Evolution of multidrug resistance during Staphylococcus aureus infection involves the essential cell wall biosynthesis regulator WalKR

The glycopeptide antibiotic vancomycin is the mainstay of treatment for serious methicillin-resistant Staphylococcus aureus (MRSA) infections. The emergence of S. aureus strains with reduced vancomycin susceptibility is thus a major antimicrobial resistance issue, further compounded by the co-non-susceptibility to daptomycin, a new class of lipopeptide antibiotic used for treating extensively-resistant Gram-positive bacteria. Here we have sequenced, fully assembled and annotated a representative Australasian strain of the locally dominant multi-locus sequence type 239 MRSA, and then used this reference genome to perform detailed high throughput comparative genomics with sequences derived from five clinical pairs of vancomycin-susceptible (VSSA) and vancomycin-intermediate S. aureus (VISA); each pair isolated from patients before and after vancomycin treatment failure. These comparisons revealed a frequent pattern of mutation among the VISA strains within the essential walKR two-component regulatory locus involved in control of cell wall metabolism. Bi-directional allelic exchange experiments in VSSA and VISA strains confirmed that single nucleotide substitutions within either walK or walR are sufficient to confer intermediate vancomycin resistance and daptomycin non-susceptibility. The single nucleotide changes also lead to the characteristic VISA phenotypes of cell-wall thickening and down regulation of virulence mechanisms, including expression of the quorum sensing system agr, biofilm formation, but enhanced surface capsule expression. These experiments highlight the dramatic impacts of single mutations that arise during persistent S. aureus infections and show for the first time the key role played by walKR to increase drug resistance and alter the virulence potential of this major pathogen.