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Antifungal resistance mechanisms in dermatophytes
Kolarczyková, Daniela ; Hubka, Vít (advisor) ; Kuklová, Ivana (referee)
For the treatment of superficial fungal infections (dermatophytosis) is available a wide spectrum of antifungals from various chemical groups. These antifungals are mainly focused on various steps of ergosterol synthesis, thereby disrupting the cell membrane (allylamine, azole and morpholine antifungals) and on microtubule function (benzofuran antifungals). Despite the fact that the treatment of dermatophytosis is often associated with long-term exposure of the fungus to antifungals (weeks or months), until recently antifungal resistance in dermatophytes has been considered rare. However, current studies have shown the incidence of chronic infections, reinfection and treatment failures due to emerging resistance to some commonly used antifungals. The most serious problem today is the spread of resistance to terbinafine where the molecular principles are founded in the structural changes in the squalene epoxidase enzyme (SQLE). The increase in the incidence of this and other resistances is currently alarming especially in India, while the situation in Europe and America is in terms of dermatophyte susceptibility still quite favorable. The aim of this thesis is to summarize our knowledge of antifungal resistance in dermatophytes and their molecular principles. The thesis further summarizes the...
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Vegetative incompatibility in filamentous ascomycetes
Glässnerová, Kateřina ; Hubka, Vít (advisor) ; Sklenář, František (referee)
Vegetative incompatibility is a process occurring during vegetative growth in filamentous fungi which can prevent fusion of hyphae between individuals. In case of hyphal fusion between two individuals with genetically incompatible combination of alleles of vegetative incompatibility genes (i.e. het or vic genes), the newly-emerged heterokaryotic cell is destroyed via programmed cell death, which can be mediated in various ways. The purpose of this mechanism can be to preserve the genetic individuality of an individual or to prevent the transmission of a deleterious cytoplasmatic elements (e.g. mycoviruses). Exploring the vegetative incompatibility genes plays an important role for example in the induction of sexual state in vitro or in the study of speciation mechanisms in fungi. On the molecular-genetic level, vegetative incompatibility has been so far described in detail only in three ascomycete species. This thesis aims to summarize our knowledge concerning the significance of vegetative incompatibility and genetical mechanisms that underlie this process. Key words: vegetative incompatibility, het genes, vic genes, mat locus, programmed cell death, Neurospora crassa, Podospora anserina, Cryphonectria parasitica
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Genetic background and spread of azole resistance in Aspergillus fumigatus
Holíková, Kristýna ; Hubka, Vít (advisor) ; Lysková, Pavlína (referee)
Aspergillus is a globally widespread saprophytic filamentous genus of fungi, including opportunistic pathogenic species that cause a disease called aspergillosis. Azole derivatives have an irreplaceable role in the treatment of aspergillosis. These drugs inhibit the biosynthetic pathway of ergosterol. That leads to disruption of the fungal plasma membrane. However, nowadays, the rate of occurrence of azole-resistant fungi is rapidly increasing, which also increases the mortality of the patients. The aim of this bachelor thesis is to summarize the current knowledge associated with azole resistance with focus on its genetic background and global distribution. The main emphasis is given on the species Aspergillus fumigatus which is the most common agent of aspergillosis. Resistance to azole derivatives arises from prolonged fungi exposure to azole drugs or fungicides. The most common origin of azole resistance relates to the use of azole fungicides in agriculture. There are several ways how the resistance works. Most of them, however, relate to changes in the cyp51A gene, which is affected by azoles, and its promoter, with the most frequent mutation being TR34/L98H. The most common mechanism of the resistance that is not associated with changes in the cyp51A gene is increased expression of efflux...
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Verification of species boundaries in clinically relevant Arthroderma species
Míková, Ivana ; Hubka, Vít (advisor) ; Dobiáš, Radim (referee)
The genus Arthroderma contains predominantly geophilic dermatophytes (naturally occuring in soil). Some species, especially those from Trichophyton terrestre complex, cause human and animal dermatomycosis. In the past, the species boundaries were determined mainly on the basis of biological species concept using in vitro mating experiments. But these nearly 70-years-old findings have not been tested by means of modern taxonomic methods. In total 194 species of the genus Arthroderma (including all available ex-type strains) originating predominantly in USA, Canada and Europe were studied in this thesis. They were mostly isolated from soil (n = 77), animals (n = 50), human clinical material (n = 41) and cave sediment (n = 9). The main goal of the thesis was to elucidate the species boundaries between species A. insingulare, A. lenticulare and A. quadrifidum, that were classified into the T. terrestre complex because of their seemingly identical asexual stage. Further, this work aimed to resolve the relationship between Arthroderma species using the multigene phylogeny and clarify which species are clinically relevant. A multigene phylogeny of the genus Arthroderma was based on the sequences of the ITS rDNA region, β-tubulin (TUB2) and translation elongation factor 1α (TEF1α) genes. The genus...
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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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