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Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

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 108-9-cfu-g−1-of-the-organism-and-(i)-stored-in-closed-plastic-containers-at-10 °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.

Although Escherichia coli O157:H7 has been isolated from sheep ( Kudva et al. 1997 ), pigs ( Chapman et al. 1997 ), deer ( Chapman & Ackroyd 1997) and pigeons ( Dell’Omo et al. 1998), it is generally accepted that cattle, with a prevalence of up to 36·8% ( Chapman et al. 1997 ), are the primary reservoir of this pathogen. Once ingested, E. coli O157:H7 persists in the rumen and colon of the animal, contaminating the faeces ( Brown et al. 1997 ), which subsequently acts as a vehicle in the horizontal transmission to uninfected animals ( Hancock et al. 1994 ; Faith et al. 1996 ). Contamination from bovine faeces has also been implicated in a number of food-borne outbreaks of E. coli O157:H7 food poisoning ( Morgan et al. 1988 ; Dev et al. 1991 ; Besser et al. 1993 ).

Once in foods or food ingredients, E. coli O157:H7 displays a remarkable ability to survive some of the conditions and processing procedures used in the food industry ( Abdul-Raouf et al. 1993 ; Reitsma & Henning 1996; Semanchek & Golden 1996; Riordan e t al. 1998). Of particular concern in this regard is its ability to survive well under refrigeration conditions ( ICMSF 1996) and its atypically high acid tolerance. Thus, for example, investigation of an outbreak of food poisoning associated with apple cider in south-eastern Massachusetts found that E. coli O157:H7 from manure used as a fertilizer in the orchard survived for 20 d at a pH value below 4, conditions previously considered sufficient to inhibit the growth and survival of bacterial pathogens and non-toxigenic strains of E. coli ( Besser et al. 1993 ).

While survival in food has been the focus of several studies, less information is available on the survival characteristics of the organism in the environment. Such information is of fundamental importance to predict accurately the risks associated with farm practices such as the spreading of manure. It is also essential for the provision of advice regarding changes in animal husbandry or grassland management necessary to limit the risks of horizontal transfer of this organism from animal to animal, and ultimately its access to the human food chain. The primary objective of this study was to investigate the survival characteristics of E. coli O157:H7 in faeces under controlled laboratory conditions and under external environmental conditions.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

Organism

An antibiotic-resistant strain of non-toxigenic E. coli O157:H7 (NCTC 12900), resistant to streptomycin sulphate (1000 μg ml−1) and nalidixic acid (50 μg ml−1), was prepared as described by Park (1978) and stored on cryoprotective beads (Technical Service Consultants Ltd, Heywood, UK) at −20 °C.

Inoculum preparation

One bead containing E. coli O157:H7 was aseptically transferred to 30 ml BHI broth and incubated at 37 °C for 24 h. From this culture, 7·0 ml were transferred to 700 ml fresh BHI and incubated for a further 18 h at 37 °C. The resultant cells were recovered by centrifugation at 3019 g (Eppendorf Centrifuge 5403, Hamburg, Germany), washed three times in Maximum Recovery Diluent (MRD, 0·85 g sodium chloride and 1·0 g bacteriological peptone l−1 distilled water) and resuspended in 25 ml MRD. This process yielded an inoculum containing approximately 9·5 log10 cfu ml−1.

Inoculation of faecal samples

Bovine faeces were collected in buckets immediately after deposition on the ground by the animal. Exactly 25 ml of inoculum was added to 500 ml faeces to form a bulk sample containing approximately 8 log10 cfu ml−1. From this mixture, 100 ml samples were transferred into two sterile plastic containers and the containers were sealed. One container was stored in the laboratory at 10 °C and the other was placed outside on an area of grassland. A further 100 ml sample of inoculated faeces was decanted onto the surface of the grassland, adjacent to the sealed plastic container, and left exposed to ambient weather conditions for 99 d, from mid-January to mid-April. Samples were removed periodically from each faeces mixture and examined as described below.

Enumeration of E. coli O157:H7

Using a sterile spoon, 1·0 g of each sample was removed, serially diluted in 9·0 ml amounts of MRD, plated on Sorbitol MacConkey Agar (SMAC, Oxoid) and incubated at 37 °C for 48 h. Aliquots from the dilution series were also plated on Tryptic Soya Agar (TSA, Oxoid), incubated at 37 °C for 2 h, overpoured with SMAC and incubated at 37 °C for a further 48 h. The latter process allowed injured cells to recover prior to selection using SMAC.

Enrichment procedure

Escherichia coli O157:H7 present in soil where faeces had been deposited were detected by enrichment. Approximately 1·0 g of the soil was added to 9·0 ml buffered glucose-brilliant green bile broth (EE broth) and incubated at 37 °C for 24 h. The enriched culture was then examined for the presence of E. coli O157:H7 by the above selective (SMAC) and non-selective (TSA) techniques.

Statistical analysis of the results

The experiment was performed in duplicate and repeated five times to give 10 replicates. Regression analysis was performed on the changes in E. coli O157:H7 levels over time. The slopes were then analysed using factorial anova (Genstat 5, Statistics Department, Rothamsted Experimental Station, UK). Significant differences between treatments were determined to be those with a 5% level of significance (P < 0·05).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

Escherichia coli O157:H7 numbers declined during storage/ exposure of inoculated faecal samples. In samples held in sealed containers at 10 °C, the SMAC counts declined from 8·23 log10 cfu g−1 (0 d) to 3·18 log10 cfu g−1 (99 d) ( Fig. 1). In these samples, TSA/SMAC counts declined from 8·15 log10 cfu g−1 (0 d) to 2·57 log10 cfu g−1 (99 d). Similar patterns of decline in E. coli O157:H7 numbers were observed in samples stored in sealed containers outside in the field, despite the considerable fluctuations in environmental temperatures (−6·5 to 19·6 °C) being applied to these samples ( Fig. 2).

image

Figure 1. Survival of Escherichia coli O157:H7 at 10 °C in the laboratory as determined on selective (▪) and non-selective (□) media, outside in field containers as determined on selective (▵) and non-selective (▵) media, and on the grass as determined on selective (&U25CF;) and non-selective (○) media

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image

Figure 2. The daily maximum (□) and minimum (▪) outdoor temperatures during the course of this experiment (mid-January to mid-April)

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In samples decanted onto grassland, E. coli O157:H7 numbers declined in a similar pattern to sealed samples. SMAC counts declined from 7·85 log10 cfu g−1 (0 d) to 2·96 log10 cfu g−1 (99 d), and TSA/SMAC counts declined from 7·47 log10 cfu g−1 (0 d) to 3·02 log10 cfu g−1 (99 d). However, the decrease in recovered E. coli O157:H7 numbers was more rapid in exposed samples, i.e. reduction of 5 log10 cfu g−1 occurred within 43–50 d in exposed samples, whereas such reductions occurred in 90–99 d in sealed samples ( Fig. 1).

After 50–57 d, the deposited material became integrated into the soil and was no longer detectable using the plating techniques. Buffered glucose-brilliant green bile broth enrichment procedures were used to determine the presence or absence of the organism in the areas where the samples were originally decanted. The results demonstrated the presence of E. coli O157:H7 in 70% of soil samples after 85 d and in 20% of samples after 99 d ( Table 1).

Table 1.  Detection of Escherichia coli O157:H7 in the soil surrounding inoculated faeces deposited on grass
SMAC (selective)TSA (non-selective)
ReplicatesReplicates
Time (d) 1234567891012345678910
57+++++++++++++++
64++++++++++++++++++
71+++++++++++++++
78++++++++++++++++
85++++++++++++++
92++++++
99++++

Regression analysis of the changes in E. coli O157:H7 numbers during storage showed that there was no significant difference between the enumeration methods (SMAC and TSA/SMAC) (P < 0·05) regardless of the storage treatment. Similarly, there was no significant difference between storage in the laboratory containers and storage in field containers (P < 0·05). However, the decline in E. coli O157:H7 in faecal samples exposed on the grass was significantly faster than either of the other two treatments.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

Current farm waste recycling practices such as the spreading of manure onto pasture land may increase the rate of carriage of undesirable commensals and/or pathogens in bovine herds. Hancock et al. (1994) reported a tentative relationship between such practices and the prevalence of E. coli O157:H7 in dairy and beef cattle. However, more recent investigations ( Hancock et al. 1997 ) have not established any correlation between the prevalence of E. coli O157:H7 in cattle and the application of bovine manure to pasture lands.

Interestingly, more recent studies reported a 36·8% incidence of E. coli O157:H7 in cattle ( Chapman et al. 1997 ). This figure is considerably higher than that previously reported ( Wells et al. 1991 ; Chapman et al. 1993 ; Hancock et al. 1994 ). While these differences may be attributed to different experimental designs or the use of more sensitive detection methods, they may also reflect an increasing distribution of E. coli O157:H7 through cattle herds over time.

The significance of manure recycling practices in the prevalence of E. coli O157:H7 in cattle grazing treated grassland is dependent on the nature and extent of the survival of this organism in bovine faeces. The E. coli O157:H7 strain used in this study was considered a safe but suitable representative strain as it differed from pathogenic strains only with respect to the absence of the verotoxin encoding genes. Indeed, the survival characteristics observed in sealed containers under laboratory and field conditions are consistent with those reported by Maule (1995) who found that a verotoxigenic E. coli O157:H7 strain survived well in bovine faeces within a range of laboratory-scale ecosystems.

The current study recovered similar numbers of E. coli O157:H7 on selective (SMAC) and resuscitation media (TSA/SMAC) from stored samples, indicating that stressed cells did not comprise a significant component of the total E. coli O157:H7 population in inoculated bovine faeces. This also supports the suggestion made by Maule (1995) that bovine faeces provide a relatively favourable environment for the survival of this organism. Variations in holding temperature, including intermittent exposure to subzero (°C) temperatures, had little impact on survival rates of the organism in sealed containers. Pathogenic E. coli have previously been reported ( ICMSF 1996) to survive better than non-pathogenic strains in foods held at low temperature (+4–+8 °C and at −20 °C). The results of this study suggest that E. coli O157:H7 also exhibits this atypically enhanced tolerance in bovine faeces, and during temperature fluctuations within these values.

This study noted a decline in E. coli O157:H7 numbers in inoculated bovine faeces deposited directly on grassland. However, correlation of this observation with the counts from samples in sealed containers suggests that these reductions were not due to cell death, but were more likely to be due to the organisms being washed out of the inoculated faecal materials by rain. This suggestion is supported by prevalent detection of the organism in soil surrounding the sample site, and the persistence of this detection for extended periods (up to 99 d) well after the faecal mass had been assimilated into the soil (50 d). The observation of extended persistence of E. coli O157:H7 in faeces/soil is in agreement with the report of Wang et al. (1996) , who noted that E. coli O157:H7 survived in bovine faeces for up to 49 d at 37 °C, 56 d at 22 °C and up to 70 d at 5 °C.

In summary, therefore, E. coli O157:H7 has been shown to survive for extended periods, i.e. several months, in bovine faeces and/or contaminated grassland. Such persistence allows the possibility of subsequent ingestion by, and infection of other previously uninfected animals, leading to increased overall carriage rates in herds, and posing increased risks to the final consumer of derived milk and meat products.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

The assistance of Tony Hegarty in performing the statistical analysis on these results is gratefully acknowledged.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References
  • Abdul-Raouf, U.M., Beuchat, L.R., Ammar, M.S. 1993Survival and growth of Escherichia coli O157: H7 on salad vegetables.Applied and Environmental Microbiology59, 1999 2006.
  • Besser, R.E., Lett, S.M., Weber, J.T et al. 1993 An outbreak of diarrhoea and haemolytic uraemic syndrome from Escherichia coli O157: H7 in fresh pressed apple cider. Journal of the American Medical Association, 269, 2217 2220.
  • Brown, C.A., Harmon, B.G., Zhoa, T., Doyle, M.P. 1997 Experimental Escherichia coli O157: H7 carriage in calves. Applied and Environmental Microbiology, 63, 27 32.
  • Chapman, P.A. & Ackroyd, H.J. 1997 Farmed deer as a potential source of verocytotoxigenic-producing Escherichia coli O157. Veterinary Record, 20, 314 315.
  • Chapman, P.A., Siddons, C.A., Cerdan-Malo, A.T., Harkin, M.A. 1997 A 1-year study of Escherichia coli O157 in cattle, sheep, pigs and poultry. Epidemiology and Infection, 119, 245 250.
  • Chapman, P.A., Siddons, C.A., Wright, D.J., Norman, P., Fox, J., Crick, E. 1993 Cattle as a possible source of verocytotoxin-producing Escherichia coli O157 infections in man. Epidemiology and Infection, 111, 439 447.
  • <dell’omo , G., Morabito, S., Quondam, R et al. 1998 Feral pigeons as a source of verocytotoxin-producing Escherichia coli. Veterinary Record, 21, 309 310.
  • Dev, V.J., Main, M., Gould, I. 1991 Waterborne outbreak of Escherichia coli O157. Lancet, 337, 1412.
  • Faith, N.G., Shere, J.A., Brosch, R et al. 1996 Prevalence and clonal nature of Escherichia coli O157: H7 on dairy farms in Wisconsin. Applied and Environmental Microbiology, 62, 1519 1525.
  • Hancock, D.D., Besser, T.E., Kinsel, M.L., Tarr, P.I., Rice, D.H., Paros, M.G. 1994 The prevalence of Escherichia coli O157: H7 in dairy and beef cattle in Washington State. Epidemiology and Infection, 113, 199 207.
  • Hancock, D.D., Rice, D.H., Herriot, D.E., Besser, T.E., Ebel, E.D., Carpenter, L.V. 1997 Effects of manure handling practices on Escherichia coli O157: H7 prevalence in cattle. Journal of Food Protection, 60, 363 366.
  • ICMSF 1996 Intestinally pathogenic. Escherichia Coli. In: Micro-Organisms in Foods, 5, Microbiological Specifications of Food Pathogens, 127 140. London: Blackie Academic & Professional.
  • Kudva, I.T., Hatfield, P.G., Hovde, C.J. 1997 Characterisation of Escherichia coli O157: H7 and other shiga toxin-producing E. coli serotypes isolated from sheep. Journal of Clinical Microbiology, 35, 892 899.
  • Maule, A. 1995 Survival of the verotoxin strain of E. coli O157: H7 in laboratory-scale microcosms. In: Coliforms and E. Coli: Problem or Solution?, eds. Kayand, D. & Fricker, C, pp. 61 65. Gateshead, UK: Athenaeum Press Ltd.
  • Morgan, G.M., Newman, C., Palmer, S.R. 1988 First recognised community outbreak of haemorrhagic colitis due to verotoxin-producing Escherichia coli O157: H7 in the UK. Epidemiology and Infection, 101, 83 91.
  • Park, R.W.A. 1978 The isolation and use of streptomycin resistant mutants for following development of bacteria in mixed populations. In: Techniques for the Study of Mixed Populations (eds Lovelock, D. & Davies, R), pp. 107 112. London: Academic Press.
  • Reitsma, C.J. & Henning, D.R. 1996 Survival of enterohemorrhagic Escherichia coli O157: H7 during the manufacture and curing of Cheddar cheese. Journal of Food Protection, 59, 460 464.
  • Riordan, D.C.R., Duffy, G., Sheridan, J.J et al. 1998 Survival of Escherichia coli O157: H7 during the manufacture of pepperoni. Journal of Food Protection, 61, 146 151.
  • Semanchek, J.J. & Golden, D.A. 1996 Survival of Escherichia coli O157: H7 during fermentation of apple cider. Journal of Food Protection, 59, 1256 1259.
  • Wang, G., Zhao, T., Doyle, M.P. 1996 Fate of enterohemorrhagic Escherichia coli O157: H7 in bovine faeces. Applied and Environmental Microbiology, 62, 2567 2570.
  • Wells, J.G., Shipman, L.D., Greene, K.D et al. 1991Isolation of Escherichia coli serotype O157: H7 and other shiga-like toxin-producing E. coli from dairy cattle. Journal of Clinical Microbiology, 29, 985 989.