Genetic Correlates of Synergy Mechanisms of Daptomycin Plus Fosfomycin in Daptomycin-Susceptible and -Resistant Methicillin-Resistant Staphylococcus aureus (MRSA)

This study elucidates potential genetic determinants and mechanisms involved in the synergistic effects of daptomycin (DAP) + fosfomycin (FOF) combination therapy. Among 33 clinically derived DAP-susceptible (S)/DAP-resistant (R) isogenic strain pairs, mutations in the <i>mprF</i> gene occurred in 30/33 DAP-R strains, including polymorphisms of L826F (33%) or T345A/L/I (15%). Strain variants of DAP-S CB1483 serially passaged in vitro for 10 days in DAP +/- FOF identified a key non-synonymous mutation in <i>mprF</i> (L826F) only in the DAP monotherapy arm. Interestingly, passage in FOF alone or DAP + FOF prevented the emergence of this <i>mprF</i> mutation following 10-day passage. This L826F mprF polymorphism, associated with a "gain-in-function" phenotype, exhibited increased amounts of lysyl-phosphatidylglycerol (L-PG) in the cell membrane (CM). Transcriptomics revealed a relatively modest number (~10) of distinct genes that were significantly up- or downregulated (≥2 log fold) in both the DAP-S and DAP-R strain pairs upon DAP + FOF exposures (vs. DAP or FOF alone). Of note, DAP + FOF decreased expression of <i>lrgAB</i> and <i>sdrE</i> and increased the expression level of <i>fosB</i>. In a rabbit infective endocarditis (IE) model, the DAP-R CB185 strain treated with DAP +/- FOF showed significantly reduced <i>lrgB</i> expression in vegetations compared with DAP treatment alone. Overall, these findings indicate that DAP + FOF therapy impacts MRSA through multiple specific mechanisms, enhancing bacterial clearance.