Rimon Amit, Belin Jonathan, Yerushalmy Ortal, Eavri Yonatan, Shapochnikov Anatoly, Coppenhagen-Glazer Shunit, Hazan Ronen, Gavish Lilach
Institute of Biomedical and Oral Research (IBOR), Faculty of Dental Medicine, The Hebrew University of Jerusalem, Jerusalem 9112102, Israel.
Tzameret, The Military Track of Medicine, The Hebrew University-Hadassah Medical School, Jerusalem 9112001, Israel.
Antibiotics (Basel). 2025 May 9;14(5):481. doi: 10.3390/antibiotics14050481.
Antibiotic-resistant () 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 in planktonic cultures and preformed biofilms. We conducted a factorial dose-response in vitro study combining 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 , 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 ( = 5 biological replicates and technical triplicates). The bacterial growth lag phase was significantly extended for phage alone or PBL alone, with a synergistic effect of up to 144% ( < 0.001 for all), achieving a 9 log CFU/mL reduction at 24 h ( < 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%, < 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. Phage therapy combined with PBL demonstrates a synergistic bactericidal effect against 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.
耐抗生素的（）菌株正日益成为发病和死亡的一个原因。脉冲蓝光（PBL）可增强卟啉诱导的活性氧，并且临床研究表明低剂量时对皮肤无害。噬菌体是感染细菌的病毒，提供了一种很有前景的非抗生素杀菌方法。本研究调查了低剂量PBL与噬菌体疗法在浮游培养物和预先形成的生物膜中针对的潜在协同作用。我们在PA14和多重耐药的PATZ2菌株上进行了一项析因剂量反应体外研究，将噬菌体与PBL（457纳米，33千赫兹）联合使用。在排除PBL对噬菌体滴度或活性的直接影响后，我们使用生长曲线分析（通过，一种新开发的、可在GitHub上获取的基于Python的工具）、CFU和PFU评估对浮游培养物的有效性。使用超声处理后的CFU、结晶紫染色以及共聚焦显微镜下的活/死染色评估生物膜疗效。最后，我们使用硝基蓝四氮唑还原试验评估活性氧（ROS）作为一种潜在机制。使用ANOVA或Kruskal-Wallis检验以及事后Tukey或Conover-Iman检验进行比较（每组有5个生物学重复和3个技术重复）。单独使用噬菌体或单独使用PBL时细菌生长的延迟期均显著延长，协同效应高达144%（所有比较P<0.001），在24小时时实现了9 log CFU/mL的减少（P<0.001）。在预先形成的生物膜中，协同组合显著降低了生物膜生物量和细菌活力（活细菌百分比，中位数（四分位间距）：对照组80%；噬菌体组40%；PBL组25%；PBL与噬菌体联合组15%，P<0.001）。从机制上讲，PBL在浮游培养物中引发了短暂的ROS，噬菌体联合处理可使其放大，而生物膜中的双相ROS模式反映了时间依赖性协同作用。噬菌体疗法与PBL联合在浮游培养物和生物膜中均显示出对的协同杀菌作用。鉴于PBL和噬菌体具有很强的安全性，这种方法可能会为患有难治性耐抗生素感染和生物膜相关感染的患者带来一种新的、与抗生素互补的安全治疗方式。
