title: Rose bengal uptake by E. faecalis and F. nucleatum and light-mediated antibacterial activity measured by flow cytometry.
abstract: Antibacterial photodynamic therapy (aPDT) using rose bengal (RB) and blue-light kills bacteria through the production of reactive oxygen derivates. However, the interaction mechanism of RB with bacterial cells remains unclear. This study investigated the uptake efficiency and the antibacterial activity of blue light-activated RB against Enterococcus faecalis and Fusobacterium nucleatum. Spectrophotometry and epifluorescence microscopy were used to evaluate binding of RB to bacteria. The antibacterial activity of RB after various irradiation times was assessed by flow cytometry in combination with cell sorting. Uptake of RB increased in a concentration dependent manner in both strains although E. faecalis displayed higher uptake values. RB appeared to bind specific sites located at the cellular poles of E. faecalis and at regular intervals along F. nucleatum. Blue-light irradiation of samples incubated with RB significantly reduced bacterial viability. After incubation with 10μM RB and 240s irradiation, only 0.01% (±0.01%) of E. faecalis cells and 0.03% (±0.03%) of F. nucleatum survived after treatment. This study indicated that RB can bind to E. faecalis and F. nucleatum in a sufficient amount to elicit effective aPDT. Epifluorescence microscopy showed a yet-unreported property of RB binding to bacterial membranes. Flow cytometry allowed the detection of bacteria with damaged membranes that were unable to form colonies on agars after cell sorting.
authors.affiliation: Endodontics Unit, Section of Dental Medicine, University of Geneva, 19 rue Barthelemy Menn, CH-1205 Geneva, Switzerland. Electronic address: Daniel.Manoil@unige.ch.
authors.forename: Daniel
authors.initials: D
authors.lastname: Manoil
authors.affiliation: Endodontics Unit, Section of Dental Medicine, University of Geneva, 19 rue Barthelemy Menn, CH-1205 Geneva, Switzerland. Electronic address: Anna.Filieri@unige.ch.
authors.forename: Anna
authors.initials: A
authors.lastname: Filieri
authors.affiliation: Service of Infectious Diseases, University Hospitals of Geneva, 24, rue Micheli-du-Crest, CH-1205 Geneva, Switzerland. Electronic address: Jacques.Schrenzel@unige.ch.
authors.forename: Jacques
authors.initials: J
authors.lastname: Schrenzel
authors.affiliation: Endodontics Unit, Section of Dental Medicine, University of Geneva, 19 rue Barthelemy Menn, CH-1205 Geneva, Switzerland. Electronic address: Serge.Bouillaguet@unige.ch.
authors.forename: Serge
authors.initials: S
authors.lastname: Bouillaguet
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D001692:Biological Transport / Q000528:radiation effects; D013293:Enterococcus faecalis / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000528:radiation effects*; D005434:Flow Cytometry; D016967:Fusobacterium nucleatum / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000528:radiation effects*; D008027:Light; D010778:Photochemotherapy; D017319:Photosensitizing Agents / Q000378:metabolism / Q000494:pharmacology; D012395:Rose Bengal / Q000378:metabolism* / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D017319:Photosensitizing Agents; D012395:Rose Bengal
keywords: Antibacterial photodynamic therapy; Bacteria; Flow cytometry; LIVE/DEAD BacLight; Photosensitizer uptake; Rose bengal