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Microbial communities along mucosal surfaces throughout the digestive tract are hypothesized as risk factors for impaired glucose regulation and the development of clinical cardiometabolic disease. We investigated whether baseline measures of subgingival microbiota predicted fasting plasma glucose (FPG) longitudinally. The Oral Infections, Glucose Intolerance and Insulin Resistance Study (ORIGINS) enrolled 230 diabetes-free adults (77% female) aged 20 to 55 y (mean ± SD, 34 ± 10 y) from whom baseline subgingival plaque and longitudinal FPG were measured. DNA was extracted from subgingival plaque, and V3 to V4 regions of the 16S rRNA gene were sequenced. FPG was measured at baseline and again at 2 y; glucose change was defined as follow-up minus baseline. Multivariable linear models regressed 2-y glucose change onto baseline measures of community diversity and abundances of 369 individual taxa. A microbial dysbiosis index (MDI) summarizing top individual taxa associated with glucose change was calculated and used in regression models. Models were adjusted for age, sex, race/ethnicity, education, smoking status, body mass index, and baseline glucose levels. Statistical significance was based on the false discovery rate (FDR; -4, derived from the initial 369 hypothesis tests for specific taxa. Mean 2-y FPG change was 1.5 ± 8 mg/dL. Baseline levels of 9 taxa predicted FPG change (all FDR -4). Subgingival microbiota predict 2-y glucose change among diabetes-free men and women.

Aim: This in vivo experimental study investigated bacterial microbiome and metabolome longitudinal changes associated with enamel caries lesion progression and arrest. Methods: We induced natural caries activity in three caries-free volunteers prior to four premolar extractions for orthodontic reasons. The experimental model included placement of a modified orthodontic band on smooth surfaces and a mesh on occlusal surfaces. We applied the caries-inducing protocol for 4- and 6-weeks, and subsequently promoted caries lesion arrest via a 2-week toothbrushing period. Lesions were verified clinically and quantitated via micro-CT enamel density measurements. The biofilm microbial composition was determined via 16S rRNA gene Illumina sequencing and NMR spectrometry was used for metabolomics. Results: Biofilm maturation and caries lesion progression were characterized by an increase in Gram-negative anaerobes, including Veillonella and Prevotella. Streptococcus was associated caries lesion progression, while a more equal distribution of Streptococcus, Bifidobacterium, Atopobium, Prevotella, Veillonella, and Saccharibacteria (TM7) characterized arrest. Lactate, acetate, pyruvate, alanine, valine, and sugars were more abundant in mature biofilms compared to newly formed biofilms. Conclusions: These longitudinal bacterial microbiome and metabolome results provide novel mechanistic insights into the role of the biofilm in caries progression and arrest and offer promising candidate biomarkers for validation in future studies.

Aims The aim of this study was to systematically review the literature on prevalence of microorganisms and their viability/activity in endodontic periapical lesions. Design Literature research was performed on five electronic biomedical databases from their start dates to June 2020. Only studies evaluating the presence of microorganisms in periapical lesions in human permanent teeth with secondary/persistent infection were included. Two reviewers independently assessed the eligibility for inclusion, extracted data and evaluated the risk of bias. Meta-analysis and binominal tests were used to analyse the resulting data. Results From the 1,313 records found, 23 full-texts were included for qualitative and quantitative analysis. The prevalence of microorganisms in endodontic periapical lesions was 87 % (95 % CI, 75–94) and the prevalence of viable/active microorganisms was 82 % (95 % CI, 66–91). There were statistical differences in the geographic area subgroup and between viable bacteria and active viruses. The most common detection method of microorganisms was the molecular one (69 %), and the most prevalent bacteria were the species Actinomyces, Fusobacterium and Prevotella (40 %). Most of the included studies had moderate risk of bias. Conclusions The prevalence of microorganisms in endodontic periapical lesions was 87 % and the prevalence of viable/active microorganisms was 82 %.

Background: Oral microbiota is believed to play important roles in systemic diseases, including cancer. Methods: We collected oral samples (tongue, buccal, supragingival, and saliva) and pancreatic tissue or intestinal samples from 52 subjects, and characterized 16S rRNA genes using high-throughput DNA sequencing. Results: Bray–Curtis plot showed clear separations between bacterial communities in the oral cavity and those in intestinal and pancreatic tissue samples. PERMANOVA tests indicated that bacterial communities from buccal samples were similar to supragingival and saliva samples, and pancreatic duct samples were similar to pancreatic tumor samples, but all other samples were significantly different from each other. A total of 73 unique Amplicon Sequence Variants (ASVs) were shared between oral and pancreatic or intestinal samples. Only four ASVs showed significant concordance, and two specific bacterial species (Gemella morbillorum and Fusobacterium nucleatum subsp. vincentii) showed consistent presence or absence patterns between oral and intestinal or pancreatic samples, after adjusting for within-subject correlation and disease status. Lastly, microbial co-abundance analyses showed distinct strain-level cluster patterns among microbiome members in buccal, saliva, duodenum, jejunum, and pancreatic tumor samples. Conclusions: Our findings indicate that oral, intestinal, and pancreatic bacterial microbiomes overlap but exhibit distinct co-abundance patterns in patients with pancreatic cancer and other gastrointestinal diseases.

Background There is a sparsity of data describing the periodontal microbiome in elderly individuals. We analyzed the association of subgingival bacterial profiles and clinical periodontal status in a cohort of participants in the Washington Heights-Inwood Columbia Aging Project (WHICAP). Methods Dentate individuals underwent a full-mouth periodontal examination at six sites/tooth. Up to four subgingival plaque samples per person, each obtained from the mesio-lingual site of the most posterior tooth in each quadrant, were harvested and pooled. Periodontal status was classified according to the Centers for Disease Control/American Academy of Periodontology (CDC/AAP) criteria as well as based on the percentage of teeth/person with pockets ≥4 mm deep. Bacterial DNA was isolated and was processed and analyzed using Human Oral Microbe Identification using Next Generation Sequencing (HOMINGS). Differential abundance across the periodontal phenotypes was calculated using the R package DESeq2. α- and β-diversity metrics were calculated using DADA2-based clustering. Results The mean age of the 739 participants was 74.5 years, and 32% were male. Several taxa including Sneathia amnii-like sp., Peptoniphilaceae [G-1] bacterium HMT 113, Porphyromonas gingivalis, Fretibacterium fastidiosum, Filifactor alocis, and Saccharibacteria (TM7) [G-1] bacterium HMT 346 were more abundant with increasing severity of periodontitis. In contrast, species such as Veillonella parvula, Veillonella dispar, Rothia dentocariosa, and Lautropia mirabilis were more abundant in health. Microbial diversity increased in parallel with the severity and extent of periodontitis. Conclusions The observed subgingival bacterial patterns in these elderly individuals corroborated corresponding findings in younger cohorts and were consistent with the concept that periodontitis is associated with perturbations in the resident microbiome.

Oral microbiota are believed to play important roles in systemic diseases, including cancer. We collected oral swabs and at least one pancreatic tissue or intestinal samples from 52 subjects, and characterized 16S ribosomal RNA genes using high-throughput DNA sequencing in a total 324 samples. We identified a total of 73 unique Amplicon Sequence Variants (ASVs) that were shared between oral and pancreatic or intestinal samples. Accounting for pairing and within-subject correlation, 7 ASVs showed significant concordance (Kappa statistics) and 5 ASVs exhibited significant or marginally significant Pairwise Stratified Association (PASTA) between oral samples and pancreatic tissue or intestinal samples. Of these, two specific bacterial species (Gemella morbillorum and Fusobacterium nucleatum subsp. vincentii) showed consistent presence or absence patterns between oral and intestinal or pancreatic samples. Lastly, our microbial co-abundance analyses showed several distinct ASVs clusters and complex correlation-networks between ASV clusters in buccal, saliva, duodenum, jejunum, and pancreatic tumor samples. Toghether, our findings suggest that oral, intestinal, and pancreatic microbiomes are correlated and bacteria of oral origin exhibit co-abundance relationships and demonstrate complex correlation patterns in the intestinal and pancreatic tumor samples. Future prospective studies should aim to uncover the co-abundance of specific microbial communities for studying etiology of microbiota-driven carcinogenesis.

Background The enterosalivary nitrate‐nitrite‐nitric oxide pathway is an alternative pathway of nitric oxide generation, potentially linking the oral microbiome to insulin resistance and blood pressure (BP). We hypothesized that increased abundance of nitrate‐reducing oral bacteria would be associated with lower levels of cardiometabolic risk cross‐sectionally. Methods and Results ORIGINS (Oral Infections, Glucose Intolerance, and Insulin Resistance Study) enrolled 300 diabetes mellitus–free adults aged 20 to 55 years (mean=34±10 years) (78% women). Microbial DNA was extracted from subgingival dental plaque (n=281) and V3–V4 regions of the 16S rRNA gene were sequenced to measure the relative abundances of 20 a priori – selected taxa with nitrate‐reducing capacity. Standardized scores of each taxon's relative abundance were summed, producing a nitrate‐reducing taxa summary score ( NO 3 TSS ) for each participant. Natural log‐transformed homeostatic model assessment of insulin resistance, plasma glucose, systolic BP, and diastolic BP were regressed on NO 3 TSS in multivariable linear regressions; prediabetes mellitus and hypertension prevalence were regressed on NO 3 TSS using modified Poisson regression models. Nitrate‐reducing bacterial species represented 20±16% of all measured taxa. After multivariable adjustment, a 1‐ SD increase in NO 3 TSS , was associated with a −0.09 (95% CI , −0.15 to −0.03) and −1.03 mg/dL (95% CI , −1.903 to −0.16) lower natural log‐transformed homeostatic model assessment of insulin resistance and plasma glucose, respectively. NO 3 TSS was associated with systolic BP only among patients without hypertension; 1‐ SD increase in NO 3 TSS was associated with −1.53 (95% CI , −2.82 to −0.24) mm Hg lower mean systolic BP. No associations were observed with prediabetes mellitus and hypertension. Conclusions A higher relative abundance of oral nitrate‐reducing bacteria was associated with lower insulin resistance and plasma glucose in the full cohort and with mean systolic BP in participants with normotension.

Inflammation might link microbial exposures to insulin resistance. We investigated the cross-sectional association between periodontal microbiota, inflammation and insulin resistance.The Oral Infections, Glucose Intolerance and Insulin Resistance Study (ORIGINS) enrolled 152 diabetes-free adults (77% female) aged 20-55 years (mean = 34 ± 10). Three hundred and four subgingival plaque samples were analysed using the Human Oral Microbe Identification Microarray to measure the relative abundances of 379 taxa. C-reactive protein, interleukin-6, tumour necrosis factor-α and adiponectin were assessed from venous blood and their z-scores were summed to create an inflammatory score (IS). Insulin resistance was defined via the HOMA-IR. Associations between the microbiota and both inflammation and HOMA-IR were explored using multivariable linear regressions; mediation analyses assessed the proportion of the association explained by inflammation.The IS was inversely associated with Actinobacteria and Proteobacteria and positively associated with Firmicutes and TM7 (p-values 0.05). Proteobacteria levels were associated with insulin resistance (p 0.05). Inflammation explained 30-98% of the observed associations between levels of Actinobacteria, Proteobacteria or Firmicutes and insulin resistance (p-values 0.05). Eighteen individual taxa were associated with inflammation (p 0.05) and 22 with insulin resistance (p 0.05). No findings for individual taxa met Bonferroni-adjusted statistical significance.Bacterial measures were related to inflammation and insulin resistance among diabetes-free adults.

Aggregatibacter actinomycetemcomitans is associated with aggressive periodontitis resulting in premature tooth loss in adolescents. Tooth adherence and biofilm persistence are prerequisites for survival in the oral domain. Here, using a rhesus monkey model, 16S rRNA sequencing, and weighted network analysis, we assessed colonization of A. actinomycetemcomitans variants and ascertained microbial interactions in biofilm communities. Variants in A. actinomycetemcomitans leukotoxin (ltx) were created, labeled, inoculated, and compared with their progenitor strain for in vivo colonization. Samples of tooth-related plaque were assessed for colonization at baseline and after debridement and inoculation of labeled strains. Null, minimal, and hyper-Ltx-producing strains were created and assessed for hydroxyapatite binding and biofilm formation in vitro. Ltx-hyperproducing strains colonized with greater prevalence and at higher levels than wild type or ltx mutants (P = 0.05). Indigenous and inoculated A. actinomycetemcomitans strains that attached were associated with lactate-producing species (i.e., Leptotrichia, Abiotrophia, and Streptoccocci). A. actinomycetemcomitans was found at 0.13% of the total flora at baseline and at 0.05% 4 wk after inoculation. In vivo data were supported by in vitro results. We conclude that hyper-Ltx production affords these strains with an attachment advantage providing a foothold for competition with members of the indigenous microbiota. Increased attachment can be linked to ltx gene expression and up-regulation of adherence-associated genes. Growth of attached A. actinomycetemcomitans in vivo was enhanced by lactate availability due to consorting species. These associations provide A. actinomycetemcomitans with the constituents required for its colonization and survival in the complex and competitive oral environment.

Background: Molecular taxonomic assignments in oral microbial communities have been made using probe-matching approaches, but never compared to those obtained by more readily accepted tree-based approaches. Objective: To compare community composition profiles obtained from a probe-matching approach (HOMINGS) to those from a closed-ended tree-based approach (QIIME using the eHOMD database). Design: HOMINGS and QIIME were used for parallel analysis of ten mock community samples, and of 119 supragingival plaque samples from ecologically unique sites (sound tooth surfaces in healthy subjects, sound tooth surfaces in patients with primary Sjögren’s Syndrome, and carious lesions in Sjögren’s Syndrome patients). Linear discriminant analysis Effective Size (LEfSe) was used to identify discriminating taxa among the natural plaque samples. Results: Community composition profiles of all samples were congruent between the two analysis aproaches. Alpha and beta diversity of the natural plaque communities were likewise similar. Communities from pSS patients and those from individuals with normal salivary flow differed in alpha and beta diversity. Both classification approaches yielded differences in composition predicted for samples from these subject cohorts, and discriminating taxa were similar between approaches. Conclusions: A direct comparison demonstrates that HOMINGS is largely equivalent to the tree-based approach as implemented here. Trial registration: ClinicalTrials.gov identifier: NCT00001196. Trial registration: ClinicalTrials.gov identifier: NCT01425892. Trial registration: ClinicalTrials.gov identifier: NCT00001390.

Periodontitis is a biofilm-induced inflammatory disease characterized by dysbiosis of the commensal periodontal microbiota. It is unclear how natural regulation of inflammation affects the periodontal biofilm. Promoters of active resolution of inflammation, including resolvin E1 (RvE1), effectively treat inflammatory periodontitis in animal models. The goals of this study were 1) to compare periodontal tissue gene expression in different clinical conditions, 2) to determine the impact of local inflammation on the composition of subgingival bacteria, and 3) to understand how inflammation impacts these changes. Two clinically relevant experiments were performed in rats: prevention and treatment of ligature-induced periodontitis with RvE1 topical treatment. The gingival transcriptome was evaluated by RNA sequencing of mRNA. The composition of the subgingival microbiota was characterized by 16S rDNA sequencing. Periodontitis was assessed by bone morphometric measurements and histomorphometry of block sections. H&E and tartrate-resistant acid phosphatase staining were used to characterize and quantify inflammatory changes. RvE1 treatment prevented bone loss in ligature-induced periodontitis. Osteoclast density and inflammatory cell infiltration in the RvE1 groups were lower than those in the placebo group. RvE1 treatment reduced expression of inflammation-related genes, returning the expression profile to one more similar to health. Treatment of established periodontitis with RvE1 reversed bone loss, reversed inflammatory gene expression, and reduced osteoclast density. Assessment of the rat subgingival microbiota after RvE1 treatment revealed marked changes in both prevention and treatment experiments. The data suggest that modulation of local inflammation has a major role in shaping the composition of the subgingival microbiota.

A urease-negative, fusiform, novel bacterium named Helicobacter saguini was isolated from the intestines and feces of cotton-top tamarins (CTTs) with chronic colitis. Helicobacter sp. was detected in 69% of feces or intestinal samples from 116 CTTs. The draft genome sequence, obtained by Illumina MiSeq sequencing, for H. saguini isolate MIT 97-6194-5, consisting of ∼2.9 Mb with a G+C content of 35% and 2,704 genes, was annotated using the NCBI Prokaryotic Genomes Automatic Annotation Pipeline. H. saguini contains homologous genes of known virulence factors found in other enterohepatic helicobacter species (EHS) and H. pylori . These include flagellin, γ-glutamyl transpeptidase ( ggt ), collagenase, the secreted serine protease htrA , and components of a type VI secretion system, but the genome does not harbor genes for cytolethal distending toxin ( cdt ). H. saguini MIT 97-6194-5 induced significant levels of interleukin-8 (IL-8) in HT-29 cell culture supernatants by 4 h, which increased through 24 h. mRNAs for the proinflammatory cytokines IL-1β, tumor necrosis factor alpha (TNF-α), IL-10, and IL-6 and the chemokine CXCL1 were upregulated in cocultured HT-29 cells at 4 h compared to levels in control cells. At 3 months postinfection, all H. saguini -monoassociated gnotobiotic C57BL/129 IL-10 −/− mice were colonized and had seroconverted to H. saguini antigen with a significant Th1-associated increase in IgG2c ( P < 0.0001). H. saguini induced a significant typhlocolitis, associated epithelial defects, mucosa-associated lymphoid tissue (MALT) hyperplasia, and dysplasia. Inflammatory cytokines IL-22, IL-17a, IL-1β, gamma interferon (IFN-γ), and TNF-α, as well as inducible nitric oxide synthase (iNOS) were significantly upregulated in the cecal tissues of infected mice. The expression of the DNA damage response molecule γ-H2AX was significantly higher in the ceca of H. saguini -infected gnotobiotic mice than in the controls. This model using a nonhuman primate Helicobacter sp. can be used to study the pathogenic potential of EHS isolated from primates with naturally occurring inflammatory bowel disease (IBD) and colon cancer.

Oral mucositis (OM) is among the most common, painful, and debilitating toxicities of cancer regimen–related treatment, resulting in the formation of ulcers, which are susceptible to increased colonization of microorganisms. Novel discoveries in OM have focused on understanding the host-microbial interactions, because current pathways have shown that major virulence factors from microorganisms have the potential to contribute to the development of OM and may even prolong the existence of already established ulcerations, affecting tissue healing. Additional comprehensive and disciplined clinical investigation is needed to carefully characterize the relationship between the clinical trajectory of OM, the local levels of inflammatory changes (both clinical and molecular), and the ebb and flow of the oral microbiota. Answering such questions will increase our knowledge of the mechanisms engaged by the oral immune system in response to mucositis, facilitating their translation into novel therapeutic approaches. In doing so, directed clinical strategies can be developed that specifically target those times and tissues that are most susceptible to intervention.

Growing research has examined the association of oral microbiome with pancreatic cancer risk, but results have been inconsistent. Our previous study showed that bacterial taxa known to inhabit the oral cavity were common in the pancreas microbiome, and that bacterial DNA profiles in the pancreas were similar to those in the duodenum tissue of the same subjects regardless of disease state. These data suggest that bacteria may be migrating from oral into the gut and pancreas. We collected oral swabs and at least one pancreatic tissue or intestinal samples from subjects who underwent surgery for pancreatic diseases or diseases and characterized 16S ribosomal RNA genes using high-throughput DNA sequencing. We then quantified bacterial communities (Amplicon Sequence Variant level) at different body sites, investigated their co-abundance patterns, and analyzed the correlations between microbiome at oral sites and that in pancreatic tissue and intestinal samples using concordance statistics and Pairwise Stratified Association (PASTA) testing. The present analysis included 52 subjects (46% with pancreatic cancer; aged from 31 to 86 years old) contributing a total 324 samples. We identified a total of 73 unique Amplicon Sequence Variants (ASVs) that were shared between oral and pancreatic or intestinal samples. Accounting for pairing and within-subject correlation, 7 ASVs showed significant concordance (Kappa statistics) and 5 ASVs exhibited significant or marginally significant PASTA between oral samples and pancreatic tissue or intestinal samples. Of these, our PASTA analyses identified two specific bacterial species (Gemella morbillorum and Fusobacterium nucleatum subsp. vincentii) that showed consistent presence or absence patterns between oral and intestinal or pancreatic samples. Lastly, our microbial co-abundance analyses showed several distinct ASVs clusters and complex correlation-networks between ASV clusters in buccal, saliva, duodenum, jejunum, and pancreatic tumor samples. Oral, intestinal, and pancreatic microbiomes are correlated. Bacteria of oral origin exhibit co-abundance relationships and demonstrate complex correlation patterns in the intestinal and pancreatic tumor samples. Growing evidence has shown that bacterial species may survive, decline, and adapt as interdependent functional groups. Future studies should aim to uncover the co-abundance of specific microbial communities for studying etiology of microbiota-driven carcinogenesis in prospective and longitudinal studies. Citation Format: Mei Chung, Naisi Zhao, Richard Meier, Devein C. Koestler, Erika Del Castillo, Guojun Wu, Bruce J. Paster, Kevin Charpentier, Jacques Izard, Karl T. Kelsey, Dominique S. Michaud. Oral, intestinal, and pancreatic microbiomes are correlated and exhibit co-abundance in patients with pancreatic cancer and other gastrointestinal diseases [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B07.

Background Microbially mediated oral diseases can signal underlying HIV/AIDS progression in HIV-infected adults. The role of the oral microbiota in HIV-infected youth is not known. The Adolescent Master Protocol of the Pediatric HIV/AIDS Cohort Study is a longitudinal study of perinatally HIV-infected (PHIV) and HIV-exposed, uninfected (PHEU) youth. We compared oral microbiome levels and associations with caries or periodontitis in 154 PHIV and 100 PHEU youth. Results Species richness and alpha diversity differed little between PHIV and PHEU youth. Group differences in average counts met the significance threshold for six taxa; two Corynebacterium species were lower in PHIV and met thresholds for noteworthiness. Several known periodontitis-associated organisms (Prevotella nigrescens, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, and Filifactor alocis) exhibited expected associations with periodontitis in PHEU youth, associations not observed in PHIV youth. In both groups, odds of caries increased with counts of taxa in four genera, Streptococcus, Scardovia, Bifidobacterium, and Lactobacillus. Conclusions The microbiomes of PHIV and PHEU youth were similar, although PHIV youth seemed to have fewer “health”-associated taxa such as Corynebacterium species. These results are consistent with the hypothesis that HIV infection, or its treatment, may contribute to oral dysbiosis. Electronic supplementary material The online version of this article (10.1186/s40168-018-0484-6) contains supplementary material, which is available to authorized users.

Summary Periodontitis is a polymicrobial inflammatory disease that results from the interaction between the oral microbiota and the host immunity. Although the innate immune response is important for disease initiation and progression, the innate immune receptors that recognize both classical and putative periodontal pathogens that elicit an immune response have not been elucidated. By using the Human Oral Microbe Identification Microarray (HOMIM), we identified multiple predominant oral bacterial species in human plaque biofilm that strongly associate with severe periodontitis. Ten of the identified species were evaluated in greater depth, six being classical pathogens and four putative novel pathogens. Using human peripheral blood monocytes (HPBM) and murine bone-marrow-derived macrophages (BMDM) from wild-type (WT) and Toll-like receptor (TLR)-specific and MyD88 knockouts (KOs), we demonstrated that heat-killed Campylobacter concisus, Campylobacter rectus, Selenomonas infelix, Porphyromonas endodontalis, Porphyromonas gingivalis, and Tannerella forsythia mediate high immunostimulatory activity. Campylobacter concisus, C. rectus, and S. infelix exhibited robust TLR4 stimulatory activity. Studies using mesothelial cells from WT and NOD1-specific KOs and NOD2-expressing human embryonic kidney cells demonstrated that Eubacterium saphenum, Eubacterium nodatum and Filifactor alocis exhibit robust NOD1 stimulatory activity, and that Porphyromonas endodontalis and Parvimonas micra have the highest NOD2 stimulatory activity. These studies allowed us to provide important evidence on newly identified putative pathogens in periodontal disease pathogenesis showing that these bacteria exhibit different immunostimulatory activity via TLR4, NOD1, and NOD2 (Clinicaltrials.gov NCT01154855).

Purpose-bred common marmosets from domestic sources housed in a US research facility, and used in multiple drug discovery programmes, were noted to have a high incidence of spontaneous inflammatory bowel disease and sporadic cholecystitis and cholangiohepatitis. Inflammatory infiltrates increased in incidence and severity with age. Because Helicobacter spp. have been linked to gastrointestinal diseases, samples from the gastrointestinal tracts of 39 marmosets were screened for Helicobacter spp. by culture and PCR. Helicobacter spp. were frequently detected in marmosets; 28.2% of the marmosets were positive for a proposed novel species, Helicobacter jaachi sp. nov., by culture, and 48.7% were positive by Helicobacter genus-specific PCR. Seventeen strains of Helicobacter sp. from 11 marmosets were cultured from various gastrointestinal sites. Older animals (age 6-11 years) had a higher helicobacter prevalence rate (57.1%) compared with younger animals (age 3-5 years), which had a 27.2% prevalence rate. Cells of H. jaachi sp. nov. were catalase, urease and oxidase positive and had fusiform morphology, with periplasmic fibres and multiple bipolar, sheathed flagella. All isolates had similar 16S and 23S rRNA sequences, which clustered as representatives of a novel Helicobacter species closely related to 'Helicobacter sanguini' (97%), a species isolated from cotton-top tamarins and 'Helicobacter callitrichis' (96%) isolated previously from the faeces of common marmosets. The whole genome sequence of one of the liver isolates, H. jaachi sp. nov. MIT 09-6949(T), had a 1.9 Mb genome length with a 41 mol% DNA G+C content. The type strain of Helicobacter jaachi sp. nov., MIT 09-6949(T), has been deposited in the BCCM/LMG Bacteria Collection as LMG 28613(T). These findings add to the increasing number of animal species with gastrointestinal disease in which novel enterohepatic Helicobacter spp. have been isolated.

Objective Studies of the fecal microbiome have implicated the gut microbiota in obesity, but few studies have examined the microbial diversity at other sites. The association between obesity and the upper gastrointestinal (UGI) microbial diversity was explored. Methods The UGI microbiome of 659 healthy Chinese adults with a measured body mass index (BMI) range of 15.0 to 35.7 was characterized using the 16S rRNA gene DNA microarray (HOMIM). Results In multivariate-adjusted models, alpha diversity was not associated with BMI. However, beta diversity, assessed by principal coordinate vectors generated from an unweighted UniFrac distance matrix of pairwise comparisons, was associated with BMI (third and fourth vectors, P = 0.01 and P = 0.03, respectively). Moreover, beta diversity, assessed by cluster membership (three clusters), was also associated with BMI; individuals in the first cluster [median BMI 22.35, odds ratio (OR) = 0.48, 95% confidence interval (CI) = 0.05-4.34] and second cluster [median BMI 22.55, OR = 0.26, 95% CI = 0.09-0.75] were significantly less likely to be obese (BMI ≥ 27.5) than those in the third cluster (median BMI 23.59). Conclusions A beta-diversity metric of the UGI microbiome is associated with a four fold difference in obesity risk in this Asian population. Future studies should address whether the UGI microbiome plays a causal role in obesity.

Background and Objective Aggressive periodontitis (AgP) is prevalent and shows a rapid course in African individuals. Although a strong focus has been placed on Aggregatibacter actinomycetemcomitans, new methods support the existence of a complex subgingival microflora in AgP. The purpose of the present study was to map the subgingival microbiota as well as explore the presence of A. actinomycetemcomitans and the JP2 clone in a group of Sudanese individuals with AgP, using different analytical methods. Material and Methods A study population consisting of 19 patients with AgP was recruited from patients seeking treatment at University of Science and Technology (UST) in Khartoum. Fifteen healthy subjects were included as controls. Plaque samples were analyzed for 272 taxa using human oral microbe identification microarrays and for 26 periodontal taxa using DNA-DNA hybridization checkerboard. Conventional polymerase chain reaction (PCR) was applied for the detection of A. actinomycetemcomitans and the JP2 clone in plaque. Saliva from patients with AgP was analyzed using quantitative PCR (qPCR) for the detection of A. actinomycetemcomitans. Results Eubacterium yurii was detected more frequently in patients with AgP than in controls, and E. nodatum was found in patients with AgP only. A. actinomycetemcomitans was found in plaque samples of two (12%) patients by human oral microbe identification microarrays and in five (29%) patients with AgP by conventional PCR, as well as in six (32%) of the AgP saliva samples by qPCR. The JP2 clone was identified in only one patient. Conclusion The classical periodontal pathogens were not present in high amounts in AgP in the population studied here. Species of Eubacterium, which are not typically associated with AgP, were often detected in individuals with disease. Using laboratory methods with different sensitivities and detection levels allowed identification of variances in microbial communities. The findings reported in this study provide a basis for the further understanding of AgP.

ObjectiveTo determine whether bacteria are present in the pancreas of pancreatic cancer and non-cancer subjects and examine whether bacterial profiles vary by site and disease phenotype.Design77 patients requiring surgery for pancreatic diseases, or diseases of the foregut, at the Rhode Island Hospital (RIH) were recruited into this study between 2014 and 2016. In addition, 36 whole pancreas were obtained from the National Disease Research Interchange (NDRI) from subjects who were of similar age as the RIH patients and had not died of cancer. The primary exposure of interest was the measurement of the relative abundance of bacterial taxa in all tissue specimens using 16S rRNA gene sequencing.ResultsNumber of bacterial reads per sample varied substantially across sample type and patients, but all demonstrated the presence of diverse gastrointestinal bacteria, including bacterial taxa typically identified in the oral cavity. Bacterial profiles were noted to be more similar within individuals across sites in the pancreas, than between individuals by site, suggesting that the pancreas as a whole has its own microbiome. Comparing the mean relative abundance of bacterial taxa in pancreatic cancer patients to those without cancer revealed differences in bacterial taxa previously linked to periodontal disease, includingPorphyromonas.ConclusionsBacterial taxa known to inhabit the oral cavity, as well as the intestine, were identified in pancreatic tissue of cancer and non-cancer subjects. Whether any of these bacteria play a causal role in pancreatic carcinogenesis, or are simply opportunistic in nature, needs to be further examined.

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 non-surgical 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. This article is protected by copyright. All rights reserved.

Specific interbacterial adhesion, termed coaggregation, is well established for three early colonizers of the plaque biofilm: streptococci, actinomyces, and veillonellae. However, little is known about interactions of other early colonizers and about the extent of interactions within the bacterial community from a single host. To address these gaps, subject-specific culture collections from two individuals were established using an intraoral biofilm retrieval device. Molecular taxonomy (Human Oral Microbe Identification Microarray [HOMIM]) analysis of biofilm samples confirmed the integrity and completeness of the collections. HOMIM analysis verified the isolation of Streptococcus gordonii and S. anginosus from only one subject, as well as isolation of a previously uncultivated streptococcal phylotype from the other subject. Strains representative of clonal diversity within each collection were further characterized. Greater than 70% of these streptococcal strains from each subject coaggregated with at least one other coisolate. One-third of the strains carry a known coaggregation mediator: receptor polysaccharide (RPS). Almost all nonstreptococcal isolates coaggregated with other coisolates. Importantly, certain Rothia strains demonstrated more coaggregations with their coisolated bacteria than did any Streptococcus or Actinomyces strain, and certain Haemophilus isolates participated in twice as many. Confocal microscopy of undisturbed biofilms showed that Rothia and Haemophilus each occur in small multispecies microcolonies. However, in confluent high-biomass regions, Rothia occurred in islands whereas Haemophilus was distributed throughout. Together, the data demonstrate that coaggregation networks within an individual's oral microflora are extensive and that Rothia and Haemophilus can be important initiators of cell-cell interactions in the early biofilm. IMPORTANCE Extensive involvement of specific interbacterial adhesion in dental plaque biofilm formation has been postulated based on in vitro coaggregation between oral bacteria from culture collections that are not subject specific. In the present study, subject-specific culture collections were obtained from early plaque biofilm of two volunteers, and coaggregations within each culture collection were assayed. Coaggregations, several of which involved a coaggregation-mediating cell surface molecule known from well-studied streptococci, were widespread. Unexpectedly, the little-studied organisms Haemophilus and Rothia participated in the greatest numbers of interactions with community members; these two organisms showed different distributions within the undisturbed biofilm. The data show that coaggregation networks encompass most organisms within the biofilm community of each individual, and they indicate prominent participation of organisms such as Haemophilus and Rothia in early plaque biofilm formation.

An association between oral bacteria and atherosclerosis has been postulated. A limited number of studies have used 16S RNA gene sequencing-based metagenomics approaches to identify bacteria at the species level from atherosclerotic plaques in arterial walls. The objective of this study was to establish detailed oral microbiome profiles, at both genus and species level, of clinically healthy coronary and femoral artery tissues from patients with atherosclerosis. Tissue specimens were taken from clinically non-atherosclerotic areas of coronary or femoral arteries used for attachment of bypass grafts in 42 patients with atherosclerotic cardiovascular disease. Bacterial DNA was sequenced using the MiSeq platform, and sequence reads were screened in silico for nearly 600 oral species using the HOMINGS ProbeSeq species identification program. The number of sequence reads matched to species or genera were used for statistical analyses. A total of 230 and 118 species were detected in coronary and femoral arteries, respectively. Unidentified species detected by genus-specific probes consisted of 45 and 30 genera in coronary and in femoral artery tissues, respectively. Overall, 245 species belonging to 95 genera were detected in coronary and femoral arteries combined. The most abundant species were Porphyromonas gingivalis, Enterococcus faecalis, and Finegoldia magna based on species probes. Porphyromonas, Escherichia, Staphylococcus, Pseudomonas, and Streptococcus genera represented 88.5% mean relative abundance based on combined species and genus probe detections. Porphyromonas was significantly more abundant than Escherichia (i.e. 46.8% vs. 19.3%; p = 0.0005). This study provides insight into the presence and types of oral microbiome bacterial species found in clinically non-atherosclerotic arteries.

A total of 31 sea otters Enhydra lutris nereis found dead or moribund (and then euthanized) were necropsied in California, USA. Stomach biopsies were collected and transected with equal portions frozen or placed in formalin and analyzed histologically and screened for Helicobacter spp. in gastric tissue. Helicobacter spp. were isolated from 9 sea otters (29%); 58% (18 of 31) animals were positive for helicobacter by PCR. The Helicobacter sp. was catalase- and oxidase-positive and urease-negative. By electron microscopy, the Helicobacter sp. had lateral and polar sheathed flagella and had a slightly curved rod morphology. 16S and 23S rRNA sequence analyses of all ‘H. enhydrae’ isolates had similar sequences, which clustered as a novel Helicobacter sp. closely related to H. mustelae (96-97%). The genome sequence of isolate MIT 01-6242 was assembled into a single ~1.6 Mb long contig with a 40.8% G+C content. The annotated genome contained 1699 protein-coding sequences and 43 RNAs, including 65 genes homologous to known Helicobacter spp. and Campylobacter spp. virulence factors. Histological changes in the gastric tissues extended from mild cystic degeneration of gastric glands to severe mucosal erosions and ulcers. Silver stains of infected tissues demonstrated slightly curved bacterial rods at the periphery of the gastric ulcers and on the epithelial surface of glands. The underlying mucosa and submucosa were infiltrated by low numbers of neutrophils, macrophages, and lymphocytes, with occasional lymphoid aggregates and well-defined lymphoid follicles. This is the second novel Helicobacter sp., which we have named ‘H. enhydrae’, isolated from inflamed stomachs of mustelids, the first being H. mustelae from a ferret. Keywords: Helicobacter; Gastritis; Otter, National Institutes of Health (U.S.) (Grant T32-OD010978), National Institutes of Health (U.S.) (Grant P01-CA028842), National Institutes of Health (U.S.) (Grant P30-ES002109), National Institutes of Health (U.S.) (Grant R01-CA093405)

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.

Aim To determine microbial profiles that discriminate periodontal health from different forms of periodontal diseases. Methods Subgingival biofilm was obtained from patients with periodontal health (27), gingivitis (11), chronic periodontitis (35) and aggressive periodontitis (24), and analysed for the presence of >250 species/phylotypes using HOMIM. Microbial differences among groups were examined by Mann–Whitney U-test. Regression analyses were performed to determine microbial risk indicators of disease. Results Putative and potential new periodontal pathogens were more prevalent in subjects with periodontal diseases than periodontal health. Detection of Porphyromonas endodontalis/Porphyromonas spp. (OR 9.5 [1.2–73.1]) and Tannerella forsythia (OR 38.2 [3.2–450.6]), and absence of Neisseria polysaccharea (OR 0.004 [0–0.15]) and Prevotella denticola (OR 0.014 [0–0.49], p

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_ot417/462) were less frequently found in the caries group (adjusted p value Solobacterium moorei and Streptococcus salivarius) and three bacterial clusters (Streptococcus parasanguinis I and II and sp. clone BE024_ot057/411/721, Streptococcus parasanguinis I and II and sinensis_ot411/721/767, Streptococcus salivarius and sp. clone FO042_ot067/755) were present at significantly higher levels (adjusted p value

Celiac disease (CD) is a chronic immune-mediated enteropathy induced by dietary gluten in genetically predisposed individuals. Saliva harbors the second highest bacterial load of the gastrointestinal (GI) tract after the colon. We hypothesized that enzymes produced by oral bacteria may be involved in gluten processing in the intestine and susceptibility to celiac disease. The aim of this study was to investigate salivary enzymatic activities and oral microbial profiles in healthy subjects versus patients with classical and refractory CD. Stimulated whole saliva was collected from patients with CD in remission ( n = 21) and refractory CD (RCD; n = 8) and was compared to healthy controls (HC; n = 20) and subjects with functional GI complaints ( n = 12). Salivary gluten-degrading activities were monitored with the tripeptide substrate Z-Tyr-Pro-Gln-pNA and the α-gliadin-derived immunogenic 33-mer peptide. The oral microbiome was profiled by 16S rRNA-based MiSeq analysis. Salivary glutenase activities were higher in CD patients compared to controls, both before and after normalization for protein concentration or bacterial load. The oral microbiomes of CD and RCD patients showed significant differences from that of healthy subjects, e.g., higher salivary levels of lactobacilli ( P < 0.05), which may partly explain the observed higher gluten-degrading activities. While the pathophysiological link between the oral and gut microbiomes in CD needs further exploration, the presented data suggest that oral microbe-derived enzyme activities are elevated in subjects with CD, which may impact gluten processing and the presentation of immunogenic gluten epitopes to the immune system in the small intestine. IMPORTANCE Ingested gluten proteins are the triggers of intestinal inflammation in celiac disease (CD). Certain immunogenic gluten domains are resistant to intestinal proteases but can be hydrolyzed by oral microbial enzymes. Very little is known about the endogenous proteolytic processing of gluten proteins in the oral cavity. Given that this occurs prior to gluten reaching the small intestine, such enzymes are likely to contribute to the composition of the gluten digest that ultimately reaches the small intestine and causes CD. We demonstrated that endogenous salivary protease activities are incomplete, likely liberating peptides from larger gluten proteins. The potentially responsible microbes were identified. The study included refractory CD patients, who have been studied less with regard to CD pathogenesis.

Despite significant advances in recent years in culture-independent molecular microbiology methods, the detailed study of individual bacterial species still relies on having pure cultures in the laboratory. Yet, more than a third of the approximately 700 bacterial taxa found in the human oral cavity are as yet uncultivated in vitro. One such taxon, Tannerella sp. HOT-286 (phylotype BU063), is the focus of much interest since it is associated with periodontal health, while Tannerella forsythia, its closest phylogenetic neighbor, is strongly associated with periodontal disease. HOT-286, however, has remained uncultivated despite the efforts of several research groups, spanning over a decade. The aim of this study was to cultivate Tannerella sp. HOT-286. A heavily diluted sample of subgingival plaque was inoculated onto culture plates supplemented with siderophores (pyoverdines-Fe complex or desferricoprogen) or a neat plaque suspension. After 8 d of anaerobic incubation, microcolonies and colonies showing satellitism were passaged onto fresh culture plates cross-streaked with potential helper strains or onto cellulose-acetate membranes placed over lawn cultures of helper strains. Subcultured colonies were identified by 16S rRNA gene sequencing, and purity was confirmed by sequencing 20 clones per library prepared from a single colony. Three colonies of interest (derived from pyoverdines- and plaque-supplemented plates) were identified as Tannerella sp. HOT-286. The isolates were found to be incapable of independent growth, requiring helpers such as Propionibacterium acnes and Prevotella intermedia for stimulation, with best growth on membranes over “helper” lawns. A representative isolate was subjected to phenotypic characterization and found to produce a range of glycosidic and proteolytic enzymes. Further comparison of this novel “periodontal health-associated” taxon with T. forsythia will be valuable in investigating virulence factors of the latter and possible health benefits of the former.

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 (HOMINGS) 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

The oral microbiome is incredibly complex with the average adult harboring about 50-100 billion bacteria in the oral cavity, which represent about 200 predominant bacterial species. Collectively, there are approximately 700 predominant taxa of which less than one-third still have not yet been grown in vitro. Compared to other body sites, the oral microbiome is unique and readily accessible. There is extensive literature available describing the oral microbiome and discussing the roles that bacteria may play in oral health and disease. However, the purpose of this review is not to rehash these detailed studies but rather to educate the reader with understanding the essence of the oral microbiome, namely that there are abundant bacteria in numbers and types, that there are molecular methods to rapidly determine bacterial associations, that there is site specificity for colonization of the host, that there are specific associations with oral health and disease, that oral bacteria may serve as biomarkers for non-oral diseases, and that oral microbial profiles may have potential use to assess disease risk.

The aim of our study was to determine the association of Helicobacter spp. that had flexispira morphology with ovine abortion, and to understand the importance of these organisms as a cause of ovine abortion in New Zealand. A retrospective diagnostic survey was carried out on laboratory submissions from ovine abortion outbreaks. A comparison was made of the proportion of laboratory submissions where Helicobacter spp. were detected from flocks that had no other agent identified (group A) with a group that had a known cause of abortion identified (group B). This latter group was considered to be a negative control, given the premise that Helicobacter spp. were not causing abortions and that Helicobacter spp. should be present at a lower rate in the group. Where no diagnosis had been made, aborted material was positive for Helicobacter spp. with flexispira morphology in 8 submissions (20%, 8/40) from 5 of the 31 survey farms (16%, 5/31). Helicobacter spp. were not detected in any of the 18 submissions from the 17 control farms (group B). Helicobacter spp. were confirmed by 16S ribosomal RNA sequencing of 3 of the Helicobacter spp. isolated by culture from the livers of aborted sheep fetuses, and 7 of the 8 where samples were positive in a Helicobacter PCR assay. The Helicobacter spp. were identified as Helicobacter trogontum ( Flexispira taxon 5 genotype) and Helicobacter bilis ( Flexispira taxon 8 genotype). The findings support Helicobacter spp. being a probable causative agent of ovine abortions in New Zealand.

Objective This study aimed to identify the oral microbial diversity of healthy Chinese Han children. Methods Dental plaques were sampled from the oral cavity of ten healthy Chinese Han children. The oral microbiome was examined using the 16S rRNA–based Human Oral Microbe Identification Microarray. The microbial diversity and similarity were analyzed using the Chao–Jaccard similarity index. Results A total of 112 species, which belonged to nine bacterial phyla and 41 genera, were detected. Each individual harbored an average of 54.1 microbial species (ranging from 37 to 69) and 26.2 genera (ranging from 21 to 31), with interindividual variations both at the species and genus level. Thirteen genera were conserved among all individuals. The Chao–Jaccard similarity index averages, at the genus and species level, were 0.642 (ranging from 0.485 to 0.871) and 0.506 (ranging from 0.338 to 0.676), respectively, suggesting that the healthy oral community was more conserved at the genus level than at the species level. Conclusion Although there was interindividual variation in the oral microflora, some bacterial genera were conserved among individuals, supporting the existence of a core microbiome in the oral cavity of healthy Chinese Han children.

Purpose Historically, the identification of microorganisms has been limited to species that could be cultured in the microbiology laboratory. The purpose of the present study was to apply molecular techniques to identify microorganisms in orofacial odontogenic infections (OIs). Materials and Methods Specimens were obtained from subjects with clinical evidence of OI. To identify the microorganisms involved, 16S rRNA sequencing methods were used on clinical specimens. The name and number of the clones of each species identified and the combinations of species present were recorded for each subject. Descriptive statistics were computed for the study variables. Results Specimens of pus or wound fluid were obtained from 9 subjects. A mean of 7.4 ± 3.7 (standard deviation) species per case were identified. The predominant species detected in the present study that have previously been associated with OIs were Fusobacterium spp, Parvimonas micra, Porphyromonas endodontalis, and Prevotella oris. The predominant species detected in our study that have not been previously associated with OIs were Dialister pneumosintes and Eubacterium brachy. Unculturable phylotypes accounted for 24% of the species identified in our study. All species detected were obligate or facultative anaerobes. Streptococci were not detected. Conclusions Molecular methods have enabled us to detect previously cultivated and not-yet-cultivated species in OIs; these methods could change our understanding of the pathogenic flora of orofacial OIs.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract, likely caused by an aberrant immune response to the microbiota and other environmental factors in genetically susceptible individuals.1,2 Oral mucosal inflammation is commonly described in patients with IBD, particularly Crohn’s disease (CD). Oral pathology has a reported prevalence of 0.5% to 80% in CD.3–8 When studied prospectively in children in collaboration with a dentist, 42% of new diagnoses of CD had oral manifestations.7 Symptoms can span from mild and nonspecific inflammation such as minor aphthous lesions, mucogingivitis, and angular cheilitis to more specific findings, such as mucosal tags, cobblestoning, deep linear ulcerations, and more severe granulomatous swelling isolated to the labia and face known as orofacial granulomatosis (OFG).3,5–7,9 Lesions of the oral mucosa may occur years before the onset of intestinal symptoms, particularly in the pediatric population.6 Specific oral mucosal findings are common in children and in 75%–100% of cases, contain disease-defining, histologic evidence of noncaseating granulomas.3,7,9 Data from animal models suggests that the microbiome is a critical factor in the pathogenesis of IBD in knockout mice that are at risk for the disease. Preliminary studies in humans have found differences in the intestinal microbial populations of IBD patients when compared with healthy controls.2,10–12 However, such studies have largely focused on the lower gastrointestinal tract. The oral cavity provides an easily accessed mucosal surface that may potentially yield valuable information about the microbiome and its interaction with the host. The mouth and its resident flora is a well-characterized microbiome with ≈600 predominant bacterial species, of which about 35% are unable to be grown in culture.13,14 This environment is unlike any other in the body, made up of diverse ecological niches including hard surfaces upon which complex biofilms flourish, anaerobic microclimates, and rapidly shedding mucosal surfaces. Given the difficulties in studying the oral microbiome using conventional, culture-based techniques, our laboratory has developed a molecular technique using a 16S rRNA-based microarray technology known as the Human Oral Microbe Identification Microarray (HOMIM). Previous investigations in our laboratory and others have implicated distinct changes in the oral microbiome in dental caries and periodontitis.13–15 Oral microbial alterations in systemic diseases have also been identified including atherosclerosis, preterm birth, and pancreatic cancer.13–21 In this case–control study, we demonstrate an overall decrease in the oral bacterial diversity of children with CD when compared with healthy controls. Furthermore, several key phyla were significantly reduced when compared with healthy subjects, as has been identified in studies of the intestinal microbiome.11,12,22–25

Oral Diseases (2012) 18, 595–601 Objective: The purpose of this study was to determine the bacterial profiles in saliva of the isolated children for studying caries etiology. Materials and methods: Samples were collected from isolated children from 6 to 8 years old including 20 caries-free (dmfs = 0) (healthy) and 30 caries-active individuals (dmfs > 8) (patients). 16S rRNA genes were amplified by PCR from bacterial DNA of saliva sample and labeled via incorporation of Cy3-dCTP in second nested PCR. After hybridization of labeled amplicons on HOMIM, the microarray slides were scanned and original data acquired from professional software. Results: Collectively, 94 bacterial species or clusters representing six bacterial phyla and 30 genera were detected. A higher bacterial diversity was observed in patients than in healthy samples. Statistical analyses revealed eight species or clusters were detected more frequently in diseased patients than in healthy samples, while six different species were detected more frequently in healthy as compared to diseased patients. Conclusion: The diversity of microbe within saliva derived from isolated population increased in caries-active status, and there are some bacteria in salivary flora can be as candidate biomarkers for caries prognosis in mixed dentition. The imbalances in the resident microflora may be the ultimate mechanism of dental caries.

Introduction The present study investigated whether bacteria infecting the root canal can activate any infiltrating T cells to produce receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL). Methods Using a mouse model of periapical lesion induced by artificial dental pulp exposure, the presence of RANKL-positive T cells and osteoclasts in the periapical lesion was examined by an immunohistochemical approach. The bacteria colonizing the exposed root canal were identified by 16S ribosomal RNA (rRNA) sequence analysis. The isolated endodontic bacteria were further immunized to normal mice, and soluble activator of NF-κB ligand (sRANKL) production by the T cells isolated from the immunized mice was evaluated by ex vivo culture system. Results RANKL-positive T cells along with TRAP+ osteoclasts were identified in periapical bone resorption lesions. The gram-negative bacterium Pasteurella pnumotropica, which was most frequently detected from the root canal of exposed pulp, showed remarkably elevated serum immunoglobulin G (IgG)-antibody response in pulp-exposed mice compared with control nontreated mice. Immunization of mice with P. pneumotropica induced not only serum IgG-antibody but also primed bacteria-reactive T cells that produced sRANKL in response to ex vivo exposure to P. pneumotropica. Conclusions T cells infiltrating the periapical region express RANKL, and the endodontic bacteria colonizing the root canal appear to induce RANKL expression from bacteria-reactive T cells, suggesting the possible pathogenic engagement of the immune response to endodontic bacteria in the context of developing bone resorptive periapical lesions.

Although the mature dental biofilm composition is well studied, there is very little information on the earliest phase of in vivo tooth colonization. Progress in dental biofilm collection methodologies and techniques of large-scale microbial identification have made new studies in this field of oral biology feasible. The aim of this study was to characterize the temporal changes and diversity of the cultivable and noncultivable microbes in the early dental biofilm. Samples of early dental biofilm were collected from 11 healthy subjects at 0, 2, 4, and 6 h after removal of plaque and pellicle from tooth surfaces. With the semiquantitative Human Oral Microbiome Identification Microarray (HOMIM) technique, which is based on 16S rRNA sequence hybridizations, plaque samples were analyzed with the currently available 407 HOMIM microbial probes. This led to the identification of at least 92 species, with streptococci being the most abundant bacteria across all time points in all subjects. High-frequency detection was also made with Haemophilus parainfluenzae , Gemella haemolysans , Slackia exigua , and Rothia species. Abundance changes over time were noted for Streptococcus anginosus and Streptococcus intermedius ( P = 0.02), Streptococcus mitis bv. 2 ( P = 0.0002), Streptococcus oralis ( P = 0.0002), Streptococcus cluster I ( P = 0.003), G. haemolysans ( P = 0.0005), and Stenotrophomonas maltophilia ( P = 0.02). Among the currently uncultivable microbiota, eight phylotypes were detected in the early stages of biofilm formation, one belonging to the candidate bacterial division TM7, which has attracted attention due to its potential association with periodontal disease.

The associations between oral diseases and increased risk of pancreatic cancer have been reported in several prospective cohort studies. In this study, we measured variations of salivary microbiota and evaluated their potential associations with pancreatic cancer and chronic pancreatitis.This study was divided into three phases: (1) microbial profiling using the Human Oral Microbe Identification Microarray to investigate salivary microbiota variation between 10 resectable patients with pancreatic cancer and 10 matched healthy controls, (2) identification and verification of bacterial candidates by real-time quantitative PCR (qPCR) and (3) validation of bacterial candidates by qPCR on an independent cohort of 28 resectable pancreatic cancer, 28 matched healthy control and 27 chronic pancreatitis samples.Comprehensive comparison of the salivary microbiota between patients with pancreatic cancer and healthy control subjects revealed a significant variation of salivary microflora. Thirty-one bacterial species/clusters were increased in the saliva of patients with pancreatic cancer (n=10) in comparison to those of the healthy controls (n=10), whereas 25 bacterial species/clusters were decreased. Two out of six bacterial candidates (Neisseria elongata and Streptococcus mitis) were validated using the independent samples, showing significant variation (p0.05, qPCR) between patients with pancreatic cancer and controls (n=56). Additionally, two bacteria (Granulicatella adiacens and S mitis) showed significant variation (p0.05, qPCR) between chronic pancreatitis samples and controls (n=55). The combination of two bacterial biomarkers (N elongata and S mitis) yielded a receiver operating characteristic plot area under the curve value of 0.90 (95% CI 0.78 to 0.96, p0.0001) with a 96.4% sensitivity and 82.1% specificity in distinguishing patients with pancreatic cancer from healthy subjects.The authors observed associations between variations of patients' salivary microbiota with pancreatic cancer and chronic pancreatitis. This report also provides proof of salivary microbiota as an informative source for discovering non-invasive biomarkers of systemic diseases.

The transmission of simian immunodeficiency and Ebola viruses to humans in recent years has heightened awareness of the public health significance of zoonotic diseases of primate origin, particularly from chimpanzees. In this study, we analyzed 71 fecal samples collected from 2 different wild chimpanzee ( Pan troglodytes ) populations with different histories in relation to their proximity to humans. Campylobacter spp. were detected by culture in 19/56 (34%) group 1 (human habituated for research and tourism purposes at Mahale Mountains National Park) and 0/15 (0%) group 2 (not human habituated but propagated from an introduced population released from captivity over 30 years ago at Rubondo Island National Park) chimpanzees, respectively. Using 16S rRNA gene sequencing, all isolates were virtually identical (at most a single base difference), and the chimpanzee isolates were most closely related to Campylobacter helveticus and Campylobacter upsaliensis (94.7% and 95.9% similarity, respectively). Whole-cell protein profiling, amplified fragment length polymorphism analysis of genomic DNA, hsp60 sequence analysis, and determination of the mol% G+C content revealed two subgroups among the chimpanzee isolates. DNA-DNA hybridization experiments confirmed that both subgroups represented distinct genomic species. In the absence of differential biochemical characteristics and morphology and identical 16S rRNA gene sequences, we propose to classify all isolates into a single novel nomenspecies, Campylobacter troglodytis , with strain MIT 05-9149 as the type strain; strain MIT 05-9157 is suggested as the reference strain for the second C. troglodytis genomovar. Further studies are required to determine whether the organism is pathogenic to chimpanzees and whether this novel Campylobacter colonizes humans and causes enteric disease.

In a biofilm such as the subgingival microflora, strain-specific properties or factors induced by the host may impart a survival advantage to some bacterial strains. Periodontal disease has been associated with chronic obstructive pulmonary disease (COPD) and we previously found high amounts of Veillonella in the subgingival microflora of COPD subjects. Differentiation of Veillonella is difficult. The aims of this study were to identify subgingival Veillonella isolates by phenotypic, genetic typing and molecular genetic methods, and further, to assess if Veillonella strain properties or identity correlated with periodontal disease or COPD. From 22 subjects, 26 subgingival Veillonella isolates and one pulmonary isolate were analysed. The majority of the subgingival Veillonella isolates were identified as Veillonella parvula. Genotyping showed heterogeneity within strains of the same species. A subgingival and pulmonary isolate in one COPD subject was found to be genetically identical strains of V. parvula. Scanning electron microscopy of the lung biopsy confirmed single small cocci adhering or coaggregating with larger cocci on the airway epithelium. Apart from a variation in cellular fatty acid composition of six subgingival isolates from periodontitis subjects, no correlation between the subgingival Veillonella strains or genotypes and the presence of either periodontitis or COPD was found. In conclusion, V. parvula was the predominant subgingival Veillonella species with high genetic variability within strains of the same species. Subgingival V. parvula can translocate to the lungs; however, Veillonella identity or genotype did not correlate with periodontal disease or COPD.

Objective : Recent reports have shown an association between periodontitis and chronic obstructive pulmonary disease (COPD). Our objective was to assess the subgingival microflora in relation to COPD status. Methods : Eighteen subjects were classified into four different groups according to clinical periodontal status and the diagnosis of COPD. DNA from subgingival plaque samples was extracted and the bacterial 16S rRNA genes were PCR amplified, cloned, and sequenced to determine species identity. Results : From a total of 1041 clones, 168 species or phylotypes were detected. Higher amounts of Actinomyces spp., Veillonella spp., and Campylobacter gracilis were detected in the subgingival microfl ora of periodontally healthy COPD subjects, as compared with controls. COPD status was found to have little influence on the subgingival microflora in periodontitis subjects. Conclusion : COPD status does seem to influence the healthy but not the diseased subgingival microflora in periodontitis. Although preliminary, these findings indicate that there may be host factors responsible for the increased susceptibility of COPD subjects to periodontal disease. Key words: Periodontitis, subgingival bacteria, 16S rRNA gene, sequence analysis

This review gives an overview of the bacterial diversity of cultivated and not-yet-cultivated bacterial species in oral biofilms. Examples are given from the healthy oral cavity of youngsters, adults, and the elderly; caries in primary and permanent teeth; root caries in the elderly; subgingival plaque; aggressive periodontitis; chronic periodontitis; necrotizing ulcerative periodontitis; halitosis; noma; endodontic infections; and spreading odontogenic infections. Transfer of biofilm bacteria to the blood is also discussed. Techniques used for identifying these organisms are mainly PCR, cloning, and 16S rRNA gene sequencing, as well as microarrays. As much as 50% or more of the microbiota in oral biofilms cannot yet be cultured. This may have significant implications for our knowledge of the pathogens in major biofilm infections in the oral cavity such as caries, periodontitis, peri-implantitis, and mucositis. Furthermore, several bacterial species not traditionally believed to be oral pathogens have also been shown to be associated with disease. Key words: oral biofilm, PCR, cloning, 16S rRNA gene sequencing, not-yet-cultivated bacteria

Objective The purpose of this study was to compare two different methods of identifying oral bacteria from blood samples after dental extractions. Study design Blood cultures were taken before, during, and after a single tooth extraction in 30 subjects and cultured using aerobic and anaerobic Bactec media. Positive cultures from 22 subjects were subcultured on selective and nonselective media, resulting in 58 isolates. Bacterial identification was performed by biochemical analysis and sequence analysis of 16S rRNA gene. Results Of the 58 isolates, only 10 (17%) were the same species by both methods. Thirty-two (55%) belonged to the same genus but different species, and 16 (28%) showed no correlation at all. There were 31 and 40 diverse species by the biochemical and the sequencing methods, respectively. Streptococci were the dominant species. Conclusions DNA sequencing results in more accurate identification and a more diverse population of bacteria from bacteremia following dental extractions.

Although Streptococcus mutans has been implicated as a major etiological agent of dental caries, our cross-sectional preliminary study indicated that 10% of subjects with rampant caries in permanent teeth do not have detectable levels of S. mutans . Our aims were to use molecular methods to detect all bacterial species associated with caries in primary and permanent teeth and to determine the bacterial profiles associated with different disease states. Plaque was collected from 39 healthy controls and from intact enamel and white-spot lesions, dentin lesions, and deep-dentin lesions in each of 51 subjects with severe caries. 16S rRNA genes were PCR amplified, cloned, and sequenced to determine species identities. In a reverse-capture checkerboard assay, 243 samples were analyzed for 110 prevalent bacterial species. A sequencing analysis of 1,285 16S rRNA clones detected 197 bacterial species/phylotypes, of which 50% were not cultivable. Twenty-two new phylotypes were identified. PROC MIXED tests revealed health- and disease-associated species. In subjects with S. mutans , additional species, e.g., species of the genera Atopobium , Propionibacterium , and Lactobacillus , were present at significantly higher levels than those of S. mutans . Lactobacillus spp., Bifidobacterium dentium , and low-pH non- S. mutans streptococci were predominant in subjects with no detectable S. mutans . Actinomyces spp. and non- S. mutans streptococci were predominant in white-spot lesions, while known acid producers were found at their highest levels later in disease. Bacterial profiles change with disease states and differ between primary and secondary dentitions. Bacterial species other than S. mutans , e.g., species of the genera Veillonella , Lactobacillus , Bifidobacterium , and Propionibacterium , low-pH non- S. mutans streptococci, Actinomyces spp., and Atopobium spp., likely play important roles in caries progression.

Oral microbes that colonize in the mouths of humans contribute to disease susceptibility, but it is unclear if host genetic factors mediate colonization. We therefore tested the hypothesis that the levels at which oral microbes colonize in the mouth are heritable. Dental plaque biofilms were sampled from intact tooth surfaces of 118 caries-free twins. An additional 86 caries-active twins were sampled for plaque from carious lesions and intact tooth surfaces. Using a reverse capture checkerboard assay the relative abundance of 82 bacterial species was determined. An integrative computational predictive model determined microbial abundance patterns of microbial species in caries-free twins as compared to caries-active twins. Heritability estimates were calculated for the relative microbial abundance levels of the microbial species in both groups. The levels of 10 species were significantly different in healthy individuals than in caries-active individuals, including,A. defectiva, S. parasanguinis, S. mitis/oralis, S. sanguinis, S. cristatus, S. salivarius, Streptococcussp. clone CH016,G. morbillorumandG. haemolysans.Moderate to high heritability estimates were found for these species (h2= 56%–80%,p< .0001). Similarity of the overall oral microbial flora was also evident in caries-free twins from multivariate distance matrix regression analysis. It appears that genetic and/or familial factors significantly contribute to the colonization of oral beneficial species in twins.

Aim: To describe and compare the predominant bacterial and fungal species associated with gingivitis, periodontitis, and linear gingival erythema (LGE), in HIV positive subjects with different immune status. Methods: Viral loads and CD4 levels determined HIV disease status. From pooled subgingival plaque, 16S and 18S rDNA were cloned and sequenced to determine species identity. Results: One hundred and nine bacterial species were identified from 14 subjects. Nearly half of the species were not cultivable. Notably, the classical putative periodontal pathogens, Treponema denticola, Porphyromonas gingivalis and Tannerella forsythia were below the limit of detection and were not detected. Species of Gemella, Dialister, Streptococcus and Veillonella were predominant. In one HIV positive subject with periodontitis and low viral load, Gemella morbillorum, a known opportunistic pathogen, constituted 84% of the clones. Saccharomyces cerevisiae was the only fungal species detected in an LGE subject and in periodontitis subjects with high viral loads. In periodontitis patients with low viral loads, Candida albicans was predominant, while S. cerevisiae was only a minor component. Conclusion: These case studies suggest that other bacterial species, rather than the classical periodontal pathogens, may be involved in periodontal diseases of subjects with HIV. These data are indicative of opportunistic infections in a highly susceptible immunocompromised host.

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.

Background : Over 700 bacterial species reside in human oral cavity, many of which are associated with local or distant site infections. Extensive characterization of the oral microbiome depends on the technologies used to determine the presence and proportions of specific bacterial species in various oral sites. Objective : The objective of this study was to compare the microbial composition of dental plaque at baseline using Human Oral Microbe Identification Microarray (HOMIM) and Human Oral Microbe Identification using Next Generation Sequencing (HOMI NGS ) technologies, which are based on 16S rRNA. Methods : Dental plaque samples were collected from 96 patients at baseline prior to a dental procedure involving manipulation of gingival tissues. The samples were surveyed for 293 and 597 oral bacterial species via HOMIM and HOMI NGS , respectively, based on 16S rRNA gene sequences. We determined the concordance between the two technologies for common species. Genus level analysis was performed using HOMI NGS -specific genus identification capabilities. Results : HOMI NGS detected twice the number of species in the same dental plaque samples compared to HOMIM. For the species detected by both HOMIM and HOMI NGS , there was no difference in relative proportions of overall bacterial composition at the species, genus or phylum levels. Additionally, there was no difference in relative proportion for total species per patient between the two technologies. Conclusion : HOMI NGS significantly expanded oral bacterial species identification compared to HOMIM. The genus and species probes, combined in HOMI NGS , provided a more comprehensive representation of oral bacterial community, critical for future characterization of oral microbes in distant site infections. Keywords: HOMI NGS; HOMIM; MiSeq; ProbeSeq; dental plaque; oral microbiome (Published: 8 April 2016) Citation: Journal of Oral Microbiology 2016, 8: 30379 - http://dx.doi.org/10.3402/jom.v8.30379