Genetic Characterization of First-Line Drug-Resistance Mutations in Multidrug-Resistant Mycobacterium tuberculosis

Background: Resistance to first-line anti-tuberculosis drugs in Mycobacterium tuberculosis represents a significant public health challenge, particularly in high-burden tuberculosis (TB) settings such as Pakistan, where multidrug-resistant (MDR) forms further complicate disease control efforts. Drug resistance is primarily associated with mutations in rpoB, inhA, katG, embA, embB, embC, and pncA. The emergence of novel, region-specific variants underscores the urgent need for integrating genomic surveillance into routine TB diagnostics and regional control programs. This study aimed to identify the spectrum of mutations contributing to first-line drug resistance in MDR-TB isolates from Khyber Pakhtunkhwa, Pakistan. Methods: Whole-genome sequencing was performed on 16 clinical isolates (12 MDR and 4 drug-susceptible) to identify resistance-associated mutations in rpoB, inhA, katG, embA, embB, embC, and pncA. Detected variants were interpreted using the World Health Organization (WHO) mutation catalogue to determine their association with drug resistance. Phylogenetic relationships were inferred using the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) platform. Results: A total of 16 M. tuberculosis isolates were analyzed to evaluate resistance to first-line anti-tuberculosis drugs. In rpoB, 76 distinct variants were identified, including canonical mutations such as Ser450Leu and His445Arg, as well as a potentially novel substitution, Ser431Phe, predicted to confer high-level rifampicin resistance. The katG and inhA genes harbored 24 and 27 mutations, respectively, including well-characterized substitutions such as Ser315Thr and Ala114Glu, which are strongly associated with isoniazid resistance. Mutations in embA and embB were linked to ethambutol resistance, with several variants localized within conserved transmembrane domains critical for drug interaction. Phylogenetic analysis revealed substantial genetic diversity and evidence of local transmission among MDR-TB isolates. Conclusions: This study suggests that the genetic landscape of drug resistance in M. tuberculosis is highly dynamic in endemic regions. The findings highlight the importance of integrating region-specific mutation profiles into molecular diagnostic frameworks to enhance early detection, guide individualized therapeutic interventions, and strengthen strategies aimed at controlling the transmission of MDR-TB.