Unpacking bedaquiline heteroresistance: the importance of intermediate profiles for phenotypic drug susceptibility...

Phenotypic drug susceptibility testing (pDST) remains a widely used standard for determination of resistance for several drugs for the Mycobacterium tuberculosis complex. 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 performed pDST using the BACTEC MGIT 960 platform (1 and 2 µg/mL BDQ concentrations) coupled with EpiCenter TB-eXtended individual drug Susceptibility Testing software. Targeted next-generation sequencing was used to quantify the mutant subpopulation 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 (i.e., when growth units in a drug-containing tube reach the threshold for resistance but only after a further week of incubation) could BDQ heteroresistance be detected at frequencies of approximately 1% by pDST at a critical concentration of 1 µg/mL. These intermediate results, commonly disregarded during routine testing, could lead to earlier detection of BDQ resistance and may avert adverse clinical outcomes. 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.