Effect of rpoB mutations conferring rifampin resistance on fitness of Mycobacterium tuberculosis

Abstract

Rifampin is a major drug used in the treatment of tuberculosis infections, and increasing rifampin resistance represents a worldwide clinical problem. Resistance to rifampin is caused by mutations in the rpoB gene, encoding the beta-subunit of RNA polymerase. We examined the effect of three different rpoB mutations on the fitness of Mycobacterium tuberculosis. Rifampin-resistant mutants were isolated from a virulent clinical isolate of M. tuberculosis (strain Harlingen) in vitro at a mutation frequency of 2.3 x 10(-8). Mutations in the rpoB gene were identified, and the growth rates of three defined mutants were measured by competition with the susceptible parent strain in laboratory medium and by single cultures in a macrophage cell line and in laboratory medium. All of the mutants showed a decreased growth rate in the three assays. The relative fitness of the mutants varied between 0.29 and 0.96 (that of the susceptible strain was set to 1.0) depending on the specific mutant and assay system. Unexpectedly, the relative fitness ranking of the mutants differed between the different assays. In conclusion, rifampin resistance is associated with a cost that is conditional.

FIG. 1.

Number of macrophages (y axis) containing the indicated number of bacteria (x axis) on days 0 (A), 3 (B), 5 (C), and 7 (D) and increases in the number of bacteria over that at day 0 (E) of the macrophage cultures for the various rifampin-resistant mutants and the susceptible control strains. (A to D) The strains used, from left to right within each group of bars, were the wild type (Harlingen), the Ser531→Trp (TCG→TGG), His526→Tyr (CAC→TAC), and Ser522→Leu (TCG→TTG) mutants, H37Ra, and H37Rv. (E) ⧫, Harlingen; ▴, Ser531→Trp (TCG→TGG) mutant; ▪, His526→Tyr (CAC→TAC) mutant; —, Ser522→Leu (TCG→TTG) mutant; ×, H37Ra; +, H37Rv.

FIG. 1.

Number of macrophages (y axis) containing the indicated number of bacteria (x axis) on days 0 (A), 3 (B), 5 (C), and 7 (D) and increases in the number of bacteria over that at day 0 (E) of the macrophage cultures for the various rifampin-resistant mutants and the susceptible control strains. (A to D) The strains used, from left to right within each group of bars, were the wild type (Harlingen), the Ser531→Trp (TCG→TGG), His526→Tyr (CAC→TAC), and Ser522→Leu (TCG→TTG) mutants, H37Ra, and H37Rv. (E) ⧫, Harlingen; ▴, Ser531→Trp (TCG→TGG) mutant; ▪, His526→Tyr (CAC→TAC) mutant; —, Ser522→Leu (TCG→TTG) mutant; ×, H37Ra; +, H37Rv.