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Technologies for Emerging Infectious Diseases

Technologies for Emerging Infectious Diseases - Poster Session C

Poster K16 - Quantifying drug effects on Mycobacteria via fluorescence on a plate reader towards development of drug susceptibility test

Friday, October 25, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the second-most infectious disease estimated to infect more than 10 million people a year. About 4.5% of new cases report drug-resistance to one or more of the four first-line drugs, isoniazid (INH), rifampicin, pyrazinamide, and ethambutol. Increased resistance is attributed to inadequate medical systems and incorrect treatments informed by phenotypic susceptibility tests through directly observed therapy. Insufficient lab capacity, resources, and repeated courses of treatment can amplify resistance patterns in the local populations. The rapid transmission of TB in these communities highlights a need for a faster, more accessible, and reliable diagnostic. Current methods include the TB skin test, blood test, chest radiography, sputum smear microscopy, and interferon gamma release assays. Rapid molecular diagnostic tests recommended by the World Health Organization include the Xpert MTB/RIF Ultra and Truenat assays, which can detect drug resistance early; however, detection of multi-drug resistant TB, latent TB, and HIV-associated TB remains expensive, complex, and difficult for high TB burden communities to access. Several trehalose-based probes have been developed utilizing metabolic pathways that uptake trehalose for lipidation and insertion into the mycomembrane, a phenotype unique to mycobacteria. Trehalose conjugated to a solvatochromic fluorophore results in the mycobacteria-specific probe 4-N,N-Dimethylamino-1,8-naphthalimide (DMN-Tre), which can report drug susceptibility to INH in Mycobacterium smegmatis (Msmeg), a non-pathogenic close relative to Mtb.

Materials and

Methods:  Wild type (WT) Msmeg was cultured to an optical density (OD₆₀₀) of 0.1, then treated with varying concentrations of INH for different incubation periods, notably 6, 9, 12, and 15 hours (corresponding with 2, 3, 4, and 5 doubling times respectively). Labeling with 100 µM DMN-Tre followed the drug treatment to investigate how probe kinetics change over time with different drug treatments. 30 minutes after labeling, we measured the OD₆₀₀ and fluorescence intensity of the samples on a plate reader. For the 9 hour incubation experiments, fluorescent readings were confirmed with flow cytometry and OD₆₀₀ was confirmed by colony forming unit (CFU/mL) assays to more thoroughly investigate drug effects, and to corroborate plate reader readout with more rigorous methods. The protocol at the 9 hour time point was repeated for INH-resistant Msmeg (∆KatG) to investigate whether DMN-Tre reports drug resistance.

Results, Conclusions, and Discussions: The plate reader can detect changes in fluorescence based on drug treatment intensity, and correlates with flow cytometry and CFU/mL. No-drug and vehicle controls exhibited highest fluorescent values of DMN-Tre treated samples as well as highest OD₆₀₀, and we observed a stepwise decrease in the OD₆₀₀ and fluorescence values from the plate reader, flow cytometer, and CFU/mL with increasing drug concentrations. We also show we can detect drug effects at a much earlier doubling period compared to the existing culture method, with detection at 9 hours, or 3 doubling times. Comparing the WT to ∆KatG at this time point shows a clear distinction in fluorescence between susceptible and resistant mycobacteria, notably at the minimum inhibitory concentration of INH. Additionally, our results indicated that the fluorescence of the samples more widely detected differing drug concentrations in comparison to OD₆₀₀, suggesting greater the probes’ ability to distinguish between different drug treatments with greater resolution. The use of a plate reader as a low-cost readout is affordable for high TB burden communities, and the simple workflow and rapid detection can allow for potential multiplexibility of testing multiple drugs at the same time. DMN-Tre is a novel probe that can detect the presence of mycobacteria and report susceptibility of Msmeg to varying drug concentrations at earlier times compared to existing culturing methods.

Acknowledgements (Optional):  I would like to thank Professor Mireille Kamariza and The Kamariza Lab for providing me with the opportunity to conduct this research, and to the UC Leadership Excellence through Advanced Degrees (UC LEADS) program for their financial support.