中仅有4份检测到fedA基因.[结论]贵州省人群、猪群和牛群致腹泻病原菌中以带F18菌毛的ETEC为主,STEC主要分布在腹泻的猪群中.     

Development of a multiplex PCR approach for the identification of Shiga toxin-producing Escherichia coli strains and their major virulence factor genes

AIMS: To develop and evaluate a multiplex PCR (mPCR) system for rapid and specific identification of Shiga toxin-producing Escherichia coli (STEC) and their main virulence marker genes. METHODS AND RESULTS: A series of mPCR assays were developed using primer pairs that identify the sequences of Shiga toxins 1 and 2 (stx1 and stx2, including the stx2c, stx2d, stx2e and stx2f variants), intimin (eaeA), and enterohaemorrhagic E. coli enterohaemolysin (ehlyA). Moreover, two additional genes (rfb O157 and fliC H7), providing the genotypic identification of the O157:H7 E. coli serotype, were detected. As an internal positive control, primers designated to amplify the E. coli 16S rRNA were included in each mPCR. All the amplified genes in the E. coli reference strains were sucessfully identified by this procedure. The method was then used for the examination of 202 E. coli isolates recovered from cattle and children. Among them, 25 (12.4%) were stx positive including the strains of O157:H7 serotype (six isolates) and O157:NM serogroup (four strains). Moreover, 20 STEC strains possessed the eaeA (intimin) and ehlyA (enterohaemolysin) genes. CONCLUSIONS: The developed mPCR-based system enabled specific detection of STEC bacteria and identification of their main virulence marker genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to identify STEC bacteria and the majority of their virulence gene markers, including four variants of Shiga toxin, as well as the differentiation of O157:H7 from non-O157 isolates represents a considerable advancement over other PCR-based methods for rapid characterization of STEC.     

Virulence markers and serotypes of Shiga toxin-producing Escherichia coli, isolated from cattle in Rio Grande do Sul, Brazil

AIMS: To determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) and serotypes and virulence markers of the STEC isolates from beef and dairy cattle in Rio Grande do Sul, Brazil. METHODS AND RESULTS: Faecal samples from beef cattle were collected at slaughterhouses. The isolates were submitted to colony hybridization assay with specific DNA probes for stx1, stx2 and eae genes, and serotyped for the identification of O and H antigens. Thirty-nine per cent of beef cattle surveyed harboured at least one STEC strain. Among the distinct serotypes identified, 10 were shared by both beef and dairy cattle. Most of the strains isolated harboured stx2. Genotypic and phenotypic profiles allowed the identification of 34 and 31 STEC strains, isolated from beef and dairy cattle, respectively. Serotypes O10:H14, O15:H21, O96:H21, O119:H4, O124:H11, O128:H21, O137:H-, O141:H19, O159:H42, O160:H2 and O177:H11, identified in this study, have not been previously reported as STEC isolated from cattle. CONCLUSIONS: Cattle are an important reservoir of STEC strains associated with human diseases in South America. SIGNIFICANCE AND IMPACT OF THE STUDY: Determining the prevalence, genotypic profile and serotypes of STEC strains isolated from cattle enables the prediction of possible risk for public health.     

Molecular analysis of shiga toxin-producing Escherichia coli strains isolated from hemolytic-uremic syndrome patients and dairy samples in France

Shiga toxin-producing Escherichia coli (STEC) has been associated with food-borne diseases ranging from uncomplicated diarrhea to hemolytic-uremic syndrome (HUS). While most outbreaks are associated with E. coli O157:H7, about half of the sporadic cases may be due to non-O157:H7 serotypes. To assess the pathogenicity of STEC isolated from dairy foods in France, 40 strains isolated from 1,130 raw-milk and cheese samples were compared with 15 STEC strains isolated from patients suffering from severe disease. The presence of genes encoding Shiga toxins (stx(1), stx(2), and variants), intimin (eae and variants), adhesins (bfp, efa1), enterohemolysin (ehxA), serine protease (espP), and catalase-peroxidase (katP) was determined by PCR and/or hybridization. Plasmid profiling, ribotyping, and pulsed-field gel electrophoresis (PFGE) were used to further compare the strains at the molecular level. A new stx(2) variant, stx(2-CH013), associated with an O91:H10 clinical isolate was identified. The presence of the stx(2), eae, and katP genes, together with a combination of several stx(2) variants, was clearly associated with human-pathogenic strains. In contrast, dairy food STEC strains were characterized by a predominance of stx(1), with a minority of isolates harboring eae, espP, and/or katP. These associations may help to differentiate less virulent STEC strains from those more likely to cause disease in humans. Only one dairy O5 isolate had a virulence gene panel identical to that of an HUS-associated strain. However, the ribotype and PFGE profiles were not identical. In conclusion, most STEC strains isolated from dairy products in France showed characteristics different from those of strains isolated from patients.     

Isolation and Characteristics of Shiga Toxin 2f-Producing Escherichia coli among Pigeons in Kyushu,Japan

An increasing number of Shiga toxin 2f-producing Escherichia coli (STEC2f) infections in humans are being reported in Europe, and pigeons have been suggested as a reservoir for the pathogen. In Japan, there is very little information regarding carriage of STEC2f by pigeons, prompting the need for further investigation. We collected 549 samples of pigeon droppings from 14 locations in Kyushu, Japan, to isolate STEC2f and to investigate characteristics of the isolates. Shiga toxin stx _2f gene fragments were detected by PCR in 16 (2.9%) of the 549 dropping samples across four of the 14 locations. We obtained 23 STEC2f-isolates from seven of the original samples and from three pigeon dropping samples collected in an additional sampling experiment (from a total of seven locations across both sampling periods). Genotypic and phenotypic characteristics were then examined for selected isolates from each of 10 samples with pulsed-field gel electrophoresis profiles. Eight of the stx _2f gene fragments sequenced in this study were homologous to others that were identified in Europe. Some isolates also contained virulence-related genes, including lpfA _O26, irp ₂, and fyuA, and all of the 10 selected isolates maintained the eae, astA, and cdt genes. Moreover, five of the 10 selected isolates contained sfpA, a gene that is restricted to Shiga toxin-producing E. coli O165:H2 and sorbitol-fermenting Shiga toxin-producing E. coli O157:NM. We document serotypes O152:HNM, O128:HNM, and O145:H34 as STEC2f, which agrees with previous studies on pigeons and humans. Interestingly, O119:H21 was newly described as STEC2f. O145:H34, with sequence type 722, was described in a German study in humans and was also isolated in the current study. These results revealed that Japanese zoonotic STEC2f strains harboring several virulence-related factors may be of the same clonal complexes as some European strains. These findings provide useful information for public health-related disease management strategies in Japan.     

Prevalence and characterization of non-O157 Shiga toxin-producing Escherichia coli on carcasses in commercial beef cattle processing plants

Beef carcass sponge samples collected from July to August 1999 at four large processing plants in the United States were surveyed for the presence of non-O157 Shiga toxin-producing Escherichia coli (STEC). Twenty-eight (93%) of 30 single-source lots surveyed included at least one sample containing non-O157 STEC. Of 334 carcasses sampled prior to evisceration, 180 (54%) were found to harbor non-O157 STEC. Non-O157 STEC isolates were also recovered from 27 (8%) of 326 carcasses sampled after the application of antimicrobial interventions. Altogether, 361 non-O157 STEC isolates, comprising 41 different O serogroups, were recovered. O serogroups that previously have been associated with human disease accounted for 178 (49%) of 361 isolates. Although 40 isolates (11%) carried a combination of virulence factor genes (enterohemorrhagic E. coli hlyA, eae, and at least one stx gene) frequently associated with STEC strains causing severe human disease, only 12 of these isolates also belonged to an O serogroup previously associated with human disease. Combining previously reported data on O157-positive samples (R. O. Elder, J. E. Keen, G. R. Siragusa, G. A. Barkocy-Gallagher, M. Koohmaraie, and W. W. Laegreid, Proc. Natl. Acad. Sci. USA 97:2999-3003, 2000) with these data regarding non-O157-positive samples indicated total STEC prevalences of 72 and 10% in preevisceration and postprocessing beef carcass samples, respectively, showing that the interventions used by the beef-processing industry effected a sevenfold reduction in carcass contamination by STEC.     

产志贺毒素大肠埃希菌的分子生物学鉴定和耐药性分析

为了解产志贺毒素大肠埃希菌 (Shigatoxin producingEscherichiacoli ,STEC)stx1,stx2 ,eaeA ,hlyA 4种毒力基因的分布情况 ,以及分离株对 18种抗生素的敏感性 ,采用多重PCR(multiplexPCR ,mPCR)法对分离株进行毒力基因的分子生物学鉴定 ;用WHO推荐的K B法对分离株进行抗生素的敏感性测定。产志贺毒素的大肠埃希菌共有 4 6株 ,其中 2种毒素均产生的有 2 2株 (4 7.8% ) ;单纯产生stx1的有 16株 (36 .9% ) ,stx2 的有 8株 (17.4 % ) ;4种毒力基因均存在的有 19株 (4 1.3% ) ,血清型为O15 7∶H7,而非O15 7∶H7血清型的菌株 (2 3/46 )中 ,4种毒力基因同时存在的仅有 3株 (6 .6 % ) ,但有 13株 (5 6 .9% )hlyA基因阳性。全部STEC对复方新诺明耐药 ,对链霉素耐药率为 2 8.3% ,氨苄西林为 30 .4 % ,红霉素为 6 9.6 % ,而且有 5株对至少 4种以上抗生素多重耐药 ,耐药谱为复方新诺明 链霉素 红霉素 氨苄西林。非O15 7型STEC耐药菌次为 12 2 ,而O15 7型为 6 3。可见 ,mPCR法可以快速检测STEC特征性毒力基因 ,以判定其致病性能。非O15 7型STEC对抗生素较易形成耐药性。     

Prevalence and characteristics of shiga toxin-producing Escherichia coli from healthy cattle in Japan

The prevalence of Shiga toxin-producing Escherichia coli (STEC) in Japan was examined by using stool samples from 87 calves, 88 heifers, and 183 cows on 78 farms. As determined by screening with stx-PCR, the prevalence was 46% in calves, 66% in heifers, and 69% in cows; as determined by nested stx-PCR, the prevalence was 100% in all animal groups. Of the 962 isolates picked by colony stx hybridization, 92 isolates from 54 farms were characterized to determine their O serogroups, virulence factor genes, and antimicrobial resistance. Of these 92 isolates, 74 (80%) could be classified into O serogroups; 50% of these 74 isolates belonged to O serogroups O8, O26, O84, O113, and O116 and 1 isolate belonged to O serogroup O157. Locus of enterocyte effacement genes were detected in 24% of the isolates, and enterohemorrhagic E. coli (EHEC) hlyA genes were detected in 72% of the isolates. Neither the bundle-forming pilus gene nor the enteropathogenic E. coli adherence factor plasmid was found. STEC strains with characteristics typical of isolates from human EHEC infections, which were regarded as potential EHEC strains, were present on 11.5% of the farms.     

Detection of Shiga toxin-producing Escherichia coli serotypes O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7 in raw-milk cheeses by using multiplex real-time PCR

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are a diverse group of food-borne pathogens with various levels of virulence for humans. In this study, we describe the use of a combination of multiple real-time PCR assays for the screening of 400 raw-milk cheeses for the five main pathogenic STEC serotypes (O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7). The prevalences of samples positive for stx, intimin-encoding gene (eae), and at least one of the five O group genetic markers were 29.8%, 37.3%, and 55.3%, respectively. The H2, H7, H8, H11, and H28 fliC alleles were highly prevalent and could not be used as reliable targets for screening. Combinations of stx, eae variants, and O genetic markers, which are typical of the five targeted STEC serotypes, were detected by real-time PCR in 6.5% of the cheeses (26 samples) and included stx-wzx(O26)-eae-β1 (4.8%; 19 samples), stx-wzx(O103)-eae-ε (1.3%; five samples), stx-ihp1(O145)-eae-γ1 (0.8%; three samples), and stx-rfbE(O157)-eae-γ1 (0.3%; one sample). Twenty-eight immunomagnetic separation (IMS) assays performed on samples positive for these combinations allowed the recovery of seven eaeβ1-positive STEC O26:H11 isolates, whereas no STEC O103:H2, O145:H28, or O157:H7 strains could be isolated. Three stx-negative and eaeβ1-positive E. coli O26:[H11] strains were also isolated from cheeses by IMS. Colony hybridization allowed us to recover STEC from stx-positive samples for 15 out of 45 assays performed, highlighting the difficulties encountered in STEC isolation from dairy products. The STEC O26:H11 isolates shared the same virulence genetic profile as enterohemorrhagic E. coli (EHEC) O26:H11, i.e., they carried the virulence-associated genes EHEC-hlyA, katP, and espP, as well as genomic O islands 71 and 122. Except for one strain, they all contained the stx1 variant only, which was reported to be less frequently associated with human cases than stx2. Pulsed-field gel electrophoresis (PFGE) analysis showed that they displayed high genetic diversity; none of them had patterns identical to those of human O26:H11 strains investigated here.     

Comparison of shiga-toxigenic Escherichia coli prevalences among dairy, feedlot, and cow-calf herds in Washington State

Shiga-toxigenic Escherichia coli (STEC) strains were isolated from 7.4% of 1,440 fecal and farm environmental samples. Shiga toxin gene and STEC prevalences were significantly associated with animal production type and season. A range of serogroups were identified. Nine percent of isolates possessed all three principal virulence markers: stx(2), eae, and ehx.     

Potentially human-pathogenic Escherichia coli O26 in Norwegian sheep flocks

A national survey of Escherichia coli O26 in Norwegian sheep flocks was conducted, using fecal samples to determine the prevalence. In total, 491 flocks were tested, and E. coli O26 was detected in 17.9% of the flocks. One hundred forty-two E. coli O26 isolates were examined for flagellar antigens (H typing) and four virulence genes, including stx and eae, to identify possible Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC). Most isolates (129 out of 142) were identified as E. coli O26:H11. They possessed eae and may have potential as human pathogens, although only a small fraction were identified as STEC O26:H11, giving a prevalence in sheep flocks of only 0.8%. Correspondingly, the sheep flock prevalence of atypical EPEC (aEPEC) O26:H11 was surprisingly high (15.9%). The genetic relationship between the E. coli O26:H11 isolates was investigated by pulsed-field gel electrophoresis (PFGE) and multilocus variable number tandem repeat analysis (MLVA), identifying 63 distinct PFGE profiles and 22 MLVA profiles. Although the MLVA protocol was less discriminatory than PFGE and a few cases of disagreement were observed, comparison by partition mapping showed an overall good accordance between the two methods. A close relationship between a few isolates of aEPEC O26:H11 and STEC O26:H11 was identified, but all the E. coli O26:H11 isolates should be considered potentially pathogenic to humans. The present study consisted of a representative sampling of sheep flocks from all parts of Norway. This is the first large survey of sheep flocks focusing on E. coli O26 in general, including results of STEC, aEPEC, and nonpathogenic isolates.     

Prevalence and Characterization of Non-O157 Shiga Toxin-Producing Escherichia coli on Carcasses in Commercial Beef Cattle Processing Plants

Beef carcass sponge samples collected from July to August 1999 at four large processing plants in the United States were surveyed for the presence of non-O157 Shiga toxin-producing Escherichia coli (STEC). Twenty-eight (93%) of 30 single-source lots surveyed included at least one sample containing non-O157 STEC. Of 334 carcasses sampled prior to evisceration, 180 (54%) were found to harbor non-O157 STEC. Non-O157 STEC isolates were also recovered from 27 (8%) of 326 carcasses sampled after the application of antimicrobial interventions. Altogether, 361 non-O157 STEC isolates, comprising 41 different O serogroups, were recovered. O serogroups that previously have been associated with human disease accounted for 178 (49%) of 361 isolates. Although 40 isolates (11%) carried a combination of virulence factor genes (enterohemorrhagic E. coli hlyA, eae, and at least one stx gene) frequently associated with STEC strains causing severe human disease, only 12 of these isolates also belonged to an O serogroup previously associated with human disease. Combining previously reported data on O157-positive samples (R. O. Elder, J. E. Keen, G. R. Siragusa, G. A. Barkocy-Gallagher, M. Koohmaraie, and W. W. Laegreid, Proc. Natl. Acad. Sci. USA 97:2999-3003, 2000) with these data regarding non-O157-positive samples indicated total STEC prevalences of 72 and 10% in preevisceration and postprocessing beef carcass samples, respectively, showing that the interventions used by the beef-processing industry effected a sevenfold reduction in carcass contamination by STEC.     

Occurrence of virulence and antimicrobial resistance genes in Escherichia coli isolates from different aquatic ecosystems within the St. Clair River and Detroit River areas

Although the number of Escherichia coli bacteria in surface waters can differ greatly between locations, relatively little is known about the distribution of E. coli pathotypes in surface waters used as sources for drinking or recreation. DNA microarray technology is a suitable tool for this type of study due to its ability to detect high numbers of virulence and antimicrobial resistance genes simultaneously. Pathotype, phylogenetic group, and antimicrobial resistance gene profiles were determined for 308 E. coli isolates from surface water samples collected from diverse aquatic ecosystems at six different sites in the St. Clair River and Detroit River areas. A higher frequency (48%) of E. coli isolates possessing virulence and antimicrobial resistance genes was observed in an urban site located downstream of wastewater effluent outfalls than in the other examined sites (average of 24%). Most E. coli pathotypes were extraintestinal pathogenic E. coli (ExPEC) pathotypes and belonged to phylogenetic groups B2 and D. The ExPEC pathotypes were found to occur across all aquatic ecosystems investigated, including riverine, estuarine, and offshore lake locations. The results of this environmental study using DNA microarrays highlight the widespread distribution of E. coli pathotypes in aquatic ecosystems and the potential public health threat of E. coli pathotypes originating from municipal wastewater sources.     

Virulence properties and antimicrobial susceptibility of Shiga toxin-producing Escherichia coli strains isolated from healthy cattle from Paraná State, Brazil

The presence of Shiga toxin-producing Escherichia coli (STEC) strains in feces samples of cattle was determined using the cytotoxicity assay on Vero cells and a screening PCR system to detect stx genes. The STEC isolates were serotyped, tested for antimicrobial susceptibility, and analyzed for virulence genes using multiplex PCR. The verocytotoxin-producing E. coli - reverse passive latex agglutination (VTEC-RPLA) assay was also used to detect Shiga toxin production. The frequency of cattle shedding STEC was 36%. The isolates belonged to 33 different serotypes, of which O10:H42, O98:H41, and O159:H21 had not previously been associated with STEC. The most frequent serotypes were ONT:H7 (10%), O22:H8 (7%), O22:H16 (7%), and ONT:H21 (7%). Most of the strains (96%) were susceptible to all antimicrobial agents tested. Shiga toxin was detected by the VTEC-RPLA assay in most (89%) of the STEC strains. The frequency of virulence markers was as follows: stx1, 10%; stx2, 43%; stx1 plus stx2, 47%; ehxA, 44%; eae, 1%; and saa, 38%. Several strains belong to serotypes associated with human disease, and most of them carried a stx2-type gene, suggesting that they represent a risk to human health. The screening PCR assay showed fewer false-negative results for STEC than the Vero-cell assay and is suitable for laboratory routine.     

Molecular Characterization of Shiga Toxin-Producing Escherichia coli Isolated from the Environment of a Dairy Farm

Environmental samples were taken from ground, cattle water troughs, and feeders from a dairy farm with different STEC prevalence between animal categories (weaning calves, rearing calves, and dairy cows). Overall, 23 % of samples were positive for stx genes, stx(2) being the most prevalent type. Isolates were analyzed by PCR monoplex to confirm generic E. coli and by two multiplex PCR to investigate the presence of stx(1), stx(2), eae, saa, ehxA, and other putative virulence genes encoded in STEC plasmids: katP, espP, subA, and stcE. The toxin genes were subtyped and the strains were serotyped. The ground and the environment of the rearing calves were the sites with the highest number of STEC-positive samples; however, cattle water troughs and the environment of cows were the places with the greater chance of finding stx(2EDL933) which is a subtype associated with serious disease in humans. Several non-O157 STEC serotypes were detected. The serotypes O8:H19; O26:H11; O26:H-; O118:H2; O141:H-; and O145:H- have been asociated with human illness. Furthermore, the emergent pathogen STEC O157:H- (stx(1)-ehxA-eae) was detected in the environment of the weaning calves. These results emphasize the risk that represents the environment as source of STEC, a potential pathogen for human and suggest the importance of developing control methods designed to prevent contaminations of food products and transmission from animal to person.