The Combined Photosensitizers in Antimicrobial Photodynamic Therapy: The Case of Methylene Blue and Photodithazine Against Klebsiella pneumoniae.

Photodynamic therapy (PDT) is a promising antimicrobial strategy whose efficacy depends largely on the photosensitizers (PSs) used. While conventional PDT relies on a single PS, recent studies suggest that combining different PSs may improve outcomes by introducing complementary mechanisms. However, such combinations also add complexity, as timing, composition, and PS interactions must be considered alongside bacterial structures, uptake pathways, and light dosimetry. This study investigated the effects of PSs, methylene blue (MB), Photodithazine (PDZ), and their combinations on the PDT of Gram-negative bacterium Klebsiella pneumoniae. MB-mediated PDT demonstrated greater antibacterial effectiveness than PDZ-PDT. The combination of MB and PDZ produced varying results. When applied simultaneously, PDZ dose-dependently decreased MB's antibacterial activity. Sequential treatment with PDZ followed by MB showed only slight antagonism compared to MB alone, while the reverse order (MB → PDZ) nearly abolished MB's activity. Since both PSs are activated at the same wavelength (660 nm), their combined use was not additive. Photobleaching was performed on individuals and combined PSs to compare inactivation results with changes in chemical properties under red light (660 nm). This study highlights the limitations of using two photosensitizers together in antimicrobial photodynamic therapy and emphasizes the need for further optimization of combination protocols.