Patterns of drug resistance, drug resistance conferring mutations and genomic DNA methylation in Mycobacterium tuberculosis isolates from Gauteng and Mpumalanga provinces in South Africa

TB, an infectious disease caused by M. tuberculosis, remains a major public health threat in South Africa. Despite the ongoing global control efforts, TB continues to pose significant challenges, particularly due to the rise in drug-resistant strains that compromise the treatment efficacy. Although M. tuberculosis strains share over 99 percent genomic homology, considerable phenotype variation exists. The mechanisms behind this phenotypic heterogeneity are not well understood, but epigenetic factors, including DNA methylation, are believed to play a role. While DNA methylation in M. tuberculosis has been previously reported, limited information is available for clinical M. tuberculosis drug-resistant strains. Given the growing threat of drug-resistant M. tuberculosis, investigating the role of DNA methylation in TB pathogenesis is crucial. In the present study, whole-genome sequencing was performed on 35 M. tuberculosis clinical isolates. The dataset included 25 short reads for detecting drug resistance and 10 long reads to assess the association between drug resistance and DNA methylation patterns. A total of 698 polymorphic sites suitable for distinguishing the selected M. tuberculosis isolates were identified. Three DNA motifs containing methylated adenine residues on one or both DNA strands were detected: CACGCaG, CtCCaG, and GaTNNNNRtAC. The methylation at each motif was associated with its respective methyltransferase (MTase): MamA, MamB, and the HsdM MTase complexed with the motif-binding protein HsdS. Additionally, a deletion spanning the 3region of the MamB MTase gene and several adjacent hypothetical proteins was discovered in many sequenced genomes. This study revealed that isolates belonging to the EAI lineage are equally capable of developing MDR-TB and may even surpass the other two observed lineages, L2 and L4, in fluoroquinolone resistance. This is particularly concerning, as the resistance patterns of the drug resistance-conferring mutations in EAI isolates differ from those in L2 and L4