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.In both veterinary and human diagnostic laboratories, correct identification of yeasts is important for the care of patients. Traditionally, yeast identification has been performed using biochemical analysis, substrate assimilation methods, morphological examination, or various combinations of the three. To increase the ease of identification, commercial tests that use these methods have been created, but despite the convenience provided by these methods, identification of yeasts by these conventional applications can still be timeconsuming and difficult. In addition, considerable variability in the efficacy of these methods has been reported for identification of clinically important yeast (11; also reviewed in references 12, 32, 36, and 42), attributed primarily to the limitations of the databases used for the comparison of clinical isolates, as well as the subjectivity involved in the interpretation of results. Recent studies have also examined the effectiveness of various molecular identification methods for yeasts by the use of rRNA genes, with the internal transcribed spacer 1 (ITS1) and ITS2 regions and the region spanning the D1 and D2 regions (D1/ D2) shown to be the most useful for species-level identification of yeasts, as a result of the variability within...

Molecular analysis and anticancer properties of two identified isolates, Fusarium solani and Emericella nidulans isolated from Wady El–Natron soil in Egypt against Caco–2 (ATCC) cell line

“…Macroconidia were formed after [4][5][6][7] days from short multi-branched conidiophores which may form sporodochia. They are 3-septate, fusiform, cylindrical, often moderately curved, with an indistinctly pedicellate foot cell and a short blunt apical cell, (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42) 毺m 暳 4-6 毺m). Microconidia are usually abundant, cylindrical to oval, oneto two-celled and formed from long lateral phialides, (8)(9)(10)(11)(12)(13)(14)(15)(16) 毺m 暳 2-4.5 毺m).…”

“…It has typically been most useful for molecular systematics at the species level, and even within species (e.g., to identify geographic races). Because of its higher degree of variation than other genic regions of rDNA (for small-and large-subunit rRNA), ITS has proven especially useful for elucidating relationships among congeneric species and closely related genera in clinically important yeast species [35] .…”

“…PCR was conducted for [30][31][32][33][34][35][36][37][38][39][40] cycles in a thermal controller (Programmable Thermal Controller; M) Research Inc., Watertown, MA). The primers used for amplification of p53 gene were shown in Table 1.…”

Retrospective study on equine uterine fungal isolates and antifungal susceptibility patterns (1999–2011)

“…Yeast identification was based on assessment of macroscopic and microscopic features and nutrient assimilation using API 20 C AUX test strips b or the Uni-Yeast-Tek system c . Yeast differentiation methods were expanded to include fatty acid profile analysis in 2007 and DNA sequencing in 2009 [3]. The DNA sequencing revealed that many of the genus and species designations assigned to yeast isolates before 2009 were imprecise (Dr C. Altier, personal communication).…”

“…The incidence of fungal infection in mares diagnosed with endometritis is significantly lower than the incidence of bacterial infection but fungal endometritis is more challenging to treat and carries a guarded-to-poor prognosis for future fertility [1][2][3][4][5][6][7][8][9][10][11][12]. Suggested doses and treatment regimens for fungal endometritis are mostly based on case studies or extrapolations from other species (Tables S1-S3).…”