Effective treatment of tuberculosis requires at least six months of combination therapy involving four antibiotics. Alterations in the physiological state of Mycobacterium tuberculosis during infection may reduce drug efficacy and prolong treatment, but these adaptations are incompletely defined. To investigate the mechanisms limiting antibiotic efficacy, I performed a comprehensive genetic study to identify M. tuberculosis genes and pathways important for bacterial survival during antibiotic treatment in vivo. First, I identified mutants in the glycerol kinase enzyme, GlpK, that promote survival under combination therapy. Similar glycerol catabolic mutants are enriched in extensively drug-resistant clinical isolates, indicating that these mutations may promote survival and the development of resistance in humans. A majority of these mutations are frameshifts within a homopolymeric region of the glpK gene, leading to the hypothesis that M. tuberculosis may reversibly produce drug-tolerant phenotypes through genetic variation introduced at homopolymer sites as a strategy for survival during antibiotic treatment. Second, I identified bacterial mutants with altered susceptibility to individual first-line anti-mycobacterial drugs. Many of these mutations did not have obvious effects in vitro, demonstrating that a wide variety of natural genetic variants can influence drug efficacy in vivo without altering standard drug-susceptibility tests. A number of these genes are enriched in drug-resistant clinical isolates, indicating that these genetic variants influence treatment outcome. Together, these data suggest new targets for improving therapy, as well as mechanisms of genetic adaptations that can reduce antibiotic efficacy and contribute to the evolution of resistance
