Unpacking bedaquiline hetero-resistance: the importance of intermediate profiles for phenotypic drug-susceptibility...

Phenotypic drug susceptibility testing (pDST) remains the gold standard for Mycobacterium tuberculosis complex drug resistance determination. Next generation sequencing technologies can identify heteroresistant populations at low frequencies, but little is known about the impact of heteroresistance on bedaquiline (BDQ) pDST results. We simulated heteroresistance using in vitro generated MmpR5 mutants mixed with the progenitor strain at various percentages (1-20%) and did pDST using MGIT960 culture (1 and 2 μg/mL BDQ concentrations). Targeted Next Generation Sequencing (tNGS) was used to quantify the mutant sub-population in growth control tubes, which were expected to maintain the mutant: wild type proportion throughout the assay. Growth units of these growth control tubes were also comparable with minor differences in time-to-positivity between ratio mixtures. Only when intermediate results were considered could BDQ heteroresistance be detected at frequencies of approximately 1% by pDST at a critical concentration of 1 μg/mL using BACTEC MGIT960 coupled with EpiCenter TBeXiST software. The ability of pDST, a widely available DST technique, to reveal the presence of BDQ-resistant subpopulations at the phenotypic testing stage could improve resistance determination and potentially reduce time to effective treatment.