Molecular identification, genotypic heterogeneity and comparative pathogenicity of environmental isolates of Papiliotrema laurentii

Introduction. Papiliotrema laurentii , formerly Cryptococcus laurentii , is typically isolated from environmental sources, but also occasionally from clinical specimens. Other close relatives may be misidentified as P. laurentii by phenotypic methods. P. laurentii usually lacks melanin; however, melanin-forming strains have also been isolated. Hypothesis/Gap Statement. Although melanin production by encapsulated budding yeasts is considered a major virulence factor, the comparative pathogenicity of melanin-forming and non-melanized environmental strains of P. laurentii has rarely been studied. Aim. We performed phenotypic and molecular identification and determined the genotypic heterogeneity among P. laurentii isolates. We also studied the pathogenicity of melanin-forming and non-melanized strains in normal and immunosuppressed mice. Methodology. Eleven environmental isolates were tested for their identity by Vitek2 and/or ID32C systems, and by PCR-sequencing of the internal transcribed spacer (ITS) region and D1/D2 domains of ribosomal DNA (rDNA). Genotypic heterogeneity was studied by sequence comparisons. The pathogenicity of melanized and non-melanized P. laurentii strains was studied in intravenously infected normal and immunosuppressed BALB/c mice. Results. Phenotypic methods identified seven of the environmental isolates, while PCR-sequencing of the ITS region and D1/D2 domains of rDNA detected two and five isolates, respectively, as P. laurentii . Sequence comparisons demonstrated genotypic heterogeneity among P. laurentii . The remaining four environmental isolates yielded expected results. None of the normal mice infected with 10 5  cells of melanized/non-melanized P. laurentii strains died. Infection of immunosuppressed mice with 10 7  cells caused higher mortality with non-melanized P. laurentii , while viable counts in brain/lung tissue were higher in mice infected with a melanized strain and were detectable for up to 14 days. Conclusion. Phenotypic methods lacked specificity, but PCR-sequencing of D1/D2 domains correctly identified P. laurentii and sequence comparisons demonstrated the genotypic heterogeneity of the isolates. Both melanized and non-melanized strains at a higher dose caused mortality in immunosuppressed mice and persisted in brain/lung tissue up to 14 days post-infection.