A Frameshift Mutation in the Methyltransferase <i>rlmN</i> Is Associated with Increased Linezolid Resistance in <i>Mycobacterium tuberculosis</i>

Background: Linezolid is a key component of treatment regimens for multidrug-resistant and extensively drug-resistant tuberculosis, which is caused by the pathogen Mycobacterium tuberculosis (MTB). Resistance to linezolid in MTB has traditionally been attributed to mutations in the 23S ribosomal RNA ( rrl ) and ribosomal protein L3 ( rplC ), but only a fraction of clinically observed linezolid resistance is explained by mutations in these 2 genes. Results: We report that an analysis of strains with paired whole-genome sequencing and linezolid minimum inhibitory concentration phenotyping from the Bacterial and Viral Bioinformatics Resource Center reveals that a relatively common frameshift mutation in MTB methyltransferase rlmN (5.3% of assessed isolates, encompassing all isolates known to be in the globally distributed MTB lineage 4.10) is significantly associated with increased linezolid minimum inhibitory concentration. In addition to statistical associations, we provide evolutionary evidence of homology to an established linezolid resistance mechanism in Staphylococcus aureus and structural evidence that the frameshift mutation likely ablates rlmN methyltransferase functionality. Conclusions: We find a novel gene associated with increased linezolid resistance in MTB, with potential implications for resistance diagnostics and therapeutic strategies.