TAXONOMIC CLASSIFICATION OF MYCOBACTERIA OF THE MYCOBACTERIUM CHELONAE-MYCOBACTERIUM ABSCESSUS COMPLEX WITHOUT CONCLUSIVE SPECIES IDENTIFICATION Christiane Lourenço Nogueira Dissertation submitted in fulfillment of the requirements for the degree of Doctor (Ph.D.) in Sciences, Biochemistry and Biotechnology (Ghent University) Doctor (Ph.D.) in Microbiology and Immunology (Universidade Federal de São Paulo) Promotors: Prof. Dr. Peter Vandamme (Ghent University) Prof. Dr. Sylvia Cardoso Leão (Universidade Federal de São Paulo) São Paulo, 24th October 2016 EXAMINATION COMMITTEE BELGIAN JURY MEMBERS Dr. Anandi Martin LM-UGent: Laboratory of Microbiology Faculty of Science, Ghent University Prof. Dr. Steven Calles Faculty of Medicine, University Hospital, Ghent University BRAZILIAN JURY MEMBERS Prof. Dr. Rafael Silva Duarte Instituto de Microbiologia Prof. Paulo de Góes Departamento de Microbiologia Médica Universidade Federal do Rio de Janeiro Prof. Dr. Aline Maria da Silva Departamento de Bioquímica Instituto de Química Universidade de São Paulo Prof. Dr. Prof. Dra Rosana Puccia Professor Associado Livre Docência Departamento de Microbiologia, Imunologia e Parasitologia Escola Paulista de Medicina Universidade Federal de São Paulo – Campus São Paulo Dr. Roxane Maria Fontes Piazza Pesquisadora Científica Laboratório de Bacteriologia Instituto Butantan ACKNOWLEDGEMENTS Firstly, I would like to thank my advisor Prof. Sylvia Leão for the opportunity to carry out my PhD at UNIFESP, for the continuous support of my study, sharing with me her expertise, and for her sincere and valuable guidance. She definitely provided me the essential tools that I needed to complete my thesis. I also would like to thank my labmate at UNIFESP Cristianne Matsumoto that always encouraged me with the experiments and helped me with her great wet-lab experience. To Vitória Dias, Mércia Vieira and all staff of Department of Microbiology, Immunology and Parasitology of UNIFESP for their various forms of supporting my study. I would like to thank my advisor Prof. Peter Vandamme who provided me an unique opportunity to join his team doing a joint PhD at UGent, and who gave me access to all facilities of the Laboratory of Microbiology. I appreciate his technical support and assistance in thesis and papers writing. In frame of my joint PhD I had gone to Ghent four times, where several people contributed to my research. I am really thankful to all Laboratory of Microbiology staff. My special thank to Anandi Martin who always warmly received me in Ghent; for her patience, motivation, expertise and friendy advices. To Juan Carlos Palomino for his insightful comments and expertise that greatly assisted my research. To Margo Cnockaert, for her parent-like support that made me feel at home during all my stay in Ghent; and of course for her support in DNA-DNA hibridization. To Cindy Snauwaert that kindly helped with her expertise in DNA extraction. And also to my labmates Joahana Monteresin, Marie Sylvianne and Beatriz Lopez for the happy time that we spent together during my stays at Ghent. I would like to thank Prof. Françoise Portaels, Prof. Denise de Freitas, Prof. Christopher Whipps and Dr. Érica Chimara that kindly provided the isolates included in this study. I am also thankful to Érica Chimara, Natália Fernandes, Romilda Aparecida Lemes and Fernanda Simeão from Instituto Adolfo Lutz, who helped me with their expertise in susceptibility testing and phenotypic analyses. I would like to thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for providing funds for this research (2012/13763-0). Finally, I must express my gratitude to my family. A special thank to my lovely husband Lucas for supporting me spiritually through the process of researching and writing this thesis and to always encouraging me to do my best. To my parents Orlando and Margarida, my brother André and my sister Thais for providing me with unfailing support and continuous encouragement through my years of study. And to my adorable nephews, Luis Henrique and Benjamin, that fill my days with so much joy and happiness. This accomplishment would not have been possible without all of them. THANK YOU! HEEL HARTELIJK BEDANKT! OBRIGADA! TABLE OF CONTENTS ACKNOWLEDGEMENTS ....................................................................................................... 3 LIST OF ABBREVIATIONS .................................................................................................... 9 OUTLINE OF THE THESIS .................................................................................................. 11 PART I: BACKGROUND AND OBJECTIVES ......................................................................... 13 Background ................................................................................................................. 14 Objectives .................................................................................................................... 16 PART II: LITERATURE ..................................................................................................... 17 1 Mycobacterium sp .................................................................................................... 18 1.1 Mycobacterium sp – General characteristics ............................................................................ 18 1.2 Mycobacterium sp – Taxonomy .................................................................................................. 19 2 Nontuberculous Mycobacteria (NTM) ..................................................................... 21 2.1 M. chelonae-M. abscessus complex .......................................................................................... 23 2.1.1 Taxonomy of M. chelonae-M. abscessus complex ........................................................... 24 3 Mycobacteria Identification Methods ..................................................................... 29 3.1 Phenotypic Methods ...................................................................................................................... 30 3.2 Mycolic acid analysis ..................................................................................................................... 31 3.3 Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI- TOF MS) .......................................................................................................................................... 31 3.4 Genotypic Methods ....................................................................................................................... 33 3.4.1 DNA Sequencing .................................................................................................................... 33 3.4.1.1 16S rRNA ......................................................................................................................... 34 3.4.1.2 rpoB ................................................................................................................................... 35 3.4.1.3 16S-23S Internal Transcribed Spacer (ITS) ............................................................... 35 3.4.1.4 hsp65 ................................................................................................................................ 36 3.4.1.5 Multilocus Sequence Analysis (MLSA) ........................................................................ 37 3.4.2 PCR (Polymerase Chain Reaction) – Restriction Enzyme Analysis (PRA) .................. 38 3.4.3 DNA strip assays .................................................................................................................... 39 3.4.3.1 INNO-LiPA® ...................................................................................................................... 39 3.4.3.2 Genotype® Mycobacterium assays .............................................................................. 41 4 Drug Susceptibility Testing (DST) for Mycobacteria ............................................. 43 5 Description of new species ..................................................................................... 45 5.1 Polyphasic approach ..................................................................................................................... 45 5.1.1 Phenotypic methods .............................................................................................................. 46 5.1.2 Genotypic methods ................................................................................................................ 46 5.1.2.1 DNA G+C content ........................................................................................................... 47 5.1.2.2 DNA–DNA Hybridization (DDH) .................................................................................... 47 5.1.2.3 DNA sequencing ............................................................................................................. 48 5.1.2.4 Whole Genome Sequencing (WGS) ............................................................................ 49 5.2 Minimal standards for assignment new species to the Mycobacterium genus .................... 51 PART III: EXPERIMENTAL WORK...................................................................................... 53 6 Samples and Isolates ............................................................................................... 54 7 Preliminary Identification of the Isolates ............................................................... 57 7.1 Introduction