A Comparison of the Survival in Feces and Water of Escherichia coli O157:H7 Grown under Laboratory Conditions or Obtained from Cattle Feces

6 Journal of Food Protection, Vol. 69, o. 1, 2006, Pages 6 11 Copyright, International Association for Food Protection A Comparison of the Survival in Feces and Water of Escherichia coli O157:H7 Grown under aboratory Conditions or Obtained from Cattle Feces. SCOTT, 1,2 * P. MCGEE, 2 J. J. SHERIDA, 2 B. EAREY, 3 AD. EOARD 1 1 Faculty of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland; 2 Teagasc, The ational Food Centre, Ashtown, Dublin, Ireland; and 3 Teagasc, Grange Research Centre, Dunsany, County Meath, Ireland MS 05-293: Received 13 June 2005/Accepted 22 August 2005 ABSTRACT Escherichia coli O157:H7 is an important foodborne pathogen that can cause hemorrhagic colitis and hemolytic uremic syndrome. Cattle feces and fecally contaminated water are important in the transmission of this organism on the farm. In this study, the survival of E. coli O157:H7 in feces and water was compared following passage through the animal digestive tract or preparation in the laboratory. Feces were collected from steers before and after oral inoculation with a marked strain of E. coli O157:H7. Fecal samples collected before cattle inoculation were subsequently inoculated with the marked strain of E. coli O157:H7 prepared in the laboratory. Subsamples were taken from both animal and laboratory-inoculated feces to inoculate 5-liter volumes of water. E. coli O157:H7 in feces survived up to 97 days, and survival was not affected by the method used to prepare the inoculating strain. E. coli O157:H7 survived up to 109 days in water, and the bacteria collected from inoculated cattle were detected up to 10 weeks longer than the laboratory-prepared culture. This study suggests that pathogen survival in low-nutrient conditions may be enhanced by passage through the gastrointestinal tract. Escherichia coli O157:H7 is an important zoonotic pathogen that can cause a range of symptoms from bloody diarrhea to hemolytic uremic syndrome. Cattle are considered the primary source of this pathogen, although other farm animals also have been implicated in outbreaks of disease (6, 22, 28, 31). Fecal material is considered an important vehicle for transmission of E. coli O157:H7, and infection may result from contact with fecal matter on the farm and through contaminated foodstuffs or water supplies (2, 8, 9, 16, 19). One of the distinguishing characteristics of this pathogen is that it has a low infective dose; ingestion of as few as 10 CFU can cause disease in humans (30). Investigation of the survival of E. coli O157:H7 in water and feces is therefore vital, because persistence of even small numbers of this organism is significant. Occasionally, E. coli O157:H7 strains derived directly from animal sources have been used in survival experiments (21); however, the strains usually used in these studies are adapted to laboratory conditions. A comparison of the survival of isolates derived directly from the animals or following preparation in the laboratory has not been performed. Enhanced bacterial virulence after passage through the gastrointestinal (GI) tract has been previously reported for a Campylobacter isolate (5, 20), and in a recent report the authors proposed that a lower inoculation dose of naturally occurring E. coli O157:H7 cells could colonize cattle (26). These findings suggest that bacteria obtained directly from the farm environment in which they have been subjected to stressful conditions may have an enhanced ability to survive when com- * Author for correspondence. Tel: 353-1-716000; Fax: 353-1-7166185; E-mail: lscott@nfc.teagasc.ie. pared with bacteria that have been cultured in the laboratory under optimal conditions. The objective of this study was to determine whether an E. coli O157:H7 isolate grown in the laboratory had survival times in feces and water similar to those of an isolate that had been ingested by cattle and survived passage through the GI tract. MATERIAS AD METHODS Organism. A bovine fecal nontoxigenic strain of E. coli O157:H7 was used throughout the study. To aid detection, the isolate was made resistant to streptomycin sulfate (1,000 gml 1 ) and nalidixic acid (50 g ml 1 ) as previously described (27). The antibiotic-resistant strain was labeled as VC 047 and stored on cryoprotective beads (Technical Services Consultants, ancashire, UK) at 20C. A comparison was previously performed between an E. coli O157:H7 strain marked as described above and the parent strain, and no differences in survival were observed (10). Under field conditions, such a comparison could not be made. Animals. Eight Holstein Friesian steers 9 to 12 months of age were used in this study. The steers were randomly assigned to one of two groups of four and adapted over 1 month to a diet of either ad lib silage or ad lib concentrates. One week before the start of the study and 24 h before the cattle were inoculated with strain VC 047, feces from each animal were collected and tested to ensure that they were not shedding E. coli O157:H7 or other enteric bacteria resistant to nalidixic acid and streptomycin sulfate. Inoculation of animals. Cells from strain VC 047 were washed and centrifuged as previously described (26). A 1-ml aliquot of the inoculum suspension was added to 50 ml of sterile distilled water, and a total of 50 ml of sterile distilled water containing approximately 10 10 CFU of E. coli O157:H7 was administered orally with a syringe (Plastitek 60 ml, Becon and David-

J. Food Prot., Vol. 69, o. 1 SURVIVA OF E. COI O157:H7 I FECES AD WATER 7 son, Dublin, Ireland) to each of the eight animals. Immediately after inoculation, each animal was given 1 liter of sterile distilled water to ensure that none of the inoculum was lost. Collection of feces preinoculation. Approximately 600 g of feces was collected from the rectum of each animal 24 h before inoculation with E. coli O157:H7. The fecal material was stored in separate covered plastic containers in a shed for 3 days at 7 to 10C and then inoculated with strain VC 047 prepared in the laboratory. Collection of feces postinoculation. Another 600 g of feces was taken from each animal 24 h postinoculation, and the concentrations of the marked strain of E. coli O157:H7 were determined. Duplicate 200-g portions were then removed from each 600-g sample and placed into sterile plastic containers; these samples are referred to hereinafter as naturally inoculated feces. Inoculation of feces with the laboratory strain. Duplicate 200-g samples derived from the eight fecal samples collected preinoculation were placed into sterile plastic containers. These samples were inoculated in the laboratory with strain VC 047 at a concentration similar to that for the naturally inoculated feces of the same animal. The organism was grown in brain heart infusion broth (Oxoid, Basingstoke, UK) to stationary phase at 37C, centrifuged, and washed as previously described (26). A 10-ml volume of maximum recovery diluent (MRD; Oxoid) containing a known concentration of E. coli O157:H7 was added to the duplicate samples from each animal and mixed with a sterile spatula. These samples were designated as laboratory inoculated and were stored with the naturally inoculated feces in a shed. Subsamples were taken once a week from each of the 32 fecal samples for the duration of the experiment. Source of water. Farm water in 5-liter volumes was collected from an untreated river water source and stored in covered plastic containers in a shed for not more than 3 days before inoculation with strain VC 047. The water was not tested for the presence of naturally occurring E. coli O157:H7. Inoculation of water samples. Feces containing E. coli O157:H7 VC 047 were used to inoculate the water samples. Duplicate 100-g samples of naturally inoculated feces were collected from six animals, three from the group fed a concentrate diet and three from the group fed a silage diet. Each sample was mixed into 12 separate 5-liter water samples (2%, wt/vol), which were subsequently referred to as naturally inoculated water. Similarly, duplicate 100-g samples of feces (from the same six animals) that had been inoculated in the laboratory were added to another 12 water samples and referred to as laboratory-inoculated water. The containers were sealed and stored in a shed for the duration of the study. Samples were taken twice weekly for 14 days and once a week thereafter. Enumeration of E. coli O157:H7 in fecal samples. A sterile wooden spatula was used to mix each 200-g fecal sample. A 5-g subsample was removed, added to 45 ml of MRD, and stomached for 90 s in a Colworth stomacher (model BA 6024, A. J. Steward & Co. td., ondon, UK). From each sample, a dilution series was performed in 10-ml volumes of MRD. Aliquots were plated onto sorbitol MacConkey agar (SMAC; Oxoid) containing nalidixic acid (50 g/ml) and streptomycin sulfate (1,000 g/ml) (SMAC-nas) and incubated for 24 h at 37C. Aliquots were also plated onto tryptone soy agar (TSA; Oxoid), incubated at 37C for 2 h, overpoured with SMAC-nas, and reincubated for a further 48 h to allow for the recovery of injured cells. The detection limit was 0.7 log CFU g 1. VC 047 cells were identified as pale, nonsorbitol-fermenting colonies on SMAC-nas. When the numbers of organisms per sample had declined such that quantitative enumeration was no longer possible, samples were examined after enrichment. Samples were enriched by adding a 5-ml volume from the original 1:10 MRD solution to 45 ml of modified tryptone soy broth (mtsb; Oxoid) containing nalidixic acid (50 g/ml) and streptomycin sulfate (1,000 g/ml) (mtsb-nas). Samples were incubated for 24 h at 37C, and aliquots from the enriched samples were plated in duplicate onto SMAC-nas. Enrichment was carried out until the organism could not be detected on two consecutive sampling days. Enumeration of E. coli O157:H7 in water samples. The 5- liter volumes of water were agitated for approximately 30 s before subsamples were removed. Sterile 150-ml containers (Bibby Sterilin, Stone, Staffordshire, UK) were used to remove 45-ml samples, which were taken immediately to the laboratory. To each sample, 5 ml of a 5% flocculant solution (Zetag 7899, General Chemicals, Dublin, Ireland) was added to enable filtering of the water samples. The water and flocculant mix was centrifuged for 1 min at 5,000 g (Eppendorf Centrifuge 5403, Hamburg, Germany). The supernatant was decanted through a 0.2-m-pore-size filter (Sartorius, Goeftingin, Germany), the filter was returned to the fecal pellet, and 15 ml of MRD was added and vortexed for 1 min. From each sample a dilution series in 9-ml volumes of MRD was made and plated as described for the fecal samples. The detection limit was 0.16 CFU ml 1. For enrichment of the water samples, 30 ml of mtsb was added to the filter and fecal pellet and incubated at 37C for 24 h. Aliquots from the enriched samples were plated in duplicate onto SMAC-nas and incubated for another 48 h. Samples were cultured by enrichment until the organism was undetectable on two consecutive sampling days. Both fecal and water survival experiments were conducted from February to the beginning of June. Throughout the experiment, the ambient temperature in the shed where the samples were stored was recorded on an hourly basis with a data logger (Testostor 175, Testo, Alton, UK). Statistical analysis. The experiment was performed in duplicate and repeated three and four times for the water and fecal bacterial survival experiments, respectively. inear regression was used to analyze the changes in E. coli O157:H7 concentrations over time. For the fecal samples, the period before the commencement of decline was omitted from analysis, with initial decline determined as the time point from which a 0.5-log reduction in concentration occurred. The comparison of the rates of decline between treatments, i.e., the slopes of the lines, was carried out using Student s t test. Differences between treatment results were considered significant at 1% (P 0.01). RESUTS Although E. coli O157:H7 counts on SMAC were generally lower than those on TSA overpoured with SMAC (TSA-SMAC), the differences were not significant (Fig. 1). Therefore, the results presented are for TSA-SMAC cultures only. The amount of time that elapsed before E. coli O157: H7 concentrations in feces began to decline ranged from 27 to 55 days, depending on diet and method of inoculation (Table 1). Although concentrations decreased more rapidly for feces from cattle fed silage than for feces from cattle fed concentrate for both methods of inoculation (Fig. 1),

8 SCOTT ET A. J. Food Prot., Vol. 69, o. 1 FIGURE 1. Survival of E. coli O157:H7 (log CFU per gram) in feces inoculated by the natural () or laboratory () method. Feces were obtained from cattle fed on a concentrate ( / ) or silage ( /) diet. Bacteria were recovered on TSA-SMAC ( /) or SMAC ( /). the differences were not significant, as indicated by the slopes of the linear regression for the different treatments (Table 1). Regression analysis indicated that there was no difference in pathogen survival between the two methods of inoculation regardless of diet. When the survival of the organism in water was examined, the line of best fit to the data was linear. Decreases in concentrations occurred during 46 days, and differences between the rates of decline for the two methods of inoculation were not significant for both diets (Fig. 2). From day 54 on, the organism was detected by enrichment only for a total of 63 days, but survival time varied depending on the method of inoculation and diet. The results in Table 2 are represented for individual replicates because insufficient quantitative data were available to permit presentation as means. During this time (day 54 to day 122), E. coli O157:H7 was detected for a longer period in TABE 1. Time to decline and rate of decrease in population of E. coli O157:H7 in fecal samples from cattle fed a concentrate or silage diet a Treatment Concentrate Concentrate Silage Silage Slope of linear regression 0.05 0.05 0.06 0.04 Standard error of Time slope R 2 (days) b 0.001 0.007 0.009 0.003 0.99 0.89 0.85 0.96 34 55 27 34 a Feces were inoculated with E. coli using natural () or laboratory () methods. b Time before a decline in counts (0.5 log CFU/g) first occurred. naturally inoculated water regardless of diet. For the concentrate treatment group, this difference was most pronounced in replicate 1, where the pathogen survived until day 109, almost 10 weeks longer than in the laboratoryinoculated water. In replicate 1 of the experiment for the silage group, E. coli O157:H7 remained in the naturally inoculated water for 49 days longer than in the laboratoryinoculated samples. Although there was considerable variation between replicates, survival was always greater in the naturally inoculated water. The average ambient temperature during the course of this experiment was 10C (4.5 to 20.3C). The average weekly maximum and minimum temperatures are presented in Figure 3. DISCUSSIO The findings of this study indicate that regardless of diet (concentrate or silage), survival of E. coli O157:H7 in water was enhanced as a result of being passaged through the bovine GI tract. However, when the pathogen was inoculated into bovine feces, survival was not significantly different for the natural and laboratory inocula. The survival times of E. coli O157:H7 in water under various conditions has ranged from 13 to 245 days (21, 23). Research investigating the survival of E. coli O157:H7 in this environment is important because of the many outbreaks associated with contaminated water (4, 14, 17, 22). Water troughs also have been implicated in the maintenance of this organism on the farm (12). Many factors have been examined in relation to E. coli O157:H7 survival in water, such as temperature, inoculum concentration, and fecal contamination (24, 33). However, the effect of passage of the

J. Food Prot., Vol. 69, o. 1 SURVIVA OF E. COI O157:H7 I FECES AD WATER 9 FIGURE 2. Survival of E. coli O157:H7 in water (log CFU per milliliter) inoculated with feces from cattle fed on a concentrate (C) or silage (S) diet. Feces were inoculated using the natural () or laboratory () method. TABE 2. Detection of E. coli O157:H7 in water inoculated with feces from cattle fed two different diets and stored from day 54 to day 122 Inoculation Replicatod meth- a 1 2 3 1 2 3 Storage time (days) b : 54 68 74 81 97 102 109 117 122 Concentrate diet Silage diet a, water inoculated with E. coli O157 using the natural method;, water inoculated with E. coli O157 using the laboratory method. b, E. coli O157 detected in the water sample by enrichment;, E. coli O157 not detected in the water sample;, not sampled. pathogen through the GI tract has not been addressed. The results from the current study indicate that in water a naturally prepared inoculum survived longer than did a laboratory-prepared inoculum during the last 10 weeks of the experiment. For some replicates, the natural inoculum was still present after 109 days, whereas the laboratory inoculum was undetectable by day 46. These results suggest that E. coli O157:H7 cells that have survived transit through the stressful conditions of the bovine digestive system are better prepared to endure harsh environments, such as water, than are cells grown in optimum conditions in the laboratory. The most severe stress that cells passed through the GI tract have to overcome is the acidity of the bovine abomasum (ph 2.5 to 3.0). Bearson et al. (1) proposed that acid stress adaptation in E. coli can provide cross-protection against a wide range of other stresses. This cross-protection may result from the induction of protective proteins on the cell membrane (7). These proteins are also essential for the maintenance of cell viability during periods of starvation, such as occurs in water (29). In the current study, the crossprotection effect may have extended the survival in water of the E. coli O157:H7 cells passaged through the cattle in agreement with previous findings of enhanced bacterial virulence after passage through the digestive system (5, 20). In this context, additional research is required to investigate the precise bacterial changes at a cellular level. The results of the current study indicate that the survival of E. coli O157:H7 strains prepared in the laboratory will not always mirror the survival of a naturally occurring pathogen, such as would be found in fecally contaminated water on a farm. This difference is important to consider when conducting experiments to accurately predict the survival E. coli O157: H7 in the farm environment. E. coli O157:H7 is well adapted to survival for extended storage periods in bovine feces and slurry, with detectable numbers often present over many months (3, 13, 25, 34). Bovine feces are considered a favorable nutrient environment for pathogen survival (23) compared with wa-

10 SCOTT ET A. J. Food Prot., Vol. 69, o. 1 FIGURE 3. Average weekly maximum and minimum shed temperatures during the course of the experiment (February to the beginning of June). ter, which is low in nutrients. In this study, up to 55 days elapsed before E. coli O157:H7 numbers in feces declined, indicating that the cells were surviving well in that environment. In contrast to survival in water, the survival of the organism in feces was not affected by the method of inoculation. This finding suggests that passage through the GI tract will not affect pathogen survival in a nutrient-rich environment such as feces. Therefore, in feces an inoculum prepared in the laboratory can be used to accurately predict survival under natural conditions. Many issues affecting the survival of E. coli O157:H7 on the farm, such as the intestinal microbial flora and immune status of the individual animal, have been reported (13, 18). In the water experiment, pathogen survival varied greatly between replicates. To address some of this variation, animals from the same cohort, age, and management conditions were used for this study. Some of the variation observed in the water experiment may have been due to the use of feces from different animals. Differences in the microflora between fecal samples may contribute to variation in microbe survival (11). In this study, there was no influence of animal diet on pathogen survival in feces. This finding is in contrast to the findings of other studies, in which that E. coli O157:H7 survived longer in slurry from animals fed either concentrate or silage diets (15, 25, 32). These divergent results indicate that the influence of diet on the survival of E. coli O157:H7 in feces is not yet clear. The effect on E. coli O157:H7 of passage through the bovine GI tract is an important factor when planning survival experiments. 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