Comparative genomic analysis of established and emerging anti-tubercular drug targets reveals evolutionary conservation and antimicrobial resistance patterns in Mycobacterium tuberculosis isolates from Indonesia

BackgroundMultidrug-resistant tuberculosis (MDR-TB) remains a major public health threat in Indonesia, which ranks second globally in tuberculosis burden. Whole-genome sequencing (WGS) studies of drug-resistant Mycobacterium tuberculosis in Indonesia have so far been analyzed largely in isolation, and no previous study has systematically compared the evolutionary conservation of classical, clinically validated resistance-associated genes with newly proposed metabolic drug targets within a single, geographically diverse Indonesian isolate collection.MethodsWe compiled 444 publicly available Mycobacterium tuberculosis whole genomes from four Indonesian whole-genome sequencing studies spanning Java and Papua and analyzed them using TBProfiler for lineage assignment and genotypic drug-resistance prediction. Following exclusion of nine isolates for which an unambiguous single lineage could not be assigned, gene-level sequences for ten classical resistance-associated genes (rpoB, katG, inhA, fabG1, embB, pncA, gyrA, gyrB, rpsL, rrs) and ten emerging metabolic drug-target genes (guaA, guaB2, glnA1, gltB, dapB, folP1, bioA, panC, aroG, ilvB1) were reconstructed for 435 isolates and compared by multiple sequence alignment, variant calling, and functional annotation.Results(East-Asian) predominated (46.2% of isolates) and was disproportionately associated with drug-resistant genotypes (χ² = 49.07, df = 2, p < 0.001; Lineage 2 versus Lineage 4 odds ratio for any genotypic resistance 2.69, 95% CI 1.77–4.07). Overall, 28.2% of isolates were genotypically multidrug-resistant and 5.4% were pre-extensively drug-resistant, with no mutations associated with bedaquiline, linezolid, or clofazimine resistance detected. All twenty target genes were highly conserved (89.4–99.6% invariant sites), consistent with the clonal population structure of M. tuberculosis. However, the two panels differed substantially: classical resistance-associated loci were significantly less conserved than emerging targets (mean 96.47% versus 99.24%; p < 0.001), carried a significantly higher SNP density (20.70 versus 6.58 SNPs per kilobase; p = 0.002) and per-site Shannon entropy (p = 0.009), and accounted for 537 of the 670 variable sites detected across the panel despite comprising a similar total coding length. The non-synonymous-to-synonymous substitution ratio was also significantly higher among classical loci (median 1.67 versus 0.96; p = 0.033), driven by the prodrug-activating enzymes pncA (49.0) and katG (10.4) and by rpsL (6.0) and rpoB (2.08). Within the emerging panel, absolute variable-site counts were uniformly low (4–47 sites per gene) and did not support discrimination between individual candidate genes.ConclusionsAcross a geographically diverse Indonesian Mycobacterium tuberculosis population, therapeutically unexploited metabolic drug targets are markedly more conserved and carry a substantially lower mutational burden than classical resistance-associated loci, whose elevated non-synonymous variation is concentrated in genes whose inactivation confers resistance. These findings support the emerging metabolic panel collectively as a low-variability target space for future anti-tubercular development, and establish evolutionary conservation combined with substitution-pattern analysis as a quantitative framework for target-space prioritization and genomic surveillance in high-burden tuberculosis settings.