Double mutation in DNA gyrase confers moxifloxacin resistance and decreased fitness of Mycobacterium smegmatis.

Objectives: 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. Methods: 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. Results: 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. Conclusions: 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.