Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Oct;41(10):569-575.
doi: 10.1089/photob.2023.0005.

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

Lixia He  1   2 , Shanlin Yang  1   2 , Weijun Xuan  3   4 , Xiumei Zhen  1 , Qi Qi  1   2 , Yongqi Qi  1   2 , Qingxiang Li  1   2 , Meixia Du  1   2 , Michael R Hamblin  5 , Liyi Huang  1
Affiliations

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

Lixia He et al. Photobiomodul Photomed Laser Surg. 2023 Oct.

Abstract

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.

PubMed Disclaimer

Conflict of interest statement

No competing financial interests exist.

Figures

FIG. 1.
FIG. 1.
The antimicrobial effect of aPDI on Pseudomonas aeruginosa at different incubation times with MB. MB-aPDI group: MB (10 μM) plus 10 J/cm2. MB group: MB (10 μM) without light. Light group: 10 J/cm2 without MB. Bacteria of each group was incubated with or without MB for 5–240 min. aPDI, antimicrobial photodynamic inactivation; CFUs, colony-forming units; MB, methylene blue.
FIG. 2.
FIG. 2.
Potentiation of aPDI on Pseudomonas aeruginosa at different MB concentrations. MB-aPDI group: Different concentrations of MB (0–20 μM) plus 10 J/cm2. MB+PAβN-aPDI group: PAβN (25 μg/mL) then MB (0–20 μM) plus 10 J/cm2. MB+PAβN group: PAβN (25 μg/mL) then MB (0–20 μM) without illumination. PAβN, phenylalanine-arginine-β-naphthylamide.
FIG. 3.
FIG. 3.
Effects of MB + PAβN-aPDI on Pseudomonas aeruginosa with different administration sequences. PAβN-first group: P. aeruginosa incubated with PAβN (25 μg/mL) first and then followed by MB (10 or 20 μM) and 10 J/cm2. PAβN-in group: P. aeruginosa incubated with PAβN (25 μg/mL) and MB (10 or 20 μM) together and then 10 J/cm2. PAβN-after group: P. aeruginosa incubated with MB (10 or 20 μM) first and then followed by PAβN (25 μg/mL) and 10 J/cm2. (nsp > 0.05, *p < 0.05, **p < 0.01.)
FIG. 4.
FIG. 4.
Effect of MB-aPDI with varying PAβN concentrations. MB+PAβN-aPDI group: Pseudomonas aeruginosa incubated with PAβN (0–100 μg/mL) first and followed by MB (10 μM) and then 10 J/cm2 red light. PAβN+L(+) group: P. aeruginosa incubated with PAβN (0–100 μg/mL) plus 10 J/cm2 red light. PAβN+L(−) group: P. aeruginosa incubated with PAβN (0–100 μg/mL) without light.
FIG. 5.
FIG. 5.
Antibacterial effects of MB+PAβN-aPDI on two clinical CRPA isolates. MB-aPDI group: MB (10 μM) plus 10 J/cm2. MB+PAβN-aPDI group: PAβN (100 μg/mL) first then MB (10 μM), plus 10 J/cm2; MB+PAβN group: PAβN (100 μg/mL) first then MB (10 μM), without light. MB group: MB 10 μM without light. (nsp > 0.05, *p < 0.05, ***p < 0.001.) CRPA, carbapenem-resistant Pseudomonas aeruginosa.
FIG. 6.
FIG. 6.
RNO absorption curves. Control group: dd H2O. MB-aPDI group: MB (1 mM). MB+ PAβN-aPDI group: MB (1 mM) plus PAβN (100 μg/mL). PAβN group: PAβN (100 μg/mL). Each group was received illumination (0–8 min). RNO, p-nitrosodimethylaniline.
See this image and copyright information in PMC

References

    1. Tuon FF, Dantas LR, Suss PH, et al. . Pathogenesis of the Pseudomonas aeruginosa biofilm: A review. Pathogens 2022;11(3):13–47; doi: 10.3390/pathogens11030300. - DOI - PMC - PubMed
    1. Shariati A, Azimi T, Ardebili A, et al. . Insertional inactivation of oprD in carbapenem-resistant Pseudomonas aeruginosa strains isolated from burn patients in Tehran, Iran. New Microbes New Infect 2018;21:75–80; doi: 10.1016/j.nmni.2017.10.013. - DOI - PMC - PubMed
    1. Pang Z, Raudonis R, Glick BR, et al. . Antibiotic resistance in Pseudomonas aeruginosa: Mechanisms and alternative therapeutic strategies. Biotechnol Adv 2019;37(1):177–192; doi: 10.1016/j.biotechadv.2018.11.013. - DOI - PubMed
    1. Sun J, Deng Z, Yan A. Bacterial multidrug efflux pumps: Mechanisms, physiology and pharmacological exploitations. Biochem Biophys Res Commun 2014;453(2):254–267; doi: 10.1016/j.bbrc.2014.05.090. - DOI - PubMed
    1. Askoura M, Mattawa W, Abujamel T, et al. . Efflux pump inhibitors (EPIs) as new antimicrobial agents against Pseudomonas aeruginosa. Libyan J Med 2011;6(1); doi: 10.3402/ljm.v6i0.5870. - DOI - PMC - PubMed

MeSH terms

LinkOut - more resources