Ex vivo killing of Enterococcus faecalis and mixed plaque bacteria in planktonic and biofilm culture by modified photoactivated disinfection

Abstract

To compare the efficacy of conventional and modified photoactivated disinfection (PAD) against Enterococcus faecalis and mixed plaque bacteria in suspension and biofilms. Enterococcus faecalis (four strains) and mixed plaque bacteria from three adult volunteers were suspended in water, added to methylene blue (MB, 15 μmol L⁻¹), MB mixed with 0.5% hydrogen peroxide and 0.05% chlorhexidine (CHX), MB mixed with 0.5% hydrogen peroxide and 0.05% EDTA or MB mixed with 0.05% EDTA and 0.05% CHX and exposed to laser irradiation from 10 s to 5 min. After exposure, samples were taken, serially diluted and grown aerobically and anaerobically on Tryptic Soy Agar plates or on blood agar plates for 24 and 72 h, respectively. For biofilm experiments, E. faecalis and mixed plaque biofilms were grown on sterile hydroxyapatite (HA) discs coated overnight with bovine dermal collagen type I for 3 weeks. After exposure to MB or MB and low concentration of EDTA with either hydrogen peroxide or CHX, the percentage of killed bacteria by PAD was evaluated using viability staining and confocal laser scanning microscope. For statistical analysis, one-way analysis of variance was performed. Conventional PAD killed from 90.76% to 100% E. faecalis for 3 min, but failed to kill all plaque bacteria even after 5 min of laser irradiation. In modified PAD, up to 100% of suspended E. faecalis and mixed plaque bacteria were killed after 1 min and 30 s of irradiation. Up to twenty times more biofilm bacteria were killed by modified PAD than by conventional PAD with 15 μmol L⁻¹ MB (P < 0.001) and up to eight times more than 2% CHX (P < 0.001) and 1% sodium hypochlorite (P < 0.001). Modified PAD was superior to conventional PAD against planktonic and biofilm bacteria.