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Vancomycin-intermediateStaphylococcus aureus (VISA) strains often arise by mutations in the essential two-component regulatorwalKR ; however their impact onwalKR function has not been definitively established. Here, we investigated 10 MRSA strains recovered serially after exposure of vancomycin-susceptibleS. aureus (VSSA) JKD6009 to simulated human vancomycin dosing regimens (500 mg to 4,000 mg every 12 h) using a 10-day hollow fiber infection model. After continued exposure to the vancomycin regimens, two isolates displayed reduced susceptibility to both vancomycin and daptomycin, developing independent IS256 insertions in thewalKR 5′ untranslated region (5′ UTR). Quantitative reverse transcription-PCR (RT-PCR) revealed a 50% reduction inwalKR gene expression in the IS256 mutants compared to the VSSA parent. Green fluorescent protein (GFP) reporter analysis, promoter mapping, and site-directed mutagenesis confirmed these findings and showed that the IS256 insertions had replaced two SigA-likewalKR promoters with weaker, hybrid promoters. Removal of IS256 reverted the phenotype to VSSA, showing that reduced expression of WalKR did induce the VISA phenotype. Analysis of selected WalKR-regulated autolysins revealed upregulation ofssaA but no change in expression ofsak andsceD in both IS256 mutants. Whole-genome sequencing of the two mutants revealed an additional IS256 insertion withinagrC for one mutant, and we confirmed that this mutation abolishedagr function. These data provide the first substantial analysis ofwalKR promoter function and show that prolonged vancomycin exposure can result in VISA through an IS256 -mediated reduction inwalKR expression; however, the mechanisms by which this occurs remain to be determined.

Decreased Vancomycin Susceptibility in Staphylococcus aureus Caused by IS 256 Tempering of WalKR Expression