Bovine feces from animals with gastrointestinal infections are a source of serologically diverse atypical enteropathogenic Escherichia coli and Shiga toxin-producing E. coli strains that commonly possess intimin

Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) cells were isolated from 191 fecal samples from cattle with gastrointestinal infections (diagnostic samples) collected in New South Wales, Australia. By using a multiplex PCR, E. coli cells possessing combinations of stx1, stx2, eae, and ehxA were detected by a combination of direct culture and enrichment in E. coli (EC) (modified) broth followed by plating on vancomycin-cefixime-cefsulodin blood (BVCC) agar for the presence of enterohemolytic colonies and on sorbitol MacConkey agar for the presence of non-sorbitol-fermenting colonies. The high prevalence of the intimin gene eae was a feature of the STEC (35 [29.2%] of 120 isolates) and contrasted with the low prevalence (9 [0.5%] of 1,692 fecal samples possessed STEC with eae) of this gene among STEC recovered during extensive sampling of feces from healthy slaughter-age cattle in Australia (M. Hornitzky, B. A. Vanselow, K. Walker, K. A. Bettelheim, B. Corney, P. Gill, G. Bailey, and S. P. Djordjevic, Appl. Environ. Microbiol. 68:6439-6445, 2002). Forty-seven STEC serotypes were identified, including O5:H-, O8:H19, O26:H-, O26:H11, O113:H21, O157:H7, O157:H- and Ont:H- which are known to cause severe disease in humans and 23 previously unreported STEC serotypes. Serotypes Ont:H- and O113:H21 represented the two most frequently isolated STEC isolates and were cultured from nine (4.7%) and seven (3.7%) animals, respectively. Fifteen eae-positive E. coli serotypes, considered to represent atypical EPEC, were identified, with O111:H- representing the most prevalent. Using both techniques, STEC cells were cultured from 69 (36.1%) samples and EPEC cells were cultured from 30 (15.7%) samples, including 9 (4.7%) samples which yielded both STEC and EPEC. Culture on BVCC agar following enrichment in EC (modified) broth was the most successful method for the isolation of STEC (24.1% of samples), and direct culture on BVCC agar was the most successful method for the isolation of EPEC (14.1% samples). These studies show that diarrheagenic calves and cattle represent important reservoirs of eae-positive E. coli.     

Phenotypic and genotypic characterization of sorbitol-negative or slow-fermenting (suspected O157) Escherichia coli isolated from milk samples in Lombardy region

AIMS: To investigate phenotypic and genotypic aspects of sorbitol-negative or slow-fermenting Escherichia coli, suspected to belong to O157 serogroup, isolated in Italy. METHODS AND RESULTS: Milk samples originating from goats and cows were screened for the presence of E. coli O157 with cultural methods. Sorbitol-negative or slow-fermenting strains were subjected to phenotypic characterization, antibiotic resistance profiles, PCR reactions for detection of toxins (stx(1) and stx(2)) and intimin (eae(GEN) and eae(O157)) genes and clustering by pulsed field gel electrophoresis (PFGE). Only one strain revealed to be O157. Susceptibility to 11 antibiotics highlighted the high resistance to tetracycline (50%), sulfonamide and streptomycin (33%). The stx(2) gene was detected in two strains; only the strain identified as O157 exhibited an amplicon for both eae genes. PFGE identified seven distinct XbaI macrorestriction patterns at a similarity level of 41%. CONCLUSIONS: The use of sorbitol fermentation as cultural method is not sufficient for STEC discrimination while PCR assay proved to be a valuable method. SIGNIFICANCE AND IMPACT OF THE STUDY: The study reports presence of Shiga toxin-producing E. coli in raw milk, signalling a potential risk for humans.     

Genetic Characterization of Escherichia coli O157:H7 Strains Isolated from the One-Humped Camel (Camelus dromedarius) by Using Microarray DNA Technology

From the Camelidae family members, several serotypes of Escherichia coli (E. coli) have recently been isolated from diarrhoeic and non-diarrhoeic faecal samples. To date Shiga toxin-producing E. coli (STEC) strains have never been typed in one-humped camel (Camelus dromedarius). In the present study, two E. coli O157:H7 strains isolated from sick dromedaries were investigated. Virulence gene profiles were determined using a custom E. coli virulence DNA microarray, composed of 70-mer oligonucleotide probes targeting 264 virulence or related genes of known E. coli pathotypes. Both strains displayed positive hybridization signals for the Locus of enterocyte effacement (LEE) gene probes (ler, eae, espA, espB, tir genes), two Shiga toxin probes (stx1 and stx2), the O157 O-antigen specific probe, various virulence plasmid (pO157) probes like katP in addition to other accessory virulence genes characterized in STEC.     

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.     

A PCR-ELISA for detecting Shiga toxin-producing Escherichia coli

A sensitive and specific PCR-ELISA was developed to detect Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) in food. The assay was based on the incorporation of digoxigenin-labeled dUTP and a biotin-labeled primer specific for Shiga toxin genes during PCR amplification. The labeled PCR products were bound to streptavidin-coated wells of a microtiter plate and detected by an ELISA. The specificity of the PCR was determined using 39 bacterial strains, including STEC, enteropathogenic E. coli, E. coli K12, and Salmonella. All of the STEC strains were positive, and non-STEC organisms were negative. The ELISA detecting system was able to increase the sensitivity of the PCR assay by up to 100-fold, compared with a conventional gel electrophoresis. The detection limit of the PCR-ELISA was 0.1-10 CFU dependent upon STEC serotypes, and genotypes of Shiga toxins. With the aid of a simple DNA extraction system, PrepMan, the PCR-ELISA was able to detect ca. 10(5) CFU of STEC per gram of ground beef without any culture enrichment. The entire procedure took about 6 h. Because of its microtiter plate format, PCR-ELISA is particularly suitable for large-scale screening and compatible with future automation.     

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.     

江苏某地健康绵羊群产志贺毒素大肠杆菌体内分离株的分子流行病学及致病力

【目的】探讨江苏某羊场健康绵羊体内产志贺毒素大肠杆菌的带菌和流行情况,同时就分离株的致病力和对Vero细胞的毒性作用作了研究。【方法】基于本实验室已经建立的EHEC O157:H7 EDL933W株的stx1、stx2、eaeA、hlyA四个基因的多重PCR检测并配合选择性增菌、平板筛选等方法对STEC进行分离鉴定。【结果】在为期6个月的连续跟踪调查中,共分离到STEC菌株107株,分离率为19.4%(107/550)。分离株属于41种O血清型、62种O:H血清型,未定型(ONT)有22株,粗糙型(OR)1株。其中属于绵羊STEC的优势血清型有O5(2株)、O91(1株)、O103(1株)。本文检测到的优势血清型为O93,stx2阳性菌株的分离率较stx1阳性菌株的分离率高,LD50测定结果表明分离株对小鼠致病力不高,受试的3个分离株均不能致小鼠死亡。对107株stx阳性分离株噬菌斑试验表明,71株阳性菌株携带噬菌体(66.3%,71/109)。受试分离株进行Vero细胞毒性试验,其中有一个菌株stx基因阳性但不能使Vero细胞产生病变。【结论】绵羊是STEC的天然宿主,可健康带菌。虽然STEC分离株对小鼠的致病力较弱,但不能排除其对人类安全的威胁。STEC携带志贺毒素基因并不意味着一定表达志贺毒素,需对志贺毒素的表达及调控机理做进一步的研究。     

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.     

Diagnosis of Shiga toxin producing Escherichia coli infection, contribution of genetic amplification technique

There has been no culture method of choice for detecting non-O157 Shiga toxin-producing Escherichia coli strains (STEC) because of their biochemical diversity The aim of this study was the assessment of verotoxin gene detection (VT1/VT2) within STEC PCR compared with the Vero cells cytotoxicity among O157 and non-O157 STEC serotypes. Stool cultures were performed on Tryptic Soy Broth and sorbitol MacConkey agar with cefixitime and tellurite supplements which were identified as Escherichia coli (E. coli) by BBL crystal. Further identifications were performed including verotoxin production assessment by Vero cells cytotoxicity assay, PCR for specific VT1/VT2 genotyping, and isolates were plated on blood agar and tested for enterohemolysis. Vero cells cytotoxicity assay revealed that 58 of E. coli isolates (71.6%) were STEC. In PCR, 33 (56.9%) of the 58 strains were positive for the VT2 gene, 24 (41.4%) were positive for the VT1 gene and one isolate was positive for both genes. In comparison to Vero cells cytotoxicity, the sensitivity, specificity of PCR were 100%. In comparative study between verotoxin assessment by Vero cells cytotoxicity and enterohemolytic activity, concordance positive results between both were 53 (91.4%). The most common serogroups of STEC were O157 (33%) and O26 (20%). From this study we can conclude that enterohemolysin production can be used as surrogate marker for STEC. The most rapid and promising approach for detection of STEC is by molecular method.     

Occurrence and virulence factors of non-O157 Shiga toxin-producing Escherichia coli in retail meat in Dunedin, New Zealand

Retail raw meat was sampled for the presence of Shiga toxin-producing Escherichia coli (STEC) using enrichment culture and Vero cell assay. The STEC obtained were serotyped and tested for enterohaemolysin (Ehly) production and the eae gene. The presence of Shiga toxin genes (stx) was confirmed by polymerase chain reaction. A total of 18 STEC were isolated accounting for 12% of beef, 17% of lamb and 4% of pork samples. Five isolates produced Ehly but none possessed the eae gene. Five isolates were identified which possessed the stx2 gene and belonged to serotypes associated with severe infection.     

Evaluation of the 'GeneDisc' real-time PCR system for detection of enterohaemorrhagic Escherichia coli (EHEC) O26, O103, O111, O145 and O157 strains according to their virulence markers and their O- and H-antigen-associated genes

Aims:  To evaluate the GeneDisc multiplex real-time PCR assay for detection of enterohaemorrhagic Escherichia coli (EHEC) O26, O103, O111, O145 and O157 strains.
Methods and Results:  GeneDiscs for detection of genes encoding Shiga toxins ( stx ), intimins ( eae ), E. coli O157 ( rfbE _O157) and H7 ( fliC _H7) antigens as well as genes specific for EHEC O26 ( wzx _O26), O103 ( wzx _O103), O111 ( wbd1 _O111), O145 ( ihp1 _O145) and O157 ( ihp1 _O157) were evaluated. The assay was run with native bacteria in 1 h in a GeneDisc Cycler. All genotypes of stx and eae , except stx _2f and eae -rho, were identified. Escherichia coli strains belonging to O-groups O26, O103, O111, O157 as well as EHEC O145:[H28] strains were specifically detected with this assay. The ihp1 _O157 gene was not found specific for EHEC O157. O-rough mutants of EHEC and non-motile EHEC O157 strains were reliably identified with the GeneDisc assay. Two to three colonies of EHEC strains were still detectable in a lawn of 50 000 apathogenic E. coli from agar plates.
Conclusions:  The GeneDisc assay is a specific and reliable assay for detection of major EHEC strains. It is robust enough to detect few EHEC colonies in mixed cultures of bacteria.
Significance and Impact of the Study:  The assay is promising for its use in EHEC diagnostics and for EHEC monitoring with different kinds of samples.     

First isolation of Shiga toxin 1d producing Escherichia coli variant strains in shellfish from coastal areas in France

AIMS: This study was carried out to evaluate the presence of Shiga toxin-producing Escherichia coli (STEC) and E. coli O157:H7 in shellfish from French coastal environments. METHODS AND RESULTS: Shellfish were collected in six growing areas or natural beds (B category) and nonfarming areas (D category) from July 2002 to August 2004. PCR detection of stx genes was performed on homogenized whole shellfish and digestive gland tissues enrichments. STEC strains were detected by colony DNA hybridization using a stx-specific gene probe and E. coli O157 strains were additionally searched by immunomagnetic separation with O157-specific magnetic beads. Stx genes were detected in 40 of 144 (27.8%) sample enrichments from mussels, oysters or cockles, 32 of 130 enrichments (24.6%) were from B-category areas and eight of 14 (57.1%) from the D-category area. Five strains carrying stx(1) or stx(1d) genes and one stx negative, eae and ehxA positive E. coli O157:H7 were isolated from six of 40 stx-positive enrichments. No relation was found between the total E. coli counts in shellfish and the presence of STEC strains in the samples. CONCLUSIONS: The STEC strains of different serotypes and stx types are present in shellfish from French coastal environments. It is the first isolation of STEC stx1d strains in France. SIGNIFICANCE AND IMPACT OF THE STUDY: Shellfish collected in coastal environments can serve as a vehicle for STEC transmission.     

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.     

Direct PCR detection of Escherichia coli O157:H7

AIMS: This paper reports a simple, rapid approach for the detection of Shiga toxin (Stx)-producing Escherichia coli (STEC). METHODS AND RESULTS: Direct PCR (DPCR) obviates the need for the recovery of cells from the sample or DNA extraction prior to PCR. Primers specific for Stx-encoding genes stx1 and stx2 were used in DPCR for the detection of E. coli O157:H7 added to environmental water samples and milk. CONCLUSIONS: PCR reactions containing one cell yielded a DPCR product. SIGNIFICANCE AND IMPACT OF THE STUDY: This should provide an improved method to assess contamination of environmental and other samples by STEC and other pathogens.     

Shiga toxin-producing Escherichia coli O171:H25 strain isolated from a patient with haemolytic uraemic syndrome

Shiga toxin-producing Escherichia coli (STEC) strains of O157:H7 serotype are a predominant cause of haemolytic uraemic syndrome (HUS) worldwide, but strains of non-O157 serotypes can also be associated with serious disease. Some of them are associated with outbreaks of HUS, others with sporadic cases of HUS, and some with diarrhoea but not with outbreaks or HUS. A large number of STEC serotypes isolated from ruminants and foods have never been associated with human disease. In this study we characterize a STEC strain belonging to serotype O171:H25 that is responsible for a case of HUS. This strain has a single Shiga toxin gene encoding Stx2 toxin, and hlyA gene, but is eae-negative.     

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.     

Characterisation of Shiga toxin-producing Escherichia coli (STEC) isolated from seafood and beef

Shiga toxin-producing Escherichia coli (STEC) strains isolated in Mangalore, India, were characterised by bead-enzyme-linked immunosorbent assay (bead-ELISA), Vero cell cytotoxicity assay, PCR and colony hybridisation for the detection of stx1 and stx2 genes. Four strains from seafood, six from beef and one from a clinical case of bloody diarrhoea were positive for Shiga toxins Stx1 and Stx2 and also for stx1and stx2 genes. The seafood isolates produced either Stx2 alone or both Stx1 and Stx2, while the beef isolates produced Stx1 alone. The stx1 gene of all the beef STEC was found to be of recently reported stx1c type. All STEC strains and one non-STEC strain isolated from clam harboured EHEC-hlyA. Interestingly, though all STEC strains were negative for eae gene, two STEC strains isolated from seafood and one from a patient with bloody diarrhoea possessed STEC autoagglutinating adhesion (saa) gene, recently identified as a gene encoding a novel autoagglutinating adhesion.     

Pre-slaughter handling of cattle and Shiga toxin-producing Escherichia coli (STEC)

AIMS: The aim of the study was to monitor the shedding and transmission of generic and Shiga toxin-producing Escherichia coli (STEC) in a consignment of cattle during lot feeding. METHODS AND RESULTS: Faecal and environmental samples were tested for total E. coli and screened with PCR specific for Shiga toxin and O157 rfb. STEC were isolated using colony hybridization and characterized by serology and genotyping. STEC prevalence initially decreased after the diet shift from pasture to grain, although there were intermittent peaks in numbers of cattle shedding STEC and E. coli O157. Water troughs and soil were intermittently contaminated. Common genotypes and serotypes were isolated from animals, water and soil in the feedlot, with additional types introduced at slaughter. CONCLUSION: STEC and E. coli O157 are endemic in cattle and intermittent peaks in shedding occur. Prevention of these peaks and/or reduction in transmission is required to reduce the risk of carcass contamination during slaughter. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings contribute to the understanding of the ecology of STEC and suggest control points for reducing STEC contamination in feedlot cattle production.     

Serotypes and virulence factors of Shiga toxin-producing Escherichia coli isolated from healthy Norwegian sheep

AIMS: To characterize a number of Shiga toxin-producing Escherichia coli (STEC) isolates from sheep and to discuss the potential of these isolates as human pathogens. METHODS AND RESULTS: Twelve different O-groups and seven different H-types were identified by standard serotyping methods. The most common serotypes were O5:NM, O6:H10, O91:NM and O128:NM. Polymerase chain reaction (PCR) was used for the detection of virulence factor genes. Of 102 isolates, 86.3% carried stx1 and 83% of these were also positive in the stx1OX3-specific PCR. stx2 was carried by 55.9% of the isolates and 77.2% of these were also positive in the stx2d-specific PCR. The Vero cell assay showed high toxin production in 70.6% of the isolates. None of the isolates carried eae. CONCLUSIONS: The study supports the animal-host relationship suggested in other studies with STEC serogroups O5, O91 and O128 strongly associated with sheep. Most sheep STEC carry stx1OX3 (except O91) and the dominating stx2 variant is stx2d. One stx profile clearly dominates within a serotype. SIGNIFICANCE AND IMPACT OF THE STUDY: In spite of the predominance of certain sheep-associated STEC, sheep cannot be excluded as carriers of human pathogenic 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.