Synergy Mechanisms of Daptomycin-Fosfomycin Combinations in Daptomycin-Susceptible and -Resistant Methicillin-Resistant Staphylococcus aureus: In Vitro, Ex Vivo, and In Vivo Metrics

Abstract

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 is not fully understood. The current study explored 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 combinations of DAP plus Fosfo (DAP+Fosfo) (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 < 0.05), (ii) quantity (P < 0.0001) and localization of cell membrane cardiolipin (CL), (iii) DAP surface binding, and (iv) membrane fluidity (P < 0.05). Furthermore, preconditioning this strain pair to DAP with or without Fosfo (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.

Keywords: MRSA; Staphylococcus; combination; daptomycin; fosfomycin.

FIG 1

Sublethal DAP+/−Fosfo (1× MIC of each antibiotic) killing of DAP-S/DAP-R MRSA strains in vitro. Arrows indicate the enhanced bactericidal synergistic killing impact of combination of DAP and Fosfo versus those of the single antibiotics.

FIG 2

DAP MICs of DAP-S/DAP-R strains serially passaged in DAP+Fosfo versus DAP alone in vitro. (A) DAP+Fosfo prevented selection of DAP-R; (B) DAP+Fosfo did not resensitize DAP-R CB185 to the DAP-S phenotype but prevented further MIC increases.

FIG 3

Fosfo facilitates synergistic killing significantly with DAP against DAP-S/DAP-R MRSA strains in an ex vivo IE model within simulated IE vegetations. Simulated model doses were DAP at 6 mg/kg every 24 h and/or Fosfo at 4 g every 8 h. *, P ≤ 0.05 for DAP+Fosfo versus the single antibiotics in both the DAP-S and DAP-R strains. Arrows highlight the increased bactericidal synergistic killing impact of combination of DAP and Fosfo versus those of the single antibiotics.

FIG 4

Exposure to DAP, Fosfo, and a combination of DAP and Fosfo at 0.5× MIC to DAP-S (CB1483) and DAP-R (CB185) MRSA strains impacts HDP (i.e., LL-37) susceptibility.

FIG 5

Surface charge of DAP-S CB1483/DAP-R CB185 S. aureus strains following exposure to 0.5× Fosfo MIC. *, P ≤ 0.05, and **, P ≤ 0.01, versus Fosfo-untreated strains. More residual cytochrome c present in the supernatant equates to a more positively charged surface.

FIG 6

(A) Quantification of BODIPY-DAP binding; (B) BODIPY-DAP binding localization by confocal microscopy in the DAP-S/DAP-R strain pair with Fosfo treatment. *, P < 0.001 in Fosfo-treated versus untreated organisms. Yellow arrows indicate areas of high BODIPY-DAP binding.

FIG 7

NAO fluorescence intensity (A) and NAO localization by microscopy (B) of DAP-S/DAP-R strain pair following subinhibitory Fosfo exposure. Green arrows indicate areas of high NAO localization.

FIG 8

CM fluidity of DAP-S CB1483/DAP-R CB185 S. aureus strains following exposure to 0.5× Fosfo MIC. *, P ≤ 0.05, and **, P ≤ 0.001, versus Fosfo-untreated strains. Lower PI values indicate higher CM fluidity.