Image-based Fluorescence Recovery After Photobleaching (FRAP) to dissect vancomycin diffusion-reaction processes in Staphylococcus aureus biofilms

Antimicrob Agents Chemother

Fontaine-Aupart

et al. 2012

Self Cite

110

eThe failure of antibiotics to inactivate in vivo pathogens organized in biofilms has been shown to trigger chronic infections. In addition to mechanisms involving specific genetic or physiological cell properties, antibiotic sorption and/or reaction with biofilm components may lessen the antibiotic bioavailability and consequently decrease their efficiency. To assess locally and accurately the antibiotic diffusion-reaction, we used for the first time a set of advanced fluorescence microscopic tools (fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and fluorescence lifetime imaging) that offer a spatiotemporal resolution not available with the commonly used time-lapse confocal imaging method. This set of techniques was used to characterize the dynamics of fluorescently labeled vancomycin in biofilms formed by two Staphylococcus aureus human isolates. We demonstrate that, at therapeutic concentrations of vancomycin, the biofilm matrix was not an obstacle to the diffusion-reaction of the antibiotic that can reach all cells through the biostructure.

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Correlative Time-Resolved Fluorescence Microscopy To Assess Antibiotic Diffusion-Reaction in Biofilms

Oubekka

Antimicrob Agents Chemother

Fontaine-Aupart

et al. 2012

Self Cite

110

“…Briefly, FRAP is based on a brief excitation of fluorescent molecules by a very intense light source in a user-defined region to irreversibly photobleach their fluorescence. Fluorescence recovery is then probed over time at a low light power in the same photobleached region (22,24). All time-resolved measurements were obtained using the same confocal microscope.…”

Section: Methodsmentioning

confidence: 99%

“…FRAP was used to measure the local diffusion of BODIPY-FL-daptomycin and its interaction with bacteria within biofilms. According to the FRAP principle (22,24,25), if the fluorescently labeled daptomycin molecules are allowed to move freely in the sample, total fluorescence recovery is observed, meaning that the fluorescence is redistributed in the defined region. Conversely, if the fluorescence recovery is not total after the photobleaching, it means that a fraction of molecules is not diffusing freely and thus interacts with its local environment.…”

Section: Susceptibility Testingmentioning

confidence: 99%

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

Boudjemaa

Antimicrob Agents Chemother

Revest

et al. 2016

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. Staphylococcus aureus is a Gram-positive bacterial species shown to be the most frequent cause of biofilm-associated infections (BAI) (1) and one of the major causes of morbidity and mortality in hospitals and communities (2). Unlike planktonic cells, biofilms exhibit specific phenotypic traits allowing them to resist host defenses and antibiotic treatments (3), which frequently leads to chronic infections such as endocarditis, sinusitis, and osteomyelitis and also to implant-associated infections (4).Among the most recent clinically used antibiotics, daptomycin is a cyclic lipopeptide approved for the treatment of serious staphylococcal infections such as bacteremia and implant-related infections (5). Daptomycin is a calcium-dependent antibiotic that acts by insertion into the Gram-positive cytoplasmic membranes where it forms oligomeric pores, causing potassium ion leakage and subsequent membrane depolarization, leading ultimately to cell death (6). As is the case for many antibiotics, daptomycin has been shown to exhibit a significant bactericidal activity against planktonic cells (7-9). However, the eradication of adherent bacteria is rarely achieved despite the large number of in vitro and animal studies in which daptomycin activity was evaluated (10-16). Besides the results of the literature that appear controversial (17), direct comparison between studies is not directly possible, since the biofilm growth and treatment protocols...

Taking Advantage of Fluorescent‐Based Tools to Puzzle out Successes and Failures of Antibiotics to Inactivate Infectious Bacteria

Boudjemaa

Encyclopedia of Analytical Chemistry

Steenkeste

et al. 2017

In microbiology, fluorescence tools have undoubtedly proven their performance to analyze in real‐time, non‐invasively and quantitatively, the structure, composition, processes, and dynamics of microbial communities. Importantly, the available fluorescence‐based methods enable to decipher multiscale events implicated in the failures and successes of antibiotics treatments. The goal of this article is to review the large range of fluorescence techniques and probes and their interest (i) to study bacterial cells viability in vitro and in vivo under antibiotics exposure, (ii) to elucidate the mechanisms of action of these drugs, and (iii) to dissect bacterial mechanisms to resist and tolerate antibiotics action.