Background: Atypical Beijing genotype Mycobacterium tuberculosis strains are widespread in South Africa and have acquired resistance to up to 13 drugs on multiple occasions. It is puzzling that these strains have retained fitness and transmissibility despite the potential fitness cost associated with drug resistance mutations. Methods: We conducted Illumina sequencing of 211 Beijing genotype M. tuberculosis isolates to facilitate the detection of genomic features that may promote acquisition of drug resistance and restore fitness in highly resistant atypical Beijing forms. Phylogenetic and comparative genomic analysis was done to determine changes that are unique to the resistant strains that also transmit well. Minimum inhibitory concentration (MIC) determination for streptomycin and bedaquiline was done for a limited number of isolates to demonstrate a difference in MIC between isolates with and without certain variants. Results: Phylogenetic analysis confirmed that two clades of atypical Beijing strains have independently developed resistance to virtually all the potent drugs included in standard (pre-bedaquiline) drug-resistant TB treatment regimens. We show that undetected drug resistance in a progenitor strain was likely instrumental in this resistance acquisition. In this cohort, ethionamide (ethA A381P) resistance would be missed in first-line drug-susceptible isolates, and streptomycin (gidB L79S) resistance may be missed due to an MIC close to the critical concentration. Subsequent inadequate treatment historically led to amplification of resistance and facilitated spread of the strains. Bedaquiline resistance was found in a small number of isolates, despite lack of exposure to the drug. The highly resistant clades also carry inhA promoter mutations, which arose after ethA and katG mutations. In these isolates, inhA promoter mutations do not alter drug resistance, suggesting a possible alternative role. Conclusion:The presence of the ethA mutation in otherwise susceptible isolates from ethionamide-naïve patients demonstrates that known exposure is not an adequate indicator of drug susceptibility. Similarly, it is demonstrated that bedaquiline resistance can occur without exposure to the drug. Inappropriate treatment regimens, due to missed resistance, leads to amplification of resistance, and transmission. We put these results into the context of current WHO treatment regimens, underscoring the risks of treatment without knowledge of the full drug resistance profile.
Resistance to the first-line antituberculosis (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 complex (MTBC) clinical isolates that lacked the common S315T or 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 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 mutations conferred resistance to INH at a MIC 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 useful tool for detecting uncommon INH resistance mutations that would otherwise be missed by current targeted molecular testing methods and suggest that its use (or use of expanded conventional or next-generation-based targeted sequencing) may provide earlier diagnosis of INH-R TB.
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