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Production of galactosidases by yeasts of genus Cryptococcus growing on lactose medium
Pavlatovská, Barbora ; Molnárová,, Jana (referee) ; Stratilová, Eva (advisor)
This work focuses on the induction of galactosidases by yeasts of genus Cryptococcus growing on lactose medium and relations between this production and growth of yeasts. At the end biotyping using mass spectrometry is described. All tested and relative yeasts, hydrolytic enzymes -, -galactosidase and used analytical methods as UV-VIS spectrophotometry and mass spectometry, especially MALDI-TOF are described in the theoretical part. The experimental part consists of description of instrumentations, preparation of necessary solutions, cultivation of microorganisms, measurement of enzymatic activity spectrophotometrically and biotyping of yeasts by mass spectrometry. Each of the 18 tested yeast strains, including species Cr. canescens (CCY 17-3-13), Cr. flavescens (CCY 17-3-6, CCY 17-3-15, CCY 17-3-29, CCY 17-3-31, CCY 17-3-33, CCY 17-3-38), Cr. flavus (CCY 17-3-5), Cr. laurentii (CCY 17-3-2, CCY 17-3-9, CCY 17-3-17, CCY 17-3-24), Cr. magnus (CCY 17-4-39, CCY 17-4-40), Cr. saitoi (CCY 17-3-18), Cr. victoriae (CCY 17-3-26) and Bulleromyces albus (CCY17-3-35, CCY 17-3-37) from the Culture Collection of Yeasts, Institute of Chemistry of SAS (CCY), was cultured for 96 hours in a liquid medium with lactose. During cultivation, the quantity of cells was determined as well as enzyme activities of - and -galactosidase in the medium and on the cell surface. Lactose medium was shown not to be suitable culture medium for all Cryptococcus strains, because some of them grew on it very slowly (CCY 17-3-29, CCY 17-3-5, CCY 17-3-26, CCY 17-3-35), or showed a long adaptation phase (CCY 17-3-2, CCY 17-3-6). A comparison of the growth and surface -galactosidase production curves showed that the link between lactose medium and production of this enzyme does not exist in generally. The results did not confirm neither the expected impact of this enzyme on the growth of strains nor the anticipated induction of -galactosidase by lactose. For instance, the fastest growth on lactose showed the strain Cr. canescens CCY 17-3-13, which exhibited a very low activity of this enzyme on its surface. Relatively increased production of this enzyme was observed on the surfaces of the type strain Cr. laurentii CCY 17-3-2, strain Cr. flavescens CCY 17-3-31 and Cr. flavus CCY 17-3-5. The production of -galactosidase by capsular yeasts was strain-dependent with the exception of the members of Cr. flavescens species. Because of this, the expected general influence of this enzyme on the re-building of the protective cryptococcal capsule can be excluded. Only Cr. flavescens strains can be generally considered as producers of lactose-inducible surface -galactosidase. In other cases, the production of this enzyme matters on single strain and not on species, as can be seen in the case of type strain Cr. laurentii and the other Cr. laurentii strains. Similar induction by lactose medium was also observed with non-cryptococcus yeasts and fungi and therefore the link with the capsules can be also excluded. In connection with the selected culture medium, the second aim of this work was to test the suitability of lactose medium for genus Cryptococcus biotyping by mass spectrometry. Normalized spectra showed very low score between strains of the same species and as a consequence, close related strains were included in the different evolutionary branches using Biotyper programme. These findings led to the conclusion that lactose medium is an unsuitable medium.
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Effect of culture medium on the identification of yeasts of the genus Cryptococcus using mass spectrometry
Jurnečková, Alena ; Vadkertiová, Renata (referee) ; Stratilová, Eva (advisor)
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).
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Biotyping of ascomycetous yeasts
Jurnečková, Alena ; Dudášová,, Hana (referee) ; Stratilová, Eva (advisor)
In total, 84 yeast strains (originated from water, plants, fruits and soil) were selected for MALDI-TOF biotyping. All strains were cultivated on malt agar and YPD medium. Samples for biotyping were processed according to methods of Bruker Daltonik GmbH company, Institute of Chemistry of SAS and combination of these methods. Single strains were identified based on the analysis of intracellular ribozomal proteins using MALDI-TOF mass spectrometry. In case of ambiguous results, the DNA was isolated and the D1/D2 26S rRNA domain sequencing was performed. The strain identification was carried out by comparing its mass spectra with spectra of sequenced strains using MALDI Biotyper 3.0 software. The mutual similarity of strains was considered by score value, which was the result of the analysis. In total, 18 strains from 84 were previously sequenced and used as model strains for comparison with unknown isolates. Altogether 51 strains were definitely taxonomically categorized into 18 phylogenetic groups at the species level. The MALDI-TOF biotyping was repeated for overall 6 strains because of ambiguity of results. The taxonomic classification of 15 strains was not clearly determined and, therefore, these strains were suggested for D1/D2 26S rRNA domain sequencing. It was not possible to identify one strain, based on the results of sequencing, therefore, the DNA isolation was repeated. In the case of 8 strains, the results were identical with originally designed taxonomic classification. Conversely, the remaining 6 strains were identified as species. For 20 selected strains the basic characteristics were determined using microbiological methods. The shape of colonies growing on solid medium and appearance of cultures in liquid medium was assessed. Furthermore, the radial growth constant and the presence of urease were determined. Finally the microscopic observation of cells and the fermentation test for carbohydrate substrates were performed.
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Yeasts of polyphyletic genus Cryptococcus - characteristics and occurence in the nature
Švecová, Natália ; Stratilová,, Eva (referee) ; Schusterová,, Hana (advisor)
Yeasts of genus Cryptococcus belong to the Basidiomycetes, a wide group with different geographical sharing in nature. Many of them rank among human pathogens that endanger immunocompromised patients. Thanks to the big diversity at the level of subspecies, various species of the genus Cryptococcus show different molecular characteristics. Their identification is difficult because of the presence of polysaccharide capsule that surrounds the cell of some species. This work deals with the identification of species occurring in meadows and gardens. The 79 yeasts samples are identified by MALDI-TOF MS. The influence of different culture media on the identification results is monitored simultaneously with the identification. Since a capsule is present in many species, another parameter to be monitored is the influence of the sample preparation method and the matrix on the identification. 51 samples of yeasts of the genus Cryptococcus are identified at the species level in the experimental part. Selected samples are further subjected to the determination of characteristics by conventional microbiological methods. The determined parameters are the presence of urease, radial growth constant, susceptibility to antimycotics, fermentation and assimilation of sugars, growth in the presence of alcohols and growth in the absence of vitamins. The yeast samples are classified into yeast species based on microbiological results. Biotyping resulted in identification of selected samples in the species Filobasidium magnum, Filobasidium oeirense and Filobasidium wieringae. Other samples showed ambiguous identification. As these species have the same properties, they could not be distinguished by the selected microbiological tests.
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Effect of medium composition on the mass spectra of the yeast species of Cryptococcus laurentii and Cryptococcus flavescens
Ledvina, Vojtěch ; Breierová, Emília (referee) ; Stratilová, Eva (advisor)
Cryptococcus laurentii and Cryptococcus flavescens are nonfermenting yeasts forming extracellular polysaccharide capsule. Both species are mainly saprofytic but Cr. laurentii is also known to be an opportunistic pathogen in immunocompromised patients. Cr. flavescens used to be considered a synonym of Cr. laurentii but nowadays it is classified as a separate species that belongs to the phylogenetic group I of the Cr. laurentii group. In the experimental part 28 strains of species Cr. laurentii, Cr. flavescens a Cr. victoriae were biotyped using MALDI-TOF MS. The yeasts were cultivated on three different media (Sabouraud, YPD and potato agar) and three methods were used for the protein extraction. The impact of growth medium composition from which the strains were inoculated on the quality of spectra was studied together with the suitability of individual methods for use on different media. Then the impact of growth medium composition on the quality of acquired spectra was evaluated. Finally, all strains were compared mutually and with the type strain of Cr. laurentii CCY 17 3-2. The composition of the medium cells were inoculated from was found to have little impact on the spectra quality. The same result was determined for the composition of the actual growth medium cells were cultivated on. Crucial for the quality of mass spectrum is the method of cells preparation. Best results were acquired when cultivating cells on YPD agar, washing the cells with ethanol and using mix of sinapinic and ferulic acid as a matrix. Potato agar was found not suitable for cultivating yeasts of the Cryptococcus genus due to significant production of extracellular polysaccharides which complicate the protein isolation process. All strains were compared to Cr. laurentii type strain CCY 17-3-2 and MSP dendrograms were created based on the spectra similarity. In the MSP dendrograms all strains were successfully divided into relevant species on all tested media. Finally sequences of D1/D2 domain of LSU gene were compared and phylogenetic tree was created. This tree was then compared to the MSP dendrograms.
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Biotyping of Cryptococcus laurentii group using mass spectrometry
Jäger, Jakub ; Breierová, Emília (referee) ; Stratilová, Eva (advisor)
Cryptococcus laurentii has been classically considered a saprophytic species, although several cases of human and animal infection have been already reported. This species is reported to be heterogenous. The taxonomy of yeast Cryptococcus laurentii was always highly ambiguous. The application of molecular biology and bioinformatic methods led to dividing of searched strains to two distinct phylogenetic groups, some varieties were recognized as species and the locution „Cryptococcus laurentii group“ was introduced. The taxonomy of this group is likely not definitive and with advancing knowledge will change. Our aim was the identification of individual species within this group based on matrix-assisted laser desorption/ionization – time of flight mass spectrometry (MALDI – TOF MS), which has recently been described as a rapid, reliable, cost-effective and powerful tool for analyzing of microorganisms, even on variety level. Generally, the yeasts of genus Cryptococcus form highly resistant polysaccharide capsules and produce large amount of extracellular polysaccharides, therefore belong to so called „difficult“ cases for biotyping. The experimental protocol has been optimized for MS analysis of this genus on the selected strains of Cr. neoformans, Cr. laurentii and Cr. magnus from the Culture Collection of Yeasts (CCY).Thirty-three strains, originally classified as Cryptococcus laurentii has been identified by chosen method. These strains were distributed into six different groups according their spectra similarities. It was selected at least one strain of each group, which was classified based on the sequence analysis of the D1/D2 domains of the LSU rRNA gene. This strain (with known sequence) became representative for its group. Type strain of Cryptococcus laurentii (CCY 17-3-2) belongs to the group I. Its MS spectrum of ribosomal proteins differ from mass spectra of all other biotyped species, even with strains identified as Cryptococcus laurentii was the similarity of the spectra low, which could be caused by identification of two different varieties. The group II is represented by Cryptococcus laurentii CCY 17-3-17. Except this strain, thirteen more strains belong to the group II. The group III represents Cryptococcus flavescens CCY 17-3-29. This group included 12 additional strains with almost identical mass spectra. Group IV included only one strain (CCY 17-3-13), which was identified as Cryptococcus carnescens based on gene sequence analysis. Similarly, one representative (CCY 17-3-5) has the group V. Strain CCY 17-3-5 was identified as Cryptococcus flavus. The last group VI of three members represents strain 17-3-35 identified as Bulleromyces albus. While Cr. laurentii and Cr. flavescens belong to phylogenetic group I and Cr. carnescens to the phylogenetic group II, four strains giving two types of different MS spectra and identified as Cr. flavus (1 strain) and Bulleromyces albus (3 strains) were excluded from „Cr. laurentii group.“
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Taxonomic classification of yeasts associated with meadow plants
Čurillová, Natália ; Ing.Hana Dudášová, Ph.D. (referee) ; Horváthová, Ágnes (advisor)
In total 60 yeast strains isolated from meadow flowers were selected for identification using MALDI-TOF biotyping. Selected yeast strains were prepared according to standard (Bruker) method and method developed at Institute of Chemistry SAS in Bratislava for MALDI-TOF yeast identification. To acquire valuable data two cultivating media were used. The principle of identification embody in comparing obtained mass spectra according to the score of selected yeast strain isolated from meadow plants, formerly identified by physicobiochemical properties (verification of their taxonomic classification) or the new isolates (their identification) with mass spectra of the reference yeast cultures. Reference yeast cultures were identified by sequencing the D1/D2 of 26S rRNA domain. In total 53 yeast strains were identified by biotyping at species level. The remaining 7 yeast strains were designed to be identified by sequencing the D1/D2 26S rRNA domain as the reference mass spectra were not available. The highest abundance of yeast species was as follows Metschnikowia pulcherrima (12 %), Rhodotorula spp. (12 %), Cystofilobasidium infirmominiatum (10 %), Metschnikowia reukaufii (7 %), Metschnikowia fructicola (7 %), Sporobolomyces metaroseus (7 %), Hanseniospora uvarum (5 %), Rhodotorula mucilaginosa (5 %), Meyerozyma guillermondii (5 %), Cystofilobasidium macerans (3 %) a Rhodotorula dairenensis (3 %), and yeast strains of species Candida bombi, Pichia kudravzievii, Cystofilobasidium capitatum, Cystofilobasidium macerans, Solicoccozyma aeria, Sporidiobolus salmonicolor, Papiliotrema flavescens, Sporidiobolus metaroseus, Sporidiobolus pararoseus and Cryptococcus wieringae were identified as well. In general, the most diverse abundance of yeast species were isolated from meadow plants comparing to the older studies.
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Yeasts of polyphyletic genus Cryptococcus - characteristics and occurence in the nature
Švecová, Natália ; Stratilová,, Eva (referee) ; Schusterová,, Hana (advisor)
Yeasts of genus Cryptococcus belong to the Basidiomycetes, a wide group with different geographical sharing in nature. Many of them rank among human pathogens that endanger immunocompromised patients. Thanks to the big diversity at the level of subspecies, various species of the genus Cryptococcus show different molecular characteristics. Their identification is difficult because of the presence of polysaccharide capsule that surrounds the cell of some species. This work deals with the identification of species occurring in meadows and gardens. The 79 yeasts samples are identified by MALDI-TOF MS. The influence of different culture media on the identification results is monitored simultaneously with the identification. Since a capsule is present in many species, another parameter to be monitored is the influence of the sample preparation method and the matrix on the identification. 51 samples of yeasts of the genus Cryptococcus are identified at the species level in the experimental part. Selected samples are further subjected to the determination of characteristics by conventional microbiological methods. The determined parameters are the presence of urease, radial growth constant, susceptibility to antimycotics, fermentation and assimilation of sugars, growth in the presence of alcohols and growth in the absence of vitamins. The yeast samples are classified into yeast species based on microbiological results. Biotyping resulted in identification of selected samples in the species Filobasidium magnum, Filobasidium oeirense and Filobasidium wieringae. Other samples showed ambiguous identification. As these species have the same properties, they could not be distinguished by the selected microbiological tests.
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Taxonomic classification of yeasts associated with meadow plants
Čurillová, Natália ; Ing.Hana Dudášová, Ph.D. (referee) ; Horváthová, Ágnes (advisor)
In total 60 yeast strains isolated from meadow flowers were selected for identification using MALDI-TOF biotyping. Selected yeast strains were prepared according to standard (Bruker) method and method developed at Institute of Chemistry SAS in Bratislava for MALDI-TOF yeast identification. To acquire valuable data two cultivating media were used. The principle of identification embody in comparing obtained mass spectra according to the score of selected yeast strain isolated from meadow plants, formerly identified by physicobiochemical properties (verification of their taxonomic classification) or the new isolates (their identification) with mass spectra of the reference yeast cultures. Reference yeast cultures were identified by sequencing the D1/D2 of 26S rRNA domain. In total 53 yeast strains were identified by biotyping at species level. The remaining 7 yeast strains were designed to be identified by sequencing the D1/D2 26S rRNA domain as the reference mass spectra were not available. The highest abundance of yeast species was as follows Metschnikowia pulcherrima (12 %), Rhodotorula spp. (12 %), Cystofilobasidium infirmominiatum (10 %), Metschnikowia reukaufii (7 %), Metschnikowia fructicola (7 %), Sporobolomyces metaroseus (7 %), Hanseniospora uvarum (5 %), Rhodotorula mucilaginosa (5 %), Meyerozyma guillermondii (5 %), Cystofilobasidium macerans (3 %) a Rhodotorula dairenensis (3 %), and yeast strains of species Candida bombi, Pichia kudravzievii, Cystofilobasidium capitatum, Cystofilobasidium macerans, Solicoccozyma aeria, Sporidiobolus salmonicolor, Papiliotrema flavescens, Sporidiobolus metaroseus, Sporidiobolus pararoseus and Cryptococcus wieringae were identified as well. In general, the most diverse abundance of yeast species were isolated from meadow plants comparing to the older studies.
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Biotyping of ascomycetous yeasts
Jurnečková, Alena ; Dudášová,, Hana (referee) ; Stratilová, Eva (advisor)
In total, 84 yeast strains (originated from water, plants, fruits and soil) were selected for MALDI-TOF biotyping. All strains were cultivated on malt agar and YPD medium. Samples for biotyping were processed according to methods of Bruker Daltonik GmbH company, Institute of Chemistry of SAS and combination of these methods. Single strains were identified based on the analysis of intracellular ribozomal proteins using MALDI-TOF mass spectrometry. In case of ambiguous results, the DNA was isolated and the D1/D2 26S rRNA domain sequencing was performed. The strain identification was carried out by comparing its mass spectra with spectra of sequenced strains using MALDI Biotyper 3.0 software. The mutual similarity of strains was considered by score value, which was the result of the analysis. In total, 18 strains from 84 were previously sequenced and used as model strains for comparison with unknown isolates. Altogether 51 strains were definitely taxonomically categorized into 18 phylogenetic groups at the species level. The MALDI-TOF biotyping was repeated for overall 6 strains because of ambiguity of results. The taxonomic classification of 15 strains was not clearly determined and, therefore, these strains were suggested for D1/D2 26S rRNA domain sequencing. It was not possible to identify one strain, based on the results of sequencing, therefore, the DNA isolation was repeated. In the case of 8 strains, the results were identical with originally designed taxonomic classification. Conversely, the remaining 6 strains were identified as species. For 20 selected strains the basic characteristics were determined using microbiological methods. The shape of colonies growing on solid medium and appearance of cultures in liquid medium was assessed. Furthermore, the radial growth constant and the presence of urease were determined. Finally the microscopic observation of cells and the fermentation test for carbohydrate substrates were performed.
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