Evidence for a Role for Coagulase-Negative Staphylococci and their Biofilms in the Historic and Future Evolution of Staphylococcus Aureus Including MRSA
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Rates of methicillin-resistant Staphylococcus aureus (MRSA) infections in Ireland are decreasing since 2006. However, MRSA and methicillin-susceptible S. aureus (MSSA) remain a major cause of healthcare-associated infections in Europe. Recent evolutionary trends indicate that MRSA lineages associated with infections in the community are causing infections in the healthcare setting with increasing frequency.
It is therefore important to investigate potential reservoirs in the community that may contribute to MRSA and S. aureus evolution. The methicillin resistance gene, mecA, is located within a transferable genetic element, staphylococcal Cassette Chromosome (SCC), referred to as SCCmec, which is also frequently found in coagulase-negative staphylococci (CoNS) including Staphylococcus epidermidis. S. epidermidis is the predominant human skin colonizer and clinically, its most important virulence trait is biofilm production. Indirect evidence suggests that MRSA clones arose from genetic transfer of SCCmec from CoNS to MSSA. Adjacent to SCCmec, other antibiotic resistance genes, most notably fusC, encoding fusidic acid resistance, have also been found associated with SCC. It is thought that CoNS may be a reservoir of these antibiotic-resistances and the close proximity of these staphylococcal species in the human nasopharynx provides opportunity for such gene acquisitions.
In this thesis, we describe the investigation of community-associated colonizing staphylococcal species as a reservoir that may underpin the evolution of S. aureus. In Chapter 3, DNA microarray analysis of nasal colonising staphylococcal isolates revealed a greater frequency of the antimicrobial resistance genes mecA, fusC, fusB, ileS2, qacA/qacC and the arginine catabolite mobile element (ACME) among colonising CoNS compared to S. aureus among 137 S. aureus-carriers with no previous healthcare exposure, identified from 444 medical students in their pre-clinical years. Among S. aureus carriers, MRSA and methicillin-resistant CoNS (MRCoNS) rates were 6.5% (9/137) and 13.1 % (18/137), respectively. Simultaneous carriage of MRSA and MRCoNS was found in 1/137 students studied. The clonal lineages of colonizing S. aureus included several clonal complexes (CC)s associated with the acquisition of SCCmec.
In Chapter 4 an in-vitro study is described in which the efficiency of three mechanisms of horizontal gene transfer (transformation, transduction and conjugation) were investigated when applied in a staphylococcal biofilm model, for the transfer of clinically relevant antibiotic resistance genes identified in Chapter 3. Within a staphylococcal biofilm of the S. aureus strain RN4220, transduction was the most efficient genetic transfer mechanism, followed by conjugation and transformation. Transfer of the SCCmec-fus element, identified among S. aureus carriers, was successful in this model when exposed to sub-inhibitory concentrations of fusidic acid.
In Chapter 5, the further detailed analysis of a subset of isolates from S. aureus-carriers (MRSA, MSSA and methicillin-susceptible S. epidermidis (MRSE)) using a combination of DNA microarray and whole genome sequence (WGS) analyses, is described. Using this approach, a high homology of SCC elements and fusC among S. aureus and S. epidermidis isolates from healthy people was revealed including 100% sequence similarity among the SCCfus elements.
Overall, investigation of the staphylococcal species among 30.8% of medical students positive for S. aureus, prior to their exposure to the clinical environment, indicates a low rate of MRSA but the presence of several lineages into which SCCmec is capable of inserting. Furthermore, the finding of the composite element SCCfus carried in association with SCCmec V in the CC1 background, is concerning as it suggests the recent expansion of this clone in the community possibly against the selective pressure of un-regulated fusidic acid use. The stable transfer potential of this element with its associated phenotypic antibiotic resistance traits, determined in an in-vitro biofilm model, particularly by transduction further supports this evolutionary trend.
Data presented support the hypothesis that the close proximity and co-evolution of CoNS and S. aureus in the human host, even in the absence of confirmed antibiotic pressure, underpinned by horizontal gene transfer events within staphylococcal biofilms plays an important and often underestimated role in antimicrobial resistance evolution in S. aureus.
Further epidemiological investigation and follow up of the co-evolution of these species in the transition from community to clinical setting may provide further insights to explain the recent but sustained success of community strains in the healthcare setting.