title: Double mutation in DNA gyrase confers moxifloxacin resistance and decreased fitness of Mycobacterium smegmatis.
abstract: Ofloxacin and moxifloxacin are the most commonly used fluoroquinolones (FQs) for the treatment of tuberculosis. As a new generation FQ, moxifloxacin has been recommended for the treatment of ofloxacin-resistant TB. However, the mechanism by which ofloxacin-resistant Mycobacterium tuberculosis further gains resistance to moxifloxacin remains unclear.
  
  
  
  We used Mycobacterium smegmatis as a model for studying FQ resistance in M. tuberculosis . Moxifloxacin-resistant M. smegmatis was selected in vitro based on strains with primary ofloxacin resistance. The gyrA and gyrB genes of the resistant strains were sequenced to identify resistance-associated mutations. An in vitro competition assay was applied to explore the influence of gyrA / gyrB mutations on bacterial fitness. Finally, we evaluated the clinical relevance of our findings by analysing the WGS data of 1984 globally collected M. tuberculosis strains.
  
  
  
  A total of 57 moxifloxacin-resistant M. smegmatis strains based on five ofloxacin-resistant strains were obtained. Sequencing results revealed that all moxifloxacin-resistant strains harboured second-step mutations in gyrA or gyrB . The relative fitnesses of the double-mutation strains varied from 0.65 to 0.93 and were mostly lower than those of their mono-mutation parents. From the genomic data, we identified 37 clinical M. tuberculosis strains harbouring double mutations in gyrA and/or gyrB and 36 of them carried at least one low-level FQ-resistance mutation.
  
  
  
  Double mutation in DNA gyrase leads to moxifloxacin resistance and decreased fitness in M. smegmatis . Under current dosing of moxifloxacin, double mutations mainly happened in M. tuberculosis strains with primary low-level resistance mutations.
authors.affiliation: Laboratory of Infection and Immunity, West China Center of Medical Sciences, Sichuan University, Chengdu 610041, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Luo
authors.affiliation: Laboratory of Infection and Immunity, West China Center of Medical Sciences, Sichuan University, Chengdu 610041, China.
authors.forename: Jinning
authors.initials: J
authors.lastname: Yuan
authors.affiliation: Laboratory of Infection and Immunity, West China Center of Medical Sciences, Sichuan University, Chengdu 610041, China.
authors.forename: Xuan
authors.initials: X
authors.lastname: Peng
authors.affiliation: Laboratory of Infection and Immunity, West China Center of Medical Sciences, Sichuan University, Chengdu 610041, China.
authors.forename: Guoping
authors.initials: G
authors.lastname: Yang
authors.affiliation: Laboratory of Infection and Immunity, West China Center of Medical Sciences, Sichuan University, Chengdu 610041, China.
authors.forename: Youjun
authors.initials: Y
authors.lastname: Mi
authors.affiliation: Laboratory of Infection and Immunity, West China Center of Medical Sciences, Sichuan University, Chengdu 610041, China.
authors.forename: Changfeng
authors.initials: C
authors.lastname: Sun
authors.affiliation: Laboratory of Infection and Immunity, West China Center of Medical Sciences, Sichuan University, Chengdu 610041, China.
authors.forename: Chuhan
authors.initials: C
authors.lastname: Wang
authors.affiliation: Laboratory of Infection and Immunity, West China Center of Medical Sciences, Sichuan University, Chengdu 610041, China.
authors.forename: Chunxi
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Laboratory of Infection and Immunity, West China Center of Medical Sciences, Sichuan University, Chengdu 610041, China.
authors.forename: Lang
authors.initials: L
authors.lastname: Bao
mesh_terms: D027081:DNA Gyrase / Q000235:genetics* / Q000378:metabolism*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D024841:Fluoroquinolones / Q000494:pharmacology*; D056084:Genetic Fitness; D023281:Genomics; D006801:Humans; D008826:Microbial Sensitivity Tests; D000077266:Moxifloxacin; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D015242:Ofloxacin / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D014376:Tuberculosis / Q000382:microbiology; D018088:Tuberculosis, Multidrug-Resistant / Q000382:microbiology
chemical_list: D024841:Fluoroquinolones; D015242:Ofloxacin; D027081:DNA Gyrase; D000077266:Moxifloxacin