Profiling of <i>Burkholderia pseudomallei</i> variants derived from Queensland’s clinical isolates

<i>Burkholderia pseudomallei</i> (<i>Bp</i>), an environmental bacterium and opportunistic pathogen endemic to tropical regions, is highly adaptive and thrives in diverse environments, from soil to human hosts. Bacterial adaptation is critical for survival, virulence modulation, and persistence during infection and can manifest as colony morphotype variation (CMV). Although <i>Bp</i> adaptation has been studied, CMV remains poorly understood. Here, we characterized five clinical <i>Bp</i> isolates exhibiting heterogeneous populations with rough and smooth colony morphologies. We used phenotypic assays, whole-genome sequencing, and proteomics to investigate the molecular pathways reflecting CMV, by comparing smooth and rough morphotypes. Although phenotypic differences in protease activity, hemolysis, mucoidy, iron uptake, and antibiotic sensitivity-including to antimicrobial agents commonly used to treat infections-were rare, these traits alone could not distinguish morphotypes or groups of isolates. Genomic comparisons revealed either no differences or limited isolate-specific mutations, which do not explain the overall difference in phenotypes. In contrast, proteomic analysis uncovered consistent shifts in protein abundance related to virulence, including quorum sensing, DNA methylation, and secretion systems. Rough variants showed higher abundance of EPS-associated proteins, the BpsI3/R3 quorum-sensing system, and the global regulator ScmR, whereas smooth variants displayed higher abundances of proteins belonging to type III/VI secretion and siderophore biosynthesis pathways. These findings suggest that CMV is driven by phase variation and regulatory mechanisms rather than punctual genomic modifications. Our study underscores the limitations of phenotype or genome-based classification alone in the context of CMV and highlights the value of integrated multi-omics approaches to uncover CMV-associated biomarkers, with potential applications in diagnostics and the development of targeted therapies against persistent and drug-resistant <i>Bp</i> infections.IMPORTANCE<i>Burkholderia pseudomallei</i> (<i>Bp</i>), the causative agent of melioidosis, is endemic to Australia, Asia, Africa, and the Americas. It predominantly affects Indigenous populations and individuals suffering from diabetes, chronic lung or kidney disease, or alcoholism. <i>Bp</i> is known for its exceptional genomic and phenotypic plasticity, enabling rapid adaptation to diverse environments. This adaptability is reflected by colony morphotype variation (CMV), including reversible phase variation between smooth and rough colonies. In this study, we report rough and smooth colonies from clinical samples and emphasize the importance of characterizing CMV through multi-omics approaches rather than relying solely on genomics and phenotypic traits. By integrating genomic, phenotypic, and proteomic data, we identified that a limited number of mutations, including one in a putative regulatory element, likely drive major molecular changes between morphotypes. These affect the expression of quorum-sensing systems, the transcriptional regulator ScmR, DNA methyltransferase, and virulence-associated genes.