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

Abstract

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 mprF 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 mprF (L826F) only in the DAP monotherapy arm. Interestingly, passage in FOF alone or DAP + FOF prevented the emergence of this mprF 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 lrgAB and sdrE and increased the expression level of fosB. In a rabbit infective endocarditis (IE) model, the DAP-R CB185 strain treated with DAP +/- FOF showed significantly reduced lrgB 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.

Keywords: MRSA; combination therapy; daptomycin; fosfomycin; transcriptomics.

Figure 1

Venn diagram of genes altered ≥ 2-fold by DAP + FOF vs. DAP or FOF alone exposures in DAP-S/DAP-R MRSA strain pair. Red and black fonts indicate whether expression was increased (black) or decreased (red) by DAP + FOF exposure.

Figure 2

In vitro lrgB gene expression was significantly decreased in the DAP-S CB1483 and DAP-R CB185 MRSA strain pairs treated either with FOF alone or DAP + FOF vs. DAP alone treatment. * p < 0.05; ** p < 0.01 vs. DAP exposure.

Figure 3

In vivo lrgB gene expression was decreased in rabbit endocarditis vegetations of DAP-R CB185 treated either with FOF alone or DAP + FOF or control (untreated) vs. DAP alone. ** p < 0.01; *** p < 0.001 vs. DAP exposure.