Combined Effects of Amikacin and Methylene Blue-Mediated Photodynamic Therapy on Pseudomonas aeruginosa Biofilms Mimicking Mono- and Polymicrobial Diabetic Foot Ulcer Infections

Diabetic foot ulcer (DFU) infections frequently involve biofilm formation and exhibit limited responsiveness to conventional antibiotic therapy. In particular, <i>Pseudomonas aeruginosa</i> often participates in mono- and polymicrobial biofilms that display high tolerance to antimicrobial agents. This study evaluated the efficacy of methylene blue-mediated antimicrobial photodynamic therapy (aPDT), alone and in combination with antibiotics, against <i>P. aeruginosa</i> biofilms formed either as single-species or in mixed communities with <i>Enterococcus faecalis</i>, under conditions mimicking DFU infections. Macrocolony biofilms were challenged with amikacin alone (for single-species biofilms) or amikacin plus ampicillin (for mixed biofilms), aPDT, or sequential combinations of these treatments, and bacterial viability was quantified by colony-forming unit enumeration. Antibiotic treatment alone produced only modest reductions in <i>P. aeruginosa</i> viability, even at high concentrations, while aPDT using methylene blue was effective only at high photosensitizer concentrations. In contrast, sequential treatment with antibiotics followed by aPDT and a second antibiotic challenge resulted in a marked reduction in <i>P. aeruginosa</i> viability in both mono- and polymicrobial biofilms. Scanning electron microscopy revealed extensive structural damage in <i>P. aeruginosa</i> cells following combined treatments, whereas <i>E. faecalis</i> remained unaffected. Overall, our findings demonstrate that combining aPDT with antibiotics significantly enhances antibiofilm activity against <i>P. aeruginosa</i>, highlighting this approach as a promising alternative for the management of biofilm-associated DFU infections.