Profiling of Burkholderia pseudomallei variants derived from Queensland’s clinical isolates

Burkholderia pseudomallei ( Bp ), 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). Here, we characterized five clinical Bp isolates exhibiting heterogeneous populations with mucoid and non-mucoid colony morphologies. We used phenotypic assays, whole-genome sequencing, and proteomics to investigate the molecular pathways impacted by CMV - by comparing smooth and rough morphotypes. While phenotypic differences in protease activity, hemolysis, mucoidy, iron uptake and antibiotic sensitivity were rare, these traits alone could not distinguish morphotypes or group of isolates. Genomic comparisons revealed either no differences or limited isolate-specific mutations, which likely does 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. These findings suggest that CMV is driven by phase variation and regulatory mechanisms rather than punctual genomic modifications. Our proof-of-concept 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 Bp infections. Importance Burkholderia pseudomallei (Bp), the causative agent of melioidosis, is endemic to Australia, Asia, Africa, and the Americas. It predominantly affects Indigenous populations and individuals with diabetes, chronic lung or kidney disease, or alcoholism. Bp 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 genomics, 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 (QS) systems, the transcriptional regulator ScmR, DNA methyltransferase, and virulence-associated genes.