Assistant Professor UCLA Los Angeles, California, United States
Introduction: In 2020, approximately 10 million people developed Tuberculosis (TB), with nearly half a million cases of drug-resistant TB (DR-TB). A major driver of DR-TB is the lack of rapid and effective diagnostics. Only 71% of newly diagnosed TB patients are tested for rifampicin resistance, and even fewer receive comprehensive drug resistance testing. Current diagnostic methods, such as bacterial culture and nucleic acid testing, have limitations, including lengthy processing times, the need for prior knowledge of resistance mutations, and high costs. There is a critical need for fast, accurate, and cost-effective drug susceptibility tests (DSTs), especially in resource-limited settings. We previously reported on 4,4-N,N-dimethylamino-1,8-naphthalimide-trehalose (DMN-Tre), a probe targeting trehalose glycolipids on the cell surface of Mycobacterium tuberculosis (Mtb), the causative agent of TB. DMN-Tre is solvatochromic, showing a >700-fold increase in fluorescence intensity when transitioning from aqueous to hydrophobic environments upon metabolic conversion to trehalose monomycolate via Ag85 enzymatic action. This specificity to live, metabolically active Mtb allows DMN-Tre to distinguish viable from dead or drug-compromised Mtb. Here, we aim to develop a DMN-Tre based DST.
Materials and
Methods: We performed fluorescence labeling experiments on bacterial cultures under various conditions and densities. Cells were incubated with DMN-Tre probe for different time points, as indicated, and fluorescence was measured using a fluorescence plate reader, microscopy, and flow cytometry. Data were analyzed using GraphPad Prism software’s ANOVA (analysis of variance) test.
Results, Conclusions, and Discussions: We found that the optimal labeling time for Mtb cells is a 16-hour (overnight) incubation with DMN-Tre. Limit of detection (LOD) analyses with cultures of varying cell densities (10² – 10⁸ CFU/mL) revealed an LOD of 10⁷ CFU/mL under these conditions. Allowing cells to grow for 36 hours longer significantly improved the signal-to-background ratio, lowering the LOD to samples initially seeded with 100 CFU/mL. These preliminary results demonstrate that our assay could detect drug susceptibility or resistance in Mycobacterium tuberculosis clinical isolates within a few doubling times from sample collection (roughly a week) for any drug of interest. Lastly, we exposed Mtb cells to the four frontline TB drugs and observed a significant decrease in mean fluorescence intensity for Mtb cells treated with drugs compared to untreated controls, suggesting plate reader analysis—commonly found in standard TB clinics—is adequate for Tre-DST. Ultimately, Tre-DST could potentially determine resistance to any drug, including established, newly approved, and upcoming TB drugs currently in preclinical assessments and clinical trials.
