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Elongation of Escherichia Coli by Cold Or Cinnamaldehyde Exposure and Transcriptomic Changes During Cinnamaldehyde Dissimilation

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Elongation of Escherichia Coli by Cold Or Cinnamaldehyde Exposure and Transcriptomic Changes During Cinnamaldehyde Dissimilation ELONGATION OF ESCHERICHIA COLI BY COLD OR CINNAMALDEHYDE EXPOSURE AND TRANSCRIPTOMIC CHANGES DURING CINNAMALDEHYDE DISSIMILATION by Jeyachchandran Visvalingam A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfilment of the requirements of the degree of DOCTOR OF PHILOSOPHY Department of Food Science University of Manitoba Winnipeg Copyright © 2012 by Jeyachchandran Visvalingam I hereby declare that I am the sole author of this thesis. I authorize the University of Manitoba to lend this thesis to other institutions or individuals for the purpose of scholarly research. Jeyachchandran Visvalingam I further authorize the University of Manitoba to reproduce this thesis by photocopying or other means, in total or in part, at the request of other institutions or individuals for the purpose of scholarly research. Jeyachchandran Visvalingam iii The University of Manitoba requires the signatures of all persons using or photocopying this thesis. Please sign below, and give address and date. iv ACKNOWLEDGEMENT I would like to express my sincere gratitude to Dr. Richard Holley for accepting me as his graduate student and for providing expert guidance. His genuine patronage during the research enabled me to reach this milestone and build the foundation for my future career. I would like to extend my gratitude to committee members, Dr. Gregory Blank and the late Dr. Denis Krause for their valuable advice throughout the progression of this research. I want to convey my kindest thanks to Dr. Teresa De Kievit for accepting to be part of the committee after the passing of Dr. Krause and for providing insightful comments. I would like to thank Dr. Richard Sparling and Dr. Michael Gaenzle (University of Alberta) for being members of the thesis examination committee. Special thanks to Dr. Colin Gill, Agriculture and Agri-Food Canada, Lacombe, Alberta for his valuable advice and providing financial support via a grant from Agriculture and Agri-Food Canada for the first two years. Financial support provided by Natural Sciences and Engineering Research Council of Canada (NSERC) and the University of Manitoba through different scholarships are gratefully acknowledged. I am profusely grateful to Namita Goswami, Kavitha Palaniappan, Chen (Dust) Wu, Chen (Monica) Yuan Liu, and Anne Marie for their technical assistance. I would like to thank Dr. R.U. Vallente and Daniel Lichtensztejn at the Genomic Centre for Cancer Research and Diagnosis, University of Manitoba for providing training and assistance with the use of the heated stage microscope. I want to extend my heartfelt thanks to Allison Cranmer, Pat Kenyon, Carola Lange, Michael Stringer, Eliana (Georgina) Mejia, Yang Qiu and Alison Ser for their assistance. I also want to thank my fellow graduate students of the Department for their support and friendship, especially Roniele Cordeiro, Anna Nilson, Kavitha Palaniappan v and Musarrat Jahan. I would like to thank all faculty members of the Department, with special thanks to Dr. Gary Fulcher for his career advice and guidance. My thanks go to undergraduate colleagues, who came from Sri Lanka to the University of Manitoba for their graduate studies, especially for organizing get-togethers and their moral support. I would like to extend my kindest gratitude to Carlos Miranda Garcia and his family for accepting me as a part of their family and for their love. It is a great pleasure to be part of their family. Finally, I would like to extend my deepest gratitude for the love, care and assistance given by my farther, brothers, and sister; without their care and support, I would not have reached this point. vi THESIS FORMAT The thesis has nine chapters of which, Chapters 3 to 7 are manuscripts at different stages of publication in peer-reviewed scientific journals. The style of each manuscript is modified for the purpose of standardized presentation of this thesis. An overall introduction to the study and a comprehensive review of literature are presented in Chapters 1 and 2, respectively. Chapter 8 provides an overall discussion to this study with suggestions for future studies. The overall conclusions are presented in Chapter 9 which is followed by bibliographical references. Chapter 3 entitled “The viabilities of cells in cultures of Escherichia coli growing with formation of filaments at 6 °C” by J. Visvalingam, C.O. Gill, R.A. Holley, 2012 was originally published in the International Journal of Food Microbiology, 153, 129-134. Chapter 4 entitled “Morphological and viability changes in Escherichia coli and E. coli O157:H7 cells upon rapid shift from 6 ºC to 37 °C” by J. Visvalingam, C.O. Gill, R.A. Holley, 2012 was originally submitted for publication in the journal Food Microbiology and was accepted for publication on 30/11/2012. Chapter 5 entitled “Adherence of cold-adapted Escherichia coli O157:H7 to stainless steel and glass surfaces by J. Visvalingam, R.A. Holley, 2013 was originally published in the journal Food Control, 30, 575-579. Chapter 6 entitled “Temperature-dependent effect of sub-lethal levels of cinnamaldehyde on viability and morphology of Escherichia coli” by J. Visvalingam, R.A. Holley, 2012 was originally published in the Journal of Applied Microbiology, 113, 591-600. vii Chapter 7 entitled “Examination of the genome-wide transcriptional response of Escherichia coli O157:H7 to cinnamaldehyde exposure” by J. Visvalingam, J.D. Hernandez- Doria, R.A. Holley, 2012 was originally submitted for publication in the journal Applied and Environmental Microbiology and was accepted for publication on 20/11/2012. viii Table of Contents Page Acknowledgement iv Thesis format v List of tables xv List of figures xvii Abstract xx Chapter 1 Introduction 1 Chapter 2 Review of literature 5 2.1 Food refrigeration 5 2.1.1 Significance of food refrigeration 5 2.1.2 Temperature control in the cold chain 6 2.1.3 Outbreaks of E. coli O157:H7 linked to refrigerated 7 foods 2.1.4 Effect of refrigeration on physiology of E. coli 9 2.1.5 Effect of refrigeration on morphology of E. coli and foodborne pathogens 14 2.2 Bacterial cell elongation under other marginal conditions 17 2.4 Application of natural antimicrobials to control foodborne pathogens in food 18 2.4.1. Application of bacteriocins 19 ix 2.4.2 Application of lactoferrin 20 2.4.3 Application of plant based antimicrobials 20 2.4.4 Mechanism of antimicrobial action of plant essential oil components 24 Chapter 3 The viabilities of cells in cultures of Escherichia coli growing with formation of filaments at 6°C 30 3.1 Abstract 30 3.2 Introduction 31 3.3 Materials and Methods 32 3.3.1 Bacterial strains and culture conditions 32 3.3.2 Treatment at 6°C 33 3.3.3 Treatment at 37°C 34 3.3.4 LIVE/DEAD staining and microscopy 34 3.3.5 Cell length measurement 35 3.3.6 Data analysis 36 3.4 Results 36 3.5 Discussion 40 Chapter 4 Morphological and viability changes in Escherichia coli and E. coli O157:H7 cells upon rapid shift from 6°C to 37°C 53 4.1 Abstract 53 4.2 Introduction 54 4.3 Materials and Methods 55 x 4.3.1 Preparation and sampling of E. coli cultures incubated at 6°C 55 4.3.2 Preparation and testing of suspensions incubated at 37°C 56 4.3.3 Response of individual 5 day, 6°C -adapted cells to 37°C 58 exposure 4.3.4 Analysis of data 59 4.4 Results 59 4.5 Discussion 62 Chapter 5 Adherence of cold-adapted Escherichia coli O157:H7 to stainless steel and glass surfaces 71 5.1 Abstract 71 5.2 Introduction 72 5.3 Materials and Methods 73 5.3.1 Bacterial strains and inoculum preparation 73 5.3.2 Stainless steel coupon and glass slide preparation 74 5.3.3 Testing adherence 75 5.3.4 Microscopy 76 5.3.5 Statistical analysis 76 5.4 Results and Discussion 77 5.5 Conclusion 79 Chapter 6 Temperature-dependent effect of sub-lethal levels of cinnamaldehyde on viability and morphology of Escherichia coli 85 xi 6.1 Abstract 85 6.2 Introduction 86 6.3 Materials and Methods 88 6.3.1 Bacterial strains and inoculum preparation 88 6.3.2 Cinnamaldehyde stock solution preparation 89 6.3.3 Minimal inhibitory concentration determination 89 6.3.4 Cinnamaldehyde treatment at 6ºC or 37ºC 90 6.3.5 Microscopy 91 3.3.6 Measurement of cell size 93 6.3.7 Statistical analysis 93 6.4 Results 93 6.4.1 Effect of cinnamaldehyde at 37ºC 93 6.4.2 Effect of cinnamaldehyde at 6ºC 96 6.5 Discussion 97 Chapter 7 Examination of the genome-wide transcriptional response of Escherichia coli O157:H7 to cinnamaldehyde exposure 111 7.1 Abstract 111 7.2 Introduction 112 7.3 Materials and Methods 114 7.3.1 Bacterial strain and growth conditions 114 7.3.2 Cinnamaldehyde treatment 115 7.3.3 Viable number and microscopy measurements 116 xii 7.3.4 RNA extraction 116 7.3.5 cDNA synthesis, labelling and hybridization 117 7.3.6 Microarray data analysis 118 7.3.7 Microarray data access 119 7.3.8 Motility assay 119 7.3.9 Sample preparation for high performance liquid chromatography 120 7.3.10 Preparation of standard solutions 121 7.3.11 RP-HPLC analysis 121 7.3.12 Determination of the minimal inhibitory concentration of cinnamic alcohol 122 7.3.13 Data analysis 122 7.4 Results 123 7.4.1 Growth and morphology 123 7.4.2 General changes in transcriptomic response 123 7.4.3 Cell shape and cell division cycle 124 7.4.4 Energy derivation and oxidation reduction 124 7.4.5 DNA metabolism 125 7.4.6 Translation/protein synthesis and amino acid metabolism 125 7.4.7 Stress response 126 7.4.8 Antibiotic resistance 128 7.4.9 Membrane/membrane transport 128 7.4.10 Transcriptional regulation 129 xiii 7.4.11 Motility and chemotaxis 129 7.4.12 Biofilm-related genes and others 130 7.4.13 Motility assay 130 7.4.14 RP-HPLC analysis 130 7.4.15 MIC value of cinnamic alcohol 131 7.5 Discussion 131 7.6 Conclusion 136 Chapter 8 General discussion 145 Chapter 9 Conclusion 159 References 161 Appendix I 194 Appendix II 195 Appendix III 196 Appendix IV 197 Appendix V 198 Appendix VI 199 Appendix VII 200 Appendix VIII 201 Appendix IX 204 Appendix X 207 xiv Appendix XI 211 xv List of Tables Table Page Table 2.1 E.
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