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The Design and Application of a Molecular Profiling Strategy to Identify Polymicrobial Acute Sepsis Infections

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The Design and Application of a Molecular Profiling Strategy to Identify Polymicrobial Acute Sepsis Infections University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2015-08-14 The Design and Application of a Molecular Profiling Strategy to Identify Polymicrobial Acute Sepsis Infections Faria Crowder, Monica Faria Crowder, M. (2015). The Design and Application of a Molecular Profiling Strategy to Identify Polymicrobial Acute Sepsis Infections (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/28047 http://hdl.handle.net/11023/2388 doctoral thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY The Design and Application of a Molecular Profiling Strategy to Identify Polymicrobial Acute Sepsis Infections by Monica Martins Pereira Faria-Crowder A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPY GRADUATE PROGRAM IN MICROBIOLOGY, IMMUNOLOGY, AND INFECTIOUS DISEASES CALGARY, ALBERTA AUGUST 2015 © Monica Martins Pereira Faria-Crowder 2015 Abstract Sepsis is a term used to describe an array of clinical presentations ranging from mild body dysfunction to multiple organ failure. These clinical signs result from a systemic inflammatory response to microbes or microbial products present in sterile sites such as blood. Current clinical diagnostics rely on culture techniques to identify systemic infections. However, culture lacks sensitivity and a positive result is only obtained in 40% of cases thereby limiting our knowledge of sepsis microbiology. This doctoral study described the development of methods for direct detection of bacteria or bacterial products in blood. A method of lysing host cells and a bacterial DNA extraction protocol was developed and evaluated on mock bacterial communities spiked into whole blood. The results indicated that viable bacteria could be recovered down to 10 CFU/ml using this method. Paired-end Illumina sequencing of the 16S rRNA gene also indicated that the bacterial DNA extraction method enabled recovery of bacterial DNA from spiked blood. This method demonstrated improved detection of systemic bacterial infections involving bacteria as well as their products in three cohorts of clinically septic patients. Application of the paired-end Illumina 16S rRNA sequencing to saponin treated blood from intensive care unit (ICU) and emergency department (ED) patients indicated there were bacterial DNA profiles present in whole blood. These patterns were examined alongside the patient’s clinical data and indicated common molecular profiling patterns were associated with the primary source of infection. Polymicrobial DNA was present in the blood samples with the taxonomic profiles suggesting commensal microbiota were implicated in addition to a principal pathogen. Bacterial DNA from Streptococcus and Staphylococcus were abundant in patients that died in the ICU. ii Overall this study identified common bacterial DNA patterns in the blood of septic patients which were associated with the patients’ primary source of infection, implicated the commensal microbiota in systemic infection and suggested that sepsis infection may not always involve persistent bacterial bloodstream infections. Rather, this study concluded that bacterial products or viable organisms are likely cyclically present and cleared from the bloodstream resulting in a robust inflammatory response. iii Acknowledgements I would like to acknowledge my supervisors, Dr. Michael Surette and Dr. John Conly, for their tireless efforts on this thesis research. Dr. Michael Surette has provided extensive support on the scientific development of this project and the technical aspects of this project. Further, his insight into microbial populations and the dynamics of the host-pathogen interactions at the interface of infection have provided me with an excellent knowledge base during my doctoral training. Dr. John Conly has provided invaluable clinical insight into this project. He has helped me interpret clinical data and understand the implications of this doctoral research in a clinical environment. Without Dr. Conly’s support on this project the interpretation of this data would have been limited and many of the important conclusions would not have been made. I would like to acknowledge my supervisory committee members Dr. Michael Parkins and Dr. Paul Kubes for their support during my doctoral work. I would like to acknowledge the many members of the Alberta Sepsis Network who facilitated the sample collection and clinical data collection for this study. In particular, Joseé Wong was instrumental in enabling the sample collection for this study. Dean Yergens assisted in the amalgamation of the immense amount of clinical data for this study. I would also like to acknowledge the research nurses and assistants who identified and enrolled patients with the ICU and ED at Foothills Medical Centre and the Alberta Children’s Hospital including Janice Hammond, Linda Knox, Christine Skinner, Dori- Ann Martin, Claudia Maki, Stacey Ruddell, and Dan Lane. Lastly, I would like to acknowledge Dr. Brent Winston for allowing us to use the CCEPTR resources and Dr. Chip Doig for allowing me to contribute to the Alberta Sepsis Network team as well as providing funding for this doctoral project. I would also like to acknowledge Derrice Knight for her efforts in organizing iv the Alberta Sepsis Network research days, annual meetings, and facilitating the knowledge transfer within the group. I would like to acknowledge members of the Surette laboratory for their support throughout the years and their assistance in the analysis of the data set. In particular, Dr. Jennifer Stearns provided extensive support on the analysis and bioinformatics for this project. I would also like to thank Dr. Christopher Sibley for teaching me many of the techniques used in this project and for instilling his knowledge of bacterial identification on me. Lastly, I want to acknowledge my family for all their support during this doctoral project. They kept me grounded and provided the strength I needed to overcome many of the challenges faced during my project. v Dedication This thesis is dedicated to my husband, Ray Crowder who provided a support base for me throughout my PhD program. He has given me the light I needed in darker times and challenges me daily to be a better person. For that I am forever grateful. vi Table of Contents Abstract ............................................................................................................................... ii Acknowledgements ............................................................................................................ iv Dedication .......................................................................................................................... vi Table of Contents .............................................................................................................. vii List of Tables ..................................................................................................................... xi List of Figures and Illustrations ........................................................................................ xii List of Symbols, Abbreviations, and Nomenclature ........................................................ xiv CHAPTER ONE: INTRODUCTION ..................................................................................1 1.1 Sepsis .........................................................................................................................1 1.1.1 Definition of Sepsis ...........................................................................................1 1.1.1.1 Sepsis vs. Bacteremia ..............................................................................2 1.1.2 Clinical Significance and Epidemiology of Sepsis ...........................................2 1.2 Sepsis Infections ........................................................................................................4 1.2.1 Underlying Sepsis Infections .............................................................................4 1.2.2 Infectious Agents in Sepsis ...............................................................................5 1.2.2.1 Bacteria ....................................................................................................5 1.2.2.2 Fungi ........................................................................................................7 1.2.2.3 Viruses .....................................................................................................8 1.2.3 Microbial Translocation into the Bloodstream ..................................................8 1.2.4 Inflammatory Response in Sepsis ...................................................................10 1.3 Sepsis Management and Therapy ............................................................................13 1.3.1 Antibiotic Therapy ...........................................................................................13 1.3.2 Supportive Therapy in Sepsis ..........................................................................16 1.3.3 Immunosuppressive/Immunomodulatory
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