The characterization of Shiga toxin-non-producing Escherichia coli serotype O157:H7 isolated from carcasses of cattle at a slaughter house.

A bacteriological investigation of Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H7 was performed on 298 carcasses of cattle at slaughter houses between July 1996 and January 1997 in Gifu Prefecture, Japan. As a result, four Stx-non-producing Escherichia coli O157:H7 strains were isolated from two slaughtered carcasses of cattle. The purpose of this study was to examine the characterization of isolates. Isolates possessed the E. coli attaching and effacing gene (eaeA), and hemolysin gene (hlyA), and harbored 3.0-MDa and 60-MDa plasmids. The Xba I pulsed-field gel electrophoresis (PFGE) pattern showed three similar patterns. Consequently, a closely related genotype of Stx-non-producing E. coli O157:H7 may widely exist in cattle.     

Presence of Shiga toxin-producing Escherichia coli O157:H7 in living layer hens

AIMS: To evaluate the presence of Shiga toxin-producing strains of Escherichia coli (STEC) of the O157:H7 serotype in living layer hens so as to analyse the role of this avian species as potential reservoir. METHODS AND RESULTS: Cloacal swabs were collected between November 2004 and November 2005 from four intensive management layer hen farms and analysed for STEC O157:H7 by immunomagnetic separation methods and multiplex polymerase chain reaction for stx1 and/or stx2, the E. coli attaching and effacing (eae) and hly genes. STEC was detected in 26 of the 720 samples. CONCLUSIONS: The layer hens analysed were shown to carry STEC O157:H7. The presence of this bacterium in living layer hen farms investigated did not result in any detectable increase in gastrointestinal disease in this species. SIGNIFICANCE AND IMPACT OF THE STUDY: Living layer hens are a novel potential reservoir of E. coli O157:H7.     

Prevalence of Shiga toxin-producing Escherichia coli stx1, stx2, eaeA, and rfbE genes and survival of E. coli O157:H7 in manure from organic and low-input conventional dairy farms

Manure samples were collected from 16 organic (ORG) and 9 low-input conventional (LIC) Dutch dairy farms during August and September 2004 to determine the prevalence of the STEC virulence genes stx(1) (encoding Shiga toxin 1), stx(2) (encoding Shiga toxin 2), and eaeA (encoding intimin), as well as the rfbE gene, which is specific for Escherichia coli O157. The rfbE gene was present at 52% of the farms. The prevalence of rfbE was higher at ORG farms (61%) than at LIC farms (36%), but this was not significant. Relatively more LIC farms were positive for all Shiga toxin-producing E. coli (STEC) virulence genes eaeA, stx(1), and stx(2), which form a potentially highly virulent combination. Species richness of Enterobacteriaceae, as determined by DGGE, was significantly lower in manure positive for rfbE. Survival of a green fluorescent protein-expressing E. coli O157:H7 strain was studied in the manure from all farms from which samples were obtained and was modeled by a biphasic decline model. The time needed to reach the detection limit was predominantly determined by the level of native coliforms and the pH (both negative relationships). Initial decline was faster for ORG manure but leveled off earlier, resulting in longer survival than in LIC manure. Although the nonlinear decline curve could theoretically be explained as the cumulative distribution of an underlying distribution of decline kinetics, it is proposed that the observed nonlinear biphasic pattern of the survival curve is the result of changing nutrient status of the manure over time (and thereby changing competition pressure), instead of the presence of subpopulations differing in the level of resistance.     

Shiga toxin 2e-producing Escherichia coli isolates from humans and pigs differ in their virulence profiles and interactions with intestinal epithelial cells

Thirteen Escherichia coli strains harboring stx2e were isolated from 11,056 human stools. This frequency corresponded to the presence of the stx2e allele in 1.7% of all Shiga toxin-producing E. coli (STEC) strains. The strains harboring stx2e were associated with mild diarrhea (n = 9) or asymptomatic infections (n = 4). Because STEC isolates possessing stx2e are porcine pathogens, we compared the human STEC isolates with stx2e-harboring E. coli isolated from piglets with edema disease and postweaning diarrhea. All pig isolates possessed the gene encoding the F18 adhesin, and the majority possessed adhesin involved in diffuse adherence; these adhesins were absent from all the human STEC isolates. In contrast, the high-pathogenicity island encoding an iron uptake system was found only in human isolates. Host-specific patterns of interaction with intestinal epithelial cells were observed. All human isolates adhered to human intestinal epithelial cell lines T84 and HCT-8 but not to pig intestinal epithelial cell line IPEC-J2. In contrast, the pig isolates completely lysed human epithelial cells but not IPEC-J2 cells, to which most of them adhered. Our data demonstrate that E. coli isolates producing Shiga toxin 2e have imported specific virulence and fitness determinants which allow them to adapt to the specific hosts in which they cause various forms of disease.     

Rapid and specific identification of Shiga toxin-producing Escherichia coli in faeces by multiplex PCR

AIMS: The object of this study was to develop a multiplex PCR system for rapid and specific identification of Shiga toxin-producing Escherichia coli (STEC) in faeces. METHODS AND RESULTS: A multiplex PCR (mPCR) protocol was developed using a primer pair specific for genes that are involved in the biosynthesis of the O157 E. coli antigen, and primers that identify the sequences of Shiga toxin 1 and 2 (stx 1 and stx1) and the intimin protein (eaeA). The mPCR assay was used for amplification of STEC genes in bacteria directly (after enrichment) in faeces. The test was very sensitive and could detect between 9 and 1 bacterial cells per gram of faeces. The mPCR was used for the examination of 69 bovine faecal samples derived from healthy cattle. The results indicated that 62 x 3% of the samples were positive, generating at least one PCR amplicon of the expected size. CONCLUSIONS: The method can be applied for rapid and specific identification of STEC bacteria in faecal samples, and for differentiation of their main virulence marker genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to sensitively detect Shiga toxin-producing E. coli directly in faeces within a short time represents a considerable advancement over more time-consuming and less sensitive methods for identification and characterization of STEC bacteria.     

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。     

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.     

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.     

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.     

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.     

Epidemiological relatedness and clonal types of natural populations of Escherichia coli strains producing Shiga toxins in separate populations of cattle and sheep.

Two separate animal populations consisting of a herd of cattle (19 animals) and a flock of sheep (25 animals) were investigated for strains of Escherichia coli producing Shiga toxins (STEC) over a time period of 6 months. Thirty-three STEC were isolated from 63.2% of cattle and grouped into 11 serotypes and eight electrophoretic types (ETs) by multilocus enzyme analysis. In sheep, 88% of the animals excreted STEC (n = 67 isolates) belonging to 17 different serotypes and 12 different ETs. STEC from cattle and sheep differed with respect to serotype, and only 4 of the 16 ETs occurred in both animal populations. In cattle, ET14 (O116:H21) strains predominated, whereas other STEC serotypes occurred only sporadically. The predominating STEC types in sheep were ET4 (O125 strains), ET11 (O128:H2 and others), and ET14 (O146:H21). In contrast to their diversity, STEC originating from the same animal population were similar with respect to Shiga toxin (stxy genes. Almost all STEC isolated from cattle were positive for stx2 and stx2c; only one was positive for stx1. In sheep, almost all STEC isolated were positive for stx1 and stx2, whereas stx2c was not found. XbaI-digested DNAs of genetically closely related O146:H21 strains have different restriction profiles which were associated with size alterations in XbaI fragments hybridizing with stx1- and stx2-specific DNA probes. Our results indicate that stx-encoding bacteriophages might be the origin of the genetic heterogeneity in STEC from animals.     

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.     

Isolation and characterization of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) from calves and lambs with diarrhoea in India

AIMS: To determine the prevalence and molecular characteristics of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) in calves and lambs with diarrhoea in India. METHODS AND RESULTS: Faecal samples originating from 391 calves and 101 lambs which had diarrhoea were screened for presence of E. coli. A total number of 309 (249 bovine and 60 ovine) E. coli strains were isolated. A total of 113 bovine and 15 ovine strains were subjected to multiplex polymerase chain reaction (m-PCR) for detection of stx1, stx2, eaeA and EHEC hlyA genes. STEC and EPEC belonging to different serogpoups were detected in 9.73% of calves studied. Six per cent and 26.66% of lambs studied were carrying STEC and EPEC, respectively. Majority of the STEC serogroups isolated in this study did not belong to those which have been identified earlier to be associated mainly with diarrhoea and enteritis in cattle and sheep outside India. The most frequent serogroup among bovine and ovine EPEC was O26 (40%). One of the most important STEC serogroup O157, known for certain life-threatening infections in humans, was isolated from both bovine and ovine faecal samples. CONCLUSIONS: A high percentage of STEC and EPEC belonging to different serogroups are prevalent in calves and lambs with diarrhoea in India and could be the cause of disease in them. SIGNIFICANCE AND IMPACT OF THE STUDY: The study reports, for the first time, the isolation and characterization of STEC and EPEC serogroups associated with diarrhoea in calves and lambs in India. Many STEC and EPEC strains belonged to serogoups known for certain life-threatening diseases in humans.     

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 shiga toxin-producing Escherichia coli in swine feces recovered in the National Animal Health Monitoring System's Swine 2000 study

A study was conducted to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) in swine feces in the United States as part of the National Animal Health Monitoring System's Swine 2000 study. Fecal samples collected from swine operations from 13 of the top 17 swine-producing states were tested for the presence of STEC. After enrichment of swine fecal samples in tryptic soy broth, the samples were tested for the presence of stx1 and stx2 by use of the TaqMan E. coli STX1 and STX2 PCR assays. Enrichments of samples positive for stx1 and/or stx2 were plated, and colony hybridization was performed using digoxigenin-labeled probes complementary to the stx1 and stx2 genes. Positive colonies were picked and confirmed by PCR for the presence of the stx1, stx2, or stx2e genes, and the isolates were serotyped. Out of 687 fecal samples tested using the TaqMan assays, 70% (484 of 687) were positive for Shiga toxin genes, and 54% (370 of 687), 64% (436 of 687), and 38% (261 of 687) were positive for stx1, stx2, and both toxin genes, respectively. Out of 219 isolates that were characterized, 29 (13%) produced stx1, 14 (6%) produced stx2, and 176 (80%) produced stx2e. Twenty-three fecal samples contained at least two STEC strains that had different serotypes but that had the same toxin genes or included a strain that possessed stx1 in addition to a strain that possessed stx2 or stx2e. The STEC isolates belonged to various serogroups, including O2, O5, O7, O8, O9, OX10, O11, O15, OX18, O20, O57, O65, O68, O69, O78, O91, O96, O100, O101, O120, O121, O152, O159, O160, O163, and O untypeable. It is noteworthy that no isolates of serogroup O157 were recovered. Results of this study indicate that swine in the United States harbor STEC that can potentially cause human illness.     

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.     

Glutamate decarboxylase genes as a prescreening marker for detection of pathogenic Escherichia coli groups.

The enzyme glutamate decarboxylase (GAD) is prevalent in Escherichia coli but few strains in the various pathogenic E. coli groups have been tested for GAD. Using PCR primers that amplify a 670-bp segment from the gadA and gadB genes encoding GAD, we examined the distribution of the gadAB genes among enteric bacteria. Analysis of 173 pathogenic E. coli strains, including 125 enterohemorrhagic E. coli isolates of the O157:H7 serotype and its phenotypic variants and 48 isolates of enteropathogenic E. coli, enterotoxigenic E. coli, enteroinvasive E. coli, and other Shiga toxin-producing E. coli (STEC) serotypes, showed that gadAB genes were present in all these strains. Among the 22 non-E. coli isolates tested, only the 6 Shigella spp. carried gadAB. Analysis of naturally contaminated water and food samples using a gadAB-specific DNA probe that was labeled with digoxigenin showed that a gadAB-based assay is as reliable as standard methods that enumerate E. coli organisms on the basis of lactose fermentation. The presence of few E. coli cells initially seeded into produce rinsates could be detected by PCR to gadA/B genes after overnight enrichment. A multiplex PCR assay using the gadAB primers in combination with primers to Shiga toxin (Stx) genes stx(1) and stx(2) was effective in detecting STEC from the enrichment medium after seeding produce rinsate samples with as few as 2 CFU. The gadAB primers may be multiplexed with primers to other trait virulence markers to specifically identify other pathogenic E. coli groups.     

Phenotypic and genotypic characterization of avian Escherichia coli O86:K61 isolates possessing a gamma-like intimin

Escherichia coli O86:K61 has long been associated with outbreaks of infantile diarrhea in humans and with diarrheal disease in many animal species. Studies in the late 1990s identified E. coli O86:K61 as the cause of mortality in a variety of wild birds, and in this study, 34 E. coli O86:K61 isolates were examined. All of the isolates were nonmotile, but most elaborated at least two morphologically distinct surface appendages that were confirmed to be type 1 and curli fimbriae. Thirty-three isolates were positive for the eaeA gene encoding a gamma type of intimin. No phenotypic or genotypic evidence was obtained for elaboration of Shiga-like toxins, but most isolates possessed the gene coding for the cytolethal distending toxin. Five isolates were selected for adherence assays performed with tissue explants and HEp-2 cells, and four of these strains produced attaching and effacing lesions on HEp-2 cells and invaded the cells, as determined by transmission electron microscopy. Two of the five isolates were inoculated orally into 1-day-old specific-pathogen-free chicks, and both of these isolates colonized, invaded, and persisted well in this model. Neither isolate produced attaching and effacing lesions in chicks, although some pathology was evident in the alimentary tract. No deaths were recorded in inoculated chicks. These findings are discussed in light of the possibility that wild birds are potential zoonotic reservoirs of attaching and effacing E. coli.     

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 comparison of two pre-enrichment media prior to immunomagnetic separation for the isolation of E. coli O157 from bovine faeces

AIMS: To compare the sensitivity of two pre-enrichment broth media prior to immunomagnetic separation for the isolation of Escherichia coli O157 from cattle faeces. METHODS AND RESULTS: One-gram portions of 721 cattle faeces collected from 43 farms were pre-enriched in buffered peptone water containing vancomycin, cefixime and cefsulodin (BPW-VCC) and buffered peptone water without additives (BPW-WOA), respectively. A total of 137 samples were positive for E. coli O157: 127 pre-enriched with BPW-WOA and 89 pre-enriched in BPW-VCC. Representative isolates were tested for phage type, verotoxin and eae (E. coli attaching and effacing) gene sequences, resulting in the recognition of eight different types. All the E. coli O157 types recognized were isolated by both methods except for three different strains, each of which were isolated only on a single occasion: two by BPW-WOA and another by BPW-VCC. CONCLUSIONS: The results clearly demonstrate, under the conditions of this study, that BPW without antibiotics was the superior pre-enrichment medium for the isolation of E. coli O157 from cattle faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of BPW-WOA in preference to BPW-VCC for the isolation of E. coli O157 from cattle faeces in future research and outbreak studies should lead to a higher number of positive isolates.