The Salivary Microbiome of Diabetic and Non-Diabetic Adults with Periodontal

Disease

Amarpreet Sabharwal,* Kevin Ganley,† Jeffrey C. Miecznikowski,† Elaine M. Haase,*

Virginia Barnes,‡ and Frank A. Scannapieco*

Correspondence: Dr. Frank A. Scannapieco, University at Buffalo, Department of

Oral Biology, School of Dental Medicine,109 Foster Hall, 3435 Main St, Buffalo, NY

14214; E-mail fas1@buffalo.edu; fax: (716)829-3946 (email and fax can be

published)

KEYWORDS: Saliva; Microbiome; Diabetes Mellitus, Type 2; Chronic Periodontitis;

RNA, ribosomal, 16S

Word count: 3630; Tables (1); Figures (3); Supplemental Table (1); References (47)

SHORT TITLE: Oral microbiome in Type 2 diabetes and chronic periodontitis

This is the author manuscript accepted for publication and has undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/JPER.18-0167.

This article is protected by copyright. All rights reserved.

SUMMARY: Salivary microbiome diversity decreased in diabetics and increased with

progression of periodontal disease compared to periodontally healthy controls.*

*
Department of Oral Biology, School of Dental Medicine, University at Buffalo,

Buffalo, NY

†
Department of Biostatistics, School of Public Health and Health Professions,

University at Buffalo, Buffalo, NY

‡
Colgate Palmolive Technology Center, Piscataway, NJ; deceased

This article is protected by copyright. All rights reserved.

Abstract

Background: A comparison of the salivary microbiome of non-diabetic and diabetic

cohorts having periodontal health, gingivitis and periodontitis could reveal microbial

signatures unique to each group that will increase understanding of the role of oral

microbiota in the pathogenesis of disease, and to assist with diagnosis and risk

assessment for both periodontal disease and diabetes.

Methods: A group of individuals diagnosed with Type 2 diabetes (T2D) was

compared to a group without T2D. For both the diabetic and non-diabetic cohorts,

three sub-groups were established: periodontal health, gingivitis, and periodontitis.

Salivary DNA was extracted (n=146), PCR was performed to amplify 16S rRNA

hypervariable region V3-V4, and constructed libraries were sequenced and

subjected to bioinformatic and statistical analyses.

Results: MIcrobiome analysis resulted in 88 different genus level operational

taxonomic units (OTUs) for differential abundance testing. Results were largely

described by two trends. Trend 1 showed OTUs that increased in abundance with

increasing periodontal disease, and in diabetics relative to non-diabetics. Trend 1

OTUs comprised a mix of primarily anaerobic commensals and potential

periodontopathogens. Trend 2 was driven primarily by genera that decreased in

diabetics relative to non-diabetics, which included other anaerobes associated with

periodontal disease. Overall, oral microbial diversity decreased in diabetics and

increased with progression of periodontal disease compared to periodontally healthy

controls.

This article is protected by copyright. All rights reserved.

Conclusions: Although select microbiota increased in both diabetes and periodontal

disease progression, these genera decreased in co-existing diabetes and

periodontal disease. These findings suggest that the genera abundance continues to

change with additional stress imposed by co-existing conditions.

This article is protected by copyright. All rights reserved.

INTRODUCTION

Type 2 diabetes mellitus (T2D), a subclass of diabetes mellitus that is not

insulin responsive or dependent, is a metabolic disorder that is characterized initially

by insulin resistance and hyperinsulinemia and eventually by glucose intolerance,

hyperglycemia and overt diabetes.1 The systemic effects and long-term

complications are serious and can result in significant morbidity and increased risk

for mortality.2 Periodontal disease is often more severe in T2D patients, and

hyperglycemia is thought to be more severe in patients with periodontal disease.3

Both diabetes and chronic periodontal disease have been shown to be associated

with microbiome changes compared to health.4, 5

It has been proposed that microbial dysbiosis, a concept earlier proposed in

the context of the gut microbiota and inflammatory bowel disease6, may influence the

initiation and/or progression of a variety of inflammatory states and chronic diseases.

A comprehensive analysis of the oral microbiome in diabetic patients is required to

better understand how the microbiota relates to both diabetes and periodontal

disease status. This information may also lead to defining microbial signatures for

each disease that may be useful for diagnosis and risk assessment.

Previously, several studies have reported that specific periodontal microbes

are associated with diabetes.7-9 In a recent report on the periodontal status and

salivary microbiome of obese children with and without T2D, differences in salivary

microbial diversity was found to be minimal between groups, with only a few

differences in bacterial genus composition noted between groups.10 Further, a

literature review from 2013 concluded that diabetes does not have a significant effect

This article is protected by copyright. All rights reserved.

on the composition of periodontal microbiota, and there is little evidence that it

influences glycemic control.11 However, conclusions drawn from studies that target

select microbiota are inherently restrictive about comprehensive biofilm composition

and system-level changes.

The microbiome is influenced by selective environmental pressures created

by both local and systemic factors.12 Information obtained through various omics

approaches, when effectively integrated, will allow for a better system level

understanding of a disease process 13 Several studies have demonstrated changes

in the oral and gut microbiome in diabetic patients.4, 7, 14, 15

Increased levels of glucose in saliva of patients with diabetes may influence

microbial diversity.16 A recent study of salivary metabolites in controlled diabetic

patients found an increase in glucose and a significant change in markers of

glycemic control and insulin resistance.17 In participants with periodontal disease, the

concentration of other selected metabolites decreased in diabetics and increased in

non-diabetics. These findings suggest an interaction between diabetes and

periodontal disease. Moreover, in the non-diabetic cohort with periodontal disease,

concentrations of salivary metabolites increased with disease progression

demonstrating a biochemical continuum.17 To extend this metabolomics investigation

using the same cohorts, the aim of the present study is to use 16S rRNA

amplification sequencing to determine if a meaningful difference exists in the

diversity of the salivary microbiome between controlled diabetic and non-diabetic

participants with periodontal health, gingivitis or periodontitis.

This article is protected by copyright. All rights reserved.

MATERIALS AND METHODS

Study Population and Selection Criteria

This cross-sectional study, the details of which have been previously described17,

was approved by the Institutional Review Board at the University at Buffalo and was

conducted in accordance with the Helsinki Declaration. The study population

included adult males and females from the Buffalo, New York area recruited over a

period of 1 year. All participants provided a written informed consent before their

enrollment in the study. Inclusion criteria comprised individuals between 18 to 65

years, in good general health or diagnosed with diabetes, and having a minimum of

20 natural teeth (excluding third molars Individuals were excluded if they were

unable or unwilling to sign the informed consent, showed a history of a medical

condition that required pre-medication prior to dental visits, showed 5 or more

decayed teeth, diseases of the hard or soft tissues, impaired salivary function, were

exposed to antimicrobial drugs within 30 days prior to the first study visit, a history of

uncharacterized systemic disease, were pregnant or nursing, participated in any

other clinical study within 1 week prior to enrollment in this study, currently used

tobacco products, were required to have dental treatment during the duration of the

study, were immune compromised, or received periodontal treatment within the last

30 days.

This article is protected by copyright. All rights reserved.

Diabetes Status

Diabetes status was established by self-reported history. Participants who reported a

history of diabetes were asked to confirm diabetic status by providing blood samples

to measure HbA1c values. An individual with T2D was defined as having HbA1c 

6.5%.

Oral Examination and Subgroup Criteria

For both the diabetic and non-diabetic cohorts, three sub-groups were established:

periodontal health, gingivitis and periodontitis. Participants were stratified according

to the gingivitis index, as described previously.17 Briefly, healthy participants had an

average full mouth Modified Gingival Index (MGI) score below 1.0, with fewer than 3

bleeding on probing (BOP) sites and minimal dental plaque present as scored by the

method of Silness and Löe.18 Participants with gingivitis had an average full mouth

MGI score between 1 and 2 with multiple BOP sites and periodontal pockets < 4 mm.

Participants with periodontitis had an average full mouth MGI of 2.0 or greater with

multiple BOP sites, and 2 or more periodontal pockets with probing depths of 5 mm

or more in at least two quadrants. Measurement of oral indices was performed as

per Lobene et al.19 as follows: 0 = normal, 1 = mild inflammation of the gingival unit

(slight change in color, little change in texture of any portion of the gingival unit), 2 =

mild inflammation of the entire gingival unit, 3 = moderate inflammation (moderate

change in texture, redness, edema and hypertrophy of the gingival unit), 4 = severe

inflammation (marked redness and edema/hypertrophy, spontaneous bleeding or

This article is protected by copyright. All rights reserved.

ulceration) of the gingival unit. Plaque at any level was evaluated. Plaque indices

were assessed for 6 surfaces on each tooth and the plaque index was the average

of the scores obtained from all teeth. The amount of plaque observed on each tooth

was scored 0 to 3 as follows: 0 = no plaque noted; 1 = plaque seen only on the tip of

an explorer passed over the tooth surface; 2 = plaque obvious with the naked eye; 3

= gross deposits of plaque present over the entire tooth.

Study Design and Sample Collection

A single dental examiner evaluated all those enrolled for inclusion/exclusion criteria.

Samples were collected as previously described.17 At the first visit, to minimize the

effect of toothpaste on variations in microbiota, participants received a tube of

standard, non-antimicrobial fluoridated toothpaste with instructions for use. A fasting

blood sample was then collected from participants self-identifying as diabetic to

confirm their diabetic status. Plasma was collected by venipuncture in a sterile blood

collection tube containing EDTA. At this visit, participants were given instructions for

saliva collection to be obtained at the next visit within 30 days. They were to refrain

from eating or drinking (excluding water) from 11:00 PM the previous evening, and to

brush their teeth and entire mouth the evening before saliva donation, but not on the

morning of collection. During the second visit, participants provided a completed

medical history questionnaire, and answered questions to assess compliance with

use of toothpaste previously provided. The oral tissues of each individual were

examined to assess periodontal health status. At this visit, a minimum of 0.5 ml of

This article is protected by copyright. All rights reserved.

unstimulated saliva was collected from each participant in a 50 mL sterile

polypropylene tube, which was immediately frozen in a dry ice bath, and stored at -

80C.

Salivary Microbiome Analysis

Frozen saliva samples were thawed and centrifuged at 4C for 15 min at 1500 x g.

Saliva samples were processed for microbiome analysis as described previously.10

The pellet obtained following centrifugation was re-suspended in 300 μL of lysis

solution (20 mg/mL lysozyme in 20 mM Tris-HCl, pH 8.0; 2 mM EDTA; 1.2% Triton

X-100) and incubated at 37°C for 1 h. Viscous samples were dissociated by adding

20 μL of 1M DTT to 300 μL of lysozyme solution, vortexed and incubated at 56°C for

15 min. Salivary DNA was obtained using an automated extraction protocol.§

Amplicon PCR was performed for the 16S rRNA hypervariable region V3-V4 using

forward V3 (5' CCTACGGGNGGCWGCAG 3') and reverse V4 primers (5'

GACTACHVGGGTATCTAATCC 3’). Constructed libraries‖ were sequenced to

obtain paired-end (2 x 300 bp) reads.¶ The raw base call files were processed

automatically to obtain two fastq files (one each for forward and reverse reads) per

sample. # DNA extraction, amplicon PCR, library construction and sequencing were

performed at the genomic core facility.** Paired-end fastq reads were processed

using the software pipeline†† as previously described.20 Briefly, taxonomic

assignments were determined by using a naive Bayesian classifier with the Human

Oral Microbiome Database (HOMD) data files (version 14.5)21 and a 98% bootstrap

This article is protected by copyright. All rights reserved.

confidence threshold. Phylotype clustering was carried out to bin sequences and for

taxonomic classification. Shared and consensus taxonomy files at various taxonomic

levels and associated metadata were used for statistical analysis.†

Statistical Analysis

Clinical data analysis. Participants (n=143) were divided into six groups based on

diabetic and periodontal health status: periodontal health and non-diabetic (C);

gingivitis and non-diabetic (G); periodontitis and non-diabetic (P); periodontal health

and diabetic (D); gingivitis and diabetic (DG); and periodontitis and diabetic (DP).

Group comparisons for age, body mass index (BMI), and hemoglobin A1c (HbA1c)

were performed using ANOVA at level 0.05. Gender differences were assessed

using Fisher’s exact test at level 0.05.

Prior to microbiome data analysis, exploratory analysis was performed to

determine outlying samples based on library size at the genus level. Participants with

raw library sizes below 100 (n=3) were considered to be quantitatively inadequate

§
QIAsymphony SP Complex-200_V6_DSP protocol, QIAGEN Sciences,
Germantown, MD
‖
16S Metagenomic Sequencing Library Preparation, Illumina, San Diego, CA
¶
Illumina MiSeq using MiSeq reagent kit V3, Illumina, San Diego, CA
#
MiSeq Reporter Generate FASTQ workflow, Illumina, San Diego, CA

**University at Buffalo Genomics and Bioinformatics Core, Buffalo, NY

††
MiSeq workflow with the MOTHUR pipeline, Illumina, San Diego, CA

This article is protected by copyright. All rights reserved.

and were removed from the analysis. Additionally, exploratory analysis to determine

outlying operational taxonomic units (OTUs) was based on the variance of an OTU.

A variance less than 1, or a low overall read count (less than 100) across all

participants or being absent (read of 0) in more than 100 participants were used as

criteria for exclusion from differential expression analysis but were retained for alpha

and beta diversity analyses.

Statistical analysis of microbiome composition consisted of three stages. The

first stage identified differentially abundant genera (OTUs at the genus level)

significantly associated with disease states via the approach originally taken in

Robinson and Smyth 2007 22, and later expanded to a generalized linear model

framework (GLM) in McCarthy, Chen & Smyth 2012.23 A negative binomial GLM was

fit to the count data and likelihood ratio tests were performed to compare abundance

levels between diabetic status levels. The full GLM model included an intercept, age,

BMI, an oral health status variable (0=periodontal health, 1=gingivitis,

2=periodontitis), a diabetes indicator variable (0=non-diabetic, 1= diabetic), and an

interaction between periodontal disease and diabetic status. The reduced GLM

model included the intercept, age, and BMI. A likelihood ratio test was performed to

compare the reduced model versus the full model for each OTU with a Benjamini-

Hochberg false discovery rate (FDR) control at level 0.05 to determine significance

across the family of OTUs at the genus level.24 A dendrogram was then used to

cluster the significant OTUs to determine groups of OTUs with similar patterns of

significance.

This article is protected by copyright. All rights reserved.

 The second stage used alpha diversity to assess group differences. Several

measures of alpha diversity (Observed, Chao, ACE, Shannon, Simpson, Inverse

Simpson) were calculated for each combination of periodontal disease progression

and diabetic status. An ANOVA F-test at level 0.05 was performed to test for

significant differences between the diabetic/non-diabetics, and significant differences

between the periodontal disease groups.

The third stage analyzed beta diversity via the Bray-Curtis dissimilarity

index.25 Tests at level 0.05 to compare diabetes and periodontal disease status were

implemented using the Multi-Response Permutation Procedure (MRPP)26 where a

non-parametric test compared the observed mean group difference to the estimated

distribution of possible differences through permutations of the data. All data

analyses were performed using the R programming language. Two different software

packages were used to perform the differential abundance analyses‡‡ and the alpha

and beta diversity analyses.§ 27

RESULTS

This study extended investigation of samples from previously published

metabolomics investigation.17 From a total of 174 participants originally enrolled, 146

contained sufficient quantity of DNA for microbiome analysis. Furthermore, after the

‡‡
edgeR, Bioconductor software package, release 3.6
§§
phyloseq, R software package, The R foundation for Statistical Computing,
Vienna, Austria

This article is protected by copyright. All rights reserved.

microbiome data processing stage it was found that 3 participants had sequence

read counts too low for taxonomic assignment, and therefore were removed from

consideration in the statistical analysis. The final population under study thus

consisted of 143 participants (n=79 diabetics, n=64 non-diabetics).

Comparison of clinical parameters yielded significant differences between

groups in age and BMI, while no significant gender differences were noted (Table 1).

HbA1c was not significantly different between diabetic sub-groups. Accordingly, to

control for differences in age and BMI, those variables were included in the reduced

and full GLM models.

Stage 1 Results

The microbiome OTU pruning resulted in 88 different genus level OTUs for

differential abundance testing (see supplementary Table1 in online Journal of

Periodontology). The likelihood ratio test for each OTU examined the significance of

the oral health status variable, the diabetes status variable or their interaction. This

resulted in 30 genus level OTUs that were significant when controlling the false

discovery rate (FDR) at 0.05. A significant OTU indicates significant increasing or

decreasing abundance when comparing groups (Healthy to Gingivitis to

Periodontitis), or that there was a difference in abundance between non-diabetics or

diabetics (diabetic status variable), or that there was a significantly different

progression of increase or decrease in abundance in periodontal disease (Healthy to

Gingivitis to Periodontitis), compared to non-diabetics and diabetics (interaction

variable). The results are summarized in a heat map of these coefficients (Figure 1).

This article is protected by copyright. All rights reserved.

The brightest bands (bright red or bright green) indicate significantly large

coefficients for that variable.

The dendrogram results at the genus level suggest statistically

significant OTUs are largely described by two trends (Figure 1). Trend 1 shows

OTUs that increase in abundance with increasing periodontal disease (red

bands in the oral health variable), and also show increased abundance in

diabetics relative to non-diabetics (red bands in the diabetic variable). In the

interaction variable for this trend, Bifidobacterium and Abiotrophia

demonstrated a decrease in abundance with progressing periodontal disease

in the diabetic group (strongly green in the interaction variable). Trend 2 is

driven primarily by genera that decrease in diabetics relative to non-diabetics,

as shown by green bands in the diabetic variable. In this trend, Fretibacterium,

Peptostreptococcaceae, Filifactor, Treponema increased in abundance with

progressing periodontal disease.

Stage 2 and 3 Results

Microbial diversity at the genus level, both within (alpha) and between (beta) groups

were measured for each participant (n=143). The Observed, Chao1, ACE, and

Shannon metrics of alpha diversity were significant when measured by diabetes

status. Overall, diabetes status was associated with lower alpha diversity (Figure 2).

Similarly, Bray-Curtis dissimilarity, the beta diversity metric used, was significant for

groupings by diabetes status (P=0.002). However, a principle coordinate analysis

(PCoA) plot (Figure 3) showed clusters that differed in variance but with similar

This article is protected by copyright. All rights reserved.

means. For periodontal disease, the Observed, Chao1, and ACE metrics where

significant, indicating that alpha diversity increases with progressing periodontal

disease. The beta diversity metric for oral health status was not significant (p=0.73).

DISCUSSION

This study was undertaken to examine the salivary microbiome of participants with

diabetes and various degrees of severity of periodontal disease. When the clinical

variables age, BMI and gender of the cohorts were compared, diabetic cohorts were

consistently older and had higher BMI than the non-diabetic cohorts (hence age and

BMI were adjusted for in the modeling process). The average age of diabetic cohorts

was 53 years and average BMI was 33.1. These data align with NHANES surveys

(1988-2012), where the largest proportion of T2D were noted in the 45-64 age group.

Similarly, the average BMI was also comparable to previously published NHANES

surveys (1988-2012).28 There were no significant differences in gender between the

diabetic and non-diabetic cohorts.

We compared microbiome diversity within each group (alpha diversity), and

between groups (beta diversity), and by periodontal health status and diabetes

status. In the exploration of OTUs by abundance at the genus level, it was noted that

a distinction between diabetic and non-diabetic participants drove the largest number

of significant group differences. These results, together with the previously published

metabolomic data17, suggest that diabetes reduces the diversity of the oral

microbiome. Similarly, in exploring microbiome alpha diversity, the strongest effect

This article is protected by copyright. All rights reserved.

was imposed by diabetes status, where diabetics tended to have an overall decrease

in diversity compared to non-diabetics. While the beta diversity metric was significant

for groupings by diabetic status, the PCoA plot showed clusterings that were

generally centralized. It is possible that the modest sample size from the same

ecological environment explains the close clustering in the PCoA plot. Similarly, the

significant MRPP tests might be more an indicator of the size of the clusterings

rather than their location.

Oral microbiome diversity is affected by several factors. In this study, we

demonstrate a significant reduction in bacterial diversity in the saliva of adult

diabetics, contrary to our previous study which did not find significant differences in

in adolescents with T2D and obesity compared to periodontally healthy controls.10

These results also differ from those reported by Casarin et al.7, who found that

microbial diversity was greater in participants with diabetes compared to non-diabetic

participants. This could be explained by the fact that only uncontrolled diabetic

participants studied by Casarin at al., while the diabetics in the present study were

controlled by the use of medication.

Periodontal disease is characterized by destruction of the periodontium

secondary to an immune response to periodontopathogenic biofilm in a susceptible

host.29 Various studies have shown that in periodontal disease, there is a

significantly higher alpha diversity in periodontitis patients, supporting our findings.30-

32
Additionally, there was increased diversity in deeper periodontal pockets and the

difference was statistically significant when compared to shallow sites.33 We show in

the present study an increase in diversity from health to gingivitis to periodontitis.

This article is protected by copyright. All rights reserved.

Our results, together with Ge et al.33, indicate that perhaps the continuum of increase

is linear with progression of disease and severity. However, while the diversity was

higher in periodontitis, there was less inter-individual variation in periodontal disease

than in healthy samples.34

Our previous study found increased glucose concentration in the saliva of

diabetic participants17, an environmental exposure that may alter the composition of

the microbiome. Goodson et al. studied a large population of adolescents and

demonstrated an overall reduction in total salivary bacterial load and in the bacterial

counts of almost every species tested with increasing salivary glucose

concentration.16 Also, dysbiosis of the gut microbiome has been demonstrated in

diabetic patients. 4, 14 In their seminal work on gut microbiota in obesity, Turnbaugh

et al. found a reduction in microbial diversity in obesity, altered metabolic pathways

and alteration in the proportion of Firmicutes to Bacteroidetes.35-37 Here we did not

see a significant alteration in the Firmicutes to Bacteroidetes ratio (data not shown)

in diabetics, presumably due to the difference in ecology of oral and gastrointestinal

microbiome.38 However, we did see alterations in the salivary metabolic pathways in

participants with T2D.17

The oral cavity has one of the most diverse microbiomes when compared to

other microbial niches in the human body. 38 While the oral microbiome in health is

distinct39, there are various local and systemic factors that may play a role in shaping

its diversity and composition.40 In the present study, in Trend 1 an increased

abundance of Olsenella, Mitsukella, Peptidiphaga, Bifidobacterium, Abiotrophia,

Firmicutes, Veillonellaceae, Neisseriaceae, Actinobacterium, and Corynebacterium,

This article is protected by copyright. All rights reserved.

which includes a mix of primarily anaerobic commensals and potential

periodontopathogens, was found in both in the progression to periodontitis and in

diabetics compared to non-diabetics. When these conditions are considered

together, these genera generally decreased in diabetics compared to non-diabetics

with periodontitis. Interestingly, Filifactor and Treponema, genera associated with

periodontal disease, were increased in relation to periodontal disease status, but not

diabetes status. Trend 2 included a mix of other anaerobes that have been

associated with periodontal disease. These findings suggest that the genera

abundance continues to change with additional stress imposed by co-exiting

conditions.

Limitations

These findings also raise a pertinent question: Is the composition of the salivary and

subgingival plaque microbiomes similarly affected in diabetes? Traditionally,

characterization of the oral microbiome of periodontal disease has been done by

sampling subgingival plaque.30, 31, 41 A study of supragingival plaque, as well as tongue

scrapings and saliva samples used metagenomics and 16S rRNA sequencing for a

more comprehensive characterization of the oral microbiome.42 However, due to

ease of sampling, salivary samples provide a distinctive advantage for screening

purposes and have been used in various studies of health and disease. 10, 16, 43-47 In

addition to sampling, the choice of hypervariable regions of the 16S rRNA gene that

have been sequenced have an impact on characterizing the diversity of the oral

microbiome.31 Another potential limitation is lack of sample replicates within our

study design and thus we are not able to assess individual variability. Also, all of the

This article is protected by copyright. All rights reserved.

participants with diabetes in the current study had their blood glucose under control.

Perhaps the addition of individuals with pre-diabetes (i.e. fasting blood sugar 100-

125 mg/dL) may have provided an additional transitional microbial data.

CONCLUSIONS

Salivary microbiome diversity decreased in diabetics and increased with

progression of periodontal disease compared to periodontally healthy controls.

Select microbiota increased in both diabetes and periodontal disease, but decreased

in co-existing diabetes and periodontal disease. Further studies of the oral

microbiome at the species level may reveal unique signatures associated with both

periodontal and diabetic status.

ACKNOWLEDGMENTS

This study was supported by a grant from Colgate Palmolive Co., New York, NY.

The authors report no conflicts of interest related to this study. At the time of the

study was conducted, Dr. Barnes was an employee of Colgate Palmolive Co., but

had no conflicts of interest related to this study. AS and KG contributed equally to

this work.

This article is protected by copyright. All rights reserved.

REFERENCES

1. Alberti KG, Zimmet PZ. Definition, diagnosis and classification of diabetes

mellitus and its complications. Part 1: diagnosis and classification of diabetes
mellitus provisional report of a WHO consultation. Diabet Med 1998;15:539-
553.

2. Nathan DM. Long-term complications of diabetes mellitus. N Engl J Med

1993;328:1676-1685.

3. Llambes F, Arias-Herrera S, Caffesse R. Relationship between diabetes and

periodontal infection. World J Diabetes 2015;6:927-935.

4. Lambeth SM, Carson T, Lowe J, et al. Composition, diversity and abundance

of gut microbiome in prediabetes and type 2 diabetes. J Diabetes Obes
2015;2:1-7.

5. Teles R, Teles F, Frias-Lopez J, Paster B, Haffajee A. Lessons learned and

unlearned in periodontal microbiology. Periodontol 2000 2013;62:95-162.

6. Seksik P, Sokol H, Lepage P, et al. Review article: the role of bacteria in

onset and perpetuation of inflammatory bowel disease. Aliment Pharmacol
Ther 2006;24 Suppl 3:11-18.

7. Casarin RC, Barbagallo A, Meulman T, et al. Subgingival biodiversity in

subjects with uncontrolled type-2 diabetes and chronic periodontitis. J
Periodontal Res 2013;48:30-36.

8. Lalla E, Kaplan S, Chang SM, et al. Periodontal infection profiles in type 1

diabetes. J Clin Periodontol 2006;33:855-862.

9. Makiura N, Ojima M, Kou Y, et al. Relationship of Porphyromonas gingivalis

with glycemic level in patients with type 2 diabetes following periodontal
treatment. Oral Microbiol Immunol 2008;23:348-351.

10. Janem WF, Scannapieco FA, Sabharwal A, et al. Salivary inflammatory

markers and microbiome in normoglycemic lean and obese children

This article is protected by copyright. All rights reserved.

 compared to obese children with type 2 diabetes. PLoS One 2017;12;
doi:10.1371/journal.pone.0172647.

11. Taylor JJ, Preshaw PM, Lalla E. A review of the evidence for pathogenic
mechanisms that may link periodontitis and diabetes. J Periodontol
2013;84:S113-S134.

12. Filoche S, Wong L, Sissons CH. Oral biofilms: emerging concepts in microbial
ecology. J Dent Res 2010;89:8-18.

13. McLean JS. Advancements toward a systems level understanding of the

human oral microbiome. Front Cell Infect Microbiol 2014;4;
doi:10.3389/fcimb.2014.00098.

14. Larsen N, Vogensen FK, van den Berg FW, et al. Gut microbiota in human
adults with type 2 diabetes differs from non-diabetic adults. PLoS One 2010;5;
doi:

15. Zhou M, Rong R, Munro D, et al. Investigation of the effect of type 2 diabetes
mellitus on subgingival plaque microbiota by high-throughput 16S rDNA
pyrosequencing. PLoS One 2013;8; doi:

16. Goodson JM, Hartman ML, Shi P, et al. The salivary microbiome is altered in
the presence of a high salivary glucose concentration. PLoS One 2017;12:
doi:

17. Barnes VM, Kennedy AD, Panagakos F, et al. Global metabolomic analysis of
human saliva and plasma from healthy and diabetic subjects, with and without
periodontal disease. PLoS One 2014;9; doi:

18. Silness J, Loe H. Periodontal disease in pregnancy. Ii. correlation between

oral hygiene and periodontal condtion. Acta Odontol Scand 1964;22:121-135.

19. Lobene RR, Weatherford T, Ross NM, Lamm RA, Menaker L. A modified
gingival index for use in clinical trials. Clin Prev Dent 1986;8:3-6.

20. Schloss PD, Westcott SL, Ryabin T, et al. Introducing mothur: open-source,
platform-independent, community-supported software for describing and
comparing microbial communities. Appl Environ Microbiol 2009;75:7537-7541.

21. Rundegren J. Calcium-dependent salivary agglutinin with reactivity to various

oral bacterial species. Infect Immun 1986;53:173-178.

22. Robinson MD, Smyth GK. Moderated statistical tests for assessing
differences in tag abundance. Bioinformatics 2007;23:2881-2887.

This article is protected by copyright. All rights reserved.

23. McCarthy DJ, Chen Y, Smyth GK. Differential expression analysis of
multifactor RNA-Seq experiments with respect to biological variation. Nucleic
Acids Res 2012;40:4288-4297.

24. Benjamini Y, Hochberg Y. Controlling the false discovery rate - a practical and
powerful approach to multiple testing. J Roy Stat Soc B Met 1995;57:289-300.

25. Bray JR, Curtis JT. An ordination of the upland forest communities of southern
Wisconsin. Ecol Monogr 1957;27:326-349.

26. Mielke P, Berry K. Description of MRPP and additional MRPP applications. In:
Media SSaB, ed. Permutation methods: a distance function approach:
Springer Series in Statistics, 2007:11-123.

27. McMurdie PJ, Holmes S. phyloseq: an R package for reproducible interactive

analysis and graphics of microbiome census data. PLoS One 2013;8:e61217.

28. Casagrande SS, Cowie CC. Trends in dietary intake among adults with type 2
diabetes: NHANES 1988-2012. J Hum Nutr Diet 2017;30:479-489.

29. Kornman KS. Mapping the pathogenesis of periodontitis: a new look. J

Periodontol 2008;79:1560-1568.

30. Abusleme L, Dupuy AK, Dutzan N, et al. The subgingival microbiome in health
and periodontitis and its relationship with community biomass and
inflammation. ISME J 2013;7:1016-1025.

31. Griffen AL, Beall CJ, Campbell JH, et al. Distinct and complex bacterial
profiles in human periodontitis and health revealed by 16S pyrosequencing.
ISME J 2012;6:1176-1185.

32. Li Y, He J, He Z, et al. Phylogenetic and functional gene structure shifts of the

oral microbiomes in periodontitis patients. ISME J 2014;8:1879-1891.

33. Ge X, Rodriguez R, Trinh M, Gunsolley J, Xu P. Oral microbiome of deep and

shallow dental pockets in chronic periodontitis. PLoS One 2013;8; doi:

34. Liu B, Faller LL, Klitgord N, et al. Deep sequencing of the oral microbiome
reveals signatures of periodontal disease. PLoS One 2012;7; doi:

35. Ley RE, Turnbaugh PJ, Klein S, Gordon JI. Microbial ecology: human gut
microbes associated with obesity. Nature 2006;444:1022-1023.

This article is protected by copyright. All rights reserved.

36. Turnbaugh PJ, Hamady M, Yatsunenko T, et al. A core gut microbiome in
obese and lean twins. Nature 2009;457:480-484.

37. Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI. An
obesity-associated gut microbiome with increased capacity for energy
harvest. Nature 2006;444:1027-1031.

38. Human Microbiome Project C. Structure, function and diversity of the healthy
human microbiome. Nature 2012;486:207-214.

39. Zaura E, Keijser BJ, Huse SM, Crielaard W. Defining the healthy "core
microbiome" of oral microbial communities. BMC Microbiol 2009;9; doi:

40. Wade WG. The oral microbiome in health and disease. Pharmacol Res
2013;69:137-143.

41. Haffajee AD, Cugini MA, Tanner A, et al. Subgingival microbiota in healthy,
well-maintained elder and periodontitis subjects. J Clin Periodontol
1998;25:346-353.

42. Galimanas V, Hall MW, Singh N, et al. Bacterial community composition of

chronic periodontitis and novel oral sampling sites for detecting disease
indicators. Microbiome 2014;2; doi: .

43. Cameron SJ, Huws SA, Hegarty MJ, Smith DP, Mur LA. The human salivary
microbiome exhibits temporal stability in bacterial diversity. FEMS Microbiol
Ecol 2015;91; doi:

44. Haririan H, Andrukhov O, Bertl K, et al. Microbial analysis of subgingival

plaque samples compared to that of whole saliva in patients with periodontitis.
J Periodontol 2014;85:819-828.

45. Ogawa T, Honda-Ogawa M, Ikebe K, et al. Characterizations of oral

microbiota in elderly nursing home residents with diabetes. J Oral Sci
2017;59:549-555.

46. Stahringer SS, Clemente JC, Corley RP, et al. Nurture trumps nature in a
longitudinal survey of salivary bacterial communities in twins from early
adolescence to early adulthood. Genome Res 2012;22:2146-2152.

47. Yang F, Zeng X, Ning K, et al. Saliva microbiomes distinguish caries-active

from healthy human populations. ISME J 2012;6:1-10.

This article is protected by copyright. All rights reserved.

Figure Legend

Figure 1. Heat map dendrogram of model coefficients from the generalized

linear model for significant genera. Red/green values indicate an

increase/decrease in genera abundance with the progression to periodontal

disease (Oral Health variable), and as a function of diabetes (Diabetic variable).

The Interaction variable compares the change in trends of genera abundance

(increase or decrease) between progression to periodontal disease (Oral

Health variable) and diabetes (Diabetic variable). Interaction colors indicate the

change in genera abundance with progressing periodontal disease relative to

diabetes: zero (yellow or orange), diabetics = non-diabetics; positive (red),

diabetics > non-diabetics; and negative (green) diabetics < non-diabetics.

Trend 1, genera associated with increasing abundance with periodontal

disease progression and larger abundance in diabetics relative to non-

diabetics; Trend 2, genera associated with primarily a decrease in abundance

in diabetics relative to non-diabetics.

This article is protected by copyright. All rights reserved.

Figure 2. Alpha diversity of microbiota. Panel A, oral health status: A significant (p <

0.05) increase in diversity (Observed, Chao1 and ACE) was noted in the progression

to periodontitis. Panel B, diabetes: A significant (p < 0.05) decrease in diversity

(Observed, Chao1, ACE, and Shannon) was noted in diabetics compared to

controls. C, Control; D, Diabetic; G, Gingivitis; P, Periodontitis.

This article is protected by copyright. All rights reserved.

Figure 3. Beta diversity of microbiota. Panel A, oral health status and Panel B,

diabetics. There was a significant difference in beta diversity between

diabetics and non-diabetics (p = 0.002), but not between groups by oral health

status (p = 0.73). C, Control; D, Diabetic; G, Gingivitis; P, Periodontitis.

This article is protected by copyright. All rights reserved.

Table 1.

Clinical C G P D DG DP P
value

variable (n=19) (n=22) (n=23) (n=26) (n=26) (n=27)

Age, (33.8±14. (35±12.4 (49.2±11. (50.5±10. (54.1±7. (54.3±6. <0.000

years 5) ) 2) 9) 4) 7) 1
(mean±S
D)

BMI (25.7±4.7) (26.5±5. (30.6±5.9) (33.5±9.7) (32.8±7. (32.6±6. 0.0003

(mean±S 5) 8) 8)
D)

Gender, 6/13 8/14 10/13 15/11 11/15 17/10 0.2123

M/F

HbA1c, NA NA NA (7.1±1.1) (7.3±1.6) (7.7±1.9) 0.4179

(mean±SD
)

Summary of clinical variables for 143 participants

NA, not applicable

Supplementary Table 1.

This article is protected by copyright. All rights reserved.