Fiziol. rast. genet. 2022, vol. 54, no. 6, 516-527, doi:

Molecular genetic identification of yeast isolate mf22_1

Fomina M.O., Filipishena O.Ya., Polishchuk L.V.

  • D.K. Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine 154 Akademika Zabolotnogo St., Kyiv, 03143, Ukraine

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 [] containing information on the nucleotide structure of fungal DNA (taxid: 4751) was studied using BLASTN []. 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.

Keywords: Candida spp., yeast, identification, ITS region, 18S rRNA, 28S rRNA, RPB2

Fiziol. rast. genet.
2022, vol. 54, no. 6, 516-527

Full text and supplemented materials

Free full text: PDF  


1. Perez, J.C. (2021). Fungi of the human gut microbiota: roles and significance. International Journal of Medical Microbiology, 311, No. 3, 151490.

2. Fiers, W.D., Leonardi, I. & Iliev, I.D. (2020). From birth and throughout life: fungal microbiota in nutrition and metabolic health. The Annual Review of Nutrition, 40, pp. 323-343.

3. Moss, B.J. & Musher, D.M. (2021). Candida species in community-acquired pneumonia in patients with chronic aspiration. Pneumonia, 13, No. 12.

4. Cannon, R.D. (2022). Oral fungal infections: past, present, and future. Frontiers in Oral Health, 3, 838639.

5. Costa, A.R., Silva, F., Henriques, M., Azeredo, J., Oliveira, R. & Faustino, A. (2010). Candida clinical species identification: molecular and biochemical methods. Annals of Microbiology, 60, pp. 105-112.

6. Khot, P.D., Ko, D.L. & Fredricks, D.N. (2009). Sequencing and analysis of fungal rRNA operons for development of broad-range fungal PCR assays. Appl Environ Microbiol., 75, No. 6, pp. 1559-1565.

7. Peay, K.G., Kennedy, P.G. & Bruns, T.D. (2008) Fungal community ecology: a hybrid beast with a molecular master. BioScience Journal., 58, No. 9, pp. 799-810.

8. Schoch, C.L., Seifert, K.A., Huhndorf, S., Robert, V., Spouge, J.L., Levesque, C.A. & Chen W. (2012). Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences of the United States of America, 109, No. 16, pp. 6241-6246.

9. Bruns, T.D., White, T.J. & Taylor, J.W. (1991). Fungal molecular systematics. Annual Review of Ecology and Systematics, 22, No. 1, pp. 525-564.

10. Couble, A., Domingo, J., Miroy, K. & Villard, J. (1996). Species-specific identification of Candida krusei by hybridization with the CkF1,2 DNA probe. Journal of Clinical Microbiology, 34, No. 7, pp. 1726-1731.

11. Kurtzman, C.P., Smiley, M.J. & Johnson, C.J. (1980). Emendation of the genus Issatchenkia kudriavzevii and comparison of species by deoxyribonucleic acid reassociation, mating reaction, and ascospore ultrastructure. International Journal of Systematic Bacteriology, 30, No. 2, pp. 503-513.

12. Douglass, A.P., Offei, B., Braun-Galleani, S., Coughlan, A.Y., Martos, A. A.R., Ortiz-Merino, R.A. & Wolfe, K.H. (2018). Population genomics shows no distinction between pathogenic Candida krusei and environmental Pichia kudriavzevii: one species, four names. PLOS Pathogens, 14, No. 7, e1007138.

13. Yamada, Y., Maeda, K. & Mikata, K. (1994). The phylogenetic relationships of the hat-shaped ascospore-forming, nitrate-assimilating Pichia species, formerly classified in the genus Hansenula Sydow et Sydow, based on the partial sequences of 18S and 26S ribosomal RNAs (Saccharomycetaceae): the proposals of three new genera, Ogataea, Kuraishia, and Nakazawaea. Bioscience, Biotechnology, and Biochemistry, 58, No. 7, pp. 1245-1257.

14. Ueda-Nishimura, K. & Mikata, K. (2001). Reclassification of Pichia scaptomyzae and Pichia galeiformis. Antonie Van Leeuwenhoek (Journal), 79, No. 3-4, pp. 371-375.

15. Zheng, X., Li, K., Shi, X., Ni, Y., Li, B. & Zhuge, B. (2018). Potential characterization of yeasts isolated from Kazak artisanal cheese to produce flavoring compounds. Microbiology open, 7, No. 1, e00533.

16. Kurtzman, C. P. (2011). Pichia E.C. Hansen (1904), (Chap. 57). In (Eds. C.P. Kurtzman, J.W. Fell, T. Boekhout) The Yeasts (pp. 685-707), Elsevier.

17. Espejo, R. T. & Plaza, N. (2018). Multiple ribosomal RNA operons in bacteria; their concerted evolution and potential consequences on the rate of evolution of their 16S rRNA. Front. Microbiol., 9, 1232.

18. Liu, Y. J., Whelen, S. & Hall, B. D. (1999). Phylogenetic relationships among ascomycetes: evidence from an RNA polymerase II subunit. Molecular Biology and Evolution, 16, No. 12, pp. 1799-1808.

19. Liu, Y.J., Hodson, M.C. & Hall, B.D. (2006). Loss of the flagellum happened only once in the fungal lineage: phylogenetic structure of Kingdom Fungi inferred from RNA polymerase II subunit genes. BMC Evolutionary Biology, 6, No. 1, p. 74.

20. Hansen, K., LoBuglio, K.F. & Pfister, D.H. (2005). Evolutionary relationships of the cup-fungus genus Peziza and Pezizaceae inferred from multiple nuclear genes: RPB2, b-tubulin, and LSU rDNA. Molecular Phylogenetics and Evolution, 36, No. 1, pp. 1-23.

21. Kurtzman, C.P. (2011). Discussion of Teleomorphic and Anamorphic Ascomycetous Yeasts and Yeast-like Taxa (Chap. 13). In (Eds. C. P. Kurtzman, J. W. Fell, T.Boekhout) The Yeasts (pp. 293-207), Elsevier.