Experimental and theoretical approaches reveal that antimicrobial blue light killing efficiency decreases with biofilm growth in a Pseudomonas aeruginosa model

Abstract

Recently, the use of antimicrobial blue light (aBL) has gained interest across various applications. However, a comprehensive framework that addresses the key factors driving bacterial photoinhibition remains lacking—particularly concerning biofilms, the predominant bacterial lifestyle. The goal of this work was to evaluate the potential of photokilling in this wide-spread microbial adherent community type, and to decipher the specific mechanisms at stake. To investigate aBL killing efficiency, we conducted experiments in a Pseudomonas aeruginosa biofilm model using a well-defined millifluidic device that allows real-time microscopy and quantitative analysis of a living biofilm under local irradiation at a defined light dose. In addition, we developed a theoretical model for light-biofilm interaction that accounts for the three-dimensional structure of the bacterial biofilm. To inform our model, we examined the light dose-response in isolated cells and found a profile indicative of a multi-target mechanism of lethality. By comparing the experimental and theoretical results, we identified a loss in killing efficiency as the biofilm grows, due in part to the increase in thickness of the living material inherent to this mode of development. Our findings also highlight a reduction in the intrinsic bacterial sensitivity to blue light as biofilm development progresses, which we attribute to the low oxygen levels typical of densely populated bacterial environments. These findings reveal new features of the photokilling mechanism and redefine the approach to designing effective antimicrobial photoinactivation strategies by integrating the key physical characteristics of bacterial biofilms.