Molecular identification of veterinary yeast isolates by use of sequence-based analysis of the D1/D2 region of the large ribosomal subunit

Abstract

Conventional methods of yeast identification are often time-consuming and difficult; however, recent studies of sequence-based identification methods have shown promise. Additionally, little is known about the diversity of yeasts identified from various animal species in veterinary diagnostic laboratories. Therefore, in this study, we examined three methods of identification by using 109 yeast samples isolated during a 1-year period from veterinary clinical samples. Comparison of the three methods-traditional substrate assimilation, fatty acid profile analysis, and sequence-based analysis of the region spanning the D1 and D2 regions (D1/D2) of the large ribosomal subunit-showed that sequence analysis provided the highest percent identification among the three. Sequence analysis identified 87% of isolates to the species level, whereas substrate assimilation and fatty acid profile analysis identified only 54% and 47%, respectively. Less-stringent criteria for identification increased the percentage of isolates identified to 98% for sequence analysis, 62% for substrate assimilation, and 55% for fatty acid profile analysis. We also found that sequence analysis of the internal transcribed spacer 2 (ITS2) region provided further identification for 36% of yeast not identified to the species level by D1/D2 sequence analysis. Additionally, we identified a large variety of yeast from animal sources, with at least 30 different species among the isolates tested, and with the majority not belonging to the common Candida spp., such as C. albicans, C. glabrata, C. tropicalis, and the C. parapsilosis group. Thus, we determined that sequence analysis of the D1/D2 region was the best method for identification of the variety of yeasts found in a veterinary population.

FIG. 1.

Comparison of identifications provided by substrate assimilation and fatty acid profile analysis to D1/D2 sequencing results. The x axis and y axis show the percentage of isolates for each species that substrate assimilation and fatty acid profile analysis identified concordantly with the D1/D2 sequencing results, respectively. The size of the bubbles is proportional to the number of yeast isolates for each species, and the numbers inside the bubbles or next to the bubbles give the exact number. Only isolates with a good identification to the species level by D1/D2 sequence analysis and where n is ≥2 were included in this comparison for a total of 82 isolates.

FIG. 2.

Phylogenetic tree of yeast isolates. A neighbor-joining phylogram of representative D1/D2 sequences was created using the Clustal W2 multiple alignment tool.