Phenylalanine-Arginine-b-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-b-naphthylamide (PAbN) 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) PAbN (10–100 lg/mL) was combined with MB (1–20 lM) in different sequences (PAbN-first, PAbN+MB, PAbN-after). Colony-forming units were then determined by serial dilution. Results: Using MB 10 lM 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+PAbN-aPDI on P. aeruginosa was better than that of MB-aPDI (p < 0.05) by up to 2 logs. PAbN-first had the best killing effect, whereas PAbN-after had the worst killing effect. The killing effect increased with PAbN concentration and at 100 lg/mL reached 5.1 logs. Conclusions: The EPI PAbN 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.