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Bordetella Pertussis: Whole Genome Sequence Analysis and Its Application to Public Health Laboratory Surveillance

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Bordetella Pertussis: Whole Genome Sequence Analysis and Its Application to Public Health Laboratory Surveillance Bordetella pertussis: Whole Genome Sequence Analysis and its Application to Public Health Laboratory Surveillance by Alexandre Marchand-Austin A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Laboratory Medicine and Pathobiology University of Toronto © Copyright by Alexandre Marchand-Austin, 2015 Bordetella pertussis: Whole Genome Sequence Analysis and its Application to Public Health Laboratory Surveillance Alexandre Marchand-Austin Master of Science Department of Laboratory Medicine and Pathobiology University of Toronto 2015 Abstract Bordetella pertussis (BP) is a Gram-negative bacterium that causes respiratory infections in humans. Multi-locus sequence types (MLST), virulence factor alleles, and antimicrobial resistance markers are important molecular characteristics of BP that can provide public health officials with information on the continued efficacy of treatment guidelines, the source and spread of infections, and vaccine effectiveness. Determining these characteristics in a rapid, reproducible fashion would be an asset to public health. Short read whole genome sequencing (WGS) of 40 BP isolates and processing by the SRST2 bioinformatic package was able to identify a novel MLST genotype, reveal a significant increase of a virulence factor type compared to previous rates, and confirm the lack of BP antimicrobial resistance to macrolides. Moreover, regions of sequence similarity were also identified that can potentially interfere with the characterization of BP by WGS. This method has potential for laboratory-based public health surveillance of BP. ii Acknowledgments A project such as this one would be simply impossible without the knowledge, help, and support of a wide group of individuals to whom I am greatly indebted. Words simply cannot express my gratitude to all those who have helped me along the way, although given this space to do so I will definitely try! Any heights that I may have achieved through this project, I have done so by being supported by giants. First and foremost, I would like to thank my wonderful parents Rick and Louise. Your unwavering love, support and encouragement throughout my academic and professional career have been and always will be appreciated. At any point when I thought I wasn’t going to make it, a simple conversation over dinner or putting on the snow tires would put my mind at ease. Whether it was direct help like taking me shopping for job interview clothes and keeping the car on the road to get me to school, or indirect help like teaching me attention to detail and punctuality, part of this project belongs to you. My other non-academic shout-out has to go to my amazing girlfriend Cassy. Clearly she is the raddest (yes I wrote raddest in a paper), smartest, funniest girl that would ever have anything to do with me, and I couldn’t have done this without her. With me since my acceptance to this program and always willing to give me advice based on your experience, your real world guidance was a life saver. Even though the practical stuff like reading over paragraphs for me, harassing me to keep writing, and talking out infectious disease nonsense helped a lot, your (and Boots’) love and companionship helped the most. I can’t wait for this thesis to be next to yours on our bookshelf! From the academic side, I must extend my unending gratitude to my supervisor Dr. Frances Jamieson who has always pushed me to better myself academically and professionally. Without your support, I would neither be in this program nor in the public health field. Thank you for your tireless efforts in helping me with this project (despite the hundreds of challenges) and for always being an amazing supervisor, colleague, and friend. Thanks to my co-supervisor from afar Dr. David Farrell whose technical expertise in molecular biology, clinical microbiology, and dry humour made this project work. Thanks also to my committee chair Dr. Natasha Crowcroft for your amazing insight into all things public health, iii and willingness to indulge in some overly “labby bits” to help make this a truly multidisciplinary project. Also thanks to Dr. Jonathan Gubbay who agreed to attend my defense on short notice. If I could one day know even a fraction as much about BP as my committee members combined, I would undoubtedly have become an expert. Thanks also to my original supervisor Dr. Don Low who agreed to take me as a student and to whom I owe my chance to undertake graduate studies. I can only hope that this project does that chance justice as he is sorely missed. Finally, to all those who lent me a direct hand in some way: Jen for helping me shake the rust off my wet-lab skills and always being up for going for lunch. Jennifer, Carla, Nathalie, Mabel, Daniel and Aimin for your lab help, expertise and patience with dumb questions. Gillian and Shelley for your help with vaccination data. Dr. Raymond Tsang and his lab for their help with pertactin. Andre for your bioinformatics help and lunches once Jen left to do her PhD. Nahuel, Taryn, and Sarah for your help with SRST2. Venkata, Gnani, Sandra, and Gustavo who were always willing to be an ear for my crazy theories. Ferzeen and Rama in the graduate office for helping me sort out my numerous supervisor changes. My apologies to anyone I may have inadvertently left out, I’ll be sure to grab you a beer should you point it out! iv Table of Contents Abstract ........................................................................................................................................... ii Acknowledgments .......................................................................................................................... iii Table of Contents ............................................................................................................................ v List of Abbreviations ................................................................................................................... viii List of Tables ................................................................................................................................. ix List of Figures ................................................................................................................................. x List of Appendices ......................................................................................................................... xi Chapter One: Introduction .............................................................................................................. 1 1.1 Overview of Bordetella pertussis Infection ........................................................................ 1 1.1.1 Disease and Diagnosis ............................................................................................ 1 1.1.2 Etiologic Agent and Pathogenesis .......................................................................... 2 1.1.3 Host Response ......................................................................................................... 6 1.2 Re-emergence of Bordetella pertussis as a Public Health Threat ....................................... 8 1.2.1 Pre-Vaccine and Whole Cell Vaccine Era .............................................................. 8 1.2.2 Acellular Component Vaccine and Contemporary Outbreaks .............................. 10 1.3 Pathogen Characterization ................................................................................................ 11 1.3.1 Antimicrobial Resistance ...................................................................................... 11 1.3.2 Multi-locus Sequence Typing ............................................................................... 13 1.3.3 Virulence Factor Typing ....................................................................................... 16 1.3.4 Whole Genome Sequencing .................................................................................. 17 1.4 Public Health Response for Infectious Diseases ............................................................... 18 1.4.1 Provincial Public Health System .......................................................................... 18 1.4.2 Public Health Investigations and Interventions .................................................... 20 1.5 Rationale, Hypothesis, and Objectives ............................................................................. 22 v Chapter Two: Materials and Methods ........................................................................................... 25 2.1 Laboratory Isolation .......................................................................................................... 25 2.2 Strain Selection ................................................................................................................. 25 2.3 Preliminary Antimicrobial Susceptibility Testing ............................................................ 28 2.3.1 Phenotypic Assessment of Minimum Inhibitory Concentration for Azithromycin and Trimethoprim/Sulfamethoxazole ............................................ 28 2.3.2 23S rRNA Gene Sequencing ................................................................................ 28 2.4 Whole Genome Sequencing .............................................................................................. 29 2.4.1 Library Preparation and Sequencing ..................................................................... 29 2.4.2 Sequence Analysis Tool Preliminary Assessment ...............................................
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