THE RELATIONSHIP BETWEEN ORAL MALODOUR AND CHRONIC PERIODONTITIS: ROLE OF VOLATILE SULFUR COMPOUNDS Abish Samuel Stephen Research Centre for Diagnostic and Oral Sciences School of Dentistry Queen Mary University of London This thesis is submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy May 2016 We are in much the same position as an observer trying to gain an idea of the life of a household by careful scrutiny of the persons and material arriving or leaving the house; we keep accurate records of the foods and commodities left at the door and patiently examine the contents of the dust- bin and endeavour to deduce from such data the events occurring within the closed doors. -Marjory Stephenson (Bacterial Metabolism, 1930) i DECLARATION I, Abish Samuel Stephen, confirm that the research included within this thesis is my own work or that where it has been carried out in collaboration with, or supported by others, that this is duly acknowledged below and my contribution indicated. Previously published material is also acknowledged below. I attest that I have exercised reasonable care to ensure that the work is original, and does not to the best of my knowledge break any UK law, infringe any third party’s copyright or other Intellectual Property Right, or contain any confidential material. I accept that the College has the right to use plagiarism detection software to check the electronic version of the thesis. I confirm that this thesis has not been previously submitted for the award of a degree by this or any other university. The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author. Signature: Date: Details of collaboration: ii SUMMARY Oral malodorous gases such as hydrogen sulfide and methanethiol are positively associated with plaque-induced periodontal diseases. It is well known that these Volatile Sulfur Compounds (VSCs) are produced by the oral microbiota during proteolysis, and the specific association of methanethiol in the breath with periodontal diseases was investigated in a clinical study described in this thesis. The association between breath methanethiol and clinical parameters of disease, abundance and prevalence of putative peridontopathic bacteria in periodontal niches were confirmed. A gas chromatographic method was developed to measure subgingival VSCs and associations with clinical parameters, microbial abundance in a range of oral niches and inflammatory markers in saliva and gingival crevicular fluid (GCF) were found. Breath methanethiol was also found to be more closely associated with a range of disease associated inflammatory markers in GCF. Tongue biofilm, subgingival and interdental plaque samples were analysed by Human Oral Microbiome Identification by Next Generation Sequencing (HOMINGS) methodology. Microbial diversity in the tongue was positively associated with breath VSCs in health, but subgingival and interdental niche diversity was more strongly associated with breath VSCs in gingivitis and chronic periodontitis. Tongue ecology in the disease associated cohorts was markedly different compared to health, especially with the increase in abundance and prevalence of putative VSC producing species. The ecology of VSC producing bacteria is described for the niches studied, finding that the methanethiol producing species are almost exclusively periodontopathic, with more dynamic population differences in these group of organisms observed from health to disease. The VSC producing potential in the periodontal niches was also found to be more dynamic compared to the tongue. The role of methanethiol production by the keystone species, P. gingivalis was investigated using a 10-species biofilm co-culture model by substituting a methionine gamma lyase (mgl) deficient strain for the wild type and marked changes in the overall biofilm composition was observed in terms of community evenness and biomass. The wild type biofilm displayed a more insidious phenotype whereas the higher biomass mutant biofilm exhibited an overtly pathogenic phenotype. P. gingivalis mgl is proposed as a potential keystone virulence factor. iii ACKNOWLEDGEMENTS I’m so glad to have seen the other end of this project and I count myself as lucky to have been part of it. Firstly, I would like to thank my academic supervisor Prof. Robert Allaker. Thanks for the freedom, encouragement and thanks for looking out for me. Without your support and assurance it would have been an impossibly uphill task given that I came fresh to this subject and didn’t know a whole lot about a lot of the things I got involved with, in this project. I would say your professional and personal guidance throughout the duration of the project has made me all round, a better person and scientist. I would also like to thank two great people who have been a source of wisdom and inspiration throughout this project. Dr. David Bradshaw and Dr. Gary Burnett: you guys have been great sport during the project meetings, and I learned a lot from you two, especially the confidence to do things I could only dream of attempting. Thanks for that impetus. I would like to thank my colleagues at QMUL: Cecilia for helping me get up to speed on a lot of things at the start of the project, Joe for being such a star in every way, Kaveh for being a good friend and looking out for me, and of course Dr. David Gillam for being an absolute rock from the clinical side. I would like to thank the very special dentists I worked with who helped collect the clinical samples, Narinder, Vam and Bryar, your help is much appreciated. I would also like to thank all the folk at CDOS who I don’t have the space to name individually for making the whole experience of this project memorable. I had a lot of great experiences during this project, and one was driving from Glasgow to Inverness in my 944 via the stunning A82 and I would like to thank Prof. Gordon Ramage for helping to make that happen. I will never forget that journey. Thanks also to the crew at Glasgow Dental School for being so warm and accommodating—Leighann, Emma, Chris, David and namma ooru Ranjith. A special mention to GSK who have made a positive impact in the project development by helping establish the collaboration with Glasgow and also in doing some extra sample analyses that have inspired me and helped me to gain a real appreciation of my subject. I would like to thank my parents, sister, uncle and aunt for accommodating me during my studies and for being sources of great strength during tough times. Finally, thanks to my loving wife Chrisma, for putting up with me constantly writing and completely ignoring everything else and for looking out for my general wellbeing. iv CONTENTS 1 INTRODUCTION ........................................................................................................ 1 1.1 ETIOPATHOGENESIS OF PERIODONTAL DISEASES ...................................................... 1 1.1.1 Initial lesion ..................................................................................................... 2 1.1.2 Early Lesion ..................................................................................................... 3 1.1.3 Established Lesion ........................................................................................... 3 1.1.4 Advanced Lesion .............................................................................................. 4 1.2 MICROBIAL ROLE IN PERIODONTITIS ........................................................................ 5 1.2.1 Microbial models of plaque-induced chronic periodontitis ............................. 6 1.3 ROLE OF THE HOST IMMUNE RESPONSE IN PERIODONTITIS ...................................... 10 1.3.1 The TH1-TH2-TH17 Paradigm ......................................................................... 12 1.4 VOLATILE SULFUR COMPOUNDS IN PERIODONTAL DISEASE .................................... 17 1.4.1 VSCs in the periodontal pocket ...................................................................... 18 1.4.2 VSCs and microbial ecology .......................................................................... 19 1.4.3 Effect of VSCs on host response ..................................................................... 20 1.5 PROJECT AIMS ........................................................................................................ 22 2 MATERIALS AND METHODS .............................................................................. 23 2.1 STUDY DESIGN AND CLINICAL PROTOCOL ............................................................... 23 2.1.1 Inclusion and exclusion criteria. .................................................................... 23 2.1.2 Clinical protocol ............................................................................................ 25 2.1.3 Clinical sample collection methods ............................................................... 27 2.2 LABORATORY PROTOCOLS FOR CLINICAL SAMPLES ................................................ 31 2.2.1 Quantitative PCR analysis ............................................................................. 31 2.2.2 Human Oral Microbe Identification using Next Generation Sequencing ...... 35 2.2.3 Multiplexed Fluorimetric Immunoassay ........................................................ 37 2.2.4 Gas chromatographic headspace analysis .................................................... 38 v 2.3 BIOFILM COCULTURE MODEL