Multiple‐Aetiology Enteric Infections Involving Non‐O157 Shiga Toxin‐Producing Escherichia coli – FoodNet, 2001–2010

We describe multiple‐aetiology infections involving non‐O157 Shiga toxin‐producing Escherichia coli (STEC) identified through laboratory‐based surveillance in nine FoodNet sites from 2001 to 2010. A multiple‐aetiology infection (MEI) was defined as isolation of non‐O157 STEC and laboratory evidence of any of the other nine pathogens under surveillance or isolation of >1 non‐O157 STEC serogroup from the same person within a 7‐day period. We compared exposures of patients with MEI during 2001–2010 with those of patients with single‐aetiology non‐O157 STEC infections (SEI) during 2008–2009 and with those of the FoodNet population from a survey conducted during 2006–2007. In total, 1870 non‐O157 STEC infections were reported; 68 (3.6%) were MEI; 60 included pathogens other than non‐O157 STEC; and eight involved >1 serogroup of non‐O157 STEC. Of the 68 MEI, 21 (31%) were part of six outbreaks. STEC O111 was isolated in 44% of all MEI. Of patients with MEI, 50% had contact with farm animals compared with 29% (P < 0.01) of persons with SEI; this difference was driven by infections involving STEC O111. More patients with non‐outbreak‐associated MEI reported drinking well water (62%) than respondents in a population survey (19%) (P < 0.01). Drinking well water and having contact with animals may be important exposures for MEI, especially those involving STEC O111.     

Application of a real-time PCR-based system for monitoring of O26, O103, O111, O145 and O157 Shiga toxin-producing Escherichia coli in cattle at slaughter

Faecal samples were collected from 573 slaughtered cattle aged between three and 24 months in seven abattoirs. After enrichment (mTSB with novobiocin), samples were screened by real‐time PCR first for stx and if positive, tested for the top‐five Shiga toxin‐producing Escherichia coli (STEC) serogroups using PCR assays targeting genes specific for serogroups O26, O103, O111, O145 and O157. Of 563 samples with available results, 74.1% tested positive for stx genes. Amongst them, the serogroups O145, O103, O26, O157 and O111 were detected in 41.9%, 25.9%, 23.9%, 7.8% and 0.8%, respectively. From 95 O26, 166 O145 and 30 O157 PCR‐positive samples, 17 O26, 28 O145 and 12 O157 strains were isolated by colony hybridization after immunomagnetic separation. The 17 O26 strains were eae‐positive, but only nine strains harboured stx (eight possessing stx1 and one stx2). Of the 28 O145 strains, ten were eae‐positive including four harbouring stx1 or stx2, whereas 18 were negative for stx and eae. Five of the 12 O157 strains harboured stx2 and eae, did not ferment sorbitol, and were identified as STEC O157:H7/H^?. The other seven O157 strains were negative for stx and eae or positive only for eae. Shiga toxin genes and the top‐five STEC serogroups were frequently found in young Swiss cattle at slaughter, but success rates for strain isolation were low and only few strains showed a virulence pattern of human pathogenic STEC.     

Applicability of a multiplex PCR to detect O26, O45, O103, O111, O121, O145, and O157 serogroups of Escherichia coli in cattle feces

Shiga toxin-producing Escherichia coli (STEC), particularly O157, are major food borne pathogens. Non-O157 STEC, particularly O26, O45, O103, O111, O121, and O145, have also been recognized as a major public health concern. Unlike O157, detection procedures for non-O157 have not been fully developed. Our objective was to develop a multiplex PCR to distinguish O157 and the 'top six' non-O157 serogroups (O26, O45, O103, O111, O121, and O145) and evaluate the applicability of the multiplex PCR to detect the seven serogroups of E. coli in cattle feces. Published sequences of O-specific antigen coding genes, rfbE (O157) and wzx and wbqE-F (non-O157), were analyzed to design serogroup-specific primers. The specificity of amplifications was confirmed with 138 known STEC strains and the reaction yielded the expected amplicons for each serogroup. In feces spiked with pooled 7 STEC strains, the sensitivity of the detection was 4.1 × 10(5)CFU/g before enrichment and 2.3 × 10(2) after 6h enrichment in E. coli broth. Additionally, 216 fecal samples from cattle were collected and tested by multiplex PCR and cultural methods. The multiplex PCR revealed a high prevalence of all seven serogroups (178 [O26], 108 [O45], 149 [O103], 30 [O111], 103 [O121], 5 [O145], and 160 [O157]) of 216 samples in fecal samples. Cultural procedures identified 33.1% (53/160) and 35.5% (11/31) of PCR-positive samples for E. coli O157 and non-O157 serogroups, respectively. Samples that were culture-positive were all positive by the multiplex PCR. The multiplex PCR can be used to identify serogroups of putative STEC isolates.     

Virulence markers and genetic relationships of Shiga toxin-producing Escherichia coli strains from serogroup O111 isolated from cattle

Shiga toxin-producing Escherichia coli (STEC) strains isolated from healthy cattle (O111:NM, seven strains; O111:H8, three strains) in Brazil were studied and compared to previously characterized human strains in regard to their phenotypic and genotypic characteristics to evaluate their pathogenic potential. Most bovine STEC O111 strains were isolated from dairy calves, and strains with genotypes stx1 alone and stx1/stx2 (variant stx2) occurred in different regions. Irrespective of the stx genotype, all strains were positive for eae theta, alpha variants of tir, espA and espB, and for ler, qseA, iha, astA and efa1 genes. Only one strain was negative for EHEC-hlyA and all strains were negative for iha, saa and espP genes and for EAF and bfpA, genetic markers of EPEC. Except for the presence of stx2, bovine strains showed the same profile of putative virulence genes found among the human strains. Similar biochemical behavior was identified among the strains analysed. Two bovine STEC strains produced the localized adherence (LA) phenotype in 6-h tests with Caco-2 (human enterocyte) cells. Intimate attachment (judged by the FAS test) was found in 9 out of 10 bovine strains as it was observed for the human STEC strains. RAPD-PCR analysis showed two distinct RAPD groups among the STEC O111 strains examined. Despite the relative low frequency of STEC O111 strains recovered from cattle no differences in their pathogenic potential were observed compared to some strains isolated from human diarrhea, suggesting that healthy cattle may be a potential source of infection for humans in Brazil.     

An investigation of shedding and super‐shedding of Shiga toxigenic Escherichia coli O157 and E. coli O26 in cattle presented for slaughter in the Republic of Ireland

Shiga toxigenic Escherichia coli (STEC) are an important group of pathogens and can be transmitted to humans from direct or indirect contact with cattle faeces. This study investigated the shedding of E. coli O157 and O26 in cattle at the time of slaughter and factors associated with super‐shedding (SS) animals. Rectoanal mucosal swab (RAMS) samples were collected from cattle (n = 1,317) at three large Irish commercial beef abattoirs over an 18 month period, and metadata were collected at the time of sampling regarding farm of origin, animal age, breed and gender. RAMS swabs were examined for the presence and numbers of E. coli O157 and O26 using a previously developed quantitative real‐time PCR protocol. Samples positive by PCR were culturally examined and isolates analysed for the presence of stx subtypes, eae and phylogroup. Any samples with counts >10⁴ CFU/swab of STEC O157 or O26 were deemed to be super‐shedders. Overall, 4.18% (55/1,317) of RAMS samples were positive for STEC O157, and 2.13% (28/1,317) were classified as STEC O157 SS. For STEC O26, 0.76% (10/1,317) of cattle were positive for STEC O26, and 0.23% (3/1,317) were classified as super‐shedders. Fewer STEC shedders and SS were noted among older animals (>37 months). There was a seasonal trend observed for STEC O157, with the highest prevalence of shedding and SS events in the autumn (August to October). The majority of E. coli O157 (50/55) isolates had stx2 and were eae positive, with no significant difference between SS and low shedders (LS). Interestingly, all STEC O26 (n = 10) were eae negative and had varied stx profiles. This study demonstrates that, while the overall shedding rates are relatively low in cattle at slaughter, among positive animals there is a high level of SS, which may pose a higher risk of cross‐contamination during slaughter.     

Antibiotic‐resistant Shiga toxin‐producing Escherichia coli: An overview of prevalence and intervention strategies

Shiga toxin‐producing Escherichia coli (STEC) are foodborne pathogens that can cause severe diseases, including bloody diarrhoea and kidney failure, in humans, while remaining harmless to its primary reservoir hosts, cattle. Antibiotics such as azithromycin, fosfomycin and meropenem are being used and recommended in the treatment of early‐stage STEC (mainly E. coli O157:H7) infections, as these are reportedly effective in preventing Shiga toxin release and kidney failure while eliminating the pathogen. However, antibiotic resistance among STEC isolates could negatively impact these and other similar treatment options while contributing towards the spread of antibiotic resistance genes especially if encoded on mobile genetic elements like plasmids. Antibiotic resistance among STEC isolates recovered from animals and patients is being reported globally. A comprehensive understanding of the prevalence of antibiotic‐resistant STEC (AR‐STEC) and the mechanisms promoting this resistance among these bacteria could help direct therapies and develop strategies to effectively reduce/eliminate these pathogens. Here, we have reviewed literature from the past three decades to gain insights on this prevalence and its impact on human infections. In addition, we have reviewed various strategies proposed by researchers to control STEC that in turn would be applicable to AR‐STEC as well.     

Passive transfer of antibodies to Shiga toxin-producing Escherichia coli O26, O111 and O157 antigens in neonatal calves by feeding colostrum

To study whether or not passive immunity of neonatal calves against Shiga toxin-producing Escherichia coli (STEC) O26, O111, and O157 was obtained by colostrum administration, serum antibodies in calves after the feeding were determined by enzyme-linked immunosorbent assay (ELISA) in comparison with antibodies in colostrum and sera from donor dams. The highest antibody titers to STEC in colostrum from dams were detected soon after parturition. The antibody titers were found to be elevated in sera of neonatal calves (4-9 hr after birth) orally administered with colostrum with high antibody titers, suggesting that passive immunity of neonatal calves to STEC infection may be obtained by feeding colostrum. These results suggest that colostrum administration to neonatal calves may play an important role in elevating serum antibodies against STEC in neonatal calves.     

Persistence of Escherichia coli O157:H7 in experimentally infected swine

These experiments determined the ability of Escherichia coli O157:H7 to colonize and persist in pigs simultaneously inoculated with other pathogenic E. coli strains. Three-months-old pigs were inoculated with a mixture of five E. coli strains. The mixture included two Shiga toxigenic E. coli (STEC) O157:H7 strains, two enterotoxigenic E. coli (ETEC) strains and one enteropathogenic E. coli (EPEC) strain. A high dose mixture with all five strains at 10(10)CFU/animal (CFU: colony forming units) and a low dose mixture with the STEC strains at 10(7)CFU and the EPEC and ETEC strains remaining at 10(10)CFU were used. The STEC strains persisted in the alimentary tracts of some pigs at 2 months post-inoculation, following inoculation with both the high and low dose mixtures. When all strains were given at 10(10)CFU (high dose) the STEC strains persisted in greater numbers and in more pigs than did the other E. coli strains. The results demonstrated that persistent colonization (> or =2 months) by E. coli O157:H7 can occur in pigs. These findings were similar to those reported from sheep inoculated with the same mixture of E. coli strains. The results are consistent with reports suggesting that pigs have the potential to be reservoir hosts for STEC O157:H7.     

1株感染多种致病性大肠杆菌噬菌体的生物学特性研究

旨在测定此前从断奶前仔猪粪样中分离得到的1株大肠杆菌噬菌体C6在致病性大肠杆菌上的生物学特性,并比较该噬菌体在不同致病菌上的感染特性.利用电镜形态观察和基因组测序确定其分类,用点滴法和双层平板法测定其在致病性大肠杆菌上的宿主谱,通过噬菌斑形态、最佳感染复数(MOI)、成斑率(EOP)、吸附率、一步生长曲线以及抑菌曲线等...     

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 stx₁ 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.     

Stochastic Simulation Model Comparing Distributions of STEC O157 Faecal Shedding Prevalence Between Cattle Vaccinated With Type III Secreted Protein Vaccines and Non‐Vaccinated Cattle

Pens of cattle with high Escherichia coli O157:H7 (STEC O157) prevalence at harvest may present a greater risk to food safety than pens of lower prevalence. Vaccination of live cattle against STEC O157 has been proposed as an approach to reduce STEC O157 prevalence in live cattle. Our objective was to create a stochastic simulation model to evaluate the effectiveness of pre‐harvest interventions. We used the model to compare STEC O157 prevalence distributions for summer‐ and winter‐fed cattle to summer‐fed cattle immunized with a type III secreted protein (TTSP) vaccine. Model inputs were an estimate of vaccine efficacy, observed frequency distributions for number of animals within a pen, and pen‐level faecal shedding prevalence for summer and winter. Uncertainty about vaccine efficacy was simulated using a log‐normal distribution (mean = 58%, SE = 0.14). Model outputs were distributions of STEC O157 faecal pen prevalence of summer‐fed cattle unvaccinated and vaccinated, and winter‐fed cattle unvaccinated. The simulation was performed 5000 times. Summer faecal prevalence ranged from 0% to 80% (average = 30%). Thirty‐six per cent of summer‐fed pens had STEC O157 prevalence >40%. Winter faecal prevalence ranged from 0% to 60% (average = 10%). Seven per cent of winter‐fed pens had STEC O157 prevalence >40%. Faecal prevalence for summer‐fed pens vaccinated with a 58% efficacious vaccine product ranged from 0% to 52% (average = 13%). Less than one per cent of vaccinated pens had STEC O157 prevalence >40%. In this simulation, vaccination mitigated the risk of STEC O157 faecal shedding to levels comparable to winter, with the major effects being reduced average shedding prevalence, reduced variability in prevalence distribution, and a reduction in the occurrence of the highest prevalence pens. Food safety decision‐makers may find this modelling approach useful for evaluating the value of pre‐harvest interventions.     

Characterization of Shiga toxin – producing Escherichia coli infections in beef feeder calves and the effectiveness of a prebiotic in alleviating Shiga toxin - producing Escherichia coli infections

Background

In the less-sensitive mouse model, Shiga toxin-producing Escherichia coli (STEC) challenges result in shedding that reflect the amount of infection and the expression of virulence factors such as Shiga toxins (Stx). The purpose of this study was to characterize the contribution of STEC diversity and Stx expression to shedding in beef feeder calves and to evaluate the effectiveness of a prebiotic, Celmanax®, to alleviate STEC shedding. Fecal samples were collected from calves at entry and after 35 days in the feedlot in spring and summer. STECs were evaluated using selective media, biochemical profile, serotyping and Stx detection. Statistical analysis was performed using repeated measures ANOVA and logistic regression.

Results

At entry, non-O157 STEC were dominant in shedding calves. In spring, 21%, 14% and 14% of calves acquired O157, non-O157 and mixed STEC infections, respectively. In contrast, 45%, 48% and 46% of calves in summer acquired O157, non-O157 and mixed STEC infections, respectively. Treatment with a prebiotic, Celmanax®, in spring significantly reduced 50% of the O157 STEC infections, 50% of the non-O157 STEC infections and 36% of the STEC co-infections (P = 0.037). In summer, there was no significant effect of the prebiotic on STEC infections. The amount of shedding at entry was significantly related to the number and type of STECs present and Stx expression (r² = 0.82). The same relationship was found for shedding at day 35 (r² = 0.85), but it was also related to the number and type of STECs present at entry. Stx - producing STEC infections resulted in 100 to 1000 × higher shedding in calves compared with Stx-negative STECs.

Conclusions

STEC infections in beef feeder calves reflect the number and type of STECs involved in the infection and STEC expression of Stx. Application of Celmanax® reduced O157 and non-O157 STEC shedding by calves but further research is required to determine appropriate dosages to manage STEC infections.     

Detection and characterisation of Shiga toxin-producing Escherichia coli other than Escherichia coli O157:H7 in wild ruminants

Shiga toxin-producing Escherichia coli (STEC) are an important group of emerging pathogens, with ruminants recognised as their main natural reservoir. The aim of this work was to establish the prevalence of non-O157 STEC in free-ranging wild ruminants in the Extremadura region of Spain and to characterise them phenogenotypically. Faecal samples were collected from 243 wild ruminants, including Cervus elaphus, Capreolus capreolus, Dama dama and Ovis musimon and were examined for STEC using both phenotypic (Vero cells) and genotypic (PCR and PFGE) methods.Shiga toxin-producing Escherichia coli were isolated from 58 (23.9%) of the samples and a total of 65 isolates were characterised. A PCR method indicated that 11 (16.9%) strains carried the stx₁ gene, 44 (67.7%) carried the stx₂ gene and 10 (15.4%) carried both these genes. The ehxA gene was detected in 37 (57%) of the isolates but none contained either the eae or saa genes. The isolates were from a total of 12 ‘O’ serogroups, although 80% were restricted to the O2, O8, O128, O146, O166 and O174 serogroups. The most commonly isolated STEC bacteria, which were from the O146 serogroup, exhibited a high degree of polymorphism as indicated by PFGE. Shiga toxin-producing Escherichia coli isolates of serogroups O20, O25, O166, O171, O174 and O176 had not previously been found in wild ruminants. This is the first study to confirm that wild ruminants in Spain are a reservoir of STEC and are thus a potential source of human infection.     

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.     

Development of a PCR‐Based Method for Specific Identification of Genotypic Markers of Shiga Toxin‐Producing Escherichia coli Strains

A simple, rapid and specific PCR‐based method for identification of shiga toxin‐producing Escherichia coli (STEC) was developed. The procedure involves amplification of the E. coli‐specific universal stress protein A (uspA) gene (uspa‐PCR), with the primer pair described by other authors, which allows differentiation of E. coli (STEC and non‐STEC) from other gram‐negative bacteria followed by identification of the main genetic virulence traits of the uspA‐positive isolates. For this purpose, two multiplex PCR assays, based on previously published primer sequences, were established. Assay 1 (mPCR‐1) uses three primer pairs and detects the genes encoding O157 (rfb), enterohemolysin (ehly) and shiga toxin (stx), generating amplification products of 420, 534 and 230 bp, respectively. Assay 2 (mPCR‐2) uses four primer pairs specific for rfb (E. coli O157), eaeA (intimin), stx1 and stx2 (shiga toxin 1 and 2, respectively), generating PCR amplicons of 420, 840, 348 and 584 bp, respectively. These two assays were validated by testing several E. coli reference strains and 202 previously characterized E. coli isolates originating from calves and from children, and 100% agreement with previous results was obtained. The method developed can be used for specific identification of STEC bacteria including those of the O157 serogroup.     

Characterization of specific egg yolk immunoglobulin (IgY) against mastitis-causing Escherichia coli

The objective of this study was to estimate the in vitro activity of egg yolk immunoglobulin (IgY) against mastitis-causing Escherichia coli. Specific IgY was produced by hens immunized with formaldehyde killed E. coli O111 in long-standing immunization response (titer > or =6400 for 100 days) and was isolated from yolks with a purity of 86% by water dilution, salt precipitations and ultrafiltration. Enzyme-linked immunosorbent assay (ELISA) indicated the produced IgY specifically targeted E. coli O111 and five other E. coli strains which were isolated from mastitic cows. The growth inhibition activity of the specific IgY to bacteria was dose-dependent with an effective concentration of 20mg purified IgY per milliliter. The phagocytic activity of E. coli either by milk macrophages (MPhi) or by polymorphonuclear neutrophil leukocytes (PMN) in the presence of specific IgY was significantly higher than that with nonspecific IgY or without IgY (p<0.05), suggesting that it enhanced phagocytic activity. The current work suggests that this specific IgY has potential as a therapeutic treatment for mastitis in dairy cows.     

Prevalence and characteristics of Shiga toxin-producing Escherichia coli (STEC) from cattle in Korea between 2010 and 2011

A total of 156 Shiga-like toxin producing Escherichia coli (STEC) were isolated from fecal samples of Korean native (100/568, 18%) and Holstein dairy cattle (56/524, 11%) in Korea between September 2010 and July 2011. Fifty-two STEC isolates (33%) harbored both of shiga toxin1 (stx1) and shiga toxin2 (stx2) genes encoding enterohemolysin (EhxA) and autoagglutinating adhesion (Saa) were detected by PCR in 83 (53%) and 65 (42%) isolates, respectively. By serotyping, six STEC from native cattle and four STEC from dairy cattle were identified as O-serotypes (O26, O111, O104, and O157) that can cause human disease. Multilocus sequence typing and pulsed-field gel electrophoresis patterns highlighted the genetic diversity of the STEC strains and difference between strains collected during different years. Antimicrobial susceptibility tests showed that the multidrug resistance rate increased from 12% in 2010 to 42% in 2011. Differences between isolates collected in 2010 and 2011 may have resulted from seasonal variations or large-scale slaughtering in Korea performed to control a foot and mouth disease outbreak that occurred in early 2011. However, continuous epidemiologic studies will be needed to understand mechanisms. More public health efforts are required to minimize STEC infection transmitted via dairy products and the prevalence of these bacteria in dairy cattle.     

Shiga toxin-producing Escherichia coli O157:H7 from extensive cattle of the fighting bulls breed

Shiga toxin-producing Escherichia coli (STEC) O157:H7 represents a major public health concern worldwide, with cattle recognized as their main natural reservoir. The aim of this work was to determine the prevalence and the pheno-genotypic characteristics of STEC O157:H7 in a herd with 268 cattle of the fighting bulls breed (De Lidia breed) managed under extensive conditions in the South-West of Spain. Rectal-anal swabs of all animals were collected and examined for STEC O157:H7 by performing an immunomagnetic concentration and separation procedure combined with PCR, and the resulting isolates were characterized by both phenotypic and genotypic methods. Overall, STEC O157:H7 was isolated from seven animals (2.6%) in the herd. The PCR procedure indicated that all seven isolates displayed stx₂, eae-γ1, ehxA, O157 rfbE, and fliCh7 genes. They belonged to phage types 4 (one isolate) and 42 (two isolates), and four isolates reacted with typing phages but did not conform to a recognized pattern. Among the seven isolates there were five indistinguishable PFGE patterns and other two which differed only in ?2 restriction fragments, supporting the existence of horizontal transmission among animals in the herd. The present study demonstrates that cattle managed under extensive conditions in Spain can excrete STEC O157:H7 with their faeces. To our knowledge this is the first isolation of this pathogen from De Lidia cattle.     

Evaluation of a PCR-restriction fragment length polymorphism (PCR-RFLP) assay for molecular epidemiological study of Shiga toxin-producing Escherichia coli

In this study, we have evaluated our recently developed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for the molecular subtyping of Shiga toxin-producing Escherichia coli (STEC). A total of 200 STEC strains including O157 (n=100), O26 (n=50), O111 (n=10), and non-O26/O111/O157 (n=40) serogroups isolated during 2005-2006 in Japan, which were identified to be clonally different by pulsed-field gel electrophoresis (PFGE) were further analyzed by the PCR-RFLP assay in comparison to PFGE. Ninety-five of O157, 48 of O26, five of O111 and 19 of non-O26/O111/O157 STEC strains yielded one to three amplicons ranging from 6.0 to 15.5 kb in size by the specific primer set targeting region V which is located in the upstream of stx genes. These strains were classified into 41 (O157), 8 (O26), 4 (O111) and 17 (non-O26/O111/O157) groups based on the RFLP patterns obtained by subsequent restriction digestion, respectively. Although the discriminatory power of PCR-RFLP assay was somewhat less than that of PFGE, it is more convenient for molecular subtyping of STEC strains especially for O157, the most important serogroup implicated in human diseases, as well as to identify the outbreak-associated isolates because of its simplicity, rapidity, ease and good reproducibility.     

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.