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Purpose: Burkholderia pseudomallei, the tier 1 agent of melioidosis, is a saprophytic microbe that causes endemic infections in tropical regions such as South-East Asia and Northern Australia. It is globally distributed, challenging to diagnose and treat, infectious by several routes including inhalation, and has potential for adversarial use. B. pseudomallei strain MSHR5848 produces two colony variants, smooth (S) and rough (R), which exhibit a divergent range of morphological, biochemical and metabolic phenotypes, and differ in macrophage and animal infectivity. We aimed to characterize two major phenotypic differences, analyse gene expression and study the regulatory basis of the variation., Methodology: Phenotypic expression was characterized by DNA and RNA sequencing, microscopy, and differential bacteriology. Regulatory genes were identified by cloning and bioinformatics.Results/Key findings. Whereas S produced larger quantities of extracellular DNA, R was upregulated in the production of a unique chromosome 1-encoded Siphoviridae-like bacteriophage, φMSHR5848. Exploratory transcriptional analyses revealed significant differences in variant expression of genes encoding siderophores, pili assembly, type VI secretion system cluster 4 (T6SS-4) proteins, several exopolysaccharides and secondary metabolites. A single 3 base duplication in S was the only difference that separated the variants genetically. It occurred upstream of a cluster of bacteriophage-associated genes on chromosome 2 that were upregulated in S. The first two genes were involved in regulating expression of the multiple phenotypes distinguishing S and R., Conclusion: Bacteriophage-associated proteins have a major role in the phenotypic expression of MSHR5848. The goals are to determine the regulatory basis of this phenotypic variation and its role in pathogenesis and environmental persistence of B. pseudomallei.

Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm), the agents of melioidosis and glanders, respectively, are Tier 1 biothreats. They infect humans and animals, causing disease ranging from acute and fatal to protracted and chronic. Chronic infections are especially challenging to treat, and the identification of in vitro phenotypic markers which signal progression from acute to persistent infection would be extremely valuable. First, a phenotyping strategy was developed employing colony morphotyping, chemical sensitivity testing, macrophage infection, and lipopolysaccharide fingerprint analyses to distinguish Burkholderia strains. Then mouse spleen isolates collected 3-180 days after infection were characterized phenotypically. Isolates from long-term infections often exhibited increased colony morphology differences and altered patterns of antimicrobial sensitivity and macrophage infection. Some of the Bp and Bm persistent infection isolates clearly displayed enhanced virulence in mice. Future studies will evaluate the potential role and significance of these phenotypic markers in signaling the establishment of a chronic infection., Competing Interests: The authors declare that they have no conflict of interest.

Burkholderia pseudomallei (Bp), the agent of melioidosis, causes disease ranging from acute and rapidly fatal to protracted and chronic. Bp is highly infectious by aerosol, can cause severe disease with nonspecific symptoms, and is naturally resistant to multiple antibiotics. However, no vaccine exists. Unlike many Bp strains, which exhibit random variability in traits such as colony morphology, Bp strain MSHR5848 exhibited two distinct and relatively stable colony morphologies on sheep blood agar plates: a smooth, glossy, pale yellow colony and a flat, rough, white colony. Passage of the two variants, designated “Smooth” and “Rough”, under standard laboratory conditions produced cultures composed of > 99.9% of the single corresponding type; however, both could switch to the other type at different frequencies when incubated in certain nutritionally stringent or stressful growth conditions. These MSHR5848 derivatives were extensively characterized to identify variant-associated differences. Microscopic and colony morphology differences on six differential media were observed and only the Rough variant metabolized sugars in selective agar. Antimicrobial susceptibilities and lipopolysaccharide (LPS) features were characterized and phenotype microarray profiles revealed distinct metabolic and susceptibility disparities between the variants. Results using the phenotype microarray system narrowed the 1,920 substrates to a subset which differentiated the two variants. Smooth grew more rapidly in vitro than Rough, yet the latter exhibited a nearly 10-fold lower lethal dose for mice than Smooth. Finally, the Smooth variant was phagocytosed and replicated to a greater extent and was more cytotoxic than Rough in macrophages. In contrast, multiple locus sequence type (MLST) analysis, ribotyping, and whole genome sequence analysis demonstrated the variants’ genetic conservation; only a single consistent genetic difference between the two was identified for further study. These distinct differences shown by two variants of a Bp strain will be leveraged to better understand the mechanism of Bp phenotypic variability and to possibly identify in vitro markers of infection. [ABSTRACT FROM AUTHOR]