FACTORS AFFECTING SURVIVAL OF BACTERIA ON ABIOTIC SURFACES ANGÉLIQUE DUDMAN A thesis submitted in partial fulfilment of the requirements of the Manchester Metropolitan University for the degree of Doctor of Philosophy Faculty of Science and Engineering the Manchester Metropolitan University 2013 © Copyright by Angélique Dudman 2013 All Rights Reserved ii Acknowledgement Many people contributed to this dissertation in innumerable ways, and I am grateful to all of them. First and foremost, I would like to thank my director of study, Professor Joanna Verran, who also was my tutor during my undergraduate studies. I am very appreciative of her generosity with her time, advice, data, and references, to name a few of her contributions. She made my move to the United Kingdom comfortable and easy by providing constant help when I needed her, and for that I am most grateful. I recognise the difficulties encountered in being an overseas student and I would like to thank Professor Joanna Verran for her patience and guidance. Without her support, this project would not have been possible. Many additional colleagues and laboratory technicians (Dr. Paul Benson, Anne Leahy-Gilmartin and Gillian Collier) provided valuable information that help my data collection, and I am grateful to all of them for their assistance. In particular, I would like to thank my supervisor Dr. Kathryn A. Whitehead for providing supporting information describing the test surfaces (along with David Wickens). She has provided me with most of the methodological knowledge used in this study as well as moral support. I am grateful for the efforts and countless hours to teach, me and my colleagues, how to present our data orally to conferences, challenge our knowledge, take information from scientific articles; these are but a few examples. I would like to extend my deepest gratitude to my husband, Thomas Dudman, for his support during the last seven years. His parents, Dr. Christopher Dudman and Dr. Clare Dudman, both PhD chemists, provided me with great help regarding the development of my PhD and my gaps in chemistry knowledge. I would like to thank his brother, Jack Dudman, for his friendship. My sister, Harmonie Laurent, and my iii maternal grandparents, Marcel and Colette Burliga, provided me with infinite support. Thanks to them, I have been able to go study abroad, learn a new language and meet my husband. Their support changed my life entirely and for that I will be eternally grateful. Thank you for believing in me. I would like to thank Dr. Nils Arneborg from the University of Copenhagen, in Denmark, with whom I have collaborated during my PhD. In this European project, my part was to examine the survival of food related pathogens on “open” surfaces such as work surfaces, whereas their part involved investigating the microbial survival in “closed” surfaces such as pipes. Brigitte Carpentier from the AFSSA (Agence Française de Sécurité Sanitaire des Aliments) kindly provided the bacterium Escherichia coli CCL 410 (a non-pathogenic O157:H7 strain) used in this study. Finally, I am appreciative of my fellow graduate students, Dr. Gavin Bingley, Sarah Jackson, James Redfern, David Wickens, Lindsay Smith, Soheyla Ostovarpour, Monika Stuczen, Dr. Malcolm Kinninmonth and Dr. Leanne Fisher for making my time at Manchester Metropolitan University a more enjoyable experience. iv Abstract The ability of pathogenic bacteria to be retained on the surfaces of processing equipment constitutes a potential health problem in the food industry. Stainless steel is commonly used in the food industry, but there is an increasing demand for surfaces with enhanced hygienic properties. One way to combat microbial surface fouling is the use of novel antimicrobial alloys. Although many metal ions in solution demonstrate a significant antimicrobial effect, in the relative absence of moisture, surface release rates and the efficacy of the antimicrobial agent are altered. The aim of this study is to define conditions that minimise survival of bacteria on stainless steel and stainless steel coated with titanium nitride, alloyed with silver, a putative antimicrobial surface. Characterisation of the test substrata revealed smooth, thus hygienic surfaces with no leaching of silver observed. A method was developed to assess survival of Escherichia coli and Listeria monocytogenes on substrata under different equilibrium relative humidities (ERH): 11%, 52% and 86%ERH. Any cells remaining on the surfaces post-swabbing were detected by epifluorescence microscopy, or by metabolic dye. The survival of both microorganisms on surfaces was recorded via live/dead staining. The effect of surface re-use was also assessed. Gram positive L. monocytogenes survived better than E. coli in the highest 86%ERH and silver concentration TiN/Ag 120W, presumably due to its thicker cell wall. The increase of humidity did not affect any antimicrobial effect, but increased concentration of silver in the surface coating reduced the viability of bacteria. Re-use of the surfaces showed similar results to first time use in all experiments. The presence of meat extract increased the time of survival of E. coli from 4 hours to 16 hours on the stainless steel but the presence of silver decreased cell numbers when compared to other coatings. Finally, a differential staining method was developed to detect live/dead E. coli cells as well as meat extract to simulate contamination in situ. This novel method may be used in future studies to investigate the survival of microorganisms in food soil. Surfaces show potential being able to retain antimicrobial activity post use, low wear and no release of silver ions. Future experiments may include the use of the silver containing coating surfaces in situ, in different food industries, to evaluate their potential for reducing outbreaks. v Table of Contents Acknowledgement.................................................................................................. iii Abstract................................................................................................................... v Table of Contents.................................................................................................... vi List of Tables........................................................................................................... xii List of Figures......................................................................................................... xiii Chapter 1: Introduction......................................................................................... 1 1.1. Hygienic surfaces in the food industry: an overview.................................. 2 1.2. Approach to maintaining good hygienic status........................................... 6 1.2.1. Cleaning and disinfecting................................................................ 6 1.2.2. Surface modification....................................................................... 9 1.3. Microorganisms of interest......................................................................... 12 1.3.1. Microorganisms of concern in food industry.................................. 12 1.3.2. Escherichia coli............................................................................... 14 1.3.3. Listeria monocytogenes................................................................... 18 1.4. Factors affecting surface properties............................................................ 24 1.4.1. Physical properties.......................................................................... 24 1.4.2. Chemical properties......................................................................... 25 1.4.3. Biological properties....................................................................... 29 1.5. Environmental conditions........................................................................... 31 vi 1.5.1. Temperature..................................................................................... 31 1.5.2. Equilibrium Relative Humidity (ERH)........................................... 35 1.6. Aim.............................................................................................................. 37 1.7. Objectives.................................................................................................... 37 1.7.1. Method developed............................................................................ 37 1.7.2. Novel investigations......................................................................... 38 Chapter 2: Characterisation of putative antimicrobial surfaces....................... 39 2.1. Introduction................................................................................................. 40 2.1.1. Metals used in surface coatings....................................................... 40 2.1.2. Preparation of metal coatings.......................................................... 41 2.1.3. Surface analysis............................................................................... 42 2.2. Aim.............................................................................................................. 43 2.3. Methods....................................................................................................... 43 2.3.1. Production of nanocomposite coatings............................................ 43 2.3.1.1. Surface characteristics and preparation of fine polished stainless steel coupons................................................................. 43 2.3.1.2. Production