Genomic loss in environmental and isogenic morphotype isolates of Burkholderia pseudomallei is associated with intracellular survival and plaque-forming efficiency.

Burkholderia pseudomallei is an environmental bacterium that causes melioidosis. A facultative intracellular pathogen, B. pseudomallei can induce multinucleated giant cells (MNGCs) leading to plaque formation in vitro. B. pseudomallei can switch colony morphotypes under stress conditions. In addition, different isolates have been reported to have varying virulence in vivo, but genomic evolution and the relationship with plaque formation is poorly understood. To gain insights into genetic underpinnings of virulence of B. pseudomallei, we screened plaque formation of 52 clinical isolates and 11 environmental isolates as well as 4 isogenic morphotype isolates of B. pseudomallei strains K96243 (types II and III) and 153 (types II and III) from Thailand in A549 and HeLa cells. All isolates except one environmental strain (A4) and K96243 morphotype II were able to induce plaque formation in both cell lines. Intracellular growth assay and confocal microscopy analyses demonstrated that the two plaque-forming-defective isolates were also impaired in intracellular replication, actin polymerization and MNGC formation in infected cells. Whole genome sequencing analysis and PCR revealed that both isolates had a large genomic loss on the same region in chromosome 2, which included Bim cluster, T3SS-3 and T6SS-5 genes. Our plaque screening and genomic studies revealed evidence of impairment in plaque formation in environmental isolates of B. pseudomallei that is associated with large genomic loss of genes important for intracellular multiplication and MNGC formation. These findings suggest that the genomic and phenotypic differences of environmental isolates may be associated with clinical infection. This study used a plaque-formation screen, as a surrogate for bacterial virulence, to identify a plaque-defective environmental isolate of B. pseudomallei that is impaired in intracellular replication, actin polymerization and MNGC formation in infected cells. Whole genome sequencing and PCR indicated that this phenotype was attributable to genomic loss. A similar event was detected in a K96243 isogenic morphotype in vitro under a laboratory stress condition. In contrast, all isolates from clinical samples induced high plaque-forming efficiency. Our data suggest that further studies are required to identify the distribution of less virulent strains in the environments and the correlation with human melioidosis.