Significance: Pseudomonas (P.) aeruginosa, a common cause of infection in burns, acquires antibiotic resistance easily and forms biofilms efficiently. Thus, it is difficult to control P. aeruginosa infection in burn wounds, which causes lethal septicemia. Antimicrobial photodynamic therapy (aPDT) is attractive as a new strategy to treat burn wound infections with drug-resistant bacteria.

Aim: We examined the efficacy of methylene blue (MB)-mediated aPDT with various additives in a tissue depth-resolved manner to find conditions that minimize the bacterial invasion.

Approach: We applied MB-mediated aPDT with LED array illumination to an extensive, full-thickness burn infected with P. aeruginosa in rats for three consecutive days (days 0, 1, and 2). On day 2, the depth distributions of bacteria were assessed based on the histological analysis using Gram staining. We examined how the addition of ethylenediaminetetraacetic acid (EDTA), ethanol, and dimethyl sulfoxide (DMSO) affected the efficacy of aPDT.

Results: Pure MB-mediated aPDT significantly reduced the numbers of bacteria with biofilms on the wound surface and in the epidermis compared with those for the control tissue (saline only). However, there were many bacteria in the deeper region of the tissue. In contrast, MB/EDTA/ethanol/DMSO-mediated aPDT minimized the numbers of bacteria in the broad depth region of the tissue. Still, a limited number of bacteria was observed in the subcutaneous tissue.

Conclusions: The depthwise analysis of bacteria demonstrated the efficacy of the MB-mediated aPDT with the addition of EDTA, ethanol, and DMSO in controlling burn wound infections. However, further improvement of the therapy is needed to suppress bacterial migration into the deep tissue completely.