Mutational Profiling of ESX/Type VII Secretion System-Associated Genes in Drug-Resistant Mycobacterium tuberculosis from Khyber Pakhtunkhwa, Pakistan

Tuberculosis (TB) remains a major global public health challenge, and the increasing burden of drug-resistant Mycobacterium tuberculosis (MTB) strains poses a significant obstacle to the success of TB control programs. This study aimed to characterize the phenotypic and genotypic drug-resistance profiles and to investigate mutations in ESX/type VII secretion system-associated secretory genes. Whole-genome sequencing (WGS) was performed to detect mutations behind resistance to rifampicin ( rpoB, rpoC ), isoniazid ( katG , inhA ), fluoroquinolones ( gyrA , gyrB ), and bedaquiline ( Rv0678 , Rv2535c (pepQ) and Rv1979c). The genomic analysis identified 16 MDR (33.3%), and 32 pre-XDR (66.6%) MTB isolates, while phenotypic DST identified slightly higher counts for MDR (18, 37.5%) and pre-XDR (28, 58.3%), with only 1 XDR (2.0%) and one pan-susceptible isolate (2.0%). The predominant lineage being Mycobacterium tuberculosis lineage 3 (n=25 occurrences) . Importantly, we identified three unreported mutations in rpoC (Leu446Gln, Arg356Ser, and Leu446Met). Two unreported mutations were identified in katG His116Arg, Val260Asp. In the fluoroquinolone target genes two mutations (His272Asn and Met530Thr) were found in gyrB which have not been reported in the WHO mutation catalogue and literature. Among the bedaquiline-resistance Rv0678 (C779125TG), and Rv1979c (TA2222732T deletion) were not reported. Analysis of ESX/Type VII secretion system-associated genes identified several novel variants, including in espA , resulting in a Thr192Ile substitution in 42 isolates, and in the PE35-encoding gene, where a Glu99STOP was observed in 37 isolates. Additional novel variants were identified in genes encoding PPE69, EsxU, EsxH, MPT64, EsxT, and PPE68 proteins. These mutations were distributed predominantly among pre-XDR isolates. Overall, WGS and phenotypic DST results were largely concordant, with minor discordance in MDR and pre-XDR classification. These results highlight the resistance monitoring using WGS to inform targeted public health interventions for MDR, pre-XDR, and XDR TB.