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.