Characterization of linezolid-resistance-associated mutations in Mycobacterium tuberculosis through WGS

OBJECTIVES: Linezolid is becoming an important antibiotic for treating MDR/XDR TB, but the mutations conferring resistance to linezolid remain inadequately characterized. Herein, we investigated the linezolid-resistance-associated mutations on a whole-genome scale through parallel selections of resistant isolates in vitro. METHODS: Ten parallel Mycobacterium tuberculosis H37Rv cultures were subjected to spontaneous mutant selection on 7H11 agar plates containing 2.5 mg/L linezolid. The linezolid resistance of resulting colonies was confirmed by growth on a second linezolid plate. WGS was then performed to identify mutations associated with linezolid resistance. RESULTS: Of 181 colonies appearing on the initial linezolid plates, 154 were confirmed to be linezolid resistant. WGS showed that 88.3% (136/154) of these isolates had a T460C mutation in rplC, resulting in a C154R substitution. The other 18 isolates harboured a single mutation in the rrl gene, with G2814T and G2270T mutations accounting for 7.8% (12/154) and 3.9% (6/154), respectively. CONCLUSIONS: No mutations in novel genes were associated with linezolid resistance in a whole-genome investigation of 154 linezolid-resistant isolates selected in vitro. These results emphasize that rrl and rplC genes should be the major targets for molecular detection of linezolid resistance.