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      Salivary Microbiome Diversity in Caries-Free and Caries-Affected Children

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          Abstract

          Dental caries (tooth decay) is an infectious disease. Its etiology is not fully understood from the microbiological perspective. This study characterizes the diversity of microbial flora in the saliva of children with and without dental caries. Children (3–4 years old) with caries ( n = 20) and without caries ( n = 20) were recruited. Unstimulated saliva (2 mL) was collected from each child and the total microbial genomic DNA was extracted. DNA amplicons of the V3-V4 hypervariable region of the bacterial 16S rRNA gene were generated and subjected to Illumina Miseq sequencing. A total of 17 phyla, 26 classes, 40 orders, 80 families, 151 genera, and 310 bacterial species were represented in the saliva samples. There was no significant difference in the microbiome diversity between caries-affected and caries-free children ( p > 0.05). The relative abundance of several species ( Rothia dentocariosa, Actinomyces graevenitzii, Veillonella sp. oral taxon 780, Prevotella salivae, and Streptococcus mutans) was higher in the caries-affected group than in the caries-free group ( p < 0.05). Fusobacterium periodonticum and Leptotrichia sp. oral clone FP036 were more abundant in caries-free children than in caries-affected children ( p < 0.05). The salivary microbiome profiles of caries-free and caries-affected children were similar. Salivary counts of certain bacteria such as R. dentocariosa and F. periodonticum may be useful for screening/assessing children’s risk of developing caries.

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          Most cited references26

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          Defining the healthy "core microbiome" of oral microbial communities

          Background Most studies examining the commensal human oral microbiome are focused on disease or are limited in methodology. In order to diagnose and treat diseases at an early and reversible stage an in-depth definition of health is indispensible. The aim of this study therefore was to define the healthy oral microbiome using recent advances in sequencing technology (454 pyrosequencing). Results We sampled and sequenced microbiomes from several intraoral niches (dental surfaces, cheek, hard palate, tongue and saliva) in three healthy individuals. Within an individual oral cavity, we found over 3600 unique sequences, over 500 different OTUs or "species-level" phylotypes (sequences that clustered at 3% genetic difference) and 88 - 104 higher taxa (genus or more inclusive taxon). The predominant taxa belonged to Firmicutes (genus Streptococcus, family Veillonellaceae, genus Granulicatella), Proteobacteria (genus Neisseria, Haemophilus), Actinobacteria (genus Corynebacterium, Rothia, Actinomyces), Bacteroidetes (genus Prevotella, Capnocytophaga, Porphyromonas) and Fusobacteria (genus Fusobacterium). Each individual sample harboured on average 266 "species-level" phylotypes (SD 67; range 123 - 326) with cheek samples being the least diverse and the dental samples from approximal surfaces showing the highest diversity. Principal component analysis discriminated the profiles of the samples originating from shedding surfaces (mucosa of tongue, cheek and palate) from the samples that were obtained from solid surfaces (teeth). There was a large overlap in the higher taxa, "species-level" phylotypes and unique sequences among the three microbiomes: 84% of the higher taxa, 75% of the OTUs and 65% of the unique sequences were present in at least two of the three microbiomes. The three individuals shared 1660 of 6315 unique sequences. These 1660 sequences (the "core microbiome") contributed 66% of the reads. The overlapping OTUs contributed to 94% of the reads, while nearly all reads (99.8%) belonged to the shared higher taxa. Conclusions We obtained the first insight into the diversity and uniqueness of individual oral microbiomes at a resolution of next-generation sequencing. We showed that a major proportion of bacterial sequences of unrelated healthy individuals is identical, supporting the concept of a core microbiome at health.
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            Fast UniFrac: Facilitating high-throughput phylogenetic analyses of microbial communities including analysis of pyrosequencing and PhyloChip data

            Next-generation sequencing techniques, and PhyloChip, have made simultaneous phylogenetic analyses of hundreds of microbial communities possible. Insight into community structure has been limited by the inability to integrate and visualize such vast datasets. Fast UniFrac overcomes these issues, allowing integration of larger numbers of sequences and samples into a single analysis. Its new array-based implementation offers orders of magnitude improvements over the original version. New 3D visualization of principal coordinates analysis (PCoA) results, with the option to view multiple coordinate axes simultaneously, provides a powerful way to quickly identify patterns that relate vast numbers of microbial communities. We demonstrate the potential of Fast UniFrac using examples from three data types: Sanger-sequencing studies of diverse free-living and animal-associated bacterial assemblages and from the gut of obese humans as they diet, pyrosequencing data integrated from studies of the human hand and gut, and PhyloChip data from a study of citrus pathogens. We show that a Fast UniFrac analysis using a reference tree recaptures patterns that could not be detected without considering phylogenetic relationships and that Fast UniFrac, coupled with BLAST-based sequence assignment, can be used to quickly analyze pyrosequencing runs containing hundreds of thousands of sequences, revealing patterns relating human and gut samples. Finally, we show that the application of Fast UniFrac to PhyloChip data could identify well-defined subcategories associated with infection. Together, these case studies point the way towards a broad range of applications and demonstrate some of the new features of Fast UniFrac.
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              Microbial ecology of dental plaque and its significance in health and disease.

              P.D. Marsh (1994)
              Dental plaque forms naturally on teeth and is of benefit to the host by helping to prevent colonization by exogenous species. The bacterial composition of plaque remains relatively stable despite regular exposure to minor environmental perturbations. This stability (microbial homeostasis) is due in part to a dynamic balance of both synergistic and antagonistic microbial interactions. However, homeostasis can break down, leading to shifts in the balance of the microflora, thereby predisposing sites to disease. For example, the frequent exposure of plaque to low pH leads to inhibition of acid-sensitive species and the selection of organisms with an aciduric physiology, such as mutans streptococci and lactobacilli. Similarly, plaque accumulation around the gingival margin leads to an inflammatory host response and an increased flow of gingival crevicular fluid. The subgingival microflora shifts from being mainly Gram-positive to being comprised of increased levels of obligately anaerobic, asaccharolytic Gram-negative organisms. It is proposed that disease can be prevented or treated not only by targeting the putative pathogens but also by interfering with the processes that drive the breakdown in homeostasis. Thus, the rate of acid production following sugar intake could be reduced by fluoride, alternative sweeteners, and low concentrations of antimicrobial agents, while oxygenating or redox agents could raise the Eh of periodontal pockets and prevent the growth and metabolism of obligately anaerobic species. These views have been incorporated into a modified hypothesis (the "ecological plaque hypothesis") to explain the relationship between the plaque microflora and the host in health and disease, and to identify new strategies for disease prevention.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                Int J Mol Sci
                Int J Mol Sci
                ijms
                International Journal of Molecular Sciences
                MDPI
                1422-0067
                25 November 2016
                December 2016
                : 17
                : 12
                : 1978
                Affiliations
                Faculty of Dentistry, The University of Hong Kong, Prince Philip Dental Hospital, 34 Hospital Road, Sai Ying Pun, Hong Kong, China; shanj@ 123456hku.hk (S.J.); ljjin@ 123456hku.hk (L.J.); hrdplcm@ 123456hku.hk (E.C.M.L.)
                Author notes
                [* ]Correspondence: gaoxl@ 123456hku.hk ; Tel.: +852-2859-0481; Fax: +852-2858-7874
                Article
                ijms-17-01978
                10.3390/ijms17121978
                5187778
                27898021
                27b0fdeb-67d8-4496-a360-a12fe4fa9d65
                © 2016 by the authors; licensee MDPI, Basel, Switzerland.

                This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 20 October 2016
                : 21 November 2016
                Categories
                Article

                Molecular biology
                etiology,16s rrna gene,early childhood caries,microbiome,saliva,children
                Molecular biology
                etiology, 16s rrna gene, early childhood caries, microbiome, saliva, children

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