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Abstract We have previously demonstrated that subgingival levels of nitrate-reducing bacteria, as well as the in vitro salivary nitrate reduction capacity (NRC), were diminished in periodontitis patients, increasing after periodontal treatment. However, it remains unclear if an impaired NRC in periodontitis can affect systemic health. To determine this, the effect of nitrate-rich beetroot juice (BRJ) on blood pressure was determined in 15 periodontitis patients before and 70 days after periodontal treatment (i.e., professional mechanical plaque removal, oral hygiene instruction, and subgingival instrumentation), as well as in a healthy control group of 15 individuals. Additionally, subgingival and tongue samples were taken to analyse the bacterial composition with Illumina sequencing of the 16S rRNA gene. In healthy individuals, the systolic and diastolic blood pressure (SBP and DPB) decreased significantly (both P

IntroductionDental caries is the most prevalent chronic infectious disease globally, with Streptococcus mutans recognized as a primary causative agent due to its acidogenicity and robust biofilm-forming ability. In S. mutans biofilm formation, the role of autoinducers has been extensively studied, while the influence of other small molecules remains largely unexplored. Mutanofactins, a class of polyketide/non-ribosomal lipopeptide secondary metabolites, are emerging as potential modulators of S. mutans biofilm development.MethodsTranscriptomic analysis was conducted to examine gene expression patterns in S. mutans NMT4863 across distinct growth phases and lifestyles, aiming to identify metabolic factors influencing biofilm formation. Transcriptomic profiles were compared between cells in early-, mid-, and late-exponential-, and stationary phase, as well as between planktonic and biofilm cells. Differentially expressed genes were identified, and pathway analyses revealed significant alterations in key metabolic and regulatory pathways. Specifically, the biosynthetic mutanofactin gene cluster was analyzed via quantitative real-time polymerase chain reaction.ResultsSeveral genes and operons were differentially expressed across the tested growth phases, with 1,095 genes showing differential expression between stationary-phase, planktonic and biofilm cells. Pathway analysis revealed significant changes in ascorbate metabolism, carbohydrate utilization and transport systems, lipoic acid metabolism, bacterial toxin pathways, two-component regulatory systems, and secondary metabolite biosynthesis. Notably, expression of the muf gene cluster, was elevated in early exponential-phase cells relative to stationary-phase cells. Additionally, the mufCDEFGHIJ genes were identified as components of a single transcriptional unit (muf operon). MufC, a transcriptional regulator of the TetR/AcrR-family, acts as a positive regulator of the muf operon in strain NMT4863. Bioinformatic analysis pinpointed a 20-bp regulatory sequence in the muf operon promoter region (5′-AAATGAGCTATAATTCATTT-3′). Interestingly, the muf operon was found to be significantly downregulated in biofilm cells.ConclusionThis study provides key insights into gene expression dynamics that drive biofilm formation in S. mutans NMT4863, with a particular emphasis on the role of the muf operon. This operon is governed by the TetR/AcrR-family regulator MufC and plays a central role in biofilm development, offering a novel perspective on the molecular basis of S. mutans biofilm formation and resilience.

Abstract In our opinion, the ‘hang-time’ of nitrate-containing products discussed in the letter by Green and Green is an interesting variable that should be considered when nitrate-based treatment or prevention strategies are designed. However, due to direct nitrate recycling after nitrate intake, products with a long ‘hang-time’ (e.g., chewing gum) may not always have an advantage compared to products with a short ‘hang-time’ (e.g., vegetable juices). We argue that extending the ‘hang-time’ is especially relevant and potentially beneficial for different applications, such as using a low nitrate dose to stimulate the oral effects, reaching oral tissues that may otherwise not be exposed to dietary nitrate (e.g., periodontal pockets), and providing a longer nitrate exposure in individuals with an impaired salivary flow. Apart from the ‘hang-time’, other important variables are the nitrate dose and source (e.g., different salts and vegetable extracts), as well as the desired effect (e.g., an oral effect versus systemic effects). Finally, we believe that the alterations in salivary microbiota observed before and after chewing three nitrate-rich gums over a period of ~5 h, as reported by Green and Green, could be considered beneficial. However, the oral microbiota composition is affected by the circadian rhythm and the effect of gum mastication should be evaluated. These results should thus be confirmed by a placebo-controlled study, where these confounding factors can be accounted for.

IntroductionDuring development of dental caries, oral biofilms undergo changes in microbial composition and phenotypical traits. The aim of this study was to compare the acid tolerance (AT) of plaque from two groups of children: one with severe caries (CA) and one with no caries experience (CF) and to correlate this to the microbial composition and metabolic profile of the biofilms.MethodsDental plaque samples from 20 children (2–5 years) in each group were studied. The AT was analyzed by viability assessment after exposure to an acid challenge (pH 3.5), using LIVE/DEAD® BacLight™ stain and confocal microscopy. Levels of acid tolerance (AT) were evaluated using a scoring system ranging from 1 (no/low AT), to 5 (high/all AT). Metabolic profiles were investigated following a 20 mM glucose pulse for one hour through Nuclear Magnetic Resonance (NMR). Microbial composition was characterized by 16S rRNA Illumina sequencing.ResultsThe mean AT score of the CA group (4.1) was significantly higher than that of the CF group (2.6, p

Oral and intestinal samples from a cohort of 93 colorectal cancer (CRC) patients and 30 healthy controls (non‐CRC) were collected for microbiome analysis. Saliva (28 non‐CRC and 94 CRC), feces (30 non‐CRC and 97 CRC), subgingival fluid (20 CRC), and tumor tissue samples (20 CRC) were used for 16S metabarcoding and/or RNA sequencing (RNAseq) approaches. A differential analysis of the abundance, performed with the ANCOM‐BC package, adjusting the P‐values by the Holm‐Bonferroni method, revealed that Parvimonas was significantly over‐represented in feces from CRC patients (P‐value

Abstract 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.

Abstract 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

Background and objectives: Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by decreased immunoglobulins and recurrent infections, with non-infectious complications such as granulomatous–lymphocytic interstitial lung disease (GLILD) affecting up to 30% of patients. Methods: Using high-throughput 16S rRNA gene sequencing, salivary, sputum, and fecal microbiome from CVID patients with GLILD, comparing them to CVID patients without GLILD—with immune dysregulation (dCVID) and only infections (iCVID)—and healthy controls was analyzed. Results: A total of 41 CVID patients, 7 with GLILD, and 15 healthy donors were included. Global fecal biodiversity was significantly lower in GLILD patients compared to CVID subgroups and controls. GLILD patients harbored different specific bacterial communities in all niches, with some keystone species common to dCVID. Conchiformibius, Micrococcales, and Capnocytophaga are more frequent in the sputum of GLILD patients. Saliva in GLILD shows higher frequencies of Conchiformibius and Haemophilusparainfluenzae. Fecal samples from GLILD patients have higher levels of Gemella morbilorum, Lacticaseibacillus, and Cellulosimicrobium. A non-assigned Conchiformibius spp. is consistently associated with GLILD across different niches and could be a potential pathobiont or relevant microbiological marker for GLILD. Cluster network and correlation analyses show profound dysbiosis in the sputum, saliva, and feces of GLILD patients. Conclusions: These findings highlight significant microbiome alterations in CVID patients with GLILD, particularly in the respiratory tract, suggesting a possible link to both local and systemic immune dysregulation.

Abstract A few studies indicate that nitrate can reduce dysbiosis from a periodontitis point of view. However, these experiments were performed on samples from healthy individuals, and it is unknown if nitrate will be effective in periodontal patients, where the presence of nitrate-reducing bacteria is clearly reduced. The aim of this study was to test the effect of nitrate and a nitrate-reducing R. aeria (Ra9) on subgingival biofilms of patients with periodontitis. For this, subgingival plaque was incubated with 5 mM nitrate for 7 h (n = 20) or 50 mM nitrate for 12 h (n = 10), achieving a ~50% of nitrate reduction in each case. Additionally, Ra9 was combined with 5 mM nitrate (n = 11), increasing the nitrate reduced and nitrite produced (both p 5 mM, both p

Candida species cause life-threatening infections with high morbidity and mortality rates and their resistance to conventional therapy is closely linked to biofilm formation. Thus, the development of new approaches to study Candida biofilms and the identification of novel therapeutic strategies could yield improved clinical outcomes. In the current study, we have set up an impedance-based in vitro system to study Candida spp. biofilms in real-time and to evaluate their sensitivity to two conventional antifungal groups used in clinical practice - azoles and echinocandins. Both fluconazole and voriconazole were unable to inhibit biofilm formation in most strains tested, while echinocandins showed biofilm inhibitory capacity at relatively low concentrations (starting from 0.625 mg/L). However, assays performed on 24 h Candida albicans and C. glabrata biofilms revealed that micafungin and caspofungin failed to eradicate mature biofilms at all tested concentrations, evidencing that once formed, Candida spp. biofilms are extremely difficult to eliminate using currently available antifungals. We then evaluated the antifungal and anti-biofilm effect of andrographolide, a natural compound isolated from the plant Andrographis paniculata with known antibiofilm activity on Gram-positive and Gram-negative bacteria. Optical density measures, impedance evaluation, CFU counts, and electron microscopy data showed that andrographolide strongly inhibits planktonic Candida spp. growth and halts Candida spp. biofilm formation in a dose-dependent manner in all tested strains. Moreover, andrographolide was capable of eliminating mature biofilms and viable cell numbers by up to 99.9% in the C. albicans and C. glabrata strains tested, suggesting its potential as a new approach to treat multi-resistant Candida spp. biofilm-related infections.

ABSTRACTBackground A growing body of evidence demonstrates a different bacterial composition in the oral cavity of patients with oral lichen planus (OLP).Patients and methods Buccal swab samples were collected from affected and non-affected sites of six patients with reticular OLP and the healthy oral mucosa of six control subjects. 16S rRNA gene MiSeq sequencing and mass spectrometry-based proteomics were utilised to identify the metataxonomic and metaproteomic profiles of the oral microbiome in both groups.Results From the metataxonomic analysis, the most abundant species in the three subgroups were Streptococcus oralis and Pseudomonas aeruginosa, accounting for up to 70% of the total population. Principal Coordinates Analysis showed differential clustering of samples from the healthy and OLP groups. ANCOM-BC compositional analysis revealed multiple species (including P. aeruginosa and several species of Veillonella, Prevotella, Streptococcus and Neisseria) significantly over-represented in the control group and several (including Granulicatella elegans, Gemella haemolysans and G. parahaemolysans) in patients with OLP. The metaproteomic data were generally congruent and revealed that several Gemella haemolysans-belonging peptidases and other proteins with inflammatory and virulence potential were present in OLP lesions.Conclusion Our data suggest that several bacterial species are associated with OLP. Future studies with larger cohorts should be conducted to determine their role in the aetiology of OLP and evaluate their potential as disease biomarkers.

ABSTRACTCurrent data on the efficacy of antiseptic mouthwashes to reduce viral load are contradictory. Firstly, in vitro data indicate very strong virucidal effects that are not replicated in clinical studies. Secondly, most clinical studies identify a limited effect, do not include a control/placebo group, or do not evaluate viral viability in an infection model. In the current manuscript, we perform a double-blind, randomized clinical trial where salivary viral load was measured before and after the mouthwash, and where saliva samples were also cultured in an in vitro infection model of SARS-CoV-2 to evaluate the effect of mouthwashes on viral viability. Our data show a 90–99% reduction in SARS-CoV-2 salivary copies with one of the tested mouthwashes, although we show that the remaining viruses are mostly viable. In addition, our data suggest that the active ingredient concentration and the overall excipients’ formulation can play an important role; and most importantly, they indicate that the effect is not immediate, being significant at 15 min and having maximum effectiveness after 1 h. Thus, we show that some oral mouthwashes can be useful in reducing viral transmission, although their efficacy must be improved through refined formulations or revised protocols.

IntroductionPeriodontitis is a biofilm-mediated disease that is usually treated by non-surgical biofilm elimination with or without antibiotics. Antibiotic treatment in periodontal patients is typically selected empirically or using qPCR or DNA hybridization methods. These approaches are directed towards establishing the levels of different periodontal pathogens in periodontal pockets to infer the antibiotic treatment. However, current methods are costly and do not consider the antibiotic susceptibility of the whole subgingival biofilm.MethodsIn the current manuscript, we have developed a method to culture subgingival samples ex vivo in a fast, label-free impedance-based system where biofilm growth is monitored in real-time under exposure to different antibiotics, producing results in 4 hours. To test its efficacy, we performed a double-blind, randomized clinical trial where patients were treated with an antibiotic either selected by the hybridization method (n=32) or by the one with the best effect in the ex vivo growth system (n=32).ResultsAntibiotic selection was different in over 80% of the cases. Clinical parameters such as periodontal pocket depth, attachment level, and bleeding upon probing improved in both groups. However, dental plaque was significantly reduced only in the group where antibiotics were selected according to the ex vivo growth. In addition, 16S rRNA sequencing showed a larger reduction in periodontal pathogens and a larger increase in health-associated bacteria in the ex vivo growth group.DiscussionThe results of clinical and microbiological parameters, together with the reduced cost and low analysis time, support the use of the impedance system for improved individualized antibiotic selection.

Background: In vitro studies have shown that several oral antiseptics have virucidal activity against SARS-CoV-2. Thus, mouthwashes have been proposed as an easy to implement strategy to reduce viral transmission. However, there are no data measuring SARS-CoV-2 viability after mouthwashes in vivo. Methods: In this randomized double-blind, five-parallel-group, placebo-controlled clinical trial, SARS-CoV-2 salivary viral load (by quantitative PCR) and its infectious capacity (incubating saliva in cell cultures) have been evaluated before and after four different antiseptic mouthwashes and placebo in 54 COVID-19 patients. Results: Contrary to in vitro evidence, salivary viral load was not affected by any of the four tested mouthwashes. Viral culture indicated that cetylpyridinium chloride (CPC) significantly reduced viral infectivity, but only at 1-hour post-mouthwash. Conclusion: These results indicate that some of the mouthwashes currently used to reduce viral infectivity are not efficient in vivo and, furthermore, that this effect is not immediate, generating a false sense of security.Trial registration: ClinicalTrials.gov identifier: NCT04707742..

Abstract Background Tooth decay is one of the most prevalent diseases worldwide, and efficient tooth brushing with a fluoride-containing dentifrice is considered fundamental to caries prevention. Fluoride-containing dentifrices have been extensively studied in relation to enamel resistance to demineralization. Arginine (Arg) has also been proposed as a promising prebiotic to promote pH buffering through ammonia production. Here, we present the first metagenomic (DNA sequencing of the whole microbial community) and metatranscriptomic (RNAseq of the same community) analyses of human dental plaque to evaluate the effect of brushing with fluoride (Fl) and a Fl+Arg containing dentifrices on oral microbial composition and activity. Fifty-three patients were enrolled in a longitudinal clinical intervention study with two arms, including 26 caries-active and 27 caries-free adults. After a minimum 1-week washout period, dental plaque samples were collected at this post-washout baseline, 3 months after the use of a 1450-ppm fluoride dentifrice, and after 6 months of using a 1450-ppm fluoride with 1.5% arginine dentifrice. Results There was a shift in both the composition and activity of the plaque microbiome after 3 months of brushing with the fluoride-containing toothpaste compared to the samples collected at the 1-week post-washout period, both for caries-active and caries-free sites. Although several caries-associated bacteria were reduced, there was also an increase in several health- and periodontitis-associated bacteria. Over 400 genes changed proportion in the metagenome, and between 180 and 300 genes changed their expression level depending on whether caries-free or caries-active sites were analyzed. The metagenome and metatranscriptome also changed after the subjects brushed with the Fl+Arg dentifrice. There was a further decrease of both caries- and periodontitis-associated organisms. In both caries-free and caries-active sites, a decrease of genes from the arginine biosynthesis pathway was also observed, in addition to an increase in the expression of genes associated with the arginine deiminase pathway, which catabolizes arginine into ammonia, thereby buffering acidic pH. Bacterial richness and diversity were not affected by either of the two treatments in the two arms of the study. Conclusions Our data demonstrate that long-term use of both assayed dentifrices changes the bacterial composition and functional profiles of human dental plaque towards a healthier microbial community, both in caries-free and caries-active sites. This observation was especially apparent for the Fl+Arg dentifrice. Thus, we conclude that the preventive benefits of tooth brushing go beyond the physical removal of dental plaque and that the active ingredients formulated within dentifrices have a positive effect not only on enamel chemistry but also on the metabolism of oral microbial populations. Video Abstract

BackgroundThe microbiota is increasingly recognized as a significant factor in the pathophysiology of many diseases, including cardiometabolic diseases, with lifestyles probably exerting the greatest influence on the composition of the human microbiome. The main objectives of the study are to analyze the association of lifestyles (diet, physical activity, tobacco, and alcohol) with the gut and oral microbiota, arterial aging, and cognitive function in subjects without cardiovascular disease in the Iberian Peninsula. In addition, the study will examine the mediating role of the microbiome in mediating the association between lifestyles and arterial aging as well as cognitive function.Methods and analysisMIVAS III is a multicenter cross-sectional study that will take place in the Iberian Peninsula. One thousand subjects aged between 45 and 74 years without cardiovascular disease will be selected. The main variables are demographic information, anthropometric measurements, and habits (tobacco and alcohol). Dietary patterns will be assessed using a frequency consumption questionnaire (FFQ) and the Mediterranean diet adherence questionnaire. Physical activity levels will be evaluated using the International Physical Activity Questionnaire (IPAQ), Marshall Questionnaire, and an Accelerometer (Actigraph). Body composition will be measured using the Inbody 230 impedance meter. Arterial aging will be assessed through various means, including measuring medium intimate carotid thickness using the Sonosite Micromax, conducting analysis with pulse wave velocity (PWA), and measuring pulse wave velocity (cf-PWV) using the Sphygmocor System. Additional cardiovascular indicators such as Cardio Ankle Vascular Index (CAVI), ba-PWV, and ankle-brachial index (Vasera VS-2000®) will also be examined. The study will analyze the intestinal microbiota using the OMNIgene GUT kit (OMR−200) and profile the microbiome through massive sequencing of the 16S rRNA gene. Linear discriminant analysis (LDA), effect size (LEfSe), and compositional analysis, such as ANCOM-BC, will be used to identify differentially abundant taxa between groups. After rarefying the samples, further analyses will be conducted using MicrobiomeAnalyst and R v.4.2.1 software. These analyses will include various aspects, such as assessing α and β diversity, conducting abundance profiling, and performing clustering analysis.DiscussionLifestyle acts as a modifier of microbiota composition. However, there are no conclusive results demonstrating the mediating effect of the microbiota in the relationship between lifestyles and cardiovascular diseases. Understanding this relationship may facilitate the implementation of strategies for improving population health by modifying the gut and oral microbiota.Trial registrationclinicaltrials.gov/ct2/show/NCT04924907, ClinicalTrials.gov, identifier: NCT04924907. Registered on 21 April 2021.

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.

Abstract Most public health measures to contain the COVID-19 pandemic are based on preventing the pathogen spread, and the use of oral antiseptics has been proposed as a strategy to reduce transmission risk. The aim of this manuscript is to test the efficacy of mouthwashes to reduce salivary viral load in vivo. This is a multi-centre, blinded, parallel-group, placebo-controlled randomised clinical trial that tests the effect of four mouthwashes (cetylpyridinium chloride, chlorhexidine, povidone-iodine and hydrogen peroxide) in SARS-CoV-2 salivary load measured by qPCR at baseline and 30, 60 and 120 min after the mouthrinse. A fifth group of patients used distilled water mouthrinse as a control. Eighty-four participants were recruited and divided into 12–15 per group. There were no statistically significant changes in salivary viral load after the use of the different mouthwashes. Although oral antiseptics have shown virucidal effects in vitro, our data show that salivary viral load in COVID-19 patients was not affected by the tested treatments. This could reflect that those mouthwashes are not effective in vivo, or that viral particles are not infective but viral RNA is still detected by PCR. Viral infectivity studies after the use of mouthwashes are therefore required. ( https://clinicaltrials.gov/ct2/show/NCT04707742 ; Identifier: NCT04707742)

IntroductionRecent studies have revealed the presence of N-acyl-homoserine lactones (AHLs) quorum sensing (QS) signals in the oral environment. Yet, their role in oral biofilm development remains scarcely investigated. The use of quorum quenching (QQ) strategies targeting AHLs has been described as efficient for the control of pathogenic biofilms. Here, we evaluate the use of a highly active AHL-targeting QQ enzyme, Aii20J, to modulate oral biofilm formation in vitro.MethodsThe effect of the QQ enzyme was studied in in vitro multispecies biofilms generated from oral samples taken from healthy donors and patients with periodontal disease. Subgingival samples were used as inocula, aiming to select members of the microbiota of the periodontal pocket niche in the in vitro biofilms. Biofilm formation abilities and microbial composition were studied upon treating the biofilms with the QQ enzyme Aii20J.Results and DiscussionThe addition of the enzyme resulted in significant biofilm mass reductions in 30 – 60% of the subgingival-derived biofilms, although standard AHLs could not be found in the supernatants of the cultured biofilms. Changes in biofilm mass were not accompanied by significant alterations of bacterial relative abundance at the genus level. The investigation of 125 oral supragingival metagenomes and a synthetic subgingival metagenome revealed a surprisingly high abundance and broad distribution of homologous of the AHL synthase HdtS and several protein families of AHL receptors, as well as an enormous presence of QQ enzymes, pointing to the existence of an intricate signaling network in oral biofilms that has been so far unreported, and should be further investigated. Together, our findings support the use of Aii20J to modulate polymicrobial biofilm formation without changing the microbiome structure of the biofilm. Results in this study suggest that AHLs or AHL-like molecules affect oral biofilm formation, encouraging the application of QQ strategies for oral health improvement, and reinforcing the importance of personalized approaches to oral biofilm control.

Ecological and genetic factors have influenced the composition of the human microbiome during our evolutionary history. We analysed the oral microbiota of the Agta, a hunter–gatherer population where some members have adopted an agricultural diet. We show that age is the strongest factor modulating the microbiome, probably through immunosenescence since we identified an increase in the number of species classified as pathogens with age. We also characterised biological and cultural processes generating sexual dimorphism in the oral microbiome. A small subset of oral bacteria is influenced by the host genome, linking host collagen genes to bacterial biofilm formation. Our data also suggest that shifting from a fish/meat diet to a rice-rich diet transforms their microbiome, mirroring the Neolithic transition. All of these factors have implications in the epidemiology of oral diseases. Thus, the human oral microbiome is multifactorial and shaped by various ecological and social factors that modify the oral environment.

Here we investigate the effects of extensive sociality and mobility on the oral microbiome of 138 Agta hunter–gatherers from the Philippines. Our comparisons of microbiome composition showed that the Agta are more similar to Central African BaYaka hunter–gatherers than to neighbouring farmers. We also defined the Agta social microbiome as a set of 137 oral bacteria (only 7% of 1980 amplicon sequence variants) significantly influenced by social contact (quantified through wireless sensors of short-range interactions). We show that large interaction networks including strong links between close kin, spouses and even unrelated friends can significantly predict bacterial transmission networks across Agta camps. Finally, we show that more central individuals to social networks are also bacterial supersharers. We conclude that hunter–gatherer social microbiomes are predominantly pathogenic and were shaped by evolutionary tradeoffs between extensive sociality and disease spread.

Abstract Background Primary prevention trials have demonstrated that the traditional Mediterranean diet is associated with a reduction in cardiovascular mortality and morbidity. However, this benefit has not been proven for secondary prevention after an acute coronary syndrome (ACS). We hypothesized that a high-intensity Mediterranean diet intervention after an ACS decreases the vulnerability of atherosclerotic plaques by complex interactions between anti-inflammatory effects, microbiota changes and modulation of gene expression. Methods The MEDIMACS project is an academically funded, prospective, randomized, controlled and mechanistic clinical trial designed to address the effects of an active randomized intervention with the Mediterranean diet on atherosclerotic plaque vulnerability, coronary endothelial dysfunction and other mechanistic endpoints. One hundred patients with ACS are randomized 1:1 to a monitored high-intensity Mediterranean diet intervention or to a standard-of-care arm. Adherence to diet is assessed in both arms using food frequency questionnaires and biomarkers of compliance. The primary endpoint is the change (from baseline to 12 months) in the thickness of the fibrous cap of a non-significant atherosclerotic plaque in a non-culprit vessel, as assessed by repeated optical coherence tomography intracoronary imaging. Indices of coronary vascular physiology and changes in gastrointestinal microbiota, immunological status and protein and metabolite profiles will be evaluated as secondary endpoints. Discussion The results of this trial will address the key effects of dietary habits on atherosclerotic risk and will provide initial data on the complex interplay of immunological, microbiome-, proteome- and metabolome-related mechanisms by which non-pharmacological factors may impact the progression of coronary atherosclerosis after an ACS. Trial registration ClinicalTrials.gov NCT03842319 . Registered on 13 May 2019

Biofilm formation and the appearance of persister cells with low metabolic rates are key factors affecting conventional treatment failure and antibiotic resistance. Using impedance-based measurements, crystal violet staining and traditional culture we have studied the biofilm growth dynamics of 13 Pseudomonas aeruginosa strains under the effect of seven conventional antibiotics. Real-time growth quantifications revealed that the exposure of established P. aeruginosa biofilms to certain concentrations of ciprofloxacin, ceftazidime and tobramycin induced the emergence of persister cells, that showed different morphology and pigmentation, as well increased antibiotic resistance. Whole-genome sequencing of wildtype and persister cells identified several SNPs, a genomic inversion and a genomic duplication in one of the strains. However, these mutations were not uniquely associated with persisters, suggesting that the persistent phenotype may be related to metabolic and transcriptional changes. Given that mannitol has been proposed to activate bacterial metabolism, the synergistic combination of mannitol and ciprofloxacin was evaluated on clinical 48 h P. aeruginosa biofilms. When administered at doses ≥320 mg/L, mannitol was capable of preventing persister cell formation by efficiently activating dormant bacteria and making them susceptible to the antibiotic. These results were confirmed using viable colony counting. As the tested ciprofloxacin-mannitol combination appeared to fully eradicate mature biofilms, we conclude that impedance-based biofilm diagnostics, which permits antibiotic susceptibility testing and the identification of persister cells, is of great potential for the clinical practice and could aid in establishing treatment breakpoints for emerging biofilm-related infections.

Objective. Whether a minimum quantity of saliva inhibit the caries process remains uncertain. This study aimed to investigate the impact of saliva dilutions on an in vitro caries model using Streptococcus mutans (S. mutans) biofilms. Methods. S. mutans biofilms were cultivated on enamel and root dentin slabs, in culture media containing different proportions of saliva (v/v): 0%, 5%, 10%, 25%, 50%, 75%, and 100% saliva, and exposed to a 10% sucrose solution (5 min, 3x/day), with appropriate controls. After 5 (enamel) and 4 (dentin) days, demineralization, biomass, viable bacteria, and polysaccharide formation were analyzed. The acidogenicity of the spent media was monitored overtime. Each assay was performed in triplicate across two independent experiments (n = 6). Results. In both enamel and dentin, an inverse relationship was observed between acidogenicity, demineralization, and the proportion of saliva. Even small quantities of saliva incorporated into the media led to a noticeable reduction in enamel and dentin demineralization. Saliva presence resulted in significant reductions in biomass, viable S. mutans cells, and polysaccharides, with the effects being concentration-dependent for both tissues. Conclusions. High quantities of saliva can almost completely inhibit sucrose-induced cariogenicity, while even small amounts exhibit a dose-dependent caries-protective effect.

ABSTRACT Biofilm community development has been established as a sequential process starting from the attachment of single cells on a surface. However, microorganisms are often found as aggregates in the environment and in biological fluids. Here, we conduct a comprehensive analysis of the native structure and composition of aggregated microbial assemblages in human saliva and investigate their spatiotemporal attachment and biofilm community development. Using multiscale imaging, cell sorting, and computational approaches combined with sequencing analysis, a diverse mixture of aggregates varying in size, structure, and microbial composition, including bacteria associated with host epithelial cells, can be found in saliva in addition to a few single-cell forms. Phylogenetic analysis reveals a mixture of complex consortia of aerobes and anaerobes in which bacteria traditionally considered early and late colonizers are found mixed together. When individually tracked during colonization and biofilm initiation, aggregates rapidly proliferate and expand tridimensionally, modulating population growth, spatial organization, and community scaffolding. In contrast, most single cells remain static or are incorporated by actively growing aggregates. These results suggest an alternative biofilm development process whereby aggregates containing different species or associated with human cells collectively adhere to the surface as “growth nuclei” to build the biofilm and shape polymicrobial communities at various spatial and taxonomic scales. IMPORTANCE Microbes in biological fluids can be found as aggregates. How these multicellular structures bind to surfaces and initiate the biofilm life cycle remains understudied. Here, we investigate the structural organization of microbial aggregates in human saliva and their role in biofilm formation. We found diverse mixtures of aggregates with different sizes, structures, and compositions in addition to free-living cells. When individually tracked during binding and growth on tooth-like surfaces, most aggregates developed into structured biofilm communities, whereas most single cells remained static or were engulfed by the growing aggregates. Our results reveal that preformed microbial consortia adhere as “buds of growth,” governing biofilm initiation without specific taxonomic order or cell-by-cell succession, which provide new insights into spatial and population heterogeneity development in complex ecosystems.

The ability of Staphylococcus aureus to colonise different niches across the human body is linked to an adaptable metabolic capability, as well as its ability to persist within specific tissues despite adverse conditions. In many cases, as S. aureus proliferates within an anatomical niche, there is an associated pathology. The immune response, together with medical interventions such as antibiotics, often removes the S. aureus cells that are causing this disease. However, a common issue in S. aureus infections is a relapse of disease. Within infected tissue, S. aureus exists as a population of cells, and it adopts a diversity of cell types. In evolutionary biology, the concept of “bet-hedging” has established that even in positive conditions, there are members that arise within a population that would be present as non-beneficial, but if those conditions change, these traits could allow survival. For S. aureus, some of these cells within an infection have a reduced fitness, are not rapidly proliferating or are the cause of an active host response and disease, but these do remain even after the disease seems to have been cleared. This is true for persistence against immune responses but also as a continual presence in spite of antibiotic treatment. We propose that the constant arousal of suboptimal populations at any timepoint is a key strategy for S. aureus long-term infection and survival. Thus, understanding the molecular basis for this feature could be instrumental to combat persistent infections.

IntroductionAntibiotics are commonly prescribed to young children for treating bacterial infections such as invasive pneumococcal disease (IPD) caused by Streptococcus pneumoniae. Despite the obvious benefits of antibiotics, little is known about their possible side effects on children’s nasopharyngeal microbiota. In other ecological niches, antibiotics have been described to perturb the balanced microbiota with short- and long-term effects on children’s health. The present study aims to evaluate and compare the nasopharyngeal microbiota of children with IPD and different degree of antibiotic exposure.MethodsWe investigated differences in nasopharyngeal microbiota of two groups of children

Abstract Streptococcus dentisani has been identified as an oral cavity probiotic due to its beneficial characteristics. One of its beneficial features is the production of bacteriocins, which inhibit the growth of cariogenic bacteria, and another is its buffering capacity through the production of ammonium from arginine. The purpose of this study was to determine the presence of S. dentisani in the dental plaque of Colombian children and whether the presence of this bacterium is related to oral health and other conditions. Dental plaque and information on diet and oral hygiene habits were collected from children between 6 and 12 years of age from four Colombian cities, divided into caries‐free children (International Caries Detection and Assessment System [ICDAS] 0, Decayed Missing Filled Teeth index [DMFT] 0), children with ICDAS 1 and 2, and children with ICDAS >3. Plaque DNA was extracted and quantified, and real‐time polymerase chain reaction was performed using specific primers. This bacterium was identified in all samples, with a median of 0.46 cells/ng DNA (interquartile range [IQR] 0.13–1.02), without finding significant differences between the groups (P > 0.05). In caries‐free children, a median of 0.45 cells/ng DNA (IQR 0.14–1.23) was found. In children with ICDAS 1 and 2, the median was 0.49 cells/ng DNA (IQR 0.11–0.97), and in children with ICDAS >3, the median was 0.35 cells/ng DNA (IQR 0.12–1.07). However, statistically significant differences were found in the origin of children (P

Supragingival dental plaque samples were collected from 40 Swedish adolescents, including 20 with caries lesions (CAR) and 20 caries-free (CF). Fresh plaque samples were subjected to an ex vivo acid tolerance (AT) test where the proportion of bacteria resistant to an acid shock was evaluated through confocal microscopy and live/dead staining, and the metabolites produced were quantified by 1H Nuclear Magnetic Resonance (1H NMR). In addition, DNA was extracted and the 16S rRNA gene was sequenced by Illumina sequencing, in order to characterize bacterial composition in the same samples. There were no significant differences in AT scores between CAR and CF individuals. However, 7 out of the 10 individuals with highest AT scores belonged to the CAR group. Regarding bacterial composition, Abiotrophia, Prevotella and Veillonella were found at significantly higher levels in CAR individuals (p=0.0085, 0.026 and 0.04 respectively) and Rothia and Corynebacterium at significantly higher levels in CF individuals (p=0.026 and 0.003). The caries pathogen Streptococcus mutans was found at low frequencies and was absent in 60% of CAR individuals. Random-forest predictive models indicate that at least 4 bacterial species or 9 genera are needed to distinguish CAR from CF adolescents. The metabolomic profile obtained by NMR showed a significant clustering of organic acids with specific bacteria in CAR and/or high AT individuals, being Scardovia wiggsiae the species with strongest associations. A significant clustering of ethanol and isopropanol with health-associated bacteria such as Rothia or Corynebacterium was also found. Accordingly, several relationships involving these compounds like the Ethanol : Lactate or Succinate : Lactate ratios were significantly associated to acid tolerance and could be of predictive value for caries risk. We therefore propose that future caries risk studies would benefit from considering not only the use of multiple organisms as potential microbial biomarkers, but also their functional adaptation and metabolic output.

Tooth decay starts with enamel demineralization due to an acidic pH, which arises from sugar fermentation by acidogenic oral bacteria. Previous in vitro work has demonstrated that nitrate limits acidification when incubating complex oral communities with sugar for short periods (e.g., 1-5 h), driven by changes in the microbiota metabolism and/or composition. To test whether a single dose of nitrate can reduce acidification derived from sugar fermentation in vivo, 12 individuals received a nitrate-rich beetroot supplement, which was compared to a placebo in a blinded crossover setting. Sucrose-rinses were performed at baseline and 2 h after supplement or placebo intake, and the salivary pH, nitrate, nitrite, ammonium and lactate were measured. After nitrate supplement intake, the sucrose-induced salivary pH drop was attenuated when compared with the placebo (p < 0.05). Salivary nitrate negatively correlated with lactate production and positively with ΔpH after sucrose exposure (r= -0.508 and 0.436, respectively, both p < 0.05). Two additional pilot studies were performed to test the effect of sucrose rinses 1 h (n = 6) and 4 h (n = 6) after nitrate supplement intake. In the 4 h study, nitrate intake was compared with water intake and bacterial profiles were analysed using 16S rRNA gene Illumina sequencing and qPCR detection of Rothia. Sucrose rinses caused a significant pH drop (p < 0.05), except 1 h and 4 h after nitrate supplement intake. After 4 h of nitrate intake, there was less lactate produced compared to water intake (p < 0.05) and one genus; Rothia, increased in abundance. This small but significant increase was confirmed by qPCR (p < 0.05). The relative abundance of Rothia and Neisseria negatively correlated with lactate production (r = -0.601 and -0.669, respectively) and Neisseria positively correlated with pH following sucrose intake (r = 0.669, all p < 0.05). Together, these results show that nitrate can acutely limit acidification when sugars are fermented, which appears to result from lactate usage by nitrate-reducing bacteria. Future studies should assess the longitudinal impact of daily nitrate-rich vegetable or supplement intake on dental health.

The increased incidence of COVID-19 cases and deaths in Spain in March 2020 led to the declaration by the Spanish government of a state of emergency imposing strict confinement measures on the population. The objective of this study was to characterize the nasopharyngeal microbiota of children and adults and its relation to SARS-CoV-2 infection and COVID-19 severity during the pandemic lockdown in Spain. This cross-sectional study included family households located in metropolitan Barcelona, Spain, with one adult with a previous confirmed COVID-19 episode and one or more exposed co-habiting child contacts. Nasopharyngeal swabs were used to determine SARS-CoV-2 infection status, characterize the nasopharyngeal microbiota and determine common respiratory DNA/RNA viral co-infections. A total of 173 adult cases and 470 exposed children were included. Overall, a predominance of Corynebacterium and Dolosigranulum and a limited abundance of common pathobionts including Haemophilus and Streptococcus were found both among adults and children. Children with current SARS-CoV-2 infection presented higher bacterial richness and increased Fusobacterium, Streptococcus and Prevotella abundance than non-infected children. Among adults, persistent SARS-CoV-2 RNA was associated with an increased abundance of an unclassified member of the Actinomycetales order. COVID-19 severity was associated with increased Staphylococcus and reduced Dolosigranulum abundance. The stringent COVID-19 lockdown in Spain had a significant impact on the nasopharyngeal microbiota of children, reflected in the limited abundance of common respiratory pathobionts and the predominance of Corynebacterium, regardless of SARS-CoV-2 detection. COVID-19 severity in adults was associated with decreased nasopharynx levels of healthy commensal bacteria.

Background: Electrolytes, proteins, and other salivary molecules play an important role in tooth integrity and can serve as biomarkers associated with caries. Objective: To determine the concentration of potential biomarkers in children without caries (CF) and children with caries (CA). Methods: Unstimulated saliva was collected, and the biomarkers quantified in duplicate, using commercial Enzyme Linked Immunosorbent Assay (ELISA) kits to determine IgA, fibronectin, cathelicidin LL-37, and statherin levels, as well as colorimetric tests to detect formate and phosphate. Results: Significantly higher concentrations of statherin was detected in the CF group (Median: 94,734.6; IQR: 92,934.6–95,113.7) compared to the CA2 group (90,875.0; IQR: 83,580.2–94,633.4) (p = 0.03). Slightly higher median IgA (48,250.0; IQR: 31,461.9–67,418.8) and LL-37 levels (56.1; IQR 43.6–116.2) and a lower concentration of formate were detected in the CF group (0.02; IQR 0.0034–0.15) compared to the group with caries (IgA: 37,776.42; IQR: 33,383.9–44,128.5; LL-37: 46.3; IQR: 40.1011–67.7; formate: 0.10; IQR: 0.01–0.18), but these differences were not statistically significant. Conclusion: The fact that these compounds have been identified as good markers for caries among European adults highlights the difficulty of identifying universal biomarkers that are applicable to all ages or to different populations.

Recent evidence indicates that the reduction of salivary nitrate by oral bacteria can contribute to prevent oral diseases, as well as increase systemic nitric oxide levels that can improve conditions such as hypertension and diabetes. The objective of the current manuscript was to isolate nitrate-reducing bacteria from the oral cavity of healthy donors and test their in vitro probiotic potential to increase the nitrate-reduction capacity (NRC) of oral communities. Sixty-two isolates were obtained from five different donors of which 53 were confirmed to be nitrate-reducers. Ten isolates were selected based on high NRC as well as high growth rates and low acidogenicity, all being Rothia species. The genomes of these ten isolates confirmed the presence of nitrate- and nitrite reductase genes, as well as lactate utilization genes, and the absence of antimicrobial resistance, mobile genetic elements and virulence genes. The pH at which most nitrate was reduced differed between strains. However, acidic pH 6 always stimulated the reduction of nitrite compared to neutral pH 7 or slightly alkaline pH 7.5 (p < 0.01). We tested the effect of six out of 10 isolates on in vitro oral biofilm development in the presence or absence of 6.5 mM nitrate. The integration of the isolates into in vitro communities was confirmed by Illumina sequencing. The NRC of the bacterial communities increased when adding the isolates compared to controls without isolates (p < 0.05). When adding nitrate (prebiotic treatment) or isolates in combination with nitrate (symbiotic treatment), a smaller decrease in pH derived from sugar metabolism was observed (p < 0.05), which for some symbiotic combinations appeared to be due to lactate consumption. Interestingly, there was a strong correlation between the NRC of oral communities and ammonia production even in the absence of nitrate (R = 0.814, p < 0.01), which indicates that bacteria involved in these processes are related. As observed in our study, individuals differ in their NRC. Thus, some may have direct benefits from nitrate as a prebiotic as their microbiota naturally reduces significant amounts, while others may benefit more from a symbiotic combination (nitrate + nitrate-reducing probiotic). Future clinical studies should test the effects of these treatments on oral and systemic health.

Streptococcus dentisani 7746, isolated from dental plaque of caries-free individuals, has been shown to have several beneficial effects in vitro which could contribute to promote oral health, including an antimicrobial activity against oral pathogens by the production of bacteriocins and a pH buffering capacity through ammonia production. Previous work has shown that S. dentisani was able to colonize the oral cavity for 2–4 weeks after application. The aim of the present work was to evaluate its clinical efficacy by a randomized, double-blind, placebo-controlled parallel group study. Fifty nine volunteers were enrolled in the study and randomly assigned to a treatment or placebo group. The treatment consisted of a bucco-adhesive gel application (2.5 109 cfu/dose) with a dental splint for 5 min every 48 h, for a period of 1 month (i.e., 14 doses). Dental plaque and saliva samples were collected at baseline, 15 and 30 days after first application, and 15 days after the end of treatment. At baseline, there was a significant correlation between S. dentisani levels and frequency of toothbrushing. Salivary flow, a major factor influencing oral health, was significantly higher in the probiotic group at day 15 compared with the placebo (4.4 and 3.4 ml/5 min, respectively). In the probiotic group, there was a decrease in the amount of dental plaque and in gingival inflammation, but no differences were observed in the placebo group. The probiotic group showed a significant increase in the levels of salivary ammonia and calcium. Finally, Illumina sequencing of plaque samples showed a beneficial shift in bacterial composition at day 30 relative to baseline, with a reduction of several cariogenic organisms and the key players in plaque formation, probably as a result of bacteriocins production. Only 58% of the participants in the probiotic group showed increased plaque levels of S. dentisani at day 30 and 71% by day 45, indicating that the benefits of S. dentisani application could be augmented by improving colonization efficiency. In conclusion, the application of S. dentisani 7746 improved several clinical and microbiological parameters associated with oral health, supporting its use as a probiotic to prevent tooth decay.

Microorganisms grown in biofilms are more resistant to antimicrobial treatment and immune system attacks compared to their planktonic forms. In fact, infections caused by biofilm-forming Staphylococcus aureus and Staphylococcus epidermidis are a large threat for public health, including patients with medical devices. The aim of the current manuscript was to test the effect of dalbavancin, a recently developed lipoglycopeptide antibiotic, alone or in combination with compounds contributing to bacterial cell disaggregation, on staphylococcal biofilm formation and elimination. We used real-time impedance measurements in microtiter plates to study biofilm growth dynamics of S. aureus and S. epidermidis strains, in the absence or presence of dalbavancin, linezolid, vancomycin, cloxacillin, and rifampicin. Further experiments were undertaken to check whether biofilm-detaching compounds such as N-acetylcysteine (NAC) and ficin could enhance dalbavancin efficiency. Real-time dose–response experiments showed that dalbavancin is a highly effective antimicrobial, preventing staphylococcal biofilm formation at low concentrations. Minimum biofilm inhibitory concentrations were up to 22 higher compared to standard E-test values. Dalbavancin was the only antimicrobial that could halt new biofilm formation on established biofilms compared to the other four antibiotics. The addition of NAC decreased dalbavancin efficacy while the combination of dalbavancin with ficin was more efficient than antibiotic alone in preventing growth once the biofilm was established. Results were confirmed by classical biofilm quantification methods such as crystal violet (CV) staining and viable colony counting. Thus, our data support the use of dalbavancin as a promising antimicrobial to treat biofilm-related infections. Our data also highlight that synergistic and antagonistic effects between antibiotics and biofilm-detaching compounds should be carefully tested in order to achieve an efficient treatment that could prevent both biofilm formation and disruption.

Objective: An insufficient mineralization (hypomineralization) in the teeth during the maturation stage of amelogenesis cause defects in 3–44% of children. Here, we describe for the first time the microbiota associated with these defects and compared it to healthy teeth within the same subjects. Methods: Supragingival dental plaque was sampled from healthy and affected teeth from 25 children with molar–incisor hypomineralization (MIH). Total DNA was extracted and the 16S rRNA gene was sequenced by Illumina sequencing in order to describe the bacterial composition. Results: We detected a higher bacterial diversity in MIH samples, suggesting better bacterial adhesion or higher number of niches in those surfaces. We found the genera Catonella, Fusobacterium, Campylobacter, Tannerella, Centipeda, Streptobacillus, Alloprevotella and Selenomonas associated with hypomineralized teeth, whereas Rothia and Lautropia were associated with healthy sites. Conclusion: The higher protein content of MIH-affected teeth could favour colonization by proteolytic microorganisms. The over-representation of bacteria associated with endodontic infections and periodontal pathologies suggests that, in addition to promote caries development, MIH could increase the risk of other oral diseases.

Objective: The oral microbiota has been deeply studied by high-throughput sequencing techniques. However, although the interproximal regions have one of the highest caries rates in the oral cavity, information about the bacterial composition at those sites is scarce. Methods: In this study, we used 16S rRNA Illumina sequencing to describe the microbiota associated to interproximal regions at two time points. In addition, dental plaque samples at the vestibular and lingual surfaces from the same teeth were also analysed at the two time points. Results: Interproximal-associated microbiota was found to be similar to already described bacterial communities in other mouth niches. Streptoccocus, Veillonella, Rothia, Actinomyces, Neisseria, Haemophilus and Fusobacterium were the most abundant genera in this oral region. Statistical analyses showed that the microbiota from interproximal sites was more similar to that sampled from the vestibular surfaces than to the lingual surfaces. Interestingly, many potentially cariogenic bacteria such as Scardovia, Atopobium or Selenomonas were over-represented in the interproximal regions in comparison with vestibular and lingual sites. Conclusion: The microbiota at interproximal regions appears to be specific and stable through time. Potentially pathogenic bacteria may increase caries development risk and gingival inflammation at those sites.

Background: Bacterial vaginosis is the most common infection in women and it has been proved that dysbiosis of vaginal microbiota can promote the infectious status. This case report shows the effect of oral and vaginal sex over the microbiota of a heterosexual couple who reported repeated problems of vaginal and oral infections after sexual intercourse.Case Presentation: A woman (32) reported to have vaginal infections and gingivitis after she had started a relationship with a man (34) and associated them with unprotected sex. No treatments successfully removed the problem and it repeated every time they had sexual encounters. Vaginal, penile and oral swabs were collected before and after sexual encounters in order to analyze changes in the respective microbiotas. DNA was extracted from all samples and the bacterial 16S rRNA gene was sequenced using Illumina MiSeq.Conclusions:Lactobacillus occupied the great majority of the vaginal microbiota in all scenarios except after unprotected sex, which caused a bacterial dysbiosis that lasted at least for a week. Similarly, the penile microbiota changed significantly after unprotected sexual relationships. Interestingly, both oral and vaginal sex increased the abundance of Lactobacillus in the male oral and penile microbiota, respectively. In conclusion, unprotected sexual intercourse influenced the genital microbiota in the couple studied and future studies with larger sample sizes should study if sex may be a factor promoting vaginal infection through dysbiosis and hampered protection by the resident microbiota.

Periodontitis (PD) shows an association with rheumatoid arthritis (RA) and systemic inflammation. Periodontal pathogens, namely Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, are proposed to be capable of inducing citrullination of peptides in the gingiva, inducing the formation of anti-citrullinated protein antibodies (ACPAs) within susceptible hosts. Here, we sought to investigate whether periodontal treatment influenced systemic inflammation and antibody titres to P. gingivalis, A. actinomycetemcomitans, Prevotella intermedia and ACPA in 42 systemically health patients with periodontal disease. Subgingival plaque and serum samples were collected from study participants before (baseline) and 90 days after treatment to analyse the abundance of specific bacteria and evaluate anti-bacterial antibodies, C-reactive protein (CRP), tumour necrosis factor α (TNF-α), interleukin 6 (IL-6) and ACPA in serum. Following treatment, all patients showed reduced periodontal inflammation. Despite observing a weak positive correlation between CRP and IL-6 with periodontal inflammation at baseline, we observed no significant reductions in any indicators of systemic inflammation 90 days after treatment. In contrast, anti-P. gingivalis IgG significantly reduced post-treatment (p < 0.001, Wilcoxon signed rank test), although no changes were observed for other antibody titres. Patients who had detectable P. gingivalis in subgingival plaques had significantly higher anti-P. gingivalis IgG and ACPA titres, suggesting a potential association between P. gingivalis colonisation and systemic antibody titres.

Background and Aims: The risk of suffering from some infectious diseases can be related to specific microbiota profiles. Specifically, the nasopharyngeal microbiota could play a role as a risk or protective factor in the development of invasive disease caused by S. pneumoniae.Methodology: We analyzed the nasopharyngeal microbiota of children with invasive pneumococcal disease (IPD) and that of healthy controls matched by age, sex, and seasonality from Catalonia, Spain. Epidemiological, microbiological and clinical variables were considered to compare microbiota profiles, analyzed by sequencing the V1–V4 region of the 16S rRNA gene.Results: Twenty-eight children with IPD (median age 43 months) and 28 controls (42.6 months) were included in the study. IPD children presented a significantly higher bacterial diversity and richness (p < 0.001). Principal coordinate analysis revealed three different microbiota profiles: microbiota A, dominated by the genus Dolosigranulum (44.3%); Microbiota B, mostly represented by Streptococcus (36.9%) and Staphylococcus (21.3%) and a high diversity of anaerobic genera including Veillonella, Prevotella and Porphyromonas; and Microbiota C, mainly containing Haemophilus (52.1%) and Moraxella (31.4%). The only explanatory factor for the three microbiotas was the classification of children into disease or healthy controls (p = 0.006). A significant negative correlation was found between Dolosigranulum vs. Streptococcus (p = 0.029), suggesting a potential antagonistic effect against pneumococcal pathogens.Conclusions: The higher bacterial diversity and richness in children with IPD could suggest an impaired immune response. This lack of immune competence could be aggravated by breastfeeding

Background and objectives: We have developed a standardized, easy-to-use in vitro model to study single- and multiple-species oral biofilms in real time through impedance technology, which elucidates the kinetics of biofilm formation in 96-well plates, without the requirement for any further manipulation. Design and Results: Using this system, biofilms of Streptococcus mutans appear to be sugar-dependent and highly resistant to amoxicilin, an antibiotic to which this oral pathogen is highly sensitive in a planktonic state. Saliva, tongue and dental plaque samples were also used as inocula to form multiple-species biofilms. DNA isolation and Illumina sequencing of the biofilms showed that the multi-species biofilms were formed by tens or hundreds of species, had a similar composition to the original inoculum, and included fastidious microorganisms which are important for oral health and disease. As an example of the potential applications of the model, we show that oral biofilms can be inhibited by amoxicilin, but in some cases they are induced by the antibiotic, suggesting the existence of responders and non-responders to a given antibiotic. Conclusions: We therefore propose the system as a valid in vitro model to study oral biofilm dynamics, including their susceptibility to antibiotics, antiseptics or anti-adhesive compounds.

Epidemiological studies use saliva on a regular basis as a non-invasive and easy-to-take sample, which is assumed to be a microbial representative of the oral cavity ecosystem. However, comparative studies between different kinds of saliva samples normally used in microbial studies are scarce. The aim of the current study was to compare oral microbiota composition between two different saliva samples collected simultaneously: non-stimulated saliva with paper points and stimulated saliva collected after chewing paraffin gum. DNA was extracted from saliva samples of ten individuals, then analyzed by 16S rRNA pyrosequencing to describe bacterial diversity. The results demonstrate significant differences between the microbiota of these two kinds of saliva. Stimulated saliva was found to contain an estimated number of species over three times higher than unstimulated saliva. In addition, bacterial composition at the class and genus level was radically different between both types of samples. When compared to other oral niches, both types of saliva showed some similarity to tongue and buccal mucosa, but they do not correlate at all with the bacterial composition described in supra- or sub-gingival dental plaque, questioning their use in etiological and epidemiological studies of oral diseases of microbial origin.

Background: Previous studies have suggested the quorum sensing signal AI-2 as a potential target to prevent the biofilm formation by Streptococcus mutans, a pathogen involved in tooth decay. Objective: To obtain inhibition of biofilm formation by S. mutans by extracts obtained from the marine bacterium Tenacibaculum sp. 20J interfering with the AI-2 quorum sensing system. Design: The AI-2 inhibitory activity was tested with the biosensors Vibrio harveyi BB170 and JMH597. S. mutans ATCC25175 biofilm formation was monitored using impedance real-time measurements with the xCELLigence system®, confocal laser microscopy, and the crystal violet quantification method. Results: The addition of the cell extract from Tenacibaculum sp. 20J reduced biofilm formation in S. mutans ATCC25175 by 40–50% compared to the control without significantly affecting growth. A decrease of almost 40% was also observed in S. oralis DSM20627 and S. dentisani 7747 biofilms. Conclusions: The ability of Tenacibaculum sp. 20J to interfere with AI-2 and inhibit biofilm formation in S. mutans was demonstrated. The results indicate that the inhibition of quorum sensing processes may constitute a suitable strategy for inhibiting dental plaque formation, although additional experiments using mixed biofilm models would be required.

Urolithins are intestinal microbial metabolites produced from ellagitannin- and ellagic acid-containing foods such as walnuts, strawberries, and pomegranates. These metabolites, better absorbed than their precursors, can contribute significantly to the beneficial properties attributed to the polyphenols ellagitannins and ellagic acid (EA). However, both the ability of producing the final metabolites in this catabolism (urolithins A, B and isourolithin A) and the health benefits associated with ellagitannin consumption differ considerably among individuals depending on their gut microbiota composition. Three human urolithin metabotypes have been previously described, i.e., metabotype 0 (urolithin non-producers), metabotype A (production of urolithin A as unique final urolithin) and metabotype B (urolithin B and/or isourolithin A are produced besides urolithin A). Although production of some intermediary urolithins has been recently attributed to intestinal species from Eggerthellaceae family named Gordonibacter urolithinfaciens and Gordonibacter pamelaeae, the identification of the microorganisms responsible for the complete transformation of EA into the final urolithins, especially those related to metabotype B, are still unknown. In the present research we illustrate the isolation of urolithin-producing strains from human feces of a healthy adult and their ability to transform EA into different urolithin metabolites, including isourolithin A. The isolates belong to a new genus from Eggerthellaceae family. EA transformation and urolithin production arisen during the stationary phase of the growth of the bacteria under anaerobic conditions. The HPLC-DAD-MS analyses demonstrated the sequential appearance of 3,8,9,10-tetrahydroxy-urolithin (urolithin M6), 3,8,9-trihydroxy-urolithin (urolithin C) and 3,9-dihydroxy-urolithin (isourolithin A) while 3,8-dihydroxy-urolithin (urolithin A) and 3-hydroxy-urolithin (urolithin B) were not detected. For the first time isourolithin A production capacity of pure strains has been described. The biological activity attributed to urolithins A and B and isourolithin A (anti-inflammatory, anti-carcinogenic, cardioprotective, and neuroprotective properties) explains the relevance of identifying these urolithin-producing bacteria as potential novel probiotics with applications in the development of functional foods and nutraceuticals. Their human administration could improve the health benefits upon ellagitannin consumption, especially in metabotype 0 individuals. However, further research is necessary to probe well-established beneficial effects on the host and safety requirements before being considered among the next-generation probiotics.

Background: Identifying the microbial species in caries lesions is instrumental to determine the etiology of dental caries. However, a significant proportion of bacteria in carious lesions have not been cultured, and the use of molecular methods has been limited to DNA-based approaches, which detect both active and inactive or dead microorganisms. Objective: To identify the RNA-based, metabolically active bacterial composition of caries lesions at different stages of disease progression in order to provide a list of potential etiological agents of tooth decay. Design: Non-cavitated enamel caries lesions (n=15) and dentin caries lesions samples (n=12) were collected from 13 individuals. RNA was extracted and cDNA was constructed, which was used to amplify the 16S rRNA gene. The resulting 780 bp polymerase chain reaction products were pyrosequenced using Titanium-plus chemistry, and the sequences obtained were used to determine the bacterial composition. Results: A mean of 4,900 sequences of the 16S rRNA gene with an average read length of 661 bp was obtained per sample, giving a comprehensive view of the active bacterial communities in caries lesions. Estimates of bacterial diversity indicate that the microbiota of cavities is highly complex, each sample containing between 70 and 400 metabolically active species. The composition of these bacterial consortia varied among individuals and between caries lesions of the same individuals. In addition, enamel and dentin lesions had a different bacterial makeup. Lactobacilli were found almost exclusively in dentin cavities. Streptococci accounted for 40% of the total active community in enamel caries, and 20% in dentin caries. However, Streptococcus mutans represented only 0.02–0.73% of the total bacterial community. Conclusions: The data indicate that the etiology of dental caries is tissue dependent and that the disease has a clear polymicrobial origin. The low proportion of mutans streptococci detected confirms that they are a minority and questions its importance as the main etiological agent of tooth decay. Future experimental work should be performed to confirm the cariogenicity of the identified bacteria.

Gut microbiota colonization is a complex, dynamic, and step-wise process that is in constant development during the first years of life. This microbial settlement occurs in parallel with the maturation of the immune system, and alterations during this period, due to environmental and host factors, are considered to be potential determinants of health-outcomes later in life. Given that host–microbe interactions are mediated by the immune system response, it is important to understand the close relationship between immunity and the microbiota during birth, lactation, and early infancy. This work summarizes the evidence to date on early gut microbiota colonization, and how it influences the maturation of the infant immune system and health during the first 1000 days of life. This review will also address the influence of perinatal antibiotic intake and the importance of delivery mode and breastfeeding for an appropriate development of gut immunity.

The gastrointestinal tract microbiota is known to play very important roles in the well being of animals. It is a complex community composed by hundreds of microbial species interacting closely among them and with their host, that is, a microbial ecosystem. The development of high throughput sequencing techniques allows studying the diversity of such communities in a realistic way and considerable work has been carried out in mammals and some birds such as chickens. Wild birds have received less attention and in particular, in the case of penguins, only a few individuals of five species have been examined with molecular techniques. We collected cloacal samples from Chinstrap penguins in the Vapour Col rookery in Deception Island, Antarctica, and carried out pyrosequencing of the V1-V3 region of the 16S rDNA in samples from 53 individuals, 27 adults and 26 chicks. This provided the first description of the Chinstrap penguin gastrointestinal tract microbiota and the most extensive in any penguin species. Firmicutes, Bacteoridetes, Proteobacteria, Fusobacteria, Actinobacteria, and Tenericutes were the main components. There were large differences between chicks and adults. The former had more Firmicutes and the latter more Bacteroidetes and Proteobacteria. In addition, adults had richer and more diverse bacterial communities than chicks. These differences were also observed between parents and their offspring. On the other hand, nests explained differences in bacterial communities only among chicks. We suggest that environmental factors have a higher importance than genetic factors in the microbiota composition of chicks. The results also showed surprisingly large differences in community composition with other Antarctic penguins including the congeneric Adélie and Gentoo penguins.

Bivalve molluscs, due to its filter-feeding mechanism, have an abundant associated bacterial microbiota composed of a large number of species of different genera. The knowledge about the composition of the microbiota associated with bivalve molluscs is based, mostly, on techniques of cultivation of microorganisms, but also on DGGE methods. However, it is estimated that less than 1% of the bacteria are culturable with current methods, which lead to an underestimation of the microbial diversity. On the other hand, DGGE methodology has a limited resolution due to the low number of DNA fragments obtained as representative of a microbial community. The introduction of new molecular approaches allowed the improvement of the knowledge about bacterial communities present in a sample [1]. In recent years, new molecular techniques known as Next Generation Sequencing (NGS) have been developed, based on high- throughput sequencing [2]. In this study, we present the analysis of the microbiota associated to reared scallop gonads before and after spawning by pyrosequencing of the 16S rRNA gene. After the DNA extraction of the samples, amplification was performed using a set of degenerate primers with low stringency conditions. Pyrosequencing of samples was carried out using the sequencer 454 GLX Titanium Plus (Roche). The data obtained was further subjected to bioinformatics analysis using Galaxy and UCHIME program and the Ribosomal Database Project. Rarefaction curves were obtained, the composition of the bacterial community was analysed after the taxonomic assignment, and the total composition of the bacterial communities of the samples were compared using the principal coordinates analysis (PCoA) by FastUnifrac tool [3]. Pyrosequencing of the samples resulted in a total of 18520 sequences (3000 per sample, approximately) with an average length of 325 bp (base pairs). The taxonomic assignment of sequences allowed the identification to the genus level, being observed a large bacterial diversity with over 110 genera. The most prevalent genera in the samples were Hydrotalea, Acinetobacter, Delftia, Sediminibacter and Pseudomonas, among others. Differences in the microbial communities were observed among the samples, and the PCoA analysis allowed their separation by means on their gender and if they proceed from sampling before or after the spawning. Nevertheless, the rarefaction curves obtained for each sample failed to reach a saturation phase, indicating that more sequencing effort would be necessary.