Blue Laser Light Exerts Potent Anti-Biofilm Activity <i> In Vitro</i> and <i>In Vivo</i> by Inducing Oxidative Stress in Bacterial Cells

Abstract

Background: The development of novel antimicrobial strategies is becoming of crucial importance due to increasing resistance to conventional antibiotics. The aim of the present study was to evaluate the efficacy of blue laser light in eradicating Pseudomonas aeruginosa infection and biofilm formation in vitro and in vivo laser light, antimicrobial therapy, biofilm, oxidative stress, as well as to shed light on its molecular mechanisms. Methods: We first compared the efficacy of multiple protocols of blue (λ 445 nm) laser irradiation, differing in power, irradiance and fluence, on P. aeruginosa grown in either planktonic state or on agar plates. The most effective protocols were also tested with multiple assays on mature P. aeruginosa biofilms, including flow cells followed by 3-dimensional reconstruction using laser scanning confocal microscopy. The role of oxidative stress was evaluated by using knock-out genomic mutant bacterial strains, anti-oxidants and measuring the levels of reactive oxygen species. Toxicity was evaluated on human epithelial cells and in vivo efficacy was confirmed in a mouse model of skin wound infection. Findings. We selected a few blue laser protocols exerting high antimicrobial and anti-biofilm activity and low toxicity, both in vitro and in vivo and identified oxidative stress as the major molecular mechanism responsible of these effects. Interpretation. Phototherapy using blue laser light stands as an innovative and powerful tool to inhibit bacterial growth and biofilm formation, thus representing a concrete treatment option for superinfected wounds. Funding Statement: This work was supported by intramural funds from ICGEB to S.Z. and from University of Trieste and IRCCS Burlo Garofolo to S.C. Declaration of Interests: G.O. has part-time employment in K-Laser d.o.o. (Sežana, Slovenia). The other authors declare none. Ethics Approval Statement: Animal care and treatment were conducted in conformity with institutional guidelines in compliance with national and international laws and policies (European Economic Community Council Directive 86/609, OJL 358, December 12, 1987) and upon approval by the Institutional Animal Care Use Committee and by the Italian Minister of Health.