Emergence of locally transmitting and drug-resistant clones shapes global expansion of Mycobacterium tuberculosis Lineage 3

Mycobacterium tuberculosis Lineage 3 (L3) is one of the major human-adapted MTBC lineages and is highly prevalent in parts of South Asia, East Africa, and Central Asia, although it remains less well characterized than other lineages. In particular, its capacity for sustained transmission and adaptation outside historically endemic regions remains unclear. We analyzed 7,472 publicly available L3 genomes from 38 countries within a global dataset of ~ 90,000 M. tuberculosis genomes. Population structure was resolved using phylogenetic reconstruction and hierarchical Bayesian clustering. Phylogeographic and phylodynamic analyses inferred dispersal history and demographic dynamics. Transmission patterns were assessed using genomic clustering, terminal branch lengths, and time-scaled haplotypic density (THD). Genome-wide scans identified signatures of positive selection and drug-resistance mutations. The dominant L3.1.1 clade (> 95% of L3 isolates) was resolved into six monophyletic sublineages (L3.1.1.1–L3.1.1.6) with distinct geographic distributions and demographic trajectories. Phylogeographic reconstruction indicated South Asia as the probable origin for most sublineages, followed by multiple intercontinental dispersal events. While many European isolates were associated with migration, genomic clustering identified 1,399 isolates consistent with potential local transmission, and sublineage-resolved selection analyses highlighted Rv1500 as consistently enriched in European local clusters. Phylodynamic analyses revealed recent expansion in L3.1.1.1, L3.1.1.5, and L3.1.1.6. Overall, 29.8% of isolates carried resistance-associated mutations, with L3.1.1.6 showing the highest burden (45.4%) and enrichment for pre-extensively drug-resistant strains. Within this sublineage, compensatory mutations in rpoA and rpoC were associated with elevated THD, indicating a potential role in restoring fitness and facilitating the spread of highly resistant clones. These findings reveal previously unrecognized diversity and heterogeneous evolutionary trajectories within L3. While global dissemination is largely driven by migration, certain sublineages exhibit patterns consistent with local amplification and expansion beyond their historical range. The emergence of highly drug-resistant and potentially transmissible clones, exemplified by L3.1.1.6, underscores the importance of sublineage–resolved genomic surveillance for TB control.