title: 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 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. 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.
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, Karnataka, India.
authors.forename: Jees
authors.initials: J
authors.lastname: Sebastian
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, Karnataka, India.
authors.forename: Sharmada
authors.initials: S
authors.lastname: Swaminath
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, Karnataka, India.
authors.forename: Rashmi Ravindran
authors.initials: RR
authors.lastname: Nair
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, Karnataka, India.
authors.forename: Kishor
authors.initials: K
authors.lastname: Jakkala
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, Karnataka, India.
authors.forename: Atul
authors.initials: A
authors.lastname: Pradhan
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, Karnataka, India ajitkpartha@gmail.com.
authors.forename: Parthasarathi
authors.initials: P
authors.lastname: Ajitkumar
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D027081:DNA Gyrase / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D024881:Drug Resistance, Bacterial / Q000187:drug effects / Q000235:genetics*; D024841:Fluoroquinolones / Q000494:pharmacology; D016680:Genome, Bacterial; D017665:Hydroxyl Radical / Q000378:metabolism; D000077266:Moxifloxacin; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development; D018384:Oxidative Stress / Q000187:drug effects; D012293:Rifampin / Q000008:administration & dosage / Q000494:pharmacology
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D024841:Fluoroquinolones; C505805:rpoB protein, Mycobacterium tuberculosis; D017665:Hydroxyl Radical; D012321:DNA-Directed RNA Polymerases; D027081:DNA Gyrase; D000077266:Moxifloxacin; D012293:Rifampin
keywords: Mycobacterium tuberculosis; antibiotic resistance; hydroxyl radical; moxifloxacin; persistence phase cells; rifampicin