Oral Bacterial Communities Research Articles

Next-generation sequencing analysis reveals high bacterial diversity in wild venomous and non-venomous snakes from India.

BackgroundThe oral cavities of snakes are replete with various types of bacterial flora. Culture-dependent studies suggest that some of the bacterial species are responsible for secondary bacterial infection associated with snakebite. A complete profile of the ophidian oral bacterial community has been unreported until now. Therefore, in the present study, we determined the complete bacterial compositions in the oral cavity of some snakes from India.MethodsTotal DNA was isolated from oral swabs collected from three wild snake species (Indian Cobra, King Cobra and Indian Python). Next, the DNA was subjected to PCR amplification of microbial 16S rRNA gene using V3-region-specific primers. The amplicons were used for preparation of DNA libraries that were sequenced on an Illumina MiSeq platform.ResultsThe cluster-based taxonomy analysis revealed that Proteobacteria and Actinobacteria were the most predominant phyla present in the oral cavities of snakes. This result indicates that snakes show more similarities to birds than mammals as to their oral bacterial communities. Furthermore, our study reports all the unique and common bacterial species (total: 147) found among the oral microbes of snakes studied, while the majority of commonly abundant species were pathogens or opportunistic pathogens to humans. A wide difference in ophidian oral bacterial flora suggests variation by individual, species and geographical region.ConclusionThe present study would provide a foundation for further research on snakes to recognize the potential drugs/antibiotics for the different infectious diseases.

Uncovering complex microbiome activities via metatranscriptomics during 24\u2009hours of oral biofilm assembly and maturation

BackgroundDental plaque is composed of hundreds of bacterial taxonomic units and represents one of the most diverse and stable microbial ecosystems associated with the human body. Taxonomic composition and functional capacity of mature plaque is gradually shaped during several stages of community assembly via processes such as co-aggregation, competition for space and resources, and by bacterially produced reactive agents. Knowledge on the dynamics of assembly within complex communities is very limited and derives mainly from studies composed of a limited number of bacterial species. To fill current knowledge gaps, we applied parallel metagenomic and metatranscriptomic analyses during assembly and maturation of an in vitro oral biofilm. This model system has previously demonstrated remarkable reproducibility in taxonomic composition across replicate samples during maturation.ResultsTime course analysis of the biofilm maturation was performed by parallel sampling every 2–3 h for 24 h for both DNA and RNA. Metagenomic analyses revealed that community taxonomy changed most dramatically between three and six hours of growth when pH dropped from 6.5 to 5.5. By applying comparative metatranscriptome analysis we could identify major shifts in overall community activities between six and nine hours of growth when pH dropped below 5.5, as 29,015 genes were significantly up- or down- expressed. Several of the differentially expressed genes showed unique activities for individual bacterial genomes and were associated with pyruvate and lactate metabolism, two-component signaling pathways, production of antibacterial molecules, iron sequestration, pH neutralization, protein hydrolysis, and surface attachment. Our analysis also revealed several mechanisms responsible for the niche expansion of the cariogenic pathogen Lactobacillus fermentum.ConclusionIt is highly regarded that acidic conditions in dental plaque cause a net loss of enamel from teeth. Here, as pH drops below 5.5 pH to 4.7, we observe blooms of cariogenic lactobacilli, and a transition point of many bacterial gene expression activities within the community. To our knowledge, this represents the first study of the assembly and maturation of a complex oral bacterial biofilm community that addresses gene level functional responses over time.

Relative Abundance Analysis of the Oral and Gastrointestinal Microbiome during Autologous Transplantation for Multiple Myeloma: Results of a Prospective Pilot Study and Association with Transplant Outcomes

BackgroundThe human microbiome has been associated with allogeneic hematopoietic cell transplantation (HCT) outcomes. To date, there are no studies describing the longitudinal changes in the oral or gastrointestinal microbiome in the setting of autologous HCT. We conducted a prospective study to determine the longitudinal microbial profile in patients undergoing HCT for multiple myeloma (MM), and whether changes in microbial abundance correlate with HCT outcomes and/or toxicities.MethodsSamples were collected from 15 MM patients on admission (baseline, T-2), during marrow aplasia (T+7) and after engraftment (T+30) (Table 1- summarizes baseline characteristics).We evaluated the bacterial and fungal microbiome of 15 patients using Ion-Torrent PGM workflow. The amplicons generated from the 16s rRNA and the ITS genes were sequenced for bacterial and fungal identification, respectively. Sequencing reads were clustered into operational taxonomic units (OTUs, 3% distance) and taxonomically classified via Qiime bioinformatics pipeline.Statistical analysis was performed using the statistical programming language R. Multivariate distance based association between communities and outcome was performed using the Adonis function as implemented in the R package vegan using Bray-curtis dissimilarities distances with and without presence/absence standardization (BrayPA). Non-parametric Spearman correlation and wilcoxon rank-sum test were used for association with continuous outcome and binary outcome respectively.ResultsRelative abundance was determined at the phylum and genus levels across each time point, in the oral and fecal microbiome, both bacterial and fungal. At the bacterial phylum level, the oral bacterial community composition significantly changed at T+30 compared to baseline (Bray-curtis, p=0.046) and T+7 (Bray-curtis, p=0.025). When examining the changes in the mycobiome composition, test for dissimilarity showed a significant difference in the fecal fungal genus between baseline and T+30 (BrayPA, p=0.025). No other statistically significant differences were noted.Next, we correlated the microbial community (Table 2) and individual composition with outcome and transplant related toxicity, the later will be reported here. In fecal samples, the bacterial phylum Bacteriodetes present at T+7 was associated with the development and severity of diarrhea, such that patients with higher abundance of Bacteroidetes experienced lower gastrointestinal toxicity (pAdj=0.03). Additionally, the 2 genera Blautia and Ruminococcus, both belonging to the Firmicutes phylum and Clostridium Class, when detected at T+7 were associated with a higher development and severity of vomiting after exposure to high dose melphan (pAdj=0.05 for both respectively). In oral samples, the presence of the genus Glomerella at T+7 was negatively associated with rates of neutrophil engraftment (pAdj=0.03).Conclusion and Future DirectionsWhile acknowledging the limitation inherent in the small sample size, our results show that oral and fecal microbiota undergo dynamic changes in their diversity (Abstract ID 120038), composition and relative abundance during the course of transplantation. This change is likely multifactorial owing to the conditioning regimen, antimicrobial exposure and immune dysregulation. Our data suggest the bacterial and fungal microbiota present specifically at count nadir could be important players in this population of patients. Interestingly, the relative abundance of particularly the anaerobic bacterial phyla, Bacteroidetes and Firciumtes (Blautia and Ruminococcus) during marrow aplasia, correlated with transplant related toxicities in our cohort. This could further contribute to our knowledge of the role anaerobic organisms play in HCT outcomes, in accordance to what has been described in the allogeneic HCT literature.Amifostine was used as a cytoprotectant before high dose melphalan for our patients. The effect of this organic thiophosphate on the microbiota is unclear and warrants future investigations. Further studies conducted on a larger scale and incorporating metabolomics and proteomics will help elucidate the interactions between the host and the microbiome and their effect on short term and long term transplantation outcomes as well as toxicities. DisclosuresLazarus:Pluristem Ltd.: Consultancy. Malek:Amgen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

Connection Between Dysbiotic Oral Microbiota and Deregulation of Salivary Gland Epithelial Cells in Sjogren's Syndrome

Background: Dysbiosis of the gut and oral microbiomes is a potential environmental factor in Sjogren's syndrome (SS), but its connection to the etiopathogenesis of SS remains unclear. This study aimed to characterize the oral microbiota in SS and to investigate its potential role in the pathogenesis of SS. Methods: Oral bacterial communities collected by whole mouthwash from 15 healthy individuals, 10 drug/radiation-induced sicca patients, and 25 primary SS patients were analyzed by pyrosequencing. Four SS-associated species and 2 control species were selected and used to challenge human submandibular gland tumor (HSG) cells. The expression of MHC I, MHC II, CD80, CD86, and IFNλ in HSG cells and the bacterial invasion into HSG cells were examined. Sections of labial salivary gland (LSG) biopsies from 8 non-SS subjects and 15 SS patients were subjected to in situ hybridization using universal and Prevotella melaninogenica-specific probes. Findings: The SS oral microbiota was characterized by an increased bacterial load and Shannon diversity. Although a substantial portion of the changes observed in SS patients overlapped with those in drug/radiation-induced sicca patients, SS-specific changes were identified. Among the selected SS-associated bacterial species, P. melaninogenica uniquely upregulated the expression of MHC molecules, CD80, and IFN λ in HSG cells. Concomitantly, P. melaninogenica efficiently invaded HSG cells. Ductal cells and the areas of infiltration were heavily infected with bacteria in the LSGs with focal lymphocytic sialadenitis. Interpretation: Dysbiotic oral microbiota is associated with deregulation of salivary gland epithelial cells and acquisition of IFN signatures through bacterial invasion into ductal cells. Funding Statement: This study was supported by a grant of the Korean Health Technology R&D Project (HI13C0016) from Ministry of Health & Welfare, Republic of Korea. Labial salivary gland biopsy specimens used in this manuscript are from the Sjogren’s International Collaborative Clinical Alliance [SICCA], funded under contract N01 DE-32636 by the National Institute of Dental and Craniofacial Research, with funding support from the National Eye Institute and Office for Research in Women’s Health. The funders had no role in study design, data collection, analysis, and interpretation, and writing of the report. Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: All procedures involving human subjects and materials were performed in accordance with the Helsinki Declaration under approved protocols from the Institutional Review Boards at Seoul St. Mary’s Hospital (KC13ONMI0646) and at Seoul National University, School of Dentistry (S-D20140022, S-D20170004). Informed consent was obtained from all subjects.

Association between Oral Candida and Bacteriome in Children with Severe ECC

Candida albicans is an opportunistic fungal organism frequently detected in the oral cavity of children with severe early childhood caries (S-ECC). Previous studies suggested the cariogenic potential of C. albicans, in vitro and in vivo, and further demonstrated its synergistic interactions with Streptococcus mutans. In combination, the 2 organisms are associated with higher caries severity in a rodent model. However, it remains unknown whether C. albicans influences the composition and diversity of the entire oral bacterial community to promote S-ECC onset. With 16s rRNA amplicon sequencing, this study analyzed the microbiota of saliva and supragingival plaque from 39 children (21 S-ECC and 18 caries-free [CF]) and 33 mothers (17 S-ECC and 16 CF). The results revealed that the presence of oral C. albicans is associated with a highly acidogenic and acid-tolerant bacterial community in S-ECC, with an increased abundance of plaque Streptococcus (particularly S. mutans) and certain Lactobacillus/Scardovia species and salivary/plaque Veillonella and Prevotella, as well as decreased levels of salivary/plaque Actinomyces. Concurrent with this microbial community assembly, the activity of glucosyltransferases (cariogenic virulence factors secreted by S. mutans) in plaque was significantly elevated when C. albicans was present. Moreover, the oral microbial community composition and diversity differed significantly by disease group (CF vs. S-ECC) and sample source (saliva vs. plaque). Children and mothers within the CF and S-ECC groups shared microbiota composition and diversity, suggesting a strong maternal influence on children’s oral microbiota. Altogether, this study underscores the importance of C. albicans in association with the oral bacteriome in the context of S-ECC etiopathogenesis. Further longitudinal studies are warranted to examine how fungal-bacterial interactions modulate the onset and severity of S-ECC, potentially leading to novel anticaries treatments that address fungal contributions.

PD127: Oral bacterial community of HIV‐infected Brazilian children revealed by next‐generation DNA sequencing

PD127: Oral bacterial community of HIV‐infected Brazilian children revealed by next‐generation DNA sequencing

Smokeless tobacco impacts oral microbiota in a Syrian Golden hamster cheek pouch carcinogenesis model

The use of smokeless tobacco products (STPs) can cause many serious health problems. The oral microbiota plays important roles in oral and systemic health, and the disruption in the oral microbial population is linked to periodontal disease and other health problems. To assess the impact of smokeless tobacco on oral microbiota in vivo, high-throughput sequencing was used to examine the oral microbiota present in Syrian Golden hamster cheek pouches. Sixteen hamsters were divided into four groups and treated with the STP Grizzly snuff (0, 2.5, 25, or 250 mg) twice daily for 4 weeks. After 0, 1, 2, 3, and 4 weeks of treatment, bacterial genomic DNA was extracted from oral swabs sampled from the cheek pouches of the hamsters. The oral bacterial communities present in different hamster groups were characterized by sequencing the hypervariable regions V1-V2 and V4 of 16S rRNA using the Illumina MiSeq platform. Fifteen phyla, 27 classes, 59 orders, 123 families, and 250 genera were identified from 4,962,673 sequence reads from the cheek pouch samples. The bacterial diversity and taxonomic abundances for the different treatment groups were compared to the non–treated hamsters. Bacterial diversity was significantly decreased after 4 weeks of exposure to 2.5 mg, and significantly increased by exposure to 250 mg STP. Treatment with 250 mg STP significantly increased Firmicutes, transiently increased Cyanobacteria and TM7, and decreased Bacteroidetes and Fusobacteria compared to the control group. At the genus level, 4 weeks of administration of 250 mg STP significantly increased Granulicatella, Streptococcus, Oribacterium, Anaerococcus, Acidaminococcus, Actinomyces, Eubacterium, Negativicoccus, and Staphylococcus, and decreased Bacteroides, Buleidia, Dialister, and Leptotrichia, and transiently decreased Arcanobacterium compared to the control group. For the first time, an animal model was used for evaluating the effects of STP on oral microbiota by metagenomic sequencing. Our results provide a view of the shift of the oral microbiota in response to STP exposure in Syrian Golden hamster. Our findings indicate that the use of smokeless tobacco significantly disrupts the oral microbiota.

Oral Microbiota Community Dynamics Associated With Oral Squamous Cell Carcinoma Staging.

Oral squamous cell carcinoma (OSCC) is a highly aggressive cancer and the fourth leading malignancy among males in Taiwan. Some pathogenic bacteria are associated with periodontitis and oral cancer. However, the comprehensive profile of the oral microbiome during the cancer's progression from the early stage to the late stage is still unclear. We profiled the oral microbiota and identified bacteria biomarkers associated with OSCC. The microbiota of an oral rinse from 51 healthy individuals and 197 OSCC patients at different stages were investigated using 16S rRNA V3V4 amplicon sequencing, followed by bioinformatics and statistical analyses. The oral microbiota communities from stage 4 patients showed significantly higher complexity than those from healthy controls. The populations also dynamically changed with the cancer's progression from stage 1 to stage 4. The predominant phyla in the oral samples showed variation in the relative abundance of Fusobacteria, Bacteroidetes, and Actinobacteria. The abundance of Fusobacteria increased significantly with the progression of oral cancer from the healthy controls (2.98%) to OSCC stage 1 (4.35%) through stage 4 (7.92%). At the genus level, the abundance of Fusobacterium increased, while the number of Streptococcus, Haemophilus, Porphyromonas, and Actinomyces decreased with cancer progression. Fusobacterium periodonticum, Parvimonas micra, Streptococcus constellatus, Haemophilus influenza, and Filifactor alocis were associated with OSCC, and they progressively increased in abundance from stage 1 to stage 4. The abundances of Streptococcus mitis, Haemophilus parainfluenzae, and Porphyromonas pasteri were inversely associated with OSCC progression. We selected a bacterial marker panel of three bacteria (upregulated F. periodonticum, down-regulated S. mitis, and P. pasteri), which had an AUC of 0.956 (95% CI = 0.925–0.986) in discriminating OSCC stage 4 from the healthy controls. Furthermore, the functional prediction of oral bacterial communities showed that genes involved in carbohydrate-related metabolism, such as methane metabolism, and energy-metabolism-related parameters, such as oxidative phosphorylation and carbon fixation in photosynthetic organisms, were enriched in late-stage OSCC, while those responsible for amino acid metabolism, such as folate biosynthesis and valine, leucine, and isoleucine biosynthesis, were significantly associated with the healthy controls. In conclusion, our results provided evidence of oral bacteria community changes during oral cancer progression and suggested the possibility of using bacteria as OSCC diagnostic markers.

Prevalence and Source of Fecal and Oral Bacteria on Infant, Child, and Adult Hands.

Modern hygienic practices are applied to avoid exposure to pathogens that spread via fecal-oral transmission. Despite this, the gastrointestinal tract is quickly colonized by fecal microbes. The hands are an important vector for the transmission of microbes, but the frequency at which fecal and oral microbes exist on hands and the source of those microbes have not been extensively described. Using data from a previous study that characterized the fecal, oral, and skin microbiota from 73 families, we found a significant incidence of fecal and oral microbes on hands. Of palms, 48.9% had fecal signal and 67.2% had oral signal. Fecal, oral, and forehead microbes were tracked to family members and an individual's own palms far more often than to unrelated individuals and showed relationships with age, gender, and parental status. For instance, oral microbes that were specifically sourced to the same individual (oneself) were most common on infant palms; mothers had more infant-child-sourced and oral-sourced microbes on their palms than nonparents. Fecal microbes on palms more often sourced to members of the family than unrelated individuals, but more often to other members of the family than oneself. This study supports that the hands are an important vector for the transfer of fecal and oral microbes within families. IMPORTANCE Bacteria live all around us, and we are constantly exposed to them during our everyday lives. Modern standards of hygiene aim to limit exposure to fecal bacteria, and yet bacteria rapidly colonize the gut in early life and following antibacterial treatment. Exposures to fecal and oral microbes provide risk of disease, but are also necessary since commensal microbes play important roles in health. This work establishes that bacteria of both fecal and oral origins are commonly found on hands. It also establishes that the uniqueness of fecal and oral bacterial communities across people can allow for determination of the likely individual from whom the fecal and oral bacteria came. These techniques allow for understanding the hands as a vector for microbial transmission within families and across populations, which has important implications for public health.

Alterations in oral bacterial communities are associated with risk factors for oral and oropharyngeal cancer

Oral squamous cell carcinomas are a major cause of morbidity and mortality, and tobacco usage, alcohol consumption, and poor oral hygiene are established risk factors. To date, no large-scale case-control studies have considered the effects of these risk factors on the composition of the oral microbiome, nor microbial community associations with oral cancer. We compared the composition, diversity, and function of the oral microbiomes of 121 oral cancer patients to 242 age- and gender-matched controls using a metagenomic multivariate analysis pipeline. Significant shifts in composition and function of the oral microbiome were observed with poor oral hygiene, tobacco smoking, and oral cancer. Specifically, we observed dramatically altered community composition and function after tooth loss, with smaller alterations in current tobacco smokers, increased production of antioxidants in individuals with periodontitis, and significantly decreased glutamate metabolism metal transport in oral cancer patients. Although the alterations in the oral microbiome of oral cancer patients were significant, they were of substantially lower effect size relative to microbiome shifts after tooth loss. Alterations following tooth loss, itself a major risk factor for oral cancer, are likely a result of severe ecological disruption due to habitat loss but may also contribute to the development of the disease.

Variations in oral microbiota associated with oral cancer

Individual bacteria and shifts in microbiome composition are associated with human disease, including cancer. To unravel the connections underlying oral bacterial dysbiosis and oral squamous cell carcinoma (OSCC), cancer lesion samples and anatomically matched normal samples were obtained from the same patients. We then profiled the bacteria within OSCC lesion surface samples at the species level using next-generation sequencing to comprehensively investigate bacterial community composition and functional genes in these samples. Significantly greater bacterial diversity was observed in the cancer samples than in the normal samples. Compared with previous studies, we identified many more taxa demonstrating remarkably different distributions between the groups. In particular, a group of periodontitis-correlated taxa, including Fusobacterium, Dialister, Peptostreptococcus, Filifactor, Peptococcus, Catonella and Parvimonas, was significantly enriched in OSCC samples. Additionally, several operational taxonomic units (OTUs) associated with Fusobacterium were highly involved in OSCC and demonstrated good diagnostic power. Our study revealed drastic changes in surface bacterial communities of OSCC. The findings enrich knowledge of the association between oral bacterial communities and oral cancer.

Establishment of oral bacterial communities in germ-free mice and the influence of recipient age.

The acquisition of the oral microbiome is a complex process. We examined how the timing of microbial exposure alters bacterial colonization of the tooth surface. Germ-free mice were conventionalized by exposure to specific pathogen-free (SPF) mice to acquire a commensal microbiome over three distinct 4-week periods, 0-4weeks of age (Conv0-4w), 4-8weeks (Conv4-8w), or 8-12weeks (Conv8-12w). Bacterial DNA was extracted from the tooth surface and analyzed by 16S rDNA sequencing. Total bacteria and inflammatory cytokine expression in gingiva were determined by quantitative real-time polymerase chain reaction. After co-housing with SPF mice, Conv0-4w and Conv4-8w mice had low bacterial diversity, whereas Conv8-12w mice had high bacterial diversity that was similar to that of SPF donor mice, as determined by both operational taxonomic units and the Shannon Index. Cluster analysis with unweighted Unifrac distance also supported these trends. This was surprising as the amount of maturation time, 4weeks, was equal in all conventionalized mice and tooth eruption was largely completed by 4weeks. This suggests that host factors that occur after tooth eruption have a significant effect on the microbial tooth colonization.

Multi-omics Analysis of Periodontal Pocket Microbial Communities Pre- and Posttreatment.

Periodontitis is a polymicrobial infectious disease that causes breakdown of the periodontal ligament and alveolar bone. We employed a meta-omics approach that included microbial 16S rRNA amplicon sequencing, shotgun metagenomics, and tandem mass spectrometry to analyze sub- and supragingival biofilms in adults with chronic periodontitis pre- and posttreatment with 0.25% sodium hypochlorite. Microbial samples were collected with periodontal curettes from 3- to 12-mm-deep periodontal pockets at the baseline and at 2weeks and 3months. All data types showed high interpersonal variability, and there was a significant correlation between phylogenetic diversity and pocket depth at the baseline and a strong correlation (rho = 0.21; P = 0.008) between metabolite diversity and maximum pocket depth (MPD). Analysis of subgingival baseline samples (16S rRNA and shotgun metagenomics) found positive correlations between abundances of particular bacterial genera and MPD, including Porphyromonas, Treponema, Tannerella, and Desulfovibrio species and unknown taxon SHD-231. At 2weeks posttreatment, we observed an almost complete turnover in the bacterial genera (16S rRNA) and species (shotgun metagenomics) correlated with MPD. Among the metabolites detected, the medians of the 20 most abundant metabolites were significantly correlated with MPD pre- and posttreatment. Finally, tests of periodontal biofilm community instability found markedly higher taxonomic instability in patients who did not improve posttreatment than in patients who did improve (UniFrac distances; t = -3.59; P = 0.002). Interestingly, the opposite pattern occurred in the metabolic profiles (Bray-Curtis; t = 2.42; P = 0.02). Our results suggested that multi-omics approaches, and metabolomics analysis in particular, could enhance treatment prediction and reveal patients most likely to improve posttreatment. IMPORTANCE Periodontal disease affects the majority of adults worldwide and has been linked to numerous systemic diseases. Despite decades of research, the reasons for the substantial differences among periodontitis patients in disease incidence, progressivity, and response to treatment remain poorly understood. While deep sequencing of oral bacterial communities has greatly expanded our comprehension of the microbial diversity of periodontal disease and identified associations with healthy and disease states, predicting treatment outcomes remains elusive. Our results suggest that combining multiple omics approaches enhances the ability to differentiate among disease states and determine differential effects of treatment, particularly with the addition of metabolomic information. Furthermore, multi-omics analysis of biofilm community instability indicated that these approaches provide new tools for investigating the ecological dynamics underlying the progressive periodontal disease process.

Members of the Oral Microbiota Are Associated with IL-8 Release by Gingival Epithelial Cells in Healthy Individuals.

The triggers for the onset of oral diseases are still poorly understood. The aim of this study was to characterize the oral bacterial community in healthy humans and its association with nutrition, oral hygiene habits, and the release of the inflammatory marker IL-8 from gingival epithelial cells (GECs) with and without stimulation by bacterial endotoxins to identify possible indicator operational taxonomic units (OTUs) associated with inflammatory marker status. GECs from 21 healthy participants (13 females, 8 males) were incubated with or without addition of bacterial lipopolysaccharides (LPSs), and the oral microbiota was profiled using 16S rRNA gene-targeted sequencing. The basal IL-8 release after 6 h was between 9.9 and 98.2 pg/ml, and bacterial communities were characteristic for healthy oral microbiota. The composition of the oral microbiota was associated with basal IL-8 levels, the intake of meat, tea, white wine, sweets and the use of chewing gum, as well as flossing habits, allergies, gender and body mass index. Additionally, eight OTUs were associated with high basal levels of IL-8 and GEC response to LPS, with high basal levels of IL-8, and 1 with low basal levels of IL8. The identification of indicator bacteria in healthy subjects with high levels of IL-8 release is of importance as they may be promising early warning indicators for the possible onset of oral diseases.

Neanderthal behaviour, diet, and disease inferred from ancient DNA in dental calculus.

Recent genomic data have revealed multiple interactions between Neanderthals and modern humans, but there is currently little genetic evidence regarding Neanderthal behaviour, diet, or disease. Here we describe the shotgun-sequencing of ancient DNA from five specimens of Neanderthal calcified dental plaque (calculus) and the characterization of regional differences in Neanderthal ecology. At Spy cave, Belgium, Neanderthal diet was heavily meat based and included woolly rhinoceros and wild sheep (mouflon), characteristic of a steppe environment. In contrast, no meat was detected in the diet of Neanderthals from El Sidrón cave, Spain, and dietary components of mushrooms, pine nuts, and moss reflected forest gathering. Differences in diet were also linked to an overall shift in the oral bacterial community (microbiota) and suggested that meat consumption contributed to substantial variation within Neanderthal microbiota. Evidence for self-medication was detected in an El Sidrón Neanderthal with a dental abscess and a chronic gastrointestinal pathogen (Enterocytozoon bieneusi). Metagenomic data from this individual also contained a nearly complete genome of the archaeal commensal Methanobrevibacter oralis (10.2× depth of coverage)-the oldest draft microbial genome generated to date, at around 48,000 years old. DNA preserved within dental calculus represents a notable source of information about the behaviour and health of ancient hominin specimens, as well as a unique system that is useful for the study of long-term microbial evolution.

Comparative pan genome analysis of oral Prevotella species implicated in periodontitis.

Prevotella is part of the oral bacterial community implicated in periodontitis. Pan genome analyses of eight oral Prevotella species, P. dentalis, P. enoeca, P. fusca, P. melaninogenica, P. denticola, P. intermedia 17, P. intermedia 17-2 and P. sp. oral taxon 299 are presented in this study. Analysis of the Prevotella pan genome revealed features such as secretion systems, resistance to oxidative stress and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems that enable the bacteria to adapt to the oral environment. We identified the presence of type VI secretion system (T6SS) in P. fusca and P. intermedia strains. For some VgrG and Hcp proteins which were not part of the core T6SS loci, we used gene neighborhood analysis and identified putative effector proteins and putative polyimmunity loci in P. fusca and polymorphic toxin systems in P. intermedia strains. Earlier studies have identified the presence of Por secretion system (PorSS) in P. gingivalis, P. melaninogenica and P. intermedia. We noted the presence of their homologs in six other oral Prevotella studied here. We suggest that in Prevotella, PorSS is used to secrete cysteine proteases such as interpain and C-terminal domain containing proteins with a "Por_secre_tail" domain. We identified subtype I-B CRISPR-Cas system in P. enoeca. Putative CRISPR-Cas system subtypes for 37 oral Prevotella and 30 non-oral Prevotella species were also predicted. Further, we performed a BLASTp search of the Prevotella proteins which are also conserved in the red-complex pathogens, against the human proteome to identify potential broad-spectrum drug targets. In summary, the use of a pan genome approach enabled identification of secretion systems and defense mechanisms in Prevotella that confer adaptation to the oral cavity.

Oral and nasal microbiota in Parkinson's disease

IntroductionParkinson's disease (PD) is associated with neuropathological changes in olfactory and gastrointestinal tissues, and PD patients frequently suffer from hyposmia, hyposalivation, and dysphagia. Since hyposmia and gastrointestinal dysfunction are frequently premotor symptoms, it has been speculated that an external, for example microbial, agent could trigger the pathologic process in the corresponding organs, subsequently spreading to the central nervous system. We recently showed evidence for compositional differences between the fecal microbiota of PD patients and control subjects. In this study, our objective was to explore a possible connection between nasal and oral microbiota and PD. MethodsWe compared the oral and nasal bacterial communities of PD patients (oral: n = 72, nasal: n = 69) and control subjects (oral: n = 76, nasal: n = 67) using a 16S rRNA gene amplicon sequencing approach. ResultsOral and nasal microbiota differed markedly from each other, with no notable similarity within subjects. Oral microbiota of PD patients and control subjects had differences in beta diversity and abundances of individual bacterial taxa. An increase in the abundance of opportunistic oral pathogens was detected in males, both with and without PD. Our data did not reveal convincing differences between the nasal microbiota of control subjects and PD patients. ConclusionThe oral microbiome deserves additional research regarding its connection to PD and its biomarker potential. The higher abundance of oral pathogens in men underlines the importance of monitoring and promoting male dental health.

Evaluating the Impact of DNA Extraction Method on the Representation of Human Oral Bacterial and Fungal Communities.

The application of high-throughput, next-generation sequencing technologies has greatly improved our understanding of the human oral microbiome. While deciphering this diverse microbial community using such approaches is more accurate than traditional culture-based methods, experimental bias introduced during critical steps such as DNA extraction may compromise the results obtained. Here, we systematically evaluate four commonly used microbial DNA extraction methods (MoBio PowerSoil® DNA Isolation Kit, QIAamp® DNA Mini Kit, Zymo Bacterial/Fungal DNA Mini PrepTM, phenol:chloroform-based DNA isolation) based on the following criteria: DNA quality and yield, and microbial community structure based on Illumina amplicon sequencing of the V3–V4 region of the 16S rRNA gene of bacteria and the internal transcribed spacer (ITS) 1 region of fungi. Our results indicate that DNA quality and yield varied significantly with DNA extraction method. Representation of bacterial genera in plaque and saliva samples did not significantly differ across DNA extraction methods and DNA extraction method showed no effect on the recovery of fungal genera from plaque. By contrast, fungal diversity from saliva was affected by DNA extraction method, suggesting that not all protocols are suitable to study the salivary mycobiome.

The Oral Microbiota in Health and Disease: An Overview of Molecular Findings.

Culture-independent nucleic acid technologies have been extensively applied to the analysis of oral bacterial communities associated with healthy and diseased conditions. These methods have confirmed and substantially expanded the findings from culture studies to reveal the oral microbial inhabitants and candidate pathogens associated with the major oral diseases. Over 1000 bacterial distinct species-level taxa have been identified in the oral cavity and studies using next-generation DNA sequencing approaches indicate that the breadth of bacterial diversity may be even much larger. Nucleic acid technologies have also been helpful in profiling bacterial communities and identifying disease-related patterns. This chapter provides an overview of the diversity and taxonomy of oral bacteria associated with health and disease.

Human neutrophils and oral microbiota: a constant tug-of-war between a harmonious and a discordant coexistence.

Neutrophils are a major component of the innate host response, and the outcome of the interaction between the oral microbiota and neutrophils is a key determinant of oral health status. The composition of the oral microbiome is very complex and different in health and disease. Neutrophils are constantly recruited to the oral cavity, and their protective role is highlighted in cases where their number or functional responses are impeded, resulting in different forms of periodontal disease. Periodontitis, one of the more severe and irreversible forms of periodontal disease, is a microbial-induced chronic inflammatory disease that affects the gingival tissues supporting the tooth. This chronic inflammatory disease is the result of a shift of the oral bacterial symbiotic community to a dysbiotic more complex community. Chronic inflammatory infectious diseases such as periodontitis can occur because the pathogens are able to evade or disable the innate immune system. In this review, we discuss how human neutrophils interact with both the symbiotic and the dysbiotic oral community; an understanding of which is essential to increase our knowledge of the periodontal disease process.