Candida albicans Biofilms in Denture Wearers Sarah Louise Jackson A thesis submitted in partial fulfilment of the requirements of the Manchester Metropolitan University for the degree of Doctor of Philosophy School of Healthcare Science of Manchester Metropolitan University in collaboration GlaxoSmithKline plc July 2013 Acknowledgements I would first and foremost like to give thanks to a very inspirational and passionate person, without whom, this work and the completion of this PhD would not have been possible. Nick-named ‘Super Jo’ for her incredible ability to not only manage, but excel at everything, all at once; Professor Joanna Verran has been a constant source of encouragement and guidance to me throughout this project. Not only has she helped me to develop my research but she has also promoted professional development and provided invaluable opportunities along the way. I do not have the words to describe just how lucky I feel to have had her support as my director of studies. She is one in a million! In addition to Professor Joanna Verran, there have been several members of the microbiology lecturing team that have provided me with support and help throughout my PhD. I would like to especially thank Dr Lisa Ann Coulthwaite for her continued support, guidance and friendship, and Dr Gordon Craig, Dr Martin Whalley and Dr Rebecca Taylor for their friendly faces and helpful advice. I would like to acknowledge the contribution of GlaxoSmithKline, whom partially funded this work. My gratitude goes to Dr Zvi Loewy as my external supervisor, for his assistance in the first half of this project. The microbiology team at MMU are a true community and it has been a real pleasure to be able to work alongside such wonderful people. My personal thanks must go to the technical staff; Dr Paul Benson, Miss Gill Collier, Mrs Anne Leahy- Gilmartin and Miss Lindsey Smith whose technical support, advice and humour has been exceptional and very much appreciated. I would also like to acknowledge the superior administrative support provided by Ms Rita Kenny and Miss Anne-Marie, their assistance and good humour has been of great value to me throughout this journey. My fellow microbiology researchers and good friends have been an additional source of advice and support along the way and I would like to especially mention three individuals; Dr Gavin Bingley, Mr David Wickens and Mr James Redfern. Together we have shared ideas, advice and experiences that will stay with me forever. Finally, I do not believe I would be where I am today without the tremendous backing of my wonderful family. Without their continued encouragement, supply of cups of tea (especially during the writing period) and constant devotion to my development, I would not have been able to achieve this goal. To Mum, Dad, Chris and Justyna, you have always been there for me and I am eternally grateful for everything you have done and continue to do. Dedication This thesis is dedicated to my late Auntie Jane whose ambition, drive and achievements have always inspired me, and to my late Granddad Jackson whose elation at my acceptance onto this course of study has given me the drive to see it through to completion. Declaration I declare that this work has not already been accepted for any degree and is not being currently submitted in candidature for any other than the degree of Doctor of Philosophy of the Manchester Metropolitan University Table of contents Abstract I Structure of thesis III List of tables IV List of figures V List of Abbreviations XIV 1. Chapter 1 - Introduction to Candida albicans biofilms in denture wearers 1 1.1. Introduction 2 1.1.1. Biofilms 2 1.1.1.1 Biofilm history 2 1.1.1.2 Stages of biofilm formation 3 1.1.1.3 Biofilm phenotype 5 1.1.1.4 Biofilm growth rate and resistance 6 1.1.1.5 Clinical significance of biofilms 7 1.1.2. Denture plaque 8 1.1.2.1. Primary colonisers 8 1.1.2.2. Secondary colonisers 9 1.1.2.3. Denture plaque as a reservoir for infection 10 1.1.2.4. The denture fitting surface 12 1.1.3. Denture stomatitis 14 1.1.3.1. Denture stomatitis aetiology and management 14 1.1.4. Denture plaque and systemic infections 16 1.1.4.1. Respiratory infections 16 1.1.4.2. Gastrointestinal infections 17 1.1.5. Denture hygiene 19 1.1.5.1. Physical characteristics of dentures 19 1.1.5.2. Abrasive denture cleansing and denture plaque 21 1.1.5.3. Chemical denture cleansers 22 1.1.5.4. Denture cleanser activity against Candida spp. 24 1.1.6. Candida albicans 25 1.1.7. Adhesion 29 1.1.7.1. Adhesion and denture topography 29 1.1.7.2. C. albicans adhesins 30 1.1.7.3. Enzymes of C. albicans 31 1.1.8. C. albicans in denture plaque 32 1.1.9. Aims 35 2. Chapter 2 - Denture surface topography and the retention of Candida albicans 36 2.1. Introduction 37 2.1.1. Factors affecting C.albicans adhesion to denture acrylic surfaces 37 2.1.2. Denture surfaces and surface topography 39 2.1.3. Microbial contamination of dentures 40 2.1.4. Measuring roughness of denture surfaces 43 2.1.5. Aims, research questions and H0 45 2.2. Materials and Methods 46 2.2.1. Maintenance of cultures 46 2.2.2. Preparation of cell suspensions 46 2.2.3. Production of heat cured Polymethy methacrylate (PMMA) 46 2.2.4. Abrasion of 1cm2 heat cured PMMA with emery paper 49 2.2.5. Abrasion of heat cured PMMA with abrasive dentifrices 49 2.2.6. Characterisation of surface topography 50 2.2.7. Preliminary retention assay 53 2.2.8. Standardising methods for retention assay 53 2.2.9. Effect of abrasion on retention; defined assay 62 2.2.10. Inducing hyphal growth in adhered cells 62 2.2.11. Statistical analysis 65 2.3. Results 66 2.3.1. Measuring roughness parameters and feature dimensions of abraded denture surfaces 66 2.3.2. Characterisation of topography with different profilometers 73 2.3.3. Retention of Candida albicans to 1cm2 denture acrylic surfaces abraded with different grit sized emery papers 76 2.3.4. Retention of Candida albicans to 2cm2 PMMA subjected to dentifrice abrasion 81 2.3.5. Measuring cell retention 84 2.3.6. Hyphal growth of adhered C.albicans on abraded surfaces 86 2.4. Discussion 88 2.4.1. Adhesion and retention of C. albicans on abraded denture surfaces 88 2.4.2. Measuring cell retention 91 2.4.3. Measuring roughness profilometry 92 2.4.4. Hyphal growth of adhered C.albicans on abraded surfaces 96 2.5. Conclusions 98 3. Chapter 3 – The development and removal of C. albicans biofilms from abraded denture surfaces 100 3.1. Introduction 101 3.1.1. Biofilms of C. albicans 101 3.1.2. Morphogenesis in C. albicans 102 3.1.3. Denture hygiene 103 3.1.4. Aims, research questions and H0 106 3.2. Materials and methods 107 3.2.1. Maintenance of cultures 107 3.2.2. Preparation of cell suspensions 107 3.2.3. Retention assays 107 3.2.4. Inducing hyphal growth in adhered cells 108 3.2.5. Biofilm development from adhered yeast or hyphal cells 108 3.2.6. Confocal Microscopy of C. albicans biofilms 108 3.2.7. Measuring biofilm mass 111 3.2.8. Retention of cells following biofilm removal 111 3.2.9. Denture cleanser testing 112 3.2.10. Time kill of planktonic C. albicans 115 3.2.11. Polident denture cleanser activity against C.albicans biofilms 115 3.2.12. Denture cleanser activity against mixed biofilms 116 3.2.13. Viability testing of biofilms 117 3.2.14. XTT assay 118 3.2.15. The penetration of C. albicans biofilms by denture cleansers 119 3.2.16. Live dead staining and confocal microscopy 120 3.2.16.1. Preliminary findings and method development 123 3.2.17. Statistical analysis 124 3.3. Results 125 3.3.1. Biofilm development from adhered C. albicans blastospores or hyphae 125 3.3.2. Measuring biofilm thickness and mass 129 3.3.3. Cells retained on surfaces following biofilm removal 132 3.3.4. Denture cleanser tests with C. albicans 135 3.3.5. Denture cleanser activity against C. albicans biofilms 138 3.3.6. Denture cleanser activity against mixed biofilms 140 3.3.7. XTT viability assay 142 3.3.8. The penetration of C. albicans biofilms by denture cleansers 145 3.4. Discussion 149 3.4.1. Morphology of C. albicans biofilms 149 3.4.2. Retention of cells following biofilms removal 151 3.4.3. C. albicans and mixed biofilm viability 152 3.4.4. Live dead staining of hyphal and blastospore biofilms of C. albicans 159 3.5. Conclusions 162 4. Chapter 4 – Quorum sensing in Candida albicans 164 4.1. Introduction 165 4.1.1. Quorum sensing in C. albicans 165 4.1.2. Non-farnesol quorum sensing molecules 167 4.1.3. Identification of quorum sensing molecules from C. albicans 169 4.1.4. Aims, research question and H0 171 4.2. Materials and Methods 172 4.2.1. Maintenance of cultures 172 4.2.2. Optical tweezer microscopy 172 4.2.3. Detection of quorum sensing in C. albicans gas chromatography 177 4.2.4. Gas chromatography programme for volatile identification 179 4.2.5. Solid phase microextraction (SPME) 179 4.2.6. Preparation and analysis of C.