Phenylalanine-Arginine-β-Naphthylamide Enhances the Photobactericidal Effect of Methylene Blue on <i>Pseudomonas aeruginosa</i>

<b><i>Objective:</i></b> 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 <i>Pseudomonas aeruginosa</i>. <b><i>Methods:</i></b> <i>P. aeruginosa</i> 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. <b><i>Results:</i></b> Using MB 10 μM plus 10 J/cm², the killing effect of MB-aPDI on <i>P. aeruginosa</i> increased first and then decreased with longer incubation time. The killing effect of MB+PAβN-aPDI on <i>P. aeruginosa</i> was better than that of MB-aPDI (<i>p</i> < 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. <b><i>Conclusions:</i></b> The EPI PAβN enhanced the bactericidal effect of MB-aPDI on <i>P. aeruginosa</i>, especially when added before MB. It is proposed that MB is a substrate of the resistance-nodulation-division family efflux pump.