Validation of novelMycobacterium tuberculosisisoniazid resistance mutations not detectable by common molecular tests

Resistance to the first-line anti-tuberculosis (TB) drug, isoniazid (INH), is widespread, and the mechanism of resistance is unknown in approximately 15% of INH-resistant (INH-R) strains. To improve molecular detection of INH-R TB, we used whole genome sequencing (WGS) to analyze 52 phenotypically INH-R Mycobacterium tuberculosis complex (MTBC) clinical isolates that lacked the common katG S315T or inhA promoter mutations. Approximately 94% (49/52) of strains had mutations at known INH-associated loci that were likely to confer INH resistance. All such mutations would be detectable by sequencing more DNA adjacent to existing target regions. Use of WGS minimized the chances of missing infrequent INH resistance mutations outside commonly targeted hotspots. We used recombineering to generate 12 observed clinical katG mutations in the pansusceptible H37Rv reference strain and determined their impact on INH resistance. Our functional genetic experiments have confirmed the role of seven suspected INH resistance mutations and discovered five novel INH resistance mutations. All recombineered katG mutations conferred resistance to INH at a minimum inhibitory concentration of ≥0.25 μg/mL and should be added to the list of INH resistance determinants targeted by molecular diagnostic assays. We conclude that WGS is a superior method for detection of INH-R MTBC compared to current targeted molecular testing methods and could provide earlier diagnosis of drug-resistant TB.