Phenylalanine-Arginine--Naphthylamide Enhances the Photobactericidal Effect of Methylene Blue on Pseudomonas aeruginosa

Objective: To investigate the effectiveness, dosing sequence, concentration, and mechanism of antimicrobial photodynamic inactivation (aPDI) using methylene blue (MB) plus phenylalanine-arginine-beta-naphthylamide (PA beta N) 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) PA beta N (10-100 mu g/mL) was combined with MB (1-20 mu M) in different sequences (PA beta N-first, PA beta N+MB, PA beta N-after). Colony-forming units were then determined by serial dilution.Results: Using MB 10 mu M plus 10 J/cm(2), the killing effect of MB-aPDI on P. aeruginosa increased first and then decreased with longer incubation time. The killing effect of MB+PA beta N-aPDI on P. aeruginosa was better than that of MB-aPDI (p < 0.05) by up to 2 logs. PA beta N-first had the best killing effect, whereas PA beta N-after had the worst killing effect. The killing effect increased with PA beta N concentration and at 100 mu g/mL reached 5.1 logs.Conclusions: The EPI PA beta N 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.