Mycobacterium smegmatis moxifloxacin persister cells produce high levels of hydroxyl radical, generating genetic resisters selectable not only with moxifloxacin, but also with ethambutol and isoniazid

Abstract

Bacterial antibiotic persister cells tolerate lethal concentrations of antibiotics but emerge as the antibiotic-sensitive population upon antibiotics withdrawal. However, the possibility of antibiotic-resistant genetic mutants emerging from the antibiotic persister population in the continued exposure to microbicidal concentrations of antibiotics needed investigation. We explored this possibility using the fast-growing <i>Mycobacterium smegmatis</i> as a model organism for <i>Mycobacterium tuberculosis</i> biology, as it is known to incur antibiotic-resistant mutations identical to and at identical target positions as found in the clinical isolates of <i>M. tuberculosis</i>. Here we report that the moxifloxacin (MXF) persister population generate significantly elevated levels of hydroxyl radical. Hydroxyl radical being a sequence-non-specific mutagen, resulted in the emergence of moxifloxacin-resistant genetic mutants at 8-log₁₀ higher frequency from the persister population. Luria-Delbruck experiment (in modified format) confirmed that MXF-resistant mutants emerged <i>de novo</i> from the persister population and were not pre-existent. The nature of the mutations in the quinolone resistance determining region indicated that they were generated due to oxidative stress. These mutations were identical to and at identical positions as found in the clinical isolates of MXF-resistant <i>M. tuberculosis</i>. Interestingly, from the MXF persister population, resisters to microbicidal concentrations of ethambutol and isoniazid could also be selected. These observations implied that the significantly high levels of hydroxyl radical might have generated genome-wide mutations, creating a pool of mutants in the MXF persister population, facilitating selection of resisters to other antibiotics also. These findings may be of clinical relevance to the emergence of drug-resistant strains during prolonged tuberculosis treatment regimen with high doses of multiple antibiotics.