Convergent evolutionary analysis identifies significant mutations in drug resistance targets of Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis adapts to the environment by selecting for advantageous single-nucleotide polymorphisms (SNPs). We studied whether advantageous SNPs could be distinguished from neutral mutations within genes associated with drug resistance. A total of 1,003 clinical isolates of M. tuberculosis were related phylogenetically and tested for the distribution of SNPs in putative drug resistance genes. Drug resistance-associated versus non-drug-resistance-associated SNPs in putative drug resistance genes were compared for associations with single versus multiple-branch outcomes using the chi-square and Fisher exact tests. All 286 (100%) isolates containing isoniazid (INH) resistance-associated SNPs had multibranch distributions, suggestive of multiple ancestry and convergent evolution. In contrast, all 327 (100%) isolates containing non-drug-resistance-associated SNPs were monophyletic and thus showed no evidence of convergent evolution (P < 0.001). Convergence testing was then applied to SNPs at position 481 of the iniA (Rv0342) gene and position 306 of the embB gene, both potential drug resistance targets for INH and/or ethambutol. Mutant embB306 alleles showed multibranch distributions, suggestive of convergent evolution; however, all 44 iniA(H481Q) mutations were monophyletic. In conclusion, this study validates convergence analysis as a tool for identifying mutations that cause INH resistance and explores mutations in other genes. Our results suggest that embB306 mutations are likely to confer drug resistance, while iniA(H481Q) mutations are not. This approach may be applied on a genome-wide scale to identify SNPs that impact antibiotic resistance and other types of biological fitness.

FIG. 1.

Phylogenetic locations of the study isolates. Study isolates were placed on a previously created phylogenetic tree (which had been based on an analysis of 212 SNPs in a global collection of 327 M. tuberculosis isolates). The SCGs, including subgroups, and the positions of the M. tuberculosis reference strains 210, CDC1551, and H37Rv and of M. bovis strain AF2122/97 are indicated. The total number of distinct strains, as defined by DNA fingerprinting, within each SCG is given in parentheses.

FIG. 2.

Distribution of INH-resistant SNPs. The location of each isolate containing the indicated drug resistance-associated SNPs was mapped to the phylogenetic tree shown in Fig. 1. The number of isolates harboring the mutation is shown for each allele (the number of DNA fingerprinting-defined strains is given in parentheses when it is less than the total number of isolates). (A) Mutations within katG315; (B) mutations within inhA (promoter and open reading frame); (C) mutations within the promoter region of ahpC (except for position −46, which is a non-drug-resistant SNP).

FIG. 3.

Distribution of non-drug-resistance-associated SNPs. The location of each isolate containing a SNP known to be found in both INH-resistant and INH-susceptible isolates is shown. The number of isolates harboring the mutation is shown for each allele (the number of DNA fingerprinting-defined strains is given in parentheses when it is less than the total number of isolates). (A) Mutations at positions 269 and 312 of kasA, −46 and 73 of ahpC, 18 of ndh, and 95 of gyrA. The colored line connecting the gyrA(T95S) isolates indicates that all isolates in these two SCGs contained this mutation, a result suggestive of a single mutational event in a common ancestor. (B) Mutations found in inhA at position 194 (I194T). The asterisk indicates the single isolate with an “intermediate” INH resistance phenotype; both of the other isolates were INH resistant.

FIG. 4.

(A) Distribution of mutant embB306 alleles. The location of each isolate containing a mutant embB306 allele is shown. The number of isolates harboring the mutation is shown for each allele (the number of DNA fingerprinting-defined strains is given in parentheses when it is less than the total number of isolates). (B) Location of isolates containing a mutant iniA(H481Q) allele. The number of isolates harboring the mutation is shown, and the number of DNA fingerprinting-defined strains is given in parentheses.