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Application and Evaluation of Bacterial Wruses in Rapid APPLICATION AND EVALUATION OF BACTERIAL WRUSES IN RAPID METHODOLOGIES FOR THE DETECTION OF FOOD-BORNE PATHOGENS A Thesis Presented to nie Faculty of Graduate Studies of The University of Guelph In partial fulfilment of requirements for the degree of Doctor of Philosophy July, 1998 O Lynn McIntyre, 1998 National Library Bibliothèque nationale 1*1 ofCamda du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Weilingîon Street 395. rue Wellington OitawaON K1AON4 Ottawa ON K1A ON4 canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant a la National Lîbrary of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microfom, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nlm, de reproduction sur papier ou sur format électronique. The author retaios ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. ABSTRACT APPLICATION AND EVALUATION OF BACTERIAL VIRUSES IN RAPID METHODOLOGIES FOR THE DETECTION OF FOOD-BORNE PATHOGENS Lynn McIntyre Advisor: University of Guelph, 1998 Dr. M. W. Griffrths Bacteriophages (or phages) are viruses that replicate only by identieing and idecting specific host bacteria. This property has facilitated their application in microbial typing schemes, and more recently in various bacterial detection methods. However, lack of specificity and sensitivity, dong with time-consuming and costly procedures have hindered their application in species-specific pathogen detection. This thesis will address these issues with regard to development of phage-based methods for the detection of Listeria rnonocytogenes, Salmonella spp., and Escherichia di. A number of commercial and environmentally-isolated phages were first evaluated, based on host specificity ranges. Four phages were selected for Merinvestigation, two demonstrating broad-range specificity for Lisieria and Salmonella spp., and two exhibiting specificity for S. Enteritidis and non-VTEC (verotoxigenic E. di)isolates. Transmission electron microscopy was used to classiQ each virus into a specific group based on tail length and head size, and their mode of infection was elucidated. Pathogen detection methods were chosen, based on phage properties. First, phage replication requires an actively metabolising host, making metabolic methods, including impedance. turbidirnetry and colorimetry, ideal candidates for evaluation of bactenophage behaviour. Additionally, phage lytic properties make them potentiaily specific biological extractânts for ATP bioluminescence, whic h ty picaily uses a non-speci fic chemicai extraction, Phage-based ATP bioluminescence was successful in specificaily identiQing pathogens, but poor sensitivity was a problem. Filtration, increased phage exposure Meand an ATP amplification method were dl evaluated as means of improving sensitivity, but were limited in their application. Phage-mediated impedimeûic and colorimetric methods were developed for the sensitive and specific detection and confirmation of bovine non-VTEC isolates in raw milk (40 CFU/mL) within 12 to 16 hours. Colorimetric analysis of artificially contarninated ground beef in combination with phage AT20 was capable of confirming 4000 CFUIg of E. coli G2-2. Phage-based irnpedimetric assays were aiso developed for differentiai detection of SalmonelZu spp. in skim milk powder, and L. rnonocytogenes in raw milk. Phage-based turbidimetric pathogen assays were also demonstrated, but diluting samples to reduce initiai turbidity detracted from the sensitivity and usefùlness of the assay. This dissertation is dedicated to the mernories of Isobel McMillan Calder Jean Greenhalgh John Phillips Craig Thornley al1 of whom died before tlieir prime, and before witnessing the successfu1 completion of my doctorate. 1 miss you al1 very much. Hrnmm ... where to begin? First of ail, 1 would like to extend my doepest thanks and admiration to my supervisor, Dr. Mansel W. Griffiths. Not only did he encourage me to pursue an interest in scientific research. but he ailowed me the opportunity to broaden my horizons by moving to Canada to undertake graduate studies with him at the University of Guelph. I have learned a great deal about science fiom him, but more than that, he has unfailingly demonstrated how to be a fair and decent individual. Thank you Mansel for your guidance, support, patience and humour (the latter being of immense important when one is a Welsh rugby fan). Many thanks to Dr. Heidi Schraft and Dr. Joseph Odumeru for their constructive cnticisms of this dissertation, their attention to detail, and their arnazing endurance in reading this entire piece of work fiom cover to cover under a tight deadline. Thanks also to Dr. Massimo Marcone, Dr. Yukio Kakuda, and my extemal examiner Dr. Michael Brodsky for making my defence experience such a rewarding ordeal. 1 can't Say enough about the many wondemil people 1 have had the opportunity to work with over the course of my Ph.D. There are of course too many to narne but honourable mentions go to Ann Toner, Veena Kau., Yolanda HWi, Doug McPhee, Dr. Luba Brovko and Stacy Favrin. Much appreciation is extended to Dr. Sabah Jassim, Dr. JinChen, Wendy Cladman, Riske Meewisse and Andrew Moore for technical assistance. A special 1 thank you to Master Bill Lachowsky for being my partner in crime during the long and ktrating weeks of writing -you kept me sane, on track, and 1 do indeed owe you a bottle of whisky! Gratitude is also extended to the faculty, staffand students of Food Science and Laboratory Services Division for support and assistance during my research. I have been fortunate to make many good &ends during my studies, most of them as a result of working for several years in the Graduate Student Lounge. Thank you Bonnie for keeping me financially solvent during some lean tirnes. Erin, thank you for being so thoughtful of others despite your own difficulties. Cheers to my bartender colleagues and the Lounge patrons, past and present, for providing such a fun environment to work in. To rny close friends Brian, David, Donna, Elizabeth, Jarnie, Jan, Laurie, Lisa D., Lisa P., Louis, and Moma thank you for being so caring and supportive - 1 love you all. To my fnends in Scotland - particularly Nicola, Magnus, Stuart, and David - thanks for coming to visit. and for continuing to participate in my life even though I've been thousands of miles away. To my housernates Nancy, Mark, Kristen, and Don, thanks for being such laid- back people - 3 1 Fountain Street won? be the same without you. Finally, this would not be complete without acknowledging the love and support of my family both in Scotland and in Michigan, USA. Leaving Scotiand kvas one of the hardest things I've ever done (apart from hting this dissertation!), but 17mglad I did because it has made me appreciate you even more than before. Thank you di. 1.2.4.2. Turbidimetry ..................................................... 45 1.2.4.3. Colonmetty ...................................................... 46 1.2.5. MISCELLANEOUS PHAGE PROBE APPLICATIONS .................... 47 1 .2.5 .1 . High-performance Iiquid chromatography .............................. 47 1.2.5 .2 . Immobilised-phage capture assay ..................................... 49 1.2.5.3. Phage-linked immunosorbant assays (PHALISA) ........................ 51 1 .2.5.4. Phage amplification assays .......................................... 51 CHAPTER TWO: ISOLATION AND CHARACTEEUSATION OF BACTERIOPHAGES ACTIVE AGAINST THE GENERA LISTERIA. SALMONELLA. AND ESCHERICHIA 55 2.2. MATERIALS AND METHODS ........................................ 56 2.2.1. Bacteriai cultures ................................................... 56 2.2.1.1. Listeria strains .................................................... 57 2.2.1.2. Salmonella strains ................................................. 59 2.2.1 .3 . Escherichia coli strains ............................................. 60 2.2 .2 . Bacteriophages and propagating strains .................................. 60 2.2.2.1. Listerin phage .................................................... 60 2.2.2.2. Salmonella phages ................................................ 61 2.2.2.3. E. coliphage ..................................................... 61 2.2.3. Propagation and enurneration of phages ................................. 61 2.2.3.1. Phage lysis of host bacteria .......................................... 62 2.2.3 .2 . Cleaning of phage lysates ........................................... 63 2.2.3 .3. Enurneration of phage preparations ...................................64 2.2.4. Phage typing of bacteria .............................................. 65 2.2.5. Transmission electron microscopy of phages ............................. 65 2.3. RESULTS AND DISCUSSION ......................................... 65 2.3.1. Phage enurneration and plaque morphology .............................. 65 2-32Phage typing of Listeria spp ........................................... 66 2.3 .3 . Microscopic charactensation of Listeria phage ATCC 23074-B 1 ............. 72 2.3
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