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Molecular Responses of Non-O157 Shiga Toxin Producing E.Coli

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Molecular Responses of Non-O157 Shiga Toxin Producing E.Coli MOLECULAR RESPONSES OF NON-O157 SHIGA TOXIN PRODUCING E.COLI WHEN EXPOSED TO ACID STRESS AND ELECTRON BEAM IRRADIATION IN STRAWBERRY MATRIX A Dissertation by SHIMA SHAYANFAR Submitted to the Office of Graduate and Professional Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Chair of Committee, Suresh D. Pillai Committee Members, Christine Alvarado Joseph Awika Bhimanagouda Patil Head of Department, Boon Chew December 2016 Major Subject: Food Science and Technology Copyright 2016 Shima Shayanfar ABSTRACT Non-O157 Shiga toxin producing E.coli (STEC) serogroups are responsible for a growing number of food-related illnesses around the world. These serogroups experience dramatic pH fluctuations either by organic acids introduced during food processing or by inorganic acids in the stomach, which induce acid resistance in the pathogens. The main non-O157 STEC serogroups were analyzed for their acid sensitivity by exposing them to acid buffer, inorganic acid buffer and strawberry puree for 24h and room temperature. The results show that bacterial inactivation depends on the nature of the acid and the strain (P<0.01). Each of the serogroups exhibits different levels of resistance to acid stress with O103 as the most resistant strain and O26 and O111 as the weakest of all to acid stress (P<0.01). The pattern of microbial inactivation of the acids is inorganic acid> strawberry > organic acid. An untargeted metabolomics analysis identified that peptidoglycan, nitrogen, and unsaturated fatty acid biosynthesis are activated in E.coli O26 when exposed to inorganic acid buffer, to protect the structural integrity of the cells. D-Glutamine/D-glutamate metabolism was activated in both strawberry puree and inorganic acid exposed cells to possibly maintain the homeostasis of the cellular pH. Application of 1kGy of eBeam results in a 4-log inactivation of a cocktail of non-O157 STEC serogroups in strawberry puree and a significant (>99.99%) reduction in public health risks. A lethal dose of 3 kGy of eBeam activated metabolic pathways related to DNA repair, virulence and glutathione metabolism in an attempt to repair the lethal ii damage. Transcriptomic analysis results indicate that when E.coli O26 cells are maintained for 24 hours in phosphate buffered saline (PBS) buffer 88% (5358 genes) of its 6089 genes were up-regulated. However, in the cells are stored at room temperature in a strawberry matrix, only 71 genes (1.1%) were up-regulated. When E.coli O26 cells were exposed to 3 kGy eBeam dose and stored in PBS buffer and strawberry matrix, 5379 and 2250 genes were upregulated respectively. Though the cells are inactivated after exposure to lethal doses of eBeam radiation, the metabolomic and transcriptomic analysis indicate that they are still metabolically active. iii DEDICATION To whom I owe the air I breathe in Maman & Baba And Shadi, my other half iv ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Pillai. Thank you for all your friendly support, fatherly guidance and lighting up my way to dare and dive into the immense ocean of microbiology, the field that I feared the most! I would also like to gratefully thank my committee members, Dr. Alvarado, Dr. Awika, and Dr. Patil, for their guidance and support throughout the course of this research. I would like to thank the staff at the National Center for Electron Beam Research specifically Mickey, Emma and Amit for helping me perform my irradiation experiments. Thanks also go to my friends Shayan, Samira, Kartheek, Tiam, Sima, Zahra, Lindsay, James, Sohini, Chandi, Harshad, Luis, Ben, Derrick and Melinda who emotionally supported me over the course my exciting experience in Aggieland. I would like to specially thank Dr. Athrey for educating me the essentials in bioinformatics and his sincere technical support. I would also like to sincerely thank Dr. Ghaffari for her insightful guidance on transcriptomic analysis. I would like to thank Dr. Mena for her assistance in QMRA analysis. I would also like to thank friends and colleagues and the department faculty and staff for making my time at Texas A&M University a great experience. Finally, thanks to my mother, father and only sister for their encouragement and love. v CONTRIBUTORS AND FUNDING SOURCES Contributors This work was supervised by a dissertation committee consisting of Professor Suresh D Pillai and Professor Christine Alvarado of the Department of Poultry Science and Professor Joseph Awika of the Department of Soil and Crop and Professor Bhimu Patil of the Department of Horticultural Sciences. The data analysis for Chapter VIII was performed by Professor Giri Athrey. The results in Chapter IV were published in 2015 in Food Control journal. All other work conducted for the dissertation was completed by the student independently. Funding Sources Graduate study was supported by a fellowship from Poultry Science Department and National Center for Electron Beam Research at Texas A&M University. vi NOMENCLATURE eBeam Electron Beam Irradiation E.coli Escherichia coli kGy kilogray STEC Shiga toxin producing Escherichia coli CFU Colony forming unit DE Differentially expressed vii TABLE OF CONTENTS Page ABSTRACT .......................................................................................................................ii DEDICATION .................................................................................................................. iv ACKNOWLEDGEMENTS ............................................................................................... v CONTRIBUTORS AND FUNDING SOURCES ............................................................. vi NOMENCLATURE .........................................................................................................vii TABLE OF CONTENTS ............................................................................................... viii LIST OF FIGURES ........................................................................................................... xi LIST OF TABLES .......................................................................................................... xiv LIST OF EQUATIONS .................................................................................................. xvi CHAPTER I INTRODUCTION ....................................................................................... 1 Relevance of Research ................................................................................................... 1 Rationale......................................................................................................................... 5 Major Objectives ............................................................................................................ 6 Specific Objectives ......................................................................................................... 6 Relevance to Food Safety ............................................................................................... 7 CHAPTER II LITERATURE REVIEW .......................................................................... 10 Overview ...................................................................................................................... 10 Shiga Toxin Producing Escherichia coli (STEC) ........................................................ 12 Electron Beam Technology .......................................................................................... 31 Quantitative Microbial Risk Assessment (QMRA) in the Context of Ionizing Radiation ...................................................................................................................... 52 viii CHAPTER III QUANTIFYING THE REDUCTION IN POTENTIAL INFECTION RISKS FROM NON-O157 SHIGA TOXIN PRODUCING E.COLI IN STRAWBERRIES BY LOW DOSE ELECTRON BEAM PROCESSING ................... 56 Overview ...................................................................................................................... 56 Introduction .................................................................................................................. 57 Material and Methods................................................................................................... 59 Results .......................................................................................................................... 63 Discussion .................................................................................................................... 67 CHAPTER IV QUANTIFYING THE EFFECTS OF VARYING SOURCES OF ACID STRESS ON NON-O157 SHIGA TOXIN PRODUCING ESCHERICHIA COLI ................................................................................................................................. 69 Overview ...................................................................................................................... 69 Introduction .................................................................................................................. 70 Materials and Methods ................................................................................................. 72 Results .......................................................................................................................... 76 Discussion .................................................................................................................... 82 CHAPTER V ACID STRESS INDUCES DIFFERENTIAL ABUNDANCE OF METABOLITES IN E.COLI O26:H11 ..........................................................................
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