Failures in clinical treatment of Staphylococcus aureus Infection with daptomycin are associated with alterations in surface charge, membrane phospholipid asymmetry, and drug binding

Abstract

Increasingly frequent reports have described the in vivo loss of daptomycin susceptibility in association with clinical treatment failures. The mechanism(s) of daptomycin resistance is not well understood. We studied an isogenic set of Staphylococcus aureus isolates from the bloodstream of a daptomycin-treated patient with recalcitrant endocarditis in which serial strains exhibited decreasing susceptibility to daptomycin. Since daptomycin is a membrane-targeting lipopeptide, we compared a number of membrane parameters in the initial blood isolate (parental) with those in subsequent daptomycin-resistant strains obtained during treatment. In comparison to the parental strain, resistant isolates demonstrated (i) enhanced membrane fluidity, (ii) increased translocation of the positively charged phospholipid lysyl-phosphotidylglycerol to the outer membrane leaflet, (iii) increased net positive surface charge (P < 0.05 versus the parental strain), (iv) reduced susceptibility to daptomycin-induced depolarization, permeabilization, and autolysis (P < 0.05 versus the parental strain), (v) significantly lower surface binding of daptomycin (P < 0.05 versus the parental strain), and (vi) increased cross-resistance to the cationic antimicrobial host defense peptides human neutrophil peptide 1 (hNP-1) and thrombin-induced platelet microbicidal protein 1 (tPMP-1). These data link distinct changes in membrane structure and function with in vivo development of daptomycin resistance in S. aureus. Moreover, the cross-resistance to hNP-1 and tPMP-1 may also impact the capacity of these daptomycin-resistant organisms to be cleared from sites of infection, particularly endovascular foci.

FIG. 1.

Population analyses of study strains upon exposure to a range of vancomycin (A) or daptomycin (B) concentrations (conc.). These data represent the means (± SD) for two separate assays. See the text for details.

FIG. 2.

Membrane depolarization by daptomycin, RP-1, and Triton X-100 by flow cytometry at 60-min exposures. These data represent the means for three separate assays. See the text for details.

FIG. 3.

Membrane permeabilization of the study strains by sublethal concentrations of two study antimicrobial peptides (tPMP-1 [A] and hNP-1 [B]) and daptomycin (C) by using the calcein release assay. These data represent the means (± SD) for three separate assays. See the text for details.

FIG. 4.

Autolysis of the study strains, as determined by the change in OD₆₀₀ over time for Triton X-100 (A), daptomycin (B), and vancomycin (C). Data represent the means for at least two separate runs. Data are depicted as the percent change from the time zero (T₀ [preexposure]) value, set at 100%. See the text for details.