Pulsed Blue Light and Phage Therapy: A Novel Synergistic Bactericide

<b>Background:</b> Antibiotic-resistant <i>Pseudomonas aeruginosa</i> (<i>P. aeruginosa</i>) strains are an increasing cause of morbidity and mortality. Pulsed blue light (PBL) enhances porphyrin-induced reactive oxygen species and has been clinically shown to be harmless to the skin at low doses. Bacteriophages, viruses that infect bacteria, offer a promising non-antibiotic bactericidal approach. This study investigates the potential synergism between low-dose PBL and phage therapy against <i>P. aeruginosa</i> in planktonic cultures and preformed biofilms. <b>Methods:</b> We conducted a factorial dose-response in vitro study combining <i>P. aeruginosa-specific</i> phages with PBL (457 nm, 33 kHz) on both PA14 and multidrug-resistant PATZ2 strains. After excluding direct PBL effects on phage titer or activity, we assessed effectiveness on planktonic cultures using growth curve analysis (via <i>growth_curve_outcomes</i>, a newly developed, Python-based tool available on GitHub) , CFU, and PFU. Biofilm efficacy was evaluated using CFU post-sonication, crystal violet staining, and live/dead staining with confocal microscopy. Finally, we assessed reactive oxygen species (ROS) as a potential mechanism using the nitro blue tetrazolium reduction assay. ANOVA or Kruskal-Wallis tests with post hoc Tukey or Conover-Iman tests were used for comparisons (<i>n</i> = 5 biological replicates and technical triplicates). <b>Results:</b> The bacterial growth lag phase was significantly extended for phage alone or PBL alone, with a synergistic effect of up to 144% (<i>p</i> < 0.001 for all), achieving a 9 log CFU/mL reduction at 24 h (<i>p</i> < 0.001). In preformed biofilms, synergistic combinations significantly reduced biofilm biomass and bacterial viability (% Live, median (IQR): Control 80%; Phage 40%; PBL 25%; PBL&Phage 15%, <i>p</i> < 0.001). Mechanistically, PBL triggered transient ROS in planktonic cultures, amplified by phage co-treatment, while a biphasic ROS pattern in biofilms reflected time-dependent synergy. <b>Conclusions:</b> Phage therapy combined with PBL demonstrates a synergistic bactericidal effect against <i>P. aeruginosa</i> in both planktonic cultures and biofilms. Given the strong safety profile of PBL and phages, this approach may lead to a novel, antibiotic-complementary, safe treatment modality for patients suffering from difficult-to-treat antibiotic-resistant infections and biofilm-associated infections.