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Title: Detection of microbial taxa in complex communities : impacts of relative abundance, gene transfer and persistence of target DNA
Author: Cleary, David William
ISNI:       0000 0004 2748 9088
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2012
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The aim of this study was to understand the ecological interactions of B. pseudomallei, a highly pathogenic potential biological warfare agent, in both soils and bio-aerosols. This study examined the impact of DNA persistence on its detection in environmental samples. The potential for members of the Burkholderiaceae to use persistent extracellular DNA in the process of natural transformation was also determined. Lastly, a comparative analysis of methods using 16S rRNA analysis and metagenomics was also undertaken to evaluate their utility for identification of pathogens in complex samples. Soil microcosms were used to determine the impacts of soil mineralogy on DNA persistence using real-time PCR. This study showed that the presence of clay minerals with contrasting adsorption affinities for DNA resulted in significantly different rates of DNA decay in soil. The capacity for Burkholderia sp. to undergo natural transformation was determined through the use of both chromosomal allelic rescue and nonhomologous plasmid uptake strategies. Transformation was suggested as a result of observations of non-homologous plasmid uptake in B. multivorans 13010. However further work is required in this area. The performance of 16S rRNA sequencing using assays targeting the V1-3 and V4-6 variable regions was assessed using soil samples taken from one location that had undergone contrasting application strategies of veterinary antibiotics. This approach was shown to be able to discern subtle shifts in the relative abundances of certain microbial taxa in response to antibiotic application. Outcomes here support previous observations regarding the increased prevalence of mobile genetic elements (integrons) that harbour antibiotic resistance determinants. For bio-aerosols, samples were acquired from regions of known endemicity of B. pseudomallei (N. Australia) and both 16S rRNA and metagenomic analysis methods were used to determine the fluctuations in microbial diversity and the prevalence of this pathogenic organism. The fluctuations of bio-aerosol microbial diversity and in particular B. pseudomallei, within wet and dry seasons of Northern Australia were identifiable using a combination of 16S rRNA and total community DNA metagenomic analyses. The importance of robust bioinformatic analysis is highlighted.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QR Microbiology