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The Correlation Between Tolerance to Low Water Activity and High Hydrostatic Pressure in Shiga Toxin Producing Escherichia Coli

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The Correlation Between Tolerance to Low Water Activity and High Hydrostatic Pressure in Shiga Toxin Producing Escherichia Coli The Correlation between Tolerance to Low Water Activity and High Hydrostatic Pressure in Shiga Toxin Producing Escherichia coli BY Fahad Bazaid A Thesis Presented to The University of Guelph In partial fulfillment of requirements for the degree of Doctor of Philosophy in Food Science Guelph, Ontario, Canada © Fahad Bazaid, March 2019 ABSTRACT THE CORRELATION BETWEEN TOLERANCE TO LOW WATER ACTIVITY AND HIGH HYDROSTATIC PRESSURE IN SHIGA TOXIN PRODUCING ESCHERICHIA COLI Fahad Bazaid University of Guelph, 2019 Advisors: Dr. Keith Warriner Dr. Andrew Kropinski Dr. Don Mercer Dr. Tatiana Koutchma High hydrostatic pressure processing (HHP) is extensively applied for the non-thermal pasteurization of foods. One notable feature of HHP mediated inactivation of microbes is the large variation in lethality between strains and the influence of the food matrix. In the following it was hypothesized that the barotolerance of Shiga Toxin producing Escherichia coli could be correlated to tolerance to low water activity environments. The study evaluated the barotolerance of members of the Top 6 STEC serotypes and found variation with O26: H11 and O45: H2 exhibiting higher tolerance to pressure compared to the other bacterial types tested. A negative correlation was found between resistance to high pressure and the NaCl concentration under which E. coli could grow under. For example, E. coli O26: H11 exhibited the highest tolerance to pressure, within 300 MPa, but could only grow under NaCl up to 2.4% w/v. In contrast, E. coli O121: H19 was sensitive to pressure, but could grow in the presence of 6% NaCl. The results suggested ii that the induction of the stress-response was the likely reason for high barotolerance in salt sensitive strains vs resistant. It was also found that supplementing the suspension medium with amino acids, sugar alcohols and starch could increase the barotolerance of E. coli. Although it is possible that the solutes acted directly as osmolytes it is also proposed that metabolic products and other indirect protective mechanisms were also operating. The study suggests that early induction of the osmotic stress response can be used to predict the resistance to high hydrostatic pressure. iii DEDICATION I would like to dedicate this thesis to the greatest and precious person in my life my mother, who passed away twelve years ago. The person who I haven’t got to spent much time with, and the person who I always missed. Even though she passed away twelve years ago, she is always present in my heart and soul. I would like to dedicate this thesis to my wonderful father, who was always there for me when I needed support and encouragement. Also, would like to dedicate this thesis to my little angels Joud and Wesaam, who will be proud of their father when they grow up. Finally would like to dedicate this thesis to my sisters, brothers, friends, and professors. iv ACKNOWLEDGMENT I would like to express my thanks to Allah who guided me and gave me blessing to complete my PhD. I would also like to express my deep appreciation and thanks to my advisor Dr. Keith Warriner for giving me the opportunity to peruse my PhD. Dr. Warriner provided me with his guidance, advice, patience and encouragement throughout my research. The door for Dr. Warriner office was open to me whenever I needed guidance and advice. I would also like to express my thanks to my advisory committee Dr. Andrew Kropinski, Dr. Don Mercer, and Dr. Tatiana Koutchma for their advice, experience and help during my research. Special thanks to the government of Saudi Arabia for giving me this scholarship and funding me to peruse my graduate studies. My thanks also go to the Department of Food Science, and all my friends in Dr. Warriener’s lab. Also, special thanks to my father, my siblings, and my kids for their unconditional support and encouragement through my entire study. Finally, thanks to all my friends who supported me during my study. v Table of Content ABSTRACT ....................................................................................................................... ii DEDICATION ................................................................................................................... iv ACKNOWLEDGMENT ..................................................................................................... v Table of Content ................................................................................................................. vi List of Tables ................................................................................................................... viii List of Figures ...................................................................................................................... x General Introduction ............................................................................................................ 1 Chapter 1 ............................................................................................................................. 3 1.Literature review: ...................................................................................................................... 3 1.1. Shiga Toxin Producing E. coli STEC O157 and non-O157 ............................................. 3 1.1.2. STEC Outbreaks: ..................................................................................................................... 6 1.1.3. STEC Outbreaks in Low Water Activity Food: ...................................................................... 8 1.2 High Hydrostatic Pressure ............................................................................................... 11 1.2.1.Components of the High Hydrostatic Pressure Equipment: ................................................... 12 1.2.2.High Pressure Process: ........................................................................................................... 13 1.2.3. The Principle of High Hydrostatic Pressure: ......................................................................... 14 1.2.4. Effect of High Pressure on Proteins: ..................................................................................... 15 1.2.5. Microbial Inactivation by HHP ............................................................................................. 15 1.3. Water Activity and Osmotic Stress ................................................................................ 18 1.3.1. Effect of Water Activity on Barotolerance and Other Stresses: ............................................ 21 1.3.2. Accumulation of Osmolytes: ................................................................................................. 23 1.4 Strain Variation in Resisting High Hydrostatic Pressure: ............................................... 24 1.5 Glycolysis (Embden-Meyerhof-Parnas pathway) and Krebs cycle Tricarboxylic acid (TCA) cycle: .......................................................................................................................... 25 1.6. Dormancy State in Bacteria: .......................................................................................... 27 1.6.1.Resuscitation of VBNC: ......................................................................................................... 29 1.6.2.Detection of Dormant Cells (VBNC): .................................................................................... 30 Research Hypothesis and Objectives ................................................................................. 32 Chapter 2 ........................................................................................................................... 33 The Correlation between Tolerance to Low Water Activity and High Pressure Processing in Shiga Toxin Escherichia coli ........................................................................................ 33 2.1 Abstract ................................................................................................................................ 33 2.2 Introduction: ........................................................................................................................ 34 2.3 Materials and Methods ........................................................................................................ 36 2.3.1 Bacteria and Cultivation ............................................................................................... 36 2.3.2 Preparation of Suspensions .......................................................................................... 36 2.3.3 Persistence of the STEC Strains under osmotic stress (Colony counts) ...................... 37 2.3.3.1 Live counts under fluorescent microscope (Viability staining) ........................................... 38 2.3.3.2 Recovery of the STEC dormant cells (Resuscitation) ......................................................... 38 2.3.3.3 Growth limits of STEC (The gradient Plate Technique) ..................................................... 39 2.3.4 High Pressure Treatment .............................................................................................. 39 2.3.4.1 High Pressure inactivation (barotolerance) ......................................................................... 40 2.3.4.2 Microscopic counts .............................................................................................................. 40 2.3.4.3 High-pressure tolerance and STEC osmotic growth limits ................................................. 41 2.3.4.4 Effect of peptone (the accumulation of osmolytes)
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