Whole-Genome Analysis of Drug Resistance and Transmission Patterns in Mycobacterium tuberculosis from Khyber Pakhtunkhwa, Pakistan

The emergence of drug-resistant Mycobacterium tuberculosis strains poses a serious challenge to tuberculosis (TB) control programs worldwide, particularly in high-burden regions like Pakistan. This study aimed to characterise 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, with the identification of variants (e.g., 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. Although all four major M. tuberculosis lineages (L1–L4) were detected, 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 common 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 , which may confer increased bedaquiline susceptibility. Phylogenetic analysis using 6,635 genome-wide identified two transmission clusters (<9 SNPs difference), suggesting ongoing local transmission. This study highlights the predominance of drug-resistant M. tuberculosis strains in Khyber Pakhtunkhwa, encompassing both globally recognised and novel resistance-associated mutations. These 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.