Survival of Escherichia coli O157:H7 in farm water: its role as a vector in the transmission of the organism within herds

AIMS: The study aimed to investigate the survival characteristics of Escherichia coli O157:H7 in farm water (FW), and in sterile distilled municipal water (SDW), stored outdoors under field conditions, with or without the addition of faeces (1% w/v), in a farmyard shed and the laboratory at 15 degrees C. METHODS AND RESULTS: Water samples were inoculated with E. coli O157:H7 at 10(3) and 10(6) ml(-1), and sampled over a 31-day period. In FW stored outdoors in a field, E. coli O157:H7 survived for 14 days at temperatures <15 degrees C, at both inoculation levels, while in the laboratory at 15 degrees C, the organism was still detectable at low levels (<1 log10 cfu ml(-1)) after 31 days. The addition of bovine faeces to water outdoors (1% w/v) resulted in survival for 24 days. In SDW inoculated at 10(6) ml(-1) and stored in the laboratory (15 degrees C), only a 2.5 log reduction was observed after 31 days, while the organism could not be detected after 17 days in the field. Preliminary screening of water samples stored outdoors isolated a bacterium which exhibited antimicrobial activity towards E. coli O157:H7. CONCLUSIONS: The survival of E. coli O157:H7 observed in this study illustrates the potential of farm water to act as a vehicle in the transfer of the organism across a herd. SIGNIFICANCE AND IMPACT OF THE STUDY: The difficulty in extrapolating results from controlled laboratory situations to on-farm conditions is also highlighted in this study.     

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.     

Persistence and metabolic activity of Escherichia coli O157:H7 in farm animal faeces

Ruminants, and to a lesser extent monogastric farm animals, are known to be natural reservoirs of Escherichia coli O157:H7, and contact with contaminated faeces has been linked to human infection. This study used a nontoxigenic, chromosomally marked, lux reporter strain to compare the persistence and activity (bioluminescence) of E. coli O157:H7 over 21 days in the faecal liquor of five farm animals: horse, sheep, cow, pig and piglet. Samples were inoculated with the lux E. coli O157:H7 (7.82 log CFU mL(-1)) and stored at 20 +/- 1 degrees C. The organism was recovered from all samples throughout the experimental period, although lower numbers were recovered from horse faecal liquor relative to all other types (P<0.001). The organisms' activity declined in all samples over time and no luminescence could be detected in any sample 21 days postinoculation. However, activity did increase greatly within pig and piglet faeces during initial stages of monitoring and overall luminescence was greater in piglet samples compared with all other samples (P<0.001). This is the first study to demonstrate how both the persistence and metabolic activity of E. coli O157:H7 notably varies within a range of ruminant and nonruminant animal faeces. Further research is needed to elucidate the factors that govern differential persistence and metabolic activity of E. coli O157:H7 within such matrices.     

Survival of verocytotoxigenic Escherichia coli O157 in soil, water and on surfaces

Cattle and sheep are major reservoirs of Escherichia coli O157 and consequently these and certain other farm animals can pass out large numbers of this organism in their faeces. Thus the ability of the organism to survive in faeces, on pastureland and in associated water systems has important implications for its spread to crops by direct application of manure, by irrigation with infected water or directly to man by contact with animals or contaminated soil. Model systems were used to determine the persistence of the organism in river water, cattle faeces, soil cores and on stainless steel work surfaces. Survival of the organism was found to be greatest in soil cores containing rooted grass. Under these conditions viable numbers were shown to decline from approximately 10(8) g(-1) soil to between 10(6) and 10(7) g(-1) soil after 130 d. When the organism was inoculated into cattle faeces it remained detectable at high levels for more than 50 d. In contrast the organism survived much less readily in cattle slurry and river water where it fell in numbers from more than 10(6) ml(-1) to undetectable levels in 10 and 27 d, respectively. The survival of E. coli O157 was also investigated on stainless steel surfaces, where as air-dried deposits, it was shown to survive for periods in excess of 60 d. It was most stable at chill temperatures (4 degrees C) and viability was only partially reduced at 18 degrees C. In addition to stainless steel, the organism was shown to survive for extended periods on domestic (plastic) cutting boards, both at room and chill temperatures. Sanitizing agents, such as hypochlorites and a compound comprising both cationic and anionic-based active ingredients were found to be effective in killing various VTEC on stainless steel surfaces.     

Optimizing enrichment conditions for the isolation of Escherichia coli O157 in soils by immunomagnetic separation

AIMS: To compare a range of enrichment broths and enrichment temperatures for the isolation of Escherichia coli O157 by immunomagnetic separation (IMS) from sandy, loam and clay soils. METHODS AND RESULTS: Soils were spiked with cocktails of four atoxigenic strains of E. coli O157 and four strains of commensal E. coli. The organisms were stressed by subjecting soils to cycles of freeze/thawing, followed by drying at 20 degrees C for up to 4 days. Nine enrichment broths were trialled based on buffered peptone water, tryptone soya broths and EC broths supplemented with a range of selective additions. Enrichments were incubated for 6 h and assessed by target recovery after IMS on cefixime tellurite sorbitol MacConkey agar (CTSMAC) incubated at 37 degrees C for 24 h. A comparison of enrichment temperatures (37 and 42 degrees C) was also performed. Buffered peptone water (with or without vancomycin) and tryptone soya broth (with or without novobiocin) gave significant increases in recovery of E. coli O157 compared to others tested. In addition, broths incubated at 42 degrees C were superior to those at 37 degrees C for the recovery of E. coli O157. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that sub-lethally damaged E. coli O157 surviving in soil can be sensitive to antimicrobial additions. The choice and concentration of these additions is vitally important to optimize target recovery. Some IMS protocols, established for the isolation of E. coli O157, may be unsuitable for the examination of soil samples.     

Waterborne Escherichia coli O157

The waterborne route of Vero cytotoxin-producing E. coli (VTEC) O157 infection was first suggested in two unconnected human cases in 1985. Since then, waterborne VTEC O157 has been identified in sporadic cases and in outbreaks of illness. Recreational waters, private and municipal supplies have been implicated from microbiological, environmental and epidemiological studies of cases. In addition, a research cohort study of farm workers identified exposure to private water supplies as a risk factor for having antibodies to E. coli O157. Sources of contamination are thought to be animal and human faeces or sewage. The presence of low numbers of target organisms in water makes microbiological confirmation difficult, therefore epidemiological evidence has been essential in outbreak investigations. Despite the potential for contamination of water with VTEC O157, waterborne infection is relatively rare largely due to the susceptibility of the organism to water treatment processes. This paper presents the evidence for waterborne VTEC O157 infection, considering current microbiological, environmental and particularly epidemiological information.     

Rapid and sensitive detection of Escherichia coli O157:H7 in bovine faeces by a multiplex PCR

Cattle are considered the major reservoir for Escherichia coli O157:H7, one of the newly emerged foodborne human pathogens of animal origin and a leading cause of haemorrhagic colitis in humans. A sensitive test that can accurately and rapidly detect the organism in the food animal production environment is critically needed to monitor the emergence, transmission, and colonization of this pathogen in the animal reservoir. In this study, a novel multiplex polymerase chain reaction (PCR) assay was developed by using 5 sets of primers that specifically amplify segments of the eaeA, slt-I, slt-II, fliC, rfbE genes, which allowed simultaneous identification of serotype O157:H7 and its virulence factors in a single reaction. Analysis of 82 E. coli strains (49 O157:H7 and 33 non-O157:H7) demonstrated that this PCR system successfully distinguished serotype O157:H7 from other serotypes of E. coli and provided accurate profiling of the shiga-like toxins and the intimin adhesin in individual strains. This multiplex PCR assay did not cross-react with the background bacterial flora in bovine faeces and could detect a single O157:H7 organism per gram of faeces when combined with an enrichment step. Together, these results indicate that the multiplex PCR assay can be used for specific identification and profiling of E. coli O157:H7 isolates, and may be applied to rapid and sensitive detection of E. coli O157:H7 in bovine faeces when combined with an enrichment step.     

The survival characteristics of a non-toxigenic strain of Escherichia coli O157:H7

The survival characteristics of a non-toxigenic, antibiotic-resistant strain of Escherichia coli O157:H7 in bovine faeces were investigated. Faecal samples were inoculated with 10(8-9) cfu g-1 of the organism and (i) stored in closed plastic containers at 10 degrees C, (ii) stored in closed plastic containers placed outside or (iii) decanted onto the surface of grazing land. Recovery and enumeration on Sorbitol MacConkey Agar (SMAC) and Tryptic Soya Agar (TSA) revealed that the E. coli O157:H7 numbers in both enclosed samples (i and ii) had decreased by 4.5-5.5 log10 cfu g-1 within 99 d. Numbers in samples decanted onto grassland (iii) decreased by 4.0-5.0 log10 cfu g-1 within 50 d but the organism was still detectable in the surrounding soil for up to 99 d. Persistence of E. coli O157:H7 in bovine faeces and contaminated pastures may therefore be an important factor in the initial infection and re-infection of cattle.     

Matrix effects, nonuniform reduction and dispersion in risk assessment for Escherichia coli O157

AIMS: To assess the importance of the variation (or spatial heterogeneity) in the doses of Escherichia coli O157 ingested by individuals when estimating the group risk. METHODS AND RESULTS: Exposure scenarios, which differed in how the same total number of E. coli O157 cells were distributed across a group of persons were simulated and the risks of illness in the population estimated using available dose-response data. Differences in the degree of spatial heterogeneity within routes such as direct contact with sheep faeces, consumption of hamburgers and drinking water could affect the magnitude of the predicted risk by over 1000-fold and contribute to an apparent matrix effect. The effect of barriers, such as cooking, on the spatial heterogeneity of E. coli O157 cells remaining in undercooked hamburgers affects the magnitude of the risk by eightfold. Furthermore, a 100-fold reduction in E. coli O157 concentrations by cooking may reduce the risk of illness from consumption of the burgers by only 12-fold (and not the 100-fold perhaps expected). CONCLUSIONS: The development of microbiological risk assessment (MRA) models for E. coli O157 requires information on the degree of dispersion of faeces within routes such as the environment, food and water, so that the proportion of persons actually exposed to faeces, and hence pathogen, can be quantified. The effect of dispersion is greater for pathogens for which there is greater variation in susceptibility within the population. SIGNIFICANCE AND IMPACT OF THE STUDY: In terms of public health protection, minimizing the dispersion of cattle/sheep faeces reduces the risks to humans by confining exposure to fewer individuals. For MRA, estimating the reduction in risk by a treatment process, e.g. cooking, is complicated by spatial heterogeneity. Regional differences in dispersion (e.g. because of rainfall) may contribute to regional differences in the observed attack rate.     

Detection and quantification of Escherichia coli O157:H7 in environmental samples by real-time PCR

AIMS: To apply the real-time Polymerase chain reaction (PCR) method to detect and quantify Escherichia coli O157:H7 in soil, manure, faeces and dairy waste washwater. METHODS AND RESULTS: Soil samples were spiked with E. coli O157:H7 and subjected to a single enrichment step prior to multiplex PCR. Other environmental samples suspected of harbouring E.coli O157:H7 were also analysed. The sensitivity of the primers was confirmed with DNA from E.coli O157:H7 strain 3081 spiked into soil by multiplex PCR assay. A linear relationship was measured between the fluorescence threshold cycle (C T ) value and colony counts (CFU ml(-1)) in spiked soil and other environmental samples. The detection limit for E.coli O157:H7 in the real-time PCR assay was 3.5 x 10(3) CFU ml(-1) in pure culture and 2.6 x 10(4) CFU g(-1) in the environmental samples. Use of a 16-h enrichment step for spiked samples enabled detection of <10 CFU g(-1) soil. E. coli colony counts as determined by the real-time PCR assay, were in the range of 2.0 x 10(2) to 6.0 x 10(5) CFU PCR (-1) in manure, faeces and waste washwater. CONCLUSIONS: The real-time PCR-based assay enabled sensitive and rapid quantification of E. coli O157:H7 in soil and other environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to quantitatively determine cell counts of E.coli O157:H7 in large numbers of environmental samples, represents considerable advancement in the area of pathogen quantification for risk assessment and transport studies.     

Development and application of a spiral plating method for the enumeration of Escherichia coli O157 in bovine faeces

AIM: To develop and validate a direct plating method applicable to epidemiological studies for enumerating Escherichia coli O157 in cattle faeces. METHODS AND RESULTS: The spiral plate count method was used to enumerate E. coli O157 in faecal samples. The accuracy and variation of counts was then assessed using faecal samples inoculated with E. coli O157. There was good agreement between inoculated levels of E. coli O157 and those recovered from faeces, particularly when counts were > 10(2) CFU g(-1) of faeces. The method was applied to a small study assessing short-term survival of E. coli O157 in naturally infected cattle faeces. E. coli O157 was found to survive in faeces for over 10 days at concentrations above 10(3) CFU g(-1) of faeces. Populations of E. coli O157 were also found to increase 100-fold in the first few hours after defecation. CONCLUSIONS: The enumeration method is easy to implement and enables a quick throughput of large numbers of samples. The method is accurate and reliable and enables the inherent variation in count data to be explored but needs to be used in combination with a more sensitive method for samples containing < 10(2) CFU g(-1) of faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The method described is appropriate for enumeration of E. coli O157 in cattle faeces in large-scale epidemiological studies.     

Enumeration of Escherichia coli O157 in cattle faeces using most probable number technique and automated immunomagnetic separation

AIMS: To determine the numbers of Escherichia coli O157 present in the faeces of naturally infected cattle. METHODS AND RESULTS: A combination of the most probable number (MPN) technique and automated immunomagnetic separation (AIMS) was used to enumerate E. coli O157 in cattle faeces from both pasture-fed and grain-fed animals. A total of 22 E. coli O157 positive faecal samples were enumerated for E. coli O157 (10 from pasture-fed and 12 from grain-fed animals). The numbers of E. coli O157 in cattle faeces varied from undetectable (<3 MPN g-1 of faeces) to 2.4 x 104 MPN g-1. There was no significant difference (P = 0.06) between the numbers of E. coli O157 in pasture-fed or grain-fed cattle faeces, although the geometric mean (antilog of the mean of log10 transformed MPN values) was higher in grain-fed (130 MPN g-1) than in pasture-fed (13 MPN g-1). CONCLUSIONS: Although the number of samples tested is small, the results indicate that E. coli O157 make up a small proportion of the total E. coli population present in cattle faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on the numbers of E. coli O157 present in cattle will assist in developing more robust quantitative risk assessments and formulating intervention strategies.     

Concentration and prevalence of Escherichia coli O157 in sheep faeces at pasture in Scotland

AIMS: To study the presence, numbers and virulence profiles of Escherichia coli O157 in sheep faeces and validate the microbiological methods used to attain these data. METHODS AND RESULTS: Flock level prevalence was found to be 40% (six from 15) and 6.5% of faecal samples tested were found to be positive. Two farms gave samples defined as high shedding (>10(4) CFU g(-1)), one of which comprised 91% positive samples with 13/33 at the high shedding level. CONCLUSIONS: These data confirmed that sheep are an important reservoir of E. coli O157. SIGNIFICANCE AND IMPACT OF THE STUDY: Sheep play a significant role in the maintenance and dispersal of E. coli O157 in the farming environment and are an important source of human infection.     

Survival of Escherichia coli O157:H7 in the rhizosphere of maize grown in waste-amended soil

AIMS: To assess whether the persistence of Escherichia coli O157:H7 in soil amended with cattle slurry and ovine stomach content waste is affected by the presence of a maize rhizosphere. METHODS AND RESULTS: Cattle slurry and ovine stomach content waste were inoculated with E. coli O157:H7. Wastes were then applied to soil cores with and without established maize plants. The pathogen survived in soil for over 5 weeks, although at significantly greater numbers in soil receiving stomach content waste in comparison to cattle slurry. Persistence of the pathogen in soil was unaffected by the presence of a rhizosphere. CONCLUSIONS: Other factors may be more influential in regulating E. coli O157:H7 persistence in waste-amended soil than the presence or absence of a rhizosphere; however, waste type did have significant affect on the survival of E. coli O157:H7 in such soil. SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli O157:H7 can be present within animal-derived organic wastes that are routinely spread on land. Introduced measures with regards to such waste disposal may decrease exposure to the organism; however, the persistence of E. coli O157:H7 for considerable periods in waste-amended soil may still pose some risk for both human and animal infection. This study has shown that whilst survival of E. coli O157:H7 in waste-amended soil is not significantly affected by the presence or absence of a maize rhizosphere; it may vary significantly with waste type. This may have implications for land and waste management.     

Prevalence and molecular characterization of Escherichia coli O157:H7 on Irish lamb carcasses, fleece and in faeces samples

AIMS: To determine the prevalence, seasonal variation and virulence characteristics of Escherichia coli O157:H7 in lambs presented for slaughter in Ireland. METHODS AND RESULTS: Over a 13-month period, pre- and postchill carcass swabs, faeces and fleece samples from 1600 lambs were examined for the presence of E. coli O157:H7. Escherichia coli O157:H7 was isolated from 5.75% (23/400) of fleece samples, 1.5% (6/400) of pre- and 1% (4/400) of postchill carcass swabs but was not isolated in faeces (0/400). The present study detected no evidence of seasonal variation. Polymerase chain reaction analysis showed that both the vt1 and vt2 genes associated with clinical illness were carried by five of the E. coli O157:H7 isolates, while 24 of the remaining isolates carried the vt2 gene only. Phage typing detected four different subtypes: PT 32 (48.48%), PT 8 (12.12%), PT 31 (12.12%) and PT 21/28 (12.12%). CONCLUSIONS: Escherichia coli O157:H7 is present in lambs at slaughter in Irish abattoirs and the virulence profiles of these isolates reveals that they are potentially harmful to humans. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study provides crucial information indicating that sheep may be a significant contributing source to human E. coli O157:H7 infection.     

Comparison of an immunochromatographic method and the TaqMan E. coli O157:H7 assay for detection of Escherichia coli O157:H7 in alfalfa sprout spent irrigation water and in sprouts after blanching

An immunochromatographic-based assay (Quixtrade mark E. coli O157 Sprout Assay) and a polymerase chain reaction (PCR)-based assay (TaqMan E. coli O157:H7 Kit) were used to detect Escherichia coli O157:H7 strain 380-94 in spent irrigation water from alfalfa sprouts grown from artificially contaminated seeds. Ten, 25, 60, or 100 seeds contaminated by immersion for 15 min in a suspension of E. coli O157:H7 at concentrations of 10(6) or 10(8) cfu/ml were mixed with 20 g of non-inoculated seeds in plastic trays for sprouting. The seeds were sprayed with tap water for 15 s every hour and spent irrigation water was collected at intervals and tested. E. coli O157:H7 was detected in non-enriched water by both the TaqMan PCR (30 of 30 samples) and the immunoassay (9 of 24 samples) in water collected 30 h from the start of the sprouting process. However, enrichment of the spent irrigation water in brain heart infusion (BHI) broth at 37 degrees C for 20 h permitted detection of E. coli O157:H7 in water collected 8 h from the start of sprouting using both methods, even in trays containing as few as 10 inoculated seeds. The TaqMan PCR assay was more sensitive (more positive samples were observed earlier in the sprouting process) than the immunoassay; however, the immunoassay was easier to perform and was more rapid. At 72 h after the start of the sprouting process, the sprouts were heated at 100 degrees C for 30 s to determine the effectiveness of blanching for inactivation of E. coli O157:H7. All of the 32 samples tested with the TaqMan assay and 16 of 32 samples tested with the Quixtrade mark assay gave positive results for E. coli O157:H7 after enrichment of the blanched sprouts at 37 degrees C for 24 h. In addition, the organism was detected on Rainbow Agar O157 in 9 of 32 samples after 24 h of enrichment of the blanched sprouts. In conclusion, E. coli O157:H7 was detected in spent irrigation water collected from sprouts grown from artificially contaminated seeds by both the TaqMan and Quixtrade mark assays. The data also revealed that blanching may not be effective to completely inactivate all the E. coli O157:H7 that may be present in sprouts.     

Effect of supplementing corn- or barley-based feedlot diets with canola oil on faecal shedding of Escherichia coli O157:H7 by steers

AIMS: This study was conducted to evaluate the effect of supplementing barley- or corn-based diets with canola oil on faecal shedding of Escherichia coli O157:H7 by experimentally inoculated feedlot cattle. METHODS AND RESULTS: Four groups of yearling steers fed on barley- or corn-based feedlot diets containing 0% (BA; CO) or 6% canola oil (BA-O; CO-O) were inoculated with 10(10) CFU of a mixture of four nalidixic acid-resistant strains of E. coli O157:H7. The inoculated strains were tracked in oral (mouth swab) and environmental (water, water bowl interface, feed, faecal pat) samples by enrichment and immunomagnetic separation (IMS) for 12 weeks, and in rectally collected faecal samples for 23 weeks (enumeration by dilution plating for 12 weeks; detection by IMS for a further 11 weeks). Levels of E. coli O157:H7 shed in faecal samples over the course of the enumeration period were similar (P = 0.14) among treatments. Disappearance of the inoculated strains from faeces was more rapid (P = 0.009) with barley than with corn, but shedding levels at the end of the enumeration period were similar (P = 0.21) across grain types. Canola oil supplementation did not affect (P = 0.71) the rate of disappearance of E. coli O157:H7 from faeces. The numbers of steers culture positive for E. coli O157:H7 during the enumeration period were similar (P = 0.57) among treatments. During the 11-week detection period, however, more (P < 0.001) steers were E. coli O157:H7-positive in the BA group (15/64) than in BA-O (two of 64), CO (two of 56), or CO-O (one of 56). The organism was present in two of 48 water samples (both CO-O), one of 48 water bowl swabs (BA-O), four of 48 feed samples (two of 12 BA; two of 12 CO-O), 30 of 48 pen floor faecal pat samples, and 296 of 540 mouth swabs (81/144 BA, 80/144 BA-O, 74/126 CO and 61/126 CO-O). CONCLUSION: Supplementing corn or barley-based diets with canola oil did not affect shedding of E. coli O157:H7 by feedlot cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: High-shedding individuals (i.e. 'super shedders') may be responsible for disseminating E. coli O157:H7 among penmates. Faeces on pen floors appears to be a more significant source of infection than are feed or drinking water.     

Selective enrichment with a resuscitation step for isolation of freeze-injured Escherichia coli O157:H7 from foods

We studied injury of Escherichia coli O157:H7 cells in 11 food items during freeze storage and methods of isolating freeze-injured E. coli O157:H7 cells from foods. Food samples inoculated with E. coli O157:H7 were stored for 16 weeks at -20 degrees C in a freezer. Noninjured and injured cells were counted by using tryptic soy agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Large populations of E. coli O157:H7 cells were injured in salted cabbage, grated radish, seaweed, and tomato samples. In an experiment to detect E. coli O157:H7 in food samples artificially contaminated with freeze-injured E. coli O157:H7 cells, the organism was recovered most efficiently after the samples were incubated in modified E. coli broth without bile salts at 25 degrees C for 2 h and then selectively enriched at 42 degrees C for 18 h by adding bile salts and novobiocin. Our enrichment method was further evaluated by isolating E. coli O157:H7 from frozen foods inoculated with the organism prior to freezing. Two hours of resuscitation at 25 degrees C in nonselective broth improved recovery of E. coli O157:H7 from frozen grated radishes and strawberries, demonstrating that the resuscitation step is very effective for isolating E. coli O157:H7 from frozen foods contaminated with injured E. coli O157:H7 cells.     

Detection, distribution and probable fate of Escherichia coli O157 from asymptomatic cattle on a dairy farm

The use of commercial anti- Escherichia coli O157-labelled magnetic beads was investigated to improve detection of E. coli O157 by immunomagnetic separation (IMS) from a range of environments on a dairy farm. Immunomagnetic separation proved effective for separation of target cells from laboratory mixtures and during stress in sterile and non-sterile pond water. The IMS procedure was possible with a range of samples (water, faeces, slurry, grass and soil). Non-specific binding of non-target bacterial cells proved problematic in a number of sample types. However, indigenous E. coli O157 cells were detected from samples with a high faecal load, and only with use of IMS. Data on the probable survival and spread of the organism around the farm environment are also discussed.     

Evaluation of a PCR detection method for Escherichia coli O157:H7/H- bovine faecal samples

AIMS: Combinations of PCR primer sets were evaluated to establish a multiplex PCR method to specifically detect Escherichia coli O157:H7 genes in bovine faecal samples. METHODS AND RESULTS: A multiplex PCR method combining three primer sets for the E. coli O157:H7 genes rfbE, uidA and E. coli H7 fliC was developed and tested for sensitivity and specificity with pure cultures of 27 E. coli serotype O157 strains, 88 non-O157 E. coli strains, predominantly bovine in origin and five bacterial strains other than E. coli. The PCR method was very specific in the detection of E. coli O157:H7 and O157:H- strains, and the detection limit in seeded bovine faecal samples was <10 CFU g(-1) faeces, following an 18-h enrichment at 37 degrees C, and could be performed using crude DNA extracts as template. CONCLUSIONS: A new multiplex PCR method was developed to detect E. coli O157:H7 and O157:H-, and was shown to be highly specific and sensitive for these strains both in pure culture and in crude DNA extracts prepared from inoculated bovine faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This new multiplex PCR method is suitable for the rapid detection of E. coli O157:H7 and O157:H- genes in ruminant faecal samples.