Bacterial persisters are a subpopulation of cells that can tolerate lethal concentrations of antibiotics. However, the possibility of the emergence of genetically resistant mutants from antibiotic persister cell population, upon continued exposure to lethal concentrations of antibiotics, remained unexplored. In the present study, we found that Mycobacterium tuberculosis (Mtb) cells exposed continuously to lethal concentrations of rifampicin (RIF) or moxifloxacin (MXF) for prolonged durations showed killing, RIF/MXF persistence, and regrowth phases. RIF-resistant or MXF-resistant mutants carrying clinically relevant mutations in the rpoB or gyrA gene, respectively, were found to emerge at high frequency from the RIF persistence phase population. Luria-Delbruck fluctuation experiment using RIF-exposed Mtb cells showed that the rpoB mutants were not pre-existent in the population, but were formed de novo from the RIF persistence phase population. The RIF persistence phase Mtb cells carried elevated levels of hydroxyl radical that inflicted extensive genome-wide mutations, generating RIF-resistant mutants. Consistent with the elevated levels of hydroxyl radical mediated genome-wide random mutagenesis, MXF-resistant Mtb gyrA de novo mutants could be selected from the RIF persistence phase cells. Thus, unlike the previous studies, which showed emergence of genetically resistant mutants upon exposure of bacteria for short duration to sub-lethal concentrations of antibiotics, our study demonstrates that continuous prolonged exposure of Mtb cells to lethal concentrations of an antibiotic generates antibiotic persistence phase cells that form a reservoir for the generation of genetically resistant mutants to that antibiotic or another antibiotic. These findings may have clinical significance in the emergence of drug-resistant tubercle bacilli.
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