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      Nitrate as a potential prebiotic for the oral microbiome

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          Abstract

          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 < 0.01). Significantly higher levels of the oral health-associated nitrate-reducing genera Neisseria (3.1 ×) and Rothia (2.9 ×) were detected in the nitrate condition already after 5 h (both p < 0.01), while several caries-associated genera ( Streptococcus, Veillonella and Oribacterium) and halitosis- and periodontitis-associated genera ( Porphyromonas, Fusobacterium, Leptotrichia, Prevotella, and Alloprevotella) were significantly reduced (p < 0.05 at 5 h and/or 9 h). In conclusion, the addition of nitrate to oral communities led to rapid modulation of microbiome composition and activity that could be beneficial for the host (i.e., increasing eubiosis or decreasing dysbiosis). Nitrate should thus be investigated as a potential prebiotic for oral health.

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          The subgingival microbiome in health and periodontitis and its relationship with community biomass and inflammation.

          The goals of this study were to better understand the ecology of oral subgingival communities in health and periodontitis and elucidate the relationship between inflammation and the subgingival microbiome. Accordingly, we used 454-pyrosequencing of 16S rRNA gene libraries and quantitative PCR to characterize the subgingival microbiome of 22 subjects with chronic periodontitis. Each subject was sampled at two sites with similar periodontal destruction but differing in the presence of bleeding, a clinical indicator of increased inflammation. Communities in periodontitis were also compared with those from 10 healthy individuals. In periodontitis, presence of bleeding was not associated with different α-diversity or with a distinct microbiome, however, bleeding sites showed higher total bacterial load. In contrast, communities in health and periodontitis largely differed, with higher diversity and biomass in periodontitis. Shifts in community structure from health to periodontitis resembled ecological succession, with emergence of newly dominant taxa in periodontitis without replacement of primary health-associated species. That is, periodontitis communities had higher proportions of Spirochetes, Synergistetes, Firmicutes and Chloroflexi, among other taxa, while the proportions of Actinobacteria, particularly Actinomyces, were higher in health. Total Actinomyces load, however, remained constant from health to periodontitis. Moreover, an association existed between biomass and community structure in periodontitis, with the proportion of specific taxa correlating with bacterial load. Our study provides a global-scale framework for the ecological events in subgingival communities that underline the development of periodontitis. The association, in periodontitis, between inflammation, community biomass and community structure and their role in disease progression warrant further investigation.
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            The oral metagenome in health and disease.

            The oral cavity of humans is inhabited by hundreds of bacterial species and some of them have a key role in the development of oral diseases, mainly dental caries and periodontitis. We describe for the first time the metagenome of the human oral cavity under health and diseased conditions, with a focus on supragingival dental plaque and cavities. Direct pyrosequencing of eight samples with different oral-health status produced 1 Gbp of sequence without the biases imposed by PCR or cloning. These data show that cavities are not dominated by Streptococcus mutans (the species originally identified as the ethiological agent of dental caries) but are in fact a complex community formed by tens of bacterial species, in agreement with the view that caries is a polymicrobial disease. The analysis of the reads indicated that the oral cavity is functionally a different environment from the gut, with many functional categories enriched in one of the two environments and depleted in the other. Individuals who had never suffered from dental caries showed an over-representation of several functional categories, like genes for antimicrobial peptides and quorum sensing. In addition, they did not have mutans streptococci but displayed high recruitment of other species. Several isolates belonging to these dominant bacteria in healthy individuals were cultured and shown to inhibit the growth of cariogenic bacteria, suggesting the use of these commensal bacterial strains as probiotics to promote oral health and prevent dental caries.
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              Inorganic nitrate is a possible source for systemic generation of nitric oxide.

              Nitrate and nitrite have been considered stable inactive end products of nitric oxide (NO). While several recent studies now imply that nitrite can be reduced to bioactive NO again, the more stable anion nitrate is still considered to be biologically inert. Nitrate is concentrated in saliva, where a part of it is reduced to nitrite by bacterial nitrate reductases. We tested if ingestion of inorganic nitrate would affect the salivary and systemic levels of nitrite and S-nitrosothiols, both considered to be circulating storage pools for NO. Levels of nitrate, nitrite, and S-nitrosothiols were measured in plasma, saliva, and urine before and after ingestion of sodium nitrate (10 mg/kg). Nitrate levels increased greatly in saliva, plasma, and urine after the nitrate load. Salivary S-nitrosothiols also increased, but plasma levels remained unchanged. A 4-fold increase in plasma nitrite was observed after nitrate ingestion. If, however, the test persons avoided swallowing after the nitrate load, the increase in plasma nitrite was prevented, thereby illustrating its salivary origin. We show that nitrate is a substrate for systemic generation of nitrite. There are several pathways to further reduce this nitrite to NO. These results challenge the dogma that nitrate is biologically inert and instead suggest that a complete reverse pathway for generation of NO from nitrate exists.
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                Author and article information

                Contributors
                mira_ale@gva.es
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                30 July 2020
                30 July 2020
                2020
                : 10
                : 12895
                Affiliations
                GRID grid.428862.2, Department of Health and Genomics, Center for Advanced Research in Public Health, , FISABIO Foundation, ; Avenida de Catalunya 21, 46020 Valencia, Spain
                Article
                69931
                10.1038/s41598-020-69931-x
                7393384
                32732931
                425902fc-620b-45a6-b6ed-4f590cc12fab
                © The Author(s) 2020

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 14 May 2020
                : 21 July 2020
                Funding
                Funded by: Ministerio de Ciencia e Innovación
                Award ID: Bio2015-68711-R
                Award ID: RTI2018-102032-B-I00
                Award Recipient :
                Funded by: European Regional Development Fund
                Award ID: RTI2018-102032-B-I00
                Award Recipient :
                Funded by: Instituto de Salud Carlos III
                Award ID: DTS16/00230
                Award Recipient :
                Categories
                Article
                Custom metadata
                © The Author(s) 2020

                Uncategorized
                microbial ecology,microbiome,dna sequencing,next-generation sequencing,biofilms
                Uncategorized
                microbial ecology, microbiome, dna sequencing, next-generation sequencing, biofilms

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