Whole-genome analysis of drug resistance and transmission patterns in Mycobacterium tuberculosis from Khyber Pakhtunkhwa, Pakistan

Abstract

Abstract The emergence of drug-resistant Mycobacterium tuberculosis strains presents a significant challenge to tuberculosis (TB) control programmes worldwide, particularly in high-burden countries, such as Pakistan. This study characterises the genomic diversity and drug resistance mutation profiles of M. tuberculosis isolates from Khyber Pakhtunkhwa using whole-genome sequencing (WGS). A total of 35 clinical M. tuberculosis isolates underwent WGS. Sequence data were quality-filtered and mapped to the H37Rv reference genome, characterising variants, including single-nucleotide polymorphisms (SNPs). Lineage classification, drug resistance profiling, and identification of resistance mutations were performed using TB-Profiler software. SNP-based clustering was used to infer recent transmission events. All four major  M. tuberculosis  lineages (L1–L4) were detected; however, more than half of the isolates belonged to lineage L3 (CAS, n  = 19; 54.3%). Almost all isolates were drug-resistant (34/35), including pre-extensively drug-resistant (pre-XDR,  n  = 15; 42.9%) and multidrug-resistant (MDR,  n  = 14; 40.0%) strains. The most frequent resistance-associated mutations included  katG S315T (27/35) and  inhA c.-777C > T (4/35) for isoniazid resistance,  rpoB S450L (24/35) for rifampicin resistance,  embB M306I/V (18/35) for ethambutol resistance, and  gyrA D94G (8/35) and A90V (5/35) for fluoroquinolone resistance. Compensatory mutations were observed in  rpoC and  ahpC . Mutations associated with bedaquiline and clofazimine resistance ( mmpR5 c.140dupA, c.138_139dupTG) were detected in single isolates. Interestingly, one isolate exhibited a deletion encompassing  mmpR5, mmpL5 , and mmpS5 , potentially increasing bedaquiline susceptibility. Phylogenetic analysis using 6,635 genome-wide SNPs identified two transmission clusters with < 9 SNPs differences, suggesting ongoing local transmission. These results highlight the presence of drug-resistant  M. tuberculosis  strains in Khyber Pakhtunkhwa, encompassing both globally recognised and novel resistance-associated mutations. Our findings underscore the critical need to integrate WGS into TB surveillance and control programmes in Pakistan to guide targeted interventions and curb the spread of resistant strains.