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

Accurate and timely drug susceptibility testing (DST) is essential for tuberculosis (TB) management, especially given the global rise of multidrug-resistant Mycobacterium tuberculosis (MTB). However, conventional phenotypic DST (pDST) is reliable but time-consuming, while molecular assays may fail to detect resistance not associated with known mutations. Here, we report a rapid pDST method using single-cell Raman spectroscopy coupled with deuterium isotope probing (Raman-DIP) to assess MTB metabolic activity when exposed to different antibiotics. The Raman-DIP workflow was specifically optimized for MTB, including antibiotic exposure time and D₂O labeling conditions. Using five MTB strains and four first-line drugs (rifampicin [RIF], isoniazid [INH], streptomycin [STR], ethambutol [EMB]), we demonstrated categorical resistance classification and determined minimum inhibitory concentrations (MICs) in about 2 days (50 h). In the tested strain set, Raman-DIP clearly discriminated drug-sensitive and drug-resistant strains, showing 100% categorical agreement with the reference broth-based DST method. In simulated sputum samples, Raman-DIP showed 100% classification accuracy in the tested strain set, supporting preliminary feasibility in a sputum-like matrix. Raman-DIP further enabled quantitative MIC determination, with 100% agreement to reference MIC values in the strain-drug combinations examined. This novel workflow directly measures antibiotic-induced metabolic suppression without cultivation, substantially reducing turnaround time to ∼2 days compared with the weeks typically required for conventional pDST. The approach provides both categorical and quantitative phenotypic readouts and is readily adaptable to point-of-care testing (POCT). In conclusion, Raman-DIP provides a rapid, phenotypic-based, and biosafe platform for MTB DST, supporting more timely and individualized TB therapy.