TAXONOMY AND PHYLOGENY OF CLINICALLY IMPORTANT MUCORALEAN FUNGI Eduardo Álvarez Duarte Dipòsit Legal: T-1721-2011 ADVERTIMENT. La consulta d’aquesta tesi queda condicionada a l’acceptació de les següents condicions d'ús: La difusió d’aquesta tesi per mitjà del servei TDX (www.tesisenxarxa.net) ha estat autoritzada pels titulars dels drets de propietat intel·lectual únicament per a usos privats emmarcats en activitats d’investigació i docència. No s’autoritza la seva reproducció amb finalitats de lucre ni la seva difusió i posada a disposició des d’un lloc aliè al servei TDX. No s’autoritza la presentació del seu contingut en una finestra o marc aliè a TDX (framing). Aquesta reserva de drets afecta tant al resum de presentació de la tesi com als seus continguts. En la utilització o cita de parts de la tesi és obligat indicar el nom de la persona autora. ADVERTENCIA. 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UNIVERSITAT ROVIRA I VIRGILI TAXONOMY AND PHYLOGENY OF CLINICALLY IMPORTANT MUCORALEAN FUNGI Eduardo Álvarez Duarte DL: T-1721-2011 UNIVERSITAT ROVIRA I VIRGILI DEPARTAMENT DE CIÈNCIES MÈDIQUES BÀSIQUES Taxonomy and phylogeny of clinically important mucoralean fungi Eduardo Alvarez Duarte Doctoral thesis 2011 UNIVERSITAT ROVIRA I VIRGILI TAXONOMY AND PHYLOGENY OF CLINICALLY IMPORTANT MUCORALEAN FUNGI Eduardo Álvarez Duarte DL: T-1721-2011 UNIVERSITAT ROVIRA I VIRGILI TAXONOMY AND PHYLOGENY OF CLINICALLY IMPORTANT MUCORALEAN FUNGI Eduardo Álvarez Duarte DL: T-1721-2011 Departament de Ciències Mèdiques Bàsiques Facultat de Medicina i Ciències de la Salut Universitat Rovira i Virgili Josep Guarro Artigas, José F. Cano Lira, and Alberto M. Stchigel, Professors of the department of Basic Health Sciences of the Universitat Rovira i Virgili. STATE THAT: The present work entitled “Taxonomy and Phylogeny of clinically important mucoralean fungi” presented by Eduardo Alvarez Duarte to obtain the degree of doctor by the University Rovira i Virgili, has been carried out under my supervision at the Unit of Microbiology of the Department of Basic Health Sciences, and that it fulfills the requirements to obtain the European Doctorate mention. Reus, July 10, 2011 Dr. Josep Guarro Dr. José F. Cano Lira Dr. Alberto M. Stchigel UNIVERSITAT ROVIRA I VIRGILI TAXONOMY AND PHYLOGENY OF CLINICALLY IMPORTANT MUCORALEAN FUNGI Eduardo Álvarez Duarte DL: T-1721-2011 UNIVERSITAT ROVIRA I VIRGILI TAXONOMY AND PHYLOGENY OF CLINICALLY IMPORTANT MUCORALEAN FUNGI Eduardo Álvarez Duarte DL: T-1721-2011 Acknowledgments I would like to end this thesis by acknowledgments the people who one way or another contributed to the success of this work. However it is difficult to recall every person that has assisted, contributed or more importantly encouraged me to pursue these studies. I would like to show my gratitude to Dr. Josep Guarro for his support, encouragement and guides during these years; and for giving me the opportunity to work in his lab. I would like to thanks to Dr. José Cano and Dr. Alberto Stchigel for giving me advices and for the guidance in these years. Thanks to Dr. Josepa Gené and all others professors of the Unit of microbiology of URV, for the assistance among these years. I would like to sincerely thanks to Dr. Luis Zaror and Dr. Eduardo Valenzuela, two Chilean mycologists from who I learned so much about the fungi. I am very grateful to Dr. Cony Decock (MUCL; Mycotheque de l'Université catholique de Louvain, Belgique) for accepting me in his research group for 3 months, and for his encouragement and sense of humor. Thanks for sharing your time with me. I also want to express my gratitude to other people who helped me during my stay at the MUCL: Prudence and the little Noora, Mario, Céline, Stéphanie, Olivier, Oscar, Mateus, Paul, Madu…..thanks to all the staff at MUCL. I really appreciate the friendship, help and encouragement of my labmates at URV: Luis, Hugo, Valentina, Fabiola, Anabel, Haybrig, Mónica, Carolina, Enrique, Mar, Margarita, Keith, Mabel, Carme……many names!!!!, in general, thanks to all people in the lab. Thanks to my friends Shiva and Max; especially for your good sense of humor and advices. Thanks to Cati Nuñez and Núria Pilas for your help during these years. UNIVERSITAT ROVIRA I VIRGILI TAXONOMY AND PHYLOGENY OF CLINICALLY IMPORTANT MUCORALEAN FUNGI Eduardo Álvarez Duarte DL: T-1721-2011 Thanks to Montse and Inma, for your help and good will. I owe my deepest gratitude to my dear parents, Eduardo and Olga, thanks for giving me solid roots from which I can grow, for being a constant and active presence in my life, for show me the way with your example. In the same way, thanks to my brother and sister, for your love and respect. Finally, I wish to thank to all people with whom I have shared experiences, to those which with the gift of their company and friendship made my days more enjoyable and worth living. UNIVERSITAT ROVIRA I VIRGILI TAXONOMY AND PHYLOGENY OF CLINICALLY IMPORTANT MUCORALEAN FUNGI Eduardo Álvarez Duarte DL: T-1721-2011 CONTENTS Page List of frequently used abbreviations ................................................................................ i 1. INTRODUCTION ...................................................................................................... 1 1.1 General features of zygomycetes……………........................................................ 3 1.2 Taxonomy and phylogeny......…………………………… .................................... 6 1.3 Epidemiology of the mucormycosis………………………………..................... 12 1.4 Clinical presentations of mucormycosis………..………………………............. 15 1.4.1 Rhino-cerebral mucormycosis………….…………………………..…….... 16 1.4.2 Pulmonary infection………….……………………………………..……. .. 16 1.4.3 Cutaneous infection………….……………………………………….. ........ 17 1.4.4 Gastrointestinal infection………….……………………..…………….……18 1.4.5 Disseminated infection………….…………………………… ..................... 18 1.4.6 Atypical presentations………….………………………………..………….19 1.5 Treatment………..………………………............................................................ 19 1.6 Molecular phylogeny of the Mucorales………..……………………….............. 22 2. INTEREST AND OBJECTIVES……………………………………………….... 25 2.1 Interest of this work and objetives………..………………………...................... 27 3. MATERIALS AND METHODS……………………….…………….................... 29 3.1 Origin of the isolates and storage………………………….………………… .... 31 3.2 Phenotypic characterization……………………………...................................... 31 3.2.1 Morphology..….…………………………………………………………….32 3.2.1.1 Colonies……………………………………….………………………..32 UNIVERSITAT ROVIRA I VIRGILI TAXONOMY AND PHYLOGENY OF CLINICALLY IMPORTANT MUCORALEAN FUNGI Eduardo Álvarez Duarte DL: T-1721-2011 3.2.1.2 Microscopic features……………………………………………….…...32 3.2.1.3 Electron microscopy…………………………………………………....33 3.2.2 Physiological tests..….………………………………………………….... .. 34 3.3 Molecular studies…………………………………………………………...……35 3.3.1 Phylogenetic studies……………………………………………………... ... 38 3.4 Mating tests……………………………………………………………………....39 3.5 Antifungal susceptibility testing…………………………………………………39 4. RESULTS AND DISCUSSION……..……………………………………………..41 4.1 Spectrum of Zygomycete Species Identified in Clinically Significant Specimens in the United States…………………………………………………………..............43 4.2 Two new species of Mucor from clinical samples…………………………….... 57 4.3 Molecular phylogenetic diversity of the emerging mucoralean fungus Apophysomyces: Proposal of three new species……………………………………..71 4.4 Molecular Phylogeny and Proposal of Two New Species of the Emerging Pathogenic Fungus Saksenaea……………………………………………………….83 5. SUMMARIZING DISCUSSION………………………………………….. .......... 93 6. CONCLUSIONS………………………………………………………………......103 7. REFERENCES………………………………………………………………… ... 107 UNIVERSITAT ROVIRA I VIRGILI TAXONOMY AND PHYLOGENY OF CLINICALLY IMPORTANT MUCORALEAN FUNGI Eduardo Álvarez Duarte DL: T-1721-2011 ANI anidulafungin ABLC amphotericin B lipid complex AFLP Amplified Fragment Length Polymorphism AMB amphotericin B ATCC American Type Culture Collection BAL bronchoalveolar lavage BI Bayesian inference BLAST Basic Local Alignment Search Tool ca. circa, Latin word meaning “around”