A forward genetic methodology identifies a metabolic determinant of mycobacterial phenotypic tolerance to antibiotics.

When challenged with an antibiotic, a bacterium can survive by acquiring resistance mutations or genes that also confer resistance on its clonal progeny or can become transiently tolerant to the antibiotic while its progeny remain susceptible. The latter phenomenon is called phenotypic tolerance or persistence and is a source of treatment failure, a major contributor to infection relapse, and likely a catalyst of bona fide resistance. This makes persisters an important target for new therapeutic approaches, but a mechanistic understanding of persistence is lacking. High-persistence (hip) mutations that cause a population of cells to form persister cells at a higher frequency than a wild-type population under the same conditions have been reported in various bacterial species, and the study of such mutations promises to lead to a better understanding of the molecular mechanisms of persistence. To isolate hip mutants in mycobacteria, we have developed a novel in vitro method in which persisters are separated spatially from resisters upon exposure to a drug. Using this method, we isolated 29 Mycobacterium smegmatis hip mutants on three distinct antibiotics and sequenced their genomes to identify candidate hip mutations. Studying the mutations present in these mutants and the mechanisms by which they bolster persistence will enhance our understanding of this phenomenon in mycobacteria.