Evolution of Daptomycin Resistance in Coagulase-Negative Staphylococci Involves Mutations of the Essential Two-Component Regulator WalKR

Coagulase-negative staphylococci (CoNS) represent one of the major causes of health care- and medical device-associated infections. Emerging antimicrobial resistance has complicated the treatment of systemic infections caused by CoNS. Here, we describe the prevalence of antimicrobial resistance in clinical CoNS strains from a tertiary care hospital over a 4-year period, and we observed a significant increase in resistance to daptomycin. Notably, Staphylococcus capitis accounted for the majority of these daptomycin-resistant (DAP-R) CoNS. To further investigate the mechanisms of daptomycin resistance in CoNS, daptomycin-susceptible clinical strains of S. capitis and Staphylococcus epidermidis underwent in vitro daptomycin exposure to generate DAP-R CoNS mutants. Unlike that seen with Staphylococcus aureus, alteration of cell surface charge was not observed in the DAP-R CoNS strains, but biofilm formation was compromised. Whole-genome sequencing analysis of the DAP-R CoNS strains identified single nucleotide polymorphisms (SNPs) in walKR, the essential two-component regulatory system controlling cell wall biogenesis. PCR and sequencing of walK and walR from 17 DAP-R CoNS clinical isolates identified seven nonsynonymous mutations. The results were confirmed by the recreation of the walK SNP in S. epidermidis, which resulted in reduced susceptibility to daptomycin and vancomycin. This study highlights the significance of CoNS in evolving daptomycin resistance and showed that walKR is shared among the staphylococcal species and is involved in antibiotic resistance development. Notably, we did not observe mutations in genes responsible for phospholipid biosynthesis or an altered cell surface charge, suggesting that reduced daptomycin susceptibility in CoNS may emerge in a fashion distinct from that in S. aureus.