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Molecular Analysis of the Bacterial Community in Table Eggs

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Molecular Analysis of the Bacterial Community in Table Eggs Molecular Analysis of the Bacterial Community in Table Eggs Mohammed Ali Alawi A thesis submitted for the degree of Doctor of Philosophy Engineering and Physical Sciences Heriot-Watt University Edinburgh, UK January 2018 The copyright in this thesis is owned by the author. Any quotation from the thesis or use of the information contained in it must acknowledge this thesis as the source of the quotation or information. Chapter 1 General introduction ABSTRACT The composition of the bacterial flora on surfaces of table eggs is an important factor in influencing the incidence of egg spoilage. Previous studies have focused on a culturing approach for determining bacterial contamination of table eggs. The main problem, however, is culture-based techniques may not adequately describe the bacterial diversity of eggs, since many type of organisms are not cultivated by this method. This study describes bacterial diversity of table eggs by using both culture-based and molecular approaches. The results of culture based techniques suggested that majority of eggs tested were contaminated with Staphylococcus species. No evidence was found for the presence of Salmonella, Escherichia coli, Campylobacter or Listeria monocytogenes, but Clostridium perfringens was found to be positive from 3 eggshells out of 16 shells tested. Methods for direct extraction of bacterial DNA from eggshell and egg content were developed. Cloning of PCR amplified rRNA resulted in the isolation of 91 clones which matched existing sequences in the GenBank database. Eighty-nine % of the isolates were matched to clones of the assigned phylotypes of Psychrobacter, Acinetobacter, Staphylococcus, Clostridium, Lactobacillus, Actinobacterium, Proteobacterium, Prevotella, Olsenella and Ralstonia. In addition Psychrobacter faecalis and Psychrobacter maritimus were isolated from eggshell on TSA at 4 °C, and the characteristics of these bacteria were studied. Interestingly, these bacteria have not been isolated from table eggs in previous studies, and they could potentially be responsible for egg spoilage particularly when the egg are stored in the fridge. The results obtained in this study will provide valuable information to the egg producers and consumers that may aid improvement of the quality of table eggs and their shelf life. More importantly, it may facilitate the control of spreading these bacteria to the food chain, in order to prevent any food outbreaks that may result from consuming contaminated eggs. II Chapter 1 General introduction DEDICATION This thesis is dedicated to my son Eyad, my daughter Lamar, my beloved wife Khadijah, and my parents for their endless loves, supports and encouragements. III Chapter 1 General introduction ACKNOWLEDGEMENT This project was performed at the biology department, School of Life Science, Heriot- Watt University, Edinburgh, during the period between 2012-2015, funded by Saudi Ministry of Municipality, and this financial support was greatly appreciated. Firstly, I would like to thank my supervisors Dr. Wilfrid Mitchell, and Dr. Peter Morris for their great supervision, support and assistance during the period of this project. This project would not have been achieved without their unlimited support and guidance. I would also like to thank very much my wife Khadijah Alqahtani, my son Eyad, my daughter Lamar, and my parents for their patient and encouragement during my PhD study. I also wish to thank SLS staffs specially Dr. Daun, Margret and Paul Cyphus for their advice and supports. Also, many thanks also go to Dr. Susan Dewar, Dr. Ruth Fowler, and Dr. Derek Ball Special thanks must go to my graduated lab mates and school friends, Dr. Naif Almkaish, Dr. Mohammed Elsalem, Dr. Ash Paradh, Dr. Omar Alsaif, Dr. Amadike Ugbogu, Dr. Hassein Alnashiri, Dr. Majed Alshaeri, Dr. Majde Estoni, Dr. Salem Rajab, Dr. Ibrahim Akasha, Dr. Mohammed Alsharif, Dr. Abdullah Althubiani, Dr. Kayode and Dr. Omar Alharbi for their help and recommendations. Also, I would like to thank all my lab mates PhD, Ibrahim Alamin, Richard Freeman, Faisal Alqahtani, Sadeqh Alshaib, Vector, Mais Rajab, Mai Al-Ghanem, Tam, Chokul Kulanaree, Camilla, Charles, David, Halo, Loris, Julien and Roy Nguyen. Finally, many thanks to everyone who has given support during this project. IV Chapter 1 General introduction ACADEMIC REGISTRY Research Thesis Submission Name: Mohammed Ali Alawi School/PGI: Engineering and Physical Sciences Version: (i.e. First, Final Degree Sought PhD (Food Science) Resubmission, Final) (Award and Subject area) Declaration In accordance with the appropriate regulations I hereby submit my thesis and I declare that: 1) the thesis embodies the results of my own work and has been composed by myself 2) where appropriate, I have made acknowledgement of the work of others and have made reference to work carried out in collaboration with other persons 3) the thesis is the correct version of the thesis for submission and is the same version as any electronic versions submitted*. 4) my thesis for the award referred to, deposited in the Heriot-Watt University Library, should be made available for loan or photocopying and be available via the Institutional Repository, subject to such conditions as the Librarian may require 5) I understand that as a student of the University I am required to abide by the Regulations of the University and to conform to its discipline. * Please note that it is the responsibility of the candidate to ensure that the correct version of the thesis is submitted. Signature Date: of Candidate: Submission Submitted By (name in capitals): Mohammed Ali ALawi Signature of Individual Submitting: Date Submitted: For Completion in the Student Service Centre (SSC) Received in the SSC by (name in capitals): Method of Submission (Handed in to SSC; posted through internal/external mail): E-thesis Submitted (mandatory for final theses) Signature: Date: V Chapter 1 General introduction TABLE OF CONTENT ABSTRACT……………………...…….……………………………………….………………II DEDICATION……………………………………………………………………………....….III ACKNOWLEDGEMENT……………….……………………………………………….…….IV DECLARATION…………………………………………………….………………….………V TABLE OF CONTENT…………………………...……………………………………….…..VI LIST OF FIGURES……………………………………………………………………………..X LIST OF TABLES……………………………………………………………………...…….XIV CHAPTER 1: General introduction .......................................................................................... 1 1.1 General introduction .................................................................................................... 2 1.2 Food-borne diseases and routes of infections .............................................................. 5 1.3 Economic impact of egg loses. .................................................................................... 5 1.4 Consumption of eggs ................................................................................................... 7 1.5 Hen’s egg formation and composition ......................................................................... 7 1.6 Antimicrobial defences .............................................................................................. 11 1.7 Route of microbial contamination of intact eggs ....................................................... 13 1.8 Extrinsic factors affecting the egg content contamination......................................... 13 1.8.1 Temperature .......................................................................................................... 13 1.8.2 Moisture ................................................................................................................ 14 1.8.3 Presence of faeces and other contaminants on the eggshell .................................. 14 1.9 Table egg housing systems ........................................................................................ 15 1.10 The microbial community in table eggs .................................................................... 16 1.10.1 Salmonella in table eggs ................................................................................... 19 1.10.2 Escherichia coli in table eggs ........................................................................... 19 1.10.3 Staphylococcus in table eggs ............................................................................ 20 1.10.4 Other bacteria in table eggs............................................................................... 20 1.11 Source of bacterial contamination ............................................................................. 21 1.12 Viable but non-cultivable bacteria ............................................................................. 23 1.13 Bacterial cross-contamination ................................................................................... 24 1.14 Types of egg products involved in outbreaks ............................................................ 25 1.15 Interventions to improving microbial quality of eggs ............................................... 26 1.15.1 Egg traceability ................................................................................................. 27 1.16 Bacterial isolation and enumeration .......................................................................... 28 1.17 Bacterial detection and identification ........................................................................ 28 1.17.1 Conventional methods ...................................................................................... 29 1.17.2 Methods based on biochemical analysis ..........................................................
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