Whole Genome Sequencing Based Characterization of Extensively Drug-Resistant <i>Mycobacterium tuberculosis</i> Isolates from Pakistan

<div><p>Improved molecular diagnostic methods for detection drug resistance in <i>Mycobacterium tuberculosis</i> (MTB) strains are required. Resistance to first- and second- line anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, these SNPs can vary between MTB lineages therefore local data is required to describe different strain populations. We used whole genome sequencing (WGS) to characterize 37 extensively drug-resistant (XDR) MTB isolates from Pakistan and investigated 40 genes associated with drug resistance. Rifampicin resistance was attributable to SNPs in the <i>rpo</i>B hot-spot region. Isoniazid resistance was most commonly associated with the <i>kat</i>G codon 315 (92%) mutation followed by <i>inh</i>A S94A (8%) however, one strain did not have SNPs in <i>kat</i>G, <i>inh</i>A or <i>oxy</i>R-<i>ahp</i>C. All strains were pyrazimamide resistant but only 43% had <i>pnc</i>A SNPs. Ethambutol resistant strains predominantly had <i>emb</i>B codon 306 (62%) mutations, but additional SNPs at <i>emb</i>B codons 406, 378 and 328 were also present. Fluoroquinolone resistance was associated with <i>gyr</i>A 91–94 codons in 81% of strains; four strains had only <i>gyr</i>B mutations, while others did not have SNPs in either <i>gyr</i>A or <i>gyr</i>B. Streptomycin resistant strains had mutations in ribosomal RNA genes; <i>rps</i>L codon 43 (42%); <i>rrs</i> 500 region (16%), and <i>gid</i>B (34%) while six strains did not have mutations in any of these genes. Amikacin/kanamycin/capreomycin resistance was associated with SNPs in <i>rrs</i> at nt1401 (78%) and nt1484 (3%), except in seven (19%) strains. We estimate that if only the common hot-spot region targets of current commercial assays were used, the concordance between phenotypic and genotypic testing for these XDR strains would vary between rifampicin (100%), isoniazid (92%), flouroquinolones (81%), aminoglycoside (78%) and ethambutol (62%); while <i>pnc</i>A sequencing would provide genotypic resistance in less than half the isolates. This work highlights the importance of expanded targets for drug resistance detection in MTB isolates.</p>