Cold atmospheric plasma degrades methylene blue and shifts bacterial inactivation during photodynamic therapy.

Photodynamic therapy (PDT) and cold atmospheric plasma can inactivate bacteria via oxidative stress. Here we examined their combined action against Staphylococcus aureus (S. aureus) using methylene blue (MB)-mediated PDT with a red organic light-emitting diode (OLED) and surface dielectric barrier discharge (SDBD). Under individual treatments, S. aureus inactivation by both PDT and SDBD varied with MB concentration and exposure time. In the presence of MB, plasma rapidly degraded MB, and the effect of microbial inactivation was reduced compared with MB-free conditions. When PDT and SDBD were applied simultaneously, bacterial reduction increased from approximately 1.4 log at 10 min to 4.8 log at 20 min, while MB decreased markedly. This suggests that early inactivation was dominated by MB-mediated photodynamic action, whereas later inactivation was sustained by secondary reactive species produced from SDBD after MB depletion. Photolysis modeling suggested that OLED emission did not substantially alter plasma-generated reactive species, supporting a mechanism driven by time-dependent changes in liquid chemistry rather than gas-phase interference. Acute NIH/3T3 screening showed reduced metabolic activity, indicating the need for cytocompatible dose optimization. These results provide a mechanistic basis for time-dependent pathway shifts during combined MB-PDT and SDBD.