Heterogeneity of genetic pathways toward daptomycin nonsusceptibility in Staphylococcus aureus determined by adjunctive antibiotics

Background: Daptomycin is increasingly used in combination with other antibiotics to enhance antimicrobial efficacy and/or to mitigate the emergence of daptomycin nonsusceptibility (DNS). This study uses clinical MRSA strains in which DNS emerged on therapy to examine the influence of antibiotic combinations on development of mutations in specific genes (mprF, rpoBC, dltA, cls2, yycFG) previously associated with DNS. Methods: Whole genome sequencing was undertaken for bacteria obtained following 28 days of in vitro exposure to daptomycin +/- adjunctive clarithromycin, linezolid, oxacillin or trimethoprim-sulfamethoxazole and were compared to the progenitor isolate. Results: Addition of oxacillin to media containing daptomycin prevented emergence of mprF mutation but did not prevent rpoBC mutation (p < 0.01). These isolates maintained susceptibility to daptomycin during the combined exposure (median MIC 1 mg/L). Daptomycin plus clarithromycin or linezolid resulted in low-level (1.5-8 mg/L) and high-level (12-96 mg/L) DNS, respectively, and did not prevent mprF mutation. However, these same combinations prevented rpoBC mutation. Daptomycin alone or combined with linezolid or trimethoprim-sulfamethoxazole resulted in high-level DNS and mutations in mprF plus rpoBC, cls2 and yycFG. Conclusion: Combining daptomycin with different antimicrobials alters the mutational space available for DNS development, thereby favoring the development of predictable collateral susceptibilities.