Navigating a Path to Rifampicin Resistance in Tuberculosis

For model bacteria, genetic drug resistance usually arises from antibiotic-tolerant subpopulations, but whether this is true for the globally important pathogen Mycobacterium tuberculosis-the cause of tuberculosis-is not known. Here, we discuss a recent article by Sebastian et al. (J. Sebastian, A. Thomas, C. Levine, R. Shrestha, et al., mBio 14:e0279522, 2023, 10.1128/mbio.02795-22) which leverages a robotic transwell microtiter experimental system coupled with deep sequencing of a barcoded library of M. tuberculosis to answer this question for rifampicin resistance. The authors investigate two distinct forms of antibiotic-tolerant subpopulations-classical tolerance, characterized by prolonged minimum duration of killing, and "differentially detectable" (DD) bacilli that are viable but can be recovered only in liquid medium as opposed to plating. They demonstrate that, indeed, resistance arises preferentially from both rifampicin-tolerant subpopulations, though earlier in the DD population. Use of barcoded libraries and parallel culture systems shows promise in investigating phenotypes mediated by minority subpopulations of bacteria such as development of antibiotic resistance.