肠出血性大肠杆菌O157：H7监测及分析

为了了解长春地区动物和人感染肠出血性大肠杆菌O157H7状况,建立流行病学监测网.采集长春市动物养殖场动物粪便和腹泻病人便样进行监测.结果在牛粪和鸡粪中共检出2株O157H7大肠杆菌.可见,在长春地区存在肠出血性大肠杆菌O157H7菌潜在污染的威胁,需要加强监测力度.     

蚯蚓粪中乳酸菌的分离及其在大肠杆菌O157:H7中的抗菌活性

本研究利用SL培养基从蚯蚓粪中分离到54株具有产酸性能的菌株,并以E. coli O157:H7 (EDL933株)作为指示菌株,采用点种法检测分离菌株的抑菌活性.结果表明其中6个菌株对指示菌具有拮抗作用,通过形态特征,结合16S rDNA序列分析,初步鉴定该6个菌株分别为食物魏斯特菌(Listeria welshimeri)、乳酸片球菌(Pediococcus acidilactici)、短乳杆菌(Lactobacillus brevis)和格氏乳球菌(Lactococcus garvieae).分离到的乳酸菌对E. coli O157:H7 (EDL933株)具有显著的抑制作用,发酵温度和初始pH值影响发酵液的抑菌作用,优化环境因子可以促进拮抗菌对E. coli O157:H7的抑制作用.本研究为进一步分离抗菌产物用于人畜共患病的预防和治疗提供了理论依据.     

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.     

Variation in the faecal shedding of Salmonella and E. coli O157:H7 in lactating dairy cattle and examination of Salmonella genotypes using pulsed-field gel electrophoresis

AIMS: To examine the variability in faecal shedding of Salmonella and Escherichia coli O157:H7 in healthy lactating dairy cattle and to evaluate the genetic relatedness of Salmonella isolates. METHODS: Faecal samples were obtained from lactating Holstein dairy cattle on four commercial farms in the southwestern US. All farms were within an 8-km radius and were sampled in August 2001, January 2002 and August 2002 (60 cows per farm per sampling; n = 720 total samples). Samples were cultured for E. coli O157:H7 and Salmonella and a portion of the recovered Salmonella isolates were examined for genetic relatedness using pulsed-field gel electrophoresis (PFGE). RESULTS: Faecal shedding of E. coli O157:H7 and Salmonella varied considerably between farms and at the different sampling times. Large fluctuations in the percentage of positive animals were observed from summer to summer for both of these pathogens. Similarly, Salmonella serotype and serotype prevalence varied from farm to farm and within farm from one sampling time to another. Multiple Salmonella genotypes were detected for a number of serotypes and identical genotypes were found on different farms with one genotype of Salmonella Senftenberg identified on three of the four farms. Significance and Impact of the STUDY: This study demonstrated the wide variability in pathogen shedding within and among dairy farms all located in a small geographical region and highlights the complexity of pathogen control at the farm level.     

Shiga-toxigenic Escherichia coli O157 and non-Shiga-toxigenic E. coli O157 respond differently to culture and isolation from naturally contaminated bovine faeces

AIM: To quantify the effect of enrichment, immunomagnetic separation (IMS), and selective plating procedures on isolation of Shiga-toxigenic Escherichia coli O157 (STEC O157) and non-Shiga-toxigenic Escherichia coli O157 (non-STEC O157) from naturally contaminated bovine faeces. METHODS AND RESULTS: Two broth enrichment times, two IMS strategies, and two selective plating media were evaluated. STEC O157 and non-STEC O157 strains were often isolated from the same faecal specimen and responded differently to the isolation protocols. A large-volume IMS system was more sensitive than a conventional small-volume IMS method, but was also more expensive. STEC O157 was more frequently isolated from 6 h enriched broth and ChromAgar plates containing 0.63 mg l(-1) potassium tellurite (TCA). Non-STEC O157 was more frequently isolated from un-enriched broth and ChromAgar plates without tellurite (CA). CONCLUSIONS: The combination of 6-h enrichment in Gram-negative broth containing vancomycin, cefixime and cefsuludin, large volume IMS and selective plating on TCA maximized STEC O157 recovery from naturally contaminated cattle faecal specimens. SIGNIFICANCE AND IMPACT OF THE STUDY: The pairing of proper enrichment with a specific plating procedure is key for STEC O157 recovery from naturally contaminated bovine faeces. Incorporating tellurite into an E. coli O157 detection strategy may select for the subset of E. coli O157 that contains the Shiga-toxin genes.     

Intermittent and persistent shedding of Escherichia coli O157 in cohorts of naturally infected calves

AIMS: We conducted two short-term studies of cohorts of naturally infected calves to determine the prevalence and concentrations of Escherichia coli O157 shed in faeces. METHODS AND RESULTS: Two cohorts of calves were sampled; in the first study 14 calves were sampled up to five times a day for 5 days; in the second study a group of 16 separate calves were sampled once or twice a day for 15 days. All cattle within the two cohorts shed E. coli O157 at some point during the respective studies. In 18% of samples, E. coli O157 could only be isolated using immunomagnetic separation after an enrichment period, suggesting concentrations <250 CFU g(-1). The highest concentrations recorded were 6.7 x 10(5) and 1.6 x 10(6) CFU g(-1) for studies 1 and 2 respectively. CONCLUSIONS: Persistent, high shedders (shedding >10(3) CFU g(-1)) were evident in both studies but, in the majority of calves, the pathogen was isolated intermittently. SIGNIFICANCE AND IMPACT OF THE STUDY: The variable patterns of shedding have important implications for the design of appropriate sampling protocols and for gaining meaningful estimates of parameters used in mathematical models of transmission.     

European starlings (Sturnus vulgaris) challenged with Escherichia coli O157 can carry and transmit the human pathogen to cattle

Aims: European starlings (Sturnus vulgaris) are an invasive species in the United States and are considered a nuisance pest to agriculture. The goal of this study was to determine the potential for these birds to be reservoirs and/or vectors for the human pathogen Escherichia coli O157:H7. Materials and Results: Under biosecurity confinement, starlings were challenged with various doses of E. coli O157:H7 to determine a minimum infectious dose, the magnitude and duration of pathogen shedding, and the potential of pathogen transmission among starlings and between starlings and cattle. Birds transiently excreted E. coli O157:H7 following low‐dose inoculation; however, exposure to greater than 10^5.5 colony‐forming units (CFUs) resulted in shedding for more than 3 days in 50% of the birds. Colonized birds typically excreted greater than 10³ CFU g^?1 of faeces, and the pathogen was detected for as long as 14 days postinoculation. Cohabitating E. coli O157:H7‐positive starlings with culture‐negative birds or 12‐week‐old calves resulted in intra‐ and interspecies pathogen transmission within 24 h. Likewise, E. coli O157:H7 was recovered from previously culture‐negative starlings following 24‐h cohabitation with calves shedding E. coli O157:H7. Conclusions: European starlings may be a suitable reservoir and vector of E. coli O157:H7. Significance and Impact of the Study: Given the duration and magnitude of E. coli O157:H7 shedding by European starlings, European starlings should be considered a public health hazard. Measures aimed at controlling environmental contamination with starling excrement, on the farm and in public venues, may decrease food‐producing animal and human exposure to this pathogen.     

Diversity of Escherichia coli O157 in a longitudinal farm study using multiple-locus variable-number tandem-repeat analysis

Aims: To perform a longitudinal study of the diversity of Escherichia coli O157 from a ruminant pasture/stream environment using multiple-locus variable-number tandem-repeat analysis (MLVA). Methods and Results: Samples of faecal droppings from grazing ruminants and from an adjacent stream were tested longitudinally for E. coli O157 by enrichment and immunomagnetic separation (IMS). Using MLVA, 24 different profiles were identified from a total of 231 E. coli O157 isolates, of which 80 were included in a similarity analysis. Four main clusters with several subclusters were observed. Although there was close contact between sheep and cattle during the study period, E. coli O157 was surprisingly not detected from cattle faeces. Conclusions: The cluster analysis indicated both unrelated and closely related E. coli O157 strains. The choice of loci to target in MLVA is important for the subtyping result, as loci with high diversities are essential for discriminating between closely related isolates. Significance and Impact of the Study: There is a lack of data available on the use of MLVA to describe E. coli O157 diversity and changes over time in the animal reservoirs and the environment. Such data are needed in order to further develop MLVA as a typing method.     

Prevalence of Escherichia coli O157 and O157:H7-infecting bacteriophages in feedlot cattle feces

AIM: To estimate the distribution and prevalence of both Escherichia coli O157 and O157:H7-infecting bacteriophages within a 50,000 head commercial beef feedlot. METHODS AND RESULTS: Escherichia coli O157 was detected in approximately 27% of the individual samples, distributed across seven of the 10 pens screened. In a simple initial screen to detect O157:H7-infecting phages, none were detected in any pen or individual sample. In contrast, after a series of enrichment procedures O157:H7-infecting phages were detected in every pen and in the majority of the samples from most pens; virulent bacteriophages active against E. coli O157:H7 were detected post-enrichment from 39/60 (65%) of the feedlot samples, and 58/60 (approximately 97%) contained phage that infected E. coli B or O157:H7. CONCLUSIONS: The data we present here indicates that we may be grossly underestimating the prevalence of O157:H7-infecting phages in livestock if we simply screen samples and that enrichment screening is required to truly determine the presence of phages in these ecosystems. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data suggest that O157:H7-infecting phages may play a role in the ecology and transient colonization of cattle by E. coli O157:H7. Further, this and previous data suggest that before starting in vivo pathogen eradication studies using phage or any other regime, test animals should be enrichment screened for phage to avoid erroneous results.     

Reliability of O157:H7 ID agar (O157 H7 ID-F) for the detection and isolation of verocytotoxigenic strains of Escherichia coli belonging to serogroup O157

AIMS: The reliability of the O157:H7 ID agar (O157 H7 ID-F) to detect verocytotoxigenic strains of Escherichia coli (VTEC) of serogroup O157 was investigated. METHODS AND RESULTS: This medium, designed to detect strains belonging to the clone of VTEC O157:H7/H-, contains carbohydrates and two chromogenic substrates to detect beta-d-galactosidase and beta-d-glucuronidase and sodium desoxycholate to increase selectivity for Gram-negative rods. A total of 347 strains of E. coli including a variety of serotypes, verocytotoxigenicity of human and animal sources were tested. The green VTEC O157 colonies were easy to detect among the other dark purple to black E. coli colonies. Of 63 O157:H7/H- strains, 59 (93.7%) gave the characteristic green colour. Three of the failed four strains of O157:H- were not verocytotoxigenic, missing only one VTEC O157. Three non-O157 strains gave the characteristic green colour on the medium and were VTEC OR:H- (2) and Ont:H- (1), possibly being degraded variants of the O157 enterohaemorrhagic E. coli clone. CONCLUSIONS: The O157:H7 ID agar (O157 H7 ID-F) was largely successful in isolating VTEC belonging to the O157:H7/H- clone. SIGNIFICANCE AND IMPACT OF THE STUDY: A medium, suitable for isolating strains of VTEC O157 was successfully evaluated and should be useful for the isolation of these pathogens.     

Persistence of Escherichia coli O157 on farm surfaces under different environmental conditions

AIMS: To compare the persistence of Escherichia coli O157 on a variety of common faecally contaminated farmyard material surfaces (wood and steel) under different moisture and temperature regimes. METHODS AND RESULTS: Samples of field-conditioned farmyard materials (galvanized steel and wood) were cut into pieces and contaminated with fresh cattle faeces inoculated with nontoxigenic E. coli O157 (strain 3704). Thereafter, they were stored at four different environmental conditions; with temperature (5 and 20 degrees C) and moisture (moist or dry) as variables. Transfer of the pathogen to hands from the surfaces was also evaluated. Escherichia coli O157 numbers declined over time on all surfaces albeit at different rates according to the sample material and environmental conditions. Persistence was greatest on moist wood samples under cooler temperatures with large population numbers remaining after 28 days. Desiccation of surfaces resulted in a more rapid decline in E. coli O157 populations under both temperature regimes. Substantial numbers of colonies may also potentially be transferred to human hands from the surfaces during brief contact. CONCLUSIONS: When environmental conditions are favourable, E. coli O157 may persist for considerable times on a range of surfaces. However, when exposed to higher temperatures and dehydration, survival is notably decreased. Overall, bacterial persistence was significantly greater on wood samples relative to steel. SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli O157 is a prevalent pathogen, common in ruminant faeces. Contact with contaminated faeces may lead to human infection, resulting in possible severe illness. Although our study used only one strain of bacteria, our findings indicates that E. coli O157 has the potential to persist for long periods of time on gates, stiles and other farmyard surfaces under a range of environmental conditions. These farmyard surfaces therefore pose a potential infection pathway particularly where there is a high risk of direct human contact (e.g. child petting zoos, open farms).     

Molecular characterization of Irish E. coli O157:H7 isolates of human, bovine, ovine and porcine origin

Aims:  To determine the degree of relatedness between isolates of Escherichia coli O157:H7 of human, bovine, ovine and porcine origin.
Methods and Results:  Escherichia coli O157:H7 isolates were compared using (i) PFGE Xba I patterns, (ii) PCR profiles of virulence genes and (iii) the DNA sequences of genes reported to play a role in pathogenicity. The 77 E. coli O157:H7 isolates demonstrated 49 different PFGE patterns of which, eight were common to multiple isolates, and the remaining 41 were distinct. Isolates of different origin did not correlate, except for one cluster consisting of two human and two beef isolates. The majority of animal isolates had the same PCR profiles of virulence genes as those isolated from clinical patients. Single nucleotide polymorphisms (SNPs) were identified in the sequence of a 255-bp region of the vtx2 subunit A gene.
Conclusions:  Six SNPs were detected in the vtx2 A gene, defining four different haplotypes. One nonsynonymous substitution encoded for an amino acid change from glutamic to aspartic acid.
Significance and Impact of the Study:  Results indicate that although E. coli O157:H7 isolates of differing origin were distinct by PFGE, the DNA sequences of the main virulence genes associated with human clinical illness were conserved.     

The effect of the native bacterial community structure on the predictability of E. coli O157:H7 survival in manure‐amended soil

Aims: The survival capability of pathogens like Escherichia coli O157:H7 in manure‐amended soil is considered to be an important factor for the likelihood of crop contamination. The aim of this study was to reveal the effects of the diversity and composition of soil bacterial community structure on the survival time (ttd) and stability (irregularity, defined as the intensity of irregular dynamic changes in a population over time) of an introduced E. coli O157:H7 gfp‐strain were investigated for 36 different soils by means of bacterial PCR‐DGGE fingerprints. Methods and Results: Bacterial PCR‐DGGE fingerprints made with DNA extracts from the different soils using bacterial 16S‐rRNA‐gene‐based primers were grouped by cluster analysis into two clusters consisting of six and 29 soils and one single soil at a cross‐correlation level of 16% among samples per cluster. Average irregularity values for E. coli O157:H7 survival in the same soils differed significantly between clusters (P = 0·05), whereas no significant difference was found for the corresponding average ttd values (P = 0·20). The irregularity was higher for cluster 1, which consisted primarily of soils that had received liquid manure and artificial fertilizer and had a significant higher bacterial diversity and evenness values (P < 0·001). Conclusions: Bacterial PCR‐DGGE fingerprints of 36 manure‐amended soils revealed two clusters which differed significantly in the stability (irregularity) of E. coli O157 decline. The cluster with the higher irregularity was characterized by higher bacterial diversity and evenness. Significance and Impact of the Study: The consequence of a high temporal irregularity is a lower accuracy of predictions of population behaviour, which results in higher levels of uncertainty associated with the estimates of model parameters when modelling the behaviour of E. coli O157:H7 in the framework of risk assessments. Soil community structure parameters like species diversity and evenness can be indicative for the reliability of predictive models describing the fate of pathogens in (agricultural) soil ecosystems.     

RAPID detection and quantification of E. coli O157/O26/O111 in minced beef by real-time PCR

AIM: To develop a real-time PCR detection procedure for Escherichia coli O111, O26 and O157 from minced meat. METHODS AND RESULTS: Strains (n = 8) of each of E. coli O26, E. coli O111 and E. coli O157 were inoculated at ca 10-20 CFU g(-1) into minced retail meat and enriched for 6 h at 41.5 degrees C as follows: E. coli O26 in tryptone soya broth (TSB) supplemented with cefixime (50 microg l(-1)), vancomycin (40 mg l(-1)) and potassium tellurite (2.5 mg l(-1)); E. coli O111 in TSB supplemented with cefixime (50 microg l(-1)) and vancomycin (40 mg l(-1)); E. coli O157 in E. coli broth supplemented with novobiocin (20 mg l(-1)). DNA was extracted from the enriched cultures, and detected and quantified by real-time PCR using verotoxin (vt1 and vt2) and serogroup (O157 per gene; O26 fliC-fliA genes and O111 wzy gene) specific primers. CONCLUSIONS: The methods outlined were found to be sensitive and specific for the routine detection of E. coli O111, O26 and O157 in minced beef. SIGNIFICANCE AND IMPACT OF THE STUDY: The enrichment, isolation and detection procedures used in this study provide a rapid routine-based molecular method for the detection and differentiation of E. coli O26, O111 and O157 from minced meat.     

Occurrence of Escherichia coli O157 on hides of slaughtered cattle

AIMS: To obtain the first information on the occurrence of Escherichia coli O157 on hides of slaughtered cattle in Serbia. METHODS AND RESULTS: A total of 355 swabs were taken on the slaughterline from five areas of hide of each of the 71 cattle in a single commercial abattoir in Serbia. Using an ISO method incorporating enrichment and immunomagnetic separation steps, E. coli O157 was isolated from the hides of 20 animals (28 x 2%). With respect to different areas of the hides, the occurrence of the pathogen was, in decreasing order: hooves (11 x 3%), brisket (8 x 4%), rump (7 x 0%), neck (4 x 2) and flank (2 x 8%). In addition, factors that had more or less effects on the occurrence included visible dirtiness of the hide, cattle's age category, geographical origin of the animals and season. CONCLUSIONS: This study revealed the presence of E. coli O157 in the beef chain in Serbia and confirmed hide as an important potential source of related contamination of beef carcasses. Therefore, incorporation of preskinning hide decontamination treatments into HACCP-based slaughterline hygiene control measures could be very useful. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will enable further optimization of necessary measures along the beef chain to reduce the E. coli O157 risks in Serbia.     

Probiotic inhibits the cytopathic effect induced by Escherichia coli O157:H7 in Vero cell line model

Aims: The effectiveness of four strains of Bifidobacteria against enterohemorrhagic Escherichiacoli O157:H7 infection was studied using a Vero cell model. Methods and results: E. coli O157 was inoculated on the Vero cell line before and after treatment with probiotic. The cytopathic effect (CPE) was evaluated during 24 h of incubation. The results indicated that Shiga toxin activity was inhibited by the probiotic. To prevent a Stx2 CPE, the probiotic needs one log more than the Stx1. Conclusion: The Vero cell assay, in particular, is a good model to evaluate the effect of Bifidobacteria inhibiting bacterial attachment because of soluble substances and the competitive aspect and could be used in a variety of foods like milk and yoghurt to protect pathogen bacteria. Significance and Impact of the Study: Probiotics could control pathogenic bacteria and Vero cell introduce as a model for evaluation of probiotics against pathogen bacteria.     

The detection of Cryptosporidium parvum and Escherichia coli O157 in UK bivalve shellfish

Optimised immunomagnetic separation methods to detect Cryptosporidium parvum and Escherichia coli O157 in UK shellfish are described. Whole tissue homogenates gave the best recoveries for C. parvum oocysts compared with gill or haemolymph extracts. The sensitivity of recovery from spiked samples was comparable to that achieved when processing water and varied from 12–34% in mussels, 48–69.5% in oysters and 30–65% in scallops. Maximum recovery of E. coli O157 was achieved by enriching in buffered peptone water supplemented with vancomycin at 42 °C. Increasing enrichment temperatures from 37 to 42 °C gave a significant increase in target number recovery. Implementation of these methods into monitoring programmes and end-product testing will enable shellfish producers to better assess product safety.     

The impact of E. coli O157 on the food industry

The relatively recent emergence of Escherichia coli O157 as a foodborne pathogen has had a significant impact on the food industry. This serovar possesses a number of undesirable characteristics that combine to make it one of the most serious threats to food safety in recent years. The widespread and sporadic occurrence in the gastrointestinal tracts of cattle, sheep, man and many other species, sometimes in the absence of any disease, and the complex nature of its ecology mean that it is not feasible to easily eradicate this serovar. In order to control and minimise foodborne disease by these organisms, it has been necessary to change primary production, processing, retailing and consumer-handling practices. Despite these new control measures, foodborne disease outbreaks caused by these microorganisms continue to occur and are accompanied by an increase in the number of cases of illness in many countries. This revised version was published online in November 2006 with corrections to the Cover Date.