Killing Mechanism by Blue Light of Bacteria Within Multi-species Biofilm

Objectives: Oral bacteria are organized in multi-species biofilm. Previous studies showed a selective inhibitory effect of blue light on Gram-negative anaerobic bacteria. The aim of the study is to characterize the selective phototoxic mechanism of blue light on bacteria within multispecies biofilm.
Methods: Multispecies biofilm was constructed with S.sanguis, A.naeslundii, P.gingivalis and F.nucleatum on hydroxyapatite. Mature biofilm was exposed to blue light for 2min (146 J/cm²) while control were left unexposed. A novel method for quantification and viability evaluation of specific bacteria within the biofilms was established using florescent staining and flow cytometry. Multispecies biofilm structure and viability was additionally evaluated using live/dead staining and confocal microscopy. Mechanism of bacterial killing was investigated on mono-species biofilms as direct phototoxic effect and indirect-paracrine effect.
Results: Blue light led to reduction in the percentage of P.gingivalis and F.nucleatum in the biofilm to 13% (compared with 23% in the unexposed biofilm). Also, F.nucleatum and P.gingivalis viability in multi-species biofilm was reduced after light exposure. Surprisingly, only P.gingivalis was affected by the exposure to blue light in a mono-species biofilm. An indirect toxic effect on F.nucleatum biofilm was achieved by exposing it to supernatant of P.gingivalis biofilm that was previously exposed to blue light. This effect was eliminated when oxygen species scavengers were added to the supernatant.
Conclusions: Blue light showed a selective phototoxic effect on the growth of P.gingivalis and F.nucleatum in the multi-species biofilm. The toxic effect was induced directly on P.gingivalis and via paracrine path on F.nucleatum, which is mediated by ROS. The phenomenon of selective phototoxic effect in multi-species biofilms may lead to a novel approach for “replacement therapy” aiming to establish a less periopathogenic biofilm.