Development and application of a high-throughput luminescent prophage induction assay for the identification of temperate bacteriophage-inducing food-grade compounds By Elizabeth Tompkins Food Science and Agricultural Chemistry, McGill University, Sainte-Anne-de-Bellevue, Quebec April 2019 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science in Food Science © Elizabeth Tompkins 2019 Table of Contents Abstract ...........................................................................................................................vi Résumé ......................................................................................................................... viii Acknowledgements .........................................................................................................xi Contribution of authors .................................................................................................. xiii List of tables ...................................................................................................................xv List of figures ................................................................................................................. xvi List of abbreviations ..................................................................................................... xvii Chapter 1: Introduction .................................................................................................... 1 1.1 General introduction .............................................................................................. 1 1.2 Research objectives .............................................................................................. 3 1.2.1 Overall objective .............................................................................................. 3 1.2.2 Specific objectives ........................................................................................... 3 Chapter 2: A review of the clean label trend in the context of food safety: Considerations for the replacement of conventionally used antimicrobials with natural compounds ....... 6 2.1 Abstract ................................................................................................................. 6 2.2 Drivers of the clean label trend .............................................................................. 8 2.2.1 Clean label implications for food safety ........................................................... 9 2.3 Natural compounds as antimicrobials for the control of bacterial foodborne pathogens .................................................................................................................. 11 2.4 Bacteriophages as natural antimicrobials ............................................................ 13 2.4.1 Bacteriophage infection ................................................................................. 14 ii 2.4.2 Bacteriophage life cycles............................................................................... 15 2.4.2.1 The lytic cycle: Virulent bacteriophages.................................................. 15 2.4.2.2 The lysogenic cycle: Temperate bacteriophages .................................... 16 2.5 Bacteriophage applications in the food industry................................................... 18 2.5.1 Phage cocktails ............................................................................................. 18 2.5.2 Prophage induction for the control of bacterial foodborne pathogens ........... 19 2.5.3 Safety considerations of bacteriophage applications .................................... 21 2.6 Conclusion ........................................................................................................... 25 2.7 Tables .................................................................................................................. 27 2.8 References .......................................................................................................... 28 Connecting text ............................................................................................................. 36 Chapter 3: Development of a high-throughput luminescent prophage induction assay for the identification of prophage inducing compounds.................................................... 37 3.1 Abstract ............................................................................................................... 37 3.2 Introduction .......................................................................................................... 39 3.3 Materials and methods ........................................................................................ 42 3.3.1 Bacterial strains ............................................................................................. 42 3.3.2 Whole genome sequencing (WGS) of Escherichia coli BR513 ..................... 42 3.3.3 Development of the high-throughput luminescent prophage induction assay 43 3.3.3.1 Data analysis .......................................................................................... 45 3.3.4 Screening of food-grade compounds ............................................................ 45 3.3.4.1 Data analysis .......................................................................................... 46 3.4 Results ................................................................................................................. 47 iii 3.4.1 Bioinformatics analysis confirms presence of cryptic phage in E. coli BR513 ............................................................................................................................... 47 3.4.2 Multivariate regression analysis characterizes change in relative light units (RLU) in antibiotic-treated cultures ......................................................................... 48 3.4.3 Antibiotics reduce the cell concentration of E. coli BR513 and E. coli K-12 .. 49 3.4.4 Food-grade compounds induce prophage in Escherichia coli BR513 ........ 50 3.5 Discussion ........................................................................................................... 51 3.6 Conclusion ........................................................................................................... 56 3.7 Tables and figures ............................................................................................... 58 3.8 References .......................................................................................................... 65 Connecting text ............................................................................................................. 70 Chapter 4: Use of the high-throughput luminescent prophage induction assay to evaluate bioactive compounds for identification of natural prophage inducers .............. 71 4.1 Abstract ............................................................................................................... 71 4.2 Introduction .......................................................................................................... 73 4.3 Materials and methods ........................................................................................ 75 4.3.1 Bacterial strain .............................................................................................. 75 4.3.2 High-throughput luminescent prophage induction assay ............................... 76 4.3.2.1 Compounds tested in the assay ............................................................. 77 4.3.2.2 Data analysis .......................................................................................... 77 4.4 Results ................................................................................................................. 79 4.4.1 The High-throughput luminescent prophage induction assay identified 61 prophage-inducing agents ...................................................................................... 79 iv 4.4.2 Dose response .............................................................................................. 80 4.5 Discussion ........................................................................................................... 82 4.6 Conclusion ........................................................................................................... 89 4.7 Tables and figures ............................................................................................... 90 4.8 References .......................................................................................................... 97 Chapter 5: Conclusion ................................................................................................. 104 5.1 General conclusion ............................................................................................ 104 5.2 Contributions to knowledge................................................................................ 105 5.3 Future research ................................................................................................. 106 References .................................................................................................................. 109 v Abstract In response to consumer trends, conventional synthetic food additives used to control the growth of foodborne bacterial pathogens are being replaced with natural alternatives. Bacteriophages (phages) are viruses of bacteria that have been successfully applied in the control of pathogenic bacteria in food. Cocktails of virulent