Photodynamic inactivation of microorganisms by protoporphyrin DC combined with pulsed light irradiation

Abstract

There is an increased interest in application of modulated light to achieve more efficient photodynamic inactivation of microorganisms. We report that 405 rati light excitation (45 mW/cm2) modulated at 5 Hz (100 ms pulses; 50 % duty cycle) could improve the photodynamic toxicity of protoporphyrin IX applied against Candida albicans and Staphylococcus aureus species. For experiments, microorganism samples were prepared from overnight cultures which were re-suspended in PBS (1.82 * 10e7 u/ml of C. albicans and 1.8 x 10e9 u/ml of S. aureus) and then incubated with protopoiphyrin IX for 10 min at 50 jiM final concentration. The light excitation was delivered by single optical fiber of the bundle positioned at the top side of the 96-well plate and the rest fibers were used for fluorescence spectra acquisition during photodynamic inactivation. The colony formation assay was employed to assess the microorganism survival in photodynamic activation groups (0.05 x 20 J/cm2; 0.5 x 20 J/cm2; 1 * 20 J/cm2) as well as in light application/or sensitizer application alone groups following 48 h of incubation on Sabouraud dextrose agar at 30 deg C for C. albicans and 18 h on LB-agar for S. aureus at 37 deg C. The highest applied light dose induced significant (p<0.001, Mann-Whitney) decrease of the sensitized microorganisms' viability, resulting up to 0.80 loglO (killing efficacy of 84.4 %) and 2.85 log 10 (killing efficacy of 99.86 %) reduction of C. albicans and S. aureus survival, respectively. The complementary results of PCB- ion binding assay demonstrated the possibility of membrane integrity loss induced by photodynamic effect in situ. However, the enhanced photodynamic killing of S. aureus could be affected by the sensitizer uptake characteristics.[...]