Full-length 16S rRNA gene sequencing by PacBio improves taxonomic resolution in human microbiome samples.

BACKGROUND: Sequencing variable regions of the 16S rRNA gene (≃300 bp) with Illumina technology is commonly used to study the composition of human microbiota. Unfortunately, short reads are unable to differentiate between highly similar species. Considering that species from the same genus can be associated with health or disease it is important to identify them at the lowest possible taxonomic rank. Third-generation sequencing platforms such as PacBio SMRT, increase read lengths allowing to sequence the whole gene with the maximum taxonomic resolution. Despite its potential, full length 16S rRNA gene sequencing is not widely used yet. The aim of the current study was to compare the sequencing output and taxonomic annotation performance of the two approaches (Illumina short read sequencing and PacBio long read sequencing of 16S rRNA gene) in different human microbiome samples. DNA from saliva, oral biofilms (subgingival plaque) and faeces of 9 volunteers was isolated. Regions V3-V4 and V1-V9 were amplified and sequenced by Illumina Miseq and by PacBio Sequel II sequencers, respectively. RESULTS: With both platforms, a similar percentage of reads was assigned to the genus level (94.79% and 95.06% respectively) but with PacBio a higher proportion of reads were further assigned to the species level (55.23% vs 74.14%). Regarding overall bacterial composition, samples clustered by niche and not by sequencing platform. In addition, all genera with > 0.1% abundance were detected in both platforms for all types of samples. Although some genera such as Streptococcus tended to be observed at higher frequency in PacBio than in Illumina (20.14% vs 14.12% in saliva, 10.63% vs 6.59% in subgingival plaque biofilm samples) none of the differences were statistically significant when correcting for multiple testing. CONCLUSIONS: The results presented in the current manuscript suggest that samples sequenced using Illumina and PacBio are mostly comparable. Considering that PacBio reads were assigned at the species level with higher accuracy than Illumina, our data support the use of PacBio technology for future microbiome studies, although a higher cost is currently required to obtain an equivalent number of reads per sample.

Gut microbiota and its relationship with early vascular ageing in a Spanish population (MIVAS study).

BACKGROUND: Gut microbiota and its by-products are increasingly recognized as having a decisive role in cardiovascular diseases. The aim is to study the relationship between gut microbiota and early vascular ageing (EVA). METHODS: A cross-sectional study was developed in Salamanca (Spain) in which 180 subjects aged 45-74 years were recruited. EVA was defined by the presence of at least one of the following: carotid-femoral pulse wave velocity (cf-PWV), cardio-ankle vascular index (CAVI) or brachial-ankle pulse wave velocity (ba-PWV) above the 90th percentile of the reference population. All other cases were considered normal vascular ageing (NVA). MEASUREMENTS: cf-PWV was measured by SphygmoCor® System; CAVI and ba-PWV were determined by Vasera 2000® device. Gut microbiome composition in faecal samples was determined by 16S rRNA Illumina sequencing. RESULTS: Mean age was 64.4 ± 6.9 in EVA group and 60.4 ± 7.6 years in NVA (p < .01). Women in EVA group were 41% and 53% in NVA. There were no differences in the overall composition of gut microbiota between the two groups when evaluating Firmicutes/Bacteriodetes ratio, alfa diversity (Shannon Index) and beta diversity (Bray-Curtis). Bilophila, Faecalibacterium sp.UBA1819 and Phocea, are increased in EVA group. While Cedecea, Lactococcus, Pseudomonas, Succiniclasticum and Dielma exist in lower abundance. In logistic regression analysis, Bilophila (OR: 1.71, 95% CI: 1.12-2.6, p = .013) remained significant. CONCLUSIONS: In the studied Spanish population, early vascular ageing is positively associated with gut microbiota abundance of the genus Bilophila. No relationship was found between phyla abundance and measures of diversity.

Streptococcus dentisani inhibits the growth of Candida albicans and Candida glabrata: in vitro assay.

BACKGROUND: Probiotic bacteria inhibit aggregation, biofilm formation, and dimorphism of Candida spp. However, the effects of a new probiotic, Streptococcus dentisani, on the growth of Candida albicans and Candida glabrata biofilms are unknown. OBJECTIVE: To determine the effect of S. dentisani on the different phases of C. albicans and C. glabrata biofilm development. METHODS: Growth quantification and ultrastructural analyses were performed on biofilms of C. albicans ATCC 90028, C. glabrata ATCC 2001, and clinical isolates of C. albicans from oral candidiasis (CA-C1), caries (CA-CR1), and periodontal pocket (CA-P1) treated with cell suspensions of S. dentisani CECT 7746. Cell viability was determined by quantifying colony-forming units (CFU/mL). The ultrastructural analyses were done with atomic force microscopy. RESULTS: S. dentisani induced a significant reduction (p < 0.05) of CFU/mL of immature and mature biofilm in all strains of C. albicans and C. glabrata. Microscopic analysis revealed that S. dentisani reduced C. albicans density in mixed biofilm. The fungus-bacteria interaction affected cell membrane integrity in yeast. CONCLUSION: For the first time, our data elucidate the antifungal effect of S. dentisani on the development of C. albicans and C. glabrata biofilms, supporting its usefulness as a niche-specific probiotic to prevent and treat oral dysbiosis.

Salivary proteins and metabolites as Caries Biomarkers in adolescents.

INTRODUCTION: The identification of salivary molecules that can be associated to dental caries could provide insights about caries risk and offer valuable information to develop caries prediction models. However, the search for a universal caries biomarker has proven elusive due to the multifactorial nature of this oral disease. We have therefore performed a systematic effort to identify caries-associated metabolites and proteins in saliva samples from adolescents that had a caries experience and those that were caries-free. METHODS: Quantification of approximately 100 molecules was performed by the use of a wide range of techniques, ranging from NMR metabolomics to ELISA, Luminex or colorimetric assays, as well as clinical features like plaque accumulation and gingival index. In addition, simplified dietary and oral hygiene habits questionnaires were also obtained. RESULTS: The caries-free group had significantly lower consumption of sweetened beverages and higher toothbrushing frequency. Surprisingly, very few compounds were found to individually provide discriminatory power between Caries-experienced and Caries-Free individuals. The data analysis revealed several potential reasons that could underly this lack of association value with caries, including differences in metabolite concentrations throughout the day, a lack of correlation between metabolite concentrations in plaque and saliva, or sex-related differences, among others. However, when multiple compounds were combined by multivariate analysis and random forest modelling, a combination of 3-5 compounds were found to provide good prediction models for morning (with an AUC accuracy of 0.87) and especially afternoon samples (AUC=0.93). CONCLUSION: While few salivary biomarker could differentiate between caries-free and caries-experienced adolescents, a combination of markers proved effective, particularcly in afternoon samples. To predict caries risk, these biomarkers should be validated in larger cohorts and longitudinal settings, considering factors such as gender differences, and variations in oral hygiene and diet.

Dysimmunity in common variable immunodeficiency is associated with alterations in oral, respiratory, and intestinal microbiota.

Common variable immunodeficiency (CVID) is the most common symptomatic primary immunodeficiency characterized by decreased immunoglobulins and recurrent infections. Its aetiology remains unknown, and some patients present with severe non-infectious autoimmune or inflammatory complications with elevated associated morbimortality. Recently, intestinal dysbiosis has been proposed as a driver of immune dysregulation. In this study, we assessed the oral, respiratory, and gastrointestinal microbiota of 41 CVID patients (24 with dysimmune and 17 with infection complications) and 15 healthy volunteers using 16S rRNA gene sequencing to explore associations between microbiome profiles and CVID phenotypes. Profound differences in the composition of the microbiota in saliva, sputum, and stool were detected between dysimmune CVID patients and healthy individuals. Globally, respiratory species diversity and faecal bacterial richness were lower in CVID individuals with immune complications. Although a single species could not be identified as a robust predictor of dysimmunity, a combination of around 5-7 bacterial species in each type of sample could predict this severe phenotype with an accuracy of around 90% in the study population. Our study provides new insights into these previously unexplored but highly interrelated ecological niches among themselves and with patient profiles. Our data suggest that this disease-related systemic dysbiosis could be implicated in the immune dysregulation associated with severe cases of CVID.

Functional mgrA Influences Genetic Changes within a Staphylococcus aureus Cell Population over Time.

Prolonged survival in the host-bacteria microenvironment drives the selection of alternative cell types in Staphylococcus aureus, permitting quasi-dormant sub-populations to develop. These facilitate antibiotic tolerance, long-term growth, and relapse of infection. Small Colony Variants (SCV) are an important cell type associated with persistent infection but are difficult to study in vitro due to the instability of the phenotype and reversion to the normal cell type. We have previously reported that under conditions of growth in continuous culture over a prolonged culture time, SCVs dominated a heterogenous population of cell types and these SCVs harbored a mutation in the DNA binding domain of the gene for the transcription factor, mgrA. To investigate this specific cell type further, S. aureus WCH-SK2-ΔmgrA itself was assessed with continuous culture. Compared to the wild type, the mgrA mutant strain required fewer generations to select for SCVs. There was an increased rate of mutagenesis within the ΔmgrA strain compared to the wild type, which we postulate is the mechanism explaining the increased emergence of SCV selection. The mgrA derived SCVs had impeded metabolism, altered MIC to specific antibiotics and an increased biofilm formation compared to non-SCV strain. Whole genomic sequencing detected single nucleotide polymorphisms (SNP) in phosphoglucosamine mutase glmM and tyrosine recombinase xerC. In addition, several genomic rearrangements were detected which affected genes involved in important functions such as antibiotic and toxic metal resistance and pathogenicity. Thus, we propose a direct link between mgrA and the SCV phenotype. IMPORTANCE Within a bacterial population, a stochastically generated heterogeneity of phenotypes allows continual survival against current and future stressors. The generation of a sub-population of quasi-dormant Small Colony Variants (SCV) in Staphylococcus aureus is such a mechanism, allowing for persistent or relapse of infection despite initial intervention seemingly clearing the infection. The use of continuous culture under clinically relevant conditions has allowed us to introduce time to the growth system and selects SCV within the population. This study provides valuable insights into the generation of SCV which are not addressed in standard laboratory generated models and reveals new pathways for understanding persistent S. aureus infection which can potentially be targeted in future treatments of persistent S. aureus infection.

Ulcerative Colitis Seems to Imply Oral Microbiome Dysbiosis.

Ulcerative colitis (UC) is a recurrent pathology of complex etiology that has been occasionally associated with oral lesions, but the overall composition of the oral microbiome in UC patients and its role in the pathogenesis of the disease are still poorly understood. In this study, the oral microbiome of UC patients and healthy individuals was compared to ascertain the possible changes in the oral microbial communities associated with UC. For this, the salivary microbiota of 10 patients diagnosed with an active phase of UC and 11 healthy controls was analyzed by 16S rRNA gene sequencing (trial ref. ISRCTN39987). Metataxonomic analysis revealed a decrease in the alpha diversity and an imbalance in the relative proportions of some key members of the oral core microbiome in UC patients. Additionally, Staphylococcus members and four differential species or phylotypes were only present in UC patients, not being detected in healthy subjects. This study provides a global snapshot of the existence of oral dysbiosis associated with UC, and the possible presence of potential oral biomarkers.

Streptococcus dentisani is a common inhabitant of the oral microbiota worldwide and is found at higher levels in caries-free individuals.

Streptococcus dentisani has been proposed as a promising probiotic against tooth decay, due to its ability to buffer acidic pH and to inhibit the growth of oral pathogens. However, it is unknown if this bacterial species has a global distribution. The current study aimed to establish the presence of S. dentisani in oral samples from different geographic locations by identifying the sequence of its 16S rRNA gene in available datasets from across the globe. In addition, an analytical and cross-sectional study was carried out to determine if the levels of this probiotic strain are higher in caries-free individuals compared to those with dental caries. Samples from various geographical sources demonstrated that S. dentisani is present in saliva and dental plaque from individuals of different continents. Typical S. dentisani levels in saliva ranged from 104 to 105 cells/ml and a total of 106-107 cells in dental plaque. Using real-time qPCR, S. dentisani was quantified from supragingival dental plaque of 25 caries-free and 29 caries-active individuals from a Mexican children population, where significantly higher proportions of S. dentisani were found in the caries-free group (p = 0.002). Finally, a negative correlation was found between caries levels (as measured by the dmft caries index) and the percentage of S. dentisani (p < 0.001). Thus, the current manuscript indicates that this species has a global distribution, can be found in saliva and dental plaque, and appears to be present in higher numbers in plaque samples from caries-free children.

Allergy development is associated with consumption of breastmilk with a reduced microbial richness in the first month of life.

BACKGROUND: Early colonization with a diverse microbiota seems to play a crucial role for appropriate immune maturation during childhood. Breastmilk microbiota is one important source of microbes for the infant, transferred together with maternal IgA antibodies. We previously observed that allergy development during childhood was associated with aberrant IgA responses to the gut microbiota already at 1 month of age, when the IgA antibodies are predominantly maternally derived in breastfed infants. OBJECTIVE: To determine the microbial composition and IgA-coated bacteria in breastmilk in relation to allergy development in children participating in an intervention trial with pre- and post-natal Lactobacillus reuteri supplementation. METHODS: A combination of flow cytometric cell sorting and 16S rRNA gene sequencing was used to characterize the bacterial recognition patterns by IgA in breastmilk samples collected one month post-partum from 40 mothers whose children did or did not develop allergic and asthmatic symptoms during the first 7 years of age. RESULTS: The milk fed to children developing allergic manifestations had significantly lower bacterial richness, when compared to the milk given to children that remained healthy. Probiotic treatment influenced the breastmilk microbiota composition. However, the proportions of IgA-coated bacteria, the total bacterial load and the patterns of IgA-coating were similar in breastmilk between mothers of healthy children and those developing allergies. CONCLUSION: Consumption of breastmilk with a reduced microbial richness in the first month of life may play an important role in allergy development during childhood.

A pilot study to assess oral colonization and pH buffering by the probiotic Streptococcus dentisani under different dosing regimes.

Bacterial colonization in the oral cavity is critical for efficient action of probiotics. However, limited colonization rates have been reported in many clinical trials. The aim of this pilot clinical study was to evaluate the colonization efficiency of Streptococcus dentisani under different dosing schedules and pre-treatment conditions. Eleven adult volunteers enrolled in the study. A professional ultrasound cleaning was performed in quadrants 1 and 4. The probiotic was applied in all four quadrants at a total dose of 1010 CFUs, administered in a buccoadhesive gel for 5 min, either in a single dose (n = 5) or daily for a week (n = 6). Dental plaque and saliva samples were collected at baseline and after 14 and 28 days of first application. Amounts of S. dentisani and the cariogenic organism Streptococcus mutans were measured by qPCR and salivary pH was measured by reflectometry. There was a significant increase in S. dentisani cells at day 14 but not at day 28 under both dosing schedules. A non-significant higher colonization was found in the half-mouth with previous professional cleaning as compared to the intact half. There was a significant increase in salivary pH at day 14 (p = 0.024) and day 28 (p = 0.014), which was stronger in multi-dose patients, and a significant decrease in S. mutans at day 28 (p < 0.01). The results indicate that S. dentisani is transiently able to colonize the oral cavity and that it buffers oral pH, especially after multiple dosing. Future randomized, placebo-controlled clinical trials should evaluate its use to prevent tooth decay.

Mycobiome Profiles in Breast Milk from Healthy Women Depend on Mode of Delivery, Geographic Location, and Interaction with Bacteria.

Recent studies report the presence of fungal species in breast milk of healthy mothers, suggesting a potential role in infant mycobiome development. In the present work, we aimed to determine whether the healthy human breast milk mycobiota is influenced by geographical location and mode of delivery, as well as to investigate its interaction with bacterial profiles in the same samples. A total of 80 mature breast milk samples from 4 different countries were analyzed by Illumina sequencing of the internal transcribed spacer 1 (ITS1) region, joining the 18S and 5.8S regions of the fungal rRNA region. Basidiomycota and Ascomycota were found to be the dominant phyla, with Malassezia and Davidiella being the most prevalent genera across countries. A core formed by Malassezia, Davidiella, Sistotrema, and Penicillium was shared in the milk samples from the different origins, although specific shifts in mycobiome composition were associated with geographic location and delivery mode. The presence of fungi in the breast milk samples was further confirmed by culture and isolate characterization, and fungal loads were estimated by quantitative PCR (qPCR) targeting the fungal ITS1 region. Cooccurrence network analysis of bacteria and fungi showed complex interactions that were influenced by geographical location, mode of delivery, maternal age, and pregestational body mass index. The presence of a breast milk mycobiome was confirmed in all samples analyzed, regardless of the geographic origin.IMPORTANCE During recent years, human breast milk has been documented as a potential source of bacteria for the newborn. Recently, we have reported the presence of fungi in breast milk from healthy mothers. It is well known that environmental and perinatal factors can affect milk bacteria; however, the impact on milk fungi is still unknown. The current report describes fungal communities (mycobiota) in breast milk samples across different geographic locations and the influence of the mode of delivery. We also provide novel insights on bacterium-fungus interactions, taking into account environmental and perinatal factors. We identified a core of four genera shared across locations, consisting of Malassezia, Davidiella, Sistotrema, and Penicillium, which have been reported to be present in the infant gut. Our data confirm the presence of fungi in breast milk across continents and support the potential role of breast milk in the initial seeding of fungal species in the infant gut.

Sputum Microbiome Dynamics in Chronic Obstructive Pulmonary Disease Patients during an Exacerbation Event and Post-Stabilization.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) affects up to 65 million people worldwide, and COPD exacerbation causes tissue damage and subsequent loss of lung function. It is a multifactorial event in which respiratory infections are involved, but little is known about its dynamics. OBJECTIVES: The objective of our study was to determine the microbiome composition during an exacerbation event and post-stabilization. METHODS: We conducted an observational analytical study of a cohort of 55 COPD patients in which 2 sputum samples (the first taken during an exacerbation event and the second during clinical post-stabilization) were submitted to 16s RNA ribosomal analysis by Illumina Miseq Next Generation Sequencing (NGS). The presence of respiratory viruses was also determined. RESULTS: Our study found a stable microbiome composition in the post-stabilization sputum samples of COPD patients, and 4 additional microbiomes in samples taken during the exacerbation, 3 of which showed a marked dysbiosis by Haemophilus, Pseudomonas, and Serratia. The fourth exacerbation microbiome had a very similar composition to post-stabilization samples, but some pathogens such as Moraxella and respiratory viruses were also found. CONCLUSIONS: Our study reveals the main protagonists involved in lung microbiome dynamics during an exacerbation event and post-stabilization in COPD patients by NGS analysis.

Aberrant IgA responses to the gut microbiota during infancy precede asthma and allergy development.

BACKGROUND: Although a reduced gut microbiota diversity and low mucosal total IgA levels in infancy have been associated with allergy development, IgA responses to the gut microbiota have not yet been studied. OBJECTIVE: We sought to determine the proportions of IgA coating together with the characterization of the dominant bacteria, bound to IgA or not, in infant stool samples in relation to allergy development. METHODS: A combination of flow cytometric cell sorting and deep sequencing of the 16S rDNA gene was used to characterize the bacterial recognition patterns by IgA in stool samples collected at 1 and 12 months of age from children staying healthy or having allergic symptoms up to 7 years of age. RESULTS: The children with allergic manifestations, particularly asthma, during childhood had a lower proportion of IgA bound to fecal bacteria at 12 months of age compared with healthy children. These alterations cannot be attributed to differences in IgA levels or bacterial load between the 2 groups. Moreover, the bacterial targets of early IgA responses (including coating of the Bacteroides genus), as well as IgA recognition patterns, differed between healthy children and children with allergic manifestations. Altered IgA recognition patterns in children with allergy were observed already at 1 month of age, when the IgA antibodies are predominantly maternally derived in breast-fed children. CONCLUSION: An aberrant IgA responsiveness to the gut microbiota during infancy precedes asthma and allergy development, possibly indicating an impaired mucosal barrier function in allergic children.

Role of microbial communities in the pathogenesis of periodontal diseases and caries.

The microbiological characteristics of both caries and periodontal disease show significant change from those in health. In both instances, there is evidence of co-association of different organisms into consortia. AIM: We review and summarize a number of issues pertinent to the community organization and functional activity of the bacterial populations resident on supra- and subgingival tooth surface and the influence of these populations on disease. METHODS: A literature review was undertaken with a particular emphasis on recent publications involving high-throughput, deep sequencing approaches to the analysis of microbial populations and their functional activity. RESULTS: There is increasing evidence to suggest that both caries and periodontal disease represent dysbiotic states of the oral microbiome. The mode of acquisition of the oral microbial communities may be less passive than previously recognized but once established remains relatively stable within an individual although there are very significant site variations. A repertoire of stable dysbiotic states may occur in both caries and periodontitis involving different microbial community structures with potentially similar functional properties. CONCLUSIONS: The processes which underlie the development and stability of microbial populations in the healthy mouth are fundamental to understanding how these populations are transformed into a dysbiotic state in disease.

Role of microbial biofilms in the maintenance of oral health and in the development of dental caries and periodontal diseases. Consensus report of group 1 of the Joint EFP/ORCA workshop on the boundaries between caries and periodontal disease.

BACKGROUND AND AIMS: The scope of this working group was to review (1) ecological interactions at the dental biofilm in health and disease, (2) the role of microbial communities in the pathogenesis of periodontitis and caries, and (3) the innate host response in caries and periodontal diseases. RESULTS AND CONCLUSIONS: A health-associated biofilm includes genera such as Neisseria, Streptococcus, Actinomyces, Veillonella and Granulicatella. Microorganisms associated with both caries and periodontal diseases are metabolically highly specialized and organized as multispecies microbial biofilms. Progression of these diseases involves multiple microbial interactions driven by different stressors. In caries, the exposure of dental biofilms to dietary sugars and their fermentation to organic acids results in increasing proportions of acidogenic and aciduric species. In gingivitis, plaque accumulation at the gingival margin leads to inflammation and increasing proportions of proteolytic and often obligately anaerobic species. The natural mucosal barriers and saliva are the main innate defence mechanisms against soft tissue bacterial invasion. Similarly, enamel and dentin are important hard tissue barriers to the caries process. Given that the present state of knowledge suggests that the aetiologies of caries and periodontal diseases are mutually independent, the elements of innate immunity that appear to contribute to resistance to both are somewhat coincidental.

The human oral metaproteome reveals potential biomarkers for caries disease.

Tooth decay is considered the most prevalent human disease worldwide. We present the first metaproteomic study of the oral biofilm, using different mass spectrometry approaches that have allowed us to quantify individual peptides in healthy and caries-bearing individuals. A total of 7771 bacterial and 853 human proteins were identified in 17 individuals, which provide the first available protein repertoire of human dental plaque. Actinomyces and Coryneybacterium represent a large proportion of the protein activity followed by Rothia and Streptococcus. Those four genera account for 60-90% of total diversity. Healthy individuals appeared to have significantly higher amounts of L-lactate dehydrogenase and the arginine deiminase system, both implicated in pH buffering. Other proteins found to be at significantly higher levels in healthy individuals were involved in exopolysaccharide synthesis, iron metabolism and immune response. We applied multivariate analysis in order to find the minimum set of proteins that better allows discrimination of healthy and caries-affected dental plaque samples, detecting seven bacterial and five human protein functions that allow determining the health status of the studied individuals with an estimated specificity and sensitivity over 96%. We propose that future validation of these potential biomarkers in larger sample size studies may serve to develop diagnostic tests of caries risk that could be used in tooth decay prevention.

Revealing microbial recognition by specific antibodies.

BACKGROUND: Recognition of microorganisms by antibodies is a vital component of the human immune response. However, there is currently very limited understanding of immune recognition of 50 % of the human microbiome which is made up of as yet un-culturable bacteria. We have combined the use of flow cytometry and pyrosequencing to describe the microbial composition of human samples, and its interaction with the immune system. RESULTS: We show the power of the technique in human faecal, saliva, oral biofilm and breast milk samples, labeled with fluorescent anti-IgG or anti-IgA antibodies. Using Fluorescence-Activated Cell Sorting (FACS), bacterial cells were separated depending on whether they are coated with IgA or IgG antibodies. Each bacterial population was PCR-amplified and pyrosequenced, characterizing the microorganisms which evade the immune system and those which were recognized by each immunoglobulin. CONCLUSIONS: The application of the technique to healthy and diseased individuals may unravel the contribution of the immune response to microbial infections and polymicrobial diseases.

Microbiota diversity and gene expression dynamics in human oral biofilms.

BACKGROUND: Micro-organisms inhabiting teeth surfaces grow on biofilms where a specific and complex succession of bacteria has been described by co-aggregation tests and DNA-based studies. Although the composition of oral biofilms is well established, the active portion of the bacterial community and the patterns of gene expression in vivo have not been studied. RESULTS: Using RNA-sequencing technologies, we present the first metatranscriptomic study of human dental plaque, performed by two different approaches: (1) A short-reads, high-coverage approach by Illumina sequencing to characterize the gene activity repertoire of the microbial community during biofilm development; (2) A long-reads, lower-coverage approach by pyrosequencing to determine the taxonomic identity of the active microbiome before and after a meal ingestion. The high-coverage approach allowed us to analyze over 398 million reads, revealing that microbial communities are individual-specific and no bacterial species was detected as key player at any time during biofilm formation. We could identify some gene expression patterns characteristic for early and mature oral biofilms. The transcriptomic profile of several adhesion genes was confirmed through qPCR by measuring expression of fimbriae-associated genes. In addition to the specific set of gene functions overexpressed in early and mature oral biofilms, as detected through the short-reads dataset, the long-reads approach detected specific changes when comparing the metatranscriptome of the same individual before and after a meal, which can narrow down the list of organisms responsible for acid production and therefore potentially involved in dental caries. CONCLUSIONS: The bacteria changing activity during biofilm formation and after meal ingestion were person-specific. Interestingly, some individuals showed extreme homeostasis with virtually no changes in the active bacterial population after food ingestion, suggesting the presence of a microbial community which could be associated to dental health.

Streptococcus dentisani sp. nov., a novel member of the mitis group.

Genomic, taxonomic and biochemical studies were performed on two strains of α-haemolytic streptococci that showed them to be clustered with major members of the Streptococcus mitis group. These Gram-stain-positive strains were isolated from tooth surfaces of caries-free humans and showed the classical spherical shape of streptococcal species growing in chains. Sequence analysis from concatenated 16S and 23S rRNA gene and sodA genes showed that these strains belonged to the mitis group, but both of them clustered into a new phylogenetic branch. The genomes of these two isolates were sequenced, and whole-genome average nucleotide identity (ANI) demonstrated that these strains significantly differed from any streptococcal species, showing ANI values under 91 % even when compared with the phylogenetically closest species such as Streptococcus oralis and S. mitis. Biochemically, the two isolates also showed distinct metabolic features relative to closely related species, like α-galactosidase activity. From the results of the present study, the name Streptococcus dentisani sp. nov. is proposed to accommodate these novel strains, which have been deposited in open collections at the Spanish type Culture Collection (CECT) and Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSMZ), being respectively identified as Streptococcus dentisani Str. 7746 ( = CECT 8313 = DSM 27089) and Streptococcus dentisani Str. 7747(T) ( = CECT 8312(T) = DSM 27088(T)).

Nitrate reduction capacity of the oral microbiota is impaired in periodontitis: potential implications for systemic nitric oxide availability.

The reduction of nitrate to nitrite by the oral microbiota has been proposed to be important for oral health and results in nitric oxide formation that can improve cardiometabolic conditions. Studies of bacterial composition in subgingival plaque suggest that nitrate-reducing bacteria are associated with periodontal health, but the impact of periodontitis on nitrate-reducing capacity (NRC) and, therefore, nitric oxide availability has not been evaluated. The current study aimed to evaluate how periodontitis affects the NRC of the oral microbiota. First, 16S rRNA sequencing data from five different countries were analyzed, revealing that nitrate-reducing bacteria were significantly lower in subgingival plaque of periodontitis patients compared with healthy individuals (P < 0.05 in all five datasets with n = 20-82 samples per dataset). Secondly, subgingival plaque, saliva, and plasma samples were obtained from 42 periodontitis patients before and after periodontal treatment. The oral NRC was determined in vitro by incubating saliva with 8 mmol/L nitrate (a concentration found in saliva after nitrate-rich vegetable intake) and compared with the NRC of 15 healthy individuals. Salivary NRC was found to be diminished in periodontal patients before treatment (P < 0.05) but recovered to healthy levels 90 days post-treatment. Additionally, the subgingival levels of nitrate-reducing bacteria increased after treatment and correlated negatively with periodontitis-associated bacteria (P < 0.01). No significant effect of periodontal treatment on the baseline saliva and plasma nitrate and nitrite levels was found, indicating that differences in the NRC may only be revealed after nitrate intake. Our results suggest that an impaired NRC in periodontitis could limit dietary nitrate-derived nitric oxide levels, and the effect on systemic health should be explored in future studies.