Antimicrobial mechanisms behind photodynamic effect in the presence of hydrogen peroxide

This study describes the use of methylene blue (MB) plus light (photodynamic inactivation, PDI) in the presence of hydrogen peroxide (H ₂O₂) to kill Staphylococcus aureus, Escherichia coli, and Candida albicans. When H₂O₂ was added to MB plus light there was an increased antimicrobial effect, which could be due to a change in the type of ROS generated or increased microbial uptake of MB. To clarify the mechanism, the production of ROS was investigated in the presence and absence of H₂O₂. It was observed that ROS production was almost inhibited by the presence of H₂O₂ when cells were not present. In addition, experiments using different sequence combinations of MB and H₂O₂ were performed and MB optical properties inside the cell were analyzed. Spectroscopy experiments suggested that the amount of MB was higher inside the cells when H₂O₂ was used before or simultaneously with PDI, and ROS formation inside C. albicans cells confirmed that ROS production is higher in the presence of H₂O₂. Moreover enzymatic reduction of MB by E. coli during photosensitizer uptake to the photochemically inactive leucoMB could be reversed by the oxidative effects of hydrogen peroxide, increasing ROS formation inside the microorganism. Therefore, the combination of a photosensitizer such as MB and H ₂O₂ is an interesting approach to improve PDI efficiency.