Efficacy of antimicrobial photodynamic therapy for multidrug resistant Pseudomonas aeruginosa in vitro

Abstract

Objective To investigate the bactericidal effect of toluidine blue O (TBO)-mediated photodynamic antimicrobial therapy (APDT) on multi-drug resistant Pseudomonas aeruginosa (P. aeruginosa). Methods Multi-drug resistant P. aeruginosa isolated from 1 patient with burn wounds was used as study subject, and so was the standard strain from ATCC (No. 27853). The study bacteria were treated with different concentrations of TBO (2.5, 5.0, 7.5 and 10.0 μmol/L) combined with different doses of light (10, 20, 30 and 40 J/cm2, Red light, wavelength of 635±5 nm). While the control bacteria were divided into blank control group, TBO group and light group. The bacterial survival rate was determined by colony counting. Morphological alterations of the bacteria were observed with laser confocal electron microscopy (CLSM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The effect of sub-lethal dose of APDT (2.5 μmol/L TBO, 10 J/cm2 lighting) on minimum inhibitory concentrations (MICs) of several antibiotics were detected, including piperacillin/tazobactam, ceftazidime, cefepime, imipenem and meropenem. The expression levels of drug-resistant genes related to the multi-drug resistant P. aeruginosa were detected using RT-qPCR before and after APDT. Results The antibacterial efficacy of APDT was improved with the increases of TBO concentrations and light dose (P < 0.05). After sub-lethal dose of APDT, the bacteria showed shrinkage, depression and perforation of bacterial cell wall by SEM, and fimbriae disappearance, cell wall discontinuity and decreased cytoplasmic electron density and nucleus mimics disorder by TEM. And the sub-lethal dose treatment resulted in enhanced sensitivity of the multi-drug resistant strain to above antibiotics, and down-regulation of efflux pump genes (P < 0.05). Conclusion TBO-mediated APDT exerts a bactericidal efficacy on multidrug-resistant P. aeruginosa. APDT could alter the expression of drug-resistant genes by attacking nucleoid DNA, and thereby improve the sensitivity of drug-resistant bacteria to antibiotics.

Keywords

• multidrug resistant bacteria
• pseudomonas aeruginosa
• antimicrobial photodynamic therapy
• toluidine blue o
• efflux pump genes