Salivary Features of Periodontitis and Gingivitis in Type 2 Diabetes Mellitus

doi:10.21203/rs.3.rs-4391317/v1

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Salivary Features of Periodontitis and Gingivitis in Type 2 Diabetes Mellitus

https://doi.org/10.21203/rs.3.rs-4391317/v1

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Type 2 diabetes mellitus (T2DM) is associated with cellular abnormalities, tissue and organ dysfunctions, and periodontitis. This investigation examined the relationship between the oral microbiome and salivary biomarkers in T2DM patients with or without periodontitis. This cohort (35–80 years) included systemically healthy non-periodontitis (NP; n = 31), T2DM without periodontitis (DWoP; n = 32) and T2DM with periodontitis (DWP; n = 29). The oral microbiome [Operational Taxonomic Units (OTUs)] (16s rRNA sequencing) and targeted host salivary biomarkers (immunoassays) were assessed. We identified 47 OTUs that were significantly different in abundance between NP samples and any disease subset or between disease subgroups. The most unique microbiome patterns were observed in the DWP group. Differences in genera/species abundance were also observed when T2DM patients were stratified by extent of periodontal inflammation and disease (i.e., generalized versus localized gingivitis/periodontitis). Salivary biomarkers showed significant elevations in MMP-8, MMP-9, resistin, IL-1β, IL-6, IFNα, and BAFF (THFSR13b) comparing generalized to localized periodontitis. Salivary analytes showed significant positive correlations with specific microbiome members, predominantly in DWP patients. Odds ratio analyses reinforced that a panel of biologic markers (IL-6, MMP-8) and bacteria (e.g., Bacteroidetes, Fusobacteria, Spirochaetes) discriminated the severity and extent of periodontal disease in this diabetic population.

Biological sciences/Immunology

Biological sciences/Microbiology

Health sciences/Biomarkers

Health sciences/Diseases

diabetes

microbiome

inflammation

periodontitis

saliva

Type 2 diabetes mellitus (T2DM) is a chronic inflammatory disease with elevated systemic biomolecules of inflammation that are central to the pathogenesis of the disease.^1–5 Beyond the identification of periodontitis as a sequelae of T2DM, localized inflammatory proteins are routinely increased in the gingival crevicular fluid and saliva of periodontitis affected T2DM patients.^6–10 Within this oral environment the host responses are driven by, and reflect, an altered microbiome with dysbiotic changes of opportunistic pathogens and pathobionts revealing a lesional process.^11–15

As variations in the characteristics of the oral microbiome and associated host responses have been related to extrinsic and intrinsic (e.g., T2DM, age) effectors of the host ecosystem ^16–19, there remain gaps in our knowledge concerning the biologic mechanisms that are controlling these features in the oral cavity. Additionally, mechanical therapeutics in systemically normal individuals have been shown to substantially impact the microbiome and help transition its characteristics to one more consistent with periodontal health.^20–22 Nevertheless, existing data indicate that the prevalence and severity of periodontitis is increased in T2DM^23–27.

This investigation cross-tabulated the microbiome and biomarkers in saliva of T2DM patients with or without periodontitis. The findings provide insight into the unique features of localized and generalized gingivitis and periodontal disease in the presence of T2DM.

Oral clinical features in diabetes

Figure 1 provides the periodontal characteristics and BMI relative to the various subgroups of T2DM and NP subjects. Only the NP group showed a significantly lower BMI compared to most of the T2DM groups (e.g., DWoP, DWP). In contrast and as expected, the clinical features of destructive disease in the generalized periodontitis patients were significantly different from both the gingivitis and localized periodontitis groups. Additionally, BOP showed significantly greater levels with increasing severity of disease. Of note, the majority of periodontally healthy individuals were in the NP group, and the majority of gingivitis cases were in the DWoP group. Based upon the disease features in affected patients, an additional perspective evaluated the characteristics of the microbiome and host response patterns related to the magnitude of disease.

Microbiome characteristics in generalized and localized disease in diabetes

Supplemental Fig. 1 provides a summary of the individual sample OTU reads for the NP, GG, LG, GP, and LP salivary microbiomes. Generally, similar numbers of overall reads were identified across the subgroups. In another report we identified the top 104 OTUs based upon levels across the three study groups (NP, DWoP, DWP), and showed that this portfolio of microorganisms provide 96–98% coverage of the microbiome reads in the samples ³⁸ (Table 1). Figure 2A presents the results for the individual NP subjects and shows Firmicutes (Bacillota) as the dominant phyla, with the relative abundance varying from ~ 40–80%. Bacteroidetes phyla was next most prevalent, being detected in 23 subjects. Proteobacteria was observed in the remaining six individuals. A subset of NP subjects demonstrated elevated levels of Spirochetes and four individuals had elevated Absconditabacteria in their salivary microbiome.

Table 1

Operational taxonomic units (OTUs) included in the analysis.
OTU	Phyla	Genera/Species
Otu150	Absconditabacteria_(SR1)	Absconditabacteria_(SR1)_[G-1] bacterium_HMT_345
Otu130	Absconditabacteria_(SR1)	Absconditabacteria_(SR1)_[G-1] bacterium_HMT_875
Otu025	Actinobacteria	Actinomyces graevenitzii
Otu029	Actinobacteria	Actinomyces sp._HMT_169
Otu007	Actinobacteria	Actinomyces sp._HMT_180
Otu005	Actinobacteria	Actinomyces_unclassified
Otu049	Actinobacteria	Atopobium_unclassified
Otu106	Actinobacteria	Bifidobacterium longum
Otu053	Actinobacteria	Corynebacterium matruchotii
Otu014	Actinobacteria	Rothia mucilaginosa
Otu080	Actinobacteria	Rothia_unclassified
Otu105	Bacillota	Selenomonas artemidis
Otu163	Bacillota	Selenomonas sp._HMT_126
Otu023	Bacillota	Selenomonas sp._HMT_136
Otu034	Bacillota	Selenomonas sputigena
Otu019	Bacillota	Selenomonas_unclassified
Otu048	Bacillota	Stomatobaculum longum
Otu087	Bacillota	Stomatobaculum sp._HMT_097
Otu064	Bacteroidetes	Alloprevotella rava
Otu058	Bacteroidetes	Alloprevotella sp._HMT_308
Otu111	Bacteroidetes	Alloprevotella sp._HMT_473
Otu042	Bacteroidetes	Alloprevotella tannerae
Otu147	Bacteroidetes	Bacteroidales_[G-2] bacterium_HMT_274
Otu098	Bacteroidetes	Bergeyella sp._HMT_322
Otu062	Bacteroidetes	Capnocytophaga gingivalis
Otu063	Bacteroidetes	Capnocytophaga granulosa
Otu065	Bacteroidetes	Capnocytophaga leadbetteri
Otu074	Bacteroidetes	Capnocytophaga sputigena
Otu083	Bacteroidetes	Porphyromonas endodontalis
Otu057	Bacteroidetes	Porphyromonas gingivalis
Otu038	Bacteroidetes	Porphyromonas pasteri
Otu173	Bacteroidetes	Prevotella dentalis
Otu054	Bacteroidetes	Prevotella denticola
Otu136	Bacteroidetes	Prevotella intermedia
Otu033	Bacteroidetes	Prevotella nanceiensis
Otu070	Bacteroidetes	Prevotella nigrescens
Otu018	Bacteroidetes	Prevotella oris
Otu068	Bacteroidetes	Prevotella oulorum
Otu017	Bacteroidetes	Prevotella pallens
Otu164	Bacteroidetes	Prevotella pleuritidis
Otu015	Bacteroidetes	Prevotella salivae
Otu092	Bacteroidetes	Prevotella sp._HMT_300
Otu073	Bacteroidetes	Prevotella sp._HMT_305
Otu011	Bacteroidetes	Prevotella veroralis
Otu001	Bacteroidetes	Prevotella_unclassified
Otu085	Bacteroidetes	Tannerella sp._HMT_286
Otu067	Firmicutes	Catonella morbi
Otu135	Firmicutes	Dialister invisus
Otu031	Firmicutes	Gemella morbillorum
Otu027	Firmicutes	Granulicatella adiacens
Otu056	Firmicutes	Lachnoanaerobaculum orale
Otu125	Firmicutes	Lachnoanaerobaculum umeaense
Otu035	Firmicutes	Lachnospiraceae_[G-2] bacterium_HMT_096
Otu022	Firmicutes	Megasphaera micronuciformis
Otu158	Firmicutes	Mogibacterium vescum
Otu252	Firmicutes	Mycoplasma faucium
Otu051	Firmicutes	Oribacterium asaccharolyticum
Otu052	Firmicutes	Oribacterium_unclassified
Otu112	Firmicutes	Parvimonas micra
Otu137	Firmicutes	Peptostreptococcaceae_[XI][G-1]_unclassified
Otu078	Firmicutes	Peptostreptococcus stomatis
Otu094	Firmicutes	Ruminococcaceae_[G-1] bacterium_HMT_075
Otu129	Firmicutes	Ruminococcaceae_[G-2] bacterium_HMT_085
Otu012	Firmicutes	Streptococcus oralis_subsp._dentisani_clade_058
Otu008	Firmicutes	Streptococcus parasanguinis_clade_411
Otu006	Firmicutes	Streptococcus salivarius
Otu004	Firmicutes	Streptococcus_unclassified
Otu003	Firmicutes	Veillonella dispar
Otu009	Firmicutes	Veillonella parvula
Otu119	Firmicutes	Veillonella sp._HMT_780
Otu069	Firmicutes	Veillonella sp._HMT_917
Otu002	Firmicutes	Veillonella_unclassified
Otu152	Firmicutes	Veillonellaceae_[G-1] bacterium_HMT_155
Otu024	Fusobacteria	Fusobacterium nucleatum_subsp._vincentii
Otu059	Fusobacteria	Fusobacterium sp._HMT_203
Otu013	Fusobacteria	Fusobacterium_unclassified
Otu044	Fusobacteria	Leptotrichia hongkongensis
Otu047	Fusobacteria	Leptotrichia sp._HMT_212
Otu020	Fusobacteria	Leptotrichia sp._HMT_215
Otu095	Fusobacteria	Leptotrichia sp._HMT_218
Otu045	Fusobacteria	Leptotrichia sp._HMT_221
Otu185	Fusobacteria	Leptotrichia sp._HMT_223
Otu046	Fusobacteria	Leptotrichia sp._HMT_392
Otu055	Fusobacteria	Leptotrichia wadei
Otu016	Fusobacteria	Leptotrichia_unclassified
Otu165	Fusobacteria	Sneathia_unclassified
Otu071	Proteobacteria	Campylobacter rectus
Otu171	Proteobacteria	Campylobacter sp._HMT_044
Otu026	Proteobacteria	Campylobacter_unclassified
Otu091	Proteobacteria	Escherichia coli
Otu110	Proteobacteria	Haemophilus influenzae
Otu010	Proteobacteria	Haemophilus_unclassified
Otu115	Proteobacteria	Kingella_unclassified
Otu097	Proteobacteria	Lautropia mirabilis
Otu028	Proteobacteria	Neisseria macacae
Otu066	Proteobacteria	Neisseria oralis
Otu039	Proteobacteria	Neisseria perflava
Otu021	Proteobacteria	Neisseria_unclassified
Otu050	Proteobacteria	Pasteurellaceae_unclassified
Otu146	Spirochaetes	Treponema denticola
Otu099	Spirochaetes	Treponema socranskii
Otu156	Synergistetes	Fretibacterium fastidiosum
Otu123	Synergistetes	Fretibacterium sp._HMT_361
Otu114	Synergistetes	Fretibacterium_unclassified

We next stratified the diabetic periodontitis (DWP) and gingivitis (DWoP) patients based on the clinical presentation (i.e., generalized or localized form) of periodontal inflammation and lesions to examine the phyla distribution. Firmicutes was the dominant phyla across the majority of DWoP patients with mean levels similar between generalized and localized gingivitis (Fig. 2B). Three individuals showed Bacteroidetes as the dominant phyla. Generally, this phylum appeared more prevalent in the GG patients, while the Proteobacteria phylum was elevated across the LG subset. An elevated abundance was seen in LG for Absconditabacteria. Figure 2C presents the phyla data for the DWP group with GP and LP. These generalized disease adults showed higher levels of Bacteroidetes, Fusobacteria, Proteobacteria, Spirochaetes, and Synergistetes compared to the localized disease group although the differences did not reach statistical significance.

The evaluation of the microbiome differences was extended to focus on the individual microbial components in the various patient groups. Figure 3 illustrates the relative abundance of the targeted set of 104 OTUs in samples from the study population based on the level of oral disease (i.e., NP [healthy], LG, GG, LP, and GP). We identified 47 OTUs (Fig. 2) that were significantly different in abundance between NP and any disease subset or between disease subgroup categories (Table 2). There also were numerous OTU abundance differences (N = 18) between LG and LP. Seven bacterial species differences in abundance were observed between GG and GP, four bacterial species abundance differences were observed between LG and GG, and only one species (P. gingivalis) that was different in relative abundance between LP and GP.

Table 2

Microbiome relative abundance differences between generalized and localized disease in T2DM patients. These taxa are significantly different between the groups determined by a *t test* (see Fig. 2).
GG vs. LG	GP vs. LP	GG vs. GP	LG vs. LP
Prevotella_unclassified M. micronuciformis Prevotella sp._HMT_305 Bergeyella sp._HMT_322	P. gingivalis	F. nucleatum_subsp._vincentii Prevotella nigrescens Rothia_unclassified Alloprevotella tannerae P. gingivalis Catonella morbi Fretibacterium fastidiosum	Veillonella_unclassified Gemella morbillorum Prevotella nigrescens Stomatobaculum sp._HMT_097 Capnocytophaga gingivalis P. endodontalis Treponema socranskii Selenomonas sp._HMT_126 Prevotella intermedia Porphyromonas gingivalis Treponema denticola Fretibacterium fastidiosum Tannerella sp._HMT_286 Fretibacterium_unclassified Kingella_unclassified Bacteroidales_[G-2]_HMT_274 Prevotella sp._HMT_300 Mycoplasma faucium
GG: generalized gingivitis, GP: generalized periodontitis, LG: localized gingivitis, LP: localized periodontitis.

Salivary biomarkers, disease severity, and microbiome in diabetes

Salivary concentrations of host response analytes by study group and periodontal disease category are depicted in Fig. 4. Here, nine host biomarkers showed major concentration differences between the NP subjects and DWP individuals, both GP and LP. MMP-8, MMP-9, TIMP-1, and MIP-1α were the only markers that significantly differed between health and the subgroups of gingivitis (DWoP) patients. Resistin concentrations were significantly increased in the DWP periodontitis subgroups, while differences in adiponectin levels between disease categories were not observed.

As the oral microbiome both drives and responds to host responses in the oral cavity, it was of interest to explore the relationship of targeted salivary biomolecules to the microbiome components and how that relationship was affected by diabetes and periodontal characteristics. Figure 5A shows the frequency of significant correlations of the individual biomarker levels with the relative abundance of the 104 OTUs in the microbiome samples. These correlations were generally higher in the NP and DWP groups, with MMP-8, BAFF, adiponectin, and resistin showing the greatest frequency of positive correlations within the 3 subject groups. In contrast, there were only a limited number of negative correlations with any of the host molecules and the array of microbial taxa identified. Figure 5B summarizes the details of the bacterial species that dominated these correlations with the 11 salivary biomarkers. As was expected few correlations with individual bacterial species were noted in the DWoP samples. The findings depicts 27 of the OTUs with extensive correlations to the salivary analytes, with 17 in the DWP patients and 10 in the NP subjects. The identified OTUs encompassed members of the Bacteroidetes, Firmicutes (Bacillota), Fusobacterium, Spirochaetes, and Synergistetes phyla.

Supplemental Fig. 2A-D summarizes features of the correlations between individual OTUs of the 104 dominant members and levels of the individual salivary analytes. In the DWoP LG group, several significant correlations were observed, with the majority of positive correlations occurring with resistin and TIMP-1 (Fig. 2A). In the DWoP GG patients, IL-1ß and adiponectin showed parallel patterns of positive correlations (Fig. 2B). Additionally, correlations with MMP-8 and IL-6 were all positive but with different OTUs. All significant correlations with MIP-1α were negative in this patient subset. Figure 2C summarizes the values for the DWP LP group that showed IFNα, TIMP-1, and adiponectin positively correlated with multiple OTUs. Finally, the largest frequency of correlations was noted in the DWP GP (Fig. 2D) subjects. Here IL-1β, IL-6, IFNα, BAFF, adiponectin, and resistin were the predominant biomarkers that correlated with specific OTU, and frequently the same OTU.

These various relationships allowed us to explore whether a targeted array of microbes and salivary biomarkers might be predictive of generalized and localized disease in T2DM patients. Odds Ratios (OR) were determined comparing levels of bacteria and host response biomolecules in periodontitis versus gingivitis subjects (Fig. 6A). Here we observed OR of > 2.5 for most of the host biomarkers in the GP group, as well as increased ORs in the LP group. A similar number of oral bacteria exhibited ORs > 2 in LP or GP when compared to the gingivitis group. However, striking was an array of the bacterial species that displayed substantially lower ORs in the GP group compared to GG group. Figure 6B provides a similar analysis for generalized versus localized inflammation or disease. Over 60% of the targeted host analytes showed ORs > 2 in LP compared with GP. Additionally, multiple bacterial species were specifically elevated in samples in the GP patients (ORs > 2). Similarly, multiple species of bacteria were specifically increased in the GG versus LG patients that were frequently different from those elevated in GP (eg. R. mucilaginosa, Prevotella sp. HMT305, V. dispar). Also, a limited number of host response markers varied in the GG compared to the LG group (i.e., IL-6, MMP-8, adiponectin).

Epidemiological studies confirm that diabetes is a significant risk factor for periodontitis, and periodontitis increases the risk of poor diabetes management and sequelae in affected individuals.^16,39-42 Thus, this interplay between these chronic inflammatory conditions reflects the difficulty in controlling glucose levels and the initiation and progression of periodontitis.^43-49 The related clinical features reflect dysregulated inflammatory responses that are linked to tissue changes in T2DM, a microbiome contribution from periodontitis, and genetic regulation of the inflammatory status of the individual. l^43,50-55 Extensive reports identify the bidirectional linkage of diabetes and periodontitis, and studies that identify biological factors that contribute to the various stages of periodontal disease are not well characterized with these comorbidities. Hence, we sought to further characterize the salivary microbiome and associated host responses in T2DM patients (i.e., T2DM patients with periodontitis [DWP] or with gingivitis [DWoP]), with the goal of better understanding the panel of biological factors involved in the different extent and categories of periodontal disease compared to a NP healthy control group. This report thus described differences in the microbiomes at the individual patient level regarding the phyla distribution, and significant differences between localized and generalized gingivitis and periodontitis. Of the 104 dominant OTUs, approximately ¼ of these bacterial identifiers showed significant differences between health and the various disease presentation of localized and generalized periodontal diseases. These findings are consistent with the pathogenic microbiome in periodontitis, and particularly with more generalized disease.

Considerable variation in inflammatory biomolecules concentrations have been observed in gingival crevicular fluid, saliva, and blood of patients with diabetes and periodontal disease.^56-60 Other studies have attempted to associate these mediators with periodontitis and the transition from health to metabolic syndrome and even T2DM.⁶¹ Substantial evidence attributes pathogenic mechanisms linking periodontitis and diabetes to dysregulated inflammation.⁶² We have shown previously in this population differences in salivary biomarkers between normal subjects and patients with T2DM, with or without periodontitis ²⁹ that was consistent with the extant literature. However, in spite of the large number of studies on inflammatory mechanisms in periodontitis and diabetes, few have documented oral microbiome features that relate with specific host responses in the presence of these comorbidities. Various classic periodontal pathogens have been identified in diabetic and non-diabetic periodontitis patients, with P. gingivalis appearing in higher levels in both T2DM and T1DM patients.^63-67 A growing body of evidence suggests that diabetes may alter the local periodontal pocket environment favoring certain bacterial species to emerge. However, existing studies suggest some subtle differences in the oral microbiomes in diabetic patients, but clear clinical relevance of these differences have not been discerned. In the present study, significant differences were observed in various salivary analytes, especially MMP-8, MMP-9, TIMP-1, IL-1b, BAFF, and resistin primarily in the DWP versus both DWoP and NP subjects. Exploration of the relationship between the salivary analytes and individual members of the oral microbiome showed a greater number of significant, generally positive, correlations with MMP-8, BAFF, IFNa, adiponectin, and resistin in the DWP individuals. As the relationship between the chronic diseases of T2DM and periodontitis have emphasized a dysregulated host response, these findings support alterations in the host-microbe interactions when these diseases are coincident.

Our previous report described differences in the oral microbiomes of normal subjects, T2DM patients without periodontitis (DWoP), and T2DM patients with periodontitis (DWP) (Ebersole et al., in review). In this analysis, we also identified several differences in the microbiomes within the DWoP and DWP groups that were stratified based upon localized or generalized clinical presentation of inflammation and destructive disease. While members of the Firmicutes (Bacillota) phyla were the dominant group of bacteria in both DWP and DWoP patients, there were clear differences in the GG (DWoP) and GP (DWP) subgroups showing elevated abundance of Bacteroidetes, Fusobacteria, Spirochaetes, and Synergistetes compared to the localized disease group. Thus, for the first time we demonstrated within the T2DM stratification, microbiomic variations appeared to relate to the magnitude (extent, severity) of oral inflammation and disease.

For oral health in systemically healthy subjects or T2DM patients, there is a critical balance between tissue homeostasis and a transition into the microbial driven disease of periodontitis. Both genetic and environmental features regulate features of an individual’s response to the burden and quality of the oral microbiome.^42,68-70 As we have shown previously, there are distinctive differences in targeted host response biomolecules in saliva of these T2DM patients, as well as the characteristics of the salivary microbiome patterns coincident with these altered host response profiles.³³ We now identified clear differences in both the microbiome and salivary analytes related to disease extent in T2DM patients. Additionally, correlations between selected salivary analytes and specific members of the microbiome appeared to vary with disease extent, with the T2DM patients who had GP showing the greatest number of significant relationships. This type of analysis does not provide any cause-and-effect understanding of the processes that occur to reach this disease susceptible milieu in the oral environment. However, the results appear to reflect certain bacterial genera/species including Actinomyces_unclassified, Pr. dentalis, F. nucleatum_ssp._vincentii, Leptotrichia sp._HMT_218, P. endodontalis, Sneathia_unclassified, T. denticola, and M. faucium that relate more directly to specific salivary analytes, such as IL-6, adiponectin, BAFF, and resistin. Consideration of these host and microbial biomarkers could be useful in portending the likelihood of more severe disease and aid in improved clinical decision-making on patient specific therapy.

Limitations of this study include an overall number of subjects that were stratified based upon periodontal disease clinical characteristics of health or extent of oral disease that reduced the individual subgroup size. As such, the findings observed from these small subgroups may not be biologically generalizable to the broader T2DM or non-T2DM population. Also, poorly controlled diabetics were not included and may manifest distinctive host and microbiome traits.^50,71 This study, as most oral microbiome reports, describe the microbial ecology based upon a discrete sample thus lacking a general assessment of the permanency or transitory nature of the salivary microbiome in individual subjects. Finally, although saliva reflected many microbiome patterns apparent in the subgingival environment, saliva may not reflect all aspects important for understanding the biology of periodontitis.

Study and Participants

This cohort clinical study, approved by the Institutional Review Board of the University of Kentucky (UK) #17-0439-F6A in accordance with the Helsinki Declaration, was performed from June 2017 to July 2019. Inclusion criteria were: 35-80 years, a minimum of 16 teeth (excluding 3rd molars), in good systemic health (excluding the case definition), or diagnosed with T2DM based on the criteria of the American Diabetes Association.²⁸ We have reported previously that T2DM was confirmed by the patient's physician with glycated hemoglobin A1c (HbA1c), body mass index (BMI), and complete head, neck, and oral examination were recorded ²⁹. All subjects provided signed informed consent reviewed and accepted by the University of Kentucky Institutional Review Board.

A full-mouth periodontal examination was conducted by a single calibrated dental examiner. Periodontal health measures included bleeding on probing (BOP), pocket depth (PD) and clinical attachment loss (CAL) at six sites per tooth.³⁰ Three cohorts were enrolled: (a) T2DM subjects with chronic periodontitis (DWP; n = 29), (b) T2DM subjects without chronic periodontitis (DWoP; n = 32) and (c) a cohort of healthier participants designated as ‘Not-Periodontitis’ (NP) (n = 31). Periodontitis was defined as having at least four teeth in two quadrants with a probing depth (PD) of ≥5 mm, clinical attachment loss (AL) of ≥2 mm and bleeding upon probing (BOP).^31,32 Participants in the NP group were defined as (a) no diabetes, (b) <20% proximal probing sites with a bleeding score of > 1 (pinpoint bleeding on gentle probing), (c) <5% of sites with PD of ≥4 mm (d) no PD >5 mm, (e) <2% of sites with CAL of > 2 mm with concurrent BOP and PD of >4 mm. The three groups were balanced for age and sex.

The extent of gingivitis and periodontitis in each patient was evaluated and used to categorize the patients as displaying localized / generalized gingivitis (LG, GG) or localized / generalized periodontitis (LP, GP). The DWoP group demonstrated gingivitis (i.e., BOP at <20% of sites [LG] or >20% BOP sites [GG] coupled with all having less than five sites with >4 mm periodontal pockets). The DWP group demonstrated either LP (4-87% BOP; <10% sites with >4 mm pockets) or GP (21-97% BOP; >10% sites with >4 mm pockets).

As reported previously ³³, medical and dental histories were obtained and subjects were excluded with alcoholism; liver, kidney or salivary gland dysfunction; inflammatory bowel disease; granulomatous diseases; immunosuppressive or cancer therapy. Additionally, acute illness (i.e., fever, sore throat, body aches and diarrhea), pregnancy or lactation, use of antibiotics within the last 6 months, need for antibiotics for dental procedures, or the presence of an oral mucosal inflammatory conditions (e.g., aphthous, lichen planus, leukoplakia, and oral cancer) and current smoker were exclusion criteria.

Biologic samples and analysis

Unstimulated whole saliva samples was obtained from each participant, managed, stored, and concentrations of interleukin (IL)-1β, interleukin-6 (IL-6), MIP-1a (macrophage inflammatory protein 1 alpha), BAFF (B-cell activating factor; TNFSF13b), IFNa (interferon alpha), adiponectin, and resistin were measured in duplicate using Luminex technology with human cytokine/chemokine multiplex kits (Millipore, St. Charles, MO, USA). Salivary concentrations of matrix metalloprotease (MMP)-8, MMP-9 and TIMP-1 (tissue inhibitor of matrix matellaoproteinases-1) were determined in duplicate for each subject using human Quantikine enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN, USA, as we have previously described.^29,34,35

Salivary microbiome characteristics were determined in each subject’s samples using 16S rRNA sequencing as we have previously described.^36-38 Sequences were assigned to their respective taxonomic classification using the Human Oral Microbiome Database (HOMD V13) (http://www.homd.org/index.php?name=seqDownload&file&type=R). The raw data are deposited at BioProject ID PRJNA516659 through the NIH NCBI.

Statistical Analysis

Descriptive statistics were performed for the clinical features, Operational Taxonomic Unit (OTU) microbiome differences, and log transformed salivary analyte comparisons through ANOVA or two sample t-test. Tukey’ honest significant difference method (HSD) was used for pairwise comparisons of the variables among groups of patients, if an ANOVA test was significant. The statistical software package (SAS 9.4, Cary, NC; IBM Inc., 2020) was used for the analysis and the statistical significance level was set at 0.05. Correlation analyses were evaluated using a Pearson correlation coefficient with a significant level of 0.05. The data for gene expression have been uploaded into GEO accession GSE180588 (https://www.ncbi.nlm.nih.gov/gds).

ACKNOWLEDGEMENTS

We thank Sabrina Braden, Dawn Dawson and Jason Stevens for their contributions in the recruitment, enrollment, data collection and laboratory analyses. This study was supported by the Delta Dental Foundation of Michigan.

Author Contributions

Author contributions include JLE and CSM contributed to conception, design, data acquisition and interpretation, drafted and critically revised the manuscript. SSK contributed to data acquisition, analysis, and interpretation, and critically reviewed the manuscript. DDIII contributed to obtaining and interpreting the clinical data and critically reviewed the manuscript. XDZ provided biostatistical evaluation for data analysis, interpretation, and critical revision of the manuscript. All authors gave their final approval and agreement to be accountable for all aspects of the work. The authors state no conflict with any information provided in the report.

Data Availability

The microbiome data are deposited at BioProject ID PRJNA516659 through the NIH NCBI. The data for gene expression have been uploaded into GEO accession GSE180588 (https://www.ncbi.nlm.nih.gov/gds).

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No competing interests reported.

Deltadentalhostsupplemental524.pptx
Supplemental Figure 1: Summation of total reads for the detected OTUs in the saliva samples from various subsets of subjects. Each point denotes the value for the sample from one subject. The horizontal bar denotes the mean reads for that group. Supplemental Figure 2: Correlations for individual microbiome OTUs and the salivary analytes in T2DM patients with generalized (A; GG) and localized (B; LG) gingivitis, as well as generalized (C; GP), and localized (D; LP) periodontitis. The points denote the correlation coefficient for a specific OTU as shown on the X-axis (see Table 1). The point above or below the shaded rectangles distinguish the salivary analyte and associated OTU that exceeded significant positive or negative correlation values (p<0.01).

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Salivary Features of Periodontitis and Gingivitis in Type 2 Diabetes Mellitus

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Abstract

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INTRODUCTION

RESULTS

Oral clinical features in diabetes

Microbiome characteristics in generalized and localized disease in diabetes

Salivary biomarkers, disease severity, and microbiome in diabetes

DISCUSSION

METHODS

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References

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Supplementary Files

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