Culture-Free Metabolic Labeling Analysis for Point-of-Care Antimicrobial Efficacy Evaluation of Tuberculosis.

Rapid antimicrobial efficacy evaluation of Mycobacterium tuberculosis (Mtb) is critical for administration of appropriate antibiotics, but traditional culturing methodsare a time-consuming process and hard toavoid overtreatment, leading to severe organfailure or even death. Herein, we developedTPAPy-Tre, a cytoderm metabolic-labeling probetargeting mycomembrane biosynthesis, todynamically visualize single-bacterium metabolic responses to antibiotics. TPAPy-Tre revealed drug-concentration-dependent metabolic fingerprints (fluorescence intensity, pole, cell length) in single Mtb. Stratification of these phenotypic fingerprints accurately assessed bacterial proliferation inhibition and antimicrobial efficacy in vitro. Validated in Mtb-infected mice and tuberculosis (TB) patients, this technique identified drug responders/non-responders within 5 days, reducing turnaround time by over 50 days compared with culture-based 8-week testing. Critically, TPAPy-Tre realized noninvasive therapy monitoring using paucibacillary samples from a suspected TB case. This culture-free metabolic labeling approach enables ultra-rapid drug efficacy evaluation at single-cell resolution, advancing personalized TB therapy and antimicrobial resistance containment.