Use of antistaphylococcal beta-lactams to increase daptomycin activity in eradicating persistent bacteremia due to methicillin-resistant Staphylococcus aureus: role of enhanced daptomycin binding

Abstract

We used daptomycin plus antistaphylococcal β-lactams (ASBL) to clear refractory MRSA bacteremia. In vitro studies showed enhanced daptomycin bactericidal activity, increased membrane daptomycin binding, and decrease in positive surface charge induced by ASBLs against daptomycin nonsusceptible MRSA. Addition of ASBLs to daptomycin may be of benefit in refractory MRSA bacteremia. (Although the official designation is "daptomycin nonsusceptiblity," we will use the term "daptomycin-resistance" in this paper for facility of presentation.).

Figures

Figure 1.

One representative experiment of kill curves of isogenic MRSA strains from case 3 as follows: daptomycin 10 mg/L (diamond); daptomycin 10 mg/L plus oxacillin 20 mg/L (square); oxacillin 20 mg/L (triangle); growth control (X). Panel A shows a representative kill curve assay using DAP-susceptible parental strain, and panel B shows a similar analysis against a subsequent DAP-R-VISA isolate that emerged on therapy. Note enhanced DAP killing in the presence of oxacillin for the VISA strain, even though the organism remained highly resistant to ASBLs and showed minimal growth inhibition by oxacillin 20 mg/L alone.

Figure 2.

Incorporation of fluorescently-labelled DAP by DAP-R-VISA grown in the presence or absence of nafcillin (case 3). The strain was labeled with 16mg/L bodipy-labelled DAP (green; A, B, D, E) for 10 minutes at 37°C after growth to log phase in either antibiotic-free LB broth (A–C) or in LB broth containing nafcillin 40 mg/L (D–F). Cells were stained with FM 4–64 (red; A, B, D, E) and DAPI (blue; B & E). Scale bar equals 1 micron. Panels C and F show a quantitative comparison of DAP-bodipy incorporation, demonstrating increased DAP labeling in the presence of nafcillin (yellow-green surface deposits). Three dimensional graphs show the pixel intensities for DAP-bodipy fluorescence for cells grown without (C) or with (F) 40 mg/L nafcillin.

Figure 3.

Surface charge evaluation using fluorescein isothiocyanate (FITC)-labeled cationic poly-L-lysine (PLL) binding to the staphylococcal surface of initial MRSA and subsequent VISA-DAP resistant isolate from case 3 determined after growth in Mueller-Hinton broth with or without oxacillin 40 mg/L. Units are expressed in relative fluorescence units, with higher fluorescence indicative of decrease in net positive charge. Note the significantly increase in PLL binding by VISA when grown in oxacillin compared with growth in antibiotic-free media (P = .00002). Also noteworthy is the decreased PLL binding accompanying VISA as compared with the parent MRSA strain.