Successful expansion of Mycobacterium tuberculosis Latin American and Mediterranean sublineage (L4.3/LAM) in Tunisia mainly driven by a single, long-established clonal complex.

Objectives: To explore the evolutionary history of Mycobacterium tuberculosis Latin American and Mediterranean (L4.3/LAM) sublineage in Tunisia, where it predominates.

Methods: High-resolution genotyping of 252 L4.3/LAM clinical strains was undertaken, and whole-genome sequencing was performed on 31 representative isolates.

Results: Genotyping data coupled with Bayesian analyses split the Tunisian L4.3/LAM strain collection into two divergent entities (65.07% vs 34.92%): a major subpopulation, dominated by a single clonal complex (CC), TUN4.3_CC1 (94.51%); and a minor subpopulation, dominated by TUN4.3_CC2 (42.04%). TUN4.3_CC1 is clearly thriving in Tunisia, accounting for 61.5% of the L4.3/LAM sublineage. TUN4.3_CC1 displayed higher mean allelic richness compared with TUN4.3_CC2 and predominated throughout the entire region, indicating a long-established history. The very low proportion of drug resistance among TUN4.3_CC1 isolates is indicative of their intrinsic ability to spread successfully in the host population. Genomic analyses further confirmed the clear genetic separation between the two main CCs (pairwise fixation index 0.56), and suggested the relatively ancient origin of TUN4.3_CC1. Consistent with its successful expansion, TUN4.3_CC1 showed reduced mean pairwise genetic distance between genomes.

Conclusions: These findings link the successful expansion of L4.3/LAM in Tunisia to a single long-established clone.