Identification of Clinical and Bacterial Genetic Risk Factors Associated with Klebsiella pneumoniae Infection by Rebekah Michal Martin A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Molecular and Cellular Pathology) in the University of Michigan 2017 Doctoral Committee: Assistant Professor Michael A. Bachman, Chair Professor Nicholas W. Lukacs Professor Harry L.T. Mobley Professor Gabriel Núñez Assistant Professor Evan S. Snitkin Rebekah Michal Martin [email protected] ORCID iD: 0000-0001-9729-0283 © Rebekah Michal Martin 2017 DEDICATION To my grandfathers: George Bernard Skidmore, who gifted me my first microscope & Robert James Martin, who believed I would achieve great things ii ACKNOWLEDGEMENTS I owe a deep debt of appreciation to several people for their assistance, guidance, and support throughout this journey. Without each and every one of them, this journey would have looked quite different. My advisor, Mike, deserves my utmost gratitude. He allowed me to pursue a complicated, fascinating project despite my limited experience in this field of research and with practically all the methods we used. Because of him, I have increased my critical thinking skills and developed into a more accomplished scientist, writer, and communicator. His impact will stay with me as I progress in my career, and I will look to him as an example of what a successful mentor should be. Being a trainee in his lab provided the best graduate experience I could have asked for. I am also appreciative to the many individuals who passed through the Bachman lab during my time there, and the ways in which they impacted me professionally. I would be remiss if I did not thank those who had a hand in preparing me for graduate school ̶ even before I knew this was the path I wished to pursue. To the Michigan State University Biomedical Laboratory Diagnostics Program (BLD) faculty, staff and students: this program and faculty are what sparked my interest in teaching. I am thankful for the passion they imparted for learning about diseases and disease processes, and the continued support and encouragement they provide in every facet of my life. BLD will always be home to me. To the Clinical Microbiology directors & medical lab scientists of Henry Ford Hospital (HFH): this is where I fell in love with clinical microbiology and with being part of the patient care team. This experience helped frame my current career aspirations, and HFH will always be full of family to me. Special thanks are iii owed to Robert Tibbetts & Linoj Samuel who continue to answer my many questions and to help me navigate the field of clinical microbiology. I am also indebted to the many other individuals who mentored me in various ways throughout graduate school: my committee, Gary Huffnagle, Nick Lukacs, Harry Mobley, Gabriel Núñez, and Evan Snitkin who provided continual advice and assistance. I am truly honored to have each of them on my committee and impacting my project. Maria Sandkvist and Lynne Shetron- Rama for their guidance and advice on how to be an effective university instructor. Duane Newton for allowing me to take up space in the University of Michigan Clinical Microbiology Lab, and for taking the time to provide career advice and share his journey. I have been fortunate to have a fantastic group of friends, both from graduate school and from other stages of my life: the MCP and PIBS students, who provided much needed stress relief through trivia nights, movie and game nights, and sharing in day-to-day struggles and adventures. My best friend Marie Tyrrell, who has been my sounding board and encouragement from afar. My family have supported my passion for science since before I can remember, and have been a constant source of encouragement through each milestone and each setback. My parents have reminded me of everything I am capable of and that they support me whatever comes. My sister, Elisa, was my first hero & role model and continues to be a source of support even from across the ocean. Without my husband and partner, Ben Dettmar, this journey undoubtedly would have never happened. I still remember the moment he told me I was capable of pursuing a graduate degree, and from that moment I never looked back. He has always encouraged me to fulfill my potential, and he has taught me to slow down and embrace life along the way. I am forever grateful for the iv support, encouragement, & advice he still provides daily. I cannot imagine this life adventure with anyone else x. v TABLE OF CONTENTS DEDICATION………………………………………………………………………………….ii ACKNOWLEDGEMENTS…………………………………………………………………...iii LIST OF TABLES……………………………………………………………………………viii LIST OF FIGURES…………………………………………………………………………....ix LIST OF ACRONYMS AND ABBREVIATIONS……………………………………...…...xi ABSTRACT……………………………………………………………………………………..1 CHAPTER I: Introduction……………………………………………………………………..3 1.1 Introduction………………………………………………………………………4 1.2 Klebsiella pneumoniae: an opportunistic, hospital-acquired infection…………..5 1.3 Emergence of antibiotic resistance in Klebsiella pneumoniae…………………13 1.4 Community-acquired hypervirulent strains…………………………………….18 1.5 Klebsiella species are distinguished by their accessory genome…………….....20 1.6 Gaps in knowledge……………………………………………………………...22 1.7 Conclusions……………………………………………………………………..23 1.8 Outline of thesis………………………………………………………………...23 CHAPTER II: Molecular Epidemiology of Colonizing and Infecting Klebsiella pneumoniae Clinical Isolates………………………………………………25 2.1 Introduction…………………………………………………………………….26 2.2 Results………………………………………………………………………….28 2.3 Discussion……………………………………………………………………...48 vi 2.4 Experimental procedures……………………………………………………....51 2.5 Notes…………………………………………………………………………...56 CHAPTER III: Identification of Pathogenicity-Associated Loci in Klebsiella pneumoniae Clinical Isolates………………………………………………………………………..57 3.1 Introduction…………………………………………………………………….58 3.2 Results………………………………………………………………………….60 3.3 Discussion…………………………………………………………………….103 3.4 Experimental procedures……………………………………………………..108 3.5 Notes………………………………………………………………………….116 CHAPTER IV: Discussion…………………………………………………………………..117 4.1 Summary of thesis……………………………………………………………117 4.2 Host factors contribute to epidemiology of infection………………………...118 4.3 Identifying at-risk patients may help guide empiric therapy………...…….…120 4.4 Identifying colonization may aid in infection prevention…………………….121 4.5 Identification and characterization of pathogenicity-associated loci……....…124 4.6 Species misidentification may impact clinical care…………………………..125 4.7 Clinical modeling and bacterial whole genome sequencing in clinical care…126 4.8 Conclusions…………………………………………………………………...128 REFERENCES……………………………………………………………………………….131 vii LIST OF TABLES Table 2.1: Demographic characteristics of patients with and without infection ………..……….30 Table 2.2: Prior colonization with K. pneumoniae vs. subsequent infection……………..……...32 Table 2.3: Association with prior colonization for each site of infection…………………...…...33 Table 2.4: Multiple logistic regression model of risk factors for infection…………..……….…35 Table 2.5: Categorical agreement of colonizing-infecting isolate pairs in case patients……...…47 Table 3.1: Clinical characteristics of cases and controls…………………………………..…….62 Table 3.2: Multivariable model for clinical infection with K. pneumoniae…………………..…63 Table 3.3: Gene alignment counts…………………………………………………………...…..68 Table 3.4: Genes present in significantly different frequencies between cases and controls……75 Table 3.5: Bacterial genes significantly and independently associated with clinical infection….78 Table 3.6: Multivariable model of factors predictive for patient infection…………………...….81 Table 3.7: Variant analysis of WT and mutant strains……………………...…………………..100 Table 3.8: Multivariable model for psicose metabolism locus association with infection……..102 viii LIST OF FIGURES Figure 2.1: Study population……………………………………………………………………..29 Figure 2.2: Receiver operator characteristic curve for multivariable model of risk factors for clinical infection………………………………………………………………………….36 Figure 2.3: wzi sequencing method has similar discriminatory power to MLST…….…….……38 Figure 2.4: Phylogenetic tree for wzi sequence of patient rectal swab isolates……………...…..39 Figure 2.5: Phylogenetic trees of rectal swab and infecting isolates based on wzi gene sequencing………………………………………………………………………………..41 Figure 2.6: Core genome similarity between infecting and colonizing strains within patients….43 Figure 2.7: Concordant colonizing-infecting isolate pairs show high core genome allelic similarity…………………………………………………………………………………45 Figure 3.1: Receiver operator characteristic curve for a multivariable model of factors predictive for patient infection………………………………………………………………………64 Figure 3.2: Pathogenicity-associated locus sequencing (PAL-Seq)………………………….….66 Figure 3.3: Phylogenetic tree of Kp phylogroups………………………...…………………...…70 Figure 3.4: Hierarchical clustering of accessory genes………………………………...…….….72 Figure 3.5: Principal component analysis of normalized count sum data…………………...…..73 Figure 3.6: Pathogenicity-associated loci (PALs)…………………………………..………...…79 Figure 3.7: Phylogenetic analysis of clinical isolates……………………………..………….….83 Figure 3.8: Deletion of terC does not affect growth in nutrient rich broth………………………86 Figure 3.9: Deletion of terC confers a tellurite sensitive phenotype………………...…………..87 ix Figure 3.10: Competitive index of terC mutant compared to WT……………………………....88 Figure 3.11: Deletion of KP1_RS12820