Synergy Mechanisms of Daptomycin-Fosfomycin Combinations in Daptomycin-Susceptible and -Resistant Methicillin-Resistant S. aureus: In vitro, Ex vivo and In vivo Metrics.

Increased usage of daptomycin (DAP) for methicillin-resistant Staphylococcus aureus (MRSA) infections has led to emergence of DAP-resistant (DAP-R) strains, resulting in treatment failures. DAP-fosfomycin (Fosfo) combinations are synergistically active against MRSA, although the mechanism(s) of this interaction are not fully understood. The current study explores four unique, but likely interrelated activities of DAP-Fosfo combinations: i) synergistic killing; ii) prevention of evolution of DAP-R; iii) resensitization of already DAP-R subpopulations to a DAP-susceptible (DAP-S) phenotype; and iv) perturbations of specific cell envelope phenotypes known to correlate with DAP-R in MRSA. Using an isogenic DAP-S (CB1483) / DAP-R (CB185) clinical MRSA strain-pair, we demonstrated that DAP + Fosfo combinations: i) enhanced killing of both strains in vitro and ex vivo; ii) increased target tissue clearances of the DAP-R strain in an in vivo model of experimental infective endocarditis (IE); iii) prevented emergence of DAP-R in the DAP-S parental strain both in vitro and ex vivo; and iv) resensitized the DAP-R strain to a DAP-S phenotype ex vivo. Phenotypically, following exposure to sub-MIC Fosfo, the DAP-S/ DAP-R strain-pair exhibited distinct modifications in: i) net positive surface charge (p<0.0001); ii) quantity (p<0.0001) and localization of cell membrane cardiolipin (CL); iii) DAP surface binding; and iv) membrane fluidity (p <0.0001). Furthermore, pre-conditioning to this strain-pair to DAP +/- Fosfo sensitized these organisms to killing by the human host defense peptide, LL37. These data underscore the notion that DAP-Fosfo combinations can impact MRSA clearances within multiple microenvironments, likely based on specific phenotypic adaptations.