Genetic and Physiological Effects of Photo-chemical Treatment on S.mutans Biofilm

Objectives: To explore the effect of non-coherent light exposure (wavelengths, 400-500 nm), combined with hydrogen peroxide (H₂O₂) on gene expression of Streptococcus mutans in biofilm, the effect of such treatment on bacterial physiology and the mechanism of phototoxicity. Methods: Biofilms of S.mutans were constructed in the presence of sucrose. Samples were exposed to the visible light for 34 or 68 J/cm² in the presence and absence of H₂O₂. cDNA was created from the total RNA extracted from each bacterial sample. Specific gene expression, associated with biofilm formation, was measured using real time RT-PCR. The antibacterial effect of the procedure was assessed by the number of colony forming units of each sample when compared with that of the control. ATP levels of the samples where measured at different times after the treatment. Confocal Laser Scanning Microscopy (CSLM) was conducted to explore the effect at the different layers of the biofilm. In order to explore the mechanism by which light may affect bacteria, samples were exposed to light also in the presence of reactive oxygen species (ROS) scavengers. Results: The above treatment caused an up-regulation of genes associated with biofilm formation. In all tested light exposures we found that in the presence of H₂O₂ at concentrations lower than the MIC, a synergic bactericidal affect was observed. CSLM showed a bactericidal effect that began in the middle layers of the biofilm and progressed for six hoers after exposure. In the presence of scavengers a significant reduction in the bactericidal effect was obtained. Conclusion: Results show that this photo-chemical treatment, at sub-lethal dosages has genomic influence on S.mutans. Affected genes have been associated with biofilm formation. Visible light affects S.mutans by ROS generation, which could explain the synergistic effect between the visible light and H₂O₂.

Supported in part by the Cabakoff Grant