A Small Amount of Singlet Oxygen Generated via Excited Methylene Blue by Photodynamic Therapy Induces the Sterilization of Enterococcus faecalis

Abstract

IntroductionThe present study aimed to clarify the relationship between the amount of singlet oxygen (1O2) generated from excited methylene blue (MB) and the bactericidal effects on Enterococcus faecalis. MethodsA diode laser was used as the laser irradiation source (λ = 660 nm, 200 mW). The laser irradiation time periods were 300, 600, and 900 seconds. In experiment 1, the amount of 1O2 generated from each concentration (0.0001%–1.0%) of excited MB was examined by using electron spin resonance to determine the optimal concentration of MB. In experiment 2, the bactericidal effects of 1O2 on E. faecalis were examined. Experimental groups were with laser irradiation, L(+); without laser irradiation, L(−); including MB, M(+); and not including MB, M(−). These were combined to form 4 groups: L(+)M(+), L(+)M(−), L(−)M(+), and L(−)M(−). After treatment, E. faecalis was incubated for 48 hours at 37°C, and the bactericidal effects of 1O2 on E. faecalis were determined on the basis of the number of colony-forming units per milliliter. ResultsThe largest amount of 1O2 was generated from 0.01% excited MB. After 300, 600, and 900 seconds of irradiation, 35.2, 87.2, and 117.1 μmol/L 1O2 were detected, respectively. In groupL(+)M(+),colony-forming units per milliliter of E. faecalis dramatically decreased depending on the amount of 1O2 generated. No other groups showed any bactericidal effects. ConclusionsOur findings suggest that 0.001%–0.01% of MB is the most effective range for generating 1O2 during the application of antimicrobial photodynamic therapy. At least 35.2 μmol/L generated 1O2 was necessary to achieve the sterilization of E. faecalis.