Ethanol in Combination with Oxidative Stress Significantly Impacts Mycobacterial Physiology

Here, we investigate the mycobacterial response to the combined stress of an organic oxidant (cumene hydroperoxide [CHP]) and a solvent (ethanol). To understand the interaction between the two stressors, we treated Mycobacterium smegmatis cells to a range of ethanol concentrations (2.5% to 10% [vol/vol]) in combination with a subinhibitory concentration of 1 mM CHP. It was observed that the presence of CHP increases the efficacy of ethanol in inducing rapid cell death. The data further suggest that ethanol reacts with the alkoxy radicals to produce ethanol-derived peroxides. These radicals induce significant membrane damage and lead to cell lysis. The ethanol-derived radicals were primarily recognized by the cells as organic radicals, as was evident by the differential upregulation of the ohr-ohrR genes that function in cells treated with the combination of ethanol and CHP. The role of organic peroxide reductase, Ohr, was further confirmed by the significantly higher sensitivity of the deletion mutant to CHP and the combined stress treatment of CHP and ethanol. Moreover, we also observed the sigma factor σ^B to be important for the cells treated with ethanol alone as well as the aforementioned combination. A ΔsigB mutant strain had significantly higher susceptibility to the stress conditions. This finding was correlated with the σ^B-dependent transcriptional regulation of ohr and ohrR In summary, our data indicate that the combination of low levels of ethanol and organic peroxides induce ethanol-derived organic radicals that lead to significant oxidative stress on the cells in a concentration-dependent manner.IMPORTANCE Bacterial response to a combination of stresses can be unexpected and very different compared with that of an individual stress treatment. This study explores the physiological and transcriptional response of mycobacteria in response to the combinatorial treatment of an oxidant with the commonly used solvent ethanol. The presence of a subinhibitory concentration of organic peroxide increases the effectiveness of ethanol by inducing reactive peroxides that destroy the membrane integrity of cells in a significantly short time span. Our work elucidates a mechanism of targeting the complex mycobacterial membrane, which is its primary source of intrinsic resistance. Furthermore, it also demonstrates the importance of exploring the effect of various stress conditions on inducing bacterial clearance.