Schwartz, L. A. | Search bioRxiv

bioRxiv2026

Expanding the palette of trehalose-based fluorophores for live mycobacterial detection

Tuberculosis (TB) remains the worlds leading infectious cause of death. Trehalose-based fluorogenic probes have emerged as powerful tools for labeling and studying mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. However, existing probes occupy a limited spectral range and require compromise between brightness, specificity, and functional readouts. Here, we report the design and characterization of two trehalose conjugates derived from Janelia Fluor(R) dyes, JF635-Tre and JF646-Tre, which extend the trehalose-based platform into the far-red region. Following NHS ester-mediated synthesis, the trehalose-conjugated analogs displayed strong far-red fluorescence, with excitation/emission maxima at 638/654 nm and 648/663 nm for JF635-Tre and JF646-Tre, respectively. Both probes exhibited concentration- and time-dependent labeling of Mycobacterium smegmatis (Msmeg) and Mtb with minimal background fluorescence from the corresponding unconjugated dyes. Furthermore, we observed reduced labeling in heat-killed cells compared to live Msmeg, particularly for JF646-Tre, consistent with sensitivity to metabolic activity. Both JF-Tre derivatives produced significant cellular labeling and JF635-Tre distinguished untreated from INH-treated samples in drug susceptible Mtb, demonstrating the potential of the JF-Tre probes to report on INH susceptibility and resistance. Together, these findings expand the toolkit of trehalose-based probes and highlight how fluorophore identity influences probe performance in mycobacterial fluorescence imaging and drug susceptibility testing.

biochemistry↗

bioRxiv2025

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 104 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.

microbiology↗