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Effect of culture medium on the identification of yeasts of the genus Cryptococcus using mass spectrometry

By Alena Jurnečková


Cryptococccus genus is known for its difficult identification and taxonomical classification in area of clinical microbiology. For this bachelor thesis, total 22 yeast strains of the Cryptococcus genus were chosen. The part of strains was firstly analyzed by D1/D2 domain LSU rRNA gene analysis. Three types of culture medium – YPD, potato dextrose agar and Sabouraud’s medium were selected for cultivation. Samples were prepared according to standardized method of Bruker Daltonik company, Institute of Chemistry of SAS and combination of these two methods. Identification was done by MALDI-TOF mass spectrometry. Obtained spectra were compared using corresponding software and evaluated on the basis of specific algorithm. The most advantageous culture medium for cultivation and biotyping with the largest percentage score was YPD (Yeasts pepton dextrose). On the other hand, the least advantageous culture medium was Sabouraud’s agar, which reached the smallest percentage success due to parameters of Bruker Daltonik algorithm. The most succesfull method of sample preparation and application was the combined method with YPD as a culture medium. The results of complete analysis are dendrograms for each medium showing the genetic similarity of yeast strains. The dendrogram shows categorization of the single strains into appropriate groups. Cr. flavescens (CCY 17-3-28, CCY 17-3-30) and Cr. laurentii (CCY 17-3-2) strains were correctly integrated into phylogenetic group Cr. laurentii I (branch in the dendrogram with designation A). Cr. flavus (CCY 17-3-5) doesn’t belong to this group, although it shows similarity with Cr. flavescens. The strains Cr. carnescens (CCY 17-3-13) and Cr. victoriae (CCY 17-3-26) belong to the phylogenetic group Cr. laurentii II (designated as B). Cr. magnus (CCY 17-4-39, CCY 17-4-40) strains show similarity with these strains, but doesn’t belong to the phylogenetic group II. The strains Cr. gastricus (CCY 17-5-1) and Cr. diffluens (non-attached) form a branch designated as C. Cr. aerius (CCY 17-4-9) strain, which was also put into this group, was proposed to sequence analysis, because its spectrum indicates that it should be rather a Cr. diffluens strain. The group D contains Bulleromyces albus (CCY 17-3-35, CCY 17-3-36) and Cr. saitoi (CCY 17-3-18, CCY 17-4-2) strains. The sequenced Cr. albidus (CCY 17-4-1), non-sequenced Cr. diffluens (CCY 17-4-13) and Cr. terreus (CCY 17-8-1) form the group E. The strain CCY 17-4-13 was proposed to sequence analysis because of occurrence of the Cr. diffluens sequenced strain in the group C. The sequenced Cr. aerius (CCY 17-25-1) is also part of this group, but it represents a separate branch. The last group is named as F and consists of Cr. macerans (CCY 17-19-3) and control strains Cr. neoformans var. neoformans (CCY 17-1-4, CCY 17-1-5)

Topics: rod Cryptococcus; MALDI-TOF MS; culture medium; biotyping; genus Cryptococcus; kultivační médium
Publisher: Vysoké učení technické v Brně. Fakulta chemická
Year: 2015
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