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Investigations into the Effects of Antibacterial Compounds on the Phenotype, Susceptibility and Virulence of Staphylococcus aureus

Bazaid, Abdulrahman

[Thesis]. Manchester, UK: The University of Manchester; 2019.

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Abstract

Staphylococcus aureus can form small colony variants (SCVs), a phenotypic variant characterised by the formation of colonies with markedly reduced diameter, following exposure to antimicrobials, including the antiseptic triclosan and the antibiotic gentamicin. Whilst SCVs have been associated with altered antimicrobial susceptibility and growth rate, their clinical and environmental significance remains unclear. The aim of the work described in this doctoral thesis was to study the effects of adaptation of S. aureus to antimicrobials by exploring phenotypic characteristics and antibiotic susceptibility of SCVs and to elucidate associated mechanisms through genomic and proteomic analyses. S. aureus was repeatedly exposed to triclosan (11 strains) and gentamicin (5 strains) using a previously validated antimicrobial gradient plating system, over ten passages (P10), and a further ten passages were conducted in the absence of antimicrobial exposure (PX10). Antimicrobial susceptibility, keratinocyte invasion, pathogenicity and biofilm formation were assessed, and potential genomic changes were determined using whole genome sequencing. SCVs exhibiting attenuated relative pathogenicity, in both cell cultures and larvae, were subjected to further proteomic analysis. Following triclosan exposure, 9/11 strains showed a significant increase in triclosan MIC and 4 of these strains formed SCVs, which reverted to pre-exposure colony size when supplemented with fatty acids. SCVs exhibited increased susceptibility to the majority of antibiotics tested, including gentamicin and trimethoprim-sulfamethoxazole, attenuated virulence in both pathogenicity models, reduced planktonic growth rate and impaired biofilm formation. All SCVs exhibited point mutations in the triclosan target gene fabI, with 2 SCVs showing mutations in both fabI and fabD. The fabI enzyme and fatty acid synthesis proteins were up-regulated in SCVs, while proteins associated with virulence were down-regulated. With respect to the antibiotic gentamicin, 3/5 strains formed SCVs upon repeated gentamicin exposure, with one of these SCVs exhibiting decreased gentamicin susceptibility. Relative pathogenicity and haemolytic activity were reversibly reduced in gentamicin-induced SCVs. In summary, sustained fabI mutation and reversion of colony size following fatty acid supplementation in triclosan-induced SCVs indicate fatty acid auxotrophy in these SCVs and lack of association between SCV and fabI mutation. SCVs induced by triclosan and gentamicin exhibited altered phenotype and reduced virulence, possibly due to down-regulation of virulence proteins in the case of triclosan, suggesting a potential link between SCVs and reduced virulence.