Background: Antibiotic-resistant Pseudomonas aeruginosa (P. aeruginosa) 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 P. aeruginosa in planktonic cultures and preformed biofilms. Methods: We conducted a factorial dose–response in vitro study combining P. aeruginosa-specific 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 growth_curve_outcomes, 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 (n = 5 biological replicates and technical triplicates). Results: The bacterial growth lag phase was significantly extended for phage alone or PBL alone, with a synergistic effect of up to 144% (p < 0.001 for all), achieving a 9 log CFU/mL reduction at 24 h (p < 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%, p < 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. Conclusions: Phage therapy combined with PBL demonstrates a synergistic bactericidal effect against P. aeruginosa 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.

Antibiotic-resistant Pseudomonas aeruginosa (PA) is a critical health threat. Novel treatment approaches are urgently required in this post-antibiotic era. In the current study, we investigated the bactericidal combinatorial potential of two non-antibiotic alternative approaches: phage therapy and pulsed blue light (PBL). Bacteriophages (phages), are viruses that specifically infect and lyse bacteria without harming eukaryotic cells. Pulsed blue light (PBL) alters bacterial membranes and was clinically shown to be innocuous to the skin in low doses. Here, using a low dose 457nm, 33KHz PBL combined with specific PA targeting phages, we demonstrated a synergistic effect that achieved complete inhibition of planktonic bacteria and a 40% reduction in formed biofilms. As part of this study, we also developed a user-friendly python-based tool for extraction of growth curve outcomes. In vivo studies are warranted for further validation of this combinatorial treatment. This approach may lead to a novel, antibiotic complementary modality to help patients suffering from difficult-to-treat antibiotic-resistant infections. One Sentence Summary: Low-dose pulsed blue light and phage therapy have a synergistic bactericidal effect on Pseudomonas aeruginosa planktonic cultures and formed biofilm