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Isolation and Characterization of Bacteriophages Against Listeria Monocytogenes and Their Applications As Biosensors for Foodborne Pathogen Detection

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Isolation and Characterization of Bacteriophages against Listeria monocytogenes and their Applications as Biosensors for Foodborne Pathogen Detection by Safaa Ahmed Othman Fallatah A Thesis Presented to The University of Guelph In partial fulfillment of requirements for the degree of Master of Science in Food Science Guelph, Ontario, Canada © Safaa Fallatah, February, 2018 ABSTRACT The Isolation and Characterization of Lytic Bacteriophages against Listeria Spp and their Applications for the Rapid Detection of L. monocytogenes in Food Contact Surface and Broth Safaa Fallatah Advisor: University of Guelph, 2018 Dr. Mansel Griffiths The aim of this study was to determine the potential application of bacteriophages for the detection of Listeria spp. on food contact surfaces (FCS). Eight phages were selected for further characterization to determine the most appropriate phage for use in a detection assay. They were characterized for host range, TEM, stability to air dying for 24 h at 25 °C, and restriction endonuclease pattern. L. monocytogenes strain C716 was very resistant to all phages, however; a mutated phage, AG13M, was able to infect L. monocytogenes strain C716. AG20 & AG23 phages were high specificity against their host (Listeria spp). AG20 phage was immobilized on ColorLok paper and used in the to detect L. monocytogenes C519 in broth and FCS. AG20 phage was able to detect as few as 50 CFU/mL of L. monocytogenes in TSB and 40 CFU/cm2 on FCS using a plaque assay to detect progeny phage within 24 h. ACKNOWLEDGMENTS In the name of Allah, the most gracious and the most merciful I would like to begin my thesis with the recognition that without his support and kindness I couldn’t done this thesis. Thanks, and praise to you my Lord for your generous, guidance, and support in every step of the way. I would like to provide a special thank to Dr. Mansel Griffiths who accepted me as graduated student and gave me the magnificent chance to be one of his phage lab groups. I was so lucky to be under Dr. Griffiths supervision who is well known as one most recognized scientists in the field of food microbiology. Dr. Griffiths was a great helper, supporter, and an amazing director who was really helped me to chive my degree. Thank you, Dr. Griffiths, for personally, scientifically advised, and for providing me with the tools to be a successful independent researcher during my period of degree and in the future. I would like also to thank to Dr. Andrew M. Kropinski, who was my advisory committee member. He was always fully supportive, informative, willing to help and has a great sense of humour. I also thank him for sharing his experience and time to teach me something new. His well-known expertise, opinions and suggestions have provided valuable ideas in contribution towards my research. I would like also to thank Dr. Jeff Farber, who became my advisor since January 2016. I really appreciate his reviewing my thesis. Thank you for welcoming, helping, and encouraging me in my thesis editing processes. Also, I would like to thank my research project manager, Dr. Hany Anany who supported me during this journey. He taught me lots of valuable lessons and skills that cannot be learned in class. iv I will always remember when he was giving me from his time to discuss experiments designing or problems troubleshooting. I would also like to thank my lab mates for their friendship, supports, and kindness. Hajar Hawsawi, Nada Alasiri, Mathew Danller, and Steven Cucic, I really appreciate all for your cooperation and support. I wouldn’t be able to do this without having my family in Saudi Arabia. I’m very grateful for the support I received from my family during my M.Sc. study. My greatest gratitude goes to my parents (Ahmad Othman Fallatah and Halimah Omar Fallatah). There is nothing I can give in return for what they have done for me. I wish this achievement would make them happy and proud. Thanks for my dear siblings Ghassan, Mohammad, Marwan, Marwah, and Banan. Last but not least, my big thanks to my wonderful family who accompanied me in this Journey here in Canada. I really cannot thank my husband (Abdullah Ahmed Noor) and my children (Mawada and Mazen) enough for their love, patience, and support, which was essential to success. v TABLE OF CONTENTS ACKNOWLEDGMENTS ........................................................................................................... iv TABLE OF CONTENTS ............................................................................................................ vi LIST OF FIGURES ..................................................................................................................... ix LIST OF ABBREVIATIONS ................................................................................................... xiv CHAPTER 1: LITERATURE REVIEW ................................................................................... 1 1.1 FOODBORNE ILLNESSES ................................................................................................. 1 1.2 OVERVIEW OF LISTERIA SPECIES ................................................................................ 4 1.2.1 L. MONOCYTOGENES INFECTIONS: EPIDEMIOLOGY AND OUTBREAKS .... 4 1.2.2 INDIVIDUALS SUSCEPTIBLE TO L. MONOCYTOGENES INFECTION ............. 8 1.2.3 MICROBIOLOGY OF L. MONOCYTOGENES........................................................ 10 1.3 MONITORING LISTERIA IN FOOD PROCESSING PLANT ........................................ 11 1.3.1 POLICY ON L. MONOCYTOGENES IN READY-TO-EAT FOODS ...................... 12 1.3.2 FOOD CONTACT SURFACES USED IN FOOD PROCESSING ENVIRONMENT ............................................................................................................................................... 15 1.3.3 SURFACE ADHESINS AND L. MONOCYTOGENES ............................................ 16 1.4 DETECTION OF FOODBORNE PATHOGENS .............................................................. 17 1.4.1 TRADITIONAL CULTURE-BASED METHODS ..................................................... 18 1.4.2 RAPID DETECTION METHODS .............................................................................. 19 1.4.3 BACTERIOPHAGES FOR DETECTION OF FOODBORNE PATHOGENS .......... 27 1.5 OVERVIEW OF BACTERIOPHAGE DISCOVERY ....................................................... 27 1.5.1 BIOLOGY OF BACTERIOPHAGES.......................................................................... 28 1.5.2 BACTERIOPHAGE LIFE CYCLE ............................................................................. 29 1.5.3 BACTERIOPHAGE BASED METHODS AS DETECTION TOOLS ....................... 31 1.6 ADVANTAGES OF USING BACTERIOPHAGES FOR DETECTION ......................... 40 1.7 IMMOBILIZATION OF BACTERIOPHAGES ................................................................ 40 RESEARCH OBJECTIVES ...................................................................................................... 43 CHAPTER2 ISOLATION AND CHARACTERIZATION OF LYTIC BACTERIOPHAGES AGAINST L. MONOCYTOGENES .................................................. 44 2.1 ABSTRACT ....................................................................................................................... 44 2.2 INTRODUCTION ............................................................................................................. 45 2.3. MATERIALS AND METHODS..................................................................................... 48 2.3.1 BACTERIA AND BACTERIOPHAGES .................................................................... 48 vi 2.3.2 BACTERIOPHAGES ISOLATED AGAINST L MONOCYTOGENES ................... 49 2.3.3 PREPARATION METHOD OF BACTERIAL LAWN .............................................. 53 2.3.4 BACTERIOPHAGE ISOLATION FROM ENVIRONMENTAL SAMPLES ............ 53 2.3.5. PURIFICATION OF ISOLATED PHAGES .............................................................. 54 2.3.6. PHAGE PROPAGATION ........................................................................................... 55 2.3.7 TITRATION OF THE PROPAGATED PHAGES ...................................................... 55 2.3.8 BACTERIOPHAGE CHARACTERIZATION ........................................................... 56 2.3.9 PHAGE EVALUATION EXPERIMENT .................................................................... 59 2.4 RESULTS ........................................................................................................................... 62 2.4.1 BACTERIOPHAGE ISOLATION AGAINST L. MONOCYTOGENES................... 62 2.4.2 BACTERIOPHAGE CHARACTERIZATION ........................................................... 63 2.4.3 PHAGE MORPHOLOGY BASED ON TEM IMAGING ........................................... 73 2.4.4 RESTRICTION ENZYME DIGESTION .................................................................... 74 2.4.5 PHAGE STABILITY ON DESICCATION ................................................................. 75 2.4.6 PHAGE EVOLUTION EXPERIMENT ...................................................................... 76 2.5 DISCUSSION .................................................................................................................... 81 CHAPTER3 RAPID DETECTION OF L. MONOCYTOGENES IN BROTH AND FOOD CONTACT SURFACES USING IMMOBILIZED BACTERIOPHAGE (OR PHAGE- BASED BIOACTIVE PAPER STRIPS) ................................................................................... 89 3.1. ABSTRACT ......................................................................................................................
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