Emergence of distinct Burkholderia pseudomallei lineages in Brazil: Phenotypic, genotypic, and phylogenetic agreement.

Burkholderia pseudomallei is a Gram-negative facultative intracellular bacterium that causes melioidosis, a neglected tropical disease that spreads beyond traditional endemic regions. This study examined B. pseudomallei isolates obtained from five melioidosis cases in Piauí, Brazil, using phenotypic, molecular, and genomic methods. Isolates were identified as B. pseudomallei using VITEK 2 system and MALDI-TOF mass spectrometry. Conventional PCR targeting the orf11, burk17, and burk475 gene regions confirmed species-level identification, with sequences showing 98.78-100% identity to GenBank B. pseudomallei references. Whole-genome sequencing and multi-locus sequence typing (MLST) revealed two novel sequence types; ST2226 (four isolates), clustering closely with strains from Ceará (ST1455 and ST1458), ST2234 (one isolate), related to previously reported ST95 and ST1355 strains, previously detected in Brazil. Phylogenetic analysis based on single nucleotide polymorphism (SNPs) and concatenated MLST data showed clear genetic distinction between the two lineages, suggesting regional spread or shared ancestry. These findings confirm the presence of at least two distinct genetic B. pseudomallei lineages in Piauí, providing insights into bacterial diversity and regional epidemiology in Brazil and reinforcing the importance of molecular surveillance and improved diagnostic capacity for melioidosis.