Remodelling of pSK1 Family Plasmids and Enhanced Chlorhexidine Tolerance in Methicillin-ResistantStaphylococcus aureus

Abstract

26Staphylococcus aureus is a significant human pathogen whose evolution and adaptation has been 27 shaped in part by mobile genetic elements (MGEs), facilitating global spread of extensive 28 antimicrobial resistance. However, our understanding of the evolutionary dynamics surrounding 29 MGEs is incomplete, in particular how changes in the structure of multidrug-resistant (MDR) 30 plasmids may influence important staphylococcal phenotypes. Here, we undertook a population-31 and functional-genomics study of 212 methicillin-resistant S. aureus (MRSA) ST239 isolates 32 collected over 32 years to explore the evolution of the pSK1 family of MDR plasmids, illustrating 33 how these plasmids have co-evolved with and contributed to the successful adaptation of this 34 persistent MRSA lineage. Using complete genomes and temporal phylogenomics we reconstructed 35 the evolution of the pSK1 family lineage from its emergence in the late 1970s, with multiple 36 structural variants arising. Plasmid maintenance and stability was linked to IS256-and IS257-37 mediated chromosomal integration and disruption of plasmid replication machinery. Overlaying 38 genomic comparisons with phenotypic susceptibility data for gentamicin and chlorhexidine, it 39 appeared that pSK1 has contributed to enhanced resistance in ST239 MRSA through two 40 mechanisms: (i) acquisition of plasmid-borne resistance mechanisms increasing rates of 41 gentamicin resistance and reduced chlorhexidine susceptibility, and (ii) changes in plasmid 42 configuration linked with further enhancement of chlorhexidine tolerance. While the exact 43 mechanism of enhanced tolerance remains elusive, this research has uncovered a clear 44 evolutionary response of ST239 MRSA to chlorhexidine, one which may contribute to the ongoing 45 persistence and adaptation of this lineage within healthcare institutions.46 4 Importance 47The biocide chlorhexidine is fundamental to infection control practices which prevent nosocomial 48 infection and it is highly effective for decolonisation of S. aureus from human skin. There have 49 been increasing reports of staphylococcal populations evolving tolerance to chlorhexidine, 50 suggesting that the increasing use and reliance on this biocide may provide a significant selection 51 pressure influencing the evolution of staphylococcal populations. Chlorhexidine tolerance in 52 S. aureus is commonly enabled by the acquisition of an efflux pump, however alternative 53 mechanisms influencing tolerance are poorly understood. In this study we demonstrate a 54 previously unrecognised phenomenon, plasmid structural remodelling, by which S. aureus may 55 developed enhanced chlorhexidine tolerance. Further, we highlight the importance of undertaking 56 a detailed exploration of the evolutionary dynamics surrounding mobile genetic elements, which 57 contribute immensely to the evolution of microbial species and provide here a framework for how 58 such an analysis can be performed.59 60Mobile genetic elements (MGEs) play a central role in microbial e...