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

Abstract

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.

Extracellular DNA Impedes the Transport of Vancomycin in Staphylococcus epidermidis Biofilms Preexposed to Subinhibitory Concentrations of Vancomycin

“…In agreement with the work of Daddi Oubekka and colleagues (40), our results show that in control, antibiotic-free biofilms, Number of cells obtained from 72-h S. epidermidis biofilms treated with 2 mg/ml vancomycin, 16 g/ml of exogenous S. epidermidis DNA, and a solution of DNA and vancomycin (at the same concentrations) preincubated prior to addition to the biofilm. All biofilms were exposed to the treatments for 2 h. Control biofilms were exposed to PBS treatment.…”

“…Therefore, we investigated if the altered matrix composition of sub-MIC-vancomycin-treated S. epidermidis ATCC 35984 biofilms could affect the penetration of vancomycin. We used confocal microscopy and a fluorescent vancomycin derivative (BODIPY FL-vancomycin) for transport studies (39,40) to show that the transport of BODIPY FL-vancomycin is significantly inhibited in sub-MIC-vancomycin-treated S. epidermidis biofilms. Moreover, a correlation between the inhibition of vancomycin transport and an increase in eDNA concentration within the biofilm matrix of sub-MIC-vancomycin-treated biofilms was observed.…”

Microbiological problems and biofilms associated with Mycobacterium chimaera in heater–cooler units used for cardiopulmonary bypass

“…When not in use the 3T HCUs are stored at ambient temperatures allowing bacterial survival and proliferation of the biofilm in the stagnant water particularly on pipe materials and inside the water tanks ( Figure 2 and 5). Biofilms produce extensive extracellular polysaccharides (EPS) which presents a physicochemical barrier to the penetration of disinfectants 58 . As such the tolerance of biofilms is thought to be associated with the quenching of disinfectants as they pass through the EPS and due to the presence of slow growing or persistor cells that have reduced susceptibility to many disinfectants and other antimicrobials [59][60][61] .…”

Alpha-lipoic acid-loaded nanostructured lipid carrier: sustained release and biocompatibility to HaCaT cellsin vitro

“…Therefore, time-lapse imaging system has been allowed to be a remarkable device to visualize cells and simultaneously follow the movements indirectly through a time progression visualization of cell behavior over a long period of time. The technique has not only shown significant improvements in consequence of the implementation in terms of spatial resolution and sensitivity, but it has also presented noninvasive circumstances of investigations (Oubekka et al, 2012).…”