Bacterial diversity in the oral cavity of 10 healthy individuals.
ABSTRACT: The composition of the oral microbiota from 10 individuals with healthy oral tissues was determined using culture-independent techniques. From each individual, 26 specimens, each from different oral sites at a single point in time, were collected and pooled. An 11th pool was constructed using portions of the subgingival specimens from all 10 individuals. The 16S ribosomal RNA gene was amplified using broad-range bacterial primers, and clone libraries from the individual and subgingival pools were constructed. From a total of 11,368 high-quality, nonchimeric, near full-length sequences, 247 species-level phylotypes (using a 99% sequence identity threshold) and 9 bacterial phyla were identified. At least 15 bacterial genera were conserved among all 10 individuals, with significant interindividual differences at the species and strain level. Comparisons of these oral bacterial sequences with near full-length sequences found previously in the large intestines and feces of other healthy individuals suggest that the mouth and intestinal tract harbor distinct sets of bacteria. Co-occurrence analysis showed significant segregation of taxa when community membership was examined at the level of genus, but not at the level of species, suggesting that ecologically significant, competitive interactions are more apparent at a broader taxonomic level than species. This study is one of the more comprehensive, high-resolution analyses of bacterial diversity within the healthy human mouth to date, and highlights the value of tools from macroecology for enhancing our understanding of bacterial ecology in human health.
Project description:The subgingival microbial ecology is complex, and little is known regarding its bacteria species composition in healthy Chinese individuals. This study aimed to identify the subgingival microbiota from 6 healthy Chinese subjects. Subgingival samples from 6 volunteers were collected, the 16S rRNA gene was amplified using broad-range bacterial primers, and clone libraries were constructed. For the initial 2,439 sequences analyzed, 383 species-level operational taxonomic units (SLOTUs) belonging to seven phyla were identified, estimated as 51% [95% confidence interval (CI) 44-55] of the SLOTUs in this ecosystem. Most (85%) of the bacterial sequences, falling into 228 types of species, corresponded to known and cultivated species. However, 146 (6%) sequences, comprising 104 phylotypes, had <97% similarity to prior database sequences. Ten bacterial genera were conserved among all 6 individuals, comprising 2,000 (82%) of the 2,439 clones analyzed. Ten species were noted in all of the 6 subjects, comprising 1,435 (58.8%) of the 2,439 clones. Streptococcus infantis was the species most frequently cloned. Furthermore, certain species which may participate in the pathogenesis of periodontal disease were present in the 6 subjects. Although the initial subgingival plaque community of each subject was unique in terms of diversity and composition, 10 common key species were found in the 6 Chinese individuals. These ten species of bacteria in the human subgingival plaque in the 6 healthy individuals may be key species which, to some extent, affect periodontal health. Destruction of these key species in subgingival bacteria may break the microbiota balance and may easily lead to over-breeding conditions resulting in pathogenic oral disease.
Project description:The oral microbiome, the complex ecosystem of microbes inhabiting the human mouth, harbors several thousands of bacterial types. The proliferation of pathogenic bacteria within the mouth gives rise to periodontitis, an inflammatory disease known to also constitute a risk factor for cardiovascular disease. While much is known about individual species associated with pathogenesis, the system-level mechanisms underlying the transition from health to disease are still poorly understood. Through the sequencing of the 16S rRNA gene and of whole community DNA we provide a glimpse at the global genetic, metabolic, and ecological changes associated with periodontitis in 15 subgingival plaque samples, four from each of two periodontitis patients, and the remaining samples from three healthy individuals. We also demonstrate the power of whole-metagenome sequencing approaches in characterizing the genomes of key players in the oral microbiome, including an unculturable TM7 organism. We reveal the disease microbiome to be enriched in virulence factors, and adapted to a parasitic lifestyle that takes advantage of the disrupted host homeostasis. Furthermore, diseased samples share a common structure that was not found in completely healthy samples, suggesting that the disease state may occupy a narrow region within the space of possible configurations of the oral microbiome. Our pilot study demonstrates the power of high-throughput sequencing as a tool for understanding the role of the oral microbiome in periodontal disease. Despite a modest level of sequencing (~2 lanes Illumina 76 bp PE) and high human DNA contamination (up to ~90%) we were able to partially reconstruct several oral microbes and to preliminarily characterize some systems-level differences between the healthy and diseased oral microbiomes.
Project description:The human oral microbiome is known to play a significant role in human health and disease. While less well studied, the feline oral microbiome is thought to play a similarly important role. To determine roles oral bacteria play in health and disease, one first has to be able to accurately identify bacterial species present. 16S rRNA gene sequence information is widely used for molecular identification of bacteria and is also useful for establishing the taxonomy of novel species. The objective of this research was to obtain full 16S rRNA gene reference sequences for feline oral bacteria, place the sequences in species-level phylotypes, and create a curated 16S rRNA based taxonomy for common feline oral bacteria. Clone libraries were produced using "universal" and phylum-selective PCR primers and DNA from pooled subgingival plaque from healthy and periodontally diseased cats. Bacteria in subgingival samples were also cultivated to obtain isolates. Full-length 16S rDNA sequences were determined for clones and isolates that represent 171 feline oral taxa. A provisional curated taxonomy was developed based on the position of each taxon in 16S rRNA phylogenetic trees. The feline oral microbiome curated taxonomy and 16S rRNA gene reference set will allow investigators to refer to precisely defined bacterial taxa. A provisional name such as "Propionibacterium sp. feline oral taxon FOT-327" is an anchor to which clone, strain or GenBank names or accession numbers can point. Future next-generation-sequencing studies of feline oral bacteria will be able to map reads to taxonomically curated full-length 16S rRNA gene sequences.
Project description:BACKGROUND:To investigate the potential association between oral health and cognitive function, a pilot study was conducted to evaluate high throughput DNA sequencing of the V3 region of the 16S ribosomal RNA gene for determining the relative abundance of bacterial taxa in subgingival plaque from older adults with or without dementia. METHODS:Subgingival plaque samples were obtained from ten individuals at least 70?years old who participated in a study to assess oral health and cognitive function. DNA was isolated from the samples and a gene segment from the V3 portion of the 16S bacterial ribosomal RNA gene was amplified and sequenced using an Illumina HiSeq1000 DNA sequencer. Bacterial populations found in the subgingival plaque were identified and assessed with respect to the cognitive status and oral health of the participants who provided the samples. RESULTS:More than two million high quality DNA sequences were obtained from each sample. Individuals differed greatly in the mix of phylotypes, but different sites from different subgingival depths in the same subject were usually similar. No consistent differences were observed in this small sample between subjects separated by levels of oral health, sex, or age; however a consistently higher level of Fusobacteriaceae and a generally lower level of Prevotellaceae was seen in subjects without dementia, although the difference did not reach statistical significance, possibly because of the small sample size. CONCLUSIONS:The results from this pilot study provide suggestive evidence that alterations in the subgingival microbiome are associated with changes in cognitive function, and provide support for an expanded analysis of the role of the oral microbiome in dementia.
Project description:Single dose of systemic antibiotics and short-term use of mouthwashes reduce bacteremia. However, the effects of a single dose of preprocedural rinse are still controversial. This study evaluated, in periodontally diseased patients, the effects of a pre-procedural mouth rinse on induced bacteremia.Systemically healthy individuals with gingivitis (n=27) or periodontitis (n = 27) were randomly allocated through a sealed envelope system to: 0.12% chlorhexidine pre-procedural rinse (13 gingivitis and 13 periodontitis patients) or no rinse before dental scaling (14 gingivitis and 15 periodontitis patients). Periodontal probing depth, clinical attachment level, plaque, and gingival indices were measured and subgingival samples were collected. Blood samples were collected before dental scaling, 2 and 6 minutes after scaling. Total bacterial load and levels of P. gingivalis were determined in oral and blood samples by real-time polymerase chain reaction, while aerobic and anaerobic counts were determined by culture in blood samples. The primary outcome was the antimicrobial effect of the pre-procedural rinse. Data was compared by Mann-Whitney and Signal tests (p<0.05).In all sampling times, polymerase chain reaction revealed higher blood bacterial levels than culture (p<0.0001), while gingivitis patients presented lower bacterial levels in blood than periodontitis patients (p<0.0001). Individuals who experienced bacteremia showed worse mean clinical attachment level (3.4 mm vs. 1.1 mm) and more subgingival bacteria (p<0.005). The pre-procedural rinse did not reduce induced bacteremia.Bacteremia was influenced by periodontal parameters. In periodontally diseased patients, pre-procedural rinsing showed a discrete effect on bacteremia control.
Project description:Dysbiotic oral bacterial communities have a critical role in the etiology and progression of periodontal diseases. The goal of this study was to investigate the extent to which smoking increases risk for disease by influencing the composition of the subgingival microbiome in states of clinical health. Subgingival plaque samples were collected from 200 systemically and periodontally healthy smokers and nonsmokers. 16S pyrotag sequencing was preformed generating 1,623,713 classifiable sequences, which were compared with a curated version of the Greengenes database using the quantitative insights into microbial ecology pipeline. The subgingival microbial profiles of smokers and never-smokers were different at all taxonomic levels, and principal coordinate analysis revealed distinct clustering of the microbial communities based on smoking status. Smokers demonstrated a highly diverse, pathogen-rich, commensal-poor, anaerobic microbiome that is more closely aligned with a disease-associated community in clinically healthy individuals, suggesting that it creates an at-risk-for-harm environment that is primed for a future ecological catastrophe.
Project description:Staphylococcus aureus and Staphylococcus epidermidis are frequent commensals of the nares and skin and are considered transient oral residents. Reports on their prevalence in the oral cavity, periodontal pockets and subgingivally around infected oral implants are conflicting, largely due to methodological limitations. The prevalence of these species in the oral cavities, periodontal pockets and subgingival sites of orally healthy individuals with/without implants and in patients with periodontal disease or infected implants (peri-implantitis) was investigated using selective chromogenic agar and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Staphylococcus epidermidis was predominant in all participant groups investigated. Its prevalence was significantly higher (P = 0.0189) in periodontal pockets (30%) than subgingival sites of healthy individuals (7.8%), and in subgingival peri-implantitis sites (51.7%) versus subgingival sites around non-infected implants (16.1%) (P = 0.0057). In contrast, S. aureus was recovered from subgingival sites of 0-12.9% of the participant groups, but not from periodontal pockets. The arginine catabolic mobile element (ACME), thought to enhance colonization and survival of S. aureus, was detected in 100/179 S. epidermidis and 0/83 S. aureus isolates screened using multiplex PCR and DNA microarray profiling. Five distinct ACME types, including the recently described types IV and V (I; 14, II; 60, III; 10, IV; 15, V; 1) were identified. ACME-positive S. epidermidis were significantly (P = 0.0369) more prevalent in subgingival peri-implantitis sites (37.9%) than subgingival sites around non-infected implants (12.9%) and also in periodontal pockets (25%) compared to subgingival sites of healthy individuals (4.7%) (P = 0.0167). To investigate the genetic diversity of ACME, 35 isolates, representative of patient groups, sample sites and ACME types underwent whole genome sequencing from which multilocus sequence types (STs) were identified. Sequencing data permitted ACME types II and IV to be subdivided into subtypes IIa-c and IVa-b, respectively, based on distinct flanking direct repeat sequences. Distinct ACME types were commonly associated with specific STs, rather than health/disease states or recovery sites, suggesting that ACME types/subtypes originated amongst specific S. epidermidis lineages. Ninety of the ACME-positive isolates encoded the ACME-arc operon, which likely contributes to oral S. epidermidis survival in the nutrient poor, semi-anaerobic, acidic and inflammatory conditions present in periodontal disease and peri-implantitis.
Project description:BACKGROUND:Several in vitro oral biofilm growth systems can reliably construct oral microbiome communities in culture, yet their stability and reproducibility through time has not been well characterized. Long-term in vitro growth of natural biofilms would enable use of these biofilms in both in vitro and in vivo studies that require complex microbial communities with minimal variation over a period of time. Understanding biofilm community dynamics in continuous culture, and whether they maintain distinct signatures of health and disease, is necessary to determine the reliability and applicability of such models to broader studies. To this end, we performed next-generation sequencing on biofilms grown from healthy and disease-site subgingival plaque for 80 days to assess stability and reliability of continuous oral biofilm growth. RESULTS:Biofilms were grown from subgingival plaque collected from periodontitis-affected sites and healthy individuals for ten eight-day long generations, using hydroxyapatite disks. The bacterial community in each generation was determined using Human Oral Microbe Identification by Next-Generation Sequencing (HOMINGS) technology, and analyzed in QIIME. Profiles were steady through the ten generations, as determined by species abundance and prevalence, Spearman's correlation coefficient, and Faith's phylogenetic distance, with slight variation predominantly in low abundance species. Community profiles were distinct between healthy and disease site-derived biofilms as demonstrated by weighted UniFrac distance throughout the ten generations. Differentially abundant species between healthy and disease site-derived biofilms were consistent throughout the generations. CONCLUSIONS:Healthy and disease site-derived biofilms can reliably maintain consistent communities through ten generations of in vitro growth. These communities maintain signatures of health and disease and of individual donors despite culture in identical environments. This subgingival oral biofilm growth and perpetuation model may prove useful to studies involving oral infection or cell stimulation, or those measuring microbial interactions, which require the same biofilms over a period of time.
Project description:We examined the subgingival bacterial biodiversity in untreated chronic periodontitis patients by sequencing 16S rRNA genes. The primary purpose of the study was to compare the oral microbiome in deep (diseased) and shallow (healthy) sites. A secondary purpose was to evaluate the influences of smoking, race and dental caries on this relationship. A total of 88 subjects from two clinics were recruited. Paired subgingival plaque samples were taken from each subject, one from a probing site depth >5 mm (deep site) and the other from a probing site depth ?3mm (shallow site). A universal primer set was designed to amplify the V4-V6 region for oral microbial 16S rRNA sequences. Differences in genera and species attributable to deep and shallow sites were determined by statistical analysis using a two-part model and false discovery rate. Fifty-one of 170 genera and 200 of 746 species were found significantly different in abundances between shallow and deep sites. Besides previously identified periodontal disease-associated bacterial species, additional species were found markedly changed in diseased sites. Cluster analysis revealed that the microbiome difference between deep and shallow sites was influenced by patient-level effects such as clinic location, race and smoking. The differences between clinic locations may be influenced by racial distribution, in that all of the African Americans subjects were seen at the same clinic. Our results suggested that there were influences from the microbiome for caries and periodontal disease and these influences are independent.
Project description:OBJECTIVES:The aim of this cross-sectional observational study was to compare the prevalence of different oral Candida spp. in patients with Type 2 Diabetes and chronic periodontitis in two oral sites: dorsal surface of the tongue and subgingival area. In order to determine subgingival areas as potential reservoirs of yeasts, this study aimed to find differences in the yeasts' detection between the dorsum of the tongue, as the oral site most commonly inhabited with microorganisms, and subgingival samples. Additionally, potential predictors for the yeasts prevalence were determined. MATERIAL AND METHODS:Subjects (N = 146) were divided into four groups: group A- healthy individuals without periodontitis, group B- healthy individuals with chronic periodontitis, group C- Type 2 Diabetes patients with good glycoregulation and Chronic periodontitis and group D- Type 2 Diabetes patients with poor glycoregulation and Chronic periodontitis. Samples were obtained from the tongue by swabbing. Subgingival plaque samples were taken by paper points and periodontal curette. Isolation and identification of different Candida spp. was done using ChromAgar medium. In addition, germ-tube production and carbohydrate assimilation tests were performed. RESULTS:The prevalence of Candida spp. was higher in diabetics with poor glycoregulation. The most frequently isolated species was Candida albicans followed by Candida glabrata and Candida tropicalis. In 15.6% of cases, Candida spp. was present in the subgingival area while absent on the tongue. Multivariate regression model showed that HbA1c was Candida spp. predictor for both locations. CONCLUSIONS:Our results confirmed that there are Candida spp. carriers among subjects with clinically healthy oral mucosa. Also, this study identified subgingival areas as potential reservoirs of these pathogenic species. Glycoregulation has been recognized as a positive predictor factor of Candida spp.