Molecular genetic identification of yeast isolate MF22_1

Abstract

Microscopic fungi including yeasts are important part of human microbiota. Among them, yeasts of genus Candida are very common and often the most predominant part of mycobiota. Candida spp., including Candida albicans and non-Candida albicans species, can be opportunistic pathogens affecting people with depressed immune system. The problem with some non-Candida albicans species, e.g. C. glabrata and C. krusei, is that they are highly resistant to antifungal therapy and, at the same time, difficult to identify accurately at the species level by widely clinically used conventional biochemical methods. The aim of this work was precise molecular genetic identification of human sputum yeast isolate MF22_1, allegedly belonging to Candida spp., by using multiple genetic markers. We used four fragments of its chromosomal DNA, which were sequenced and deposited by us in GenBank NCBI database: 1) ITS (GenBank OM479428, 548 bp), 2) 28S rRNA (OM479513, 607 bp), 3) 18S rRNA (OM4794321, 737 bp), and 4) RNA polymerase II gene -RPB2 (OM524388, 1,217 bp). The part of Nucleotide collection database [http://www.ncbi.nlm.nih.gov/nuccore/] containing information on the nucleotide structure of fungal DNA (taxid: 4751) was studied using BLASTN [http://blast.ncbi.nlm.nih.gov/Blast.cgi]. The BLASTN analysis established that all four sequenced genetic markers: ITS, 28S rRNA, 18S rRNA, and RPB2 of the studied isolate MF22_1 had the maximum similarity to the corresponding sequences of the type strains of Pichia kudriavzevii species. Therefore, it was confirmed that the isolate belonged to species Pichia kudriavzevii, which anamorph (or non-ascosporic state) is Candida krusei. This ubiquitous in the environment species is a common clinical isolate responsible for about 2 % incidences of yeast infections caused by Candida species in humans. Comparative analysis of a primary structure of ITS region demonstrated the great similarity of the ITS sequence of Pichia kudriavzevii MF22_1 to the majority of other Pichia kudriavzevii clinical isolates from human sputum, stool, blood etc., which are preserved in different culture collection of the institutions specializing in medical studies of yeasts around the world. For these clinical isolates of Pichia kudriavzevii, no clear correlation was observed between the similarity of ITS sequences and the type of biomaterial sampled from humans.