Despite the administration of multiple drugs that are highly effective in vitro,
tuberculosis (TB) treatment requires prolonged
drug administration and is confounded by the emergence of
drug-resistant strains. To understand the mechanisms that limit
antibiotic efficacy, we performed a comprehensive genetic study to identify Mycobacterium tuberculosis genes that alter the rate of bacterial clearance in
drug-treated mice. Several functionally distinct bacterial genes were found to alter bacterial clearance, and prominent among these was the glpK gene that encodes the glycerol-3-kinase
enzyme that is necessary for
glycerol catabolism. Growth
on glycerol generally increased the sensitivity of M.
tuberculosis to
antibiotics in vitro, and glpK-deficient bacteria persisted during
antibiotic treatment in vivo, particularly during exposure to
pyrazinamide-containing regimens. Frameshift mutations in a hypervariable homopolymeric region of the glpK gene were found to be a specific marker of multidrug resistance in clinical M.
tuberculosis isolates, and these loss-of-function alleles were also enriched in extensively
drug-resistant clones. These data indicate that frequently observed variation in the glpK coding sequence produces a
drug-tolerant phenotype that can reduce
antibiotic efficacy and may contribute to the evolution of resistance.IMPORTANCE TB control is limited in part by the length of
antibiotic treatment needed to prevent recurrent disease. To probe mechanisms underlying survival under
antibiotic pressure, we performed a genetic screen for M.
tuberculosis mutants with altered susceptibility to treatment using the mouse model of TB. We identified multiple genes involved in a range of functions which alter sensitivity to
antibiotics. In particular, we found
glycerol catabolism mutants were less susceptible to treatment and that common variation in a homopolymeric region in the glpK gene was associated with drug resistance in clinical isolates. These studies indicate that reversible high-frequency variation in
carbon metabolic pathways can produce phenotypically
drug-tolerant clones and have a role in the development of resistance.