De Novo Emergence of Genetically Resistant Mutants of Mycobacterium tuberculosis from the Persistence Phase Cells Formed against Antituberculosis Drugs In Vitro

Abstract

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 populations, upon continued exposure to lethal concentrations of antibiotics, remained unexplored. In the present study, we found that Mycobacterium tuberculosis cells exposed continuously to lethal concentrations of rifampin (RIF) or moxifloxacin (MXF) for prolonged durations showed killing, RIF/MXF persistence, and regrowth phases. RIF-resistant or MXFresistant 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. A Luria-Delbruck fluctuation experiment using RIF-exposed M. tuberculosis cells showed that the rpoB mutants were not preexistent in the population but were formed de novo from the RIF persistence phase population. The RIF persistence phase M. tuberculosis 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 M. tuberculosis gyrA de novo mutants could be selected from the RIF persistence phase cells. Thus, unlike previous studies, which showed emergence of genetically resistant mutants upon exposure of bacteria for short durations to sublethal concentrations of antibiotics, our study demonstrates that continuous prolonged exposure of M. tuberculosis cells to lethal concentrations of an antibiotic generates antibiotic persistence phase cells that form a reservoir for the generation of genetically resistant mutants to the same antibiotic or another antibiotic. These findings may have clinical significance in the emergence of drug-resistant tubercle bacilli.

Unveiling the physiological mechanisms that drive the emergence of antibiotic resisters from antibiotic persister population of mycobacteria

“…Background information and experimental strategy. We had earlier found that M. tuberculosis (Mtb) and M. smegmatis (Msm) cells, which were exposed to minimum bactericidal concentrations (MBCs) of rifampicin and moxifloxacin for prolonged duration in vitro such as 18 days for Mtb cells and 96 hrs for Msm cells, showed a sequential killing, persistence and regrowth phases (Sebastian et al, 2017;Swaminath, 2017). In these studies, rifampicin-resistant and moxifloxacin-resistant genetic mutants of Mtb and Msm were found to emerge from the persister population, regrow and populate the culture.…”

“…Bacteria, both pathogenic and non-pathogenic, of diverse genera and habitat have shown the phenomenon of persistence. With mycobacteria being no exception to this trait, large number of studies have been carried out on Mycobacterium tuberculosis persisters McCune et al, 1966a, b;Hu et al, 2000;Lenaerts et al, 2007;Hoff et al, 2011;Sebastian et al, 2017). High density of acidfast bacilli has been found in the tubercles resected months after the patients on chemotherapy had become sputum negative (Medlar et al, 1952;Beck and Yegian, 1952).…”

“…It has been proposed that the antibiotic persister cells could behave as an evolutionary reservoir for the emergence of antibiotic-resistant mutants (Cohen et al, 2013;Sebastian et al, 2017). This has been experimentally demonstrated in diverse bacterial systems (reviewed in Cohen et al, 2013), including M. tuberculosis and M.…”

“…smegmatis (Cohen et al, 2013;Sebastian et al, 2017;Swaminath, 2017). The emergence of M. smegmatis persisters in vitro upon treatment with rifampin, INH, ciprofloxacin, and ofloxacin has been reported Swaminath, 2017).…”

“…We have recently shown the de novo emergence of rifampin/moxifloxacin-resistant genetic mutants of M. tuberculosis and M. smegmatis from the respective antibiotic persistence phase cells due to mutagenesis from elevated levels of hydroxyl radical (Sebastian et al, 2017;Swaminath, 2017). The genetically resistant mutants emerged from the persister population of cells despite the continuous presence of microbicidal concentrations of the antibiotic for prolonged duration, such as for 20 days for M. tuberculosis and 96 hrs for M. smegmatis cells in vitro (Sebastian et al, 2017;Swaminath, 2017). The antibiotic-resistant genetic mutants regrew and populated the culture in the continued presence of microbicidal concentrations of the antibiotic.…”