Combined Addition of Ethanol and Ethylenediaminetetraacetic Acid Enhances Antibacterial and Antibiofilm Effects in Methylene Blue‐Mediated Photodynamic Treatment against Pseudomonas aeruginosa In Vitro

Antimicrobial photodynamic treatment (aPDT) for infection with drug-resistant bacteria has received much attention. For P. aeruginosa, however, efficient formation of biofilms and the nature of Gram-negative bacteria often limit the efficacy of aPDT. In this study, we investigated the effects of ethanol and ethylenediaminetetraacetic acid (EDTA) as additives on bacterial viability, biofilm biomass, and structures of bacteria and biofilms in methylene blue (MB)-mediated aPDT in vitro. Matured P. aeruginosa biofilms were incubated with 32-µm MB solutions with different concentrations of additives and then illuminated with 665-nm light from an LED array. The combined addition of 10% ethanol and 10 mm EDTA to MB resulted in significantly greater bactericidal effects than those of MB alone and of MB with 10% ethanol or 10 mm EDTA. Crystal violet assays showed significant reductions in biofilm biomass by aPDT with addition of both ethanol and EDTA compared to that in the case of aPDT with MB alone. Scanning electron microscopy showed broken bacterial cells and reduction in the cell density and amount of biofilm under those conditions. Ethanol addition alone did not improve aPDT efficacy. Reduced amount of biofilm by EDTA addition would have improved the transportation of MB and ethanol to bacteria.