Bacterial Diversity in Human Subgingival Plaque
15 Jun 2001-Journal of Bacteriology (American Society for Microbiology)-Vol. 183, Iss: 12, pp 3770-3783
TL;DR: The purpose of this study was to determine the bacterial diversity in the human subgingival plaque by using culture-independent molecular methods as part of an ongoing effort to obtain full 16S rRNA sequences for all cultivable and not-yet-cultivated species of human oral bacteria.
Abstract: The purpose of this study was to determine the bacterial diversity in the human subgingival plaque by using culture-independent molecular methods as part of an ongoing effort to obtain full 16S rRNA sequences for all cultivable and not-yet-cultivated species of human oral bacteria. Subgingival plaque was analyzed from healthy subjects and subjects with refractory periodontitis, adult periodontitis, human immunodeficiency virus periodontitis, and acute necrotizing ulcerative gingivitis. 16S ribosomal DNA (rDNA) bacterial genes from DNA isolated from subgingival plaque samples were PCR amplified with all-bacterial or selective primers and cloned into Escherichia coli. The sequences of cloned 16S rDNA inserts were used to determine species identity or closest relatives by comparison with sequences of known species. A total of 2,522 clones were analyzed. Nearly complete sequences of approximately 1,500 bases were obtained for putative new species. About 60% of the clones fell into 132 known species, 70 of which were identified from multiple subjects. About 40% of the clones were novel phylotypes. Of the 215 novel phylotypes, 75 were identified from multiple subjects. Known putative periodontal pathogens such as Porphyromonas gingivalis, Bacteroides forsythus, and Treponema denticola were identified from multiple subjects, but typically as a minor component of the plaque as seen in cultivable studies. Several phylotypes fell into two recently described phyla previously associated with extreme natural environments, for which there are no cultivable species. A number of species or phylotypes were found only in subjects with disease, and a few were found only in healthy subjects. The organisms identified only from diseased sites deserve further study as potential pathogens. Based on the sequence data in this study, the predominant subgingival microbial community consisted of 347 species or phylotypes that fall into 9 bacterial phyla. Based on the 347 species seen in our sample of 2,522 clones, we estimate that there are 68 additional unseen species, for a total estimate of 415 species in the subgingival plaque. When organisms found on other oral surfaces such as the cheek, tongue, and teeth are added to this number, the best estimate of the total species diversity in the oral cavity is approximately 500 species, as previously proposed.
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TL;DR: The purposes were to utilize culture-independent molecular techniques to extend the knowledge on the breadth of bacterial diversity in the healthy human oral cavity, including not-yet-cultivated bacteria species, and to determine the site and subject specificity of bacterial colonization.
Abstract: More than 700 bacterial species or phylotypes, of which over 50% have not been cultivated, have been detected in the oral cavity. Our purposes were (i) to utilize culture-independent molecular techniques to extend our knowledge on the breadth of bacterial diversity in the healthy human oral cavity, including not-yet-cultivated bacteria species, and (ii) to determine the site and subject specificity of bacterial colonization. Nine sites from five clinically healthy subjects were analyzed. Sites included tongue dorsum, lateral sides of tongue, buccal epithelium, hard palate, soft palate, supragingival plaque of tooth surfaces, subgingival plaque, maxillary anterior vestibule, and tonsils. 16S rRNA genes from sample DNA were amplified, cloned, and transformed into Escherichia coli. Sequences of 16S rRNA genes were used to determine species identity or closest relatives. In 2,589 clones, 141 predominant species were detected, of which over 60% have not been cultivated. Thirteen new phylotypes were identified. Species common to all sites belonged to the genera Gemella, Granulicatella, Streptococcus, and Veillonella. While some species were subject specific and detected in most sites, other species were site specific. Most sites possessed 20 to 30 different predominant species, and the number of predominant species from all nine sites per individual ranged from 34 to 72. Species typically associated with periodontitis and caries were not detected. There is a distinctive predominant bacterial flora of the healthy oral cavity that is highly diverse and site and subject specific. It is important to fully define the human microflora of the healthy oral cavity before we can understand the role of bacteria in oral disease.
2,683 citations
Cites background or result from "Bacterial Diversity in Human Subgin..."
...CAG-3 ; reverse primer, 5 -GAA GGA GGT GWT CCA RCC GCA-3 ) (21)....
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...As we have previously asserted (21), to rigorously assess the association of specific species or phylotypes with oral health or disease, it is necessary to analyze larger numbers of clinical samples for the levels of essentially all oral bacteria in wellcontrolled clinical studies....
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...By using culture-independent molecular methods, we previously detected over 500 species or phylotypes in subgingival plaque of healthy subjects and subjects with periodontal diseases (21), necrotizing ulcerative periodontitis in human immunodeficiency virus-positive subjects (23), dental plaque in children with rampant caries (3), noma (22), and on the tongue dorsum of subjects with and without halitosis (15)....
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...As determined in our previous studies using culture-independent molecular techniques (15, 21, 22), over 60% of the bacterial flora was represented by not-yet-cultivated phylotypes....
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TL;DR: The HOMD is the first curated description of a human-associated microbiome and provides tools for use in understanding the role of the microbiome in health and disease.
Abstract: The human oral cavity contains a number of different habitats, including the teeth, gingival sulcus, tongue, cheeks, hard and soft palates, and tonsils, which are colonized by bacteria. The oral microbiome is comprised of over 600 prevalent taxa at the species level, with distinct subsets predominating at different habitats. The oral microbiome has been extensively characterized by cultivation and culture-independent molecular methods such as 16S rRNA cloning. Unfortunately, the vast majority of unnamed oral taxa are referenced by clone numbers or 16S rRNA GenBank accession numbers, often without taxonomic anchors. The first aim of this research was to collect 16S rRNA gene sequences into a curated phylogeny-based database, the Human Oral Microbiome Database (HOMD), and make it web accessible (www.homd.org). The HOMD includes 619 taxa in 13 phyla, as follows: Actinobacteria, Bacteroidetes, Chlamydiae, Chloroflexi, Euryarchaeota, Firmicutes, Fusobacteria, Proteobacteria, Spirochaetes, SR1, Synergistetes, Tenericutes, and TM7. The second aim was to analyze 36,043 16S rRNA gene clones isolated from studies of the oral microbiota to determine the relative abundance of taxa and identify novel candidate taxa. The analysis identified 1,179 taxa, of which 24% were named, 8% were cultivated but unnamed, and 68% were uncultivated phylotypes. Upon validation, 434 novel, nonsingleton taxa will be added to the HOMD. The number of taxa needed to account for 90%, 95%, or 99% of the clones examined is 259, 413, and 875, respectively. The HOMD is the first curated description of a human-associated microbiome and provides tools for use in understanding the role of the microbiome in health and disease.
2,598 citations
Cites background from "Bacterial Diversity in Human Subgin..."
...oral taxon 345 as clone X112 in phylum OP11 (47)....
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...It has been estimated that less than half of the bacterial species present in the oral cavity can be cultivated using anaerobic microbiological methods and that there are likely 500 to 700 common oral species (47)....
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...ulcerative gingivitis (ANUG) (47); dental plaque from children with caries (7);...
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...In brief, 16S rRNA gene clone libraries were created from and analyzed in unpublished studies and the following published studies: treponemes in a subject with severe destructive periodontitis (10); treponemes from several subjects with periodontitis and acute necrotizing ulcerative gingivitis (ANUG) (18); subgingival plaque from healthy subjects and subjects with periodontitis, HIV periodontitis, and acute necrotizing ulcerative gingivitis (ANUG) (47); dental plaque from children with caries (7); endodontic lesions (45); subjects with advanced noma lesions (49); subjects with necrotizing ulcerative periodontitis in HIV-positive subjects (1, 50); dorsum tongue microbiota in subjects with halitosis (36); dental caries in adults (44); normal biota of healthy subjects at subgingival, supragingival, dorsal tongue, ventral tongue, hard palate, vestibule, and tonsil sites (2); periodontitis in adults (15); aggressive periodontitis (22); caries-active and caries-free twins (14); root caries in elderly subjects (52); ventilator-associated pneumonia (5)....
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...Earlier publications referred to SR1 sp. oral taxon 345 as clone X112 in phylum OP11 (47)....
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TL;DR: In this article, the authors updated the recommendations by the American Heart Association (AHA) for the prevention of infective endocarditis that were last published in 1997, and the purpose of this statement is to update the recommendations.
Abstract: Background— The purpose of this statement is to update the recommendations by the American Heart Association (AHA) for the prevention of infective endocarditis that were last published in 1997. Met...
2,132 citations
TL;DR: The committee concluded that only an extremely small number of cases of IE might be prevented by antibiotic prophylaxis for dental procedures even if suchProphylactic therapy were 100 percent effective.
Abstract: Background The purpose of this statement is to update the recommendations by the American Heart Association (AHA) for the prevention of infective endocarditis, which were last published in 1997. Methods and Results A writing group appointed by the AHA for their expertise in prevention and treatment of infective endocarditis (IE) with liaison members representing the American Dental Association, the Infectious Diseases Society of America and the American Academy of Pediatrics. The writing group reviewed input from national and international experts on IE. The recommendations in this document reflect analyses of relevant literature regarding procedure-related bacteremia and IE; in vitro susceptibility data of the most common microorganisms, which cause IE; results of prophylactic studies in animal models of experimental endocarditis; and retrospective and prospective studies of prevention of IE. MEDLINE database searches from 1950 through 2006 were done for English language articles using the following search terms: endocarditis, infective endocarditis, prophylaxis, prevention, antibiotic, antimicrobial, pathogens, organisms, dental, gastrointestinal, genitourinary, streptococcus, enterococcus, staphylococcus, respiratory, dental surgery, pathogenesis, vaccine, immunization and bacteremia. The reference lists of the identified articles were also searched. The writing group also searched the AHA online library. The American College of Cardiology/AHA classification of recommendations and levels of evidence for practice guidelines were used. The article subsequently was reviewed by outside experts not affiliated with the writing group and by the AHA Science Advisory and Coordinating Committee. Conclusions The major changes in the updated recommendations include the following. (1) The committee concluded that only an extremely small number of cases of IE might be prevented by antibiotic prophylaxis for dental procedures even if such prophylactic therapy were 100 percent effective. (2) IE prophylaxis for dental procedures should be recommended only for patients with underlying cardiac conditions associated with the highest risk of adverse outcome from IE. (3) For patients with these underlying cardiac conditions, prophylaxis is recommended for all dental procedures that involve manipulation of gingival tissue or the periapical region of teeth or perforation of the oral mucosa. (4) Prophylaxis is not recommended based solely on an increased lifetime risk of acquisition of IE. (5) Administration of antibiotics solely to prevent endocarditis is not recommended for patients who undergo a genitourinary or gastrointestinal tract procedure. These changes are intended to define more clearly when IE prophylaxis is or is not recommended and to provide more uniform and consistent global recommendations.
1,674 citations
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"Bacterial Diversity in Human Subgin..." refers background in this paper
...The definition of a species is controversial, particularly when only molecular sequence data exist (14, 46)....
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TL;DR: The purpose of the present investigation was to attempt to define communities using data from large numbers of plaque samples and different clustering and ordination techniques, which related strikingly to clinical measures of periodontal disease particularly pocket depth and bleeding on probing.
Abstract: It has been recognized for some time that bacterial species exist in complexes in subgingival plaque. The purpose of the present investigation was to attempt to define such communities using data from large numbers of plaque samples and different clustering and ordination techniques. Subgingival plaque samples were taken from the mesial aspect of each tooth in 185 subjects (mean age 51 +/- 16 years) with (n = 160) or without (n = 25) periodontitis. The presence and levels of 40 subgingival taxa were determined in 13,261 plaque samples using whole genomic DNA probes and checkerboard DNA-DNA hybridization. Clinical assessments were made at 6 sites per tooth at each visit. Similarities between pairs of species were computed using phi coefficients and species clustered using an averaged unweighted linkage sort. Community ordination was performed using principal components analysis and correspondence analysis. 5 major complexes were consistently observed using any of the analytical methods. One complex consisted of the tightly related group: Bacteroides forsythus, Porphyromonas gingivalis and Treponema denticola. The 2nd complex consisted of a tightly related core group including members of the Fusobacterium nucleatum/periodonticum subspecies, Prevotella intermedia, Prevotella nigrescens and Peptostreptococcus micros. Species associated with this group included: Eubacterium nodatum, Campylobacter rectus, Campylobacter showae, Streptococcus constellatus and Campylobacter gracilis. The 3rd complex consisted of Streptococcus sanguis, S. oralis, S. mitis, S. gordonii and S. intermedius. The 4th complex was comprised of 3 Capnocytophaga species, Campylobacter concisus, Eikenella corrodens and Actinobacillus actinomycetemcomitans serotype a. The 5th complex consisted of Veillonella parvula and Actinomyces odontolyticus. A. actinomycetemcomitans serotype b, Selenomonas noxia and Actinomyces naeslundii genospecies 2 (A. viscosus) were outliers with little relation to each other and the 5 major complexes. The 1st complex related strikingly to clinical measures of periodontal disease particularly pocket depth and bleeding on probing.
4,143 citations
"Bacterial Diversity in Human Subgin..." refers background or methods in this paper
...Campylobacter rectus has long been associated with periodontal disease (44)....
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...(44) was based on analysis of the microbial community of over 13,000 plaque samples from 185 subjects by using DNA probes in checkerboard hybridization assays....
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...The remaining cultivable species of this phylum have not been typically considered periodontal pathogens (44)....
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...Eubacterium saphenum and species of Megasphaera have not been considered as putative periodontal pathogens (44)....
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