Nationwide phylogenomic surveillance of Mycobacterium tuberculosis in Mexico reveals pathogenic and drug resistant signatures of the prevailing L4 sublineage

• Genetic patterns of MTB isolates from Mexico within a global context are analyzed. • Through phylogenetics and pangenomics the genetic features of L4.1.1.3 sublineage are revealed. • L4.1.1.3 has a higher frequency of drug-resistant strains compared to other sublineages. • Specific variants of genes such as ppe, pe , and esx-1 were identified in L4.1.1.3 clade. • L4.1.1.3 clade has genes linked to pathogenicity, virulence, and drug resistance to delamanid. Tuberculosis disease is a major global health concern. In Mexico, information regarding the genomic variants of Mycobacterium tuberculosis (MTB) prevailing in the country and the existence of specific biogeographical patterns remains extremely scarce. This study aimed to identify the genotypic patterns of MTB isolates in Mexico and determine the genes and specific single nucleotide polymorphisms involved in the evolution of these populations. Phylogenomic and pan-genomic analyses were performed using publicly available Mexican MTB genomes along with 33 newly sequenced genomes from Jalisco, considering a global context. The L4 sublineages of MTB, such as L4.1.1 (X), L4.1.2 (H), and L4.3 (LAM), were the most prevalent in Mexico. We found exclusive mutations and gene clusters in a virulent sublineage L4.1.1.3 (X3), which is endemic to Mexico. These genes encoded three PE/PPE family proteins: a multidrug transporter, thioredoxin domain-containing protein, quinone-dependent l -lactate dehydrogenase, DUF1725 domain-containing protein, amidase, poly (A) polymerase, and six hypothetical/uncharacterised proteins. Additionally, the genes encode an ESX-1 secretion-associated protein and a deazaflavin-dependent nitroreductase ( ddn ). X3 was distinguished from the rest of the sublineages by containing genes related to pathogenicity and virulence, as well as a gene linked to delamanid, an antibiotic for active multidrug-resistant tuberculosis. These findings provide valuable insight into the circulation and spread of MTB in Mexico.