Cleary, David William (2012) Detection of microbial taxa in complex communities : impacts of relative abundance, gene transfer and persistence of target DNA. PhD thesis, University of Warwick.

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Abstract

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.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QR Microbiology
Library of Congress Subject Headings (LCSH):	Microbial ecology, Microbial genetics, Genetic transformation, Biology -- Classification, DNA, Ralstonia solanacearum
Official Date:	April 2012
Dates:	Date Event April 2012 Submitted
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-; Gaze, William H.; Bishop, Alistair H.
Extent:	xxxii, 284 leaves : illustrations, charts.
Language:	eng

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