Naghra, H
(2016)
Application of whole genome sequencing to the study of Pseudomonas aeruginosa.
PhD thesis, University of Nottingham.
Abstract
The reduction in cost and increase in throughput of whole genome sequencing (WGS) technologies, and the advent of benchtop WGS instruments such as the Illumina MiSeq, means that WGS is no longer restricted to large genome centres and consortia. The number of microbial genomes in public repositories is ever increasing due to the availability of WGS technologies to research groups, with individual genera having their own dedicated genome databases. Pseudomonas aeruginosa is an opportunistic pathogen and a major cause of healthcare associated infection in immunocompromised individuals and cystic fibrosis (CF) patients. The first complete genome sequence of a P. aeruginosa strain was that of the most commonly studied laboratory strain P. aeruginosa PAO1, sequenced in 2000. It was found in a study, published in 2010, that there were differences between the chromosomal sequences of two isolates of P. aeruginosa PAO1 originating from two different public strain collections, as well as differences compared to the reference sequence. It was therefore proposed that P. aeruginosa PAO1 exists as variable sublines in strain collections, whose differences included a 2.2~Mb chromosomal inversion and a prophage insertion compared to the reference sequence, as well as single nucleotide polymorphisms (SNPs) and short insertions and deletions (INDELs) which are unique to individual sublines. Since the current genomic reference sequence is based on one of these variable sublines, this study aims to deduce the sequence of the original P. aeruginosa Strain 1 (PAO) from which all the PAO1 sublines are derived, using sequence information from several laboratory sublines and from P. aeruginosa strains PAO2 and PAO3, which were directly derived from the PAO progenitor strain. This could be used as a more universal reference which is representative of a range of PAO1 sublines, to which they are all more closely related.
Although most genetic studies of P. aeruginosa are carried out in PAO1, this strain is in no way the archetype for this diverse species. As whole genomes of other P. aeruginosa strains became available, comparative genomics revealed that PAO1 shared only 80% of its genome with other strains. P. aeruginosa is a highly ubiquitous organism which is able to adapt to a variety of environmental niches, as well as to human and animal hosts. This is thought to be related to the large genome size and genetic complexity of the organism, with 10% of its genes devoted to regulatory functions. Mutations in quorum sensing (QS) genes have been reported in multiple studies of host adapted P. aeruginosa isolates. QS is the mechanism by which a bacterium adapts from the lifestyle of an individual cell to a multicellular community via the regulation of specific target genes, and has been shown to regulate key virulence factors. A study of 49 clinical isolates, collected from various wound sites from distinct inpatients and outpatients at the Queen's Medical Centre in Nottingham within a few days of one another, revealed that this set of isolates displayed a diverse range of QS phenotypes. Two of these isolates were identified as producing high levels of N-acylhomoserine lactone (AHL) QS signal molecules (QSSMs) and low levels of alkyl quinolone (AQ) QSSMs, which had not previously been found in clinical isolates of P. aeruginosa, at the time of the study. WGS of these 49 clinical isolates was carried out in this study, where the aim was to; (1) determine the genomic diversity of these isolates, and relate this to previously sequenced P. aeruginosa strains, and (2) analyse the key QS genotypes of these isolates, and attempt to relate these to the phenotypes observed, in particular for the two isolates which were AHL-proficient and AQ-deficient.
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