Correlative time-resolved fluorescence microscopy to assess antibiotic diffusion-reaction in biofilms

S S. Daddi Oubekka (1, 2) , Romain Briandet (3) , M-P M.-P. Fontaine-Aupart (1, 2) , K K. Steenkeste (1, 2)

1 ISMO - Institut des Sciences Moléculaires d'Orsay
2 Centre de Photonique Biomédicale - Fédération LUMAT FR 2764
3 MICALIS - MICrobiologie de l'ALImentation au Service de la Santé

The 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.

Mots clés: staphylococcus epidermidis biofilm; correlation spectroscopy; temporal patterns; aureus; vancomycin; recovery

Domaines: Sciences agricoles

S S. Daddi Oubekka, Romain Briandet, M-P M.-P. Fontaine-Aupart, K K. Steenkeste. Correlative time-resolved fluorescence microscopy to assess antibiotic diffusion-reaction in biofilms. Antimicrobial Agents and Chemotherapy, 2012, 56 (6), pp.3349-3358. ⟨10.1128/AAC.00216-12⟩. ⟨hal-01000631⟩