Emerging Rifampicin Resistance in M. tuberculosis: First Report on Characterization of a Novel rpoB(Ser431Phe) Mutation and Phylogenetic Lineages in Khyber Pakhtunkhwa, Pakistan

Background First-line drug resistance in Mycobacterium tuberculosis (MTB) is a major challenge in high-burden settings like Pakistan, primarily linked to mutations in rpoB, inhA, katG, embA, embB, embC , and pncA . The emergence of novel, region-specific variants underscores the need for comprehensive genomic profiling to enhance diagnostics, inform treatment, and strengthen MDR-TB control strategies. This study aimed to unravel the genetic landscape of first-line drug Resistance in multidrug-resistant Mycobacterium tuberculosis. Methods Whole-genome sequencing (WGS) of 16 Multi-Drug Resistant (MDR-TB) isolates from Khyber Pakhtunkhwa, Pakistan, was performed to identify resistance-conferring mutations in rpoB,inhA, katG, embA, embB, embC , and pncA genes . Mutations were checked for structural impacts, including polarity, hydrophobicity, and charge, and validated through the WHO 2023 mutation catalogue. Phylogenetic analysis was performed using BV-BRC. Results The mutational landscape of M. tuberculosis (MTB) isolates was analyzed to characterize resistance to first-line anti-TB drugs. In the rpoB gene, A total of 76 distinct mutations were identified, including missense substitutions, frameshift mutations, and in-frame deletions. Among these, notable variants such as Ser450Leu, His445Arg, Val243Thr, and Asp435Tyr were observed, along with potentially novel mutations like Ser431Phe, which has not been reported in the WHO 2023 catalog. Frameshift mutations such as 1917delA and 1029_1030ins are of particular concern, as they may confer high-level drug resistance. The katG gene, associated with isoniazid resistance, harbored 24 mutations, including the well-characterized Ser315Thr and Arg463Leu variants. Similarly, 27 mutations were detected in the inhA gene, with Ala114Glu and Ala235Pro contributing significantly to isoniazid resistance. For ethambutol resistance, the embA gene carried 21 mutations, while the embB gene exhibited multiple substitutions and frameshifts. Phylogenetic analysis further revealed substantial genetic diversity and evidence of regional transmission, underscoring the urgent need for improved molecular diagnostics and tailored treatment strategies that account for both common and novel resistance mutations in endemic regions. Conclusion This study reveals genetic diversity and novel mutations linked to first-line drug resistance in Mycobacterium tuberculosis from Khyber Pakhtunkhwa, offering insights to improve diagnostics, treatment, and TB control.