Molecular analysis of isoniazid-resistant Mycobacterium tuberculosis isolates from England and Wales reveals the phylogenetic significance of the ahpC -46A polymorphism

Abstract

The present study investigated the prevalence and diagnostic potential of the most commonly reported mutations associated with isoniazid resistance, katG 315Thr, katG 315Asn, inhA -15T, inhA -8A, and the oxyR-ahpC intergenic region, in a population sample of 202 isoniazid-resistant Mycobacterium tuberculosis isolates and 176 randomly selected fully sensitive isolates from England and Wales identified by using a directed oligonucleotide array and limited DNA sequencing. The strains were recovered from patients originating from 29 countries; 41 isolates were multidrug resistant. Mutations affecting katG 315, the inhA promoter, and the oxyR-ahpC intergenic region were found in 62.7, 21.9, and 30% of 169 genotypically distinct isoniazid-resistant isolates, respectively, whereas they were found in 0, 0, and 8% of susceptible strains, respectively. The frequency of mutation at each locus was unrelated to the resistance profile or previous antituberculous drug therapy. The commonest mutation in the oxyR-ahpC intergenic region, ahpC -46A, was present in 23.7% of isoniazid-resistant isolates and 7.5% of susceptible isolates. This proved to be a phylogenetic marker for a subgroup of M. tuberculosis strains originating on the Indian subcontinent, which shared IS6110-based restriction fragment length polymorphism and spoligotype features with the Delhi strain and Central Asian strain CAS1; and this marker is strongly associated with isoniazid resistance and the katG 315Thr mutation. In total, 82.8% of unrelated isoniazid-resistant isolates could be identified by analysis of just two loci: katG 315 and the inhA promoter. Analysis of the oxyR-ahpC intergenic region, although phylogenetically interesting, does not contribute significantly to further identification of isoniazid-resistant isolates.

FIG. 1.

Hybridization patterns obtained with the directed low-density oligonucleotide PCR and hybridization array. Probe numbers are indicated at the bottom of the array. Probes 1 to 3 target inhA (probe 1, inhA −8A; probe 2, inhA −15T; probe 3, wild-type inhA promoter). Probes 4 to 6 target the oxyR-ahpC intergenic region (probe 4, positions −50 to −27; probe 5, positions −30 to −10; probe 6, positions −12 to +9 with respect to the transcriptional start site). Probes 7 to 8 target the katG 463 polymorphism [probe 7, katG 463Leu(CTG); probe 8, katG 463Arg(CCG)], and probes 9 to 11 target katG 315 [probe 9, katG 315Thr(ACC); probe 10, katG 315Asn(AAC); probe 11, katG 315Ser(AGC)]. Strip inh 11 is the negative control, and strips inh 12 to 18 are examples of hybridization patterns obtained with test isolates.

FIG. 2.

IS6110 RFLP and spoligotype patterns for isolates possessing the oxyR-ahpC intergenic mutation −46A.