Population genetics study of isoniazid resistance mutations and evolution of multidrug-resistant Mycobacterium tuberculosis

Abstract

The molecular basis for isoniazid resistance in Mycobacterium tuberculosis is complex. Putative isoniazid resistance mutations have been identified in katG, ahpC, inhA, kasA, and ndh. However, small sample sizes and related potential biases in sample selection have precluded the development of statistically valid and significant population genetic analyses of clinical isoniazid resistance. We present the first large-scale analysis of 240 alleles previously associated with isoniazid resistance in a diverse set of 608 isoniazid-susceptible and 403 isoniazid-resistant clinical M. tuberculosis isolates. We detected 12 mutant alleles in isoniazid-susceptible isolates, suggesting that these alleles are not involved in isoniazid resistance. However, mutations in katG, ahpC, and inhA were strongly associated with isoniazid resistance, while kasA mutations were associated with isoniazid susceptibility. Remarkably, the distribution of isoniazid resistance-associated mutations was different in isoniazid-monoresistant isolates from that in multidrug-resistant isolates, with significantly fewer isoniazid resistance mutations in the isoniazid-monoresistant group. Mutations in katG315 were significantly more common in the multidrug-resistant isolates. Conversely, mutations in the inhA promoter were significantly more common in isoniazid-monoresistant isolates. We tested for interactions among mutations and resistance to different drugs. Mutations in katG, ahpC, and inhA were associated with rifampin resistance, but only katG315 mutations were associated with ethambutol resistance. There was also a significant inverse association between katG315 mutations and mutations in ahpC or inhA and between mutations in kasA and mutations in ahpC. Our results suggest that isoniazid resistance and the evolution of multidrug-resistant strains are complex dynamic processes that may be influenced by interactions between genes and drug-resistant phenotypes.

FIG. 1.

Alleles screened in this study. The ORF of each gene is shown as a thick line. Intergenic regions are indicated by thin lines. The position of each allele examined in this study is indicated to the left of the lines. Alleles tested inside an ORF are indicated by their codon positions; alleles tested within an intergenic region are indicated by their nucleotide positions relative to the start of the following ORF, except for ahpC, which is shown relative to the transcription start. The specific mutations examined in the HP assays are indicated in capital letters to the right of each gene. Several mutations are indicated when more than one mutant allele was tested at the same codon or nucleotide position. Deletions are indicated with the symbol “Δ,” and insertions are indicated with colons. Question marks (?) indicate the presence of a mutation detected and confirmed by a molecular beacon assay, but the exact nucleotide change was not identified because the sequencing results are inconclusive at that position. The total sequence examined by each molecular beacon is indicated by a line labeled with the name of that molecular beacon, starting with an MB prefix. Sequence accession numbers for katG, ahpC, kasA, ndh, and inhA are Z97193, Z79701, Z70692, BX842577, and Z79701, respectively. Genes and upstream regions are not drawn to scale. The sequences of the primers, molecular beacons, and sequencing primers used to detect or confirm these alleles are shown in Tables S1 to S3 in the supplemental material.

FIG. 2.

Distribution of INH resistance-associated mutations in drug-resistant clinical M. tuberculosis isolates. The proportion of M. tuberculosis isolates with detectable INH resistance-associated mutations and the number of mutations detected in each isolate are shown according to the drug susceptibility pattern of the M. tuberculosis isolate.

FIG. 3.

Distributions of mutations in specific genes. The proportions of isolates with INH resistance-associated mutations in the katG, inhA, and ahpC genes are shown according to the drug susceptibility patterns of the M. tuberculosis isolates. (A) Results using all isolates in the study; (B) results using all unique isolates plus one from each cluster. In cases where a cluster contained isolates with different drug susceptibility profiles and/or mutations, each variation was counted as a new cluster. *, P < 0.01; **, P = 0.034.