Cefixime-tellurite rhamnose MacConkey agar for isolation of Vero cytotoxin-producing Escherichia coli serogroup O26 from Scottish cattle and sheep faeces.

AIMS: To compare rhamnose MacConkey agar supplemented with cefixime and tellurite (CT-RMac) and tryptone bile X-glucuronide (TBX) agars as isolation media for Vero cytotoxin-producing Escherichia coli (VTEC) serogroup O26 from animal faeces. METHODS AND RESULTS: Nine VTEC O26 were isolated from sheep faeces; out of which six were isolated only on CT-RMac and one was isolated only on TBX. One hundred and twelve VTEC O26 were isolated from calf faeces; out of which 97% were from CT-RMac and 52% were from TBX. In a study of E. coli O26 strains, 84% of VT-positive O26 did not ferment rhamnose when compared with 16% of VT-negative O26. VT-positive (19%) and VT-negative (39%) E. coli O26 strains did not grow on CT-RMac agar. CONCLUSIONS: It is important to consider that VTEC O26 strains either may ferment rhamnose or may be sensitive to the CT supplement of CT-RMac agar. SIGNIFICANCE AND IMPACT OF THE STUDY: This work compares CT-RMac and TBX agars as isolation medium for VTEC O26 from Scottish animal faeces and highlights that VTEC O26 may be missed if only CT-RMac agar is used.

Detection of genetic diversity by pulsed-field gel electrophoresis among Escherichia coli O157 isolated from bovine faecal samples by immunomagnetic separation technique.

AIMS: Escherichia coli O157 is considered to be one of most important human pathogens of animal origin which causes serious clinical complications. One of the most common methods to isolate E. coli O157 is the immunomagnetic separation (IMS) technique which employs specific antibodies coupled to magnetic beads to bind and extract cells from enrichment broths followed by plating onto sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (CT-SMAC) plates. The aim of this study was to determine strain variation by pulsed-field gel electrophoresis (PFGE) among E. coli O157 on IMS/CT-SMAC plates. METHODS AND RESULTS: Every suspect colony of E. coli O157 was tested following isolation by the IMS/CT-SMAC technique. From 124 colonies detected; six XbaI-PFGE profiles were identified. CONCLUSIONS: Our results demonstrate that mixed populations of E. coli O157 with distinguishable PFGE profiles that are simultaneously present in bovine faeces can be isolated with IMS/CT-SMAC technique. SIGNIFICANCE AND IMPACT OF THE STUDY: If the aim of the study was to analyse diversity of PFGE profiles of E. coli O157 in a faecal sample following isolation by the IMS/CT-SMAC technique, at least five colonies per sample should be analysed to detect different PFGE subtypes if present.

Sensitivity of an immunomagnetic-separation-based test for detecting Escherichia coli O26 in bovine feces.

The sensitivity of a test for cattle shedding Escherichia coli serogroup O26 was estimated using several fecal pats artificially inoculated at a range of concentrations with different E. coli O26 strains. The test involves the enrichment of fecal microflora in buffered peptone water, the selective concentration of E. coli O26 using antibody-coated immunomagnetic-separation beads, the identification of E. coli colonies on Chromocult tryptone bile X-glucuronide agar, and confirmation of the serogroup with E. coli serogroup O26-specific antisera using slide agglutination. The effective dose of E. coli O26 for an 80% test sensitivity (ED(80)) was 1.0 x 10(4) CFU g(-1) feces (95% confidence interval, 4.7 x 10(3) to 2.4 x 10(4)). Differences in test sensitivity between different E. coli O26 strains and fecal pats were also observed. Individual estimates of ED(80) for each strain and fecal pat combination ranged from 4.2 x 10(2) to 4.8 x 10(5) CFU g(-1). These results suggest that the test is useful for identifying individuals shedding a large number of E. coli O26 organisms or, if an appropriate number of individuals in a herd are sampled, for identifying affected herds. The study also provides a benchmark estimate of sensitivity that can be used to compare alternative tests for E. coli O26 and a methodological approach that can be applied to tests for other pathogenic members of the Enterobacteriaceae and other sample types.

Molecular characterisation of bovine faecal Escherichia coli shows persistence of defined ampicillin resistant strains and the presence of class 1 integrons on an organic beef farm.

Antimicrobial use is heavily restricted on organic farms; however, few studies have been conducted to investigate the impact this has on the epidemiology of resistance in pathogenic and commensal bacteria. We investigated the persistence of antimicrobial resistant Escherichia coli within an organic beef herd over a period of 28 months. Faecal samples collected monthly from three calf cohorts and annually from adult cattle and environmental samples, were screened for the presence of ampicillin, apramycin and nalidixic acid resistant E. coli. The prevalence of ampicillin resistance ranged from 27.3 to 40.7% in the annual herd and environmental samplings (n=22-55) and was greater in the calf cohorts, with a peak cohort prevalence of >47% in all 3 years (n=16-18). Apramycin and nalidixic acid resistant E. coli were rare. Pulsed-field gel electrophoresis (PFGE) identified 10 main genotype groups within the herd, with evidence of strain transmission between different livestock groups, animal species and years. Multiple resistance was found in >44% of isolates tested, with ampicillin, neomycin, sulphamethoxazole and tetracycline carriage the commonest phenotype identified. PCR detected the presence of class 1 integrons in <5% of resistant isolates, 6/7 of which were of cattle origin. These data demonstrate that ampicillin resistant E. coli was common on the farm despite restricted antimicrobial use, although strain diversity was low. Persistence of defined genotype groups was observed between years, together with the transmission of resistant strains between different animal species on the farm.

Prevalence and virulence factors of Escherichia coli serogroups O26, O103, O111, and O145 shed by cattle in Scotland.

A national survey was conducted to determine the prevalence of Escherichia coli O26, O103, O111, and O145 in feces of Scottish cattle. In total, 6,086 fecal pats from 338 farms were tested. The weighted mean percentages of farms on which shedding was detected were 23% for E. coli O26, 22% for E. coli O103, and 10% for E. coli O145. The weighted mean prevalences in fecal pats were 4.6% for E. coli O26, 2.7% for E. coli O103, and 0.7% for E. coli O145. No E. coli O111 was detected. Farms with cattle shedding E. coli serogroup O26, O103, or O145 were widely dispersed across Scotland and were identified most often in summer and autumn. However, on individual farms, fecal shedding of E. coli O26, O103, or O145 was frequently undetectable or the numbers of pats testing positive were small. For serogroup O26 or O103 there was clustering of positive pats within management groups, and the presence of an animal shedding one of these serogroups was a positive predictor for shedding by others, suggesting local transmission of infection. Carriage of vtx was rare in E. coli O103 and O145 isolates, but 49.0% of E. coli O26 isolates possessed vtx, invariably vtx1 alone or vtx1 and vtx2 together. The carriage of eae and ehxA genes was highly associated in all three serogroups. Among E. coli serogroup O26 isolates, 28.9% carried vtx, eae, and ehxA-a profile consistent with E. coli O26 strains known to cause human disease.

Distribution of Escherichia coli O157 in bovine fecal pats and its impact on estimates of the prevalence of fecal shedding.

The distribution of Escherichia coli O157 in bovine feces was examined by testing multiple samples from fecal pats and determining the density of E. coli O157 in immunomagnetic separation (IMS)-positive fecal samples. The density of E. coli O157 in bovine feces was highly variable, differing by as much as 76,800 CFU g(-1) between samples from the same fecal pat. The density in most positive samples was <100 CFU g(-1), the limit of reliable detection by IMS. Testing only one 1-g sample of feces per pat with IMS may result in a sensitivity of detection as low as 20 to 50%. It is therefore probable that most surveys have greatly underestimated the prevalence of E. coli O157 shedding in cattle and the proportion of farms with shedding cattle. The sensitivity of the detection of E. coli O157 in bovine feces can be as much as doubled by testing two 1-g samples per pat rather than one 1-g sample.

Temporal shedding patterns and virulence factors of Escherichia coli serogroups O26, O103, O111, O145, and O157 in a cohort of beef calves and their dams.

This study investigated the shedding of Escherichia coli O26, O103, O111, O145, and O157 in a cohort of beef calves from birth over a 5-month period and assessed the relationship between shedding in calves and shedding in their dams, the relationship between shedding and scouring in calves, and the effect of housing on shedding in calves. Fecal samples were tested by immunomagnetic separation and by PCR and DNA hybridization assays. E. coli O26 was shed by 94% of calves. Over 90% of E. coli O26 isolates carried the vtx(1), eae, and ehl genes, 6.5% carried vtx(1) and vtx(2), and one isolate carried vtx(2) only. Serogroup O26 isolates comprised seven pulsed-field gel electrophoresis (PFGE) patterns but were dominated by one pattern which represented 85.7% of isolates. E. coli O103 was shed by 51% of calves. Forty-eight percent of E. coli O103 isolates carried eae and ehl, 2% carried vtx(2), and none carried vtx(1). Serogroup O103 isolates comprised 10 PFGE patterns and were dominated by two patterns representing 62.5% of isolates. Shedding of E. coli O145 and O157 was rare. All serogroup O145 isolates carried eae, but none carried vtx(1) or vtx(2). All but one serogroup O157 isolate carried vtx(2), eae, and ehl. E. coli O111 was not detected. In most calves, the temporal pattern of E. coli O26 and O103 shedding was random. E. coli O26 was detected in three times as many samples as E. coli O103, and the rate at which calves began shedding E. coli O26 for the first time was five times greater than that for E. coli O103. For E. coli O26, O103, and O157, there was no association between shedding by calves and shedding by dams within 1 week of birth. For E. coli O26 and O103, there was no association between shedding and scouring, and there was no significant change in shedding following housing.

Detection of Escherichia coli serogroups O26, O103, O111 and O145 from bovine faeces using immunomagnetic separation and PCR/DNA probe techniques.

AIMS: The aim of this study was to isolate Escherichia coli O26, O103, O111 and O145 from 745 samples of bovine faeces using (i) immunomagnetic separation (IMS) beads coated with antibodies to lipopolysaccharide, and slide agglutination (SA) tests and (ii) PCR and DNA probes for the detection of the Verocytotoxin (VT) genes. METHODS AND RESULTS: IMS-SA tests detected 132 isolates of presumptive E. coli O26, 112 (85%) were confirmed as serogroup O26 and 102 had the VT genes. One hundred and twenty-two strains of presumptive E. coli O103 were isolated by IMS-SA, 45 (37%) were confirmed as serogroup O103 but only one of these strains was identified as Verocytotoxin-producing E. coli (VTEC). Using the PCR/DNA probe method, 40 strains of VTEC O26 and three strains of VTEC O103 were isolated. IMS-SA identified 21 strains of presumptive E. coli O145, of which only four (19%) were confirmed as serogroup O145. VTEC of this serogroup was not detected by either IMS-SA or PCR/DNA probes. E. coli O111 was not isolated by either method. CONCLUSION: IMS beads were 2.5 times more sensitive than PCR/DNA probe methods for the detection of VTEC O26 in bovine faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: IMS-SA is a sensitive method for detecting specific E. coli serogroups. However, the specificity of this method would be enhanced by the introduction of selective media and the use of tube agglutination tests for confirmation of the preliminary SA results.

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.

Molecular typing of Salmonella serotype Thompson strains isolated from human and animal sources.

One-hundred-and-thirteen isolates of Salmonella serotype Thompson from diverse sources in seven countries were characterized by PvuII ribotyping and IS200 fingerprinting. Ten PvuII ribotypes were observed. The predominant PvuII ribotype 1 represented a major clone of world-wide distribution but was not found in Australia; PvuII ribotypes 2 and 3 represented minor clones. HincII ribotyping discriminated subtypes within PvuII ribotype 1: HincII ribotype 1 was distributed widely but HincII ribotype 2 was found mainly in Scottish isolates. None of 101 isolates of PvuII ribotypes 1-3 contained copies of IS200. All 12 isolates of PvuII ribotypes 4-10 were from Australia and 7 of them contained copies of IS200 of 5 different profiles. These results suggest the existence of at least two lineages of Salmonella Thompson with a different geographical distribution. The finding that most isolates from man and poultry in Scotland belonged to the same ribotype (PvuII 1/HincII 2) and were IS200-negative suggests that poultry is an important source of human infection in Scotland.