Byrne-Bailey, Kathryne Greta (2006) Bacterial antibiotic resistance and horizontal gene transfer in slurries and slurry amended agricultural soils. PhD thesis, University of Warwick.

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Abstract

A two-year investigation was performed to assess the environmental impact of veterinary medicines released into the environment through the application of pig slurries to agricultural soils. Concern has been growing about the use of antibiotics in livestock husbandry and the possible selection and horizontal transfer of antibiotic resistance genes in bacteria.

The fate and mobility of sulphachloropyridazine, oxytetracycline and tylosin was measured, when added to soils via slurry. Plate counts and bacterial isolations were performed to calculate the numbers of antibiotic resistant culturable bacteria present in the soils before and after the application of slurry, from tylosin-fed pigs amended with sulphachloropyridazine and oxytetracycline. Soil cores were collected in years 1 and 2 after slurry application at days 1,21, 90, 120 and 289 or 240 in year 2. The year 1, day 289 soil cores were also used as the pre-application samples for year 2. In total 583 bacterial isolates were collected from all slurries, soil time points and drainflow samples from non-selective Iso-Sensitest agar plates and selective plates containing a range of concentrations of sulphachloropyridazine, oxytetracycline and tylosin.
The 583 bacterial isolates were screened by PCR for the sulphonamide resistance genes; sull, sulll and su/3 and the intll and intI2 genes for the Class 1 and 2 integrons. PCR screening enabled an assessment of the prevalence and distribution of these mobile genetic elements and their potential and degree of horizontal gene transfer in the slurry and soil environments of this study, where the integrons were localised to a conjugative plasmid. PCR positive for the intll gene were 5.83 % of bacterial isolates and 9.7 % were positive for the intI2 gene. Of the intll positive isolates, 57.56 % were collected from selective plates containing 50 pg/ml tylosin. suggesting a link between tylosin

resistance and Class 1 intégrons. The intll and intI2 positive isolates were identified by 16S rRNA and a number of new bacterial genera encoding intégrons were described, including a number of Psychrobacter spp. These integrase genes were also identified in number of putative Gram positive organisms including Bacillus sp. and Arthrobacter sp., providing evidence of horizontal gene transfer events from the Enterobacteriacae hosts commonly demonstrated to carry Class 1 and 2 intégrons. Furthermore, the transfer of a plasmid with high similarity to that from Corynebacterium glutamicum, encoding a novel Class 1 integron, was demonstrated from an Arthrobacter sp. to Pseudomonas putida and Escherichia coli recipients.

Despite clinical studies confirming the intll and sull genes are commonly located together on a Class 1 intégrons, this environmental study demonstrates no link between the two genes in cultured bacterial isolates from both antibiotic amended slurries and slurry amended soils.

The data presented in this study demonstrated there maybe a pool of antibiotic resistant bacteria, in the soil and pig slurry environments, which was detected both in the presence and absence of a selective pressure. Of the total number of isolates collected in this study, 31.05 % were PCR positive for one of the three known alternative DHPS genes conferring sulphonamide resistance. Of these 181 sul gene containing bacterial isolates (a number of which were identified by 16S rRNA), 12 encoded all three genes; sull, sulll and su!3. These 12 isolates were characterised in detail, including the potential for transfer of the sulphonamide resistance genes. These 12 isolates belonged to the genera of Acinetobacter, Psychrobacter and Bacillus. No correlation was demonstrated between sul carriage and MIC levels for sulphachloropyridazine within the 12 sull, II and 3 positive isolates or within Acinetobacter sp. which were PC'R positive for none, one or more sul genes.

Data is presented for the long-term survival of enteric bacteria in the soil environment as a result of release through slurry applications to the soils. A phenomenon was investigated in which MIC values were reduced through bacterial removal from soils and laboratory repeated subculture but increased on return back into soil microcosms.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QR Microbiology S Agriculture > S Agriculture (General) S Agriculture > SF Animal culture
Library of Congress Subject Headings (LCSH):	Medical wastes -- Environmental aspects, Antibiotics in animal nutrition, Drug resistance in microorganisms, Genetic transformation, Soils -- Analysis, Soils -- Agricultural chemical content
Official Date:	April 2006
Dates:	Date Event April 2006 UNSPECIFIED
Institution:	University of Warwick
Theses Department:	School of Life Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Wellington, E. M. H. (Elizabeth M. H.), 1954- ; Hawkey, P. M. (Peter M.)
Format of File:	pdf
Extent:	xxv, 353 leaves
Language:	eng

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