Summary

Our paper offers a leap forward in TB research by addressing the need for rapid phenotypic drug susceptibility testing (pDST). While DNA-based methods have improved TB diagnostics worldwide, they fundamentally depend on prior pDST data. We developed TRACeR-TB, an RNA-based pDST distinguishing resistant from susceptible strains by measuring transcriptional stress after a 6-hour antibiotic exposure. TRACeR-TB demonstrates higher sensitivity than MGIT for rifampicin and bedaquiline, identifying resistant phenotypes undetected by genotypic methods. While our method has clear diagnostic implications, we believe its power lies in accelerating TB research. Its speed facilitates high-throughput screening of anti-TB compounds or drug-resistant strains. The assay's broad applicability is shown by testing novel drug-boosting compounds and detecting intracellular drug-inflicted stress, supporting hit-to-lead optimization in and ex vivo. As a rapid, mutation-independent platform, TRACeR-TB accelerates the development of new therapeutic strategies and the discovery of resistance mechanisms, offering a complementary DST to bridge existing gaps in TB diagnostics and research.