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2014 Kistler James 1041095 This electronic thesis or dissertation has been downloaded from the King’s Research Portal at https://kclpure.kcl.ac.uk/portal/ Defining oral microbiological health Kistler, James Oliver 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: 06. Oct. 2021 DEFINING ORAL MICROBIOLOGICAL HEALTH James Oliver Kistler A thesis submitted in accordance with the requirements of the degree of Doctor of Philosophy in King’s College London January 2014 1 Abstract Background: The composition of the oral microbiota in periodontal health and the microbial changes associated with the early stages of gingivitis are poorly defined. Aims: The aims of this work were to: (i) characterise the bacterial composition of dental plaque in subjects with experimentally-induced gingivitis; (ii) identify candidate oral probiotic taxa; (iii) evaluate alternative approaches for oral Neisseria spp. differentiation. Methods: Twenty volunteers abstained from oral hygiene in the mandible for two weeks. Clinical indicators of inflammation were monitored and samples of plaque were analysed, together with 20 control samples from periodontitis patients, by pyrosequencing of 16S rRNA genes and culture. A panel of oral bacterial isolates were screened for inhibition of six indicator organisms using a deferred antagonism assay. Sequencing of seven ‘housekeeping’ genes and a ribosomal protein gene (rplF) were evaluated as methods to differentiate Neisseria species. Results: All volunteers developed gingivitis after two weeks. 344,267 16S rDNA sequences were clustered into a median of 299 species-level Operational Taxonomic Units (OTUs) per sample. Principal Coordinate Analysis plots revealed shifts in community structure with gingivitis development, and the mean Simpson’s inverse diversity index increased from 32 at baseline to 47.5 after two weeks (P<0.0001). Fusobacterium nucleatum subsp. polymorphum, Lautropia sp. HOTA94, Lachnospiraceae sp. HOT100 and Prevotella oulorum were significantly associated with gingivitis, whilst Rothia dentocariosa was health-associated. Of 80 isolates screened, two strains of Streptococcus cristatus and a Streptococcus sp. HOT071 strain, inhibited the growth of one or more of Streptococcus anginosus, 2 Solobacterium moorei, Porphyromonas gingivalis and Filifactor alocis. rplF gene sequences were found to reliably differentiate oral Neisseria species, although some taxonomic revision to the genus is indicated. Conclusions: A highly species-rich bacterial community in health-associated plaque was revealed and new health- and gingivitis-associated taxa were identified. Three strains were found with potential for use as oral probiotics. 3 Table of Contents Abstract .......................................................................................................................... 2 Table of Contents .......................................................................................................... 4 Table of Figures ............................................................................................................. 8 Table of Tables ............................................................................................................ 13 Acknowledgements ...................................................................................................... 15 Chapter 1: Introduction ............................................................................................. 17 1.1 The oral cavity and its resident microbiome ........................................................... 18 1.2 Dental plaque .............................................................................................................. 24 1.3 Methods for the characterisation and analysis of the oral microbiome ................ 29 1.3.1 Characterisation of human-associated microbial communities ............................ 29 1.3.2 Microscopy and culture methods .......................................................................... 31 1.3.3 Molecular methods ................................................................................................ 35 1.3.3.1 Culture-independent sequencing techniques ........................................................... 35 1.3.3.2 Other molecular methods ........................................................................................ 46 1.4 The oral microbiome in health .................................................................................. 48 1.4.1 Function of the oral microbiome in health ............................................................ 48 1.4.2 Current knowledge of the composition of the oral microbiome in health ............. 50 1.5 The oral microbiome in periodontal diseases .......................................................... 61 1.5.1 Gingivitis ............................................................................................................... 61 1.5.2 Periodontitis .......................................................................................................... 64 1.6 New approaches to maintain periodontal health ..................................................... 71 1.6.1 Conventional approaches for the prevention and control of periodontal diseases71 1.6.2 Oral probiotics and prebiotics .............................................................................. 72 1.6.2.1 Clinical trials of oral probiotics ............................................................................... 78 1.7 Oral bacterial commensals of the genus Neisseria .................................................. 84 1.7.1 Neisseria and the oral microbiome ....................................................................... 84 4 1.7.2 Taxonomy of the genus Neisseria .......................................................................... 85 1.7.3 Neisseria species found in the human oral cavity ................................................. 88 1.7.4 Molecular approaches to the identification and classification of oral Neisseria . 95 1.8 Aims ............................................................................................................................. 99 1.8.1 Specific aims .......................................................................................................... 99 Chapter 2: Analysis of the oral microbiota in experimental gingivitis ................ 101 2.1 Introduction .............................................................................................................. 101 2.2 Methods ..................................................................................................................... 103 2.2.1 Study design ......................................................................................................... 103 2.2.2 Subject recruitment.............................................................................................. 104 2.2.2.1 Periodontally healthy volunteers ........................................................................... 104 2.2.2.2 Patients with chronic periodontitis ........................................................................ 104 2.2.3 Experimental gingivitis........................................................................................ 105 2.2.4 Sample collection ................................................................................................ 106 2.2.4.1 Experimental gingivitis ......................................................................................... 106 2.2.4.2 Chronic periodontitis ............................................................................................. 107 2.2.5 DNA extraction .................................................................................................... 107 2.2.6 16S rRNA gene PCR and 454 pyrosequencing .................................................... 108 2.2.7 Sequence analysis ................................................................................................ 109 2.2.8 Statistical analysis ............................................................................................... 111 2.2.9 Bacterial culture analysis .................................................................................... 112 2.2.10 Investigation of potentially novel oral taxa ......................................................... 114 2.3 Results ......................................................................................................................
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