Enterococci are a leading cause of infective endocarditis, bacteraemia and urinary tract infections. Unfortunately, an increasing proportion of these organisms display resistance to front-line antimicrobial agents. The aim of the work described in this thesis was to assess the efficacy of antibiotics against clinical strains of enterococci and evaluate their ability to trigger adaptive mechanisms (stringent response) associated with drug tolerance and virulence. Enterococcus faecalis and Enterococcus faecium isolates grown in planktonic and biofilm states were assessed for susceptibility to vancomycin, rifampicin, linezolid and tigecycline. As a potential avenue to overcome resistance and preserve antibiotic activity, combinations of drugs were evaluated for synergy against sensitive and drug-resistant strains of enterococci using in vitro and in vivo models. Transcriptome analysis of parental E. faecalis OG1RF and mutants defective or void of stringent response activity were examined to determine if this response was induced by exposure to antibiotics and if virulence gene expression was modulated. The majority of enterococci tested planktonically were susceptible to all four antibiotics, while biofilm-associated cells were refractory to killing. Rifampicin in combination with vancomycin, linezolid and tigecycline demonstrated enhanced killing properties against enterococci in planktonic and biofilm growth states compared to killing by individual agents. When tested in vivo, synergistic combinations improved Galleria mellonella survival compared to monotherapy. Exposure of parental E. faeca/is OG1RF to vancomycin and linezolid triggered upregulation of stringent response genes (re/A and re/Q). In contrast, tigecycline did not induce modulation of these genes, which suggests that the stringent response was not activated. Virulence genes associated with biofilm formation were upregulated by all antibiotics during exposure, independent of stringent response induction, while increased modulation of an endocarditis-associated adhesin and proteases were only apparent in linezolid exposed cells. Comparison of stringent response deletion mutants of E. faecalis OG1RF revealed that expression of re/A was necessary for an antibiotic induced stringent response, while re/A and re/Q worked synergistically to enhance virulence gene modulation. Combination therapy is widely used when difficult-to-treat infections are identified, though the clinical outcome is not always predictable. Additionally, antibiotics have properties other than those related to their therapeutic use. Specifically, they act as signalling molecules that provoke adaptive responses in bacteria, including modulation of gene expression. The findings of this thesis suggest that tigecyclinebased combinations may prove useful in therapy against enterococci when antibiotic resistance is detected. Additionally, tigecycline did not appear to trigger a stringent response thorough modulation of associated genes, while vancomycin and linezolid induced expression of stringent response and virulence factor associated genes. The clinical relevance of an active stringent response in E. faecalis has not been fully elucidated, though it may underlie patient treatment failure.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:658615 |
| Date | January 2013 |
| Creators | Skinner, Kirsty Gail |
| Publisher | Glasgow Caledonian University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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