The oral microbiome of patients undergoing treatment for severe aplastic anemia: a pilot study.

The microbiome, an intriguing component of the human body, composed of trillions of microorganisms, has prompted scientific exploration to identify and understand its function and role in health and disease. As associations between microbiome composition, disease, and symptoms accumulate, the future of medicine hinges upon a comprehensive knowledge of these microorganisms for patient care. The oral microbiome may provide valuable and efficient insight for predicting future changes in disease status, infection, or treatment course. The main aim of this pilot study was to characterize the oral microbiome in patients with severe aplastic anemia (SAA) during their therapeutic course. SAA is a hematologic disease characterized by bone marrow failure which if untreated is fatal. Treatment includes either hematopoietic stem cell transplantation (HSCT) or immunosuppressive therapy (IST). In this study, we examined the oral microbiome composition of 24 patients admitted to the National Institutes of Health (NIH) Clinical Center for experimental SAA treatment. Tongue brushings were collected to assess the effects of treatment on the oral microbiome. Twenty patients received standard IST (equine antithymocyte globulin and cyclosporine) plus eltrombopag. Four patients underwent HSCT. Oral specimens were obtained at three time points during treatment and clinical follow-up. Using a novel approach to 16S rRNA gene sequence analysis encompassing seven hypervariable regions, results demonstrated a predictable decrease in microbial diversity over time among the transplant patients. Linear discriminant analysis or LefSe reported a total of 14 statistically significant taxa (p < 0.05) across time points in the HSCT patients. One-way plots of relative abundance for two bacterial species (Haemophilus parainfluenzae and Rothia mucilaginosa) in the HSCT group, show the differences in abundance between time points. Only one bacterial species (Prevotella histicola) was noted in the IST group with a p value of 0.065. The patients receiving immunosuppressive therapy did not exhibit a clear change in diversity over time; however, patient-specific changes were noted. In addition, we compared our findings to tongue dorsum samples from healthy participants in the Human Microbiome Project (HMP) database and found among HSCT patients, approximately 35% of bacterial identifiers (N = 229) were unique to this study population and were not present in tongue dorsum specimens obtained from the HMP. Among IST-treated patients, 45% (N = 351) were unique to these patients and not identified by the HMP. Although antibiotic use may have likely influenced bacterial composition and diversity, some literature suggests a decreased impact of antimicrobials on the oral microbiome as compared to their effect on the gut microbiome. Future studies with larger sample sizes that focus on the oral microbiome and the effects of antibiotics in an immunosuppressed patient population may help establish these potential associations.

A practical guide to the oral microbiome and its relation to health and disease.

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.

Helicobacter saguini, a Novel Helicobacter Isolated from Cotton-Top Tamarins with Ulcerative Colitis, Has Proinflammatory Properties and Induces Typhlocolitis and Dysplasia in Gnotobiotic IL-10-/- Mice.

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-1ß, 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-1ß, 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.

Microbial Diversity in the Early In Vivo-Formed Dental Biofilm.

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.

Bacterial composition in whole saliva from patients with severe hyposalivation--a case-control study.

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.

Salivary microbiota and caries occurrence in Mutans Streptococci-positive school children.

AIM: To compare the composition of the salivary microbiota in caries-affected vs. caries-free mutans streptococci (MS)- positive children with mixed dentition. MATERIALS AND METHODS: Twenty eight healthy, 11-12-year-old schoolchildren with high MS counts (>10⊃5 CFU/mL) were included in this study. The children were screened with the Dentocult SM Strip Mutans test (Orion Diagnostica, Espoo, Finland) and examined using the International Caries Detection and Assessment System (ICDAS). The microbial composition of the saliva was assessed using the Human Oral Microbe Identification Microarray (HOMIM). Microbial differences between caries-affected (n=18) and caries-free children (n=10) were compared by Mann-Whitney analysis. RESULTS: The microbiota of the caries-affected vs. caries-free children was rather similar. Abiotrophia defectiva and Actinomyces meyeri/A. odontolyticus were significantly higher in caries-affected than in caries-free children (p=0.006, 0.046, respectively). Shuttleworthia satelles was significantly higher in caries-free compared to caries-affected children (p=0.031). A. defectiva and A. meyeri/A. odontolyticus correlated positively with caries severity measured by ICDAS Caries Index (p = 0.494, 0.454, 0.400 respectively) while S. satelles was negatively correlated with caries severity (p= -0.489). CONCLUSIONS: Salivary A. defectiva and A. meyeri/A. odontolyticus and are associated with caries occurrence in MS-positive children with mixed dentition.

Subgingival microbial profiles of Sudanese patients with aggressive periodontitis.

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.

Altered bacterial profiles in saliva from adults with caries lesions: a case-cohort study.

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 <0.01) while two bacterial taxa (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 <0.01). The principal component analysis displayed a marked difference in the bacterial community profiles between groups. Presence of manifest caries was associated with a reduced diversity and an altered salivary bacterial community profile. Our data support recent theories that ecological stress-induced changes of commensal microbial communities are involved in the shift from oral health to tooth decay.

Microbial diversity in the oral cavity of healthy Chinese Han children.

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.

A physiologically relevant culture platform for long-term studies of in vitro gingival tissue.

There is a clinical need to understand the etiologies of periodontitis, considering the growing socio-economic impact of the disease. Despite recent advances in oral tissue engineering, experimental approaches have failed to develop a physiologically relevant gingival model that combines tissue organization with salivary flow dynamics and stimulation of the shedding and non-shedding oral surfaces. Herein, we develop a dynamic gingival tissue model composed of a silk scaffold, replicating the cyto-architecture and oxygen profile of the human gingiva, along with a saliva-mimicking medium that reflected the ionic composition, viscosity, and non-Newtonian behavior of human saliva. The construct was cultured in a custom designed bioreactor, in which force profiles on the gingival epithelium were modulated through analysis of inlet position, velocity and vorticity to replicate the physiological shear stress of salivary flow. The gingival bioreactor supported the long-term in vivo features of the gingiva and improved the integrity of the epithelial barrier, critical against the invasion of pathogenic bacteria. Furthermore, the challenge of the gingival tissue with P. gingivalis lipopolysaccharide, as an in vitro surrogate for microbial interactions, indicated a greater stability of the dynamic model in maintaining tissue homeostasis and, thus, its applicability in long-term studies. The model will be integrated into future studies with the human subgingival microbiome to investigate host-pathogen and host-commensal interactions. STATEMENT OF SIGNIFICANCE: The major societal impact of human microbiome had reverberated up to the establishment of the Common Fund's Human Microbiome Project, that has the intent of studying the role of microbial communities in human health and diseases, including periodontitis, atopic dermatitis, or asthma and inflammatory bowel disease. In addition, these chronic diseases are emergent drivers of global socioeconomic status. Not only common oral diseases have been shown to be directly correlated with several systemic conditions, but they are differentially impacting some racial/ethnic and socioeconomic groups. To address this growing social disparity, the development of in vitro gingival model would provide a time and cost-effective experimental platform, able to mimic the spectrum of periodontal disease presentation, for the identification of predictive biomarkers for early-stage diagnosis.

Prevalence of streptococci and increased polymicrobial diversity associated with cystic fibrosis patient stability.

Diverse microbial communities chronically colonize the lungs of cystic fibrosis patients. Pyrosequencing of amplicons for hypervariable regions in the 16S rRNA gene generated taxonomic profiles of bacterial communities for sputum genomic DNA samples from 22 patients during a state of clinical stability (outpatients) and 13 patients during acute exacerbation (inpatients). We employed quantitative PCR (qPCR) to confirm the detection of Pseudomonas aeruginosa and Streptococcus by the pyrosequencing data and human oral microbe identification microarray (HOMIM) analysis to determine the species of the streptococci identified by pyrosequencing. We show that outpatient sputum samples have significantly higher bacterial diversity than inpatients, but maintenance treatment with tobramycin did not impact overall diversity. Contrary to the current dogma in the field that Pseudomonas aeruginosa is the dominant organism in the majority of cystic fibrosis patients, Pseudomonas constituted the predominant genera in only half the patient samples analyzed and reported here. The increased fractional representation of Streptococcus in the outpatient cohort relative to the inpatient cohort was the strongest predictor of clinically stable lung disease. The most prevalent streptococci included species typically associated with the oral cavity (Streptococcus salivarius and Streptococcus parasanguis) and the Streptococcus milleri group species. These species of Streptococcus may play an important role in increasing the diversity of the cystic fibrosis lung environment and promoting patient stability.

New approaches for isolation of previously uncultivated oral bacteria.

A significant number of microorganisms from the human oral cavity remain uncultivated. This is a major impediment to the study of human health since some of the uncultivated species may be involved in a variety of systemic diseases. We used a range of innovations previously developed to cultivate microorganisms from the human oral cavity, focusing on anaerobic species. These innovations include (i) in vivo cultivation to specifically enrich for species actively growing in the oral cavity (the "minitrap" method), (ii) single-cell long-term cultivation to minimize the effect of fast-growing microorganisms, and (iii) modifications of conventional enrichment techniques, using media that did not contain sugar, including glucose. To enable cultivation of obligate anaerobes, we maintained strict anaerobic conditions in most of our cultivation experiments. We report that, on a per cell basis, the most successful recovery was achieved using minitrap enrichment (11%), followed by single-cell cultivation (3%) and conventional plating (1%). Taxonomically, the richest collection was obtained using the single-cell cultivation method, followed by minitrap and conventional enrichment, comprising representatives of 13, 9, and 4 genera, respectively. Interestingly, no single species was isolated by all three methods, indicating method complementarity. An important result is the isolation and maintenance in pure culture of 10 strains previously only known by their molecular signatures, as well as representatives of what are likely to be three new microbial genera. We conclude that the ensemble of new methods we introduced will likely help close the gap between cultivated and uncultivated species from the human oral cavity.

Microbial profiles in saliva from children with and without caries in mixed dentition.

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 8years 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.

Diversity and site-specificity of the oral microflora in the elderly.

The purpose of the present study was to describe the bacterial diversity in the oral cavity of the elderly without root caries using bacterial microarrays, and to determine the site- and subject-specificity of bacterial colonization. Samples were collected from the tongue dorsum, mucosa of the buccal fold, hard palate, supragingival plaque from sound root surfaces, and subgingival plaque from the same roots. A new 16 S rRNA gene-based microarray method was used for the simultaneous detection of approximately 300 bacterial species. Overall, 175 species and clusters were detected, representing eight phyla. Species belonging to the genera Streptococcus, Veillonella, and Fusobacterium were common in all sites. The number of species per subject varied from 51 to 81. Statistical analyses revealed about 40 species or clusters with significant associations with at least one of the sites. The bacterial diversity was highest in the cheek and palate regions. Species typically associated with caries and periodontitis were detected rarely or not at all. The oral bacterial flora of the elderly appears to be diverse, and, to a large extent, site- rather than subject-specific.

Microarray analysis of the microflora of root caries in elderly.

The present study used a new 16S rRNA-based microarray with probes for over 300 bacterial species to better define the bacterial profiles of healthy root surfaces and root caries (RC) in the elderly. Supragingival plaque was collected from 20 healthy subjects (Controls) and from healthy and carious roots and carious dentin from 21 RC subjects (Patients). Collectively, 179 bacterial species and species groups were detected. A higher bacterial diversity was observed in Controls than in Patients. Lactobacillus casei/paracasei/rhamnosus and Pseudoramibacter alactolyticus were notably associated with most RC samples. Streptococcus mutans was detected more frequently in the infected dentin than in the other samples, but the difference was not significant. Actinomyces was found more frequently in Controls. Thus, species other than Actinomyces and S. mutans may play a role as pathogens of RC. The results from this study were in general agreement with those of our previous study based on 16S rRNA gene sequencing.

Impact of Oral Hygiene Discontinuation on Supragingival and Salivary Microbiomes.

The purpose of the present study was to characterize and compare supragingival and salivary microbiotas during a 10-d period of oral hygiene discontinuation. We tested the hypothesis that the composition of the salivary microbiota will reflect local microbial changes associated with accumulated biofilm formation and maturation. Pooled supragingival plaque (n = 145) and stimulated saliva (n = 145) samples were collected and plaque and gingival indices were recorded from 29 orally healthy individuals at baseline, during oral hygiene discontinuation (days 4, 7, and 10), and 14 d after resumption of oral hygiene. Supragingival and salivary microbiotas were processed by next-generation sequencing (Human Oral Microbe Identification using Next Generation Sequencing) and microbial community profiles were compared. Microbial composition of supragingival plaque samples collected after 4, 7, and 10 d of oral hygiene discontinuation, as well as 14 d after reuptake of oral hygiene, differed significantly from baseline samples, by a 3-fold increase in relative abundance Leptotrichia species and a 2-fold decrease in Streptococcus species (adjusted P < 0.01). In saliva samples, a significant increase in relative abundance of Leptotrichia species (adjusted P < 0.01) was evident at day 7 but completely reversed 14 d after resumption of oral hygiene. While the salivary microbiota was resistant to accumulated local biofilm formation, data from this study showed that compositional changes of supragingival microbiotas were not reversed 14 d after resumption of oral hygiene, despite the restoration of plaque to baseline levels. (ClinicalTrials.gov UCPH_OI_002, NCT02913235). Knowledge Transfer Statement: Data from this study showed compositional changes of supragingival microbiotas as a consequence of a 10-d period of oral hygiene discontinuation, that was not reversed 14 d after resumption of oral hygiene. Notably, oral hygiene discontinuation was associated with a significant increase in relative abundance of potential cariogenic Leptotrichia species and a decrease in Streptococcus species. Thus, findings from this study highlight the necessity of regular oral hygiene in the maintenance of oral homeostasis.

Ligature-associated bacterial profiles are linked to type 2 diabetes mellitus in a rat model and influenced by antibody treatment against TNF-α or RAGE.

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

Clinical and microbiological parameters of naturally occurring periodontitis in the non-human primate Macaca mulatta.

Background: Non-human primates appear to represent the most faithful model of human disease, but to date the oral microbiome in macaques has not been fully characterized using next-generation sequencing. Objective: In the present study, we characterized the clinical and microbiological features of naturally occurring periodontitis in non-human primates (Macaca mulatta). Design: Clinical parameters of periodontitis including probing pocket depth (PD) and bleeding on probing (BOP) were measured in 40 adult macaques (7-22 yrs), at six sites per tooth. Subgingival plaque was collected from diseased and healthy sites, and subjected to 16S rDNA sequencing and identification at the species or higher taxon level. Results: All macaques had mild periodontitis at minimum, with numerous sites of PD ≥ 4 mm and BOP. A subset (14/40) had moderate-severe disease, with >2 sites with PD ≥ 5mm, deeper mean PD, and more BOP. Animals with mild vs moderate-severe disease were identical in age, suggesting genetic heterogeneity. 16S rDNA sequencing revealed that all macaques had species that were identical to those in humans or closely related to human counterparts, including Porphyromonas gingivalis which was present in all animals. Diseased and healthy sites harboured distinct microbiomes; however there were no significant differences in the microbiomes in moderate-severe vs. mild periodontitis. Conclusions: Naturally occurring periodontitis in older macaques closely resembles human adult periodontitis, thus validating a useful model to evaluate novel anti-microbial therapies.

Porphyromonas gingivalis is the most abundant species detected in coronary and femoral arteries.

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.

Subgingival and tongue microbiota during early periodontitis.

Periodontal infections have a microbial etiology. Association of species with early disease would be useful in determining which microbes initiate periodontitis. We hypothesized that the microbiota of subgingival and tongue samples would differ between early periodontitis and health. A cross-sectional evaluation of 141 healthy and early periodontitis adults was performed with the use of oligonucleotide probes and PCR. Most species differed in associations with sample sites; most subgingival species were associated with subgingival samples. Few species were detected more frequently in early periodontitis by DNA probes. Porphyromonas gingivalis and Tannerella forsythia (Tannerella forsythensis) were associated with early periodontitis by direct PCR. In conclusion, the microbiota of tongue samples was less sensitive than that of subgingival samples in detecting periodontal species, and there was overlap in species detected in health and early periodontitis. Detection of periodontal pathogens in early periodontitis suggests an etiology similar to that of more advanced disease.