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Biocontrol of Foodborne Bacterial Pathogens Using BIOCONTROL OF FOODBORNE BACTERIAL PATHOGENS USING IMMOBILIZED BACTERIOPHAGES A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by HANY EL-SAID MOHAMAD ANANY In partial fulfillment of requirements for the degree of Doctor of Philosophy August, 2010 ©HanyAnany, 2010 Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 OttawaONK1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-67847-3 Our file Notre reference ISBN: 978-0-494-67847-3 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extra its substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. ••I Canada ABSTRACT BIOCONTROL OF FOODBORNE BACTERIAL PATHOGENS USING IMMOBILIZED BACTERIOPHAGE Hany ElSaid Mohamad Anany Advisor: Dr. Mansel W. Griffiths University of Guelph, 2010 The goal of the present research was to develop a simple technique to immobilize isolated lytic phages and explore the potential use of these immobilized phages to control certain foodborne pathogens in a real food system. More than one hundred phages were isolated from different environmental samples against different strains of four major foodborne pathogens; E. coli 0157:H7, Salmonella, Listeria monocytogenes and Shigella. A turbidimetric method in high throughput format using the Bioscreen C was used to monitor phage lytic activity and determine the host range of the isolated phages. This enabled identification of isolated phages with similar characteristics and the selection of twelve good phage candidates for biocontrol purposes. These phages were characterized by TEM, restriction endonuclease pattern, one-step growth curve, BIM (bacteriphage insensitve mutant) development, cross infectivity and determination of their stability and infectivity under different conditions. A novel Shigella phage, OSboM- AG3, was isolated and its genome sequenced. Its genome did not show any homology to any reported virulent or lysogenic genes and it was considered as a member of the "T4 superfamily". Phage cocktails made from the isolated phages were very effective for specific control of the four target pathogens in both broth media and contaminated food stored under different environmental conditions. The phages were immobilized using positively charged carrier substrates, which allowed specific binding of phages through their heads and leaving tail fibers free to interact with bacteria. These immobilized phages retained infectivity and immobilized Listeria and E. coli phage cocktails were able to control the growth of L. monocytogenes and E. coli 0157:H7, respectively, in food under different temperatures and packaging conditions. ACKNOWLEDGEMENTS In the first place, I would like to express my sincerest gratitude to Allah, who provided me the blessing to complete this work. He showed me numerous times that HIS support and guidance exist in every step of the way. Also, I would like to express my deepest gratitude to my advisor, Dr. Mansel W. Griffiths, for his supervision, guidance, support, kindness and patience from the very early stage of this research as well as giving me extraordinary experiences throughout the work. Above all and the most needed, he provided me with so much encouragement and support in various ways on both the scientific and personnel levels. His truly scientist nature has made him as a constant source of ideas and passions in science, which exceptionally inspire and enrich my growth as a student, a researcher and a scientist want to be. I am indebted to him more than he knows. One simply could not wish for a better or kind supervisor! I am heartily thankful to my advisory committee members, Dr. Andrew Kropinski and Dr. Parviz Sabour for their invaluable advice, support and words of encouragement. They gave me from their precious times to gain from their exceptional experience in phage biology and genomic studies. Really, without their help, I could not have completed this research. I also would like to thank Dr. Robert Pelton and Dr. Micheal Brook for their collaboration in this research and providing the positively charged cellulose membranes and silica beads. Many thanks should also go to Dr.Yi-Min She for performing the mass spectrometry analysis of O SboM-AG3 proteins. This work has benefited from the insights, directions and help of many people: i H.-W. Ackermann, Lynn Mclntyre, Luba Brovko, Ann Blake, Haifeng Wang, Kieth Warriner, Milena Corredig, Robert Harris, Sandy Smith, Mona Tolba and Erika Lingohr. Each of them provided me with invaluable comments, advices and help which substantially improved the finished product. I am grateful to my friends and lab mates from Dr. Griffiths's lab; they made our lab such a convivial place to work, providing me with the excellent atmosphere for doing my research. In particular, I would like to thank Tarek El-Arabi for his friendship, help and support in the past four years. My special thanks go to all my Egyptian friends in Guelph, especially Mumdooh Ahmed and his respectful family, for their support they offered to my family and to me during my study. I would like also to thank the Egyptian government and Sentinel bioactive network for the financial support throughout my PhD program. My deepest gratitude goes to my family for their love and support throughout my life and considerably extended academic career. I am indebted to my father, El-Said Anany and my mother, Khyria Ahmed for their care, love and support. I have no enough words that can fully describe what they have done for my little family and for me. They are simply perfect parents! My brothers, Bassem and Ahmad have always been a consistent source of encouragement and support. Finally, yet importantly, I am so grateful to my soul mate and my wife, Nahed Anany, she has made her love and support available in a number of ways throughout my life, especially during my PhD study. This dissertation is simply impossible without her! I would like also to thank my beautiful kids, Omar, Aly and Yara for their love and prayers for me. They were the motive force that inspired me throughout this research. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS I LIST OF TABLES VII LIST OF FIGURES IX CHAPTER 1: INTRODUCTION 1 1.1 RESEARCH INTRODUCTION 1 1.2 FOODBORNE ILLNESSES AND PATHOGENIC BACTERIA 2 1.2.1 Escherichia coli 0157:H7 4 1.2.2 Salmonella spp 6 1.2.3 Shigella spp 9 1.2.4 Listeria monocytogenes 10 1.3 OVERVIEW OF BACTERIOPHAGE 13 1.3.1 Bacteriophage Discovery 13 1.3.2 Bacteriophage Biology 14 1.3.3 Bacteriophage Taxonomy 15 1.3.4 Lytic and Lysogenic Pathway of Bacteriophage 18 1.4 BACTERIOPHAGE AS BIOCONTROL TOOLS FOR FOODBORNE PATHOGENS 24 1.4.1 An overview and considerations for phage application in food 24 1.4.2 Examples for the experimental proof of concept for phage application as a biocontrol agent 35 1.5 IMMOBILIZATION OF PHAGES ON SOLID SURFACES: A NEW PROPOSED TECHNIQUE FOR FOOD APPLICATION 42 1.6 RESEARCH OBJECTIVES. 52 CHAPTER 2: ISOLATION AND CHARCTERIZATION OF LYTIC BACTERIOPHAGES AGAINST IMPORTANT FOODBORNE PATHOGENS ...54 2.1 ABSTRACT 54 2.2 INTRODUCTION 55 2.3 MATERIALS AND METHODS 57 2.3.1 Bacteria and Bacteriophage 57 in 2.3.2 Enrichment and isolation of phages 57 2.3.3 Purification of phages 60 2.3.4 Propagation and stock preparation 60 2.3.5 Host Range Determination using Bioscreen C 61 2.3.6 Phage DNA Isolation and Restriction Endonuclease Digestion 62 2.3.7 Transmission Electron Microscopy 63 2.3.8 Stability of Phages under Different Temperatures and pH's 63 2.3.9 Infectivity of Phages under Different Environmental Conditions 64 2.3.10 Determination of the Frequency of Emergence of Bacteriophage Insensitive Mutants (BIM) and Lysogenic Potential 65 2.3.11 Cross Infectivity 65 2.3.12 Determination of the Phage Genome Size using PFGE 66 2.3.13 One-step growth curve 66 2.3.14 Statistical analysis 67 2.4 RESULTS 67 2.4.1 Isolation of phages 67 2.4.2 Host range pattern and determination of the identical isolated phages using Bioscreen
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