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One Health approach to understand emerging zoonotic pathogens in the Trichophyton benhamiae complex
Čmoková, Adéla ; Hubka, Vít (advisor) ; Buchta, Vladimír (referee) ; Gené, Josepa (referee)
The Trichophyton benhamiae complex encompasses several zoonotic pathogens that are of increasing concern to the scientific community due to their epidemic spread among pets and their owners. Of particular concern is the sudden appearance and rapid spread of T. benhamiae yellow phenotype strains in Europe. Considerable genetic and phenotypic variability has been revealed in the pathogens from the complex, yet the species limits and host spectra have not been clearly elucidated. To explore the diversity, epidemiology, and taxonomy of the T. benhamiae complex, my colleagues and I formed an international, cross-disciplinary team and applied a holistic One Health approach. We collected a large dataset of strains from several continents, along with associated data about host, clinical picture, and locality. Due to the high level of clonality in commonly used DNA sequence markers, I developed a new typing schema based on ten microsatellite markers and four DNA sequence markers. We then used a polyphasic approach combining data from independent analyses to decide on species limits in the T. benhamiae complex, including phylogenetic and population-genetic analyses, phenotypic and physiological analyses, mating-type gene characterization, ecological data and MALDI- TOF mass spectrometry analysis. Thanks to...
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Implementing modern species delimitation techniques into fungal taxonomy: Aspergillus charting the course
Sklenář, František ; Hubka, Vít (advisor) ; Haelewaters, Danny (referee) ; Janko, Karel (referee)
This thesis focuses on the implementation of modern methods and approaches for species delimitation in the genus Aspergillus, an economically important fungal genus and model organism. So far, taxonomic studies in Aspergillus regularly included multiple DNA loci (typically four), and the phylogenetic analysis itself mostly consisted of calculating a phylogenetic tree based on individual loci and the concatenated dataset, or less commonly on genealogical concordance principles. Additionally, the datasets usually contained only a limited number of strains per species. The inclusion of more sophisticated phylogenetic species delimitation methods (mostly based on Multispecies coalescent model) in Aspergillus taxonomy adds a whole new layer to the analysis that ensures much higher objectivity of taxonomic decisions. Each of the eight presented papers contains a taxonomic treatment of a particular part of the genus with phylogenetic analyses based on datasets containing DNA sequences from three to six loci or whole genome data (series Nigri). The available molecular data are analyzed by a gradually refined set of phylogenetic species delimitation methods reflecting progress made between years 2017 and 2022. The individual papers also examine some specific characteristics that suitably complement the...
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Taxonomic overview of the genus Arthroderma with focus on species associated with reptiles and bats
Kolarczyková, Daniela ; Hubka, Vít (advisor) ; Dobiáš, Radim (referee)
The genus Arthroderma belongs to a group of geophilic dermatophytes that primarily degrade keratin in soil and occasionally cause infections in humans and animals. This species-rich genus has received particular attention in recent years in terms of species occurring in human clinical material or clinical samples isolated from rodents and other mammals, which has been associated with a significant increase in the number of its species. In this thesis, the species diversity of the genus Arthroderma was investigated in material isolated from snakes, bats and cave sediment, a substrate that has been almost unexplored in terms of the occurrence of this genus. The strains studied were isolated during research on bats diseased by "white nose syndrome" and snakes by "snake fungal disease". In the past, the genus Arthroderma has been studied either on the basis of morphology or, after the advent of molecular methods, in terms of multigene phylogeny. However, no monographs have simultaneously combined phenotypic and molecular genetic criteria to assess species boundaries across the genus or a significant part of it. This is one of the reasons why species boundaries are often unclearly defined. Thus, another aim of this work was to improve the multigene phylogeny of the genus by incorporating new strains,...
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Azole resistance in Aspergillus fumigatus: genetic background and mechanisms of spread in the Czech Republic
Holíková, Kristýna ; Hubka, Vít (advisor) ; Chrenková, Vanda (referee)
Aspergillus fumigatus is a widespread saprophytic fungus. As an opportunistic pathogen it can also cause aspergillosis, which can manifest itself as a life-threatening infection in immunocompromised patients. Azole derivatives constitute first-line therapy of aspergillosis, but the increase worldwide of azole-resistant A. fumigatus strains represents a serious clinical problem. So far most of the molecular mechanisms causing azole resistance are dependent on the cyp51A gene. Studies of the last decade have shown that primarily because of the use of azole fungicides in agriculture, azole-resistant strains of A. fumigatus are selected in the environment. This subsequently complicates the treatment of patients with aspergillosis, and the incidence of infections unresponsive to treatment with azole antifungals increases. Increasing incidence of azole antifungals-resistant infections has become a growing concern: yet the prevalence of azole resistance has not yet been assessed in Czech Republic. Therefore, the first objective of this thesis was to determine Czech Republic's prevalence of azole resistance in clinical strains of A. fumigatus. Those findings permitted the observation of the molecular-genetic mechanisms of this resistance. Second aim was to study environmental prevalence of azole resistance...
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Species delimitation in Aspergillus section Candidi
Glässnerová, Kateřina ; Hubka, Vít (advisor) ; Caboň, Miroslav (referee)
Aspergillus section Candidi encompasses white- or yellow-sporulating species mostly isolated from indoor and cave environments, soil, food, feed, clinical material and dung. Their identification is non-trivial due to largely uniform morphology. This work aims to re- evaluate the species boundaries in the section Candidi and present an overview of all existing species along with information on their ecology. For this work, a set of 113 strains of different origins was gathered. DNA sequences of the genes for β-tubulin (benA), calmodulin (CaM), and the second largest subunit of RNA polymerase (RPB2) were used for molecular analyses, and species delimitation methods based on a multispecies coalescent model were applied. Classical phylogenetic methods and the genealogical concordance species recognition approach were used for comparison. Phenotypic studies involved comparisons of macromorphology on four cultivation media, seven micromorphological characters and growth in temperatures ranging from 10 to 45 řC. For the final decision about species boundaries, an integrative approach consisting of four criteria was defined, based on which seven existing and two undescribed species were supported. The revised section Candidi comprises nine species, some of which manifest a high level of intraspecific...
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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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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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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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