Objective: To investigate the effectiveness, dosing sequence, concentration, and mechanism of antimicrobial photodynamic inactivation (aPDI) using methylene blue (MB) plus phenylalanine-arginine-β-naphthylamide (PAβN) against Pseudomonas aeruginosa. Methods: P. aeruginosa bacterial suspension was incubated with MB for different times (5-240 min), and then, 10 J/cm2 red light was irradiated. The efflux pump inhibitor (EPI) PAβN (10-100 μg/mL) was combined with MB (1-20 μM) in different sequences (PAβN-first, PAβN+MB, PAβN-after). Colony-forming units were then determined by serial dilution. Results: Using MB 10 μM plus 10 J/cm2, the killing effect of MB-aPDI on P. aeruginosa increased first and then decreased with longer incubation time. The killing effect of MB+PAβN-aPDI on P. aeruginosa was better than that of MB-aPDI (p < 0.05) by up to 2 logs. PAβN-first had the best killing effect, whereas PAβN-after had the worst killing effect. The killing effect increased with PAβN concentration and at 100 μg/mL reached 5.1 logs. Conclusions: The EPI PAβN enhanced the bactericidal effect of MB-aPDI on P. aeruginosa, especially when added before MB. It is proposed that MB is a substrate of the resistance-nodulation-division family efflux pump.
Keywords: Pseudomonas aeruginosa; antimicrobial photodynamic inactivation; efflux pump inhibitor; methylene blue; phenylalanine-arginine-β-naphthylamide; resistance-nodulation-division family efflux pump.