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DEVELOPMENT of MOLECULAR TOOLS to ASSESS WHETHER ARCOBACTER BUTZLERI IS an ENTERIC PATHOGEN of HUMAN BEINGS ANDREW L WEBB Bachel

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DEVELOPMENT of MOLECULAR TOOLS to ASSESS WHETHER ARCOBACTER BUTZLERI IS an ENTERIC PATHOGEN of HUMAN BEINGS ANDREW L WEBB Bachel DEVELOPMENT OF MOLECULAR TOOLS TO ASSESS WHETHER ARCOBACTER BUTZLERI IS AN ENTERIC PATHOGEN OF HUMAN BEINGS ANDREW L WEBB Bachelor of Science, University of Lethbridge, 2011 A Thesis Submitted to the School of Graduate Studies of the University of Lethbridge in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE Department of Biological Sciences University of Lethbridge LETHBRIDGE, ALBERTA, CANADA © Andrew Lawrence Webb, 2016 DEVELOPMENT OF MOLECULAR TOOLS TO ASSESS WHETHER ARCOBACTER BUTZLERI IS AN ENTERIC PATHOGEN OF HUMAN BEINGS ANDREW LAWRENCE WEBB Date of Defence: June 27, 2016 G. Douglas Inglis Research Scientist Ph.D. Thesis Co-Supervisor L. Brent Selinger Professor Ph.D. Thesis Co-Supervisor Eduardo N. Taboada Research Scientist Ph.D. Thesis Examination Committee Member Robert A. Laird Associate Professor Ph.D. Thesis Examination Committee Member Sylvia Checkley Associate Professor Ph.D., DVM External Examiner University of Calgary Calgary, Alberta, Canada Tony Russell Assistant Professor Ph.D. Chair, Thesis Examination Committee DEDICATION This thesis is dedicated to my partner Jen, who has been a source of endless patience and support. Furthermore, I dedicate this thesis to my parents, for their unwavering confidence in me and their desire to help me do what I love. iii ABSTRACT The pathogenicity of Arcobacter butzleri remains enigmatic, in part due to a lack of genomic data and tools for comprehensive detection and genotyping of this bacterium. Comparative whole genome sequence analysis was employed to develop a high throughput and high resolution subtyping method representative of whole genome phylogeny. In addition, primers targeting a taxon-specific gene (quinohemoprotein amine dehydrogenase) were designed to detect and quantitate A. butzleri. The application of these methods showed that A. butzleri is present at high frequencies but low densities in diarrheic and healthy people, and specific strains are associated with human enteritis. The developed tools were also used to determine that A. butzleri is common in wastewater, survives tertiary wastewater treatment, and may be transmitted to people via ingestion of contaminated surface water. Diverse subtypes of A. butzleri occur in the environment, but pathogenicity is likely strain-specific and/or dependent on other factors such as host resistance. iv ACKNOWLEDGEMENTS Foremost, I thank my supervisors, Dr. G. Douglas Inglis and Dr. L. Brent Selinger; thank you for your guidance and encouragement, this thesis would not have been possible without your involvement. Additionally, I thank my committee members; Dr. Eduardo Taboada and Dr. Robert Laird; thank you for your positive feedback and patience throughout the course of this thesis. I would also like to acknowledge the technicians and students at Agriculture and Agri-Food Canada for their contributions to projects of which my thesis is only a small part: Philip Kubara and Randy Wilde collected surface water and wastewater samples; Randy Wilde, Amy Wright, and Skip Inglis collected fecal samples from non-human animals; Jenny Gusse, Philip Kubara and Xhevrie Qelag processed water samples; Kathaleen House processed human stool samples; and Greg Frick, Gwen Leusink, Rachel Poberznick, Xhevrie Qelag, Amy Wright, and Mitchel Stevenson completed preliminary identification of isolates. Finally, I would like to acknowledge Dr. Valerie Boras and the staff of the Microbiology Diagnostic Laboratory at the Chinook Regional Hospital, without whom we would not have access to stools from diarrheic individuals. v TABLE OF CONTENTS Title . i Examination Committee Members . ii Dedication . iii Abstract . iv Acknowledgements. v Table of Contents . vi List of Tables . x List of Figures . xi List of Abbreviations . xiii Chapter 1 Review of Relevant Literature . 1 1.1. Introduction . 1 1.2. Growth and morphology . 1 1.3. Ecology . 3 1.3.1. Arcobacter butzleri in animals and animal products . 3 1.3.2. Arcobacter butzleri in water sources . 6 1.3.3. Arcobacter butzleri associated with plants and plant products . 6 1.3.4. Arcobacter butzleri in human beings . 7 1.4. Isolation and detection . 7 1.4.1. Microbiological detection and isolation . 7 1.4.2. Molecular detection and identification . 8 1.5. Etiology . 10 1.5.1. Foodborne infection . 10 1.5.2. Waterborne infection . 10 1.5.3. Mechanism of infection . 11 1.6. Genomics . 11 1.6.1 Data availability . 11 1.6.2 Genome annotation and variation . 11 1.7. Pathogenicity . 12 1.7.1 Ascertaining pathogenicity . 12 1.7.2. Arcobacter butzleri as a potential pathogen . 14 1.7.3. Animal models of infection . 14 1.8. Molecular epidemiology, population structure, and species subtyping . 16 1.8.1. Molecular epidemiology . 16 1.8.2. Population structure . 16 1.8.3. Species subtyping . 17 1.9. Southwestern Alberta as a model agroecosystem . 18 1.10. Knowledge gaps . 19 1.10.1. Colonization versus infection . 19 1.10.2. Strain-based pathogenicity . 20 1.10.3. Population structure . 20 1.11. Study goal and hypothesis . 21 1.12. Objectives . 21 1.13. Introduction to chapters . 21 vi Chapter 2 Development of a comparative genomic fingerprinting assay for rapid and high resolution genotyping of Arcobacter butzleri . 23 2.1. Abstract . 23 2.2. Introduction . 24 2.3. Materials and methods . 26 2.3.1. Primer design and in silico evaluation . 26 2.3.2. Primer evaluation . 27 2.3.3. Detection and quantification of A. butzleri in diarrheic and non- diarrheic stools . 29 2.4. Results . 33 2.4.1. Primer design and in silico evaluation . 33 2.4.2. Primer evaluation . 33 2.4.3. Detection and quantification of A. butzleri in diarrheic and non- diarrheic stools . 34 2.5. Discussion . 36 2.5.1. Efficiency of A. butzleri detection methods . 36 2.5.2. Prevalence of A. butzleri in human stools . 39 2.5.3. Comparative detection of A. butzleri in diarrheic and non- 40 diarrheic stools . 2.5.4. Comparative quantification of A. butzleri in diarrheic and non- 41 diarrheic stools . 2.5.5. Epidemiology of diarrheic individuals infected with A. butzleri . 41 2.5.6. Co-isolation of A. butzleri with recognized pathogens . 42 2.6. Conclusions . 42 Chapter 3 Comparative detection and quantification of Arcobacter butzleri in stools from diarrheic and non-diarrheic human beings in southwestern Alberta, Canada . 44 3.1. Abstract . 44 3.2. Introduction . 45 3.3. Materials and methods . 47 3.3.1. Ethics statement . 47 3.3.2. Arcobacter butzleri isolation and DNA extraction . 47 3.3.3. Whole genome sequencing and assembly . 47 3.3.4. Detection and identification of coding sequences . 48 3.3.5. Identification of candidate accessory genes for CGF assay development . 48 3.3.6. Optimization of markers for development of final CGF assay . 50 3.3.7. CGF assay development . 50 3.3.8. Assessment of CGF discrimination and concordance . 51 3.4. Results . 53 3.4.1. Whole genome sequence assembly and comparison . 53 3.4.2. A ‘reference phylogeny’ for a sample population of A. butzleri isolates . 54 3.4.3. Analysis of CGF40 concordance with reference phylogeny . 56 3.4.4. Analysis of CGF40 reproducibility . 57 3.5. Discussion . 57 vii 3.6. Conclusions . 63 Chapter 4 Prevalence and diversity of waterborne Arcobacter butzleri in southwestern Alberta, Canada . 64 4.1. Abstract . 64 4.2. Introduction . 65 4.3. Materials and methods . 66 4.3.1. Ethics Statement . 66 4.3.2. Diarrheic stool collection and isolation of A. butzleri . 66 4.3.3. Wastewater sample collection and processing . 67 4.3.4. Fecal coliform enumeration . 68 4.3.5. Isolation of A. butzleri from wastewaters . 68 4.3.6. Quantitative PCR . 68 4.3.7. Subtyping of A. butzleri isolates . 69 4.4. Results and discussion . 70 4.4.1. Densities in untreated in wastewater . 70 4.4.2. Wastewater treatment efficacy . 70 4.4.3. Comparative genomic analysis . 75 4.5. Conclusions . 75 Chapter 5 Efficacy of biological treatment and UVB irradiation on the reduction of Arcobacter butzleri and viability in municipal.
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