Development of two molecular approaches for differentiation of clinically relevant yeast species closely related to Candida guilliermondii and Candida famata

Abstract

The emerging pathogens Candida palmioleophila, Candida fermentati, and Debaryomyces nepalensis are often misidentified as Candida guilliermondii or Candida famata in the clinical laboratory. Due to the significant differences in antifungal susceptibilities and epidemiologies among these closely related species, a lot of studies have focused on the identification of these emerging yeast species in clinical specimens. Nevertheless, limited tools are currently available for their discrimination. Here, two new molecular approaches were established to distinguish these closely related species. The first approach differentiates these species by use of restriction fragment length polymorphism analysis of partial internal transcribed spacer 2 (ITS2) and large subunit ribosomal DNA with the enzymes BsaHI and XbaI in a double digestion. The second method involves a multiplex PCR based on the intron size differences of RPL18, a gene coding for a protein component of the large (60S) ribosomal subunit, and species-specific amplification. These two methods worked well in differentiation of these closely related yeast species and have the potential to serve as effective molecular tools suitable for laboratory diagnoses and epidemiological studies.

FIG 1

Neighbor-joining tree for Candida strains determined through analysis of ITS sequences. The Saccharomyces cerevisiae type strain was used as an outgroup. Bootstrap values of >70% are indicated for the main branches. Clusters containing the C. famata complex, the C. guilliermondii complex, and C. palmioleophila are shown in bold type.

FIG 2

Agarose gel electrophoresis of enzyme-digested products (a) and amplicons from the multiplex PCR assay (b). Lanes M, DL2000 ladder; lanes 1, CBS2030 (C. guilliermondii); lanes 2, Y-27403 (C. fermentati); lanes 3, Y-17323 (C. palmioleophila); lanes 4, CBS1795 (C. famata); lanes 5, CBS796 (D. fabryi); lanes 6, CBS2334 (D. nepalensis). Bands of <100 bp in the RFLP analysis are not shown.