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To isolate model bacterial strains possessing transferable antibiotic resistance, and their fate and behaviour studied under conditions reflecting those potentially encountered in the field. <P>
The strains will be attempted to be isolated from pig waste. The resistance of these organisms to oxytetracycline and sulphamezathine and tylosin will be sought. The resistant strains will then be used to model storage and spread of wastes in different conditions. The possibility of transfer of resistance to salmonellae is also being studied. The fate and behaviour of strains in pig slurry in the farm will also be modelled
Progress: During the early stages of this project, it was found by examination of antibiotic resistant bacterial isolates from the pigs kept at Askham Bryan Farm, that there were bacteria in the pig faeces which possessed transferable tetracycline resistance. Transfer of tetracycline resistance was observed from these indigenous bacterial strains to each of three recipient strains (E. coli DB1, Salmonella enterica var. Typhimurium 19R689, S. Typhimurium 48R626) under highly artificial laboratory conditions (e.g. high nutrient availability, optimal growth temperature, no competing microflora).
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In the work described in this report, laboratory and field studies were performed to determine whether the recipient strains could acquire tetracycline resistance from the indigenous manure or soil microflora under natural conditions, through in situ experiments on a farm and in vitro experiments in the laboratory.
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In the in vitro experiments, microcosms containing manure, and soil-amended manure were inoculated with E. coli and S. Typhimurium recipient strains, and incubated at various temperatures. In situ experiments were performed with stored manure, and manure spread on to field lysimeters.
When the recipient strains were introduced into manure and stored in situ on farm, or spread onto soil in the field lysimeters, or incubated in the laboratory microcosms, no tetracycline-resistant recombinants were ever observed. Thus, no transfer of antibiotic resistance took place with any observable frequency.
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In the stored manure, survival of the recipient strains was observed over several weeks, especially over the winter months. Different persistence patterns were observed, depending upon the season of inoculation / storage. This was probably due to an influence of temperature, and this was reflected in the behaviour of the recipient strains in the laboratory microcosms, where the strains survived better at lower temperatures than at higher. This was possibly due to more vigorous growth of the indigenous manure microflora at these temperatures, resulting in outcompetition of the introduced strains for nutrients.
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It was observed that the recipient strains survived longer at the higher temperatures in the manure-amended soil microcosms than in the microcosms which contained manure only. In the field lysimeters there was a rapid die-off of the recipient strains, but survivors persisted, albeit at very low numbers, from inoculation in Spring 2002 through to at least early Spring 2003. This may have been due to a protective effect of the sandy clay soil used in the lysimeters.
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The overall conclusion from this series of studies is, that although transferable antibiotic resistance exists amongst bacterial strains in pig manure, transfer of antibiotic resistance does not occur in the environment of stored and spread manure, at least at any observable frequency.
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