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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.govNCT03842319. Registered on 13 May 2019

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 10(4) to 10(5) cells/ml and a total of 10(6)-10(7) 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.

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 studyiin vitro/idue 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,imgrA/i. To investigate this specific cell type further, S. aureus WCH-SK2-ΔimgrA/iitself was assessed with continuous culture. Compared to the wild type, theimgrA/imutant strain required fewer generations to select for SCVs. There was an increased rate of mutagenesis within the ΔimgrA/istrain compared to the wild type, which we postulate is the mechanism explaining the increased emergence of SCV selection. TheimgrA/iderived 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 mutaseiglmM/iand tyrosine recombinaseixerC/i. 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 betweenimgrA/iand the SCV phenotype.bIMPORTANCE/bWithin 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.

Ancestral humans evolved a complex social structure still observed in extant hunter-gatherers. Here we investigate the effects of extensive sociality and mobility on the oral microbiome of 138 Agta hunter-gatherers from the Philippines. Comparisons of microbiome composition showed that the Agta are more similar to Central African Bayaka hunter-gatherers than to neighboring 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 interaction networks covering large areas, and their strong links between close kin, spouses, and even unrelated friends, can significantly predict bacterial transmission networks across Agta camps. Finally, more central individuals to social networks are also bacterial supersharers. We conclude that hunter-gatherer social microbiomes, which are predominantly pathogenic, were shaped by evolutionary tradeoffs between extensive sociality and disease spread.

Ecological and genetic factors have influenced the composition of the human microbiome during our evolutionary history. We analyzed the oral microbiota of the Agta, a hunter-gatherer population where part of its members is adopting an agricultural diet. We show that age is the strongest factor modulating the microbiome, likely through immunosenescence as there is an increase of pathogenicity with age. Biological and cultural processes generate 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 suggests that shifting from a fish/meat to a rice-rich diet transforms their microbiome, mirroring the Neolithic transition. All 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.

Acute respiratory infections (ARIs) constitute one of the leading causes of antibiotic administration, hospitalization and death among children Barcelona, Spain) from 2014 to 2018. This study included three groups of children Streptococcus pneumoniae , higher frequency of invasive pneumococcal serotypes, increased rate of viral infection and underrepresentation of potential protective bacterial species such as Dolosigranulum pigrum and Moraxella lincolnii . Microbiota-based classification models differentiated cases from controls with moderately high accuracy. These results demonstrate the close relationship existing between a child’s nasopharyngeal microbiota and respiratory health, and provide initial evidence of the potential of microbiota-based diagnostics for differential diagnosis of severe ARIs using non-invasive samples.

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

[Background]: Periodontal diseases are of high prevalence globally and are characterized by an exacerbated inflammatory response which leads to oral tissue destruction. The use of probiotics is widely extended in the case of gastrointestinal disorders; however, their use in microbial‐origin oral diseases is still preliminary., [Methods]: We used quantitative polymerase chain reaction to determine the levels of the oral bacterium Streptococcus dentisani 7746 in the tongue, saliva, supragingival, and subgingival plaque. We explore the potential benefits of this probiotic by measuring inhibition of the periodontal pathogens Porphyromonas gingivalis and Fusobacterium nucleatum growth and attachment to human gingival fibroblasts. In addition, its anti‐inflammatory activity against cytokines secretion induced by these pathogens was determined in an in vitro model by enzyme‐linked immunosorbent assay., [Results]: We report that S. dentisani is found at high levels in the gingival crevice. Data show a strong inhibitory action of S. dentisani supernatant against the periodontal pathogens in pure culture. S. dentisani attached to gingival cells in vitro, inhibiting periodontal pathogens by competition, adherence, and displacement mechanisms. Finally, in a simple in vitro model, the oral probiotic strongly increased the secretion of the anti‐inflammatory cytokine IL‐10 after incubations with P. gingivalis and F. nucleatum, as well as significantly reduced the expression of interferon‐γ induced by F. nucleatum., [Conclusion]: Altogether, these results highlight the potential of S. dentisani as adjuvant therapy in the management of periodontal diseases, whose efficacy will need to be tested in clinical studies., This work was funded by the Spanish MINECO (AGL2015‐64522‐C2‐R, BIO2015‐68711‐R, and RTC‐2015‐4292‐1 projects) and Madrid Regional Government (ALIBIRD‐CM S2013/ABI‐2728). Adelaida Esteban‐Fernández and Irene Zorraquín‐Peña are recipients of fellowships from the FPI‐MINECO program.

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

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 &lt, 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: 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.

Periodontitis is characterized by subgingival biofilm dysbiosis, inflammation and tissue destruction. Current treatment involves mechanical biofilm disruption known as non-surgical periodontal therapy (NSPT). This study sought to characterise the impact of treatment on microbial diversity and overall community, and the parallel impact on host inflammation in the oral cavity. Fourty-two periodontitis patients were included in this study, with periodontal clinical parameters, subgingival plaque and saliva samples collected at baseline and 90 days after treatment. Salivary cytokines were quantified, and subgingival plaque was analysed using 16S rRNA sequencing. After treatment, there were marked health-associated alterations in microbial composition and diversity, including differential abundance of 42 genera and 61 species. These changes were accompanied by substantial clinical improvement (pockets ≥ 5 mm, 27.50% to 9.00%, p p Fusobacterium, Prevotella, Tanenerella, Treponema) remained present and formed a distinct subnetwork associated with residual disease. Collectively, this study shows that current periodontal treatment results in partial restoration of a healthy microbial ecosystem, but features of biofilm dysbiosis and host inflammation remain in some patients, which were surprisingly independent of clinical response.

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.

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.

Dental diseases are now viewed as a consequence of a deleterious shift in the balance of the normally stable resident oral microbiome. It is known that frequent carbohydrate consumption or reduced saliva flow can lead to caries, and excessive plaque accumulation increases the risk of periodontal diseases. However, when these "disease drivers" are present, while some individuals appear to be susceptible, others are more tolerant or resilient to suffering from undesirable changes in their oral microbiome. Health-maintaining mechanisms that limit the effect of disease drivers include the complex set of metabolic and functional interrelationships that develop within dental biofilms and between biofilms and the host. In contrast, "positive feedback loops" can develop within these microbial communities that disrupt resilience and provoke a large and abrupt change in function and structure of the ecosystem (a microbial "regime shift"), which promotes dysbiosis and oral disease. For instance, acidification due to carbohydrate fermentation or inflammation in response to accumulated plaque select for a cariogenic or periopathogenic microbiota, respectively, in a chain of self-reinforcing events. Conversely, in tolerant individuals, health-maintaining mechanisms, including negative feedback to the drivers, can maintain resilience and promote resistance to and recovery from disease drivers. Recently studied health-maintaining mechanisms include ammonia production, limiting a drop in pH that can lead to caries, and denitrification, which could inhibit several stages of disease-associated positive feedback loops. Omics studies comparing the microbiome of, and its interaction with, susceptible and tolerant hosts can detect markers of resilience. The neutralization or inhibition of disease drivers, together with the identification and promotion of health-promoting species and functions, for example, by pre- and probiotics, could enhance microbiome resilience and lead to new strategies to prevent disease.

A correction to this article has been published in https://doi.org/10.1038/s41598-017-13270-x, Human breastmilk contains a variety of bacteria that are transmitted to the infant and have been suggested to contribute to gut microbiota development and immune maturation. However, the characterization of fungal organisms in milk from healthy mothers is currently unknown although their presence has been reported in the infant gut and also in milk from other mammals. Breastmilk samples from healthy lactating mothers (n = 65) within 1 month after birth were analyzed. Fungal presence was assessed by different techniques, including microscopy, growth and identification of cultured isolates, fungal load estimation by qPCR, and fungal composition using 28S rRNA gene high-throughput sequencing. In addition, milk macronutrients and human somatic cells were quantified by spectrophotometry and cytometry. qPCR data showed that 89% of samples had detectable levels of fungal DNA, at an estimated median load of 3,5 × 105 cells/ml, potentially including both viable and non-viable fungi. Using different culture media, 33 strains were isolated and identified, confirming the presence of viable fungal species. Pyrosequencing results showed that the most common genera were Malassezia (44%), followed by Candida (19%) and Saccharomyces (12%). Yeast cells were observed by fluorescence microscopy. Future work should study the origin of these fungi and their potential contribution to infant health., This work was funded by ERC Starting Grant 639226-MAMI

Novel approaches using OMICS techniques enable a collective assessment of multiple related biological units, including genes, gene expression, proteins, and metabolites. In the past decade, next-generation sequencing (NGS) technologies were improved by longer sequence reads and the development of genome databases and user-friendly pipelines for data analysis, all accessible at lower cost. This has generated an outburst of high-Throughput data. The application of OMICS has provided more depth to existing hypotheses as well as new insights in the etiology of dental caries. For example, the determination of complete bacterial microbiomes of oral samples rather than selected species, together with oral metatranscriptome and metabolome analyses, supports the viewpoint of dysbiosis of the supragingival biofilms. In addition, metabolome studies have been instrumental in disclosing the contributions of major pathways for central carbon and amino acid metabolisms to biofilm pH homeostasis. New, often noncultured, oral streptococci have been identified, and their phenotypic characterization has revealed candidates for probiotic therapy. Although findings from OMICS research have been greatly informative, problems related to study design, data quality, integration, and reproducibility still need to be addressed. Also, the emergence and continuous updates of these computationally demanding technologies require expertise in advanced bioinformatics for reliable interpretation of data. Despite the obstacles cited above, OMICS research is expected to encourage the discovery of novel caries biomarkers and the development of next-generation diagnostics and therapies for caries control. These observations apply equally to the study of other oral diseases.

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 salivary glands actively concentrate plasma nitrate, leading to high salivary nitrate concentrations (5–8 mM) after a nitrate-rich vegetable meal. Nitrate is an ecological factor that can induce rapid changes in structure and function of polymicrobial communities, but the effects on the oral microbiota have not been clarified. To test this, saliva of 12 healthy donors was collected to grow in vitro biofilms with and without 6.5 mM nitrate. Samples were taken at 5 h (most nitrate reduced) and 9 h (all nitrate reduced) of biofilm formation for ammonium, lactate and pH measurements, as well as 16S rRNA gene Illumina sequencing. Nitrate did not affect biofilm growth significantly, but reduced lactate production, while increasing the observed ammonium production and pH (all p Neisseria (3.1 ×) and Rothia (2.9 ×) were detected in the nitrate condition already after 5 h (both p Streptococcus, Veillonella and Oribacterium) and halitosis- and periodontitis-associated genera (Porphyromonas, Fusobacterium, Leptotrichia, Prevotella, and Alloprevotella) were significantly reduced (p

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.

The opportunistic pathogen Staphylococcus aureus is responsible for causing infections related to indwelling medical devices, where this pathogen is able to attach and form biofilms. The intrinsic properties given by the self-produced extracellular biofilm matrix confer high resistance to antibiotics, triggering infections difficult to treat. Therefore, novel antibiofilm strategies targeting matrix components are urgently needed. The Biofilm Associated Protein, Bap, expressed by staphylococcal species adopts functional amyloid-like structures as scaffolds of the biofilm matrix. In this work we have focused on identifying agents targeting Bap-related amyloid-like aggregates as a strategy to combat S. aureus biofilm-related infections. We identified that the flavonoids, quercetin, myricetin and scutellarein specifically inhibited Bap-mediated biofilm formation of S. aureus and other staphylococcal species. By using in vitro aggregation assays and the cell-based methodology for generation of amyloid aggregates based on the Curli-Dependent Amyloid Generator system (C-DAG), we demonstrated that these polyphenols prevented the assembly of Bap-related amyloid-like structures. Finally, using an in vivo catheter infection model, we showed that quercetin and myricetin significantly reduced catheter colonization by S. aureus. These results support the use of polyphenols as anti-amyloids molecules that can be used to treat biofilm-related infections., This research was supported by Grants RTI2018-096011-B-I00 from Ministry of Science and Innovation and PI011 KILL-BACT from the Government of Navarra.

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.

Human milk represents a source of bacteria for the initial establishment of the oral (and gut) microbiomes in the breastfed infant, however, the origin of bacteria in human milk remains largely unknown. While some evidence points towards a possible endogenous enteromammary route, other authors have suggested that bacteria in human milk are contaminants from the skin or the breastfed infant mouth. In this work 16S rRNA sequencing and bacterial culturing and isolation was performed to analyze the microbiota on maternal precolostrum samples, collected from pregnant women before delivery, and on oral samples collected from the corresponding infants. The structure of both ecosystems demonstrated a high proportion of taxa consistently shared among ecosystems, Streptococcus spp. and Staphylococcus spp. being the most abundant. Whole genome sequencing on those isolates that, belonging to the same species, were isolated from both the maternal and infant samples in the same mother-infant pair, evidenced that in 8 out of 10 pairs both isolates were >99.9% identical at nucleotide level. The presence of typical oral bacteria in precolostrum before contact with the newborn indicates that they are not a contamination from the infant, and suggests that at least some oral bacteria reach the infant’s mouth through breastfeeding., This research was supported by grant AGL2016-75476-R. LR was funded by grant 624773 (FP-7-PEOPLE-2013IEF, European Commission) and is currently supported by the Juan de la Cierva Postdoctoral Trainee Program of the Spanish Ministry of Economy and Competitiveness (MINECO; IJCI-2015-23196).

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., A.B.-A. and M.C.C. acknowledge the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (ERC Starting Grant project no. 639226). F.P.-S. was supported by Ministerio de Economía y Competitividad (MINECO) grant no. CTM2016-80095-C2-1-R (NOVAMAR). The Chinese group acknowledges support from Key Projects of Beijing Science and Technology (D141100004814002) and the Natural Scientific Foundation of Beijing (Z140001).

Helicobacter pylori colonizes the human stomach of approximately 50% of the world's population, and increases the risk of several gastric diseases. The goal of this study is to compare the gastric microbiota in pediatric patients with and without H. pylori colonization.We studied 51 children who underwent gastric endoscopy because of dyspeptic symptoms (18 H. pylori positive and 33 negative). Gastric biopsies were obtained for rapid urease test, culture, histology and DNA extraction. H. pylori was quantified by quantitative polymerase chain reaction and the gastric microbiome studied by V4-16S ribosomal RNA gene high-throughput sequencing.Bacterial richness and diversity of H. pylori-positive specimens were lower than those of negative, and both groups were clearly separated according to beta diversity. Taxonomic analysis confirmed that H. pylori-positive subjects had a higher relative abundance of Helicobacter genus (66.3%) than H. pylori-negative subjects (0.45%). Four phyla (proteobacteria, bacteroidetes, firmicutes and actinobacteria) accounted for97% of all reads in both groups. Within proteobacteria, gamma- and betaproteobacteria were the most abundant for H. pylori-negative patients, whereas epsilonproteobacteria was for H. pylori positive. H. pylori-positive patients were associated with low body mass index. In the group of underweight patients (body mass index,18.5), there were 46.1% of H. pylori-positive patients compared with 24% in the nonunderweight group (P = 0.049). Patients with active superficial gastritis in H. pylori-positive patients had the lowest alpha diversity (P = 0.035).We characterized the gastric microbiota for the first time in children with and without H. pylori and observed that when H. pylori is present, it tends to dominate the microbial community. In the H. pylori-negative patients, there was more relative abundance of gammaproteobacteria, betaproteobacteria, bacteroidia and clostridia classes and a higher bacterial richness and diversity.

Nowadays, due to worldwide water shortage, water utilities are forced to re-evaluate treated wastewater. Consequently, wastewater treatment plants need to conduct biomonitoring. Coking wastewater (CWW) has toxic, mutative and carcinogenic components with threatening effect on the environment. CWW was selected as a model for complex highly toxic industrial wastewater that should be treated. CWW from Egypt was treated in a nine-liter photobioreactor using an algal-bacterial system. The photobioreactor was operated for 154 days changing different parameters (toxic load and light duration) for optimization. Optimized conditions achieved significant reduction (45%) in the operation cost. The algal-bacterial system was monitored using chemical assays (chemical oxygen demand and phenol analysis), bioassays (phytotoxicity, Artemia toxicity, cytotoxicity, algalbacterial ratio and settleability) and Illumina-MiSeq sequencing of 16S rRNA gene. The algal-bacterial system detoxified (in terms of phytotoxicity, cytotoxicity and Anemia-toxicity) CWW introduced as influent through all phases. A significant difference was recorded in the microbial diversity between influent and effluent samples. Four phyla dominated influent samples; Proteobacteria (77%), Firmicutes (11%), Bacteroidetes (5%) and Deferribacteres (3%) compared to only two in effluent samples; Proteobacteria (66%) and Bacteroidetes (26%). The significant relative-abundance of versatile aromatic degraders (Comamonadaceae and Pseudomonadaceae families) in influent samples conformed to the nature of CWW. Microbial community shifted and promoted the activity of catabolically versatile and xenobiotics degrading families (Chitinophagaceae and Xanthomonadaceae). Co-culture of microalgae had a positive effect on the biodegrading bacteria that was reflected by enhanced treatment efficiency, significant increase in relative abundance of bacterial genera with cyanide-decomposing potential and negative effect on waterborne pathogens. (C) 2019 Elsevier B.V. All rights reserved.

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., This study was supported by the Spanish Ministry of Economy and Competitiveness (Grant No. BIO2015‐68711‐R) to Alex Mira. This study was supported by the Swedish Research Council (2016‐01698), the Swedish Heart and Lung Foundation (20140321 and 20170365) and the Cancer and Allergy Foundation to Maria C. Jenmalm. This study was supported by European Research Council (ERC‐starting grant 639226) to M. Carmen Collado.

Human breastmilk contains a variety of bacteria that are transmitted to the infant and have been suggested to contribute to gut microbiota development and immune maturation. However, the characterization of fungal organisms in milk from healthy mothers is currently unknown although their presence has been reported in the infant gut and also in milk from other mammals. Breastmilk samples from healthy lactating mothers (n = 65) within 1 month after birth were analyzed. Fungal presence was assessed by different techniques, including microscopy, growth and identification of cultured isolates, fungal load estimation by qPCR, and fungal composition using 28S rRNA gene high-throughput sequencing. In addition, milk macronutrients and human somatic cells were quantified by spectrophotometry and cytometry. qPCR data showed that 89% of samples had detectable levels of fungal DNA, at an estimated median load of 3,5 × 10

The objective of the present study was to determine cytokine thresholds derived from predictive models for the diagnosis of chronic periodontitis, differentiating by smoking status. Seventy-five periodontally healthy controls and 75 subjects affected by chronic periodontitis were recruited. Sixteen mediators were measured in gingival crevicular fluid (GCF) using multiplexed bead immunoassays. The models were obtained using binary logistic regression, distinguishing between non-smokers and smokers. The area under the curve (AUC) and numerous classification measures were obtained. Model curves were constructed graphically and the cytokine thresholds calculated for the values of maximum accuracy (ACC). There were three cytokine-based models and three cytokine ratio-based models, which presented with a bias-corrected AUC > 0.91 and > 0.83, respectively. These models were (cytokine thresholds in pg/ml for the median ACC using bootstrapping for smokers and non-smokers): IL1alpha (46099 and 65644); IL1beta (4732 and 5827); IL17A (11.03 and 17.13); IL1alpha/IL2 (4210 and 7118); IL1beta/IL2 (260 and 628); and IL17A/IL2 (0.810 and 1.919). IL1alpha, IL1beta and IL17A, and their ratios with IL2, are excellent diagnostic biomarkers in GCF for distinguishing periodontitis patients from periodontally healthy individuals. Cytokine thresholds in GCF with diagnostic potential are defined, showing that smokers have lower threshold values than non-smokers. This work was supported by the Instituto de Salud Carlos III (General Division of Evaluation and Research Promotion, Madrid, Spain) and co-financed by FEDER (“A way of making Europe”) under Grant ISCIII/PI17/01722, and the Consellería de Cultura, Educación e Ordenación Universitaria da Xunta de Galicia (Spain) under Grant ED431B 2017/029 and A. Regueira-Iglesias support ED481A-2017 SI

This study aimed to compare the bacterial viability and diversity of a substrate-formed biofilm (SF-biofilm) in situ to a supragingival tooth-formed biofilm (TF-biofilm) in the same group of individuals. The impact of the device/disc position and toothbrushing during the formation of SF-biofilm was also assessed. Two tests were run. In test 1, 15 volunteers wore two hemi-splints carrying six discs of human enamel, glass, and hydroxyapatite for 2 days, and were instructed to not perform any oral hygiene measure. Biofilm samples were collected from the substrates and the contralateral tooth and were analysed using CLSM. In five volunteers, half of the biofilm present on the discs and their contralateral teeth were scraped and analysed using 16S pyrosequencing. In test 2, the microscopic analysis was repeated only on the SF-biofilm samples, and the volunteers were allowed to brush their teeth. Multivariate analyses revealed that the donors had a significant effect on the composition of the biofilm, confirming its subject-dependent character. The bacterial composition of the SF-biofilm was similar to the TF-biofilm, with significant differential abundance detected in very few taxa of low abundance. The toothbrushing during the formation of SF-biofilm was the only factor that conditioned the thickness or bacterial viability This work was supported by the Instituto de Salud Carlos III (General Division of Evaluation and Research Promotion, Madrid, Spain) and co-financed by FEDER (‘A way of making Europe’) under Grant ISCIII/PI17/ 01722 and the Consellería de Cultura, Educación e Ordenación Universitaria da Xunta de Galicia (Spain) under Grant ED431B 2017/029. Specifically, the development of the Matlab toolbox called the Dentius Biofilm received financial support from the Consellería de Cultura, Educación e Ordenación Universitaria (accreditation 2016-2019) under Grant ED431G/08 SI

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 authors would like to acknowledge the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Programme (ERC Starting Grant, project no. 639226) and the grants AGL2015-707487-P and BIO2015-68711-R from the Spanish Ministerio de Economía, Industria y Competitividad (MINECO). MSR would like to acknowledge the support from the Ayudas para la Contratación de Personal Investigador en Formación (ACIF) program of Generalitat Valenciana with the European Social Fund (ESF).

Breast milk constitutes one of the most important sources of postnatal microbes. However, the influence of perinatal factors on the milk microbiome is still poorly understood. The aim of our study was to assess the impact of mode of delivery on the microbiome composition and diversity present in breast milk of healthy mothers. Mature milk samples (n=10) were taken from mothers after 1 month of exclusively breastfeeding. Microbiomes from milk samples were analyzed with 16S ribosomal RNA gene pyrosequencing and targeted quantitative polymerase chain reaction (PCR). Despite inter-individual variability in bacterial composition, The Principal Coordinates Analysis clearly separated milk microbiome from mothers with vaginal delivery (n=6) from those who undergo C-section (n=4). In addition, higher bacterial diversity and richness was found in milk samples from vaginal deliveries. Quantitative PCR data showed that higher levels of Bifidobacterium spp. were related significantly to lower levels of Staphylococcus spp. Despite the low sample size, our data suggest that mode of delivery has an important impact on milk microbiome composition. Further studies with larger sample sizes are needed to confirm these results and to understand the biological effects of C-section associated microbes on infant’s health.

The application of Spectral Imaging FISH to oral biofilm samples has permitted the direct, simultaneous observation of up to nine different bacterial taxa. This has revealed a complex yet organized microbial architecture, identifying the key microorganisms in the community and detecting the existing interspecies physical interactions at the micron scale.

An ability to monitor cells in real time has the potential to help elucidate the mechanisms underlying health and disease in diverse areas of biomedical research and clinical diagnostics. In the specific contexts of microbial, viral, and parasitic infections, being able to continuously track the structural and functional changes that occur in the host or pathogen cells can be used for clinical diagnosis, monitoring treatment efficacy, etc. The xCELLigence® Real-Time Cell Analysis (RTCA) instrument from ACEA Biosciences is a label-free microelectronic sensor-based technology that enables live cell analysis for these purposes. Using the principle of cellular impedance, RTCA dynamically monitors changes in cell number, cell size, cell-cell interactions (i.e., barrier function), and cell-substrate attachment quality to provide a wealth of information that is missed using traditional endpoint assays. In this chapter, we describe using RTCA to identify, quantify, and dynamically monitor bacterial, viral, and parasitic infections. Using recent publications, we demonstrate the application of RTCA for diverse aspects of microbiology, including bacterial toxin detection, virus-induced cytopathic effect quantification, neutralizing antibody detection, bacterial biofilm antibiotic susceptibility screening, and anthelmintic drug screening, as well as functional analysis of host immune cell activity. RTCA is shown to display great promise both for clinical detection of toxins/pathogens and for ex vivo theranostic purposes.

Several benefits have been described for red wine polyphenols and probiotic strains in the promotion of colonic metabolism and health. On the contrary, knowledge about their role in the management of oral health is still scarce. In this work, the antiadhesive capacity of selected red wine polyphenols and oenological extracts against the oral pathogens Porphyromonas gingivalis, Fusobacterium nucleatum, and Streptococcus mutans in an in vitro model of human gingival fibroblasts has been explored as well as their complementary action with the candidate oral probiotic Streptococcus dentisani. Results highlighted the antiadhesive capacity of caffeic and p-coumaric acids as well as grape seed and red wine oenological extracts. Both, caffeic and p-coumaric acids increased their inhibition potential against S. mutans adhesion when combined with S. dentisani. Additionally, UHPLC-MS/MS analysis demonstrated the oral metabolism of wine phenolics due to both, cellular and bacterial activity., This work was funded by the Spanish MINECO (Grant AGL2015-64522-C2-R project) and Comunidad de Madrid (Grant ALIBIRD-CM S2013/ABI-2728).

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 This work was supported by the non-profit organization Fundación Ramón Areces [CIVP16A1814] and by the grant ‘Axudas do Programa de Consolidación e Estructuración de Unidades de Investigación Competitivas (GPC)’ from the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia (ED431B2017/53). A.M. was supported by a predoctoral fellowship from the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia (ED481A2015/311) SI

Information on how the oral microbiome develops during early childhood and how external factors influence this ecological process is scarce. We used high-throughput sequencing to characterize bacterial composition in saliva samples collected at 3, 6, 12, 24 months and 7 years of age in 90 longitudinally followed children, for whom clinical, dietary and health data were collected. Bacterial composition patterns changed through time, starting with “early colonizers”, including Streptococcus and Veillonella; other bacterial genera such as Neisseria settled after 1 or 2 years of age. Dental caries development was associated with diverging microbial composition through time. Streptococcus cristatus appeared to be associated with increased risk of developing tooth decay and its role as potential biomarker of the disease should be studied with species-specific probes. Infants born by C-section had initially skewed bacterial content compared with vaginally delivered infants, but this was recovered with age. Shorter breastfeeding habits and antibiotic treatment during the first 2 years of age were associated with a distinct bacterial composition at later age. The findings presented describe oral microbiota development as an ecological succession where altered colonization pattern during the first year of life may have long-term consequences for child´s oral and systemic health., A.M.: Spanish Ministry of Economy and Competitiveness (grant no. BIO2015-68711-R). M.S.: The Research Council for the South-East Sweden (grant no: 79001). M.C.J.: The Swedish Research Council (2016-01698); the Swedish Heart and Lung Foundation (20140321); the Medical Research Council of Southeast Sweden (FORSS-573471); the Cancer and Allergy Foundation. M.C.C.: European Research Council (ERC-starting grant 639226).

Background: Allergic diseases have become a major public health problem in affluent societies. Microbial colonization early in life seems to be critical for instructing regulation on immune system maturation and allergy development in children. Even though the oral cavity is the first site of encounter between a majority of foreign antigens and the immune system, the influence of oral bacteria on allergy development has not yet been reported. Objective: We sought to determine the bacterial composition in longitudinally collected saliva samples during childhood in relation to allergy development. Methods: Illumina sequencing of the 16S rDNA gene was used to characterize the oral bacterial composition in saliva samples collected at 3, 6, 12, 24 months, and 7 years of age from children developing allergic symptoms and sensitization (n = 47) and children staying healthy (n = 33) up to 7 years of age. Results: Children developing allergic disease, particularly asthma, had lower diversity of salivary bacteria together with highly divergent bacterial composition at 7 years of age, showing a clearly altered oral microbiota in these individuals, likely as a consequence of an impaired immune system during infancy. Moreover, the relative amounts of several bacterial species, including increased abundance of Gemella haemolysans in children developing allergies and Lactobacillus gasseri and L. crispatus in healthy children, were distinctive during early infancy, likely influencing early immune maturation. Conclusion: Early changes in oral microbial composition seem to influence immune maturation and allergy development. Future experiments should test the probiotic potential of L. gasseri and L. crispatus isolates., Funding information: Alex Mira: Spanish Ministry of Economy and Competitiveness (grant no. BIO2015‐68711‐R). Maria C. Jenmalm: The Swedish Research Council (2016‐01698); the Swedish Heart and Lung Foundation (20140321); the Medical Research Council of Southeast Sweden (FORSS‐573471); and the Cancer and Allergy Foundation. Maria Carmen Collado: European Research Council (ERC‐starting grant 639226).

For decades, the sugar-fermenting, acidogenic species Streptococcus mutans has been considered the main causative agent of dental caries and most diagnostic and therapeutic strategies have been targeted toward this microorganism. However, recent DNA- and RNA-based studies from carious lesions have uncovered an extraordinarily diverse ecosystem where S. mutans accounts only a tiny fraction of the bacterial community. This supports the concept that consortia formed by multiple microorganisms act collectively, probably synergistically, to initiate and expand the cavity. Thus, antimicrobial therapies are not expected to be effective in the treatment of caries and other polymicrobial diseases that do not follow classical Koch's postulates.

Objectives: To Characterize the microbiota in COPD patients during exacerbation and posterior stabilization. Methods: A good quality expectorated sputum during exacerbation and posterior stabilization was obtained from each patient. A region between position 27 and 533 of the 16S rRNA gene was amplified and sequenced by NGS illumina miseq. Detection of Influenza A and B, MPV, RhV, EV, Parainfluenza A and RSV were also carried out in exacerbated samples. The sequences were filtered by length and quality and taxonomically assigned using rdp database. Only sequences over 400 bp were used for the study. Core diversity analyses were carried with QIIME v18.0. Results: 3D PCoA revealed that all 116 samples were grouped into four cluster (C). The cluster 1,2 and 3 corresponded to N=7, 11 and 3 exacerbated samples respectively. C1 was composed of Haemophilus (73% relative abundance). C2 was composed of Haemophilus (35%) and Streptococcus (19%) and C3 comprised Pseudomomonas (50%), Serratia (22%), Streptococcus (8%) mainly. In C4, all the stable and the rest of the exacerbated sputum samples (N=37) were found. Microbiota consisted mainly of Streptococcus (33%), Prevotella (10%), Veillonella (5%). In 10/37 exacerbated samples of this cluster, at least one respiratory virus was found. Conclusions: The microbiotas 1, 2 and 3 are specific for exacerbated patients and seems to be the cause of the exacerbation. The role of microbiota of C4 on exacerbated samples is less clear being the same as the stable patients. In 10/37 exacerbated patients with microbiota 4, exacerbation seems to be of viral origin and in 27/37 were probably due to a non-infectious etiology.

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.