New Insight into Daptomycin Bioavailability and Localization in Staphylococcus aureus Biofilms by Dynamic Fluorescence Imaging

Abstract

Staphylococcus aureus is one of the most frequent pathogens responsible for biofilm-associated infections (BAI), and the choice of antibiotics to treat these infections remains a challenge for the medical community. In particular, daptomycin has been reported to fail against implant-associated S. aureus infections in clinical practice, while its association with rifampin remains a good candidate for BAI treatment. To improve our understanding of such resistance/tolerance toward daptomycin, we took advantage of the dynamic fluorescence imaging tools (time-lapse imaging and fluorescence recovery after photobleaching [FRAP]) to locally and accurately assess the antibiotic diffusion reaction in methicillin-susceptible and methicillin-resistant S. aureus biofilms. To provide a realistic representation of daptomycin action, we optimized an in vitro model built on the basis of our recently published in vivo mouse model of prosthetic vascular graft infections. We demonstrated that at therapeutic concentrations, daptomycin was inefficient in eradicating biofilms, while the matrix was not a shield to antibiotic diffusion and to its interaction with its bacterial target. In the presence of rifampin, daptomycin was still present in the vicinity of the bacterial cells, allowing prevention of the emergence of rifampin-resistant mutants. Conclusions derived from this study strongly suggest that S. aureus biofilm resistance/tolerance toward daptomycin may be more likely to be related to a physiological change involving structural modifications of the membrane, which is a strain-dependent process.

FIG 1

Absorption and fluorescence spectra of daptomycin (20 μg/ml) alone and combined with rifampin (20 μg/ml). The excitation wavelength was 360 nm.

FIG 2

FRAP acquisitions for BODIPY-FL-daptomycin inside S. aureus biofilms. (a) Kymogram representation (x,t) of FRAP acquisitions. The line along which the kymogram was done is 38 μm. (b) Typical fluorescence recovery curves representative of six different zones for each condition: BODIPY-FL-daptomycin inside biofilms (black) and inside TSBpc without biofilm (gray). The kymogram and fluorescence recovery curve presented here are the ones obtained for MSSA ATCC 27217 biofilms, since they were representative of the data obtained for the other strains in the presence or absence of rifampin.

FIG 3

Fluorescence imaging of BODIPY-FL-daptomycin (green channel) and FM4-64 (red channel) in S. aureus biofilms. Merged images are also shown. In the top panels (Without proteins), the surrounding medium is an aqueous NaCl (150 mM) solution supplemented with calcium ions (50 mg/liter). In the bottom panels (With proteins), the surrounding medium is TSB enriched with proteins (36 g/liter) and calcium ions (50 mg/liter). Only images of MSSA ATCC 27217 biofilms are represented, since they were representative of all biofilms visualized for other strains in the presence or absence of rifampin.

FIG 4

(a) Visualization of MSSA ATCC 27217 and MRSA BCB8 biofilms using 3D reconstruction to observe biofilm thickness. Images were collected without any drug exposure (control) and after 72-h exposure to unlabeled daptomycin (20 μg/ml) alone and in association with rifampin (20 μg/ml). Dead cells were stained red with propidium iodide, and all bacteria were stained green with Syto9. The acquisition was performed on the whole biofilm thickness with an axial displacement of 1 μm. The dimensions of the images are 82 by 82 μm². The mean thickness values of the biofilms over time (from 24 to 72 h) are written in white on each image. Bar, 20 μm. (b) Percentage of dead cells over time calculated from three series of biofilm images. The biofilms were not treated with daptomycin (control) or were treated with daptomycin (20 μg/ml) alone or combined with rifampin (20 μg/ml). The values are means ± standard deviations (error bars).

FIG 5

Time-kill curves of daptomycin (20 μg/ml) alone or combined or with rifampin (20 μg/ml) against MSSA ATCC 27217 and MRSA BCB8 biofilms. The values are means ± standard deviations (error bars).

FIG 6

Number of rifampin-resistant mutants determined in MSSA ATCC 27217 biofilms counted on rifampin-containing TSA plates. The biofilms were treated with rifampin alone (20 μg/ml) or with the daptomycin-rifampin combination (20 μg/ml for both antibiotics). Above each bar is shown the percentage of rifampin-resistant mutants in S. aureus biofilms among the total bacterial population. Error bars represent the standard deviations.