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Molecular Tools for the Study of Fungal Aerosols

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Molecular tools for the study of fungal aerosols Thèse Hamza Mbareche Doctorat en microbiologie Philosophiæ doctor (Ph. D.) Québec, Canada © Hamza Mbareche, 2019 Molecular tools for the study of fungal aerosols Thèse Hamza Mbareche Sous la direction de : Caroline Duchaine, directrice de recherche Guillaume Bilodeau, codirecteur de recherche Université Laval ii Résumé Depuis le développement rapide des méthodes de séquençage à haut débit (SHD) en écologie moléculaire, les moisissures ont eu moins d’attention que les bactéries et les virus, en particulier dans les études de bioaérosols. Les études d'exposition aux moisissures dans différents environnements sont limitées par les méthodes de culture traditionnelles qui sous- estiment le large spectre de moisissures pouvant être présentes dans l'air. Bien que certains problèmes de santé soient déjà associés à une exposition fongique, le risque peut être sous- estimé en raison des méthodes utilisées. L’application du séquençage à haut débit dans des échantillons de sol par exemple a permis de mieux comprendre le rôle des moisissures dans les écosystèmes. Cependant, la littérature n'est pas clair quant à la région génomique à utiliser comme cible pour l'enrichissement et le séquençage des moisissures. Cette thèse vise à déterminer laquelle des deux régions universellement utilisées, ITS1 et ITS2, convient le mieux pour étudier les moisissures dans l’air. Durant le développement de la méthode moléculaire, un autre défi, touchant la perte de cellules fongiques lors de la centrifugation d'échantillons d'air liquide à des fins de concentration, s’est rajouté. Ainsi, cette thèse décrit une nouvelle méthode de filtration pour remédier à la perte due à la centrifugation. Ces deux objectifs représentent la première partie de la thèse qui se concentre sur le développement de méthodes: le traitement des échantillons d’air avant extraction de l’ADN et la meilleure région à cibler avec la méthode SHD. La deuxième partie consiste à appliquer la méthodologie développée pour caractériser l'exposition aux moisissures dans trois environnements de travail différents: le compost, la biométhanisation et les fermes laitières. Les résultats montrent que la région d’ITS1 a surpasser ITS2 en couvrant davantage de diversité dans les bioaérosols. En raison de profils taxonomiques complémentaires, l'auteur de la thèse suggère d'utiliser les deux régions pour couvrir la plupart des taxons lorsque la taxonomie constitue le principal intérêt de l'étude. Cependant, ITS1 devrait être le premier choix dans les autres études, principalement en raison de la grande diversité et de la similarité des profils taxonomiques obtenus par l’approche métagénomique et l’approche ciblant ITS1. De plus, la nouvelle approche de filtration proposée constitue une meilleure alternative pour compenser la perte fongique due à la centrifugation. Ensemble, ces méthodes ont permis une meilleure description de l’exposition aux moisissures en milieu professionnel. iii Abstract Since the rapid development of high-throughput sequencing methods in molecular ecology, fungi have been the underdogs of the microbial world, especially in bioaerosol studies. Particularly, studies describing fungal exposure in different occupational environments have been limited by traditional culture methods that underestimate the broad spectrum of fungi present in the air. There are potential risks in the human inhalation of fungal spores in an occupational scenario where the quantity and diversity of fungi is high. Although some health problems are already known to be associated with fungal exposure in certain work environments, the risk may be underestimated due to the methods used. Applying high-throughput sequencing in soil samples has helped the explanation of the fungal role in ecosystems. However, the literature is not decisive in terms of the genomic region to use as target for the enrichment and sequencing of fungi. The present thesis deals with the challenge of determining which region from the two universally used regions, ITS1 and ITS2, is best suited for study of fungal aerosols. In tandem with this challenge came another of addressing the loss of fungal cells during the centrifugation of liquid impaction air samples for purposes of concentration. This thesis describes a new filtration-based method to circumvent such losses during centrifugation. These two challenges represent the first part of the thesis, which focuses on methodology development. In synopsis, the treatment of air samples prior to DNA extraction is considered, along with the identification of the best region to target in amplicon-based high throughput sequencing. In the second part of the thesis, the focus turns to the application of the developed methodology to characterize fungal exposure in three different work environments: compost, biomethanization, and dairy farms. All three are of special interest due to potentially high fungal exposure. Results show that ITS1 outperformed ITS2 in disclosing higher levels of fungal diversity in aerosol samples. Due to complementarity in the taxonomic profiles disclosed by the two regions, the author suggests the use of both regions to cover the greatest possible number of taxa when taxonomy is the main interest of the study. However, ITS1 should be the first choice in other studies, mainly because of the high diversity it reveals and its concordance with results obtained via shotgun metagenomic profiling. In addition, the new filtration-based approach proposed in this work might be the best alternative available for compensating the loss of propagules in iv centrifugation done prior to DNA extraction. Taken together, these methods allowed a profound characterization of fungal exposure in occupational environments. v Table of Contents Résumé ............................................................................................................................. iii Abstract .............................................................................................................................iv List of Figures ................................................................................................................... ix List of Tables .................................................................................................................... xii List of Abbreviations ....................................................................................................... xiv Acknowledgements ......................................................................................................... xvii Preface ............................................................................................................................ xix Introduction ...................................................................................................................... 1 Chapter 1: Literature Review ............................................................................................ 5 1.1 Bioaerosols .......................................................................................................................... 5 1.2 Air Sampling .................................................................................................................... 10 1.3 Fungal Biology .................................................................................................................. 12 1.4 Health Outcomes after Fungal Exposure ........................................................................ 16 1.5 Occupational Exposure to Fungi ..................................................................................... 22 1.6 Cultivating Fungi ............................................................................................................. 26 1.7 Microbiota Analyses ......................................................................................................... 30 1.8 Bioinformatics .................................................................................................................. 37 1.9 Future of Bioaerosols ....................................................................................................... 41 1.10 Specific Aims of the Thesis ............................................................................................. 42 Part one: Methods for the Characterization of Fungal Aerosols .................................... 44 Chapter 2: Comparison of the performance of ITS1 and ITS2 as barcodes in amplicon- based sequencing of bioaerosols ..................................................................................... 45 2.1 Résumé ............................................................................................................................. 45 2.2 Summary of the Paper ..................................................................................................... 46 2.3 Abstract ............................................................................................................................ 48 2.4 Introduction...................................................................................................................... 48 2.5 Materials and Methods .................................................................................................... 52 2.6 Results .............................................................................................................................. 60 2.7 Discussion ........................................................................................................................
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