Assessment of the Impact of Antimicrobial Photodynamic Therapy Using a 635 nm Diode Laser and Toluidine Blue on the Susceptibility of Selected Strains of Candida and Staphylococcus aureus: An In Vitro Study

Yeasts of the genus Candida (C.) and the bacterium Staphylococcus aureus (S. aureus) are among the most common pathogens responsible for infections that are difficult to treat, including those resistant to standard therapy. In recent decades, this has become an increasing clinical problem. In response to the limitations of traditional procedures, antimicrobial photodynamic therapy (aPDT), which combines light, a photosensitizer, and oxygen, is gaining growing interest. The aim of this study was to evaluate the in vitro effectiveness of aPDT using a 635 nm diode laser in combination with toluidine blue O (TBO) against Candida spp. and S. aureus. Reference strains of C. albicans, C. glabrata, C. krusei, and S. aureus were subjected to aPDT. In phase I of this study, the optimal TBO incubation time was assessed with constant laser parameters. In phase II, the impact of the physical parameters of the laser, irradiation time, and output power, was analyzed, with the TBO incubation time set based on the phase I results, to evaluate the degree of microbial reduction (CFU/mL). Statistical analyses were then conducted to assess significance. TBO-mediated aPDT significantly reduced microbial viability, depending on incubation time and laser settings. The minimal effective incubation times were 10 min for Candida spp. and 5 min for S. aureus. The highest pathogen inactivation efficacy was observed at an output power of 400 mW and an irradiation time of 120 s. The use of the photosensitizer or laser alone did not result in significant antimicrobial effects. TBO-mediated aPDT may serve as an effective complement to conventional antimicrobial therapy and, in selected cases (e.g., drug resistance), has the potential to partially or fully replace it. The observed minimal effective incubation times provide a practical baseline, but further statistical comparisons are required to determine whether these durations are truly optimal.