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We introduce single-cell analysis for isoniazid-treated  Mycobacterium smegmatis  mutant,  msm1946-NADH pyrophosphatase , using microfluidics and automated time-lapse microscopy. Mycobacterial NADH pyrophosphatase isoforms play an important role for the mechanism of isoniazid and ethionamide activation. Our single-cell analysis revealed important insights on isoniazid killing mechanism that was masked by traditional killing assays, raised significant questions related to viable but non-culturable subpopulation of cells, and existing methods that defines minimum inhibitory concentration of drugs. The major goal of this study was quantitatively analyze bacterial cell parameters to obtain high-resolution data for the time evolution of antibiotic killing at the single-cell level. The presented tools and methods could be applied to the closely related organisms to provide more detailed information for the design and employment of antibiotic treatments.

Isoniazid Killing of Mycobacterium smegmatis NADH Pyrophosphatase Mutant at Single-Cell Level using Microfluidics and Time-Lapse Microscopy