Patterns of Drug Resistance, Drug Resistance Conferring Mutations and Genomic DNA Methylation Revealed in Mycobacterium tuberculosis From South Africa

ABSTRACT Tuberculosis remains a major public health threat globally, with drug‐resistant strains undermining treatment efficacy. We analyzed 126 Mycobacterium tuberculosis ( M. tuberculosis ) isolates with diverse drug resistance spectra and selected 35 for whole genome sequencing (WGS) using Illumina NextSeq, SMRT PacBio Onso and SMRT PacBio Revio sequencing platforms. The study aimed to characterize drug resistance profiles, compare short‐ and long‐read sequencing performance, identify lineages among South African isolates, detect known drug resistance mutations and their lineage‐specific patterns, and utilize long‐read SMRT platforms for epigenetic profiling. Multiple drug resistance mutations were identified, some lineage‐specific, and notably, East‐African‐Indian (EAI) Lineage 1 isolates often considered less pathogenic, showed significant potential for multidrug‐resistance development, including higher fluoroquinolone resistance as compared to other lineages. Three DNA motifs with methylated adenines, namely CACGCaG, CtCCaG and GaTNNNNRtAC, were detected, with methylation patterns varying by lineage and strain due to mutations in the corresponding methyltransferases (MTases). A particularly notable finding was the stable maintenance of a genetic heterogeneity in the mamB MTase, performing methylation at CACGC a G motifs. These results highlight the combined role of genetic and epigenetic variation in M. tuberculosis adaptive evolution and underscore the value of integrating long‐read sequencing into TB surveillance and research.