Origin and Dynamics of Mycobacterium tuberculosis Subpopulations That Predictably Generate Drug Tolerance and Resistance

Tuberculosis is unusual among bacterial diseases in that treatments which can rapidly resolve symptoms do not predictably lead to a durable cure unless treatment is continued for months after all clinical and microbiological signs of disease have been eradicated. Using a novel steady-state antibiotic exposure system combined with chromosomal barcoding, we identified small hidden Mycobacterium tuberculosis subpopulations that repeatedly enter a state of drug tolerance with a predisposition to develop fixed drug resistance after first developing a cloud of unfixed resistance mutations. Initial responses to tuberculosis treatment are poor predictors of final therapeutic outcomes in drug-susceptible disease, suggesting that treatment success depends on features that are hidden within a small minority of the overall infecting Mycobacterium tuberculosis population. We developed a multitranswell robotic system to perform numerous parallel cultures of genetically barcoded M. tuberculosis exposed to steady-state concentrations of rifampicin to uncover these difficult-to-eliminate minority populations. We found that tolerance emerged repeatedly from at least two subpopulations of barcoded cells, namely, one that could not grow on solid agar media and a second that could form colonies, but whose kill curves diverged from the general bacterial population within 4 and 16 days of drug exposure, respectively. These tolerant subpopulations reproducibly passed through a phase characterized by multiple unfixed resistance mutations followed by emergent drug resistance in some cultures. Barcodes associated with drug resistance identified an especially privileged subpopulation that was rarely eliminated despite 20 days of drug treatment even in cultures that did not contain any drug-resistant mutants. The association of this evolutionary scenario with a defined subset of barcodes across multiple independent cultures suggested a transiently heritable phenotype, and indeed, glpK phase variation mutants were associated with up to 16% of the resistant cultures. Drug tolerance and resistance were eliminated in a Delta ruvA mutant, consistent with the importance of bacterial stress responses. This work provides a window into the origin and dynamics of bacterial drug-tolerant subpopulations whose elimination may be critical for developing rapid and resistance-free cures.IMPORTANCE Tuberculosis is unusual among bacterial diseases in that treatments which can rapidly resolve symptoms do not predictably lead to a durable cure unless treatment is continued for months after all clinical and microbiological signs of disease have been eradicated. Using a novel steady-state antibiotic exposure system combined with chromosomal barcoding, we identified small hidden Mycobacterium tuberculosis subpopulations that repeatedly enter a state of drug tolerance with a predisposition to develop fixed drug resistance after first developing a cloud of unfixed resistance mutations. The existence of these difficult-to-eradicate subpopulations may explain the need for extended treatment regimen for tuberculosis. Their identification provides opportunities to test genetic and therapeutic approaches that may result in shorter and more effective TB treatments.

Automated summary

Tuberculosis requires continued treatment for months to achieve a durable cure. A study identified small hidden subpopulations of Mycobacterium tuberculosis that repeatedly enter a state of drug tolerance and have a predisposition to develop drug resistance. These difficult-to-eliminate subpopulations may explain why tuberculosis treatment requires extended regimens. The findings provide opportunities for genetic and therapeutic approaches to develop shorter and more effective tuberculosis treatments.