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Metatranscriptomics of the Human Oral Microbiome during Health and Disease

ABSTRACT The human microbiome plays important roles in health, but when disrupted, these same indigenous microbes can cause disease. The composition of the microbiome changes during the transition from health to disease; however, these changes are often not conserved among patients. Since microbiome-associated diseases like periodontitis cause similar patient symptoms despite interpatient variability in microbial community composition, we hypothesized that human-associated microbial communities undergo conserved changes in metabolism during disease. Here, we used patient-matched healthy and diseased samples to compare gene expression of 160,000 genes in healthy and diseased periodontal communities. We show that health- and disease-associated communities exhibit defined differences in metabolism that are conserved between patients. In contrast, the metabolic gene expression of individual species was highly variable between patients. These results demonstrate that despite high interpatient variability in microbial composition, disease-associated communities display conserved metabolic profiles that are generally accomplished by a patient-specific cohort of microbes. IMPORTANCE The human microbiome project has shown that shifts in our microbiota are associated with many diseases, including obesity, Crohn’s disease, diabetes, and periodontitis. While changes in microbial populations are apparent during these diseases, the species associated with each disease can vary from patient to patient. Taking into account this interpatient variability, we hypothesized that specific microbiota-associated diseases would be marked by conserved microbial community behaviors. Here, we use gene expression analyses of patient-matched healthy and diseased human periodontal plaque to show that microbial communities have highly conserved metabolic gene expression profiles, whereas individual species within the community do not. Furthermore, disease-associated communities exhibit conserved changes in metabolic and virulence gene expression. The human microbiome project has shown that shifts in our microbiota are associated with many diseases, including obesity, Crohn’s disease, diabetes, and periodontitis. While changes in microbial populations are apparent during these diseases, the species associated with each disease can vary from patient to patient. Taking into account this interpatient variability, we hypothesized that specific microbiota-associated diseases would be marked by conserved microbial community behaviors. Here, we use gene expression analyses of patient-matched healthy and diseased human periodontal plaque to show that microbial communities have highly conserved metabolic gene expression profiles, whereas individual species within the community do not. Furthermore, disease-associated communities exhibit conserved changes in metabolic and virulence gene expression.

Salivary Antioxidants in Patients With Type 1 or 2 Diabetes and Inflammatory Periodontal Disease: A Case-Control Study

BACKGROUND The purpose of this study was to evaluate and compare salivary concentrations of reduced, oxidized glutathione, uric acid, ascorbic acid, and total antioxidant capacity in subjects with diabetes and systemically healthy subjects with inflammatory periodontal disease. METHODS Sixteen patients with type 1 diabetes mellitus (DM), 25 patients with type 2 DM, and 24 systemically healthy patients, all with inflammatory periodontal disease, were recruited. Whole-saliva samples were obtained, and full-mouth clinical periodontal measurements, including plaque index, probing depth, gingival recession, clinical attachment level, and bleeding on probing, were recorded at six sites per tooth. Saliva flow rate and salivary levels of reduced and oxidized glutathione, vitamin C, uric acid, and total antioxidant capacity were determined. Data were analyzed statistically by non-parametric tests. RESULTS The subjects with type 2 DM had fewer teeth and more sites with probing depths >4 mm than the patients with type 1 DM (both P <0.01). The mean salivary reduced-glutathione concentration was lower in patients with type 1 DM than in the other two groups (both P <0.05). No significant differences in the salivary concentrations of the other antioxidants measured were found among the groups (P >0.05). Oxidized glutathione levels in the patients with type 1 DM were significantly lower than in the systemically healthy group (P = 0.007). In both groups with diabetes, salivary reduced-glutathione levels correlated positively with probing depth, and total antioxidant capacity correlated with salivary flow rate (P <0.01). CONCLUSION The decrease in salivary reduced-glutathione levels in patients with type 1 DM may have a role in periodontal tissue destruction by predisposing tissues to oxidative stress.

Gingival crevicular fluid, serum levels of receptor activator of nuclear factor-κB ligand, osteoprotegerin, and interleukin-17 in patients with rheumatoid arthritis and osteoporosis and with periodontal disease.

BACKGROUND This study is performed to evaluate gingival crevicular fluid (GCF) and serum levels of soluble receptor activator of nuclear factor-κB ligand (sRANKL), interleukin (IL)-17A, IL-17E, IL-17F, IL-17A/F, and osteoprotegerin (OPG) in women with rheumatoid arthritis (RA), osteoporosis (OPR), and those who are systemically healthy (SH), all with periodontal disease. METHODS GCF and serum samples were obtained before any periodontal intervention from 17 women with RA, 19 with OPR, and 13 who were SH with periodontitis. Full-mouth clinical periodontal measurements were recorded. sRANKL, OPG, and IL-17 levels were determined by enzyme-linked immunosorbent assay. RESULTS Clinical periodontal measurements were similar in the three study groups. Although the total amounts of GCF albumin, OPG, IL-17A, and IL-17A/F were similar in the study groups, there were statistically significant differences in GCF concentrations of sRANKL, OPG, IL-17A, IL-17E, IL-17F, and IL-17A/F. The sRANKL/OPG ratios were significantly higher in the RA group than in the OPR and SH groups (P <0.05). Serum sRANKL, sRANKL/OPG, and IL-17A/IL-17E ratios were significantly higher, whereas OPG concentrations were significantly lower in the RA group compared to other groups (P <0.05). Serum IL-17A concentrations were significantly higher in the RA and OPR groups than in the SH group (P <0.05). CONCLUSION Increased inflammatory mediator levels in patients with RA, despite the long-term use of various anti-inflammatory drugs, suggest that these patients may have a propensity to overproduce these inflammatory mediators.

Serum and Salivary Matrix Metalloproteinases, Neutrophil Elastase, Myeloperoxidase in Patients with Chronic or Aggressive Periodontitis

Salivary, serum matrix metalloproteinase-8 (MMP-8), tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), neutrophil elastase (NE), and myeloperoxidase (MPO) levels were investigated in generalized chronic periodontitis (GCP), generalized aggressive periodontitis (GAgP), and healthy groups. Whole-mouth clinical periodontal measurements were recorded. Salivary, serum concentrations of MMP-8, MPO, TIMP-1, and NE were determined by immunofluorometric assay or ELISA in 18 patients with GCP, 23 patients with GAgP, 18 individuals with healthy periodontium. Patients in the GAgP group were younger than the other groups (p < 0.05). The study groups were similar in gender, smoking status. Plaque index was higher in GCP than GAgP group (p < 0.05). Biochemical data were similar in periodontitis groups. Salivary, serum MPO, and salivary NE concentrations were higher; TIMP-1 concentrations were lower in the periodontitis groups than the controls (p < 0.05). The present data support a close relationship between salivary, serum protease content and clinical periodontal parameters in patients with generalized periodontitis.

Saliva and Serum Levels of Pentraxin-3 and Interleukin-1β in Generalized Aggressive or Chronic Periodontitis

BACKGROUND Pentraxin-3 (PTX3) is a multifactorial protein involved in immunity and inflammation, which is rapidly produced and released by several cell types in response to inflammatory signals. The aim of the present study is to evaluate saliva, serum levels of PTX3, interleukin (IL)-1β in patients with generalized chronic periodontitis (CP) or aggressive periodontitis (AgP), and periodontally healthy individuals. METHODS A total of 94 participants (25 patients with AgP, 25 patients with CP, and 44 periodontally healthy individuals matched with AgP and CP groups) were recruited. Saliva and serum samples were collected. Clinical periodontal measurements were recorded. PTX3, IL-1β levels in serum, and saliva samples were determined by enzyme-linked immunosorbent assay. Data were tested statistically using Kruskal-Wallis, Mann-Whitney U, and Spearman ρ rank test. RESULTS Serum and saliva data were similar in CP and AgP groups. Saliva levels of IL-1β were significantly higher in the AgP and CP groups than controls (P <0.05). Salivary PTX3 levels were similar in the CP and control groups. Significantly higher salivary concentrations of PTX3 were detected in the AgP group than the control group (P <0.05). Saliva PTX3 levels correlated with plaque index and bleeding on probing in the CP group (P <0.05). Serum and saliva PTX3 levels correlated with those of IL-1β in the AgP group (P <0.05). CONCLUSIONS It may be suggested that PTX3 is related with periodontal tissue inflammation. Its salivary concentrations may have a diagnostic potential. Additional intervention and follow-up studies coupling PTX3 concentrations with microbiologic analysis would better clarify its role in periodontal diseases.

Increased infection with key periodontal pathogens during gestational diabetes mellitus

AIM Gestational diabetes mellitus (GDM), gingivitis, infection with specific periodontal pathogens and systemic inflammation each increase the risk for poor pregnancy outcome. We set out to monitor the interactions of gingivitis and GDM with respect to oral infection and the systemic inflammatory burden. MATERIALS AND METHODS Four case-control groups (n = 117) were recruited, (1) No gingivitis, No GDM (n = 27); (2) Gingivitis, No GDM (n = 31); (3) No gingivitis, GDM (n = 21); and (4) Gingivitis, GDM (n = 38). Oral infection with three key periodontal pathogens was determined by PCR. Systemic inflammation was determined by quantification of CRP by EIA. RESULTS Gingivitis during pregnancy was associated with oral infection with Porphyromonas gingivalis, Filifactor alocis and Treponema denticola and combinations thereof (all p < 0.01). GDM was also associated with increased infection with individual and multiple oral pathogens (all p < 0.05). Gingivitis during pregnancy led to a 325% increase in systemic CRP (mean, 2495 versus 8116 ng/ml, p < 0.01). CONCLUSIONS Diabetes and gingivitis act in concert to increase risk biomarkers for poor pregnancy outcome.

Altered antigenic profiling and infectivity of Porphyromonas gingivalis in smokers and non-smokers with periodontitis.

BACKGROUND Cigarette smokers are more susceptible to periodontal diseases and are more likely to be infected with Porphyromonas gingivalis than non-smokers. Furthermore, smoking is known to alter the expression of P. gingivalis surface components and compromise immunoglobulin (Ig)G generation. The aim of this study is to evaluate whether the overall IgG response to P. gingivalis is suppressed in smokers in vivo and whether previously established in vitro tobacco-induced phenotypic P. gingivalis changes would be reflected in vivo. METHODS The authors examined the humoral response to several P. gingivalis strains as well as specific tobacco-regulated outer membrane proteins (FimA and RagB) by enzyme-linked immunosorbent assay in biochemically validated (salivary cotinine) smokers and non-smokers with chronic periodontitis (CP: n = 13) or aggressive periodontitis (AgP: n = 20). The local and systemic presence of P. gingivalis DNA was also monitored by polymerase chain reaction. RESULTS Smoking was associated with decreased total IgG responses against clinical (10512, 5607, and 10208C; all P <0.05) but not laboratory (ATCC 33277, W83) P. gingivalis strains. Smoking did not influence IgG produced against specific cell-surface proteins, although a non-significant pattern toward increased total FimA-specific IgG in patients with CP, but not AgP, was observed. Seropositive smokers were more likely to be infected orally and systemically with P. gingivalis (P <0.001), as determined by 16S RNA analysis. CONCLUSION Smoking alters the humoral response against P. gingivalis and may increase P. gingivalis infectivity, strengthening the evidence that mechanisms of periodontal disease progression in smokers may differ from those of non-smokers with the same disease classification.

Association between Polycystic Ovary Syndrome, Oral Microbiota and Systemic Antibody Responses

Polycystic ovary syndrome (PCOS) is a hormonal disorder of women that not only is the leading cause of infertility but also shows a reciprocal link with oral health. This study aimed to investigate the hypothesis that the levels of putative periodontal pathogens in saliva and their antibody response in serum are elevated in PCOS, compared to systemic health. A total of 125 women were included in four groups; 45 women with PCOS and healthy periodontium, 35 women with PCOS and gingivitis, 25 systemically and periodontally healthy women, 20 systemically healthy women with gingivitis. Salivary levels of seven putative periodontal pathogens were analyzed by quantitative real-time polymerase chain reaction and serum antibody levels were analyzed by ELISA. In women with PCOS, salivary Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus oralis and Tannerella forsythia levels were higher than matched systemically healthy women, particularly in the case of gingivitis. Aggregatibacter actinomycetemcomitans and Treponema denticola levels were similar among study groups. The presence of PCOS also enhanced P. gingivalis, Prevotella intermedia and S. oralis serum antibody levels, when gingivitis was also present. Gingival inflammation correlated positively with levels of the studied taxa in saliva, particularly in PCOS. The presence of P. gingivalis and F. nucleatum in saliva also exhibited a strong positive correlation with the corresponding serum antibody levels. In conclusion, as an underlying systemic endocrine condition, PCOS may quantitatively affect the composition of oral microbiota and the raised systemic response to selective members of this microbial community, exerting a confounding role in resultant gingival inflammation and periodontal health. The most consistent effect appeared to be exerted on P. gingivalis.

Evaluation of Biochemical Parameters and Local and Systemic Levels of Osteoactive and B-Cell Stimulatory Factors in Gestational Diabetes in the Presence or Absence of Gingivitis

BACKGROUND Gestational diabetes mellitus (GDM) is defined as varying glucose intolerance, with first onset or recognition in pregnancy. This study evaluates clinical and biochemical parameters in a possible association between GDM and gingivitis. METHODS A total of 167 pregnant females was included in the study. There were 101 females with GDM and 66 females without GDM. Subgroups were created according to the presence or absence of gingival inflammation. Plaque index, bleeding on probing, and probing depth were recorded at four sites per tooth. Serum, saliva, and gingival crevicular fluid (GCF) levels of interleukin (IL)-6, IL-8, soluble receptor activator of nuclear factor-kappa B ligand (sRANKL), osteoprotegerin (OPG), B-cell activating factor (BAFF), and a proliferation-inducing ligand (APRIL) were determined by enzyme-linked immunosorbent assay. Data were analyzed by Kruskal-Wallis and Mann-Whitney U tests and Spearman correlation analysis. RESULTS Age and anthropometric indices were higher in the GDM than non-GDM group (P <0.0001). Clinical periodontal recordings, serum BAFF, IL-8, and saliva sRANKL levels were higher in the GDM group (P <0.05). Saliva IL-6 level was higher in the GDM with gingivitis group than non-GDM with gingivitis group (P = 0.044). Serum and GCF BAFF (P <0.0001), serum, saliva, and GCF APRIL (P <0.0001; P <0.0001; P = 0.032, respectively), GCF OPG (P = 0.036), and serum and saliva sRANKL (P <0.0001) were higher in the GDM with gingivitis group than GDM without gingivitis group. CONCLUSIONS The inflammatory response seems to be more pronounced in females with GDM. The observed increase in both local and systemic levels of inflammatory cytokines may suggest an interaction between gingivitis and GDM.

Gingival crevicular fluid and serum levels of APRIL, BAFF and TNF-alpha in rheumatoid arthritis and osteoporosis patients with periodontal disease

BACKGROUND This study was performed to evaluate gingival crevicular fluid (GCF) and serum levels of a proliferation inducing ligand (APRIL) and B cell activating factor (BAFF) and compare this to differences between TNF-alpha levels in rheumatoid arthritis (RA), osteoporosis (OPR) and systemically healthy women with periodontal disease (SH). DESIGN Gingival crevicular fluid (GCF) and serum samples were obtained before any periodontal intervention from 17 RA, 19 OPR patients and 13 SH women with periodontitis. Full-mouth clinical periodontal measurements were recorded. APRIL, BAFF and TNF-α levels were determined by ELISA. Statistical analysis was performed using multivariate analysis, ANOVA and Spearman correlation. RESULTS Pocket depths differed in site-specific comparisons, but otherwise clinical measurements were similar in the three study groups. Multivariate least squares regression ANOVA adjusted for age and for plaque index indicated that total amounts of TNF-α and concentrations of TNF-α, BAFF and APRIL were significantly greater in the RA patients than in the SH group (p<0.05), and GCF concentrations of BAFF were greater in OPR patients than in SH. Serum TNF-α and BAFF were significantly higher in the RA group compared to SH (p<0.05) and serum TNF-α was greater in RA than in OPR (p<0.05). APRIL and BAFF correlated with RANKL levels in GCF and serum (p<0.05). CONCLUSION Despite long-term usage of anti-inflammatory drugs in the RA and OPR patients, increased TNF-family cytokines, might suggest that these patients have a propensity to overproduce these inflammatory mediators but whether this results from greater disease activity or contribute to greater disease activity remains moot.