Fitness cost of chromosomal drug resistance-conferring mutations

Abstract

To study the cost of chromosomal drug resistance mutations to bacteria, we investigated the fitness cost of mutations that confer resistance to different classes of antibiotics affecting bacterial protein synthesis (aminocyclitols, 2-deoxystreptamines, macrolides). We used a model system based on an in vitro competition assay with defined Mycobacterium smegmatis laboratory mutants; selected mutations were introduced by genetic techniques to address the possibility that compensatory mutations ameliorate the resistance cost. We found that the chromosomal drug resistance mutations studied often had only a small fitness cost; compensatory mutations were not involved in low-cost or no-cost resistance mutations. When drug resistance mutations found in clinical isolates were considered, selection of those mutations that have little or no fitness cost in the in vitro competition assay seems to occur. These results argue against expectations that link decreased levels of antibiotic consumption with the decline in the level of resistance.

FIG. 1.

Determination of bacterial fitness by competitive growth. (A) rpsL mutants; (B) streptomycin resistance-conferring 16S rRNA mutations (spontaneous mutants); (C) streptomycin resistance-conferring 16S rRNA mutations (mutations introduced by site-directed mutagenesis). The mean of at least two independent experimental determinations is given. (A) RpsL 42 Lys→Arg (•; strain 1674); RpsL 42 Lys→Asn (▪; strain 1644); RpsL 42 Lys→Thr (▴; strain 1646). (B) 16S rRNA 522C→T (⧫ strains 1634 and 1636); 16S rRNA 523A→C (•; strain 1632); 16S rRNA 524G→C (▴; 1647); 16S rRNA 526C→T (▪; strains 1592 and 1630). (C) 16S rRNA 523A→C (•; strains 1699 and 1700); 16S rRNA 524G→C (▴; strains 1684 and 1683); 16S rRNA 526C→T (▪; strains 1687, 1688, and 1689). Cultures were inoculated with equal numbers of the drug-resistant mutant and the drug-susceptible parental strain, and the ratio of the number of resistant organisms/number of susceptible organisms recovered was determined after the indicated number of generations; M. smegmatis has a doubling time of 3 h.

FIG. 2.

Relationship between frequency of streptomycin resistance mutations and relative fitness. (A) Clinical M. tuberculosis isolates: ⧫, RpsL 42 Lys→Thr; ▾, RpsL 42 Lys→Arg; •, 16S rRNA 522C→T; ▪, 16S rRNA 523A→C; ▴, 16S rRNA 526C→T; data were tabulated from previous reports (10, 11, 15, 23, 26, 28, 40). (B) In vitro rpsL mutants of M. smegmatis mc²155: ▾, 42 Lys→Arg; ⧫, 42 Lys→Thr; •, 42 Lys→Asn; data were tabulated from reference . (C) In vitro 16S rRNA mutants of M. smegmatis rrnB rpsL3⁺: ⧫, 524G→C; ▴, 526C→T; •, 522C→T; ▪, 523A→C.