Antimicrobial Photodynamic Inactivation Using Riboflavin 5′-Phosphate and a 450 nm Diode Laser: An In Vitro Dose-Optimisation Study

Background: Rising antifungal and antibiotic resistance among Candida species, Staphylococcus aureus, and Enterococcus faecalis has renewed interest in antimicrobial photodynamic therapy (aPDT) as a resistance-independent strategy. Riboflavin 5′-phosphate is a biocompatible, blue-light-activated photosensitizer, but standardized dosing across fungal and bacterial targets is lacking. Objective: The aim of this study was to systematically optimize pre-irradiation incubation time, photosensitizer volume, irradiation time, and laser power for riboflavin 5′-phosphate aPDT (450 nm diode laser) against C. albicans, C. glabrata, C. krusei, S. aureus, and E. faecalis, and compare species susceptibility under optimized conditions. Methods: ATCC strains were treated with 0.1% riboflavin 5′-phosphate across four groups (photodynamic, photosensitizer-only, laser-only, control) in a staged design optimizing incubation (1–30 min), photosensitizer volume (50–150 µL), irradiation time (10–120 s), and power (50–400 mW). Viable counts (CFU/mL) were quantified. Results: Significant reductions occurred only with combined light-plus-photosensitizer treatment. Optimal parameters were 15 min incubation, 100 µL photosensitizer for Candida spp. (50 µL for bacteria), and 120 s at 400 mW, though C. albicans and C. krusei plateaued by 60 s. Maximum reductions were modest: 53.5% (C. albicans), 46.7% (S. aureus), 37.9% (C. glabrata), 35.9% (C. krusei), and 26.5% (E. faecalis), all below 1 log10. A significant light × photosensitizer interaction, confirming photodynamic specificity, was seen for C. albicans, C. glabrata, and S. aureus, but not C. krusei or E. faecalis. Conclusions: Riboflavin 5′-phosphate aPDT under 450 nm light produces reproducible, dose-dependent, species-specific antimicrobial activity, best suited as an adjunctive rather than stand-alone therapy pending biofilm and in vivo validation.