Genomic divergence events in methicillin resistant 'Staphylococcus aureus' (MRSA): an in silico approach to investigate the evolutionary epidemiology of 'S. aureus'

Staphylococci commonly form a part of the normal micro-flora for humans and a wide variety of animals but also represent a group of clinically significantly pathogens. However, it has also been recognised that for bacteria to become pathogens, they must have the ability to accomplish infection and consequently cause disease in the host. This is known as pathogenicity and virulence is a degree of the pathogenicity observed. Virulence factors are proteins made through the expression of virulence factor genes produced by a pathogen which contribute to the pathogenicity, and literature research identified a variety of virulence factor genes. The predominant pathogenic staphylococcal species is Staphylococcus aureus, with methicillin-resistant Staphylococcus aureus (MRSA) recognised as significant in both veterinary and medical contexts. The role of the S. aureus in human disease is clear; however, the organism is also well recognised as a cause of animal disease with animal specific strains such as ST398 becoming increasingly common. The approach taken to determine how similar or different the S. aureus strains are at genomic level as well as investigate the virulence factors that may be under positive selection was undertaken by employing the wealth of nucleotide sequences data available for a number methicillin-resistant and methicillin-sensitive S. aureus strains. This helped to establish if there was genomic variation between strains and investigate the potential relationship of these alterations in host/ niche preferences. In total, 53 strains of S. aureus were investigated; including, three novel isolates of S. aureus from human patients that were provided by Prof. Valerie Edward-Jones (Manchester Metropolitan University) and 3 novel isolates from various farm animals (cattle, chicken & swine) provided by Dr Patrick Butaye (The Veterinary & Agrochemical Research Centre, Belgium). The novel isolates were sequenced with Ion Torrent Next-Generation sequencing methodologies. Literature research has classified the strains of S. aureus as either hospital-associated (HA), community-associated (CA) or livestockassociated (LA); these classifications were further investigated. Virulence factor gene sequences, if present, were isolated from the S. aureus strains and a comparative analysis was carried out; this includes initial MAUVE analysis and more detailed maximum-likelihood phylogenetic analysis, using MEGA. A bioinformatics software called DnaSP was used for the analysis of polymorphism from aligned virulence factor nucleotide sequences and the dN/dS (synonymous vs nonsynonymous mutations) ratio (¤ë) was investigated to infer the evolutionary mechanisms that may be acting on the genes. Haplotype networks were also created to investigate the S. aureus population to determine the diversity of the population, determine any geographical relationship and further investigate the classification HA, CA & LA that has been given in literature. This study concluded that the presence of functional PantonValentine Leukocidin (PVL) a bi-component virulence factor encoded by the lukS-PV and lukF-PV genes, typically found in highly virulent CA S. aureus strains, were detected in these virulent strains, but surprisingly also in the novel livestock-associated strains. This study also concluded that the population of S. aureus was very diverse and a geographical relationship was not observed. It was further concluded that the selective pressures on individual virulence factor genes causes S. aureus to evolve, with a number of genes recognised as undergoing positive selection; lukS-PV, lekF-PV, chp, sspA, vwb, lip, lip1, nucA, hysA, hysA1, aur, fnb, ebpS, bbp, entE, seb and tst. It was also suggested that the 3 classifications denoted in the literature may possibly be artificial and at a molecular level the strains are in fact very similar and do not represent genuine divisions. This has also been recently published by Bal et al. (2016) and this thesis supports their hypothesis; they describe the blurring of each of the 3 epidemiological groups (HA, CA & LA), demonstrating the limited relevance of this classification.