Genotyping, antifungal susceptibility testing, and biofilm formation of Trichosporon spp. isolated from urine samples in a University Hospital in Bangkok, Thailand.

The basidiomycetes yeast Trichosporon is widespread in the natural environment, but can cause disease, mainly in immunocompromised patients. However, there have been only few studies about this infection in Thailand. In this study, we characterized 53 Trichosporon spp. isolated from urine samples from patients admitted to a single hospital in Bangkok, Thailand over a one-year period from 2019 to 2020. The strains were identified using colony morphology, microscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and nucleotide sequence analysis of intergenic spacer 1 (IGS1). Fifty-one isolates were Trichosporon asahii, and the remaining isolates were Trichosporon inkin and other Trichosporon species. Three genotypes of IGS1-1, 3, and 7 were observed among T. asahii. The sensitivity of the yeasts to the antifungal drugs amphotericin B, fluconazole, and voriconazole ranged from 0.25 to >16 µg ml-1, 0.5-8 µg ml-1, and 0.01-0.25 µg ml-1, respectively. We investigated biofilm formation by the isolates, and no biofilm production was found in one isolate, low biofilm production in forty-four isolates, and medium biofilm production in six isolates. T. inkin produced biofilms at low levels, and Trichosporon spp. produced biofilms at medium levels. This research increases our understanding of the molecular epidemiology of Trichosporon spp. isolated from one university hospital in Bangkok, Thailand, and reveals their genetic diversity, antifungal susceptibility profiles, and capacity for in vitro biofilm production.

Tetraspanins are involved in Burkholderia pseudomallei-induced cell-to-cell fusion of phagocytic and non-phagocytic cells.

Tetraspanins are four-span transmembrane proteins of host cells that facilitate infections by many pathogens. Burkholderia pseudomallei is an intracellular bacterium and the causative agent of melioidosis, a severe disease in tropical regions. This study investigated the role of tetraspanins in B. pseudomallei infection. We used flow cytometry to determine tetraspanins CD9, CD63, and CD81 expression on A549 and J774A.1 cells. Their roles in B. pseudomallei infection were investigated in vitro using monoclonal antibodies (MAbs) and recombinant large extracellular loop (EC2) proteins to pretreat cells before infection. Knockout of CD9 and CD81 in cells was performed using CRISPR Cas9 to confirm the role of tetraspanins. Pretreatment of A549 cells with MAb against CD9 and CD9-EC2 significantly enhanced B. pseudomallei internalization, but MAb against CD81 and CD81-EC2 inhibited MNGC formation. Reduction of MNGC formation was consistently observed in J774.A1 cells pretreated with MAbs specific to CD9 and CD81 and with CD9-EC2 and CD81-EC2. Data from knockout experiments confirmed that CD9 enhanced bacterial internalization and that CD81 inhibited MNGC formation. Our data indicate that tetraspanins are host cellular factors that mediated internalization and membrane fusion during B. pseudomallei infection. Tetraspanins may be the potential therapeutic targets for melioidosis.

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

BACKGROUND: 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. METHODOLOGY/PRINCIPLE FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.