Mechanism of the Development of Genetic Resistance to Antibiotics Mediated by three Reactive Oxygen Species in Mycobacterium smegmatis

Abstract

Bacteria use different means to survive in the continued presence of antibiotics. We find that upon prolonged exposure to antibiotics, mycobacteria put up a triphasic response comprised of a logarithmic reduction of the population, followed by the maintenance of an antibiotic surviving population (ASP), from which genetic resisters to the antibiotic emerge de novo. We had earlier reported that the cells in the ASP are at a high oxidative state compared to the cells in the mid-log phase (MLP) population. These cells were found to generate significantly high levels of hydroxyl radical that inflicted genome-wide mutations due to its high reactivity, enabling the selection of antibiotic-resistant strains.

The generation of hydroxyl radical involves an array of biochemical events, beginning with the conversion of molecular oxygen to superoxide radical, and its dismutation to hydrogen peroxide, which finally participates in Fenton reaction with labile iron to generate hydroxyl radical. Reactive oxygen species (ROS) are known to be extremely unstable and chemically highly reactive molecules, which makes the cellular coordination and regulation of their production extremely important from therapeutic point of view. In this study we demonstrate a well-coordinated pattern of ROS generation by Mycobacterium smegmatis upon prolonged exposure to two different antibiotics. The physiological relevance of this event was demonstrated by the addition of ROS scavenger, which significantly affected the survival fitness of the bacterial population and the subsequent generation of resisters against antibiotic.

In the second part of this study, we shed light on a possible mechanism of this cellular event. Superoxide anion radical is known to be the primary reactant to participate in the array of reactions producing subsequent species ROS. Among other pathways, NADH oxidase is known to be the major source of superoxide radical generation in Mycobacterium smegmatis. Upon progression, this study confirms the temporal expression and activity of NADH oxidase in Msm cells upon continuous antibiotic exposure. Finally, the involvement of NADH oxidase in the survival of bacterial population and subsequent generation of resisters were established by exposing the Msm cells to a specific inhibitor to NADH oxidase.

Altogether, the findings of this study elucidated the pathway involved in the production and utilisation of ROS by Mycobacterium smegmatis to acquire a survival

benefit against antibiotics stress and identified some major enzyme systems as potential therapeutic targets.