Tre-DST: A new drug susceptibility test for Mycobacterium tuberculosis using solvatochromic trehalose probes

Tuberculosis (TB) is the most lethal cause of death from a single infectious agent. In 2024, an estimated 10 million people developed TB, nearly half a million of which were infected with drug-resistant tuberculosis (DR-TB). Early detection of infection and drug resistance is critical to controlling DR-TB as this enables rapid engagement into effective care. Currently, bacterial culture and nucleic acid testing remain the primary methods for diagnosing infection, with smear microscopy being phased out. However, these methods present significant limitations for diagnosing drug resistance such as lengthy time-to-result for phenotypic tests, as well as the need for prior knowledge of resistance mutations and prohibitive cost for molecular tests. To address this, we developed a rapid phenotypic TB drug susceptibility test, termed Tre-DST, based on novel trehalose probes, which upon metabolic conversion emit enhanced fluorescence signal, giving them their unique ability to specifically detect live mycobacteria. We used the nonpathogenic Mycobacterium smegmatis and the virulence-attenuated Mycobacterium tuberculosis (Mtb) H37Ra or auxotrophic Mtb to demonstrate a strong correlation between cost-effective plate reader results and flow cytometry data, suggesting the fluorescence plate reader is a suitable fluorescence detector for Tre-DST. We determined that adding a one-week incubation step for Mtb allowed samples originally seeded at 10 ⁴ CFU/mL to become detectable, over two weeks earlier than colony forming unit analysis. Importantly, we found that Tre-DST reports on drug susceptibility in a drug-agnostic manner, demonstrating loss of fluorescence with frontline TB drugs rifampicin (RIF), isoniazid (INH), and ethambutol, as well as the newer drug bedaquiline. Finally, Tre-DST distinguished RIF-and INH-resistant auxotrophs from susceptible controls and accurately reported resistance activity. Ultimately, because Tre-DST is agnostic to mechanisms of drug resistance, this assay is likely compatible with all WHO-recommended DR-TB drugs as well as any future TB drugs as a diagnostic in reference laboratories.