Rapid detection of treatment-emergent resistance to bedaquiline and pretomanid in Mycobacterium tuberculosis using TM4::GeNL reporter mycobacteriophages.

UNLABELLED: There is an urgent need for rapid, affordable diagnostic tools capable of detecting baseline and treatment-emergent resistance among patients with drug-resistant tuberculosis (DR-TB). Reporter mycobacteriophages provide a phenotypic solution enhanced by brighter luminescent reporters compatible with simple readouts. We applied the TM4::GeNL reporter mycobacteriophage for rapid phenotypic drug susceptibility testing (pDST) on serial Mycobacterium tuberculosis isolates from six DR-TB patients, including four with treatment-emergent resistance and two with baseline bedaquiline (BDQ)-resistant subpopulations. Susceptibility to BDQ, pretomanid (PMD), delamanid (DLM), and linezolid (LZD) was assessed by luminescence retention after TM4::GeNL infection compared to no-drug control, with conventional pDST and whole-genome sequencing as the reference standard. Isolates with baseline and treatment-emergent resistance to BDQ and PMD had a mean relative luminescence unit (RLU) retention of 83.8% (95% CI, 52.1-115.6), while susceptible clinical isolates retained 1.5% mean RLU (95% CI, 0.2-2.8). The TM4::GeNL DST results were consistent with conventional pDST in detecting BDQ and PMD resistance. Three patients with BDQ treatment-emergent resistance had mutations in mmpR5 (Rv0678), with a range of variant frequencies (12.6%-92.3%). One isolate with treatment-emergent DLM/PMD resistance had mutations in the fbiC gene, with A487fs and S534stop variants at 25.0% and 12.0%, respectively. Sequencing of serial isolates from one patient on a regimen that included LZD identified an emergent mutation in rrl (n.2814G > T, 53.8% frequency); however, the isolate was LZD susceptible by both TM4::GeNL DST and conventional pDST. TM4::GeNL reporter mycobacteriophage accurately detected baseline and treatment-emergent resistance to BDQ and PMD compared to conventional pDST. This rapid, phenotypic approach shows promise for accelerating detection of resistance to new and repurposed DR-TB drugs in clinical settings.

IMPORTANCE: Ongoing transmission of drug-resistant tuberculosis (DR-TB), combined with limited access to rapid diagnostics for resistance to key drugs in modern regimens, poses a major challenge to global TB control. We applied TM4::GeNL reporter mycobacteriophage for rapid phenotypic drug susceptibility testing on Mycobacterium tuberculosis (Mtb) clinical isolates with baseline and treatment-emergent resistance to DR-TB drugs (bedaquiline, pretomanid, and delamanid), generating results within 48 h from a positive culture. Our findings provide evidence on the utility of the TM4::GeNL mycobacteriophage to infect Mtb clinical isolates and distinguish between drug-susceptible and resistant isolates. TM4::GeNL reporter mycobacteriophage detected emerging resistance to bedaquiline and pretomanid within 48 h from a positive culture, with our results correlating well with conventional phenotypic drug susceptibility and whole-genome sequencing data. These findings support TM4::GeNL reporter mycobacteriophage as a rapid, multiplexed alternative drug-susceptibility test for DR-TB.