Abstract
Reactive oxygen and nitrogen species (RONS) are emerging as a novel antibacterial strategy to combat the alarming increase in antimicrobial resistance (AMR). RONS can inhibit bacterial growth through reactions with cellular molecules, compromising vital biological functions and leading to cell death. While their mechanisms of action have been studied, many remain unclear, especially in biologically relevant environments. In this study, we exposed Gram-positive and Gram-negative bacteria to varying RONS ratios, mimicking what microbes may naturally encounter. A ratio in favour of RNS induced membrane depolarization and pore formation, resulting in an irreversible bactericidal effect. By contrast, ROS predominance caused membrane permeabilization and necrotic-like responses, leading to biofilm formation. Furthermore, bacterial cells exposed to more RNS than ROS activated metabolic processes associated with anaerobic respiration, DNA & cell wall/membrane repair, and cell signalling. Our findings suggest that the combination of ROS and RNS can be an effective alternative to inhibit bacteria, but only under higher RNS levels, as ROS dominance might foster bacterial tolerance, which in the context of AMR could have devastating consequences.