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Background: Gingivitis in response to biofilm formation may exhibit different trajectories. The purposes of the present study were to characterize the composition of the supragingival microbiota and salivary cytokine and protein levels in healthy individuals with different gingivitis patterns, to test the hypothesis that manifestations of gingivitis associate with specific profiles in terms of supragingival microbiota, salivary cytokines, and proteins., Methods: Forty orally and systemically healthy individuals refrained from all oral hygiene procedures for a period of 14 days, followed by a resolution period of 14 days with regular oral care. Supragingival plaque level and bleeding on probing (BOP) were recorded, and supragingival plaque as well as saliva samples were collected at baseline, day 14, and day 28. Based on change in BOP% from baseline to day 14, rapid ( n  = 15), moderate ( n  = 10), and slow ( n  = 15) responders were identified. Supragingival microbiota composition, salivary cytokine, and protein levels were compared between groups at baseline, day 14, and day 28., Results: A significantly higher baseline abundance of Capnocytophaga, Eikenella , and Campylobacter species were recorded in rapid responders, whereas a significantly higher baseline abundance of Streptococcus species were detected in slow responders. Slow responders expressed a high degree of resilience, with minimal difference in microbial composition at baseline and after 14 days of resolution (day 28). On the contrary, significant differences in relative abundance of members of the core microbiota, Streptococcus , Actinomyces , and Rothia species, was noted in baseline samples versus day 28 samples in rapid responders. Comparable baseline cytokine and protein levels were recorded in all groups., Conclusion: Supragingival microbiota composition, but not saliva cytokine and protein profiles, seems to influence the extent of the inflammatory response during development of gingivitis in systemically healthy individuals., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

The microbial communities of the oral cavity are important elements of oral and systemic health. With emerging evidence highlighting the heritability of oral bacterial microbiota, this study aimed to identify host genome variants that influence oral microbial traits. Using data from 16S rRNA gene amplicon sequencing, we performed genome-wide association studies with univariate and multivariate traits of the salivary microbiota from 610 unrelated adults from the Danish ADDITION-PRO cohort. We identified six single nucleotide polymorphisms (SNPs) in human genomes that showed associations with abundance of bacterial taxa at different taxonomical tiers (P < 5 × 10 -8 ). Notably, SNP rs17793860 surpassed our study-wide significance threshold (P < 1.19 × 10 -9 ). Additionally, rs4530093 was linked to bacterial beta diversity (P < 5 × 10 -8 ). Out of these seven SNPs identified, six exerted effects on metabolic traits, including glycated hemoglobin A1c, triglyceride and high-density lipoprotein cholesterol levels, the risk of type 2 diabetes and stroke. Our findings highlight the impact of specific host SNPs on the composition and diversity of the oral bacterial community. Importantly, our results indicate an intricate interplay between host genetics, the oral microbiota, and metabolic health. We emphasize the need for integrative approaches considering genetic, microbial, and metabolic factors., (© 2024. The Author(s).)

Data from systematic reviews and meta-analyses show that probiotics positively impact clinical parameters of oral diseases such as gingivitis, dental caries, and periodontitis. However, the working mechanism of probiotics is not fully understood, but is hypothesized to be mediated by direct and indirect interactions with the oral microbiota and the human host. In the present narrative review, we focused on the microbiological effect of probiotic supplements based on data retrieved from randomized clinical trials (RCTs). In addition, we assessed to what extent contemporary molecular methods have been employed in clinical trials in the field of oral probiotics. Multiple RCTs have been performed studying the potential effect of probiotics on gingivitis, dental caries, and periodontitis, as evaluated by microbial endpoints. In general, results are conflicting, with some studies reporting a positive effect, whereas others are not able to record any effect. Major differences in terms of study designs and sample size, as well as delivery route, frequency, and duration of probiotic consumption, hamper comparison across studies. In addition, most RCTs have been performed with a limited sample size using relatively simple methods for microbial identification, such as culturing, qPCR, and DNA-DNA checkerboard, while high-throughput methods such as 16S sequencing have only been employed in a few studies. Currently, state-of-the-art molecular methods such as metagenomics, metatranscriptomics, and metaproteomics have not yet been used in RCTs in the field of probiotics. The present narrative review revealed that the effect of probiotic supplements on the oral microbiota remains largely uncovered. One important reason is that most RCTs are performed without studying the microbiological effect. To facilitate future systematic reviews and meta-analyses, an internationally agreed core outcome set for the reporting of microbial endpoints in clinical trials would be desirable. Such a standardized collection of outcomes would most likely improve the quality of probiotic research in the oral context., Competing Interests: The authors declare no conflicts of interest.

The aim was to test if probiotics counteract oral dysbiosis during 14 days of sugar stress and subsequently help restore oral homeostasis. Eighty healthy individuals received either probiotics ( n = 40) or placebo lozenges ( n = 40) for 28 days and rinsed with a 10% sucrose solution 6-8 times during the initial 14 days of the trial. Saliva and supragingival samples were collected at baseline, day 14, and day 28. Saliva samples were analyzed for levels of pro-inflammatory cytokines, albumin, and salivary enzyme activity. The supragingival microbiota was characterized according to the Human Oral Microbiome Database. After 14 days of sugar stress, the relative abundance of Porphyromonas species was significantly higher ( p = 0.03) and remained significantly elevated at day 28 in the probiotic group compared to the placebo group ( p = 0.004). At day 28, the relative abundance of Kingella species was significantly higher in the probiotic group ( p = 0.03). Streptococcus gordinii and Neisseria elongata were associated with the probiotic group on day 28, while Streptococcus sobrinus was associated with the placebo group on day 14 and day 28. On day 28, the salivary albumin level was significantly lower in the probiotic group. The present study demonstrates a potential stabilizing effect on the supragingival microbiota mediated by consumption of probiotics during short-term sugar stress.

The present study aims to test whether probiotics protect against experimental gingivitis incited by 14 days of oral hygiene neglect and/or subsequently support the restoration of oral homeostasis. Eighty systemically and orally healthy participants refrained from oral hygiene procedures for 14 days, followed by 14 days with regular oral hygiene procedures. Additionally, participants consumed either probiotics ( n = 40) or placebo ( n = 40) throughout the trial. At baseline, day 14, and day 28, supragingival plaque score and bleeding-on-probing percentage (BOP %) were registered, and supragingival plaque and saliva samples were collected. The supragingival microbiota was characterized using 16S sequencing, and saliva samples were analyzed for levels of pro-inflammatory cytokines and proteases. At day 28, the relative abundance of Lautropia ( p = 0.014), Prevotella ( p = 0.046), Fusobacterium ( p = 0.033), and Selenomonas ( p = 0.0078) genera were significantly higher in the placebo group compared to the probiotics group, while the relative abundance of Rothia ( p = 0.047) species was associated with the probiotics group. Streptococcus sanguinis was associated with the probiotics group, while Campylobacter gracilis was associated with the placebo group. No difference was observed in salivary cytokines, albumin, or any enzyme activity. The present study suggests that probiotics support the resilience of the oral microbiota in the resolution period after gingivitis.

Background: Since 1972 The National Child Odontology Registry has collected data on the oral health of most of all Danish children and adolescents. However, comprehensive information on the registry has not previously been available, making it difficult to approach and use the registry for research purposes., Methods: By combining historical documentation and simple descriptive statistics we provide an overview of major events in the timeline of The National Child Odontology Registry and discuss how they impact the available data. We provide a broad overview of the dental variables in the registry, and how the registration criteria for some of the core dental variables (gingivitis, periodontitis, and dental caries) have changed over time. We then provide examples of how aggregate variables for the core dental diseases, allowing for comparison across registration criteria, can be created., Results: Most of the Danish population born during or after 1965 have a least one entry in the National Child Odontology Registry, with 68% having entries spanning their entire childhood and adolescence. The prevalence of gingivitis and periodontitis seem to increase significantly in the years immediately following changes in how registration criteria for these variables, raising questions as to whether these diseases are generally underreported, or subject to overreporting in the years following the registration changes. The mandatory ages of registration instituted in 2003, do not appear to have had a strong impact on the ages at which registrations are made. For variables not directly comparable across datasets due to changes in registration criteria aggregate variables of measurements can be computed in most cases., Conclusions: The National Child Odontology Registry provides a unique opportunity to study the impact of childhood oral health on life trajectories, but using the registry is not without issues, and we strongly recommend consulting with experts in the field of odontology to ensure the best use of available data., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Frequent intake of free sugars is a major risk factor for dental caries, but the immediate influence of sugar intake on the supragingival microbiota remains unknown. We aim to characterize the effect of 14 days of sugar rinsing on the supragingival microbiota. Forty orally and systemically healthy participants rinsed their mouth with a 10% sucrose solution, 6-8 times a day, for 14 days, followed by 14 days without sugar stress. Supragingival plaque samples were collected at baseline, and after 14, and 28 days. The supragingival microbiota was analyzed using 16S rDNA sequencing. Taxonomic classification was performed using the Human Oral Microbiome Database. After 14 days of sugar stress induced by the daily sugar rinses, a significant loss of α-diversity ( p  = 0.02) and a significant increase in the relative abundance of Actinomyces (6.5% to 9.6%, p  = 0.006) and Corynebacterium (6.2% to 9.1%, p  = 0.03) species were recorded. In addition, a significant decrease in Streptococcus (10.3% to 6.1%, p  = 0.001) species was observed. Sugar-mediated changes returned to baseline conditions 14 days after the last sugar rinse. The present study shows that temporary sugar stress induces loss of diversity and compositional changes to the supragingival microbiota, which are reversible if oral care is maintained., Competing Interests: No potential conflict of interest was reported by the authors., (© 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

This narrative review summarizes the collective knowledge on periodontal microbiology, through a historical timeline that highlights the European contribution in the global field. The etiological concepts on periodontal disease culminate to the ecological plaque hypothesis and its dysbiosis-centered interpretation. Reference is made to anerobic microbiology and to the discovery of select periodontal pathogens and their virulence factors, as well as to biofilms. The evolution of contemporary molecular methods and high-throughput platforms is highlighted in appreciating the breadth and depth of the periodontal microbiome. Finally clinical microbiology is brought into perspective with the contribution of different microbial species in periodontal diagnosis, the combination of microbial and host biomarkers for this purpose, and the use of antimicrobials in the treatment of the disease., (© 2023 The Authors. Periodontology 2000 published by John Wiley & Sons Ltd.)

Background: Hyposalivation and xerostomia (dry mouth), are the leading site-effects to treatment of head and neck cancer. Currently, there are no effective therapies to alleviate radiation-induced hyposalivation. Adipose tissue-derived mesenchymal stem/stromal cells (AT-MSCs) have shown potential for restoring salivary gland function. However, the mode of action is unknown. The purpose of the present study was therefore to characterize the effect of AT-MSC therapy on the salivary proteome in previously irradiated head and neck cancer patients., Methods: Whole saliva was collected from patients with radiation-induced salivary gland hypofunction (n = 8) at baseline, and 120 days after AT-MSC treatment, and from healthy controls (n = 10). The salivary proteome was characterized with mass spectrometry based proteomics, and data was compared within the AT-MSC group (baseline versus day 120) and between AT-MSC group and healthy controls. Significance levels between groups were determined by using double-sided t-test, and visualized by means of principal component analysis, volcano plots and cluster analysis., Results: Here we show that 140 human proteins are significantly differentially expressed in saliva from patients with radiation-induced hypofunction versus healthy controls. AT-MSC treatment induce a significant impact on the salivary proteome, as 99 proteins are differentially expressed at baseline vs. 120 days after treatment. However, AT-MSC treatment does not restore healthy conditions, as 212 proteins are significantly differentially expressed in saliva 120 days after AT-MSCs treatment, as compared to healthy controls., Conclusion: The results indicate an increase in proteins related to tissue regeneration in AT-MSCs treated patients. Our study demonstrates the impact of AT-MSCs on the salivary proteome, thereby providing insight into the potential mode of action of this novel treatment approach., (© 2022. The Author(s).)

Introduction: Previous research indicates that the salivary microbiota may be a biomarker of oral as well as systemic disease. However, clarifying the potential bias from general health status and lifestyle-associated factors is a prerequisite of using the salivary microbiota for screening., Materials & Methods: ADDDITION-PRO is a nationwide Danish cohort, nested within the Danish arm of the Anglo-Danish-Dutch Study of Intensive treatment in People with Screen-Detected Diabetes in Primary Care. Saliva samples from n=746 individuals from the ADDITION-PRO cohort were characterized using 16s rRNA sequencing. Alpha- and beta diversity as well as relative abundance of genera was examined in relation to general health and lifestyle-associated variables. Permutational multivariate analysis of variance (PERMANOVA) was performed on individual variables and all variables together. Classification models were created using sparse partial-least squares discriminant analysis (sPLSDA) for variables that showed statistically significant differences based on PERMANOVA analysis (p < 0.05)., Results: Glycemic status, hemoglobin-A 1c (HbA 1c ) level, sex, smoking and weekly alcohol intake were found to be significantly associated with salivary microbial composition (individual variables PERMANOVA, p < 0.05). Collectively, these variables were associated with approximately 5.8% of the observed differences in the composition of the salivary microbiota. Smoking status was associated with 3.3% of observed difference, and smoking could be detected with good accuracy based on salivary microbial composition (AUC 0.95, correct classification rate 79.6%)., Conclusions: Glycemic status, HbA 1c level, sex, smoking and weekly alcohol intake were significantly associated with the composition of the salivary microbiota. Despite smoking only being associated with 3.3% of the difference in overall salivary microbial composition, it was possible to create a model for detection of smoking status with a high correct classification rate. However, the lack of information on the oral health status of participants serves as a limitation in the present study. Further studies in other cohorts are needed to validate the external validity of these findings., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Poulsen, Nygaard, Constancias, Stankevic, Kern, Witte, Vistisen, Grarup, Pedersen, Belstrøm and Hansen.)

Objectives: To determine the diagnostic potential of simultaneous presence of serum anti-SSA/Ro and upregulated salivary protein biomarkers in patients with primary Sjögren's syndrome (pSS)., Methods: Previous proteomics data on the intensity of neutrophil elastase, calreticulin, tripartite motif containing protein 29 (TRIM29), clusterin and vitronectin provided basis for performing extended analysis. Protein data was obtained by liquid chromatography tandem mass spectrometry technique in whole saliva from 24 patients with pSS and 16 patients having symptoms of pSS, but not fulfilling the American College of Rheumatology/European League against Rheumatism classification criteria (non-pSS). Serum anti-SSA/Ro antibody was measured using enzyme-linked immunosorbent assays. Receiver operating characteristic curve (ROC) value was calculated for combined biomarkers., Results: Simultaneous presence of serum anti-SSA/Ro and upregulated salivary TRIM29 provided the most optimal combination with an area under curve (AUC) of 0.995 (95% CI 0.98-1.00, p = 2.0E-7 and standard error 0.007) and combinations of sensitivity and specificity within the interval of 91-100%. ROC analysis showed that salivary levels of TRIM29 alone enabled differentiation between pSS and non-pSS with an area under curve (AUC) of 0.88 (95%CI 0.77-1.00). All patients with pSS and 3 non-pSS patients were serum anti-SSA/Ro positive., Conclusions: Simultaneous presence of serum anti-SSA/Ro and upregulated salivary TRIM29 provided a high diagnostic accuracy exceeding that of currently available tools used in pSS diagnostics. This biomarker combination represents a promising less invasive diagnostic tool for pSS. The clinical applicability of TRIM29 needs further testing in independent cohorts using relevant analytical techniques., Competing Interests: The authors have declared that no competing interests exist.

The purpose of the present investigation was to characterize species-specific bacterial activity of the oral microbiota in periodontitis. We tested the hypotheses that chronic inflammation, i.e., periodontitis, associates with bacterial gene expression of the oral microbiota. Oral microbial samples were collected from three oral sites-subgingival plaque, tongue, and saliva from patients with periodontitis and healthy controls. Paired metagenomics and metatranscriptomics were used to perform concomitant characterization of taxonomic composition and to determine species-specific bacterial activity as expressed by the ratio of specific messenger RNA reads to their corresponding genomic DNA reads. Here, we show the association of periodontitis with bacterial gene expression of the oral microbiota. While oral site was the main determinant of taxonomic composition as well as bacterial gene expression, periodontitis was significantly associated with a reduction of carbohydrate metabolism of the oral microbiota at three oral sites (subgingival plaque, tongue, and saliva). Data from the present study revealed the association of periodontitis with bacterial gene expression of the oral microbiota. Conditions of periodontitis was associated with bacterial activity of local subgingival plaque, but also on tongue and the salivary microbiota. Collectively, data suggest that periodontitis associates with impaired carbohydrate metabolism of the oral microbiota. Future longitudinal and interventional studies are warranted to evaluate the potential pathogenic role of impaired bacterial carbohydrate metabolism not only in periodontitis but also in other diseases with low-grade inflammation, such as type 2 diabetes mellitus., (© 2021. The Author(s).)

Background: Streptococcus species are predominant members of the oral microbiota in both health and diseased conditions. The purpose of the present study was to explore if different ecological characteristics, such as oxygen availability and presence of periodontitis, associates with transcriptional activity of predominant members of genus Streptococcus. We tested the hypothesis that genetically closely related Streptococcus species express different transcriptional activities in samples collected from environments with critically different ecological conditions determined by site and inflammatory status., Methods: Metagenomic and metatranscriptomic data was retrieved from 66 oral samples, subgingival plaque (n=22), tongue scrapings (n=22) and stimulated saliva (n=22) collected from patients with periodontitis (n=11) and orally healthy individuals (n=11). Species-specific transcriptional activity was computed as Log2(RNA/DNA), and transcriptional activity of predominant Streptococcus species was compared between multiple samples collected from different sites in the same individual, and between individuals with different oral health status., Results: The predominant Streptococcus species were identified with a site-specific colonization pattern of the tongue and the subgingival plaque. A total of 11, 4 and 2 pathways expressed by S. parasanguinis, S. infantis and S. salivarius , respectively, were recorded with significantly higher transcriptional activity in saliva than in tongue biofilm in healthy individuals. In addition, 18 pathways, including pathways involved in synthesis of peptidoglycan, amino acid biosynthesis, glycolysis and purine nucleotide biosynthesis expressed by S. parasanguinis and 3 pathways expressed by S. salivarius were identified with significantly less transcriptional activity in patients with periodontitis., Conclusion: Data from the present study significantly demonstrates the association of site-specific ecological conditions and presence of periodontitis with transcriptional activity of the predominant Streptococcus species of the oral microbiota. In particular, pathways expressed by S. parasanguinis being involved in peptidoglycan, amino acid biosynthesis, glycolysis, and purine nucleotide biosynthesis were identified to be significantly associated with oral site and/or inflammation status., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Belstrøm, Constancias, Markvart, Sikora, Sørensen and Givskov.)

Background: The purpose of the present investigation was to characterize the effect of probiotics on the composition of the salivary microbiota and salivary levels of inflammation-related proteins during short-term sugar stress. We tested the hypotheses that consumption of probiotics may partly counteract the detrimental influence of sugar stress on oral homeostasis., Methods: The present study was a five-week, blinded, randomized controlled trial with four study arms-A: sucrose and probiotic ( n = 20); B: sucrose and placebo ( n = 20); C: xylitol and probiotic ( n = 20); D: xylitol and placebo ( n = 20). Saliva samples were collected at baseline and after two and five weeks. The salivary microbiota was characterized by means of 16S rDNA sequencing, and sequences were referenced against the Human Oral Microbiome Database (HOMD). Neutrophil gelatinase-associated lipocalin (NGAL) and transferrin levels were quantified using immunoassays., Results: Sugar stress induced a significant increase in the relative abundance of the genus Streptococcus from 29.8% at baseline to 42.9% after two weeks. Changes were transient and were completely reversed three weeks after discontinuation of sugar stress. Xylitol and probiotics alone had no effect on the salivary microbiota, whereas the combination of xylitol and probiotics induced a significant decrease in the relative abundance of Streptococcus species from 37.6% at baseline to 23.0% at week 2. Sugar stress significantly increased salivary transferrin levels, and the effect was partly counteracted by concomitant use of probiotics., Conclusions: The data clearly demonstrate an impact of combined consumption of xylitol and probiotics on the composition of the salivary microbiota. Future studies are needed to evaluate whether the combined use of xylitol and the probiotic strains tested could have clinically protective effects during periods of sugar stress.

Background: The purpose of the present study was to longitudinally characterize the supragingival microbiota throughout a three months period in orally healthy individuals. We tested the hypothesis that the supragingival microbiota shows a high degree of compositional stability, which is resilient against the external perturbation of regular use of probiotics, as long as oral health is maintained., Methods: The present study was a double-blinded, randomized, placebo-controlled clinical trial. The study population comprised a total of 110 oral and systemic healthy individuals, distributed in a probiotic ( n = 55) and placebo ( n = 55) group, where the test group consumed tablets with the probiotic strains Lacticaseibacillus rhamnosus (formerly Lactobacillus ) PB01 DSM14870 and Latilactobacillus curvatus (formerly Lactobacillus ) EB10 DSM32307 for a period of 12 weeks. Supragingival plaque samples and clinical registrations were performed at baseline, and after 4, 8, and 12 weeks, respectively. The supragingival microbiota was characterized by means of 16S rDNA sequencing. Sequences were referenced against the HOMD database., Results: No significant changes of the core microbiota, as expressed by relative abundance of predominant genera and species were evident during the three months observation period in the probiotic or the placebo group., Conclusions: Data from the present study clearly demonstrate long term compositional stability of the supragingival microbiota as long as oral health is maintained. In addition, the tested probiotics had no augmenting effect on the supragingival microbiota in oral health.

Background and Objective: The complement system is engaged in inflammatory reactions both in the periodontal pockets and in the periodontium itself, where it can mediate tissue destruction. The aim of this study was, first, to compare salivary levels of the total complement system protein C3 and its split product, fluid-phase C3c in patients with periodontitis and periodontally healthy controls. Next, to determine if C3 and C3c levels had biomarker potential in diagnosing and monitoring periodontitis and its treatment. We hypothesized that salivary levels of total C3 and the split product C3c associated with the severity of periodontitis and reflected decreased inflammatory activity after periodontal treatment., Methods: At baseline, stimulated saliva samples were collected from patients with periodontitis (n = 18) and periodontally healthy controls (n = 15). Subsequently, non-surgical periodontal treatment was performed in the patients, and saliva sampling from patients was repeated two-, six-, and twelve weeks post-treatment (NCT02913248 at clinicaltrials.gov). The patients were grouped as good and poor responders to treatment according to the achieved reduction in bleeding on probing (BOP). Salivary levels of C3 and C3c were quantified using sandwich ELISA., Results: Patients with periodontitis had higher baseline levels of both total C3 and the split product C3c in saliva than did periodontally healthy controls (P < .0001). Receiver operating curve (ROC) analyses discriminated patients with periodontitis from controls based on both C3 (AUC (area under curve) = 0.91, P < .001) and C3c levels (AUC = 0.84, P < .001) in saliva. Periodontal treatment improved all clinical parameters (P < .01). Good responders (n = 10) had lower baseline levels of C3c than poor responders (n = 8), (P < .05), and baseline levels of C3c discriminated between good and poor responders (AUC = 0.80, P < .05)., Conclusion: In conclusion, patients with periodontitis had higher salivary levels of C3c, and the C3c levels were predictive of reductions in BOP, that is, the poor responders. This suggests that salivary C3c levels possess potential to serve as a biomarker predicting the clinical response to non-surgical periodontal treatment., (© 2020 The Authors. Journal of Periodontal Research published by John Wiley & Sons Ltd.)

Objectives: Increasing evidence suggests that aberrant expression of microRNAs (miRNAs) is involved in the pathogenesis of primary Sjögren's syndrome (pSS). The aim was thus to characterize the miRNA profile in saliva, salivary gland tissue, and plasma from patients with pSS and compare findings with those of patients having Sjögren-like disease (non-pSS). In addition, to correlate miRNA levels and clinicopathological features of pSS., Methods: miRNA real-time quantitative polymerase chain reaction was performed on saliva, plasma, and salivary gland tissue samples from 24 patients with pSS and 16 non-pSS in 384-well plates. T test was used for comparison of miRNA profiles, followed by Benjamini-Hochberg correction. The discriminatory power of miRNAs was evaluated by receiver-operating characteristic curves, and Pearson/Spearman correlation was used for correlation analyses., Results: In saliva, 14 miRNAs were significantly differentially expressed between pSS and non-pSS, including downregulation of the miR-17 family in pSS. In salivary gland tissue of patients with pSS, miR-29a-3p was significantly upregulated. Plasma miRNAs did not differ between the two groups, although the miR-17 family tended to be downregulated. The combination of miR-17-5p and let-7i-5p in saliva yielded an area under curve of 97% (CI 92%-100%). Several miRNAs correlated significantly with one another and with salivary flow rates and histopathology., Conclusion: Our findings indicate that the miRNA expression profile in saliva may enable to discriminate between pSS and non-pSS patients. However, further validation in larger cohorts is needed as well as functional analyses of the miRNAs of interest., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Aim was to profile salivary total protease, Porphyromonas gingivalis gingipain, and neutrophil elastase activities in relation to the resolution of periodontal inflammation, salivary macrophage-derived chemokine (MDC), and macrophage inflammatory protein-1α concentrations. Nonsurgical periodontal treatment was performed in 24 periodontitis patients in a prospective interventional study design. Periodontal clinical parameters were recorded, and stimulated saliva samples were collected at baseline and 2, 6, and 12 weeks after treatment. Salivary total protease and gingipain activities were determined using fluorogenic substrates, elastase activity by chromogenic substrates, and cytokine concentrations by Luminex immunoassay. For statistical analyses, generalized linear mixed models for repeated measures were used. Salivary total protease activity elevated, while gingival inflammation and plaque accumulation decreased 2 and 6 weeks after periodontal therapy. Salivary MDC concentration was elevated 12 weeks after periodontal treatment. Patients with elevated protease activities at baseline in comparison to patients with low baseline total protease activities, had higher levels of gingival inflammation before and after periodontal treatment. In conclusion, elevations in salivary total protease activity seem to be part of periodontal healing at its early phases. Higher levels of salivary total protease activities before periodontal treatment may predict the severity and steadiness of unresolved gingival inflammation.

Objective: The aim of this study was to characterize and compare the proteome in whole saliva, plasma, and salivary gland tissue in patients with primary Sjögren's syndrome (pSS) and patients having symptoms of pSS, but not fulfilling the classification criteria, and to search for diagnostic biomarker candidates for pSS., Methods: Liquid chromatography tandem mass spectrometry was conducted on whole saliva, plasma, and labial salivary gland tissue samples from 24 patients with pSS and 16 non-Sjögren control subjects (non-pSS). Gene Ontology (GO)-terms and Kyoto Encyclopedia of Genes and Genomes (KEGG)-pathways were applied for functional annotation., Results: 1013 proteins were identified in whole saliva, 219 in plasma, and 3166 in salivary gland tissue. In saliva, 40 proteins differed significantly between the two groups. In pSS, proteins involved in immunoinflammatory processes were upregulated, whereas proteins related to salivary secretion were downregulated. The combination of neutrophil elastase, calreticulin, and tripartite motif-containing protein 29 yielded a receiver-operating characteristic (ROC) value of 0.97 (CI 0.93-1.00). Protein expression in plasma and salivary gland tissue did not differ between the patient groups., Conclusion: The salivary proteome of patients with pSS differed from that of non-pSS patients, indicating that saliva proteomics represents a promising non-invasive diagnostic tool for pSS., Significance: Primary Sjögren's syndrome (pSS) is a chronic systemic autoimmune disease, which clinically may present with a wide variety of symptoms and signs. Symptoms of dry eyes and dry mouth due to lacrimal and salivary gland dysfunction are prominent, but not pathognomonic, and an extensive diagnostic work-up including blood tests and labial salivary gland biopsy is often required to distinguish pSS from non-pSS. In this study, we used high throughput proteomics and identified a non-invasive biomarker candidate comprising a combination of three different upregulated salivary proteins, which enables differentiation between patients with pSS and non-pSS patients with an accuracy of 97%. In the future, this could contribute to earlier, more accurate and less costly diagnosis of pSS., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: The purpose of the present study was to characterize the composition of the salivary microbiota and quantify salivary levels of inflammation-related proteins (neutrophil gelatinase-associated lipocalin [NGAL] and transferrin) in patients with psoriasis and compare data to those obtained in patients with periodontitis and orally healthy controls, respectively., Materials and Methods: Stimulated saliva samples from patients with psoriasis (n = 27), patients with periodontitis (n = 58), and orally healthy controls (n = 52) were characterized by means of next-generation sequencing of the 16S rRNA gene. Salivary levels of NGAL and transferrin were quantified using immunoassays., Results: Linear discriminant effect size analysis showed that 52 (22 psoriasis-associated and 30 periodontitis-associated) and 21 (8 psoriasis-associated and 13 orally healthy control-associated) bacterial taxa differentiated the salivary microbiota in patients with psoriasis from that of patients with periodontitis and orally healthy controls, respectively. Significantly lower mean salivary levels of NGAL (psoriasis: 996 [std. error 320], periodontitis: 2,072 [295], orally healthy controls: 2,551 [345] ng/ml, p < .0001) and transferrin (psoriasis: 4.37 [0.92], periodontitis: 7.25 [0.88], orally healthy controls: 10.02 [0.94] ng/ml, p < .0001) were identified in patients with psoriasis., Conclusions: Psoriasis associates with characteristics of the salivary microbiota and salivary levels of inflammation-related proteins, which are different from characteristics in patients with periodontitis and orally healthy controls, respectively., (© 2020 The Authors. Oral Diseases published by John Wiley & Sons Ltd. All rights reserved.)

The salivary microbiota (SM), comprising bacteria shed from oral surfaces, has been shown to be individualized, temporally stable and influenced by diet and lifestyle. SM reflects local bacterial alterations of the supragingival and subgingival microbiota, and periodontitis and dental-caries associated characteristics of SM have been reported. Also, data suggest an impact of systemic diseases on SM as demonstrated in patients with a wide variety of systemic diseases including diabetes, cancer, HIV and rheumatoid arthritis. The presence of systemic diseases seems to influence salivary levels of specific bacterial species, as well as α- and β-diversity of SM. The composition of SM might thereby potentially mirror oral and general health status. The contentious development of advanced molecular techniques such as metagenomics, metatranscriptomics and metabolomics has enabled the possibility to address bacterial functions rather than presence in microbial samples. However, at present only a few studies have employed such techniques on SM to reveal functional and metabolic characteristics in oral health and disease. Future studies are therefore warranted to illuminate the possible impact of metabolic functions of SM on oral and general health status. Ultimately, such an approach has the possibility to reveal novel and personalized therapeutic avenues in oral and general medicine., (© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Background: During periodontal inflammation, bacteria induces chemokine expression and migration of various inflammatory cells. The aim of the study was to learn if periodontal treatment alters salivary concentrations of macrophage activation-related chemokines and if such alterations correlate with abundance of periodontitis-associated bacteria. Methods: Twenty-five patients with periodontitis completed the study (NCT02913248 at clinicaltrials.gov). Periodontal parameters and stimulated saliva samples were obtained at baseline and 2, 6 and 12 weeks after non-surgical periodontal treatment. Salivary concentrations of monocyte chemoattractant proteins (MCP-1-4), macrophage-derived chemokine (MDC), macrophage migration inhibitory factor (MIF), monokine induced by interferon-gamma (MIG), macrophage inflammatory protein (MIP-1α) and interferon-inducible protein (IP-10) were quantified using the Luminex® xMAP™ technique and abundance of bacteria was quantified using next-generation sequencing. Results: The treatment improved all periodontal parameters and caused an increase in the concentrations of MCP-2, MDC and MIP-1α at week 12 compared to baseline, week 2 and week 6, respectively. Salivary concentrations of MCP-1-2, MDC, MIG, MIP-1α and IP-10 correlated with the abundance of specific periodontitis-associated bacteria. Conclusions: Periodontal treatment impacts salivary concentrations of MCP-2, MDC and MIP-1α, which correlate with the abundance of specific periodontitis-associated bacteria. This indicates that these chemokines reflect periodontal status and possess potential in illustrating a response to treatment., (© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Objective : To characterize and compare the salivary microbiota in patients with pSS and patients with non-Sjögren's-related sicca, and to relate the findings to their oral health status and saliva flow rates. Methods : Twenty-four patients fulfilled the 2016 classification criteria for pSS and 34 did not (non-pSS). A clinical examination included registration of decayed, missing and filled teeth/-surfaces and collection of whole saliva. The microbiota was characterized using next-generation sequencing of the V1-V3 region of the 16S rRNA gene. Data were annotated against the eHOMD database. Results : A total of 509 different bacterial taxa were identified. There were no statistically significant differences between the groups with regard to the abundance of predominant genera, bacterial diversity and relative abundance on the genus or species level. The two groups did not differ with regard to general health, including intake of xerogenic medication and polypharmacy, oral health status or unstimulated and stimulated whole saliva flow rates. Conclusion : The salivary microbiota and oral health status, as well as salivary flow rate in patients with pSS resemble that of non-pSS patients. Our findings indicate that changes in the salivary microbiota do not appear to be determined by the disease entity pSS itself.

Objective : To characterize the salivary microbiota of patients with aggressive periodontitis, patients with chronica periodontitis and orally healthy individuals. Methods : A total of 81 unstimulated saliva samples from aggressive periodontitis patients (n = 31), chronic periodontitis patients (n = 25), and orally healthy controls (n = 25) were examined. The V1-V3 region of the 16S rDNA gene was sequenced with Illumina® MiSeq TM , and sequences were annotated to the expanded Human Oral Microbiome Database (eHOMD). Results : A mean percentage of 97.6 (range: 89.8-99.7) of sequences could be identified at species level. Seven bacterial species, including Porphyromonas gingivalis, were identified with significantly higher relative abundance in saliva from aggressive periodontitis patients than in saliva from orally healthy controls. Salivary abundance of P. gingivalis could discriminate aggressive (AUC: 0.80, p = 0.0001) and chronic periodontitis (AUC: 0.72, p = 0.006) from healthy controls. Likewise, salivary presence of P. gingivalis was significantly associated with aggressive (p < 0.0001, RR: 8.1 (95% CI 2.1-31.2)) and chronic periodontitis (p = 0.002, RR: 6.5 (95% CI: 1.6-25.9)). Conclusion : Salivary presence and relative abundance of P. gingivalis associate with aggressive and chronic periodontitis, but do not discriminate between aggressive and chronic periodontitis.

Objectives: To examine the relationship between the medium term use (>1 year) of a toothpaste containing natural enzymes and proteins (Zendium™) upon gingival index, plaque index and bleeding index compared to medium term use of toothpastes without antimicrobial/antiinflammatory ingredients., Methods: A total of 305 participants eligible for inclusion were grouped according to their toothpaste use and matched with regard to gender and age (18-30, 31-55 and 56+ years of age). A total of 161 persons were using a toothpaste which contained enzymes and proteins (Zendium™, test group), and 144 persons were using a toothpaste without these ingredients (control group). The amount of dental plaque and the gingival condition were assessed at six sites of each tooth using the modified gingival index (MGI), plaque index (Modified Quigley and Hein plaque index, PI), and bleeding index (BI). Mean values of MGI, PI and BI were compared using analysis of covariance., Results: The test group had significantly less gingival inflammation than the control group (adjusted mean scores (SD); 1.80 (0.65) vs. 2.27 (0.63),p < 0.0001), as well as lower levels of plaque (2.03 (0.33) vs. 2.12 (0.33), p = 0.0168) and gingival bleeding (0.74 (0.45) vs. 1.08 (0.45), p < 0.0001). Females had significantly less gingival inflammation (p < 0.0001), plaque (p = 0.0005) and bleeding (p = 0.0118) than males. Participants aged 18-30 years had significantly higher levels of inflammation and bleeding than the older age groups (p < 0.001), and also higher plaque levels compared to participants aged 31-55 years (p = 0.0069). Potential confounding factors including oral hygiene practices and consistency of dental visits did not differ between groups., Conclusions: Our findings indicate that medium term use of fluoride toothpaste containing enzymes and proteins (Zendium™) is associated with a better gingival health than the use of other types of fluoride toothpastes without antimicrobial active ingredients., Clinical Significance: Medium term (> 1 year) use of toothpaste containing naturally occurring enzymes and proteins (Zendium™) in an unsupervised home setting is associated with better gingival health compared to the unsupervised use of other commercially available toothpastes without antimicrobial/antiinflammatory active ingredients., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objectives: To provide an update on our current understanding of how saliva and its various constituents directly and indirectly affect oral bacteria and thereby play a role in the modulation and maintenance of a healthy oral microbiota and also the associations with symbiosis and dysbiosis., Methods: The search for biomedical literature on saliva and its antimicrobial activities (years 1966 to 2017) was conducted in PubMed, Embase and Web of Science databases., Results: This review underlines that saliva plays an essential role in shaping and maintaining the ecological equilibrium of the resident oral microbiota. Saliva contributes to the formation of the salivary pellicle, which covers the oral hard and soft tissues, and thereby determines the initial adhesion and colonisation of microorganisms. Saliva facilitates clearance of dietary carbohydrates and microorganisms from the oral cavity, but also supplies bacteria with nutrients through enzymatic breakdown of dietary starch and proteins and salivary glycoproteins. In addition, saliva comprises proteins such as mucins, which block the adherence of certain microorganisms to oral surfaces through binding and aggregating mechanisms. Saliva also provides antimicrobial activity through numerous proteins and peptides including mucins, lactoferrin, lysozyme, lactoperoxidase, statherin, histatins and secretory immunoglobulin A., Conclusions: A balanced oral microbiome is important for the maintenance of oral health and symbiosis. Conditions associated with salivary gland hypofunction, impaired oral clearance, low salivary pH and altered salivary composition, often lead to perturbation of the function and composition of the oral microbiome causing dysbiosis, and an associated risk of oral disease., Clinical Significance: Saliva plays a significant role in keeping the relationship between the host and oral microbiota in a symbiotic state. In conditions with salivary gland dysfunction, the natural balance of the oral microbiome is often disturbed, leading to dysbiosis and associated risks of gingivitis, caries and fungal infection., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The composition of ancient oral microbiomes has recently become accessible owing to advanced biomolecular methods such as metagenomics and metaproteomics, but the utility of metaproteomics for such analyses is less explored. Here, we use quantitative metaproteomics to characterize the dental calculus associated with the remains of 21 humans retrieved during the archeological excavation of the medieval (ca. 1100-1450 CE) cemetery of Tjærby, Denmark. We identify 3671 protein groups, covering 220 bacterial species and 81 genera across all medieval samples. The metaproteome profiles of bacterial and human proteins suggest two distinct groups of archeological remains corresponding to health-predisposed and oral disease-susceptible individuals, which is supported by comparison to the calculus metaproteomes of healthy living individuals. Notably, the groupings identified by metaproteomics are not apparent from the bioarchaeological analysis, illustrating that quantitative metaproteomics has the potential to provide additional levels of molecular information about the oral health status of individuals from archeological contexts.

Background: The purpose of this study was to characterize and compare subgingival and salivary microbiotas before and after periodontal treatment to learn if any changes of the subgingival microbiota were reflected in saliva. We tested the hypothesis that salivary levels of specific periopathogens correlate with corresponding subgingival levels before and after periodontal treatment., Methods: Twenty-five patients with generalized chronic periodontitis completed the study. Stimulated saliva samples and subgingival plaque samples were collected at baseline and 2, 6, and 12 weeks after nonsurgical periodontal therapy. Subgingival and salivary microbiotas were processed by means of the Human Oral Microbe Next Generation Sequencing (HOMINGS) technique and characterized based on relative abundance. Spearman signed rank test was used to test correlation of periopathogens in subgingival and saliva samples., Results: Periodontal treatment resulted in significantly higher relative abundance of Streptococcus, Rothia and Actinomyces in combination with a significant decrease in Porphyromonas and Treponema in subgingival plaque samples. Relative abundance of the overall predominant genera in saliva was not influenced by periodontal treatment. However, there was a positive correlation between samples of subgingival plaque and saliva before and after periodontal treatment (p < 0.0001) with respect to relative abundance of specific periopathogens, such as Porphyromonas gingivalis (r = 0.68), Prevotella intermedia (r = 0.72), Filifactor alocis (r = 0.58), Treponema denticola (r = 0.51), Tannerella forsythia (r = 0.45) and Parvimonas micra (r = 0.45)., Conclusions: Subgingival and salivary abundance of periodontal pathogens correlated before and after treatment. Thus, data from this study suggest that periopathogens identified in saliva may be spill-over from the subgingival microbiota., (© 2018 American Academy of Periodontology.)

The purpose of the present study was to characterize and compare supragingival and salivary microbiotas during a 10-d period of oral hygiene discontinuation. We tested the hypothesis that the composition of the salivary microbiota will reflect local microbial changes associated with accumulated biofilm formation and maturation. Pooled supragingival plaque ( n = 145) and stimulated saliva ( n = 145) samples were collected and plaque and gingival indices were recorded from 29 orally healthy individuals at baseline, during oral hygiene discontinuation (days 4, 7, and 10), and 14 d after resumption of oral hygiene. Supragingival and salivary microbiotas were processed by next-generation sequencing (Human Oral Microbe Identification using Next Generation Sequencing) and microbial community profiles were compared. Microbial composition of supragingival plaque samples collected after 4, 7, and 10 d of oral hygiene discontinuation, as well as 14 d after reuptake of oral hygiene, differed significantly from baseline samples, by a 3-fold increase in relative abundance Leptotrichia species and a 2-fold decrease in Streptococcus species (adjusted P < 0.01). In saliva samples, a significant increase in relative abundance of Leptotrichia species (adjusted P < 0.01) was evident at day 7 but completely reversed 14 d after resumption of oral hygiene. While the salivary microbiota was resistant to accumulated local biofilm formation, data from this study showed that compositional changes of supragingival microbiotas were not reversed 14 d after resumption of oral hygiene, despite the restoration of plaque to baseline levels. (ClinicalTrials.gov UCPH&#95;OI&#95;002, NCT02913235). Knowledge Transfer Statement: Data from this study showed compositional changes of supragingival microbiotas as a consequence of a 10-d period of oral hygiene discontinuation, that was not reversed 14 d after resumption of oral hygiene. Notably, oral hygiene discontinuation was associated with a significant increase in relative abundance of potential cariogenic Leptotrichia species and a decrease in Streptococcus species. Thus, findings from this study highlight the necessity of regular oral hygiene in the maintenance of oral homeostasis., Competing Interests: The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

The taxonomic composition of the salivary microbiota has been reported to differentiate between oral health and disease. However, information on bacterial activity and gene expression of the salivary microbiota is limited. The purpose of this study was to perform metagenomic and metatranscriptomic characterization of the salivary microbiota and test the hypothesis that salivary microbial presence and activity could be an indicator of the oral health status. Stimulated saliva samples were collected from 30 individuals (periodontitis: n  = 10, dental caries: n  = 10, oral health: n  = 10). Salivary microbiota was characterized using metagenomics and metatranscriptomics in order to compare community composition and the gene expression between the three groups. Streptococcus was the predominant bacterial genus constituting approx. 25 and 50% of all DNA and RNA reads, respectively. A significant disease-associated higher relative abundance of traditional periodontal pathogens such as Porphyromonas gingivalis and Filifactor alocis and salivary microbial activity of F . alocis was associated with periodontitis. Significantly higher relative abundance of caries-associated bacteria such as Streptococcus mutans and Lactobacillus fermentum was identified in saliva from patients with dental caries. Multiple genes involved in carbohydrate metabolism were significantly more expressed in healthy controls compared to periodontitis patients. Using metagenomics and metatranscriptomics we show that relative abundance of specific oral bacterial species and bacterial gene expression in saliva associates with periodontitis and dental caries. Further longitudinal studies are warranted to evaluate if screening of salivary microbial activity of specific oral bacterial species and metabolic gene expression can identify periodontitis and dental caries at preclinical stages.

Objectives: The purpose of this study was to compare microbial profiles of saliva, pooled and site-specific subgingival samples in patients with periodontitis. We tested the hypotheses that saliva can be an alternative to pooled subgingival samples, when screening for presence of periopathogens., Design: Site specific subgingival plaque samples (n = 54), pooled subgingival plaque samples (n = 18) and stimulated saliva samples (n = 18) were collected from 18 patients with generalized chronic periodontitis. Subgingival and salivary microbiotas were characterized by means of HOMINGS (Human Oral Microbe Identification using Next Generation Sequencing) and microbial community profiles were compared using Spearman rank correlation coefficient., Results: Pronounced intraindividual differences were recorded in site-specific microbial profiles, and site-specific information was in general not reflected by pooled subgingival samples. Presence of Porphyromonas gingivalis, Treponema denticola, Prevotella intermedia, Filifactor alocis, Tannerella forsythia and Parvimona micra in site-specific subgingival samples were detected in saliva with an AUC of 0.79 (sensitivity: 0.61, specificity: 0.94), compared to an AUC of 0.76 (sensitivity: 0.56, specificity: 0.94) in pooled subgingival samples., Conclusions: Site-specific presence of periodontal pathogens was detected with comparable accuracy in stimulated saliva samples and pooled subgingival plaque samples. Consequently, saliva may be a reasonable surrogate for pooled subgingival samples when screening for presence of periopathogens. Future large-scale studies are needed to confirm findings from this study.

Salivary protein levels have been studied in periodontitis. However, there is lack of information on salivary cytokine levels in early gingival inflammation. The aim of this study was to determine salivary levels of vascular endothelial growth factor (VEGF), interleukin (IL)-8, monocyte chemoattractant protein (MCP)-1, IL-1β, and IL-1 receptor antagonist (IL-1Ra) in gingival inflammation. Twenty-eight systemically and orally healthy nonsmokers abstained from oral hygiene protocols for 10 days. After that, self-performed cleaning was resumed for 14 days. Plaque and gingival indexes were measured, and saliva samples were collected at days 1, 4, 7, 10, and 24. Salivary cytokines were detected with Luminex®-xMAP™. Salivary IL-1β, IL-1Ra, and VEGF levels decreased after 10 days' development of experimental gingivitis and reached baseline levels at the end of the 2-week resolution period. Salivary IL-8 levels decreased and remained low during development and resolution of experimental gingivitis. Initial inflammation in gingival tissues is associated with a decrease in inflammatory cytokines in saliva. Further studies are needed to evaluate if inflammatory cytokines bind to their functional receptors within the gingival tissue during early gingivitis, which may limits their spillover to the gingival crevice and ultimately saliva.

The aim was to compare the oral microbial profiles in young adults with an intake of free sugars above or below the current recommendations by the WHO for sugar consumption. Seventy subjects completed a Quantitative Food Frequency Questionnaire to establish the proportion of free sugars in relation to the total energy intake (% E). Subjects with <5% E ( n  = 30) formed the low-sugar group, while those with ≥5% E ( n  = 40) were regarded as reference group. Saliva and plaque samples were analyzed by qPCR, and 52 of the plaque samples were assayed by HOMI NGS . The HOMI NGS analysis revealed a comparable core microbiota in plaque samples with Streptococcus , Leptotrichia , Actinobaculum , and Veillonella as predominant. No major differences between groups were revealed by α-diversity testing ( p  = 0.83), principal component analysis, or correspondence analysis. Higher relative abundance of Streptococcus sobrinus and Prevotella melaninogenica was observed in plaque samples in the reference group. By qPCR, Scardovia wiggsiae was associated with elevated sugar intake. The findings suggests that the amount of ingested sugars had a marginal influence on microbial profiles in dental plaque and saliva. However, some caries-associated species were less abundant in the dental plaque of the low sugar group.

There is a bidirectional relationship between periodontal disease (PD) and type 2 diabetes mellitus (T2D). T2D may lead to ecological perturbations in the oral environment, which may facilitate an altered microbiota. However, previous studies have been inconclusive in determining the effect of T2D on oral bacterial profiles. Therefore, we aimed to evaluate the influence of T2D on the ligature-associated bacterial profile in a diabetic rat model with PD and investigated the impact of blocking inflammatory pathways with antibodies targeting either Tumor Necrosis Factor α (TNF-α) or the receptor of advanced glycation end-products (RAGE). A total of 62 Zucker obese rats (45 T2D) and 17 lean (non-T2D) were divided into 4 treatment groups; lean with PD, obese with PD, obese with PD and anti-TNF-α treatment, and obese with PD with anti-RAGE treatment. Periodontal disease was ligature induced. Ligature-associated bacterial profiles were analyzed using Human Oral Microbe Identification Microarray (HOMIM). Ligature-associated bacterial profiles differed between lean and obese rats. Furthermore, treatment with antibodies against TNF-α or RAGE had an impact on subgingival bacterial profiles. T2D phenotypes are associated with different ligature-associated bacterial profiles and influenced by treatment with antibodies against TNF-α or RAGE.

This study compared salivary bacterial profiles in two groups having a 10-fold difference in levels of caries experience, as it was hypothesized that the composition of the salivary microbiota might associate with the levels of caries experience. Bacterial profiles in stimulated saliva samples from 85 individuals with low levels of caries experience (healthy group) and 79 individuals with high levels of caries experience (caries group) were analyzed by means of the Human Oral Microbiome Identification Next Generation Sequencing (HOMI NGS ) technique. Subsequently, saliva samples from caries-free individuals in the healthy group ( n  = 57) and the caries group ( n  = 31) were compared. A significantly higher α-diversity ( p  < 0.0001) and a twofold higher relative abundance of Neisseria , Haemophilus , and Fusobacterium were recorded in saliva samples from the healthy group compared with the caries group. Differences observed were more pronounced when limiting the analyses to caries-free individuals in each group. Data from this cross-sectional analysis suggest that low levels of caries experience might associate with a characteristic salivary bacterial composition different from that in individuals with high caries experience. Consequently, longitudinal studies are required to determine if the composition of the salivary microbiota might be a predictive factor of caries risk at the individual level.

Chronic infection is associated with an increased risk of atherothrombotic disease and direct bacterial infection of arteries has been suggested to contribute to the development of unstable atherosclerotic plaques. In this study, we examined coronary thrombi obtained in vivo from patients with ST-segment elevation myocardial infarction (STEMI) for the presence of bacterial DNA and bacteria. Aspirated coronary thrombi from 22 patients with STEMI were collected during primary percutaneous coronary intervention and arterial blood control samples were drawn from radial or femoral artery sheaths. Analyses were performed using 16S polymerase chain reaction and with next-generation sequencing to determine bacterial taxonomic classification. In selected thrombi with the highest relative abundance of Pseudomonas aeruginosa DNA, peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) with universal and species specific probes was performed to visualize bacteria within thrombi. From the taxonomic analysis we identified a total of 55 different bacterial species. DNA from Pseudomonas aeruginosa represented the only species that was significantly associated with either thrombi or blood and was >30 times more abundant in thrombi than in arterial blood (p<0.0001). Whole and intact bacteria present as biofilm microcolonies were detected in selected thrombi using universal and P. aeruginosa-specific PNA-FISH probes. P. aeruginosa and vascular biofilm infection in culprit lesions may play a role in STEMI, but causal relationships remain to be determined., Competing Interests: The authors have declared that no competing interests exist.

Background: The composition of the salivary microbiota has been reported to differentiate between patients with periodontitis, dental caries and orally healthy individuals. To identify characteristics of diseased and healthy saliva we thus wanted to compare saliva metaproteomes from patients with periodontitis and dental caries to healthy individuals., Methods: Stimulated saliva samples were collected from 10 patients with periodontitis, 10 patients with dental caries and 10 orally healthy individuals. The proteins in the saliva samples were subjected to denaturing buffer and digested enzymatically with LysC and trypsin. The resulting peptide mixtures were cleaned up by solid-phase extraction and separated online with 2 h gradients by nano-scale C18 reversed-phase chromatography connected to a mass spectrometer through an electrospray source. The eluting peptides were analyzed on a tandem mass spectrometer operated in data-dependent acquisition mode., Results: We identified a total of 35,664 unique peptides from 4,161 different proteins, of which 1,946 and 2,090 were of bacterial and human origin, respectively. The human protein profiles displayed significant overexpression of the complement system and inflammatory markers in periodontitis and dental caries compared to healthy controls. Bacterial proteome profiles and functional annotation were very similar in health and disease., Conclusions: Overexpression of proteins related to the complement system and inflammation seems to correlate with oral disease status. Similar bacterial proteomes in healthy and diseased individuals suggests that the salivary microbiota predominantly thrives in a planktonic state expressing no disease-associated characteristics of metabolic activity., Competing Interests: The authors declare that they have no competing interests.

Objective: The purpose of this study was to compare the microbiota of stimulated whole saliva samples from patients with severe hyposalivation to samples from individuals with normal whole saliva flow rates. It was hypothesized that the two groups differ with regard to salivary bacterial profiles., Methods: This cross-sectional study included 36 participants (24 females and 12 males, mean age 58.5 years) with severe hyposalivation and 36 gender-, age-, and geographically matched participants with normal salivary secretion from the Danish Health Examination Survey (DANHES). The microbiota of stimulated whole saliva samples was characterized by HOMINGS., Results: The two groups had comparable caries experience measured by decayed, missed, filled surfaces/teeth and decayed, missed, filled root surfaces as well as active caries lesions. In addition, no single probe target was present with a significant difference in frequency or proportional presence between groups. Furthermore, data reduction by principal component analysis and correspondence analysis showed comparable bacterial community profiles between groups., Conclusions: The results indicate that the salivary bacterial profiles of patients with severe hyposalivation do not differ from those of individuals with normal salivary secretion, when there are virtually no untreated active caries lesions present in the oral cavity., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: It is estimated that 3.6% and 13.6% of the Danish population have undiagnosed type 2 diabetes mellitus and prediabetes, respectively. Periodontitis is an established complication associated with diabetes (DM). Identification of individuals with DM and prediabetes is important to reduce DM-related complications, including periodontitis. The objective of this study is to identify individuals with undiagnosed DM or prediabetes among patients attending a dental setting for diagnosis and treatment., Methods: A total of 291 adults with no history of DM was included in the study (periodontitis patients: n = 245; non-periodontitis control individuals: n = 46). Participants answered questionnaires concerning general health, including family history of DM. Body mass index, waist circumference, fat percentage, and glycated hemoglobin (HbA1c) level were recorded chairside. Periodontal examination was performed and radiographic bone level measured. All individuals were informed about their HbA1c test result and were referred to their physician if HbA1c level was above guideline levels for DM or prediabetes according to the American Diabetes Association., Results: Nine (3.1%) and 79 (27.1%) individuals were identified with HbA1c levels corresponding to guideline levels for DM and prediabetes, respectively. Higher proportions of patients with undiagnosed DM and prediabetes were observed in the periodontitis group (32.7%) than in the control group (17.4%) (P = 0.054). Identification of DM and prediabetes based on a diagnosis of periodontitis yielded a sensitivity of 0.91 and a specificity of 0.19., Conclusions: This study confirms that individuals with undiagnosed DM and prediabetes can be identified in the dental office by chairside recordings of HbA1c levels. Routine measurement of HbA1c levels in dental offices, eventually restricted to those at risk, may help identification of individuals with DM and prediabetes at early stages of disease, which may prevent future complications.

Background and Objective: The microbial profiles of stimulated saliva samples have been shown to differentiate between patients with periodontitis, patients with dental caries, and orally healthy individuals. Saliva was stimulated to allow for easy and rapid collection; however, microbial composition may not reflect the more natural, unstimulated state. The purpose of this study was to validate whether stimulated saliva is an adequate surrogate for unstimulated saliva in determining salivary microbiomes., Design: Unstimulated (n=20) and stimulated (n=20) saliva samples were collected from 20 orally and systemically healthy, non-smoking participants. Salivary bacterial profiles were analyzed by means of the Human Oral Microbe Identification using Next Generation Sequencing (HOMINGS), and statistical analysis was performed using Mann-Whitney test with Benjamini-Hochberg's correction for multiple comparison, cluster analysis, principal component analysis, and correspondence analysis., Results: From a total of 40 saliva samples, 496 probe targets were identified with a mean number of targets per sample of 203 (range: 146-303), and a mean number of probe targets of 206 and 200 in unstimulated and stimulated saliva samples, respectively (p=0.62). Based on all statistical methods used for this study, the microbial profiles of unstimulated and stimulated saliva samples collected from the same person were not statistically significantly different., Conclusions: Analysis of bacterial salivary profiles in unstimulated and stimulated saliva samples collected from the same individual showed comparable results. Thus, the results verify that stimulated saliva is an adequate surrogate of unstimulated saliva for microbiome-related studies.

Objectives: Saliva is a biological fluid suitable for biomarker analysis, and differences in the salivary microbiota in oral health and disease have been reported. For such comparative analyses, time of sampling is critical since the bacterial composition may vary throughout the day, i.e., diurnal variation. The purpose of this study is to compare the salivary microbiome over time to determine the optimal time for sampling., Design: Stimulated saliva samples were collected from 5 orally healthy individuals in 4 h intervals for 24 h, and collection was repeated 7 days later (number of samples per person, n = 12, total number of samples, n = 60). Salivary microbiota was analyzed using the Human Oral Microbe Identification using Next Generation Sequencing (HOMINGS), and statistical analysis was performed using the Kruskal-Wallis test with Benjamini-Hochberg's correction for multiple comparisons, cluster analysis, principal component analysis and correspondence analysis., Results: From a total of 60 saliva samples, 477 probe targets were collectively identified with a mean number of probes per sample of 207 (range: 153-307). Little or no variation in microbial profiles within subjects was observed over time., Conclusions: Although there was considerable variation between subjects, microbial profiles within subjects were stable throughout a 24 hour period and after 1 week. Since there is little or no evidence of diurnal variation of the salivary microbiome, time of sampling of saliva is not critical for perturbation or other microbial studies.

Background and Objective: The composition of the salivary microbiota, as determined using various molecular methods, has been reported to differentiate oral health from diseases. Thus, the purpose of this study was to utilize the newly developed molecular technique HOMINGS (Human Oral Microbe Identification using Next Generation Sequencing) for comparison of the salivary microbiota in patients with periodontitis, patients with dental caries, and orally healthy individuals. The hypothesis was that this method could add on to the existing knowledge on salivary bacterial profiles in oral health and disease., Design: Stimulated saliva samples (n=30) were collected from 10 patients with untreated periodontitis, 10 patients with untreated dental caries, and 10 orally healthy individuals. Salivary microbiota was analyzed using HOMINGS and statistical analysis was performed using Kruskal-Wallis test with Benjamini-Hochberg's correction., Results: From a total of 30 saliva samples, a mean number of probe targets of 205 (range 120-353) were identified, and a statistically significant higher mean number of targets was registered in samples from patients with periodontitis (mean 220, range 143-306) and dental caries (mean 221, range 165-353) as compared to orally healthy individuals (mean 174, range 120-260) (p=0.04 and p=0.04). Nine probe targets were identified with a different relative abundance between groups (p<0.05)., Conclusions: Cross-sectional comparison of salivary bacterial profiles by means of HOMINGS analysis showed that different salivary bacterial profiles were associated with oral health and disease. Future large-scale prospective studies are needed to evaluate if saliva-based screening for disease-associated oral bacterial profiles may be used for identification of patients at risk of acquiring periodontitis and dental caries.

Bacterial profiles of saliva in subjects with periodontitis and dental caries have been demonstrated to differ from that of oral health. The aim of this comparative analysis of existing data generated by the Human Oral Microbe Identification Microarray (HOMIM) from 293 stimulated saliva samples was to compare bacterial profiles of saliva in subjects with periodontitis and dental caries.

Background and Objective: The bacterial profile of saliva is composed of bacteria from different oral surfaces. The objective of this study was to determine whether different diet intake, lifestyle, or socioeconomic status is associated with characteristic bacterial saliva profiles., Design: Stimulated saliva samples from 292 participants with low levels of dental caries and periodontitis, enrolled in the Danish Health Examination Survey (DANHES), were analyzed for the presence of approximately 300 bacterial species by means of the Human Oral Microbe Identification Microarray (HOMIM). Using presence and levels (mean HOMIM-value) of bacterial probes as endpoints, the influence of diet intake, lifestyle, and socioeconomic status on the bacterial saliva profile was analyzed by Mann-Whitney tests with Benjamini-Hochberg's correction for multiple comparisons and principal component analysis., Results: Targets for 131 different probes were identified in 292 samples, with Streptococcus and Veillonella being the most predominant genera identified. Two bacterial taxa (Streptococcus sobrinus and Eubacterium [11][G-3] brachy) were more associated with smokers than non-smokers (adjusted p-value<0.01). Stratification of the group based on extreme ends of the parameters age, gender, alcohol consumption, body mass index (BMI), and diet intake had no statistical influence on the composition of the bacterial profile of saliva. Conversely, differences in socioeconomic status were reflected by the bacterial profiles of saliva., Conclusions: The bacterial profile of saliva seems independent of diet intake, but influenced by smoking and maybe socioeconomic status.

Aim: Periodontitis is a multifactorial disease in which subgingival bacteria play an important role in the pathogenesis of the disease. The objective of this study was to determine if periodontitis is associated with a characteristic salivary bacterial profile. This was accomplished by comparing the bacterial profile of saliva from subjects with chronic periodontitis with that of saliva from a control cohort., Materials and Methods: Stimulated saliva samples from 139 chronic periodontitis patients and 447 samples from a control cohort were analysed using the Human Oral Microbe Identification Microarray (HOMIM). Frequency and levels (mean HOMIM-value) of around 300 bacterial taxa/clusters in samples were used as parameters for investigation. Differences at taxon/cluster values between groups were analysed using Mann-Whitney U-test with Benjamini-Hochberg correction for multiple comparisons. Principal component analysis was used to visualize bacterial community profiles obtained by the HOMIM., Results: Eight bacterial taxa, including putative periodontal pathogens as Parvimonas micra and Filifactor alocis, and four bacterial clusters were identified statistically more frequently and at higher levels in samples from periodontitis patients than in samples from the control cohort. These differences were independent of the individuals' smoking status., Conclusions: Periodontitis is associated with a characteristic bacterial profile of saliva different from that of a control cohort., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The aim of this study was to learn whether presence of caries in an adult population was associated with a salivary bacterial profile different from that of individuals without untreated caries. Stimulated saliva samples from 621 participants of the Danish Health Examination Survey were analyzed using the Human Oral Microbe Identification Microarray technology. Samples from 174 individuals with dental caries and 447 from a control cohort were compared using frequency and levels of identified bacterial taxa/clusters as endpoints. Differences at taxon/cluster level were analyzed using Mann-Whitney's test with Benjamini-Hochberg correction for multiple comparisons. Principal component analysis was used to visualize bacterial community profiles. A reduced bacterial diversity was observed in samples from subjects with dental caries. Five bacterial taxa (Veillonella parvula, Veillonella atypica, Megasphaera micronuciformis, Fusobacterium periodontium and Achromobacter xylosoxidans) and one bacterial cluster (Leptotrichia sp. clones C3MKM102 and GT018&#95;ot417/462) were less frequently found in the caries group (adjusted p value <0.01) while two bacterial taxa (Solobacterium moorei and Streptococcus salivarius) and three bacterial clusters (Streptococcus parasanguinis I and II and sp. clone BE024&#95;ot057/411/721, Streptococcus parasanguinis I and II and sinensis&#95;ot411/721/767, Streptococcus salivarius and sp. clone FO042&#95;ot067/755) were present at significantly higher levels (adjusted p value <0.01). The principal component analysis displayed a marked difference in the bacterial community profiles between groups. Presence of manifest caries was associated with a reduced diversity and an altered salivary bacterial community profile. Our data support recent theories that ecological stress-induced changes of commensal microbial communities are involved in the shift from oral health to tooth decay., (© 2014 S. Karger AG, Basel.)

Inflammation is believed to play a central part in the pathogenesis of atherosclerosis, and much attention has been paid to the possible association between atherosclerosis and other inflammatory diseases. Periodontal disease is a common inflammatory disease affecting up to 50% of the adult population, and during the past two decades much research has focused on a possible association between periodontitis and cardiovascular disease. Here, we review the existing literature on the association between the two diseases., (Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

A relationship between periodontitis and coronary heart disease has been investigated intensively. A pathogenic role for the oral bacterium Porphyromonas gingivalis has been suggested for both diseases. We examined whether complement activation by P. gingivalis strain ATCC 33277 allows the bacterium to adhere to human red blood cells (RBCs) and thereby evade attack by circulating phagocytes. On incubation with normal human serum, the P. gingivalis strain efficiently fixed complement component 3 (C3). Incubation of bacteria with washed whole blood cells suspended in autologous serum resulted in a dose- and time-dependent adherence to RBCs. The adherence required functionally intact complement receptor 1 (CR1; also called CD35) on the RBCs and significantly inhibited the uptake of P. gingivalis by neutrophils and B cells within 1 min of incubation (by 64% and 51%, respectively) and that by monocytes after between 15 min and 30 min of incubation (by 66% and 53%, respectively). The attachment of C3b/iC3b to bacterium-bearing RBCs decreased progressively after 15 min, indicating that conversion of C3 fragments into C3dg occurred, decreasing the affinity for CR1 on RBCs. We propose that P. gingivalis exploits RBCs as a transport vehicle, rendering it inaccessible to attack by phagocytes, and by doing so plays a role in the development of systemic diseases.