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Taxonomy, diversity and clinical relevance of the genus Aspergillus
Hubka, Vít ; Kolařík, Miroslav (advisor) ; Labuda, Roman (referee) ; de Beer, Wilhelm (referee)
Aspergillus is a speciose genus encompassing nearly 400 species that has significant economic impacts on human health, the food industry, biotechnology and pharmacology. The research included in this thesis focuses on current issues related to the generic concept, subgeneric classification and species delimitation in Aspergillus. It addresses the need for revisions of several sections or species complexes. It provides novel information regarding etiology of aspergillosis as well as the antifungal susceptibilities of several less common opportunistic pathogens. The taxonomic section of the thesis contributes to the taxonomic stability and the new concept of the genus Aspergillus, which changed in response to the discontinuation of dual nomenclature in fungi. Sufficient arguments were collected (e.g., verification of monophyly, unifying phenotypic characters) for maintaining a broad concept of the genus and avoiding splitting it into several genera. All genera typified by sexual morphs and having Aspergillus asexual states were synonymized with Aspergillus and the appropriate names adopted; new combinations were made for teleomorphic species that lacked Aspergillus names. This thesis also contributed to infrageneric taxonomy of the genus via the proposal of four new sections in the subg. Circumdati,...
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In-vivo and ex-vivo models for superficial mycosis
Míková, Ivana ; Hubka, Vít (advisor) ; Julák, Jaroslav (referee)
12. 5. 2016 In vivo a ex vivo modely povrchových mykóz (bakalářská práce) Ivana Míková Abstract In my bachelor thesis I have dealt with the importance of using the laboratory animals (in vivo models) and artificial tissue (ex vivo models) for studying the superficial mycosis. The purpose of using these models is especially to clarify the pathogenesis of illness (the pathogenʼs penetration into the organism, the clinical, histopathological and immunological changes) or testing of new treatments. Selection of a suitable and susceptible model, methodics of preparation of the inoculum and preparation of the model before inoculation are crucial for the successful creation of the experiment. Small rodents (guinea-pig, mouse, rat), skin equivalents and keratin films made from human hair, nails and animal hooves are the most used in vivo and ex vivo models. This bachelor thesis summarizes their advantages and disadvantages. Furthermore, the work deals with the types of dermatophytes and thermally dimorphic fungi used for successful inoculation and with the specific purpose of their use. The dermatophytes (Trichophyton, Microsporum, Epidermophyton, Arthroderma) and thermally dimorphic fungi (Blastomyces and Sporothrix) are the most often researched originators of the superficial mycosis. Both groups cause the animal...
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Species limits within Aspergillus section Restricti inferred from multiple gene phylogenies and phenotype analysis
Sklenář, František ; Hubka, Vít (advisor) ; Janko, Karel (referee)
Section Restricti is one of the last sections of the genus Aspergillus that hasn't been revised in the modern taxonomic era. All species are osmophilic, have simple morphology and they are mainly important because of the food, feed and seed spoilage, some of them also have negative influence to the indoor air. The section consists of seven species according to the last taxonomic revision from 2008 based only on molecular data. It currently consists of six asexual and one homothalic species. 126 isolates from the section coming from four continents were studied in this thesis, including ex-type strains. Revision of the section was carried out combining molecular phylogenetic analysis and conventional taxonomic methods. Modern methods of species delimitation based on multispecies coalescent model were used for the phylogenetic reconstruction. From the conventional methods analysis of morphology (macro- and micromorphology including scanning electron microscopy) and physiology (ability of growing in osmotic gradient and several different temperatures) was performed. Apart from the seven known species, eight new undescribed species were discovered. Majority of the new species belongs to the Aspergillus penicillioides species complex. Key words: Aspergillus restrictus, osmophilic fungi, species...
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Molecular typization of isolates from Arthroderma benhamiae complex, a zoonotic agent of epidemic dermatophytosis in Europe
Čmoková, Adéla ; Hubka, Vít (advisor) ; Hrabák, Jaroslav (referee)
Arthroderma benhamiae has been almost unknown among clinical mycologists but it is a cause of around 40 % of dermatophytic infections according to current studies in the Czech Republic. The species is primarily transmitted to humans from guinea pigs and other rodents. The epidemiological situation is similar in other countries in Central and Western Europe. The reason of significant increase in the incidence of infections has not been identified yet, and no sufficiently informative molecular markers have been developed for typification of the species that could help to resolve the cause of this problem. For the purposes of this study, isolates (n=268) from cases of human and animal dermatophytoses from the Czech Republic, Germany, Belgium, Switzerland, Italy, Japan and USA were selected. Ten variable microsatellite markers were developed and sequence analysis of two genetic loci (ITS rDNA and gpdh gene) were performed to reveal intraspecific variation. Phenotype was also studied at the level of micro- and macromorphology of the strains and growth parameters at several temperatures and on several cultivation media. Mating type idiomorph of each isolate was determined and mating experiments were performed by crossing pairs of genetically related as well as genetically distant strains. Sequence...
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Mating system and species concept in heterothallic species of Aspergillus section Fumigati
Dudová, Zuzana ; Hubka, Vít (advisor) ; Mencl, Karel (referee)
The aim of this study was to determine the species boundaries within the Aspergillus viridinutans species complex. The species belonging to this complex are predominantly soil- inhabiting organisms that are increasingly reported as opportunistic human and animal pathogens. A total number of 98 isolates from various substrates and countries was subjected to morphological, physiological and molecular analysis (calmodulin, β-tubulin, actin and RNA polymerase II subunit 2 gene) and mating experiments were provided on different media and temperatures. Some other heterothallic species from section Fumigati with unknown sexual state were analysed using similar method as well. Key words: Aspergillus viridinutans, Aspergillus turcosus, phylogenetic analysis, mating- type genes, anamorph, teleomorph, mating experiments, MAT1-1, MAT1-2
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Mechanisms of water stress adaptations of the filamentous fungi
Sklenář, František ; Hubka, Vít (advisor) ; Nováková, Alena (referee)
Some species of the filamentous fungi can grow on the substrates with low water activity that have either low water content or high concentration of osmotically active substances. These fungi are usually called osmotolerant or xerotolerant. They occur particularly in the soil of arid areas and in hypersaline environments. Economically important species cause spoilage of stored food, seed and feed. This thesis brings the summary of physiological and morphological adaptations of fungal organisms to water stress and deals with the molecular genetic background of these adaptations. The current taxonomic classification of the fungal organisms adapted to grow at low water activity is summarized. Separate chapters are dedicated to the influence of different osmotically active substances on the phenotype of the fungi and to the possibilities of using media with osmotically active substances for taxonomic purposes.
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Molecular methods used for taxonomy and epidemiology of dermatophytes
Čmoková, Adéla ; Hubka, Vít (advisor) ; Hamal, Petr (referee)
In my bachelor thesis I have dealt with the role and aplication of the molecular methods in the taxonomy and epidemiology of the dermatophytes and the question of the species concepts in dermatophytes. In the first part, I focused on the evaluation of the advantages and disadvantages of the recent species concepts and their problematic application in dermatophytes. The second part is focused on the molecular methods that used genetic markers for phylogenetic analysis and species delineation within dermatophytes, e.g. the PCR-RFLP method, PCR-fingerprinting and DNA sequencing. I have evaluated the dicrimination power of the particular DNA sequence loci to distinguish closely related species. In the last part, I have summarized the molecular methods that have been used in the typization at the intraspecific level, e.g. microsatelite analysis, PCR-fingerprinting, multilocus sequence typing.
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β-tubulin paralogs in Aspergillus: taxonomical importance and molecular tools for distinguishing
Hubka, Vít ; Kolařík, Miroslav (advisor) ; Bunček, Martin (referee)
A beta-tubulin gene (benA) is widely used in taxonomy and identification of Aspergillus spp. and other Fungi.Across Aspergillus spp. There is either one (benA) or two beta-tubulin paralogs (benA and tubC). The risk ofcontemporary use of sequences of paralogous genes with non-homologous function in the same phylogeneticanalysis is well known. It is evident that it had happened repeatedly in Aspergillus section Nigri. It is alarmingthat conventional primers for amplification of partial benA sequence can specifically amplify tubC paralog insome species. In this work, both paralogs were characterised in a set of species. The beta-tubulin primers in usewere revised and new, more benA specific primers were designed. Applicability of some markers such as basecomposition, codon usage and length of introns for distinguishing -tubulin paralogs benA and tubC is tested. Alarge study on molecular diversity of 349 isolates of Aspergillus (PCR-fingerprint, sequence data - ITS, benA,rpb2, caM) originating from Czech culture collections and from clinical material is also included. 82 specieswere identified, togetherwith nine tentative new taxa belonging to sections with high economic impact - Nigri,Fumigati or Aspergillus (Eurotium spp.). Five species from Section Aspergillus could be synonymised withexisting taxa. A study...
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