1

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

2

Copyrighted by

Najla Sani Kasabreh

2019

ii

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.

iii

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.

iv

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.

v

Dedication

Dedicated to my dad, my mum and my sisters (Nadine, Nora and Natalie) for their

tremendous love and support.

vi

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.

vii

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

viii

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

ix

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 ......................................................................................................... 79

x

List of Tables

Table 1 Demographic, biometric and clinical characteristics. .......................................... 46 Table 2 Ethnicity of individuals participating in the study. .............................................. 47 Table 3 Significant difference in adipokines and cytokines levels between BMI groups. 47 Table 4 Significant difference in metabolomic concentrations between BMI-smoking groups. ............................................................................................................................... 48 Table 5 Subgingival bacteria and metabolomes network in normalweight nonsmoker individuals. ........................................................................................................................ 48 Table 6 Subgingival bacteria and metabolomes network in normalweight smoker individuals. ........................................................................................................................ 49 Table 7 Subgingival bacteria and metabolomes network in overweight nonsmoker individuals. ........................................................................................................................ 64 Table 8 Subgingival bacteria and metabolomes network in overweight smoker individuals. ........................................................................................................................ 67 Table 9 Subgingival bacteria and metabolomes network in obese nonsmoker individuals............................................................................................................................................ 73 Table 10 Subgingival bacteria and metabolomes network in obese smoker individuals. 75

xi

List of Figures

Figure 1 Linear discriminant analysis, Shannon index and DESeq for BMI groups. ....... 79 Figure 2 Cytokine discriminants and Cytokine discriminants For BMI groups. Cytokine discriminants (2A) and Cytokine discriminants (2B) between normalweight (green), overweight (orange) and obese (yellow) individuals. ....................................................... 80 Figure 3 Co-occurrence networks of BMI groups. ........................................................... 80 Figure 4 Linear discriminant analysis, Shannon index and DESeq for BMI sub-grouped according gender. .............................................................................................................. 81 Figure 5 Linear discriminant analysis, Shannon index and DESeq for smoking groups. 82 Figure 6 Linear discriminant analysis, Shannon index and DESeq for smoking sub-grouped according gender. ................................................................................................ 83 Figure 7 Linear discriminant analysis, Shannon index and DESeq for BMI-smoking groups. ............................................................................................................................... 84 Figure 8 Representative NMR spectrum. .......................................................................... 85 Figure 9 Metabolic profile of the BMI-Smoking groups. ................................................. 85 Figure 10 Co-occurrence networks in normalweight nonsmoker and normalweight smoker individuals. ........................................................................................................... 86 Figure 11 Co-occurrence networks in overweight nonsmoker and overweight smoker individuals. ........................................................................................................................ 87 Figure 12 Co-occurrence networks in obese nonsmoker and obese smoker individuals. 88

1

Chapter 1. Introduction

One-fourth of Americans are obese, and one-fifth are smokers 1. Together, 81 million

American adults are either obese or smoke. Even more importantly, 9 million Americans

are obese smokers 1. Obesity and smoking are often listed as risk factors for many

chronic conditions and diseases both in the medical and oral fields 2 3 4 5 6 7. These two

factors are thought to share a common etiopathogenetic mechanism of chronic

inflammation, causing detrimental effects on tissues. While some of these effects are

reversible, others are not. Importantly, the reversible outcomes take a very long time to

return to baseline following elimination of these factors. Smoking is listed as risk factor

in the 2017 classification of periodontal disease 8, and obesity is classified as a metabolic

and endocrine disorder that affects the periodontal tissues 9 .

Obesity

Obesity, defined as an excess or abnormal accumulation of fat, is associated with

increased risk for many systemic diseases including hypertension, type II diabetes,

cardiovascular diseases and stroke 10 . One metric used to numerically quantify the level

of obesity is the body mass index (BMI); which is the result of the person’s weight in

kilograms divided by the square of his/her height in meters. Accordingly, a person with a

2

BMI of <18.5 is considered underweight, ≥ 18.5 and < 25 is normal, ≥ 25 and < 30 is

overweight and with a BMI of ≥ 30, the person is considered obese 11.

A systematic analysis for the Global Burden of Disease Study in 2010 revealed that

overweight and obesity caused approximately 3.4 million deaths, 3.9% of years of life

lost and 3.8% of disability adjusted life years 10 . Annually, the medical expenditure

attributable to obesity in the US ranges between $147 and 210 million 12.

Globally, the prevalence of overweight and obesity combined has risen by 27.5% for

adults between 1980 and 2013, increasing the number of individuals from 921 million in

1980 to 2.1 billion in 2013 13. The proportion of overweight and obese male adults has

increased from 28.8% in 1980 to 36.9% in 2013. For female adults, the proportion

increased from 29.8% to 38.0% 13.

In developed countries, males exhibit higher rates of overweight and obesity than

females; the peak was around the age 55 years in males, with 70.9 % overweight and 31.7

% obese. For females the peak was 60 years with 61.9 % overweight and 33.9% obese 13.

More than 50% of the obese individuals in the world live in 10 countries, 13% of which

are in the USA 13.

According to NHANES report in 2017 14, 39.8% of US adults are obese. Obesity was

higher among older adults (40-59) compared to younger adults (20-39) and there was no

3

difference in prevalence between females and males. Hispanic (47.0%) and non-Hispanic

black (46.8%) adults had the highest prevalence of obesity, followed by non-Hispanic

white adults (37.9%). The prevalence was the lowest among non-Hispanic Asian adults

(12.7%).

The cause of obesity is complex and unknown; there appear to be biologic, psychosocial,

and behavioral factors. Obesity has been linked to gut microbiota, epigenetics, increasing

maternal age, greater fecundity, sleep deprivation, endocrine disruptors, pharmaceutical

products, reduction in variability of ambient temperatures and intrauterine and

intergenerational effects 15.

Many pathogens have been reported to have adipogenic effects; Canine distemper virus

was the first virus to be reported to induce obesity in mice 16 which was confirmed by

subsequent studies. Chlamydia pneumoniae was the first microorganism linked to

increased BMI in humans 17. Rous-associated virus-7, Scrapie agent, Borna disease virus

and Adenoviruses (Avian adenovirus, SMAM-1, Adenovirus type 5,36 and 37) were

among the other pathogens to be related to obesity.

In addition, several studies have reported a relationship between obesity and gut

microbiota. It was reported that a decrease in Bacteroides and an increase in Firmicutes

was seen in obese mice 18. In their study, Backhed el al., 2004 19 found that

conventionalized germ free mice developed more body fat and insulin resistance, even

4

with lower food consumption. The microbiota contributed to a 2.3-fold increase in

hepatic triglyceride content, no change in cholesterol, increased synthesis of de novo fatty

acid and decreased expression of fasting-induced adipocyte factor (Fiaf). The authors

suggested that suppression of Fiaf by the gut microbiota led to an increase hepatic

lipogenesis and adipose tissue 19.

Preadipocytes are believed to function like macrophages 20 and adipocytes to secrete

many cytokines and chemokines such as tissue necrosis factor (TNF), leptin, interleukin

(IL)-1 21 and IL-6 22. Moreover, Vendrell el al., 2004 23 found elevated levels of plasma

(soluble) TNF receptors in obese patients.

Obesity and periodontal disease

Periodontists and researchers have been long trying to establish whether a correlation

exists between these chronic inflammatory diseases. It started in 1977 when Perlstein and

Bissada 24 tested the hypotheses: “Can hypertension and obesity be risk factors for

periodontal disease?” Normalweight and obese rats were fed with standard diet

containing 5% fat. Gingival irritation was produced using stainless steel wire that was

placed around the maxillary second molar. The other maxillary second molar was used

as a control. 7 weeks later, animals were killed, and specimens were prepared for

histologic examination. The answer to that question was that obesity does not induce

periodontal destruction, but periodontal inflammation and destruction was greater in the

presence of bacterial plaque.

5

The NHANES III report in 2014 25 showed that waist to hip ratio (WHR), BMI, fat-free

mass, and log sum of subcutaneous fat had significant correlations with periodontal

disease, suggesting that abnormal fat metabolism may be an important factor in the

pathogenesis of periodontal disease. Subsequently, many studies were conducted to

investigate more in this linkage. Epidemiological studies noticed an increase in

periodontal disease with an increase in obesity. For example, in a Jordanian population,

the prevalence of periodontal disease was found to be 14% among normalweight

individuals, 29.6% among overweight and 51.9% among obese individuals 26. Moreover,

it was found that the risk of experiencing an increase in attachment loss in 5 years was

36% higher in obese individuals compared to non-obese 27. Independent of other factors

such as gender, ethnicity, smoking status and dental plaque level; an odd ratio (OR) for

overweight/obesity of 1.12 per increase of 1 kg/m2 was found in periodontitis group. The

OR of periodontitis was 2.5 in overweight and 3 in obese 28.

The next step was to find an answer to these observations; why there is such an

association? How can the individuals’ increase in weight affect their oral health and how

does oral health affect the individuals’ weight? Answering this question is quite

complicated as general health and oral health have similar causal mechanisms and share

similar personal behaviors, socioeconomic and lifestyle factors. Some pointed to the fact

that obese individuals don’t take care of their general health and as an extension, will

subsequently not take care of their oral health; in fact, regular dental visits were found to

6

be lower among obese individuals when compared to non-obese individuals 29 30. After

adjustment of obesity risk factors, such as diet and energy intake, Saxlin et al 2011.,

concluded that the association between periodontitis and obesity is not influenced by

these factors 31. Ostberg et al., 2012 32 suggested inflammation (i.e. periodontitis) as a

possible factor.

More can be learnt about this relation through the obesity and gastrointestinal studies; the

microbiota can influence host adiposity through energy extraction and by affecting

metabolism. This will depend on the bacterial community composition. Gut bacteria can

initiate inflammation by inducing the production of TNF- 𝛼 and insulin resistance 33.

Another plausible mechanism is bacterial lipopolysaccharide (LPS). Saxlin et al 2011 31

suggested that LPS, endotoxins of gram-negative bacteria in the deepened periodontal

pockets might have an effect on obesity; they found an exposure-response among the

study population as more individuals with BMI≥30 had more teeth with deeper pocket

depth. Experimental studies had in fact linked endotoxemia to weight gain in mice

infused subcutaneously with LPS for 4 weeks but fed with a normal diet 34. This weight

gain was even similar to the weight gained by mice fed with higher fat diet for the same

period of time. Goodson et al., in 2009 suggested that this relation might be due to a shift

in the oral microbiota 35. In this study; it was suggested that this causal relation could be

attributed to ingestion of oral pathogens such as Selenomonas noxia.

7

Positive correlation between severity of periodontal attachment loss and BMI has been

identified in the literature 36 37 26 38 39 40 41 42 43. Subsequent studies described a 76%

higher prevalence of periodontal disease among young individuals with a BMI ≥30 with

an adjusted OR of 1.76 5. A systematic review and meta-analysis found about a 33%

increase in prevalence of obesity in individuals with periodontal disease, and a higher

mean attachment loss in obese individuals 44. Adipokine induced inflammation has been

proposed as the underlying etiopathogenic mechanism 45.

Smoking

A smoker is a person who smoked at least 100 cigarettes during his/her lifetime and is

currently smoking according to CDC guidelines 46. Smoking causes cancer,

cardiovascular disease, pulmonary diseases, immune diseases and stroke 7. Annually, the

medical expenditure attributable to smoking is $170 million 47. Worldwide, the

prevalence of daily tobacco smoking for males declined from 41.2% to 31.1% and for

females declined from 10.6 to 6.2% between 1980 and 2012 48 . In the U.S., 14% of the

adult population are smokers (15.8 % of males and 12.2% of females) 49.

Smoking and periodontal disease

The prevalence of periodontitis in smokers was about 4 times higher than in persons who

had never smoked; with about 41.9% of periodontitis cases in the U.S. adult population

being attributable to smok50. Current smokers had significantly more attachment loss,

missing teeth, deeper pockets and fewer sites exhibiting bleeding on probing than past or

8

never smokers 51. Classical papers have demonstrated that the effects of smoking are

associated with suppression of salivary and tissue neutrophil activity and increased levels

of IL-1ß 52.

Our long-standing research on tobacco has demonstrated that smoking promotes

acquisition of periodontal and oropharyngeal pathogens within the disease-naïve oral

microbiome. Smokers demonstrated higher abundances of anaerobes and lower levels of

aerobes when compared with nonsmokers. The subgingival microbiome of smokers was

enriched for periodontal and systemic pathogens Fusobacterium nucleatum, F.

naviforme, Filifactor alocis, Dialister microaerophilus, Desulfobulbus sp. clone R004,

Megasphaera sueciensis, M. geminatus, M. elsdenii, M. micronuciformis, Acinetobacter

johnsonii, A. guillouiae, A. schindleri, A. baumannii, A. haemolyticus, Pseudomonas

pseudoalcaligenes and Pseudoramibacter alactolyticus 5353. In addition, our lab found

that there were significantly lower levels of several health-compatible commensals, such

as: Streptococcus sanguinis, S. parasanguinis, S. oralis, Granulicatella elegans, G.

adiacens, Actinomyces viscosus, A. israelii, A. dentalis, Neisseria subflava and

Hemophilus parainfluenzae 53 .

Obesity, smoking and periodontal disease

Studies that examine the combined impact of both factors are scarce. Wood and Johnson

2008 54 found that smokers with a Periodontal Screening and Recording (PSR) code of 4

(indicating that they had severe periodontitis in at least one quadrant of their dentition)

9

had a significant risk of being obese or overweight. Classification and regression analysis

of 372 Japanese workers demonstrated that both pack-years and BMI exhibited clear

dose-response relationships with periodontitis 55.

Specific Aims

Based on a review of the literature and our own prior work, we hypothesized that both

smoking and BMI>25 will impact the subgingival microbial ecosystem. We tested this

hypothesis using the following aims:

Aim 1: To characterize the subgingival community of periodontally and systemically

healthy individuals who smoke, are obese or are obese smokers.

Aim 2: To stratify the salivary metabolic profiles of these individuals according to their

BMI and smoking status.

Aim 3: To assess the inflammatory burden imposed by the microbiome in obese,

overweight and normalweight individuals.

10

Chapter 2. Materials and Methods

Subjects and site selection

Approval for this study was obtained from the Office of Responsible Research Practices

at The Ohio State University (Protocol number: 2016H0438) and the study was

conducted in accordance with the approved guidelines. 183 subjects were recruited from

those visiting the Bariatric Surgery Clinics and College of Dentistry at The Ohio State

University and College of Dentistry at the University of Jordan and informed consent

obtained. Participants were divided into groups according to their smoking status (smoker

or nonsmoker) and their BMI status (normalweight, overweight or obese). Current

smokers were those who had smoked more than 100 cigarettes in their lifetime (CDC

guidelines). Nonsmokers were defined as those with a lifetime exposure of less than 100

cigarettes and not currently smoking. Normalweight were individuals with BMI<25,

overweight was 25≤BMI<30 and obesity was BMI ≥30. Inclusion criteria were

systemically (ASA I and ASA II) and periodontally healthy subjects (attachment loss ≤ 1,

less than 3 sites with 4mm of probe depths (PD), bleeding on probing (BOP) ≤ 20%,

who were 18 years or older with at least 20 natural teeth in their dentition. Exclusion

criteria for all groups included those who had had antibiotic therapy or professional

cleaning within the previous 3 months, required antibiotic coverage before dental

11

treatment, used immunosuppressant medications, bisphosphonates or steroids, reported a

history of controlled or uncontrolled diabetes or HIV or had fewer than 20 teeth in the

dentition.

Subgingival plaque collection

Comprehensive periodontal examination consisting of clinical attachment levels, plaque

index (PI), gingival index (GI), pocket depth (PD) and bleeding on probing was done on

all participants. Subgingival plaque samples were collected and pooled from 15 mesial

sites on tooth numbers 6,7,8,9,22,23 and 24 using sterile endodontic paper-points (Caulk-

Dentsply, Milford, DE, USA).

DNA isolation and sequencing

Bacterial DNA was isolated using a Qiagen DNA MiniAmp kit (Qiagen, Valencia, CA,

USA) according to instructions. Two regions of the 16S rRNA genes were sequenced as

previously described 56 . Analyses were conducted using the QIIME 57 and PhyloToAST

58. Sequences with an average quality score of 30 over a sliding window of 50bp and

length >200 bp were assigned a taxonomic identity by alignment to the HOMD database

59 using the Blastn algorithm at 99% identity.

Comparative metataxonomic

Both phylogenetic (UniFrac) and non-phylogenetic (Bray–Curtis) distance matrices were

utilized to estimate beta diversity. Linear discriminant analysis (LDA) was performed on

12

distance matrices and on variance-stabilized relative abundances of species-level OTUs.

Significance of clustering was interrogated using Adonis with 999 permutations. The

Bioconductor package for R, DESeq2, was used to test differences in OTU abundances,

and p-values were adjusted for multiple testing (FDR < 0.1, FDR-adjusted Wald Test).

FDR-corrected significance and overlapping pairwise Spearman’s ρ and Kendall-τ

correlations were used to create the graph structures using NetworkX and Gephi. A

correlation value of ≥0.75 and a p-value <0.05 were used.

NMR spectroscopy

Saliva was collected using a methodology described by Navazesh 60. Briefly, subjects

were asked to collect saliva in their mouth for 3 minutes and then continuously drool into

a tube for 3 minutes. This method allowed us to collect unstimulated saliva. NMR

spectroscopy was performed on 500 μl of saliva. Saliva samples were analyzed using the

first increment of NOESY pulse sequence with presaturation and the CPMG pulse

sequence. 1H NMR spectra were acquired at 298K using 128 scans and 64K data points.

2D NMR was applied on selected samples to confirm the identity of the specific

metabolites. All free induction decays (FIDs) were multiplied by a decaying exponential

function with a 1 Hz line broadening factor prior to Fourier transformation. The 1H NMR

spectra were corrected manually for phase and a polynomial fourth-order function was

applied for base-line correction in order to achieve accurate and reproducible

measurements upon integration of the signals of interest. Chemical shifts are reported in

ppm as referenced to TSP (δ = 0). All spectra were processed and analyzed using Topspin

13

3.2. Prior to statistical data analysis, each bucketed region was normalized to the total

sum of the spectral intensities to compensate for the overall concentration differences.

Inflammatory analysis

Gingival crevicular fluid (GCF) samples were collected and pooled from 6 distal sites on

tooth numbers 6,7,8,22,23 and 24 using sterile absorbing paper strips. Levels of

interferon (IFN)-γ, IL-1ß, IL2, IL-4, IL-5, IL-6, IL-10, IL-12 and (TNF)-α as well as

selected adipokines (resisitin, leptin, adipsin, ghrelin, gastro-inhibitory peptide (GIP),

adiponectin, C-peptide, plasminogen activator inhibitor 1 (PAI-1), glucagon like peptide

1 (GLP-1),visfatin, glucagon and insulin) in GCF were determined using a multiplexed

bead-based assay. Assays were carried out according to the manufacturer’s

recommendations using the MAGpix™ instrument (MiraiBio, Alameda, CA) and

concentrations were estimated from the standard curve using a five-parameter polynomial

equation using Xponent® software (Millipore, Corporation, Billerica, MA).

14

Chapter 3: Results

Demographic

183 individuals were included in the cross-sectional study (Table 1); 131 were

nonsmokers. 53 were normalweight (BMI average of 21.5), 54 overweight (BMI average

= 26.8) and 76 were obese (BMI average= 42.1). The age ranged from 19-63 (mean 32.1

years). Mean PI and GI were < 1 and mean PD was < 3 mm for all groups. 121

individuals were females. 22 were African American, 14 Asian, 105 Caucasian, 6

Hispanic and 35 were Middle Eastern (Table 2). 32.3 million sequences were generated.

They represented 579 species-level OTUs.

BMI≥30 is a discriminant of the subgingival microbiome

LDA showed significant group separation between obese and normalweight individuals

with a (p-value =0.026, 0.009, 0.001, Adonis test of weighted UniFrac, unweighted

UniFrac and Bray-Curtis respectively), but no differences could be identified between

either obese and overweight or overweight and normalweight individuals (p-value > 0.05,

Figure 1A). Alpha diversity (as measured by Shannon) was not significantly different

between the 3 groups (p-value > 0.05, Figure 1B). 138 species were significantly

different between obese and normalweight individuals (Figure 1C) , 73 were lower in the

15

obese individuals, notably species related to genera Actinomyces, Alloprevotella,

Capnocytophaga, Cardiobacterium, Enterococcus, Fusobacterium, Gemella,

Haemophilus, Kingella, Leptotrichia, Neisseria, Streptococcus, TM7 and Treponema and

65 species were higher, notably, Atopobium, Bacteroidetes, Dialister, Lactobacillus,

Prevotella, Selenomonas, Stomatobaculum, and Veillonellaceae; constituting 38.1% of

the species.

Subgingival microbiome is influenced by adipokine levels

Multivariate discriminant analysis (Tukey HSD, p-value < 0.05, Table 3) showed a

difference in adipokine and cytokine profiles among individuals based on BMI. Resitin,

leptin, adipsin, TNF- 𝛼,IL-6,andIL-10weresignificantlyhigher,whileGLP-1was

significantlylowerinobeseindividualscomparedtonormalweightindividual.

Higher concentrations of ghrelin, leptin, GIP, adipsin, glucagon and insulin characterized

the profiles of obese individuals, while elevated resistin, adiponectin, C-peptide PAI-1,

visfatin were characteristics of the overweight group. Elevated GLP-1 characterized the

normalweight group. All groups shared similar levels of cytokines such as IL-5, IL-12,

IL-2 and IFN-g, however, elevated TNF- 𝛼 and IL-6 characterized obese group, IL-10

was characteristic of overweight group and IL-4 discriminated the normalweight controls

(Figure 2A-B).

Network analysis revealed a robust, highly connected hub anchored by ghrelin, GIP,

glucagon, insulin, adipsin and IL-5 in obese individuals. Most for the significant

16

correlations for these molecules were with species in the genera Porphyromonas,

Capnocytophaga and Actinomyces. Similar hubs anchored by Ghrelin, GLP-1, C-Peptide

were observed in overweight individuals, Resistin correlated positively with subgingival

community in normalweight individuals (Figure 3 A-C). These adipokines also emerged

as discriminants of the subgingival environment in these individuals. Strong negative

correlations were seen between inflammatory cytokines and bacteria more often in

normalweight and overweight but not obese individuals, suggesting an uncoupled host-

bacterial equilibrium.

Men are from Mars, and women from Venus (unless they are obese)

Subgroup analysis by gender revealed that normalweight and overweight, but not obese,

women are microbially distinct from men in the same categories (p-value=0.001, 0.01

respectively, Adonis of Unweighted Unifrac, p-value=0.007, 0.005 respectively, Adonis

of Bray Curtis Dissimilarity distances), indicating that BMI outweighs gender as a

determinant of the microbiome once BMI is ≥ 30 (Figure 4A). Furthermore, females

exhibited significant group separation between obese and normalwieght and between

obese and overweight females (p-value = 0.019, 0.004, 0.001, Adonis test of weighted

UniFrac, unweighted UniFrac and Bray-Curtis respectively). Alpha diversity (as

measured by Shannon, Ace, Chao 1) was not significantly different once BMI was ≥ 30.

(p-value > 0.05 between obese groups, Figure 4B).

17

84 species out of 529 were significantly different between females and males in

normalweight category (Figure 4C). We noted 46 taxa that were significantly lower in

females, which belonged to genera Actinomyces, Fretibacterium, Peptostreptococcaceae

and Treponema. Species belonging to genera Capnocytophaga, Leptotrichia, Prevotella,

Rothia, Selenomonas, Streptococcus and TM7 were significantly higher contributing to

16.5% of the abundant species.

86 species out of 521 were significantly different between overweight females and males,

(Figure 4D). Overweight females demonstrated significantly lower levels of 42 species

belonging to genera Aggregatibacter, Alloprevotella, Prevotella and Treponema and

higher abundances of species belonging to genera Actinomyces, Capnocytophaga,

Corynebacterium, Haemophilus and Streptococcus contributing to 21.8% of the species.

Interestingly, species that were abundant in normalweight females were found in higher

abundances in overweight males. For example, species of the genera Eikenella,

Leptotrichia, SR1, Provotella, TM7, Actinomyces, Desulfobulbus, Haemophilus, and

Megasphaera were higher in normalweight females when compared to normalweight

males. However, they were higher in overweight males when compared to overweight

females.

Out of the 551 species, 135 were significantly different between normalweight and obese

females (Figure 4E), 70 species were significantly less in obese females, notably species

belonging to genera Alloprevotella, Capnocytophaga, Cardiobacterium, Fusobacterium,

18

Gemella, Leptotrichia, Neisseria, Peptostreptococcaceae and TM7. However, species

belonging to genera Actinomyces, Atopobium, Bifidobacterium, Dialister, Lactobacillus,

Mycoplasma, Prevotella, Selenomonas, Treponema and Veillonellaceae were higher in

this cohort constituting 8.1% of the abundant species.

Smoking: an old story that still holds true

Significant clustering was seen between smokers and nonsmokers as showed by LDA of

weighted UniFrac, unweighted UniFrac and Bray-Curtis with p= 0.001, 0.001, 0.001

respectively, Adonis test) (Figure 5A). Alpha diversity (as measured by Shannon) was

significantly different between the 2 groups (p<0.0001, 0.0003, 0.0008 respectively

(Figure 5B). 244 species were significantly different between nonsmokers and smokers

(Figure 5C); 84 species were lower in abundance in nonsmokers, these species belonged

to genera Bifidobacterium, Dialister, Fretibacterium, Lactobacillus, Mycoplasma,

Prevotella and Treponema. On the other hand, Actinobaculum, Actinomyces, Bergeyella,

Capnocytophaga, Cardiobacterium, Enterococcus, Fusobacterium, Gemella, GN02,

Granulicatella, Haemophilus, Kingella, Lachnospiraceae, Leptotrichia, Neisseria,

Oribacterium, Parvimonas, Porphyromonas, Propionibacterium, Rothia, Selenomonas,

SR1, Streptococcus, Tannerella and TM7 were significantly higher in nonsmokers.

Constituting to 57.6 % of the species.

LDA showed clustering between subgroups; which was significant between female

nonsmoker vs female smoker (weighted UniFrac, unweighted UniFrac and Bray-Curtis

19

with p= 0.005, 0.001, 0.001 respectively) and between male nonsmoker vs male smoker

(weighted UniFrac, unweighted UniFrac and Bray-Curtis with p= 0.032, 0.001, 0.001

respectively, (Figure 6A) . Alpha diversity (as measured by Shannon) was significantly

different between the male smokers and female nonsmokers and between male smokers

and male nonsmokers with p-value <0.00001 (Figure 6B).

From the 562 species between female nonsmoker and female smoker (Figure 6C), 146

were significantly different; 45 species were less in female nonsmoker group notably

species belonging to genera Bifidobacterium, Lactobacillus, Mycoplasma, Prevotella and

Selenomonas. Actinomyces, Aggregatibacter, Bergeyella, Capnocytophaga,

Cardiobacterium, Haemophilus, Leptotrichia, Neisseria, Rothia, SR1, Streptococcus,

Tannerella and TM7 were the species genera higher in female nonsmokers with a relative

abundance of 44.0 % in female nonsmokers.

Out of the 531 species between male nonsmoker and male smoker (Figure 6D), 192 were

significantly different; 58 species were less in male nonsmoker group notably species

belonging to genera Bacteroidaceae, Bifidobacterium, Lactobacillus, Prevotella,

Stomatobaculum and Treponema. Actinomyces, Aggregatibacter, Alloprevotella,

Campylobacter, Capnocytophaga, Cardiobacterium, Enterococcus, Fusobacterium,

Gemella, Granulicatella, Haemophilus, Kingella, Leptotrichia, Neisseria, Parvimonas,

Peptostreptococcaceae, Porphyromonas, Propionibacterium, Rothia, Selenomonas, SR1,

20

Streptococcus, TM7 and Veillonella were the species genera higher in male nonsmokers

with a relative abundance of 10.4% in male nonsmokers.

The subgingival microbiome at the intersection of BMI and smoking

LDA revealed significant group separation between the individuals when they were

grouped according to their BMI and smoking status (Figure 7A); for instance, weighted

UniFrac, unweighted UniFrac and Bray-Curtis were significant between normalweight

nonsmokers and smokers (p= 0.007, 0.001, 0.001 respectively), overweight nonsmokers

and smokers (p= 0.011, 0.002, 0.003 respectively), obese nonsmokers and smokers (p=

0.02, 0.001, 0.001 respectively), normalweight nonsmoker and obese nonsmokers (p=

0.072, 0.036, 0.002 respectively), normalweight smoker and obese smoker (p= 0.039,

0.033, 0.006 respectively), normalweight nonsmoker and obese smoker (p= 0.001, 0.001,

0.001 respectively), and normalweight smoker and obese nonsmoker (p= 0.021, 0.001,

0.001 respectively). Alpha diversity (as measured by Shannon, Ace, Chao 1) was

significantly different between obese smoker and nonsmoker, overweight smoker and

nonsmoker, and between obese smoker and normalweight nonsmoker, with p-value <0.05

(Figure 7B).

144 species out of 529 were significantly different between normalweight nonsmokers

and smokers. 54 species were less in normalweight nonsmoker, such as; Bacteroidetes,

Fretibacterium and Treponema. 90 species were higher, and they belonged to genera

Capnocytophaga, Cardiobacterium, Fusobacterium, Gemella, Leptotrichia, Neisseria,

21

Parvimonas, Propionibacterium, Rothia, Selenomonas, SR1, Streptococcus and TM7. The

relative abundance in normalweight nonsmoker was 51.8%, indicating that they were not

rare taxa, but dominant members of the community.

66 species out of 521 were significantly different between overweight nonsmokers and

smokers. 17 species were less in overweight nonsmoker, such as Leptotrichia,

Mitsuokella, Prevotella and Treponema. 49 species were higher, and they belonged to

genera Actinomyces, Bergeyella, Capnocytophaga, Haemophilus, Neisseria, Parvimonas,

Propionibacterium, Pseudomonas, Rothia and Streptococcus. The relative abundance of

these species in overweight nonsmoker was 27.0%.

183 species out of 550 were significantly different between obese nonsmokers and

smokers. 58 species were less in obese nonsmoker, such as Bifidobacterium,

Lactobacillus, Mycoplasma and Prevotella. 125 species were higher, and they belonged

to genera Actinomyces, Aggregatibacter, Capnocytophaga, Cardiobacterium,

Fretibacterium, Gemella, Granulicatella, Haemophilus, Lachnospiraceae, Leptotrichia,

Neisseria, Parvimonas, Peptostreptococcaceae, Porphyromonas, Propionibacterium,

Rothia, Selenomonas, Streptococcus, Tannerella, TM7 and Veillonella. The relative

abundance of these species in obese nonsmoker was 47.5%, indicating that they were not

rare taxa, but dominant members of the community.

22

116 species out of 551 were significantly different between normalweight and obese

nonsmokers. 58 species were less in normalweight nonsmoker, such as Actinomyces,

Atopobium, Dialister, Fretibacterium, Lachnoanaerobaculum, Megasphaera, Prevotella,

Selenomonas, Treponema and Veillonellaceae. 58 species were higher, and they belonged

to genera Alloprevotella, Capnocytophaga, Cardiobacterium, Enterococcus,

Fusobacterium, Haemophilus, Kingella, Leptotrichia, Neisseria and SR1. Relative

abundance in normalweight nonsmoker was 38.2% indicating that they were not rare

taxa, but dominant members of the community.

102 species out of 503 were significantly different between normalweight and obese

smokers. 53 species were less in normalweight smoker, such as Atopobium,

Bifidobacterium, Dialister, Lactobacillus, Parvimonas, Prevotella and Streptococcus. 48

species were higher, and they belonged to genera Aggregatibacter, Fretibacterium,

Leptotrichia, Neisseria, Tannerella, TM7 and Treponema. Relative abundance in

normalweight smoker was 20.7%.

104 species out of 542 were significantly different between normalweight smoker and

obese nonsmoker. 65 species were less in normalweight smoker, such as Actinomyces,

Leptotrichia, Parvimonas, Prevotella, Rothia, Selenomonas, Streptococcus and TM7. 39

species were higher, and they belonged to genera Neisseria and Treponema. Relative

abundance in normalweight smoker was 16.5%.

23

Smoking thirties: Obese smokers present a distinct microbiome when compared to normalweight nonsmokers

The differences revealed by alpha and beta diversity between normalweight nonsmoker

and obese smoker were driven by the levels of 233 species out of 535 (Figure 7C). Obese

smokers were enriched for putative pathogens belonging to the genera Atopobium,

Bacteroidaceae, Bifidobacterium, Dialister, Lactobacillus, Mycoplasma, Prevotella,

Treponema and Veillonellaceae. Species belonging to genera Actinomyces,

Aggregatibacter, Campylobacter, Capnocytophaga, Cardiobacterium, Eikenella,

Enterococcus, Fusobacterium, Gemella, Granulicatella, Haemophilus, Kingella,

Lachnospiraceae, Leptotrichia, Mycoplasma, Neisseria, Porphyromonas,

Propionibacterium, Selenomonas, SR1, Streptococcus, TM7 and Veillonella were higher

in normalweight nonsmokers. Relative abundance in normalweight nonsmoker was

67.4%.

Metabolic correlations explain microbial differences in BMI-smoking groups

NMR spectroscopy identified the metabolic profile of each individual (Figure 8). Linear

discriminant analysis showed differences in metabolic profiles were identified between

the 6 groups (Figure 9). 31 metabolites were identified in the saliva samples collected.

Formate, lactic acid, phenylalanine, and methanol were the metabolites found to be

significantly different between groups (Tukey HSD, p-value < 0.05, Table 4). Lactic acid

was significantly higher in obese smokers compared to normalwieght nonsmoker (p-

value =0.0401) and obese nonsmoker (p-value = 0.0192) while methanol was

significantly lower in obese smoker compared to normalweight nonsmoker (p-

24

value=0.0165). Phenylalanine was significantly higher in nonsmoker groups

(normalweight nonsmoker vs overweight smoker with p-value= 0.0067, obese nonsmoker

vs overweight smoker with p-value= 0.0065, and overweight nonsmoker vs overweight

smoker with p-value =0.0394). Formate was significantly higher in overweight smoker

compared to overweight nonsmoker (p-value = 0.0283) and normalweight nonsmoker (p-

value=0.0468).

Significant correlations were observed between these metabolites and bacterial

community networks (Figures 10-12). SCFA were undetectable in normalweight

nonsmokers (Figure 10A) but some amino acids correlated to some species with only 6

correlations (Campylobacter gracilis corelated negatively with Lysine, Bergeyella sp.

oral taxon 900 corelated negatively with Glycose, Streptococcus australis corelated

positively with Leucine, Streptococcus mitis corelated positively with Lysine and

Leptotrichia wadei corelated positively with Glutamine (Table 5). On the other hand,

472 significant correlations were identified in normalweight smokers with robust positive

hubs anchored by acetic acid, acetoin, alanine, butyrate, formate, fucose, galactose,

leucine, phenylalanine and tyrosine (Figure 10B); 53.8% were negative correlations

(Table 6). In overweight nonsmoker, 68 correlations were identified (Table 7), of which,

44.1% were negative. Positive correlations were anchored by Acetoin, aspartate, butyrate,

fucose, galactose, glutamine, histidine, leucine and proline (Figure 11A). 159

correlations were identified in overweight smokers (Table 8), of which, 44.7 % were

negative. Positive correlation was anchored by acetic acid, alanine, butyrate,

25

ethanolamine, fucose, galactose, glucose, glutamine, histidine, isoleucine, leucine, lysine,

methanol, propionate and valine. 38 correlation were identified in obese nonsmoker

group (Figure11B) with 52.8% being negative correlations (Table 9). Positive

correlations were anchored by alanine, aspartate, formate, fucose, galactose, leucine,

phenylalanine, propionate sugars polyols, ethyl propionate and proline (Figure 12A). In

obese smokers, 119 correlations were identified and 52.9 % were negative (Table 10).

Robust hubs were anchored by ethanolamine, formate, glucose, lactic acid, methanol,

propylene glycerol, succinic acid, ethyl propionate and proline (Figure 12B).

26

Chapter 4: Discussion

This study had 3 objectives; the first one was to investigate whether obesity poses a

potential risk factor for periodontal disease through its effects on the subgingival

microbiome. A metataxonomic approach was used to characterize the microbiome in

these individuals. The second objective was to identify a correlation between adipokines

levels and the subgingival bacterial community. The last objective was to identify the

effect of smoking and obesity on the subgingival community and to identify the

metabolic correlations between different bacterial taxonomies and metabolites present in

saliva.

There are several indices that can be used to quantify obesity; BMI, waist circumference

(WC), Hip conference (HC), waist to hip ratio (WHR) and body fat percentage (BF%).

These indices were tested to identify which one can be used to correlate between obesity

oral health. Morbidly and mortality studies proved similar significance when using these

indices 61 62. As an indicator for oral health, Ostberg et al., 2012 32 found that both

central and total obesity indices showed a significance, particularly for number of teeth

but not for numbers of carious lesions thus emphasizing careful consideration when

choosing an index. Saxlin et al., 2011 31 found an association between the number of

teeth with pocket depth ≥4mm when BMI, BF% and WC were used. Moreover, BMI,

27

WC, WHR and BF% were significantly associated with increased odds of periodontal

disease or were identified as risk indicator for periodontal disease by some authors 4 26.

Thus, we may conclude that using BMI as an index in our study allowed to identify an

association between obesity and periodontal health/disease.

Maciel et al., 2016 63 studied the influence of obesity on the subgingival community; they

reported that obese individuals with chronic periodontitis had higher levels of several

periodontal pathogens compared to normalweight individuals. These pathogens were

Aggregatibacter actinomycetemcomitans, Eubacterium nodatum, Fusobacterium

nucleatum ss vincentii, Parvimonas micra, Prevotella intermedia, Tannerella forsythia,

Prevotella melaninogenica and Treponema socranskii. The pathogens were present more

in deeper sites of the obese than normalweight individuals. In addition, the shallow

pockets of the obese individuals, whether they had chronic periodontitis or not, harbored

higher proportions of pathogens as compared to shallow pockets of normalweight

individuals. In our study, we noticed the presence of periodontal pathogens in obese,

smokers or obese smokers even though they were periodontally healthy.

The present investigation establishes associations between BMI and subgingival

microbial profiles and provides evidence that these are not simply explained by dietary

differences in obese and nonobese individuals. Although all individuals in this study had

healthy periodontium, the subgingival plaque composition of obese smokers was similar

to that of subjects with periodontal disease; some of these had correlations with SCFA or

28

with amino acids that can be used to form SCFA such as Olsenella uli 64 and

Pseudoramibacter alactolyticus 65 suggesting that this shift in the subgingival

microbiome is moving towards the dysbiosis seen in peridontontitis and risking a future

attachment changes.

With regard to adipokines; several studies have shown comparable concentrations of

different adipokines between obese and non-obese individuals such as adiponectin, leptin,

resistin, vaspin and adpisin IL-1𝛽 , IL-6,IL-8, IL-10 and TNF- 𝛼 66 67 68 69 70. However,

other studies have actually found that lipocaline-2 71 TNF- 𝛼 72 , IL-1𝛽 and IL-8 73

concentrations to be higher in obese individuals. Virto et al., in 2017 74 in an animal study

found a statistically significant difference between high fat diet rats versus normal diet

groups for IL-1𝛽 when periodontitis was induced. Insulin leptin, visfatin, and resistin

were significantly higher in obese rats with periodontitis. However, adiponectin levels

were lower in this group. Thanakakun et al., 2017 75 showed that leptin levels were higher

and adiponectin levels were lower in individuals with BMI ≥ 23.0. In our study, utilizing

multiplexed bead-based assay, we found highly connected hubs anchored by ghrelin,

leptin, adipsin, glucagon, insulin, GIP, IL-2, IL-6 and TNF- 𝛼 in obese individuals.

Adipokines can be produced by the adipose tissue. TNF- 𝛼 and IL-6 are also considered

proinflammatory adipokines. Circulating TNF- 𝛼 levels increase in obese individuals and

it is believed that this increase contributes to poor health due to production in C-reactive

peptide 76. TNF- 𝛼 inhibits adiponectin as well, an important anti-inflammatory

adipokine. Leptin, an adipokine that is secreted by adipocytes, enhances the proliferation

29

and activation of human circulating T lymphocytes and stimulation of cytokine

production, suppresses appetite, increases energy expenditure and increases glucose

uptake in muscle and adipose tissue. Ghrelin is a peptide hormone secreted by the

stomach and other tissues including teeth, salivary glands, immune cells and osteoblasts.

Ghrelin levels were found to be significantly increased in individuals with chronic

periodontitis 77. In our population, Ghrelin was found to be higher in the obese group as

well. Resistin, a hormone derived from adipocytes, plays a detrimental role in the

development of insulin resistance. Resistin levels were found to be higher in GCF

samples of overweight or obese individuals with periodontitis 78. In our study, resistin

characterized the adipokine profiles of overweight individuals. Adipsin, also known as

complement factor D initiates complement activation via the alternative pathway.

Schenkein in 1991 79 proposed that P. gingivalis protease is able to activate the

alternative pathway by mimicking complement factor D.

It has long been a question to whether gender is associated with periodontal disease or

not. It is known that the percentage of males with periodontal disease is greater than the

percentage of females. However, the mean attachment loss per year between males and

females 80 is not significantly different. Chaffee and Wetson 2010 44 found the association

between periodontal disease and obesity to be stronger in women, in nonsmokers, and in

younger individuals. Dalla Vecchia et al., 2005 81 observed a modification in the

association between obesity and periodontal disease due to gender; they concluded no

association between obesity and periodontitis in males while obese females had higher

30

likelihood of periodontitis than normalweight females. A five-year follow up of Dalla

Vechhia et al study, Gaio et al., 2016 27 revealed a significant progression of attachment

loss among obese females compared to normalweight females with 64% higher risk of

experiencing attachment loss progression. Again, this was not significant among males.

Suvan et al., 2015 28 found the odds ratio to have periodontitis to be ~ 2 in obese

compared to normalweight females and ~3 in obese nonsmoker females. Analysis

showed no such a significant correlation among males. Silva-Boghossian et al., 2018 82

found Porphyromonas gingivalis and Leptotrichia buccalis to be significantly higher in

obese females compared to normalweight. Our analysis revealed that Leptotrichia

buccalis is significantly lower in obese females while no significant difference was found

for Porphyromonas gingivalis. These observations may be explained by two factors: one

is the difference in methods for microbial characterization, and the other is that sub-group

analysis of our data based on males and females revealed that the subgingival

microbiome is significantly different between normalweight and obese females and such

difference was not noticeable between males. These species were again SCFA producing

such as Lactobacillus, Prevotella and Selenomonas. Another observation was that even

between the same BMI category, there was a difference in species levels. This difference

was no longer present once the individual was categorized as obese. Providing an

evidence that gender can be a determinant for subgingival bacteria but is outweighed by

obesity at BMI≥30.

31

SCFA are the products of the bacterial anaerobic fermentation. The glycolysis pathway

starts with glucose 6-phosphate and produces pyruvate and ATP; pyruvate can be

metabolized into lactate, acetate, ethanol, and formate. Streptococcus, Actinomyces, and

Lactobacillus are saccharolytic oral bacteria that share this pathway 83. Free fatty acids in

plasma were found to be higher in obese with periodontitis suggesting an association 74.

In the gut, most of these SCFA are beneficial; they can be a source of energy, they

regulate gene expression and recently, Louis et al 2014 84 pointed out that SCFA can also

regulate immune cell development and act as anti-inflammatory molecules. The story is

however different in the oral cavity. Niederman et al., (1996,1997) 85 86 cross-sectionally

examined human subjects with and without periodontal disease, both on subject and site

levels showing a significant correlation between SCFA and gingival inflammation.

Butyrate and propionate effect on the periodontium has been extensively studied in vitro;

They are believed to induce reactive oxygen production, suppress proliferation of

endothelial cells, gingival epithelial cells and fibroblasts proliferation, induce epithelia

cell apoptosis, inhibit leukocyte apoptosis and cytokines release 86 87 88 89 90 91. While in

the large intestine, butyrate and propionate act as an anticarcinogenic and anti-

inflammatory SCFA 92 93 94 95.

Lactic acid is an end product of the glycolytic cascade. Macfarlane and Gibson in 1995 96

have shown that lactate and acetate is produced by Actinomyces, Bacteroides, Eubacteria,

32

and Fusobacteria. Butyrate is produced by Eubacteria, Fusobacteria and Peptococci and

formate by Lactobacilli.

Propionate and butyrate can be produced from peptides and amino acids fermentation. In

vitro study revealed that propionate was formed form aspartate, alanine, threonine and

methionine and that butyrate was formed from glutamate, lysine, histidine, cysteine,

serine and methionine 97. Common sugar metabolic pathways are shared by periodontal

pathogens such as Fusobacterium and Prevotella, which can be utilized to produce SCFA

98. Fusobacterium was found in the gut to produce butyrate by utilizing lysine 99.

Veillonella species utilize lactate and oxaloacetate producing pyruvate and succinate,

respectively 100. Pyruvate is then converted into acetate and formate, and succinate into

propionate. Porphyromonas gingivalis utilize glutamate to produce butyrate and

propionate 101.The subgingival community is unique in how they can interact with each

other in a synergism and antagonism. This relationship is also evident when amino acids

are present or absent. For example, it was found that coaggregation between F. nucleatum

and P. gingivalis in the presence of galactose was reduced by 58% and by 46% in the

presence of lactose 102.

We thus provide the first evidence that subgingival adipokine and metabolites play

important roles in shaping this ecosystem in obese individuals. The significant

differences in their levels between normalweight and obese individuals were attributable

33

to some SCFA producing bacteria that were present in higher levels in obese, namely

Lactobacillus.

34

Chapter 5: Summary and Conclusions

In summary, by combining a metataxonomic approach with targeted proteomics and

untargeted metabolomics, we report the following:

1. BMI ≥30 is significantly associated with a dysbiotic subgingival microbiome.

2. These dysbiotic shifts correlate strongly with subgingival adipokine levels.

3. These dysbiotic shifts also correlate strongly with increased levels of pro-

inflammatory cytokines in the gingival crevicular fluid.

4. There are critical gender-based differences in the association between BMI and

microbial community assemblages, these differences mimic the epidemiology of

periodontitis in males and females.

5. Short chain fatty acids of bacterial origin were also evident at higher levels in obese

individuals and correlated with levels of bacteria known to produce SCFA.

6. Smoking is associated with a dysbiotic subgingival microbiome, however, the

community assemblages are different in smokers and obese individuals

7. Obese smokers have a microbiome that is substantially different from both

normalweight smokers and obese nonsmokers, indicating that when the two factors

intersect, the whole is greater than the sum of the parts.

35

These studies have important implications for clinical therapeutics and biomarker

discovery. To the best of our knowledge, this is the first study reporting on the divergent

effects of two pro-inflammatory influences on the subgingival microbiome, underscoring

the need for developing a personalized risk assessment strategy that assigns differential

weightage to each risk factor. It also underscores the importance of integrating microbial

community-level metrics into periodontal risk assessments, rather than targeting

individual species

36

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Appendix A: Tables

Table 1 Demographic, biometric and clinical characteristics.

Control BMI <25

Overweight ≥ 𝟐𝟓, <30

Obese ≥ 𝟑𝟎

Total

Fisher's exact test

(n=53) (n= 54) (n= 76) (p-value)

Demographics

Mean age (years) 25.8 31.1 38 / /

Female (n) 37 35 49 121 0.0029*

Nonsmoker (n) 40 38 53 131 0.765

Biometric Characteristics

BMI (Mean) 21.5 26.8 42.1 / /

Clinical characteristics

Mean PI 0.14 0.46 0.37 / 0.9618

Mean GI 0.14 0.49 0.8 / 0.2997

Mean PD (mm) 2.15 2.15 2.35 /

* Significant p value PI=plaque index, GI=gingival index, PD=pocket depth

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Table 2 Ethnicity of individuals participating in the study.

Ethnicity Total

African American 22

Asian 14 Caucasian 105 Hispanic 6 Middle eastern 35 Total 182

Table 3 Significant difference in adipokines and cytokines levels between BMI groups.

Cytokine Level - Level Difference Std Err Dif P-value Resistin Obese Normalweight 0.0807584 0.0235475 0.0033 Leptin Obese Normalweight 0.8370301 0.2658139 0.0074 TNFa Obese Normalweight 0.009696 0.0033212 0.0139 IL-6 Obese Normalweight 0.0327602 0.0131399 0.0411 IL-10 Obese Normalweight 0.1117162 0.0455926 0.0455 Adipsin Obese Normalweight 0.0387214 0.0158874 0.0469 GLP-1 Normalweight Obese 0.0700006 0.0288383 0.048

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Table 4 Significant difference in metabolomic concentrations between BMI-smoking groups.

Metabolite Level - Level Difference Std Err Dif p-Value

Formate Overweight smoker

Overweight nonsmoker

0.0016779 0.0005401 0.0283281

Formate Overweight smoker

Normalweight nonsmoker

0.0014413 0.0004928 0.0468407

Lactic acid Obese smoker Obese nonsmoker

0.0284503 0.0087827 0.0192131

Lactic acid Obese smoker Normalweight nonsmoker

0.0249012 0.0083518 0.0401481

Methanol Normalweight nonsmoker

Obese smoker 0.0010662 0.0003240 0.0164726

Phenyl-alanine

Normalweight nonsmoker

Overweight smoker

0.0027836 0.0007786 0.00672778

Phenyl-alanine

Obese nonsmoker

Overweight smoker

0.0029342 0.0008187 0.00654446

Pheny-lalanine

Overweight nonsmoker

Overweight smoker

0.0025502 0.0008534 0.03941315

Table 5 Subgingival bacteria and metabolomes network in normalweight nonsmoker individuals.

Metabolite Taxonomy Correlation P-value

Lysine Campylobacter gracilis -0.654282536 8.78415E-05 Glycose Bergeyella sp. oral taxon 900 -0.640338561 0.000138281 Leucine Streptococcus australis 0.615681724 0.00029277 Lysine Streptococcus mitis 0.628921023 0.000197268 Glutamine Leptotrichia wadei 0.680685557 3.48284E-05

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Table 6 Subgingival bacteria and metabolomes network in normalweight smoker individuals.

Metabolite Taxonomy Correlation P-value Acetic acid Aggregatibacter sp. oral taxon 898 -0.668588526 0.012476478 Acetic acid Parvimonas sp. oral taxon 110 -0.651528814 0.01584155 Acetic acid Lactococcus lactis -0.628970902 0.021283066 Acetic acid Actinomyces naeslundii -0.616065063 0.024960997 Acetic acid Prevotella sp. oral taxon 306 0.621717036 0.023296787 Acetic acid Shuttleworthia satelles 0.628970902 0.021283066 Acetic acid Actinomyces israelii 0.631868132 0.020516043 Acetic acid Oribacterium sinus 0.635867064 0.019491365 Acetic acid Prevotella salivae 0.679731668 0.010590347 Acetic acid Eggerthia catenaformis 0.731494203 0.004487683 Acetic acid Streptococcus salivarius 0.731775108 0.004464581 Acetic acid Johnsonella sp. oral taxon 166 0.751764017 0.003041751 Acetic acid Erysipelotrichaceae [G-1] sp. oral

taxon 904 0.801881618 0.00097669

Acetoin Prevotella sp. oral taxon 942 0.609349723 0.027050591 Acetoin Streptococcus mutans 0.609724736 0.026930616 Acetoin Lactobacillus gasseri 0.628970902 0.021283066 Acetoin Neisseria bacilliformis 0.635368813 0.019616911 Acetoin Prevotella sp. oral taxon 820 0.638999414 0.018715757 Acetoin Streptococcus tigurinus 0.67032967 0.01216622 Alanine Capnocytophaga sp. oral taxon 902 0.631868132 0.020516043 Alanine Lachnoanaerobaculum orale 0.646302241 0.016997058 Aspartate TM7 [G-3] sp. oral taxon 351 -0.704832551 0.007136094 Aspartate Streptococcus sp. oral taxon 066 -0.650220741 0.016125043 Aspartate Streptococcus oligofermentans -0.620879121 0.023538167 Aspartate TM7 [G-1] sp. oral taxon 347 0.615658601 0.025083971 Aspartate Fusobacterium nucleatum subsp.

vincentii 0.692307692 0.008730318

Butyrate SR1 [G-1] sp. oral taxon 345 -0.679719965 0.010592207 Butyrate Leptotrichia sp. oral taxon 879 -0.620235195 0.023724919 Butyrate Streptococcus gordonii 0.60989011 0.026877834 Butyrate Prevotella histicola 0.615384615 0.025167117 Butyrate Veillonella atypica 0.620879121 0.023538167 Butyrate Neisseria flavescens 0.632737875 0.020289849 Butyrate Fretibacterium sp. oral taxon 361 0.770558117 0.002051945 Butyrate Veillonella dispar 0.774725275 0.001871362

50

Butyrate Veillonella parvula 0.807692308 0.00083916 Butyrate Streptococcus tigurinus 0.818681319 0.000620988 Ethanol Veillonella rogosae -0.880330957 7.15568E-05 Ethanol Johnsonella ignava -0.852820614 0.000211317 Ethanol Actinomyces johnsonii -0.796703297 0.001113653 Ethanol Capnocytophaga gingivalis -0.741758242 0.003701314 Ethanol Streptococcus sp. oral taxon 056 -0.74040847 0.003798131 Ethanol Corynebacterium durum -0.703296703 0.00731857 Ethanol Propionibacterium sp. oral taxon

194 -0.695381715 0.008316154

Ethanol Neisseria flava -0.692886237 0.008651196 Ethanol Prevotella sp. oral taxon 475 -0.66693282 0.012777015 Ethanol Leptotrichia sp. oral taxon 212 -0.642857143 0.017792451 Ethanol Aggregatibacter paraphrophilus -0.626801743 0.021871129 Ethanol Fusobacterium nucleatum subsp.

polymorphum -0.626373626 0.021988602

Ethanol Streptococcus sp. oral taxon 064 -0.626373626 0.021988602 Ethanol Capnocytophaga sputigena -0.615384615 0.025167117 Ethanol Prevotella loescheii -0.613480635 0.025750543 Ethanol Propionibacterium acnes -0.610808263 0.026586175 Ethanol Neisseria sp. oral taxon 018 -0.609349723 0.027050591 Ethanol Porphyromonas sp. oral taxon 284 -0.607744082 0.027568723 Ethanol Cardiobacterium hominis -0.604395604 0.028672706 Ethanol Eikenella corrodens -0.604395604 0.028672706 Ethanol Lachnospiraceae [G-8] sp. oral

taxon 500 0.602219136 0.029407481

Ethanol Leptotrichia hofstadii 0.613269028 0.025815995 Ethanol Prevotella sp. oral taxon 526 0.621283499 0.023421447 Ethanol Mogibacterium timidum 0.627235807 0.0217525 Ethanol Fretibacterium sp. oral taxon 360 0.631868132 0.020516043 Ethanol Fretibacterium fastidiosum 0.637362637 0.019118114 Ethanol Olsenella sp. oral taxon 807 0.640893759 0.018258008 Ethanol Dialister invisus 0.664835165 0.013165576 Ethanol Prevotella oralis 0.738233164 0.003958271 Ethanolamine Ottowia sp. oral taxon 894 -0.824295696 0.000528367 Ethanolamine Selenomonas sp. oral taxon 892 -0.771759939 0.001998527 Ethanolamine Stenotrophomonas maltophilia -0.609349723 0.027050591 Ethanolamine Capnocytophaga sp. oral taxon 380 0.605634176 0.028260636 Ethanolamine Lactococcus lactis 0.628970902 0.021283066 Ethanolamine Fusobacterium sp. oral taxon 203 0.703296703 0.00731857

51

Ethyl Propionate and proline

Prevotella sp. oral taxon 317 -0.846153846 0.000266012

Ethyl Propionate and proline

Prevotella sp. oral taxon 315 -0.810663587 0.000774999

Ethyl Propionate and proline

Prevotella maculosa -0.708791209 0.006681784

Ethyl Propionate and proline

Oribacterium sp. oral taxon 078 -0.686813187 0.009509496

Ethyl Propionate and proline

Peptococcus sp. oral taxon 168 -0.674003389 0.01153074

Ethyl Propionate and proline

Selenomonas sp. oral taxon 134 -0.664835165 0.013165576

Ethyl Propionate and proline

Peptostreptococcaceae [XI][G-1] [Eubacterium] infirmum

-0.638672955 0.018795499

Ethyl Propionate and proline

Mitsuokella sp. oral taxon 521 -0.620205314 0.023733611

Ethyl Propionate and proline

Capnocytophaga sp. oral taxon 336 -0.618793975 0.024146872

Ethyl Propionate and proline

Actinomyces sp. oral taxon 897 -0.61623167 0.02491072

Ethyl Propionate and proline

Peptostreptococcaceae [XI][G-9] [Eubacterium] brachy

0.653846154 0.015348517

Formate GN02 [G-1] sp. oral taxon 872 -0.637540819 0.019074003 Formate Prevotella loescheii -0.627235807 0.0217525 Formate Treponema sp. oral taxon 517 0.61041309 0.026711418 Formate Dialister micraerophilus 0.620235195 0.023724919 Formate Prevotella sp. oral taxon 396 0.628970902 0.021283066 Formate Prevotella multiformis 0.639127822 0.018684461 Formate Bacteroidetes [G-3] sp. oral taxon

365 0.645100979 0.017271317

Formate Lachnoanaerobaculum sp. oral taxon 089

0.654661164 0.015177884

Formate Peptostreptococcaceae [XI][G-3] sp. oral taxon 495

0.657673687 0.014559509

Formate Prevotella dentalis 0.683888739 0.009945171 Formate Solobacterium moorei 0.708791209 0.006681784 Formate Treponema sp. oral taxon 246 0.734698337 0.004229568 Fucose Neisseria oralis 0.607301244 0.0277129 Fucose Porphyromonas pasteri 0.607978567 0.027492604 Fucose Eikenella sp. oral taxon 011 0.61306527 0.025879135 Fucose Bergeyella sp. oral taxon 907 0.616065063 0.024960997 Fucose SR1 [G-1] sp. oral taxon 875 0.620205314 0.023733611

52

Fucose GN02 [G-1] sp. oral taxon 871 0.620235195 0.023724919 Fucose Leptotrichia sp. oral taxon 217 0.627181508 0.021767314 Fucose Peptostreptococcaceae [XI][G-7]

[Eubacterium] yurii subsps. yurii & margaretiae

0.637362637 0.019118114

Fucose Neisseria flava 0.645100979 0.017271317 Fucose Porphyromonas sp. oral taxon 284 0.646418706 0.016970643 Fucose Leptotrichia sp. oral taxon 225 0.651943652 0.015752429 Fucose Cardiobacterium valvarum 0.653846154 0.015348517 Fucose Leptotrichia sp. oral taxon 212 0.659340659 0.014225579 Fucose Leptotrichia sp. oral taxon 392 0.662999252 0.013512902 Fucose Leptotrichia sp. oral taxon 219 0.667190565 0.012729875 Fucose Fusobacterium periodonticum 0.675824176 0.011225246 Fucose Lachnoanaerobaculum orale 0.679731668 0.010590347 Fucose Prevotella sp. oral taxon 472 0.701668168 0.00751593 Fucose TM7 [G-1] sp. oral taxon 346 0.71978022 0.005536116 Fucose Propionibacterium propionicum 0.741758242 0.003701314 Fucose Streptococcus dentisani 0.741758242 0.003701314 Galctose Prevotella sp. oral taxon 306 0.61041309 0.026711418 Galctose Neisseria flava 0.615235193 0.025212547 Galctose Bacteroides zoogleoformans 0.620235195 0.023724919 Galctose Prevotella veroralis 0.626373626 0.021988602 Galctose Cardiobacterium valvarum 0.631868132 0.020516043 Galctose Streptococcus salivarius 0.638239944 0.018901659 Galctose Scardovia wiggsiae 0.645264114 0.017233879 Galctose Streptococcus infantis 0.663020451 0.013508852 Galctose Selenomonas sputigena 0.664628859 0.013204266 Galctose Leptotrichia sp. oral taxon 223 0.693525766 0.008564371 Galctose Atopobium parvulum 0.745540199 0.003440183 Glucose Peptococcus sp. oral taxon 168 -0.701513731 0.007534855 Glucose Streptococcus sp. oral taxon 431 -0.638621643 0.018808056 Glucose Prevotella sp. oral taxon 315 -0.635159099 0.019669933 Glucose Peptostreptococcaceae [XI][G-9]

[Eubacterium] brachy 0.620879121 0.023538167

Glutamine Actinomyces radicidentis -0.736263736 0.004107709 Glutamine Johnsonella sp. oral taxon 166 -0.685984665 0.009631424 Glutamine Selenomonas sp. oral taxon 442 -0.67032967 0.01216622 Glutamine Lachnoanaerobaculum sp. oral

taxon 083 -0.644401025 0.01743264

Glutamine Treponema pectinovorum -0.642789647 0.017808307

53

Glutamine Leptotrichia goodfellowii 0.609724736 0.026930616 Glutamine Streptococcus lactarius 0.60989011 0.026877834 Glutamine Streptococcus mitis 0.697802198 0.008000717 Glycose Prevotella salivae -0.819020944 0.000615045 Glycose Selenomonas noxia -0.734526142 0.004243141 Glycose Johnsonella sp. oral taxon 166 -0.698514065 0.007909707 Glycose Oribacterium sinus -0.698514065 0.007909707 Glycose Prevotella sp. oral taxon 306 -0.689540712 0.009116373 Glycose Prevotella denticola -0.629843867 0.021049749 Glycose Eggerthia catenaformis -0.617407951 0.024557876 Glycose Leptotrichia goodfellowii 0.655959787 0.014908948 Glycose Actinomyces naeslundii 0.689540712 0.009116373 Histidine Clostridiales [F-1][G-1] sp. oral

taxon 093 -0.743804735 0.003558176

Histidine Actinobaculum sp. oral taxon 848 -0.72151845 0.005369704 Histidine Desulfobulbus sp. oral taxon 041 -0.72151845 0.005369704 Histidine Tannerella forsythia -0.659340659 0.014225579 Histidine Fusobacterium nucleatum subsp.

animalis -0.653846154 0.015348517

Histidine Treponema maltophilum -0.642857143 0.017792451 Histidine SR1 [G-1] sp. oral taxon 345 -0.632734714 0.020290668 Histidine TM7 [G-5] sp. oral taxon 356 -0.629843867 0.021049749 Histidine Treponema socranskii -0.626373626 0.021988602 Histidine Neisseria bacilliformis 0.602219136 0.029407481 Histidine Streptococcus oralis 0.648351648 0.016536699 Histidine Streptococcus pneumoniae 0.659340659 0.014225579 Isoleucine Campylobacter showae -0.862637363 0.000147448 Isoleucine SR1 [G-1] sp. oral taxon 345 -0.798749268 0.001057846 Isoleucine Actinobaculum sp. oral taxon 848 -0.752162091 0.003017545 Isoleucine Fusobacterium nucleatum subsp.

animalis -0.631868132 0.020516043

Isoleucine Actinomyces meyeri -0.607744082 0.027568723 Isoleucine Prevotella pallens 0.604395604 0.028672706 L Glutamic acid Actinomyces israelii -0.873626374 9.5289E-05 L Glutamic acid Actinomyces cardiffensis -0.842143985 0.000303973 L Glutamic acid Treponema vincentii -0.836492012 0.000364655 L Glutamic acid Peptostreptococcaceae [XI][G-2]

sp. oral taxon 091 -0.825188066 0.000514712

L Glutamic acid Clostridiales [F-1][G-1] sp. oral taxon 093

-0.819020944 0.000615045

54

L Glutamic acid Peptostreptococcaceae [XI][G-6] [Eubacterium] nodatum

-0.802580173 0.000959279

L Glutamic acid Filifactor alocis -0.802197802 0.000968779 L Glutamic acid Desulfobulbus sp. oral taxon 041 -0.785591518 0.001458406 L Glutamic acid Veillonellaceae [G-1] sp. oral taxon

150 -0.779017414 0.001698588

L Glutamic acid Anaeroglobus geminatus -0.77854269 0.001717059 L Glutamic acid TM7 [G-5] sp. oral taxon 356 -0.767967522 0.002170877 L Glutamic acid Prevotella micans -0.753783382 0.002920496 L Glutamic acid Mogibacterium timidum -0.737277176 0.004030276 L Glutamic acid Fretibacterium sp. oral taxon 360 -0.730769231 0.004547734 L Glutamic acid Treponema sp. oral taxon 257 -0.718732665 0.0056383 L Glutamic acid Treponema denticola -0.718019936 0.005708648 L Glutamic acid Actinomyces radicidentis -0.708791209 0.006681784 L Glutamic acid Fretibacterium fastidiosum -0.708791209 0.006681784 L Glutamic acid Peptostreptococcaceae [XI][G-7]

sp. oral taxon 081 -0.699305148 0.007809499

L Glutamic acid Actinomyces meyeri -0.690618275 0.008964517 L Glutamic acid Treponema socranskii -0.67032967 0.01216622 L Glutamic acid Peptostreptococcaceae [XI][G-4]

sp. oral taxon 369 -0.665750286 0.012994983

L Glutamic acid Treponema maltophilum -0.664835165 0.013165576 L Glutamic acid Prevotella buccae -0.662993544 0.013513992 L Glutamic acid Alloprevotella tannerae -0.659340659 0.014225579 L Glutamic acid Mycoplasma faucium -0.651074136 0.015939663 L Glutamic acid Selenomonas sp. oral taxon 134 -0.648351648 0.016536699 L Glutamic acid Veillonellaceae [G-1] sp. oral taxon

148 -0.647290715 0.016773835

L Glutamic acid Prevotella denticola -0.646418706 0.016970643 L Glutamic acid Treponema sp. oral taxon 270 -0.645264114 0.017233879 L Glutamic acid Actinomyces sp. oral taxon 414 -0.640893759 0.018258008 L Glutamic acid Bacteroidetes [G-3] sp. oral taxon

280 -0.638672955 0.018795499

L Glutamic acid Atopobium rimae -0.638239944 0.018901659 L Glutamic acid Actinomyces sp. oral taxon 172 -0.635358553 0.019619502 L Glutamic acid Treponema sp. oral taxon 517 -0.633020982 0.020216623 L Glutamic acid Fretibacterium sp. oral taxon 362 -0.631868132 0.020516043 L Glutamic acid Dialister micraerophilus -0.628970902 0.021283066 L Glutamic acid Treponema medium -0.627235807 0.0217525 L Glutamic acid Treponema sp. oral taxon 247 -0.627181508 0.021767314

55

L Glutamic acid Peptostreptococcaceae [XI][G-4] sp. oral taxon 103

-0.626801743 0.021871129

L Glutamic acid Prevotella sp. oral taxon 300 -0.626373626 0.021988602 L Glutamic acid Prevotella sp. oral taxon 396 -0.620235195 0.023724919 L Glutamic acid TM7 [G-1] sp. oral taxon 349 -0.618982704 0.024091304 L Glutamic acid Prevotella salivae -0.615658601 0.025083971 L Glutamic acid Fusobacterium nucleatum subsp.

animalis -0.604395604 0.028672706

L Glutamic acid Treponema sp. oral taxon 237 -0.602476498 0.029319883 L Glutamic acid Streptococcus sanguinis 0.626373626 0.021988602 L Glutamic acid Actinomyces massiliensis 0.631868132 0.020516043 L Glutamic acid Streptococcus mitis 0.631868132 0.020516043 L Glutamic acid Streptococcus oralis 0.637362637 0.019118114 L Glutamic acid Streptococcus lactarius 0.659340659 0.014225579 L Glutamic acid Lautropia mirabilis 0.71978022 0.005536116 L Glutamic acid Aggregatibacter sp. oral taxon 898 0.729875807 0.004622587 L Glutamic acid Haemophilus sp. oral taxon 036 0.812025675 0.000746912 L Glutamic acid Actinomyces naeslundii 0.842143985 0.000303973 Lactic acid Actinomyces johnsonii -0.879120879 7.54456E-05 Lactic acid Veillonella rogosae -0.855571648 0.000191546 Lactic acid Capnocytophaga leadbetteri -0.774725275 0.001871362 Lactic acid Corynebacterium durum -0.752747253 0.002982233 Lactic acid Capnocytophaga gingivalis -0.747252747 0.003326741 Lactic acid Streptococcus sp. oral taxon 056 -0.734756497 0.004224991 Lactic acid Neisseria elongata -0.731775108 0.004464581 Lactic acid Cardiobacterium hominis -0.692307692 0.008730318 Lactic acid Neisseria meningitidis -0.677806555 0.010899646 Lactic acid Johnsonella ignava -0.674003389 0.01153074 Lactic acid Rothia aeria -0.64073067 0.018297085 Lactic acid Leptotrichia sp. oral taxon 212 -0.637362637 0.019118114 Lactic acid Streptococcus sp. oral taxon 423 -0.631868132 0.020516043 Lactic acid Aggregatibacter paraphrophilus -0.626801743 0.021871129 Lactic acid Alloprevotella sp. oral taxon 914 -0.624318921 0.022558931 Lactic acid Leptotrichia sp. oral taxon 392 -0.61623167 0.02491072 Lactic acid SR1 [G-1] sp. oral taxon 874 -0.615658601 0.025083971 Lactic acid Streptococcus sp. oral taxon 064 -0.615384615 0.025167117 Lactic acid Bergeyella sp. oral taxon 322 -0.60989011 0.026877834 Lactic acid Fusobacterium nucleatum subsp.

polymorphum -0.60989011 0.026877834

Lactic acid Prevotella pleuritidis 0.604395604 0.028672706

56

Lactic acid Dialister invisus 0.615384615 0.025167117 Lactic acid Fretibacterium fastidiosum 0.642857143 0.017792451 Leucine Prevotella intermedia -0.674003389 0.01153074 Leucine Capnocytophaga granulosa 0.615384615 0.025167117 Leucine Neisseria lactamica 0.623337664 0.022835136 Leucine Porphyromonas pasteri 0.635488909 0.019586595 Leucine Neisseria oralis 0.651873812 0.015767406 Leucine Kingella sp. oral taxon 012 0.683888739 0.009945171 Leucine Peptostreptococcaceae [XI][G-7]

[Eubacterium] yurii subsps. yurii & margaretiae

0.785714286 0.00145419

Lysine Megasphaera sp. oral taxon 123 -0.672584793 0.011773079 Lysine Fusobacterium nucleatum subsp.

vincentii -0.637362637 0.019118114

Lysine Streptococcus sp. oral taxon 431 0.601055664 0.02980589 Lysine Neisseria flava 0.615235193 0.025212547 Lysine Alloprevotella sp. oral taxon 308 0.618793975 0.024146872 Lysine Prevotella salivae 0.626801743 0.021871129 Lysine Prevotella histicola 0.637362637 0.019118114 Lysine Treponema sp. oral taxon 269 0.642131764 0.017963407 Lysine Actinomyces johnsonii 0.642857143 0.017792451 Lysine Leptotrichia sp. oral taxon 225 0.657468598 0.014600997 Lysine Selenomonas noxia 0.665750286 0.012994983 Lysine Prevotella melaninogenica 0.67032967 0.01216622 Lysine Streptococcus sp. oral taxon 056 0.678236766 0.01082994 Lysine Johnsonella ignava 0.682256491 0.010194832 Lysine Bergeyella sp. oral taxon 907 0.712148605 0.006314124 Lysine Prevotella sp. oral taxon 306 0.712148605 0.006314124 Lysine Veillonella rogosae 0.748281313 0.003260015 Methanol Prevotella sp. oral taxon 315 -0.752162091 0.003017545 Methanol Peptococcus sp. oral taxon 168 -0.751032347 0.003086636 Methanol Streptococcus constellatus -0.692307692 0.008730318 Methanol Streptococcus sp. oral taxon 431 -0.641511334 0.0181106 Methanol Prevotella sp. oral taxon 317 -0.631868132 0.020516043 Methanol Campylobacter gracilis -0.604395604 0.028672706 Methanol Propionibacterium propionicum 0.620879121 0.023538167 Methanol Fusobacterium periodonticum 0.714285714 0.006088305 Phenylalanine Fusobacterium nucleatum subsp.

animalis -0.714285714 0.006088305

Phenylalanine Tannerella forsythia -0.703296703 0.00731857

57

Phenylalanine Desulfobulbus sp. oral taxon 041 -0.702017952 0.007473202 Phenylalanine Campylobacter showae -0.697802198 0.008000717 Phenylalanine SR1 [G-1] sp. oral taxon 345 -0.685984665 0.009631424 Phenylalanine Filifactor alocis -0.675824176 0.011225246 Phenylalanine Treponema socranskii -0.626373626 0.021988602 Phenylalanine Tannerella sp. oral taxon 916 -0.618793975 0.024146872 Phenylalanine Capnocytophaga sputigena 0.60989011 0.026877834 Phenylalanine Catonella morbi 0.60989011 0.026877834 Phenylalanine Capnocytophaga sp. oral taxon 324 0.624318921 0.022558931 Phenylalanine Porphyromonas pasteri 0.624484772 0.022512492 Phenylalanine Gemella sanguinis 0.626373626 0.021988602 Phenylalanine Prevotella scopos 0.626373626 0.021988602 Phenylalanine TM7 [G-1] sp. oral taxon 952 0.626373626 0.021988602 Phenylalanine TM7 [G-4] sp. oral taxon 355 0.62706288 0.021799703 Phenylalanine Streptococcus gordonii 0.631868132 0.020516043 Phenylalanine Enterococcus italicus 0.633020982 0.020216623 Phenylalanine Veillonella sp. oral taxon 780 0.633020982 0.020216623 Phenylalanine Capnocytophaga sp. oral taxon 380 0.650220741 0.016125043 Phenylalanine Fusobacterium sp. oral taxon 203 0.653846154 0.015348517 Phenylalanine Neisseria oralis 0.682517453 0.010154601 Phenylalanine Granulicatella elegans 0.696011663 0.008233163 Phenylalanine Streptococcus pneumoniae 0.708791209 0.006681784 Phenylalanine Kingella sp. oral taxon 012 0.774320308 0.001888345 Proline Actinomyces radicidentis -0.785714286 0.00145419 Proline Treponema vincentii -0.746060443 0.003405409 Proline Actinomyces sp. oral taxon 172 -0.717100589 0.005800382 Proline Actinomyces cardiffensis -0.712148605 0.006314124 Proline Peptostreptococcaceae [XI][G-2]

sp. oral taxon 091 -0.700844659 0.007617265

Proline Actinomyces israelii -0.697802198 0.008000717 Proline Prevotella sp. oral taxon 301 -0.691228467 0.008879383 Proline TM7 [G-5] sp. oral taxon 356 -0.679568383 0.01061632 Proline Actinomyces sp. oral taxon 897 -0.66850132 0.012492173 Proline Johnsonella sp. oral taxon 166 -0.667190565 0.012729875 Proline Treponema sp. oral taxon 257 -0.651873812 0.015767406 Proline Prevotella salivae -0.632373314 0.020384433 Proline Selenomonas sp. oral taxon 442 -0.631868132 0.020516043 Proline Prevotella micans -0.624484772 0.022512492 Proline Treponema sp. oral taxon 230 -0.623337664 0.022835136

58

Proline Peptostreptococcaceae [XI][G-7] sp. oral taxon 081

-0.621283499 0.023421447

Proline Treponema denticola -0.618982704 0.024091304 Proline Clostridiales [F-1][G-1] sp. oral

taxon 093 -0.618444386 0.024250054

Proline Capnocytophaga granulosa -0.615384615 0.025167117 Proline Peptostreptococcaceae [XI][G-4]

sp. oral taxon 369 -0.610729601 0.026611071

Proline Veillonellaceae [G-1] sp. oral taxon 150

-0.607744082 0.027568723

Proline Alloprevotella tannerae -0.604395604 0.028672706 Proline Streptococcus lactarius 0.615384615 0.025167117 Proline Neisseria meningitidis 0.624118907 0.022615029 Proline Haemophilus parainfluenzae 0.642857143 0.017792451 Proline Lautropia mirabilis 0.659340659 0.014225579 Proline Haemophilus sp. oral taxon 908 0.660248217 0.014046214 Proline Actinomyces naeslundii 0.757364389 0.002714729 Proline Streptococcus mitis 0.758241758 0.002666079 Propionate Aggregatibacter sp. oral taxon 898 -0.791163089 0.001276439 Propionate Actinomyces naeslundii -0.746060443 0.003405409 Propionate Streptococcus oralis -0.664835165 0.013165576 Propionate Actinobaculum sp. oral taxon 183 -0.657468598 0.014600997 Propionate Lautropia mirabilis -0.642857143 0.017792451 Propionate Veillonella denticariosi -0.632737875 0.020289849 Propionate Haemophilus sp. oral taxon 036 -0.617407951 0.024557876 Propionate Neisseria bacilliformis -0.602219136 0.029407481 Propionate Filifactor alocis 0.631868132 0.020516043 Propionate Tannerella forsythia 0.631868132 0.020516043 Propionate Streptococcus salivarius 0.635488909 0.019586595 Propionate Treponema socranskii 0.637362637 0.019118114 Propionate Clostridiales [F-1][G-1] sp. oral

taxon 093 0.637944885 0.018974253

Propionate Johnsonella sp. oral taxon 166 0.651528814 0.01584155 Propionate Prevotella salivae 0.674160097 0.011504203 Propionate Desulfobulbus sp. oral taxon 041 0.710375308 0.006506327 Propionate Peptostreptococcaceae [XI][G-6]

[Eubacterium] nodatum 0.717800578 0.005730434

Propionate Erysipelotrichaceae [G-1] sp. oral taxon 904

0.739234616 0.003883912

Propionate Eggerthia catenaformis 0.771759939 0.001998527

59

Propionate Actinomyces israelii 0.78021978 0.001652501 Propylene glycerol

Prevotella loescheii -0.896837162 3.2688E-05

Propylene glycerol

Corynebacterium durum -0.879120879 7.54456E-05

Propylene glycerol

Actinomyces johnsonii -0.840659341 0.000319071

Propylene glycerol

Cardiobacterium hominis -0.82967033 0.000450286

Propylene glycerol

Porphyromonas sp. oral taxon 284 -0.82874193 0.000463075

Propylene glycerol

Capnocytophaga gingivalis -0.807692308 0.00083916

Propylene glycerol

Fusobacterium nucleatum subsp. polymorphum

-0.802197802 0.000968779

Propylene glycerol

Neisseria flava -0.800403067 0.001014364

Propylene glycerol

Veillonella rogosae -0.797799929 0.00108346

Propylene glycerol

Leptotrichia sp. oral taxon 212 -0.78021978 0.001652501

Propylene glycerol

Streptococcus sp. oral taxon 056 -0.768668335 0.002138186

Propylene glycerol

Streptococcus sp. oral taxon 064 -0.741758242 0.003701314

Propylene glycerol

Prevotella sp. oral taxon 472 -0.740342791 0.003802892

Propylene glycerol

Johnsonella ignava -0.737277176 0.004030276

Propylene glycerol

Capnocytophaga sputigena -0.736263736 0.004107709

Propylene glycerol

Propionibacterium acnes -0.707911116 0.006780817

Propylene glycerol

Propionibacterium propionicum -0.697802198 0.008000717

Propylene glycerol

Bergeyella sp. oral taxon 322 -0.675824176 0.011225246

Propylene glycerol

Selenomonas sp. oral taxon 937 -0.674043437 0.011523954

Propylene glycerol

Leptotrichia sp. oral taxon 392 -0.674003389 0.01153074

Propylene glycerol

Propionibacterium sp. oral taxon 194

-0.670322915 0.012167413

60

Propylene glycerol

SR1 [G-1] sp. oral taxon 875 -0.664058215 0.013311731

Propylene glycerol

SR1 [G-1] sp. oral taxon 874 -0.663016955 0.01350952

Propylene glycerol

Streptococcus sanguinis -0.659340659 0.014225579

Propylene glycerol

Alloprevotella sp. oral taxon 308 -0.657468598 0.014600997

Propylene glycerol

Actinomyces gerencseriae -0.653846154 0.015348517

Propylene glycerol

Gemella sanguinis -0.648351648 0.016536699

Propylene glycerol

Leptotrichia sp. oral taxon 219 -0.642131764 0.017963407

Propylene glycerol

Porphyromonas catoniae -0.640893759 0.018258008

Propylene glycerol

Fusobacterium periodonticum -0.637362637 0.019118114

Propylene glycerol

Aggregatibacter paraphrophilus -0.629587528 0.021118062

Propylene glycerol

Gemella morbillorum -0.620879121 0.023538167

Propylene glycerol

Lautropia mirabilis -0.60989011 0.026877834

Propylene glycerol

Neisseria sp. oral taxon 018 -0.609349723 0.027050591

Propylene glycerol

Prevotella nanceiensis -0.607744082 0.027568723

Propylene glycerol

Peptostreptococcaceae [XI][G-6] [Eubacterium] nodatum

0.61041309 0.026711418

Propylene glycerol

Peptostreptococcaceae [XI][G-4] sp. oral taxon 103

0.612872815 0.025938878

Propylene glycerol

Filifactor alocis 0.615384615 0.025167117

Propylene glycerol

Treponema sp. oral taxon 237 0.61623167 0.02491072

Propylene glycerol

Prevotella sp. oral taxon 396 0.620235195 0.023724919

Propylene glycerol

Fretibacterium sp. oral taxon 360 0.626373626 0.021988602

Propylene glycerol

Dialister micraerophilus 0.628970902 0.021283066

61

Propylene glycerol

Treponema sp. oral taxon 247 0.663020451 0.013508852

Propylene glycerol

Lachnospiraceae [G-8] sp. oral taxon 500

0.66851849 0.012489082

Propylene glycerol

Atopobium rimae 0.682256491 0.010194832

Propylene glycerol

Prevotella dentalis 0.700844659 0.007617265

Propylene glycerol

Olsenella sp. oral taxon 807 0.701668168 0.00751593

Propylene glycerol

Fretibacterium fastidiosum 0.703296703 0.00731857

Propylene glycerol

Megasphaera sp. oral taxon 123 0.712148605 0.006314124

Propylene glycerol

Treponema sp. oral taxon 517 0.717800578 0.005730434

Propylene glycerol

Dialister invisus 0.71978022 0.005536116

Succinic acid Campylobacter showae -0.620879121 0.023538167 Succinic acid SR1 [G-1] sp. oral taxon 345 -0.617072964 0.024657982 Succinic acid Neisseria bacilliformis 0.696143221 0.008215911 Sugars polyols TM7 [G-3] sp. oral taxon 351 -0.603288879 0.02904463 Sugars polyols Selenomonas sp. oral taxon 146 0.620235195 0.023724919 Sugars polyols Bulleidia extructa 0.621717036 0.023296787 Sugars polyols Fusobacterium nucleatum subsp.

vincentii 0.686813187 0.009509496

Tyrosine Desulfobulbus sp. oral taxon 041 -0.77166259 0.002002813 Tyrosine Treponema socranskii -0.747252747 0.003326741 Tyrosine Clostridiales [F-1][G-1] sp. oral

taxon 093 -0.738233164 0.003958271

Tyrosine Filifactor alocis -0.736263736 0.004107709 Tyrosine Actinomyces sp. oral taxon 172 -0.717100589 0.005800382 Tyrosine Peptostreptococcaceae [XI][G-6]

[Eubacterium] nodatum -0.689540712 0.009116373

Tyrosine Eggerthia catenaformis -0.684517511 0.009850245 Tyrosine Treponema denticola -0.682256491 0.010194832 Tyrosine Actinomyces sp. oral taxon 414 -0.635368813 0.019616911 Tyrosine Tannerella forsythia -0.631868132 0.020516043 Tyrosine Veillonellaceae [G-1] sp. oral taxon

150 -0.629843867 0.021049749

Tyrosine Erysipelotrichaceae [G-1] sp. oral taxon 904

-0.623337664 0.022835136

62

Tyrosine Fretibacterium sp. oral taxon 360 -0.620879121 0.023538167 Tyrosine Treponema sp. oral taxon 231 -0.620879121 0.023538167 Tyrosine Treponema sp. oral taxon 230 -0.620205314 0.023733611 Tyrosine Actinomyces israelii -0.615384615 0.025167117 Tyrosine Capnocytophaga sp. oral taxon 380 0.605634176 0.028260636 Tyrosine Actinomyces gerencseriae 0.615384615 0.025167117 Tyrosine Alloprevotella sp. oral taxon 473 0.618982704 0.024091304 Tyrosine Abiotrophia defectiva 0.626801743 0.021871129 Tyrosine Lactococcus lactis 0.628970902 0.021283066 Tyrosine Propionibacterium acnes 0.632734714 0.020290668 Tyrosine Granulicatella elegans 0.632737875 0.020289849 Tyrosine Neisseria sp. oral taxon 018 0.635358553 0.019619502 Tyrosine Gemella sanguinis 0.637362637 0.019118114 Tyrosine Enterococcus italicus 0.638672955 0.018795499 Tyrosine Porphyromonas pasteri 0.640990978 0.018234744 Tyrosine Neisseria oralis 0.651873812 0.015767406 Tyrosine Aggregatibacter sp. oral taxon 898 0.663016955 0.01350952 Tyrosine Lautropia mirabilis 0.664835165 0.013165576 Tyrosine Kingella sp. oral taxon 012 0.672584793 0.011773079 Tyrosine Catonella morbi 0.681318681 0.010340409 Tyrosine TM7 [G-1] sp. oral taxon 952 0.681318681 0.010340409 Tyrosine Leptotrichia sp. oral taxon 219 0.711043466 0.006433389 Tyrosine Streptococcus sanguinis 0.71978022 0.005536116 Tyrosine TM7 [G-4] sp. oral taxon 355 0.733981436 0.004286298 Tyrosine Veillonella denticariosi 0.734526142 0.004243141 Tyrosine Streptococcus lactarius 0.736263736 0.004107709 Tyrosine Propionibacterium sp. oral taxon

194 0.751764017 0.003041751

Tyrosine Streptococcus pneumoniae 0.763736264 0.002376615 Tyrosine, Creatine

Campylobacter gracilis -0.747252747 0.003326741

Tyrosine, Creatine

Megasphaera sp. oral taxon 123 -0.746060443 0.003405409

Tyrosine, Creatine

Neisseria bacilliformis -0.629843867 0.021049749

Tyrosine, Creatine

Neisseria flava 0.639127822 0.018684461

Tyrosine, Creatine

Veillonella rogosae 0.640990978 0.018234744

63

Tyrosine, Creatine

Johnsonella ignava 0.662999252 0.013512902

Tyrosine, Creatine

Fusobacterium nucleatum subsp. polymorphum

0.703296703 0.00731857

Valine Neisseria bacilliformis -0.651943652 0.015752429 Valine Streptococcus sp. oral taxon 058 -0.635368813 0.019616911 Valine Prevotella intermedia -0.632737875 0.020289849 Valine Tyrosine, Creatine 0.736263736 0.004107709 β-Glucose TM7 [G-3] sp. oral taxon 351 -0.848188324 0.000248251 β-Glucose Leptotrichia buccalis -0.774725275 0.001871362 β-Glucose Lachnoanaerobaculum saburreum -0.714285714 0.006088305 β-Glucose Streptococcus oligofermentans -0.708791209 0.006681784 β-Glucose Corynebacterium matruchotii -0.675824176 0.011225246 β-Glucose Streptococcus cristatus -0.664835165 0.013165576 β-Glucose Capnocytophaga sp. oral taxon 336 -0.635368813 0.019616911 β-Glucose Capnocytophaga sp. oral taxon 324 -0.618793975 0.024146872 β-Glucose Bacteroidetes [G-3] sp. oral taxon

365 0.609262036 0.0270787

β-Glucose Lachnospiraceae [G-7] sp. oral taxon 086

0.609262036 0.0270787

β-Glucose Selenomonas sp. oral taxon 146 0.620235195 0.023724919 β-Glucose Schlegelella aquatica 0.679568383 0.01061632 β-Glucose Parvimonas micra 0.692307692 0.008730318 β-Glucose Prevotella sp. oral taxon 304 0.698514065 0.007909707

64

Table 7 Subgingival bacteria and metabolomes network in overweight nonsmoker individuals.

Metabolite Taxonomy Correlation P-value B-Glucose Gemella sanguinis -0.6333333 0.00360264 Acetic acid Lachnoanaerobaculum

saburreum -0.6473684 0.00273199

Acetic acid Actinomyces dentalis -0.6361295 0.00341305 Acetoin Streptococcus sp. oral taxon 431 -0.60091 0.00650919 Acetoin Mycoplasma faucium 0.60390003 0.00617952 Acetoin Treponema lecithinolyticum 0.62457479 0.00425351 Acetoin Alloprevotella rava 0.64408911 0.0029179 Aspartate Neisseria flava 0.61369071 0.00519402 Aspartate Kluyvera ascorbata 0.62417604 0.0042853 Butyrate Stomatobaculum sp. oral taxon

097 0.60149826 0.00644322

Butyrate Corynebacterium mucifaciens 0.60898592 0.00565013 Butyrate Lachnoanaerobaculum

umeaense 0.6520221 0.00248508

Ethanol Granulicatella adiacens -0.7438596 0.00026109 Ethanol Capnocytophaga sp. oral taxon

334 -0.624176 0.0042853

Ethanol Actinomyces sp. oral taxon 448 -0.6145321 0.00511572 Ethanol Stomatobaculum sp. oral taxon

910 0.60014114 0.00659624

Ethanol Fusobacterium periodonticum 0.64414223 0.00291481 Ethanol Veillonellaceae [G-1] sp. oral

taxon 135 0.65747375 0.00221965

Ethanolamine Kingella oralis -0.6807018 0.00133707 Ethanolamine Campylobacter curvus 0.76286173 0.00014556 Formate Neisseria elongata -0.6315789 0.00372596 Fucose Kluyvera ascorbata 0.62693788 0.00406907 Fucose Neisseria flava 0.74174055 0.00027782 Galctose Neisseria flava 0.74079203 0.00028559 Glucose Veillonellaceae [G-1] sp. oral

taxon 135 -0.6620003 0.00201757

Glutamine Peptostreptococcaceae [XI][G-2] sp. oral taxon 091

0.62228167 0.004439

Glutamine Bacteroidetes [G-5] sp. oral taxon 505

0.62693788 0.00406907

65

Glutamine Johnsonella sp. oral taxon 166 0.62693788 0.00406907 Glutamine Treponema sp. oral taxon 269 0.62693788 0.00406907 Glutamine Bacteroidetes [G-3] sp. oral

taxon 365 0.64728914 0.00273636

Glutamine Veillonellaceae [G-1] sp. oral taxon 148

0.67146939 0.00164395

Glutamine Prevotella dentalis 0.68622696 0.00117749 Histidine Campylobacter curvus 0.61064338 0.00548585 Histidine Peptostreptococcaceae [XI][G-7]

[Eubacterium] yurii subsps. yurii & margaretiae

0.6211514 0.00453285

Histidine Capnocytophaga sp. oral taxon 338

0.62329516 0.00435622

Histidine Prevotella sp. oral taxon 317 0.70820542 0.00069088 Lactic acid TM7 [G-1] sp. oral taxon 352 -0.6998622 0.00085045 Lactic acid Actinomyces cardiffensis -0.635457 0.00345787 Lactic acid Leptotrichia hofstadii -0.6264378 0.00410754 Lactic acid Prevotella pallens -0.6116002 0.00539281 Lactic acid TM7 [G-1] sp. oral taxon 348 -0.6112101 0.00543058 Lactic acid Actinomyces lingnae [NVP] -0.6030254 0.00627451 Lactic acid TM7 [G-3] sp. oral taxon 351 -0.6014094 0.00645315 Leucine Kingella sp. oral taxon 012 0.61938181 0.00468304 Leucine Neisseria flava 0.6677562 0.00178291 Lysine Prevotella sp. oral taxon 306 -0.6117937 0.00537416 Proline Veillonellaceae [G-1] sp. oral

taxon 148 0.61735831 0.00485977

Proline Peptostreptococcaceae [XI][G-2] sp. oral taxon 091

0.66586166 0.00185749

Propionate Streptococcus gordonii -0.7508772 0.00021167 Propionate Capnocytophaga sp. oral taxon

324 -0.655537 0.00231111

Propionate Bacteroidetes [G-5] sp. oral taxon 511

-0.6136907 0.00519402

Propionate Treponema sp. oral taxon 237 -0.6136907 0.00519402 Succinic acid Neisseria flava -0.6611166 0.00205577 Succinic acid Kingella sp. oral taxon 012 -0.60136 0.00645868 Succinic acid Alloprevotella sp. oral taxon 473 0.70732784 0.00070638 Succinic acid Veillonella sp. oral taxon 780 0.73733379 0.00031553 Sugars polyols Atopobium sp. oral taxon 199 -0.6737562 0.00156295

66

Sugars polyols Tannerella forsythia -0.6389011 0.00323333 Sugars polyols Campylobacter sp. oral taxon

044 -0.6291831 0.00389997

Sugars polyols Prevotella pallens -0.6010554 0.00649283 Tyrosine Prevotella salivae -0.6371931 0.00334313 Tyrosine Campylobacter curvus 0.62657321 0.00409709 Tyrosine, Creatine Capnocytophaga sp. oral taxon

326 0.6000235 0.00660964

Tyrosine, Creatine Campylobacter curvus 0.78056154 8.04E-05 Valine Atopobium rimae -0.636244 0.00340546 Valine Prevotella loescheii -0.6353665 0.00346395 Valine Neisseria sicca 0.66081621 0.00206889 Valine Neisseria flava 0.76355644 0.00014234

67

Table 8 Subgingival bacteria and metabolomes network in overweight smoker individuals.

Metabolite Taxonomy Correlation P-value Acetic acid Veillonella denticariosi 0.60603766 0.01283105 Acetic acid Aggregatibacter segnis 0.62941177 0.00898809 Acetic acid Neisseria lactamica 0.64720556 0.00672549 Acetic acid Neisseria oralis 0.64783693 0.00665448 Acetic acid Pseudoramibacter alactolyticus 0.66513464 0.00492948 Acetic acid Prevotella oralis 0.6785401 0.00385611 Acetic acid Streptococcus dentisani 0.74172206 0.00100514 Acetoin Capnocytophaga haemolytica -0.6428628 0.00723043 Acetoin Stomatobaculum sp. oral taxon

910 -0.6079188 0.01248092

Acetoin Leptotrichia sp. oral taxon 463 0.60485249 0.01305557 Alanine Actinomyces johnsonii 0.60294118 0.01342413 Alanine Mogibacterium pumilum 0.66666957 0.00479567 Alanine Campylobacter gracilis 0.69117647 0.00302472 Alanine TM7 [G-5] sp. oral taxon 356 0.70075997 0.00249612 Alanine Lachnospiraceae [G-7] sp. oral

taxon 086 0.76341147 0.00057941

Aspartate Pseudoramibacter alactolyticus -0.7652624 0.00055136 Aspartate Streptococcus dentisani -0.7579104 0.00066978 Aspartate Aggregatibacter segnis -0.6382353 0.00780114 Aspartate Lachnospiraceae [G-8] sp. oral

taxon 500 -0.6236098 0.00984337

Aspartate Veillonella denticariosi -0.6090231 0.01227889 B-Glucose Streptococcus sp. oral taxon

056 -0.8037726 0.00017567

B-Glucose Lachnoanaerobaculum umeaense

-0.7490804 0.00083886

B-Glucose GN02 [G-1] sp. oral taxon 872 -0.7264158 0.00143797 B-Glucose Treponema sp. oral taxon 253 -0.6870959 0.00327558 B-Glucose Fusobacterium sp. oral taxon

203 -0.6647059 0.00496739

B-Glucose Johnsonella ignava -0.6514447 0.00626007 B-Glucose Streptococcus dentisani -0.6475351 0.00668835 B-Glucose Fusobacterium nucleatum

subsp. polymorphum -0.6382353 0.00780114

68

B-Glucose Treponema maltophilum -0.6004417 0.01391834 B-Glucose Actinomyces sp. oral taxon 180 0.60076677 0.01385326 Butyrate Bulleidia extructa 0.60603766 0.01283105 Butyrate Haemophilus sp. oral taxon 036 0.6256047 0.00954235 Butyrate Clostridiales [F-1][G-1] sp. oral

taxon 093 0.64496271 0.00698263

Butyrate Aggregatibacter sp. oral taxon 458

0.69016659 0.00308531

Ethanol Mogibacterium pumilum -0.8023634 0.00018395 Ethanol Lachnospiraceae [G-7] sp. oral

taxon 086 -0.7461326 0.00090253

Ethanol Ottowia sp. oral taxon 894 -0.7399464 0.00104906 Ethanol Actinomyces gerencseriae -0.6647059 0.00496739 Ethanol Mogibacterium diversum -0.6487427 0.00655365 Ethanol Campylobacter gracilis -0.6441176 0.00708152 Ethanol Fusobacterium naviforme -0.6411765 0.00743442 Ethanol Leptotrichia goodfellowii -0.6314638 0.00870008 Ethanol Actinomyces johnsonii -0.6176471 0.01078799 Ethanol Streptococcus intermedius -0.6176471 0.01078799 Ethanol Actinomyces sp. oral taxon 897 -0.6110433 0.01191594 Ethanol Prevotella shahii -0.6106221 0.01199091 Ethanol Shuttleworthia satelles -0.6079228 0.01248017 Ethanol Leptotrichia sp. oral taxon 223 0.6093115 0.01222655 Ethanolamine Prevotella sp. oral taxon 396 0.61049838 0.012013 Ethanolamine Fusobacterium periodonticum 0.62352941 0.00985565 Ethanolamine Alloprevotella tannerae 0.63235294 0.00857758 Ethanolamine Gemella sanguinis 0.64117647 0.00743442 Ethyl Propionate and proline

Actinomyces meyeri -0.6693291 0.00457071

Ethyl Propionate and proline

Streptococcus sp. oral taxon 431

-0.6505829 0.00635254

Ethyl Propionate and proline

Actinomyces timonensis -0.6235457 0.00985316

Ethyl Propionate and proline

Selenomonas sp. oral taxon 133 0.60076677 0.01385326

Ethyl Propionate and proline

Prevotella pleuritidis 0.62693551 0.00934562

Ethyl Propionate and proline

Treponema amylovorum 0.64010269 0.00756668

69

Formate Peptostreptococcaceae [XI][G-1] [Eubacterium] infirmum

-0.7404162 0.00103729

Formate Parvimonas sp. oral taxon 393 -0.674702 0.00414206 Formate Tannerella sp. oral taxon 286 -0.6509072 0.00631762 Formate Actinomyces massiliensis -0.6342901 0.00831542 Formate Dialister invisus -0.6147059 0.01127947 Formate Treponema amylovorum -0.6007668 0.01385326 Formate Pseudoramibacter alactolyticus 0.63295071 0.008496 Formate Actinomyces meyeri 0.66625883 0.00483119 Formate Streptococcus oralis 0.72352941 0.00153448 Fucose Neisseria oralis 0.6079228 0.01248017 Fucose Lachnospiraceae [G-8] sp. oral

taxon 500 0.61104334 0.01191594

Fucose Cardiobacterium hominis 0.6136867 0.01145383 Fucose Capnocytophaga sp. oral taxon

326 0.61947172 0.01049165

Fucose Prevotella oralis 0.67546978 0.00408354 Fucose Fusobacterium nucleatum

subsp. vincentii 0.67647059 0.00400825

Fucose Actinomyces sp. oral taxon 897 0.76184067 0.00060413 Galctose Actinomyces sp. oral taxon 897 0.62203898 0.01008562 Galctose Fusobacterium nucleatum

subsp. vincentii 0.62941177 0.00898809

Galctose Prevotella shahii 0.64012078 0.00756444 Galctose Erysipelotrichaceae [G-1] sp.

oral taxon 904 0.66871064 0.00462226

Galctose Prevotella oralis 0.68775105 0.00323422 Glucose Lachnospiraceae [G-7] sp. oral

taxon 086 0.60318931 0.01337582

Glucose Actinomyces gerencseriae 0.61470588 0.01127947 Glucose Campylobacter gracilis 0.68235294 0.00358797 Glucose TM7 [G-5] sp. oral taxon 356 0.73039041 0.00131322 Glutamine Oribacterium sinus -0.7215245 0.00160458 Glutamine Veillonellaceae [G-1] sp. oral

taxon 483 0.6256047 0.00954235

Glutamine Erysipelotrichaceae [G-1] sp. oral taxon 905

0.73233101 0.0012556

Glycose Prevotella oralis -0.755298 0.00071659 Glycose Actinomyces sp. oral taxon 897 -0.6817296 0.00363075

70

Glycose Selenomonas artemidis -0.6773729 0.00394134 Glycose Capnocytophaga sp. oral taxon

326 -0.6755192 0.0040798

Glycose Neisseria oralis -0.6263447 0.00943255 Glycose Veillonella denticariosi -0.6060377 0.01283105 Glycose Aggregatibacter sp. oral taxon

949 -0.6027706 0.01345742

Histidine Neisseria lactamica -0.6143623 0.01133802 Histidine SR1 [G-1] sp. oral taxon 875 0.60833728 0.01240405 Histidine Fusobacterium periodonticum 0.60882353 0.0123152 Isoleucine Lachnoanaerobaculum sp. oral

taxon 083 0.60157797 0.01369193

Isoleucine Fusobacterium periodonticum 0.63529412 0.00818207 Isoleucine Selenomonas sp. oral taxon 937 0.7093453 0.00208857 L Glutamic acid Neisseria lactamica -0.6104984 0.012013 Lactic acid Ottowia sp. oral taxon 894 -0.7583683 0.00066185 Leucine Prevotella oralis 0.64476661 0.00700548 Leucine Treponema sp. oral taxon 236 0.6488931 0.00653702 Leucine Actinobaculum sp. oral taxon

183 0.65487011 0.00590307

Leucine Peptostreptococcaceae [XI][G-1] [Eubacterium] sulci

0.66652513 0.00480814

Leucine Prevotella enoeca 0.66748118 0.0047261 Leucine Prevotella pleuritidis 0.67171661 0.00437605 Leucine Tannerella forsythia 0.68285523 0.0035538 Leucine Fusobacterium nucleatum

subsp. vincentii 0.72647059 0.00143618

Leucine Dialister pneumosintes 0.74816867 0.00085814 Lysine Prevotella oralis 0.62327439 0.0098947 Lysine Capnocytophaga sp. oral taxon

326 0.65192024 0.00620951

Lysine Actinomyces sp. oral taxon 897 0.84352256 4.03E-05 Methanol Actinomyces gerencseriae 0.61764706 0.01078799 Methanol Campylobacter curvus 0.62096469 0.01025399 Methanol Aggregatibacter sp. oral taxon

898 0.62693174 0.00934617

Methanol Campylobacter gracilis 0.66470588 0.00496739 Methanol Actinomyces sp. oral taxon 525 0.66764706 0.00471198 Methanol Shuttleworthia satelles 0.696962 0.00269584

71

Phenylalanine TM7 [G-1] sp. oral taxon 952 -0.7617647 0.00060534 Phenylalanine Veillonella dispar -0.6617647 0.00523375 Phenylalanine Streptococcus parasanguinis I -0.65 0.00641571 Propionate Selenomonas artemidis 0.61903456 0.01056206 Propionate Pseudoramibacter alactolyticus 0.63295071 0.008496 Propionate Lachnospiraceae [G-8] sp. oral

taxon 500 0.63303462 0.0084846

Propionate Streptococcus dentisani 0.6990436 0.00258484 Propionate Neisseria lactamica 0.70902818 0.00210259 Propionate Prevotella oralis 0.74608708 0.00090354 Propylene glycerol Ottowia sp. oral taxon 894 -0.7307355 0.00130281 Propylene glycerol Leptotrichia goodfellowii -0.7162873 0.00180005 Propylene glycerol Actinomyces johnsonii -0.6941176 0.00285371 Succinic acid Fusobacterium nucleatum

subsp. vincentii -0.8323529 6.33E-05

Succinic acid Catonella morbi -0.7088235 0.00211168 Succinic acid Fusobacterium naviforme -0.7088235 0.00211168 Succinic acid Cardiobacterium valvarum -0.6725693 0.00430817 Succinic acid Treponema maltophilum -0.61663 0.01095599 Succinic acid Prevotella shahii -0.6017725 0.01365345 Succinic acid Fusobacterium nucleatum

subsp. polymorphum -0.6 0.01400712

Sugars polyols Pseudoramibacter alactolyticus -0.7223505 0.00157539 Sugars polyols Streptococcus dentisani -0.6813836 0.00365467 Sugars polyols Veillonella denticariosi -0.6657458 0.00487585 Sugars polyols Streptococcus parasanguinis II -0.6202041 0.01037454 Tyrosine Selenomonas sp. oral taxon 137 -0.7608538 0.00062009 Tyrosine Leptotrichia hofstadii -0.6776874 0.00391822 Tyrosine Streptococcus parasanguinis I -0.6647059 0.00496739 Tyrosine Selenomonas noxia -0.6058824 0.0128603 Tyrosine Ruminococcaceae [G-1] sp. oral

taxon 075 -0.6047224 0.0130804

Tyrosine Erysipelotrichaceae [G-1] sp. oral taxon 905

0.60247438 0.01351536

Tyrosine, Creatine Tannerella forsythia 0.60191333 0.01362566 Tyrosine, Creatine Actinomyces sp. oral taxon 525 0.60882353 0.0123152 Tyrosine, Creatine Actinobaculum sp. oral taxon

183 0.66666957 0.00479567

72

Tyrosine, Creatine Prevotella oralis 0.73073549 0.00130281 Valine Prevotella pleuritidis 0.61200847 0.01174555 Valine Dialister pneumosintes 0.65186974 0.00621486 Valine Prevotella enoeca 0.67424048 0.00417756 Valine Actinobaculum sp. oral taxon

183 0.69026849 0.00307915

Valine Peptostreptococcaceae [XI][G-1] [Eubacterium] sulci

0.69550448 0.00277583

Valine Prevotella oralis 0.71845422 0.00171695 Valine Fusobacterium nucleatum

subsp. vincentii 0.77941177 0.00037168

73

Table 9 Subgingival bacteria and metabolomes network in obese nonsmoker individuals.

Metabolite Taxonomy Correlation P value Acetoin Bergeyella sp. oral taxon 900 -0.6447677 0.00089571 Acetoin Lachnospiraceae [G-8] sp. oral

taxon 500 -0.630563 0.00125732

Acetoin Capnocytophaga sp. oral taxon 332

-0.619272 0.00162751

Alanine Campylobacter showae 0.6027668 0.00233324 Alanine Selenomonas dianae 0.63533306 0.00112406 Alanine Lachnospiraceae [G-8] sp. oral

taxon 500 0.65299644 0.00073022

Alanine Selenomonas sp. oral taxon 138 0.76273865 2.32E-05 Aspartate Aggregatibacter aphrophilus 0.61783043 0.00168085 B-Glucose Prevotella intermedia -0.7029833 0.00018321 B-Glucose Veillonella rogosae -0.6627131 0.00056924 B-Glucose Selenomonas sp. oral taxon 937 -0.6449297 0.00089216 B-Glucose Lachnospiraceae [G-8] sp. oral

taxon 500 -0.6317756 0.00122223

Butyrate Bergeyella sp. oral taxon 900 -0.7295022 7.82E-05 Butyrate Capnocytophaga sp. oral taxon

332 -0.6878218 0.00028658

Ethanol Aggregatibacter paraphrophilus -0.7395517 5.52E-05 Ethanolamine TM7 [G-2] sp. oral taxon 350 -0.6715557 0.00045029 Ethanolamine Actinomyces cardiffensis -0.6523211 0.00074273 Ethyl Propionate and proline

Fusobacterium sp. oral taxon 203

0.61462451 0.00180484

Formate Actinomyces viscosus 0.61247949 0.00189206 Fucose Porphyromonas pasteri 0.61675317 0.00172168 Fucose Fretibacterium sp. oral taxon

360 0.62860298 0.00131586

Fucose Prevotella baroniae 0.69863868 0.00020884 Galctose Fretibacterium sp. oral taxon

360 0.63879103 0.00103518

Galctose Prevotella baroniae 0.67450899 0.00041567 Glutamine Dialister pneumosintes -0.6363689 0.00109678 Glutamine Eikenella sp. oral taxon 011 -0.6087358 0.00205288 Glycose Prevotella baroniae -0.7048114 0.00017327

74

Glycose Fretibacterium sp. oral taxon 360

-0.6224901 0.0015136

L Glutamic acid TM7 [G-2] sp. oral taxon 350 -0.6053296 0.00220913 Leucine Selenomonas dianae 0.61629311 0.00173937 Phenylalanine Capnocytophaga sputigena 0.62944664 0.00129038 Proline Alloprevotella sp. oral taxon

913 -0.6367534 0.00108679

Propionate Stomatobaculum sp. oral taxon 097

0.64157587 0.00096804

Propionate Streptococcus sp. oral taxon 057 0.64848395 0.00081737 Propylene glycerol Megasphaera sp. oral taxon 123 -0.6097422 0.00200855 Succinic acid Selenomonas dianae -0.6754172 0.0004055 Sugars polyols Selenomonas sp. oral taxon 137 0.63636364 0.00109691 Sugars polyols Selenomonas sputigena 0.70548024 0.00016976

75

Table 10 Subgingival bacteria and metabolomes network in obese smoker individuals.

Metabolite Taxonomy Correlation P-value Acetic acid Oribacterium asaccharolyticum -0.6508307 0.00632585 Acetoin Streptococcus gordonii -0.6294118 0.00898809 Acetoin Streptococcus sp. oral taxon 070 -0.6048525 0.01305557 Acetoin Selenomonas sp. oral taxon 937 0.60247438 0.01351536 Alanine Capnocytophaga sp. oral taxon

902 -0.7941183 0.00023919

Alanine Bergeyella sp. oral taxon 900 -0.6447666 0.00700548 Alanine Leptotrichia hongkongensis -0.6205882 0.01031352 Alanine TM7 [G-4] sp. oral taxon 355 0.62981795 0.00893049 Aspartate Corynebacterium durum -0.6843688 0.0034524 Aspartate Bacteroidetes [G-5] sp. oral taxon

511 0.62210387 0.01007552

B-Glucose Campylobacter showae -0.6235294 0.00985565 B-Glucose Streptococcus sp. oral taxon 423 -0.6029412 0.01342413 B-Glucose Veillonellaceae [G-1] sp. oral

taxon 148 0.63423373 0.00832296

B-Glucose Veillonellaceae [G-1] sp. oral taxon 483

0.65149179 0.00625505

B-Glucose Actinomyces cardiffensis 0.68099979 0.00368136 Butyrate Olsenella uli -0.6157935 0.01109571 Butyrate TM7 [G-1] sp. oral taxon 488 0.61697567 0.01089866 Ethanol Pseudoramibacter alactolyticus -0.7131065 0.00192793 Ethanol Porphyromonas uenonis -0.6773789 0.0039409 Ethanol Bacteroidetes [G-7] sp. oral taxon

911 -0.6047025 0.0130842

Ethanol Prevotella melaninogenica 0.69315692 0.00290869 Ethanolamine Staphylococcus epidermidis 0.61266115 0.01163141 Ethanolamine Lachnospiraceae [G-7] sp. oral

taxon 163 0.62692536 0.0093471

Ethyl Propionate and proline

Rothia dentocariosa 0.6740252 0.00419421

Ethyl Propionate and proline

Streptococcus salivarius 0.70640196 0.0022217

Formate Prevotella sp. oral taxon 313 0.62058824 0.01031352 Formate TM7 [G-3] sp. oral taxon 351 0.64169629 0.00737105 Formate Prevotella enoeca 0.67086083 0.00444505 Formate Lachnoanaerobaculum orale 0.68579857 0.00335876

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Formate Stomatobaculum sp. oral taxon 910

0.68709595 0.00327558

Formate Ruminococcaceae [G-2] sp. oral taxon 085

0.86822749 1.30E-05

Fucose Actinomyces oris -0.6882353 0.00320391 Fucose Rothia dentocariosa -0.6622518 0.00518887 Fucose Selenomonas sp. oral taxon 138 0.60157797 0.01369193 Fucose Selenomonas infelix 0.60834664 0.01240233 Fucose Prevotella melaninogenica 0.72553368 0.00146692 Galctose Actinomyces oris -0.6647059 0.00496739 Galctose Rothia dentocariosa -0.6607802 0.00532543 Galctose Pseudomonas aeruginosa -0.6294118 0.00898809 Galctose Veillonella denticariosi -0.6117647 0.01178841 Galctose Oribacterium asaccharolyticum -0.6114948 0.01183601 Galctose Erysipelotrichaceae [G-1] sp. oral

taxon 904 0.64213379 0.00731806

Galctose Prevotella melaninogenica 0.70051527 0.00250862 Galctose Selenomonas sp. oral taxon 138 0.708037 0.00214692 Glucose Selenomonas artemidis -0.6911544 0.00302603 Glucose Bacteroidetes [G-3] sp. oral taxon

281 0.64286276 0.00723043

Glucose Streptococcus parasanguinis I 0.65 0.00641571 Glucose Actinomyces lingnae [NVP] 0.68944892 0.00312896 Glucose Propionibacterium acnes 0.69374258 0.00287507 Glucose Streptococcus parasanguinis II 0.69927844 0.00257255 Glucose Slackia exigua 0.70447056 0.00231274 Glutamine Streptococcus sp. oral taxon 070 -0.6754698 0.00408354 Histidine SR1 [G-1] sp. oral taxon 345 -0.6346054 0.00827336 Histidine Treponema sp. oral taxon 238 -0.6186467 0.01062483 Isoleucine SR1 [G-1] sp. oral taxon 345 -0.7744071 0.00042866 Isoleucine Treponema sp. oral taxon 238 -0.6544067 0.00595039 Isoleucine Centipeda periodontii -0.6312712 0.0087268 L Glutamic acid Streptococcus sp. oral taxon 070 -0.6079228 0.01248017 L Glutamic acid Prevotella sp. oral taxon 472 0.65781997 0.00560881 Lactic acid Alloprevotella sp. oral taxon 913 -0.6779724 0.00389737 Lactic acid Treponema sp. oral taxon 951 0.60277055 0.01345742 Lactic acid TM7 [G-1] sp. oral taxon 347 0.63418356 0.00832967 Lactic acid Streptococcus salivarius 0.69315692 0.00290869

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Lactic acid Actinomyces cardiffensis 0.70465735 0.0023038 Leucine Prevotella shahii -0.70076 0.00249612 Leucine Leptotrichia buccalis -0.6518697 0.00621486 Leucine Lachnospiraceae [G-7] sp. oral

taxon 163 0.73169456 0.00127426

Leucine Dialister micraerophilus 0.7330785 0.00123396 Lysine Erysipelotrichaceae [G-1] sp. oral

taxon 905 -0.7021501 0.00242609

Lysine Treponema sp. oral taxon 951 -0.6993684 0.00256786 Lysine Prevotella sp. oral taxon 300 -0.6176471 0.01078799 Lysine Treponema socranskii -0.6147059 0.01127947 Lysine Fusobacterium sp. oral taxon 203 0.65 0.00641571 Lysine Prevotella loescheii 0.73451647 0.0011932 Methanol Schlegelella aquatica 0.60305225 0.01340249 Phenylalanine Veillonellaceae [G-1] sp. oral

taxon 155 -0.703723 0.00234878

Phenylalanine Centipeda periodontii -0.6725693 0.00430817 Phenylalanine Capnocytophaga sp. oral taxon

332 -0.6611678 0.00528918

Phenylalanine Prevotella nanceiensis -0.629893 0.00891988 Phenylalanine Treponema sp. oral taxon 242 -0.6169757 0.01089866 Phenylalanine Leptotrichia sp. oral taxon 225 -0.6017822 0.01365155 Phenylalanine Actinomyces sp. oral taxon 169 0.64140969 0.00740594 Proline Prevotella baroniae -0.6958668 0.00275576 Proline Streptococcus sp. oral taxon 070 -0.6846807 0.00343179 Proline Streptococcus gordonii -0.6205882 0.01031352 Propionate Olsenella uli -0.6806139 0.00370834 Propylene glycerol Treponema pectinovorum 0.61822621 0.01069322 Propylene glycerol Treponema sp. oral taxon 951 0.62402208 0.00978055 Propylene glycerol Prevotella multiformis 0.62579872 0.00951347 Propylene glycerol Selenomonas noxia 0.6429806 0.00721634 Propylene glycerol TM7 [G-1] sp. oral taxon 347 0.64324332 0.00718501 Propylene glycerol Treponema vincentii 0.64496271 0.00698263 Propylene glycerol Neisseria bacilliformis 0.64928316 0.00649407 Propylene glycerol Capnocytophaga sp. oral taxon

338 0.65144474 0.00626007

Propylene glycerol Leptotrichia sp. oral taxon 221 0.67230476 0.00432914 Propylene glycerol Mitsuokella sp. oral taxon 521 0.67737888 0.0039409

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Propylene glycerol Veillonellaceae [G-1] sp. oral taxon 135

0.686442 0.0033173

Propylene glycerol Leptotrichia shahii 0.7075415 0.00216936 Propylene glycerol Treponema parvum 0.71877453 0.00170494 Succinic acid Lachnoanaerobaculum umeaense 0.63116363 0.00874175 Sugars polyols Corynebacterium durum -0.7404162 0.00103729 Sugars polyols Capnocytophaga leadbetteri -0.7088235 0.00211168 Sugars polyols Porphyromonas sp. oral taxon

284 -0.6902685 0.00307915

Sugars polyols Prevotella sp. oral taxon 472 -0.6755192 0.0040798 Sugars polyols Campylobacter showae -0.6352941 0.00818207 Sugars polyols Prevotella loescheii -0.634221 0.00832466 Sugars polyols Capnocytophaga sputigena -0.6133502 0.01151187 Tyrosine Streptococcus tigurinus -0.7352941 0.00117162 Tyrosine Prevotella baroniae -0.6063309 0.01277596 Tyrosine, Creatine Treponema sp. oral taxon 951 -0.7070962 0.00218969 Tyrosine, Creatine Erysipelotrichaceae [G-1] sp. oral

taxon 905 -0.6990084 0.00258668

Tyrosine, Creatine Prevotella multiformis -0.6615586 0.00525283 Tyrosine, Creatine Veillonellaceae [G-1] sp. oral

taxon 483 -0.6514918 0.00625505

Tyrosine, Creatine Veillonellaceae [G-1] sp. oral taxon 148

-0.6342337 0.00832296

Tyrosine, Creatine TM7 [G-1] sp. oral taxon 347 -0.6115341 0.01182905 Valine Prevotella shahii -0.6681665 0.00466799 Valine Olsenella sp. oral taxon 807 -0.659775 0.00542034 Valine Prevotella micans -0.6221039 0.01007552 Valine Dialister micraerophilus 0.70804655 0.00214649

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Appendix B: Figures

Figure 1 Linear discriminant analysis, Shannon index and DESeq for BMI groups.

(A) Linear discriminant analysis of unweighted and weighted UniFrac and Bray-Curtis for normalweight (yellow), overweight (light purple) and obese (dark purple) individuals. (B) Shannon index of alpha diversity. (C) Deseq plot of the species that were different between obese and normalweight individuals. Each dot represents a species. Dots above the red line were more abundant in obese individuals.

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Figure 2 Cytokine discriminants and Cytokine discriminants For BMI groups. Cytokine discriminants (2A) and Cytokine discriminants (2B) between normalweight (green), overweight (orange) and obese (yellow) individuals.

Figure 3 Co-occurrence networks of BMI groups.

Co-occurrence networks of normalweight group is shown in figure (3A), overweight in (3B) and obese group in (). Each network graph contains nodes (circles sized by relative abundance per group) and edges (lines). Nodes represent species-level OTU’s and adipokines; edges represent Spearman’s ρ. Edges are colored green for positive correlation and red for negative correlation. Only significant correlations (p < 0.05, t-test) with a coefficient of at least 0.75 are shown.

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Figure 4 Linear discriminant analysis, Shannon index and DESeq for BMI sub-grouped according gender.

(A) Linear discriminant analysis of unweighted and weighted UniFrac and Bray-Curtis for normalweight, obese and obese individuals according to gender. (B) Shannon index of alpha diversity. (C) Deseq plot of the species that were different between Female normalweight and male normalweight (D) female and male overweight and (E) female obese and female normalweight. Each dot represents a species. Dots above the red line were more abundant in Female normalweight (C), female overweight (D) and female obese (E).

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Figure 5 Linear discriminant analysis, Shannon index and DESeq for smoking groups.

(A) Linear discriminant analysis of unweighted and weighted UniFrac and Bray-Curtis for nonsmoker (green) and smoker (red) individuals. (B) Shannon index of alpha diversity. (C) Deseq plot of the species that were different. Each dot represents a species. Dots above the red line were more abundant in nonsmoker individuals.

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Figure 6 Linear discriminant analysis, Shannon index and DESeq for smoking sub-grouped according gender.

(A) Linear discriminant analysis of unweighted and weighted UniFrac and Bray-Curtis for Smoking gender subgroups. (B) Shannon index of alpha diversity. (C) Deseq plot of the species that were different between female smoker and female nonsmoker and (D) male smoker and male nonsmoker. Dots above the red line were more abundant in female smoker (C) and male smoker (D)

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Figure 7 Linear discriminant analysis, Shannon index and DESeq for BMI-smoking groups.

(A) Linear discriminant analysis of unweighted and weighted UniFrac and Bray-Curtis for individuals by BMI and smoking status. (B) Shannon index of alpha diversity. (C) Deseq plot of the species that were different between normalweight nonsmokers and obese smokers. Each dot represents a species. Dots above the red line were more abundant in normalweight nonsmoker individuals.

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Figure 8 Representative NMR spectrum.

NMR spectroscopy of a representative sample showing the metabolomes identified in the saliva sample collected.

Figure 9 Metabolic profile of the BMI-Smoking groups.

Metabolic profile of the 6 groups, showing a distinct profile for obese smoker group compared to Normalweight nonsmoker individuals. the profiles also show similar profiles between normalweight smoker and obese nonsmoker.

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Figure 10 Co-occurrence networks in normalweight nonsmoker and normalweight smoker individuals.

Co-occurrence networks of normalweight nonsmokers (A) and normalweight smokers (B). Nodes represent species- level OTU’s and metabolomes; edges represent Spearman’s ρ. Edges are colored green for positive correlation and red for negative correlation. Only significant correlations (p-value < 0.05, t-test) with a coefficient of at least 0.60 are shown.

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Figure 11 Co-occurrence networks in overweight nonsmoker and overweight smoker individuals.

Co-occurrence networks of overweight nonsmokers (A) and overweight smokers (B). Nodes represent species- level OTU’s and metabolomes; edges represent Spearman’s ρ. Edges are colored green for positive correlation and red for negative correlation. Only significant correlations (p-value < 0.05, t-test) with a coefficient of at least 0.60 are shown.

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Figure 12 Co-occurrence networks in obese nonsmoker and obese smoker individuals.

Co-occurrence networks of obese nonsmokers (A) and obese smokers (B). Nodes represent species-level OUT’s and metabolomes. Edges represent Spearman’s ρ. Edges are colored green for positive correlation and red for negative correlation. Only significant correlations (p-value < 0.05, t-test) with a coefficient of a least 0.60 are shown.