Smoking Thirties: How Tobacco & BMI Shape the Subgingival Microbiome Thesis Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By NaJla Sani Kasabreh Graduate Program in Dentistry The Ohio State University 2019 Thesis Committee Dr. Purnima Kumar, Advisor Dr Dimitris Tatakis Dr Emmanouil Chatzakis Dr Shareef Dabdoub 1 Copyrighted by NaJla Sani Kasabreh 2019 2 Abstract Background: 25% of Americans are obese, 20% are smokers and 9 million are obese smokers. It has been demonstrated that both smokers and obese individuals demonstrate a greater susceptibility for periodontitis, a bacterially-driven disease that leads to destruction of tooth supporting structures and eventual tooth loss. While early research attributed the link between obesity and periodontitis to the inflammatory effects of adipocytes on subgingival host response, evidence is emerging that the etiology is a multi-factorial. Our research group and others have previously demonstrated that smoking promotes acquisition of periodontal pathogens within oral microbiome, and that this dysbiotic shift contributes to increased risk for periodontitis. Therefore, we aimed to test the hypothesis that both obesity and smoking increase the risk for periodontitis by disrupting the oral microbiome, albeit in different ways. Methods: 183 subjects were recruited following informed consent and divided into groups based on body mass index and smoking status. Bacterial 16S rDNA genes were sequenced from subgingival plaque samples. Sequence analyses were conducted using QIIME and PhyloToAST. Metabolomic analysis was performed on saliva samples using NMR spectroscopy. Levels of selected cytokines and adipokines in gingival crevicular fluid were determined by multiplexed bead-based assay. ii Results: Linear Discriminant Analysis (LDA) showed significant group separation between obese and normalweight individuals (p= 0.026, 0.009, 0.001 Adonis test of weighted, unweighted UniFrac distances and Bray-Curtis Dissimilarity Index). 138 species were significantly different between obese and normalweight individuals. 73 species belonging to the genera Actinomyces, Alloprevotella, Capnocytophaga, Cardiobacterium, Enterococcus, Fusobacterium, Gemella, Haemophilus, Kingella, Leptotrichia, Neisseria, Streptococcus, TM7 and Treponema were lower in the obese individuals ; 65 species of the genera Atopobium, Bacteroidetes, Dialister, Lactobacillus, Prevotella, Selenomonas, Stomatobaculum, and Veillonellaceae were higher in abundance. Network analysis revealed a highly connected hub anchored by ghrelin, GIP- 1, adipsin, glucagon and insulin in obese, but not normalweight subjects. These adipokines emerged as discriminants of the subgingival environment in obese subjects. LDA also revealed significant group separation between obese smokers and normalweight nonsmokers (p =0.021,0,001, 0.001 Adonis test of weighted, unweighted UniFrac distances and Bray Curtis Dissimilarity index). Obese smokers were enriched for putative pathogens belonging to the genera Atopobium, Bacteroidaceae, Bifidobacterium, Dialister, Lactobacillus, Mycoplasma, Prevotella, Treponema and Veillonellaceae. Salivary metabolic profiles also differed significantly between obese smokers and normalweight nonsmokers. Obese smokers presented with higher levels of lactatic acid and lower levels of methanol compared to normalweight nonsmokers. Significant correlations were observed between these metabolites and bacterial community networks. iii Conclusion: By overlaying untargeted metabolomics over an open-ended method for bacterial characterization, we demonstrate that obesity and smoking impact the subgingival microbiome in significant and distinct ways, leading to enrichment of specific bacterial species, as well as the metabolites produced by them. We also provide the first evidence that adipokines and cytokines play important roles in shaping this ecosystem. When obesity intersects with smoking, the whole shift is greater than the sum of the parts. iv Dedication Dedicated to my dad, my mum and my sisters (Nadine, Nora and Natalie) for their tremendous love and support. v Acknowledgments I wish to thank Dr. Purnima Kumar for her unlimited guidance, unique mentorship, huge support and her continuous encouragement through all the times. I also wish to thank my committee members Dr. Dimitris Tatakis, Dr. Emmanouil Chatzakis and Dr. Shareef Dabdoub for their contribution and guidance. I also want to mention Dr. Khaled Altabtbaei, Dr. Shareef Dabdoub, Dr. Sukirth Ganesm, Dr. Vinayak Joshi, Dr. Shweta Saraswat and Dr.Naveen Dasari who were very generous to teach me how to perform clinical and laboratory steps, manipulation and data analysis. I would like to thank all the members of the Ohio State University Periodontology department, Dr. Omar Karadsheh from the University of Jordan and Mr. Andrew Suzo from the bariatric clinic. Without their assistance, Patient collection would have been difficult. Finally, I would like to thank my family, my friends and my faculty at University of Jordan. vi Vita 2012 ... Obtained D.D.S.: University of Jordan Publications Hassona Y, Kasabreh N, Hammoudeh H, Scully C. Oral healthcare management in Bardet Biedl syndrome. Spec Care Dentist. 2017 Jan;37(1):47–50. Fields of Study MaJor Field: Dentistry vii Table of Contents Abstract ............................................................................................................................... ii Dedication ........................................................................................................................... v Acknowledgments .............................................................................................................. vi Vita .................................................................................................................................... vii List of Tables ...................................................................................................................... x List of Figures .................................................................................................................... xi Chapter 1. Introduction ....................................................................................................... 1 Obesity ............................................................................................................................ 1 Obesity and periodontal disease ...................................................................................... 4 Smoking .......................................................................................................................... 7 Smoking and periodontal disease .................................................................................... 7 Obesity, smoking and periodontal disease ...................................................................... 8 Specific Aims .................................................................................................................. 9 Chapter 2. Materials and Methods ................................................................................... 10 Subjects and site selection ............................................................................................ 10 Subgingival plaque collection ....................................................................................... 11 DNA isolation and sequencing ..................................................................................... 11 Comparative metataxonomic ........................................................................................ 11 NMR spectroscopy ........................................................................................................ 12 Inflammatory analysis ................................................................................................... 13 Chapter 3: Results ............................................................................................................. 14 Demographic ................................................................................................................. 14 BMI≥30 is a discriminant of the subgingival microbiome ........................................... 14 Subgingival microbiome is influenced by adipokine levels ......................................... 15 Men are from Mars, and women from Venus (unless they are obese) ......................... 16 viii Smoking: an old story that still holds true .................................................................... 18 The subgingival microbiome at the intersection of BMI and smoking ........................ 20 Smoking thirties: Obese smokers present a distinct microbiome when compared to normalweight nonsmokers ............................................................................................ 23 Metabolic correlations explain microbial differences in BMI-smoking groups ........... 23 Chapter 4: Discussion ....................................................................................................... 26 Chapter 5: Summary and Conclusions .............................................................................. 34 Bibliography ..................................................................................................................... 36 Appendix A: Tables .......................................................................................................... 46 Appendix B: Figures ........................................................................................................