Synergistic enhancement of photodynamic inactivation of pathogens using methylene blue, gold nanoparticles, and efflux pump inhibitors

The increasing prevalence of antibiotic resistance in Escherichia coli, particularly mechanisms involving limited permeability and active efflux, highlights the need for alternative antimicrobial strategies. Photodynamic therapy (PDT) employs photosensitizers, light, and molecular oxygen to generate reactive oxygen species (ROS) that damage bacterial cells through multi-target pathways. Methylene blue (MB) is an effective photosensitizer; however, its PDT efficacy against E. coli is often reduced by inefficient intracellular accumulation and efflux-mediated clearance. In this study, gold nanoparticles (AuNPs) synthesized via pulsed laser ablation in liquid (PLAL) were combined with MB and the efflux pump inhibitor INF-55 to enhance PDT performance. AuNPs of varying sizes increased ROS generation through plasmonic interactions with MB, while INF-55 significantly enhanced bacterial photoinactivation without altering ROS production, indicating a mechanism associated with increased intracellular MB retention. Notably, INF-55 potentiation was consistent across different AuNP sizes, supporting a size-independent mode of action. The combined use of MB, AuNPs, and inhibitor enhanced photoinactivation, showing promise for PDT against efflux-competent E. coli.