Escherichia coli O157:H7 in Environments of Culture-Positive Cattle

Outbreaks of Escherichia coli O157:H7 disease associated with animal exhibits have been reported with increasing frequency. Transmission can occur through contact with contaminated haircoats, bedding, farm structures, or water. We investigated the distribution and survival of E. coli O157:H7 in the immediate environments of individually housed, experimentally inoculated cattle by systematically culturing feed, bedding, water, haircoat, and feed bunk walls for E. coli O157:H7 for 3 months. Cedar chip bedding was the most frequently culture-positive environmental sample tested (27/96 or 28.15%). Among these, 12 (44.0%) of positive bedding samples were collected when the penned animal was fecal culture negative. Survival of E. coli O157:H7 in experimentally inoculated cedar chip bedding and in grass hay feed was determined at different temperatures. Survival was longest in feed at room temperature (60 days), but bacterial counts decreased over time. The possibility that urine plays a role in the environmental survival of E. coli O157:H7 was investigated. Cedar chip bedding moistened with sterile water or bovine urine was inoculated with E. coli O157:H7. Bedding moistened with urine supported growth of E. coli O157:H7, whereas inoculated bedding moistened with only water yielded decreasing numbers of bacteria over time. The findings that environmental samples were frequently positive for E. coli O157:H7 at times when animals were culture negative and that urine provided a substrate for E. coli O157:H7 growth have implications for understanding the on-farm ecology of this pathogen and for the safety of ruminant animal exhibits, particularly petting zoos and farms where children may enter animal pens.     

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.     

Isolation of Escherichia coli O157:H7 strains that do not produce Shiga toxin from bovine, avian and environmental sources

AIMS: To determine the potential for naturally occurring Shiga toxin-negative Escherichia coli O157 to acquire stx(2) genes. METHODS AND RESULTS: Multiple E. coli O157:H7 isolates positive for eae and ehxA, but not for stx genes, were isolated from cattle, water trough sediment, animal bedding and wild bird sources on several Ohio dairy farms. These isolates were experimentally lysogenized by stx(2)-converting bacteriophage. CONCLUSIONS: Shiga toxin-negative strains of E. coli O157 are present in multiple animal and environmental sources. SIGNIFICANCE AND IMPACT OF THE STUDY: Shiga toxin-negative strains of E. coli O157 present in the food production environment are able to acquire the stx genes, demonstrating their potential to emerge as new Shiga toxin-producing E. coli strains.     

Effect of diet on the shedding of Escherichia coli O157:H7 in a sheep model.

The purpose of this study was to develop a sheep model to investigate reproduction, transmission, and shedding of Escherichia coli O157:H7 in ruminants. In addition, we investigated the effect of diet change on these parameters. Six groups of twin lambs given oral inoculations of 10(5) or 10(9) CFU of E. coli O157:H7 and their nondosed mothers were monitored for colonization by culture of fecal samples. A modified selective-enrichment protocol that detected E. coli O157:H7 at levels as low as 0.06 CFU per g of ovine feces was developed. Horizontal transmission of infection occurred between the lambs and most of the nondosed mothers. When animals were kept in confinement and given alfalfa pellet feed, lambs receiving the higher dose shed the bacteria sooner and longer than all other animals. However, when the animals were released onto a sagebrush-bunchgrass range, every animal, regardless of its previous status (dosed at one of the inoculum levels tested or nondosed) shed E. coli O157:H7 uniformly. Shedding persisted for 15 days, after which all animals tested negative. E. coli O157:H7 reproduction and transmission and the combined effect of diet change and feed withholding were also investigated in a pilot study with experimentally inoculated rams. Withholding feed induced animals to shed the bacteria either by triggering growth of E. coli O157:H7 present in the intestines or by increasing susceptibility to infection. Introduction of a dietary change with brief starvation caused uniform shedding and clearance of E. coli O157:H7, and all animals then tested negative for the bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of Escherichia coli O157:H7 in soil and water using multiplex PCR

AIMS: To evaluate the suitability of a multiplex PCR-based assay for sensitive and rapid detection of Escherichia coli O157:H7 in soil and water. METHODS AND RESULTS: Soil and water samples were spiked with E. coli O157:H7 and subjected to two stages of enrichment prior to multiplex PCR. Detection sensitivities were as high as 1 cfu ml(-1) drinking water and 2 cfu g(-1) soil. Starvation of E. coli O157:H7 for 35 d prior to addition to soil did not affect the ability of the assay to detect initial cell numbers as low as 10 cfu g(-1) soil. Use of an 8-h primary enrichment enabled detection of as few as 6 cfu g(-1) soil, and 10(4) cfu g(-1) soil with a 6-h primary enrichment. When soil was inoculated with 10(5) cfu g(-1), the PCR assay indicated persistence of E. coli O157:H7 during a 35 d incubation. However, when soil was inoculated with lower numbers of pathogen, PCR amplification signals indicated survival to be dependent on cell concentration. CONCLUSIONS: A multiplex PCR-based assay, in combination with an enrichment strategy enabled sensitive and rapid detection of E. coli O157:H7 in soil and water. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to sensitively detect E.coli O157:H7 in environmental material within one working day represents a considerable advancement over alternative more time-consuming methods for detection of this pathogen.     

Differences in growth of Salmonella enterica and Escherichia coli O157:H7 on alfalfa sprouts

Sprout producers have recently been faced with several Salmonella enterica and Escherichia coli O157:H7 outbreaks. Many of the outbreaks have been traced to sprout seeds contaminated with low levels of human pathogens. Alfalfa seeds were inoculated with S. enterica and E. coli O157:H7 strains isolated from alfalfa seeds or other environmental sources and sprouted to examine growth of these human pathogens in association with sprouting seeds. S. enterica strains grew an average of 3.7 log(10) on sprouting seeds over 2 days, while E. coli O157:H7 strains grew significantly less, an average of 2.3 log(10). The initial S. enterica or E. coli O157:H7 inoculum dose and seed-sprouting temperature significantly affected the levels of both S. enterica and E. coli O157:H7 on the sprouts and in the irrigation water, while the frequency of irrigation water replacement affected only the levels of E. coli O157:H7. Colonization of sprouting alfalfa seeds by S. enterica serovar Newport and E. coli O157:H7 strains transformed with a plasmid encoding the green fluorescent protein was examined with fluorescence microscopy. Salmonella serovar Newport colonized both seed coats and sprout roots as aggregates, while E. coli O157:H7 colonized only sprout roots.     

Fate of Escherichia coli O157:H7 in irrigation water on soils and plants as validated by culture method and real-time PCR

One of the most common vehicles by which Escherichia coli O157:H7 may be introduced into crops is contaminated irrigation water. Water contamination is becoming more common in rural areas of the United States as a result of large animal operations, and up to 40% of tested drinking-water wells are contaminated with E. coli. In this study, 2 contrasting soil samples were inoculated with E. coli O157:H7 expressing green fluorescent protein through irrigation water. Real-time PCR and culture methods were used to quantify the fate of this pathogen in phyllosphere (leaf surface), rhizosphere (volume of soil tightly held by plant roots), and non-rhizosphere soils. A real-time PCR assay was designed with the eae gene of E. coli O157:H7. The probe was incorporated into real-time PCR containing DNA extracted from the phyllosphere, rhizosphere, and non-rhizosphere soils. The detection limit for E. coli O157:H7 quantification by real-time PCR was 1.2 x 10(3) in the rhizosphere, phyllosphere, and non-rhizosphere samples. E. coli O157:H7 concentrations were higher in the rhizosphere than in the non-rhizosphere soils and leaf surfaces, and persisted longer in clay soil. The persistence of E. coli O157:H7 in phyllosphere, rhizosphere, and non-rhizosphere soils over 45 days may play a significant part in the recontamination cycle of produce in the environment. Therefore, the rapidity of the real-time PCR assay may be a useful tool for quantification and monitoring of E. coli O157:H7 in irrigation water and on contaminated fresh produce.     

Growth and survival of uninjured and sublethally heat-injured Escherichia coli O157:H7 on beef extract medium as influenced by package atmosphere and storage temperature.

The effect of atmospheric composition and storage temperature on growth and survival of uninjured and sublethally heat-injured Escherichia coli O157:H7, inoculated onto brain heart infusion agar containing 0.3% beef extract (BEM), was determined. BEM plates were packaged in barrier bags in air, 100% CO2, 100% N2, 20% CO2: 80% N2, and vacuum and were stored at 4, 10, and 37 degrees C for up to 20 days. Package atmosphere and inoculum status (i.e., uninjured or heat-injured) influenced (P < 0.01) growth and survival of E. coli O157:H7 stored at all test temperatures. Growth of heat-injured E. coli O157:H7 was slower (P < 0.01) than uninjured E. coli O157:H7 stored at 37 degrees C. At 37 degrees C, uninjured E. coli O157:H7 reached stationary phase growth earlier than heat-injured populations. Uninjured E. coli O157:H7 grew during 10 days of storage at 10 degrees C, while heat-injured populations declined during 20 days of storage at 10 degrees C. Uninjured E. coli O157:H7 stored at 10 degrees C reached stationary phase growth within approximately 10 days in all packaging atmospheres except CO2. Populations of uninjured and heat-injured E. coli O157:H7 declined throughout storage for 20 days at 4 degrees C. Survival of uninjured populations stored at 4 degrees C, as well as heat-injured populations stored at 4 and 10 degrees C, was enhanced in CO2 atmosphere. Survival of heat-injured E. coli O157:H7 at 4 and 10 degrees C was not different (P > 0.05). Uninjured and heat-injured E. coli O157:H7 are able to survive at low temperatures in the modified atmospheres used in this study.     

Effect of sand and sawdust bedding materials on the fecal prevalence of Escherichia coli O157:H7 in dairy cows

Farm management practices that reduce the prevalence of food-borne pathogens in live animals are predicted to enhance food safety. To ascertain the potential role of livestock bedding in the ecology and epidemiology of Escherichia coli O157:H7 on farms, the survival of this pathogen in used-sand and used-sawdust dairy cow bedding was determined. Additionally, a longitudinal study of mature dairy cattle housed on 20 commercial dairy farms was conducted to compare the prevalence of E. coli O157:H7 in cattle bedded on sand to that in cattle bedded on sawdust. E. coli O157:H7 persisted at higher concentrations in used-sawdust bedding than in used-sand bedding. The overall average herd level prevalence (3.1 versus 1.4%) and the number of sample days yielding any tests of feces positive for E. coli O157:H7 (22 of 60 days versus 13 of 60 days) were higher in sawdust-bedded herds. The choice of bedding material used to house mature dairy cows may impact the prevalence of E. coli O157:H7 on dairy farms.     

Survival of Escherichia coli O157:H7 in feces from corn- and barley-fed steers

The survival of Escherichia coli O157:H7 in feces from steers fed corn (CO) or barley (BA) was evaluated at -10, +4 and +22 degrees C. Fecal pats were inoculated with a four-strain mixture of nalidixic-acid resistant E. coli O157:H7 at two levels: 10(3) CFU g(-1) (low, L) and 105 CFU g(-1) (high, H). At -10 degrees C, duration of survival of E. coli O157:H7 was reduced (p < 0.05) in CO-L (35 days) compared to BA-L (49 days), likely due to the effects of fecal volatile fatty acids in combination with a fecal pH of <6.5. At 4 degrees C, E. coli O157:H7 was detected in BA-H, CO-H, CO-L and BA-L for 77, 77, 56 and 63 days, respectively, with no difference (p > 0.05) observed in the duration of survival or rate of decline of E. coli O157:H7 among treatments. Survival of E. coli O157:H7 was twice as likely (p < 0.05) at 22 degrees C than at 4 degrees C and -10 degrees C. While pH and dry matter content increased, and volatile fatty acid concentrations decreased over 84 days at all three temperatures, these changes were most pronounced at 22 degrees C. Survival of E. coli O157:H7 for extended periods of time in feces from both corn- and barley-fed animals was demonstrated, thus fecal material may serve as a vector for the transmission of the organism. The greater survival of E. coli O157:H7 at 22 degrees C suggests that temperature may play a role in the seasonality of transmission and prevalence of this bacterium in feedlot cattle. The reported greater prevalence of E. coli O157:H7 in cattle fed barley as compared to those fed corn does not appear to be related to elevated risk of transmission arising from differential survival of the bacterium in feces.     

Reduction of Escherichia coli O157:H7 populations in cattle by addition of colicin E7-producing E. coli to feed

A cattle trial using artificially inoculated calves was conducted to determine the effect of the addition of colicinogenic Escherichia coli strains capable of producing colicin E7 (a 61-kDa DNase) to feed on the fecal shedding of serotype O157:H7. The experiment was divided into three periods. In period 1, which lasted 24 days, six calves were used as controls, and eight calves received 10(7) CFU of E. coli (a mixture of eight colicinogenic E. coli strains) per g of feed. Both groups were orally inoculated with nalidixic acid-resistant E. coli O157:H7 strains 7 days after the treatment started. In periods 2 and 3, the treatment and control groups were switched, and the colicinogenic E. coli dose was increased 10-fold. During period 3, which lasted as long as period 1, both groups were reinoculated with E. coli O157:H7. The numbers of E. coli O157:H7 were consistently greater in the control groups during the three periods, but comparisons within each time period determined a statistically significant (P < 0.05) difference only at day 21 of period 1. However, when the daily average counts were compared between the period 1 control group and the period 3 treatment group that included the same six animals, an overall reduction of 1.1 log(10) CFU/g was observed, with a maximum decrease of 1.8 log(10) CFU/g at day 21 (overall statistical significance, P = 0.001). Serotype O157:H7 was detected in 44% of the treatment group's intestinal tissue samples and in 64% of those from the control group (P < 0.04). These results indicated that the daily addition of 10(8) CFU of colicin E7-producing E. coli per gram of feed could reduce the fecal shedding of serotype O157:H7.     

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.     

Persistence of Escherichia coli O157:H7 in soil and on plant roots

Soil microcosms were inoculated with Escherichia coli O157:H7 to test persistence in fallow soil, on roots of cover crops and in presence of manure. In fallow soils, E. coli O157:H7 persisted for 25-41 days, on rye roots for 47-96 days and on alfalfa roots, in a silt loam soil, for 92 days whereas on other legumes persistence ranged from 25-40 days, similar to fallow soil. Manure did not seem to affect the persistence of E. coli O157:H7 in these soils. Indigenous and manure-applied coliform populations often decreased faster when E. coli O157:H7 was applied, indicating possible competition between microflora. Coliform populations in microcosms not inoculated with E. coli O157:H7 decreased more slowly or increased. Microbial community analyses showed little effect for E. coli O157:H7 inoculation or addition of manure. Microbial community metabolic activity was enhanced from rye roots after 14 days and by 63 days from alfalfa roots. Microbial community lactose utilization increased over time on rye roots in all soils and on alfalfa roots in a silt loam soil when E. coli O157:H7 was inoculated. Lactose utilization also increased for uninoculated rye roots, soil around rye roots and in some fallow soils. Our data suggest that clay increases persistence and activity of E. coli O157:H7 and other coliforms. In frozen soil stored for over 500 days, E. coli O157:H7 was viable in 37% of tested samples. In summary, E. coli O157:H7 persisted longer and activity was enhanced with some cover crops in these soils due to plant roots, the presence of clay and freezing.     

Effect of forage or grain diets with or without monensin on ruminal persistence and fecal Escherichia coli O157:H7 in cattle

Twelve ruminally cannulated cattle, adapted to forage or grain diet with or without monensin, were used to investigate the effects of diet and monensin on concentration and duration of ruminal persistence and fecal shedding of E. coli O157:H7. Cattle were ruminally inoculated with a strain of E. coli O157:H7 (10(10) CFU/animal) made resistant to nalidixic acid (Nal(r)). Ruminal and fecal samples were collected for 11 weeks, and then cattle were euthanized and necropsied and digesta from different gut locations were collected. Samples were cultured for detection and enumeration of Nal(r) E. coli O157:H7. Cattle fed forage diets were culture positive for E. coli O157:H7 in the feces for longer duration (P < 0.05) than cattle fed a grain diet. In forage-fed cattle, the duration they remained culture positive for E. coli O157:H7 was shorter (P < 0.05) when the diet included monensin. Generally, ruminal persistence of Nal(r) E. coli O157:H7 was not affected by diet or monensin. At necropsy, E. coli O157:H7 was detected in cecal and colonic digesta but not from the rumen. Our study showed that cattle fed a forage diet were culture positive longer and with higher numbers than cattle on a grain diet. Monensin supplementation decreased the duration of shedding with forage diet, and the cecum and colon were culture positive for E. coli O157:H7 more often than the rumen of cattle.     

Clonal dissemination of Escherichia coli O157:H7 subtypes among dairy farms in northeast Ohio

To ascertain the extent to which indistinguishable strains of Escherichia coli O157:H7 are shared between farms, molecular characterization was performed on E. coli O157:H7 isolates recovered during a longitudinal study of 20 dairy farms in northeast Ohio. Of the 20 dairy farms sampled, 16 were located in a primary area and 4 were located in two other distant geographical areas. A total of 92 E. coli O157:H7 isolates obtained from bovine fecal samples, water trough sediment samples, free-stall bedding, and wild-bird excreta samples were characterized. Fifty genetic subtypes were observed among the isolates using XbaI and BlnI restriction endonucleases. Most restriction endonuclease digestion profiles (REDPs) were spatially and temporally clustered. However, four REDPs from multiple sources were found to be indistinguishable by pulsed-field gel electrophoresis between four pairs of farms. The geographical distance between farms which shared an indistinguishable E. coli O157:H7 REDP ranged from 9 to 50 km, and the on-farm sources sharing indistinguishable REDPs included cattle and wild bird feces and free-stall bedding. Within the study population, E. coli O157:H7 REDP subtypes were disseminated with considerable frequency among farms in close geographic proximity, and nonbovine sources may contribute to the transmission of this organism between farms.     

Prevalence and clonal nature of Escherichia coli O157:H7 on dairy farms in Wisconsin.

A survey was conducted between March and October of 1994 to determine the prevalence and identify the sources of serotype O157:H7 isolates of Escherichia coli in Wisconsin dairy herds. A stratified sample of 400 farms was identified, and 70 farms with weaned calves less than 4 months old were included in the study. During the prevalence study, 5 of the 70 farms (herd prevalence, 7.1 +/- 4.5%) and fecal samples from 10 of 560 calves (animal prevalence, 1.8%) tested positive for serotype O157:H7. In a follow-up study, the five O157:H7-positive farms and seven of the O157:H7-negative farms identified in the prevalence study were visited again. An additional 517 fecal samples from cattle of various ages were tested, and a total of 15 animals from four of the five herds that were previously positive and 4 animals from two of seven herds that were previously negative tested positive for E. coli O157:H7. Observations made during the follow-up study suggested that horizontal transmission was an important means of E. coli O157:H7 dissemination on the farms. A total of 302 environmental samples, were examined, and 2 animal drinking water samples from one previously negative farm and 1 animal drinking water sample from a previously positive farm contained E. coli O157:H7. Analyses by the pulsed-field gel electrophoresis technique of contour-clamped homogeneous electric field electrophoresis revealed that isolates from the same farm displayed identical or very similar XbaI restriction endonuclease digestion profiles (REDP), whereas isolates from different farms typically displayed different REDP. However, more than one REDP was usually observed for a given herd over the 8-month sampling period. Analyses of multiple isolates from an animal revealed that some animals harbored O157:H7 strains that had different REDP, although the REDP of isolates obtained from the same fecal sample were very similar. Collectively, 160 bovine isolates obtained from 29 different animals and three water isolates displayed 20 distinct XbaI REDP. Our data revealed that there are several clonal types of serotype O157:H7 isolates in Wisconsin and indicated that there is probably more than one source of this pathogen on the dairy farms studied. However, animal drinking water was identified as one source of E. coli O157:H7 on one farm.     

Prevalence of Escherichia coli O157 in cattle feeds in Midwestern feedlots

Comparisons of enrichment methods (with or without antibiotics and with or without a preenrichment step) using gram-negative (GN) broth or tryptic soy broth (TSB) were conducted with feeds inoculated with Escherichia coli O157:H7. TSB was more sensitive than GN broth, and TSB with a preenrichment step followed by TSB with antibiotics was more sensitive than plain TSB enrichment, in detecting E. coli O157 in inoculated feeds. Feed samples were collected from feed bunks from 54 feedlots to determine the prevalence of E. coli O157 in cattle feeds. TSB preenrichment followed by TSB with antibiotics and the standard GN broth enrichment were used for each feed sample. All samples underwent immunomagnetic separation and were plated onto sorbitol MacConkey agar with cefixime and potassium tellurite. Identification of E. coli O157 was based on indole production, positive latex agglutination for O157 antigen, API 20E test strip results, PCR for the eaeA gene, and the presence of at least one Shiga toxin. E. coli O157 was detected in 52 of 504 feed samples (10.3%) by using GN broth enrichment and in 46 of 504 feed samples (9.1%) by using TSB followed by TSB supplemented with cefixime and vancomycin. E. coli O157 was detected in 75 of 504 feed bunk samples (14.9%) by one or both methods. There was no correlation between E. coli O157 prevalence and generic coliform counts in feeds. The prevalence of E. coli O157 in cattle feed warrants further studies to increase our knowledge of the on-farm ecology of E. coli O157 in order to develop strategies to prevent food-borne disease in humans.     

Thermal death of Escherichia coli O157:H7 in cattle feeds

AIMS: To determine if the temperatures used in feed manufacture are likely to destroy Escherichia coli O157. METHODS AND RESULTS: Two commercial feeds were ground and inoculated with E. coli O157 cells. The feeds were heated to 50, 55, 60, 65 or 70 degrees C. Heating produced quadratic survivor curves, with rapid initial decreases. The survival characteristics of E. coli O157 differed in the two feeds. The reductions observed in one feed may not have been due to heat alone. There was evidence that indigenous anti-E. coli O157 factor(s) in one feed acted with the heat and contributed to the observed rates of bacterial death. Heating at 70 degrees C for 20 or 120 s resulted in approx. 1.3 and 2.2 log reductions in E. coli O157 numbers respectively. Lesser reductions were observed at lower temperatures. CONCLUSIONS: The time/temperature combinations used in commercial pelleting processes would not effectively kill high numbers of E. coli O157. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to look at the survival of E. coli O157 strains after heat treatment within concentrated animal feed. The study provides information on the likely risk of E. coli O157 surviving the animal feed manufacturing process.     

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.     

Survival of Escherichia coli O157:H7 in bottled natural mineral water

A non-verotoxin-producing isolate of Escherichia coli O157:H7 was inoculated at final concentrations of 10(3) or 10(6) ml-1 into natural non-carbonated mineral water (MW), sterile natural mineral water (SMW) and sterile distilled deionized water (SDDW) and stored at 15 degrees C for 10 weeks. Samples were examined every 7 d for the presence of E. coli O157:H7 using a resuscitative/selective agar procedure. The MW samples were also plated onto a non-selective agar, R2A, to enumerate E. coli O157:H7 and the autochthonous flora. There was a significant difference in the survival of E. coli O157:H7 (10(3) ml-1 inoculum) between the MW and the SDDW at time periods 0, 7, 14 (P < 0.005) 21, 28, 35 (P < 0.001) and 42 d (P < 0.05) and between the MW and the SMW at time periods 7, (P < 0.05) 14, 21 (P < 0.005) 28 (P < 0.01) and 35 d (P < 0.05), with the pathogen surviving longest in the MW samples. In contrast, at 10(6) ml-1, no significant differences in the survival of E. coli O157:H7 were observed between the water types. The presence of E. coli O157:H7 (10(3) ml-1) in the MW samples did not have an antagonistic effect on the recovery of the autochthonous flora. Transmission electron microscopy analysis demonstrated that the E. coli O157:H7 cells lyse during storage, releasing their contents into the surrounding environment. These substances may have been utilized by the autochthonous flora and thereby explain why the numbers of flora recovered from the inoculated MW samples were higher than those recovered from the uninoculated samples.