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This electronic thesis or dissertation has been downloaded from the King’s Research Portal at https://kclpure.kcl.ac.uk/portal/ Molecular characterisation of the bacterial community in dentinal caries Schulze-Schweifing, Kathrin Awarding institution: King's College London The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without proper acknowledgement. END USER LICENCE AGREEMENT Unless another licence is stated on the immediately following page this work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International licence. https://creativecommons.org/licenses/by-nc-nd/4.0/ You are free to copy, distribute and transmit the work Under the following conditions: Attribution: You must attribute the work in the manner specified by the author (but not in any way that suggests that they endorse you or your use of the work). Non Commercial: You may not use this work for commercial purposes. No Derivative Works - You may not alter, transform, or build upon this work. Any of these conditions can be waived if you receive permission from the author. Your fair dealings and other rights are in no way affected by the above. Take down policy If you believe that this document breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 10. Oct. 2021 Molecular characterisation of the bacterial community in dentinal caries Kathrin Schulze-Schweifing A thesis submitted in accordance with the requirements of the degree of Doctor of Philosophy for King's College London August 2012 Department of Clinical and Diagnostic Sciences, Dental Institute King's College London Abstract Abstract Dental caries remains the major cause of loss of tooth function and associated morbidity. The development of new prevention and treatment measures requires a better understanding of the disease process, particularly the role of the associated microbiota. The aim of this study was to perform a comprehensive characterisation of the microbiota in dental caries using culture-independent and next generation sequencing methods, with a focus on improving the detection of bacteria with DNA of high G+C content. Oligonucleotide probes for taxa of interest were designed and evaluated using fluorescent in situ hybridisation (FISH). DNA extraction methods, PCR polymerases and primer pairs were compared for their ability to isolate and amplify 16S rRNA genes from species with low and high G+C content. DNA extraction method and polymerase were not found to significantly influence the detection of high G+C species but primer sequence was found to be of critical importance. Five “universal” PCR primer sets and culture were used to characterise the dentine caries microbiota of six subjects. From 3240 clones and isolates, 228 taxa representing eight phyla were identified by Sanger sequencing. Detection frequency of the high G+C Actinobacteria was 33% using culture, but 2.6-11.1% in the different clone libraries. The samples were analysed further by 454 pyrosequencing and 25758 sequences were identified to 264 taxa representing 11 phyla. Pyrosequencing allowed an analysis of greater depth, although the composition of the samples was comparable to those obtained by the Sanger-based method. Total and specific bacteria were detected successfully in excavated dentine by FISH with universal and specific probes, while in bisected teeth only total bacteria were seen with the universal probe. In conclusion, this study has revealed a highly diverse caries microbiota. Pyrosequencing increased detection of taxa and overall coverage, but all methods had associated biases which affected the results obtained. - 2 - Acknowledgements Acknowledgements I share the credit of my work with many people who have helped, encouraged and supported me in a myriad of ways. I would like to express my sincere gratitude to my supervisors Prof William Wade and Prof Avijit Banerjee, who have encouraged and criticised as and when needed and helped me navigate the sometimes cloudy, sometimes stormy, waters that is a research thesis. My deepest gratitude to all patients who have kindly provided the samples on which this research is based. A very big thank you also goes to the lovely Mona Hajatdoost-Sanj, who helped recruit volunteers and co-ordinated collection of all patient samples. In addition, Pete Pilecki gave much appreciated practical support in bisecting the collected teeth and Ahmed Almahdy kindly helped excavate caries scoops. Fellow PhD student James Kistler organised and carried out the experimental work needed for the 454 pyrosequencing in collaboration with the Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London. Many thanks not just for the work, but also the numerous questions you’ve patiently answered. I would also like to thank Dr Dave Moyes for descending on my writing with a sharpened red pen, giving very helpful and much appreciated constructive criticism. Many thanks also go to my friends and colleagues (present and past), not just of the Micro Department, but also the Salivary Research and Mucosal Immunity, Fungal and HIV infection groups – thank you guys for your friendship, many pieces of advice and generally for making the labs a fun place to work. I am also grateful to be blessed with a fabulous (extended) family and many friends outside work who have boosted my morale on various occasions over the last few years. Finally, I would like to dedicate this thesis to my wonderful husband, without whom this thesis would simply have remained a dream. Christian, your encouragement, support and understanding has made me believe in myself and enabled me to chase my dream. The love and gratitude I feel for you and our precious children cannot be expressed in mere words. - 3 - Table of Contents Table of Contents Abstract ......................................................................................................................... - 2 - Acknowledgements ....................................................................................................... - 3 - Table of Contents .......................................................................................................... - 4 - List of Figures .............................................................................................................. - 12 - List of Tables ................................................................................................................ - 16 - List of Abbreviations ................................................................................................... - 18 - Chapter 1 Introduction ................................................................................................ - 21 - 1.1 Microbiota of the human oral cavity in health ........................................... - 22 - 1.2 Oral diseases and their associated microbiota ........................................... - 24 - 1.3 Shifts in the oral microbiota as a result of dynamic interactions with the host and/or environment ................................................................................................ - 27 - 1.4 Oral surfaces and microbial habitats .......................................................... - 30 - 1.4.1 Saliva, the acquired pellicle and the oral microbiota ............................. - 30 - 1.5 Plaque formation ........................................................................................ - 33 - 1.5.1 Reversible adhesion ................................................................................ - 33 - 1.5.2 Irreversible adhesion ............................................................................... - 34 - 1.6 Sequence of plaque formation ................................................................... - 36 - 1.7 Bacterial interactions in the process of biofilm formation ......................... - 41 - 1.8 Biofilms ........................................................................................................ - 43 - 1.8.1 Biofilm structure ..................................................................................... - 43 - 1.8.2 Matrix functions ...................................................................................... - 46 - 1.9 Dental caries ................................................................................................ - 47 - 1.9.1 Structure of enamel and dentine ............................................................ - 47 - 1.9.1.1 Enamel ........................................................................................... - 47 - 1.9.1.2 Dentine .......................................................................................... - 48 - 1.9.1.2.1 Types of dentine ........................................................................ - 49 - - 4 - Table of Contents 1.9.2 Clinical aspects of caries.......................................................................... - 50 - 1.9.3 Clinical lesion development .................................................................... - 51 - 1.9.4 Microbiology of caries ............................................................................. - 52 - 1.10 Molecular microbial ecology methodologies .............................................. - 60 - 1.10.1 Unculturability of bacterial species....................................................... - 60 - 1.10.2 Culture-independent methods ............................................................
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