The interface between antibiotic resistance and virulence in staphylococcus aureus
thesisposted on 27.02.2017 by Cameron, David Robert
In order to distinguish essays and pre-prints from academic theses, we have a separate category. These are often much longer text based documents than a paper.
Staphylococcus aureus is an extremely versatile bacterial pathogen. The organism colonises a significant proportion of the population, and for the most part, causes no problems. However, when given the opportunity, S. aureus can cause devastating infections such as bacteraemia, osteomyelitis, septic arthritis and endocarditis to name a few. Its clinical success is compounded by its remarkable capacity to develop antibiotic resistance. In fact, S. aureus has developed resistance mechanisms to combat virtually all antibiotics in the clinician’s arsenal. Reduced susceptibility to antibiotics considered ‘last-line’ such as vancomycin and daptomycin, has further complicated the treatment of invasive S. aureus infection. The genetic correlates for resistance to vancomycin and daptomycin are incompletely defined. Intriguingly, a number of the mechanisms described to date also appear to impact on S. aureus virulence. As such, the major focus of this thesis is twofold: 1) to define the pathogenic consequences of reduced antibiotic susceptibility in clinical S. aureus isolates; and 2) to describe novel genetic mechanisms that contribute to the interface between antibiotic susceptibility and virulence in this pathogen. In order to understand the pathogenic costs associated with reduced antibiotic susceptibility, we compared the virulence of multiple antibiotic-exposed clinical strains with that of their antibiotic naïve progenitors using a murine bacteraemia model. Strains with reduced susceptibility to vancomycin were significantly and consistently attenuated for virulence, however they showed a remarkable capacity to persist within the host. Similarly, virulence attenuation was observed for strains resistant to daptomycin. We employed next-generation whole genome sequencing and comparative genomics, identifying multiple gene mutations between paired isolates that appeared to impact upon susceptibility and virulence phenotypes. The first gene targeted was stp1, encoding a serine/threonine phosphatase. Deletion of stp1 led to an increase in MIC for vancomycin from 1.5 μg/mL to 3.0 μg/mL and this increase was found to be mediated by increased cell wall biosynthesis. Transcriptomic analysis revealed down-regulation of a number of virulence factors, and this was reflected by virulence attenuation for the stp1 deletion strain in vivo. Taken together, Stp1 was found to be critical to the interface between antibiotic susceptibility and pathogenicity in S. aureus. The second molecular system targeted in this thesis was the WalKR two-component system, and more specifically, its putative regulatory proteins YycH and YycI. Deletion of yycH and/or yycI resulted in an increase in MIC for vancomycin into the non-susceptible range (>2 μg/mL) and this phenotype was returned to wild-type levels upon gene complementation for each. YycH, YycI and WalK appeared to exist in a ternary protein complex and mutations associated with VISA were found to disrupt this interaction. Along with the physical interaction, removal of YycH and YycI impacted on known WalKR phenotypes - including autolysis - suggesting a regulatory role for the proteins. Using RNA-seq we observed down-regulation of multiple WalKR regulated genes including autolysins, suggesting YycH and YycI have a positive effect on the WalKR regulon, as opposed to a negative effect observed in other bacterial species. In addition, the deletion strains showed altered expression of key virulence factors (including surface proteins, complement inhibitors and polysaccharide capsule), which likely impact on host-pathogen interactions.