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The oral cavity is an unique ecosystem formed by different structures, tissues, and a complex microbial community formed by hundreds of different species of bacteria, fungi, viruses, phages, and the candidate phyla radiation (CPR) group, all living in symbiosis with healthy individuals. In an opposite state, dental caries is a biofilm-mediated dysbiosis that involves changes in the core microbiome composition and function, which leads to the demineralization of tooth tissues due to the fermentation of dietary carbohydrates, producing acid by select oral bacteria. The cariogenic biofilm is typically characterized by bacterial species with the ability of adhering to the saliva-coated tooth surface, production of exopolysaccharides-rich matrix (which will limit the diffusion of acidic products of carbohydrate fermentation), and the ability of surviving in this acidic environment. Besides years of research and dental treatment, dental caries remains the most common chronic disease in children worldwide. This article aims to bring an insightful discussion about important questions that remain unanswered in the Cariology and Oral Microbiology fields, to move Science forward, characterize the interrelationships of these communities, and understand mechanistic functions between microorganisms and the host, therefore leading to translatable knowledge that benefits the provision of care to our pediatric patients.

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. 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. 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. 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.

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. 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. 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. 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.

The human oral microbiome is potentially related to diverse health conditions and high-throughput technology provides the possibility of surveying microbial community structure at high resolution. We compared two oral microbiome survey methods: broad-based microbiome identification by 16S rRNA gene sequencing and targeted characterization of microbes by custom DNA microarray. Oral wash samples were collected from 20 individuals at Memorial Sloan-Kettering Cancer Center. 16S rRNA gene survey was performed by 454 pyrosequencing of the V3-V5 region (450 bp). Targeted identification by DNA microarray was carried out with the Human Oral Microbe Identification Microarray (HOMIM). Correlations and relative abundance were compared at phylum and genus level, between 16S rRNA sequence read ratio and HOMIM hybridization intensity. The major phyla, Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria were identified with high correlation by the two methods (r = 0.70∼0.86). 16S rRNA gene pyrosequencing identified 77 genera and HOMIM identified 49, with 37 genera detected by both methods; more than 98% of classified bacteria were assigned in these 37 genera. Concordance by the two assays (presence/absence) and correlations were high for common genera (Streptococcus, Veillonella, Leptotrichia, Prevotella, and Haemophilus; Correlation = 0.70-0.84). Microbiome community profiles assessed by 16S rRNA pyrosequencing and HOMIM were highly correlated at the phylum level and, when comparing the more commonly detected taxa, also at the genus level. Both methods are currently suitable for high-throughput epidemiologic investigations relating identified and more common oral microbial taxa to disease risk; yet, pyrosequencing may provide a broader spectrum of taxa identification, a distinct sequence-read record, and greater detection sensitivity.

Dental caries is the most prevalent disease in humans globally. Efforts to control it have been invigorated by an increasing knowledge of the oral microbiome composition. This study aimed to evaluate the bacterial diversity in occlusal biofilms and its relationship with clinical surface diagnosis and dietary habits. Anamneses were recorded from thirteen 12-year-old children. Biofilm samples collected from occlusal surfaces of 46 permanent second molars were analyzed by 16S rRNA amplicon sequencing combined with the BLASTN-based search algorithm for species identification. The overall mean decayed, missing and filled surfaces modified index [DMFSm Index, including active white spot lesions (AWSL)] value was 8.77±7.47. Biofilm communities were highly polymicrobial collectively, representing 10 bacterial phyla, 25 classes, 29 orders, 58 families, 107 genera, 723 species. Streptococcus sp_Oral_Taxon_065, Corynebacterium matruchotii, Actinomyces viscosus, Actinomyces sp_Oral_Taxon_175, Actinomyces sp_Oral_Taxon_178, Actinomyces sp_Oral_Taxon_877, Prevotella nigrescens, Dialister micraerophilus, Eubacterium_XI G 1 infirmum were more abundant among surfaces with AWSL, and Streptococcus gordonii, Streptococcus sp._Oral_Taxon_058, Enterobacter sp._str._638 Streptococcus australis, Yersinia mollaretii, Enterobacter cloacae, Streptococcus sp._Oral_Taxon_71, Streptococcus sp._Oral_Taxon_F11, Centipeda sp._Oral_Taxon_D18 were more abundant among sound surfaces. Streptococcus mutans was detected on all surfaces in all patients, while Streptococcus sobrinus was detected only in three patients (mean relative abundances 7.1% and 0.6%, respectively). Neither species differentiated healthy from diseased sites. Diets of nine of the subjects were scored as high in fermentable carbohydrates (≧2X/day between meals). A direct association between relative abundances of bacteria and carbohydrate consumption was observed among 18 species. High consumption of fermentable carbohydrates and sound surfaces were associated with a reduction in bacterial diversity. PCoA plots displayed differences in bacterial community profiles between sound and diseased surfaces. Our study showed that, in addition to mutans streptococci, other species may be associated with the initiation of dental caries on occlusal surfaces, and that biofilm diversity of tooth surfaces is influenced by carbohydrate consumption and a surface's health status.

Objectives Microglial activation is critical for modulating the neuroinflammatory process and the pathological progression of neurodegenerative diseases, such as Alzheimer's disease (AD). Microglia are involved in forming barriers around extracellular neuritic plaques and the phagocytosis of β-amyloid peptide (Aβ). In this study, we tested the hypothesis that periodontal disease (PD) as a source of infection alters inflammatory activation and Aβ phagocytosis by the microglial cells. Methods Experimental PD was induced using ligatures in C57BL/6 mice for 1, 10, 20, and 30 days to assess the progression of PD. Animals without ligatures were used as controls. Maxillary bone loss and local periodontal tissue inflammation associated with the development of PD were confirmed by morphometric bone analysis and cytokine expression, respectively. The frequency and the total number of activated microglia (CD45 + CD11b + MHCII + ) in the brain were analyzed by flow cytometry. Mouse microglial cells (1 × 10 5 ) were incubated with heat-inactivated bacterial biofilm isolated from the ligatures retrieved from the teeth or with Klebsiella variicola , a relevant PD-associated bacteria in mice. Expression of pro-inflammatory cytokines, toll-like receptors (TLR), and receptors for phagocytosis was measured by quantitative PCR. The phagocytic capacity of microglia to uptake β-amyloid was analyzed by flow cytometry. Results Ligature placement caused progressive periodontal disease and bone resorption that was already significant on day 1 post-ligation ( p  < 0.05) and continued to increase until day 30 ( p  < 0.0001). The severity of periodontal disease increased the frequency of activated microglia in the brains on day 30 by 36%. In parallel, heat-inactivated PD-associated total bacteria and Klebsiella variicola increased the expression of TNFα, IL-1β, IL-6, TLR2, and TLR9 in microglial cells (1.6-, 83-, 3.2-, 1.5-, 1.5-fold, respectively p  < 0.01). Incubation of microglia with Klebsiella variicola increased the Aβ-phagocytosis by 394% and the expression of the phagocytic receptor MSR1 by 33-fold compared to the non-activated cells ( p  < 0.0001). Conclusions We showed that inducing PD in mice results in microglia activation in vivo and that PD-associated bacteria directly promote a pro-inflammatory and phagocytic phenotype in microglia. These results support a direct role of PD-associated pathogens in neuroinflammation.

Recent studies uncovered that Fusobacterium nucleatum (Fn), a common, opportunistic bacterium in the oral cavity, is associated with a growing number of systemic diseases, ranging from colon cancer to Alzheimer's disease. However, the pathological mechanisms responsible for this association are still poorly understood. Here, we leverage recent technological advances to study the interactions between Fn and neutrophils. We show that Fn survives within human neutrophils after phagocytosis. Using in vitro microfluidic devices, we determine that human neutrophils can protect and transport Fn over large distances. Moreover, we validate these observations in vivo by showing that neutrophils disseminate Fn using a zebrafish model. Our data support the emerging hypothesis that bacterial dissemination by neutrophils is a mechanistic link between oral and systemic diseases. Furthermore, our results may ultimately lead to therapeutic approaches that target specific host-bacteria interactions, including the dissemination process.

BackgroundOral pathology is a commonly reported extraintestinal manifestation of Crohn's disease (CD). The host–microbe interaction has been implicated in the pathogenesis of inflammatory bowel disease (IBD) in genetically susceptible hosts, yet limited information exists about oral microbes in IBD. We hypothesize that the microbiology of the oral cavity may differ in patients with IBD. Our laboratory has developed a 16S rRNA-based technique known as the Human Oral Microbe Identification Microarray (HOMIM) to study the oral microbiome of children and young adults with IBD.MethodsTongue and buccal mucosal brushings from healthy controls, CD, and ulcerative colitis (UC) patients were analyzed using HOMIM. Shannon Diversity Index (SDI) and Principal Component Analysis (PCA) were employed to compare population and phylum-level changes among our study groups.ResultsIn all, 114 unique subjects from the Children's Hospital Boston were enrolled. Tongue samples from patients with CD showed a significant decrease in overall microbial diversity as compared with the same location in healthy controls (P = 0.015) with significant changes seen in Fusobacteria (P < 0.0002) and Firmicutes (P = 0.022). Tongue samples from patients with UC did not show a significant change in overall microbial diversity as compared with healthy controls (P = 0.418).ConclusionsAs detected by HOMIM, we found a significant decrease in overall diversity in the oral microbiome of pediatric CD. Considering the proposed microbe–host interaction in IBD, the ease of visualization and direct oral mucosal sampling of the oral cavity, further study of the oral microbiome in IBD is of potential diagnostic and prognostic value.

Leukocyte Adhesion Deficiency I (LAD-I) is a primary immunodeficiency caused by single gene mutations in the CD18 subunit of β2 integrins which result in defective transmigration of neutrophils into the tissues. Affected patients suffer from recurrent life threatening infections and severe oral disease (periodontitis). Microbial communities in the local environment (subgingival plaque) are thought to be the triggers for inflammatory periodontitis, yet little is known regarding the microbial communities associated with LAD-I periodontitis. Here we present the first comprehensive characterization of the subgingival communities in LAD-I, using a 16S rRNA gene-based microarray, and investigate the relationship of this tooth adherent microbiome to the local immunopathology of periodontitis. We show that the LAD subgingival microbiome is distinct from that of health and Localized Aggressive Periodontitits. Select periodontitis-associated species in the LAD microbiome included Parvimonas micra, Porphyromonas endodontalis, Eubacterium brachy and Treponema species. Pseudomonas aeruginosa, a bacterium not typically found in subgingival plaque is detected in LAD-I. We suggest that microbial products from LAD-associated communities may have a role in stimulating the local inflammatory response. We demonstrate that bacterial LPS translocates into the lesions of LAD-periodontitis potentially triggering immunopathology. We also show in in vitro assays with human macrophages and in vivo in animal models that microbial products from LAD-associated subgingival plaque trigger IL-23-related immune responses, which have been shown to dominate in patient lesions. In conclusion, our current study characterizes the subgingival microbial communities in LAD-periodontitis and supports their role as triggers of disease pathogenesis.