Rapid Phenotypic Drug Susceptibility Testing of Mycobacterium tuberculosis Using Raman-Deuterium Isotope Probing

Area of Science:

• Microbiology
• Spectroscopy
• Drug Discovery

Background:

• Accurate drug susceptibility testing (DST) is crucial for managing tuberculosis (TB), particularly with rising multidrug-resistant Mycobacterium tuberculosis (MTB).
• Conventional phenotypic DST (pDST) is accurate but slow, while molecular methods may miss novel resistance mechanisms.
• There is a need for rapid, reliable DST methods to guide timely TB treatment.

Purpose of the Study:

• To develop and validate a rapid phenotypic DST method for Mycobacterium tuberculosis using single-cell Raman spectroscopy and deuterium isotope probing (Raman-DIP).
• To assess the performance of Raman-DIP for classifying drug-sensitive and drug-resistant MTB strains and determining minimum inhibitory concentrations (MICs).
• To evaluate the feasibility of Raman-DIP for TB diagnostics in simulated clinical samples.

Main Methods:

• Optimized a Raman-DIP workflow for MTB, including antibiotic exposure and D₂O labeling.
• Tested the method with five MTB strains and four first-line drugs (RIF, INH, STR, EMB).
• Compared Raman-DIP results with conventional broth-based DST and determined MICs.

Main Results:

• Raman-DIP achieved categorical resistance classification and MIC determination in approximately 50 hours (2 days).
• Demonstrated 100% categorical agreement with reference DST methods for the tested strains.
• Showed 100% classification accuracy in simulated sputum samples and 100% agreement for MIC values.

Conclusions:

• Raman-DIP provides a rapid, phenotypic, and biosafe platform for MTB DST, significantly reducing turnaround time compared to conventional methods.
• The method offers both categorical and quantitative phenotypic readouts, adaptable for point-of-care testing.
• Raman-DIP supports more timely and individualized TB therapy decisions.