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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Intraindividual variation in core microbiota in peri-implantitis and periodontitis</title><author><name sortKey="Maruyama, Noriko" sort="Maruyama, Noriko" uniqKey="Maruyama N" first="Noriko" last="Maruyama">Noriko Maruyama</name><affiliation><nlm:aff id="a1"><institution>Department of Periodontology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University</institution></nlm:aff></affiliation></author><author><name sortKey="Maruyama, Fumito" sort="Maruyama, Fumito" uniqKey="Maruyama F" first="Fumito" last="Maruyama">Fumito Maruyama</name><affiliation><nlm:aff id="a2"><institution>Department of Microbiology, Graduate School of Medicine, Kyoto University</institution></nlm:aff></affiliation></author><author><name sortKey="Takeuchi, Yasuo" sort="Takeuchi, Yasuo" uniqKey="Takeuchi Y" first="Yasuo" last="Takeuchi">Yasuo Takeuchi</name><affiliation><nlm:aff id="a1"><institution>Department of Periodontology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University</institution></nlm:aff></affiliation></author><author><name sortKey="Aikawa, Chihiro" sort="Aikawa, Chihiro" uniqKey="Aikawa C" first="Chihiro" last="Aikawa">Chihiro Aikawa</name><affiliation><nlm:aff id="a2"><institution>Department of Microbiology, Graduate School of Medicine, Kyoto University</institution></nlm:aff></affiliation></author><author><name sortKey="Izumi, Yuichi" sort="Izumi, Yuichi" uniqKey="Izumi Y" first="Yuichi" last="Izumi">Yuichi Izumi</name><affiliation><nlm:aff id="a1"><institution>Department of Periodontology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University</institution></nlm:aff></affiliation></author><author><name sortKey="Nakagawa, Ichiro" sort="Nakagawa, Ichiro" uniqKey="Nakagawa I" first="Ichiro" last="Nakagawa">Ichiro Nakagawa</name><affiliation><nlm:aff id="a2"><institution>Department of Microbiology, Graduate School of Medicine, Kyoto University</institution></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">25308100</idno><idno type="pmc">4194447</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4194447</idno><idno type="RBID">PMC:4194447</idno><idno type="doi">10.1038/srep06602</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">000071</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000071</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Intraindividual variation in core microbiota in peri-implantitis and periodontitis</title><author><name sortKey="Maruyama, Noriko" sort="Maruyama, Noriko" uniqKey="Maruyama N" first="Noriko" last="Maruyama">Noriko Maruyama</name><affiliation><nlm:aff id="a1"><institution>Department of Periodontology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University</institution></nlm:aff></affiliation></author><author><name sortKey="Maruyama, Fumito" sort="Maruyama, Fumito" uniqKey="Maruyama F" first="Fumito" last="Maruyama">Fumito Maruyama</name><affiliation><nlm:aff id="a2"><institution>Department of Microbiology, Graduate School of Medicine, Kyoto University</institution></nlm:aff></affiliation></author><author><name sortKey="Takeuchi, Yasuo" sort="Takeuchi, Yasuo" uniqKey="Takeuchi Y" first="Yasuo" last="Takeuchi">Yasuo Takeuchi</name><affiliation><nlm:aff id="a1"><institution>Department of Periodontology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University</institution></nlm:aff></affiliation></author><author><name sortKey="Aikawa, Chihiro" sort="Aikawa, Chihiro" uniqKey="Aikawa C" first="Chihiro" last="Aikawa">Chihiro Aikawa</name><affiliation><nlm:aff id="a2"><institution>Department of Microbiology, Graduate School of Medicine, Kyoto University</institution></nlm:aff></affiliation></author><author><name sortKey="Izumi, Yuichi" sort="Izumi, Yuichi" uniqKey="Izumi Y" first="Yuichi" last="Izumi">Yuichi Izumi</name><affiliation><nlm:aff id="a1"><institution>Department of Periodontology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University</institution></nlm:aff></affiliation></author><author><name sortKey="Nakagawa, Ichiro" sort="Nakagawa, Ichiro" uniqKey="Nakagawa I" first="Ichiro" last="Nakagawa">Ichiro Nakagawa</name><affiliation><nlm:aff id="a2"><institution>Department of Microbiology, Graduate School of Medicine, Kyoto University</institution></nlm:aff></affiliation></author></analytic><series><title level="j">Scientific Reports</title><idno type="eISSN">2045-2322</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The oral microbiota change dramatically with each part of the oral cavity, even within the same mouth. Nevertheless, the microbiota associated with peri-implantitis and periodontitis have been considered the same. To improve our knowledge of the different communities of complex oral microbiota, we compared the microbial features between peri-implantitis and periodontitis in 20 patients with both diseases. Although the clinical symptoms of peri-implantitis were similar to those of periodontitis, the core microbiota of the diseases differed. Correlation analysis revealed the specific microbial co-occurrence patterns and found some of the species were associated with the clinical parameters in a disease-specific manner. The proportion of <italic>Prevotella nigrescens</italic> was significantly higher in peri-implantitis than in periodontitis, while the proportions of <italic>Peptostreptococcaceae</italic> sp. and <italic>Desulfomicrobium orale</italic> were significantly higher in periodontitis than in peri-implantitis. The severity of the peri-implantitis was also species-associated, including with an uncultured <italic>Treponema</italic> sp. that correlated to 4 clinical parameters. These results indicate that peri-implantitis and periodontitis are both polymicrobial infections with different causative pathogens. Our study provides a framework for the ecologically different bacterial communities between peri-implantitis and periodontitis, and it will be useful for further studies to understand the complex microbiota and pathogenic mechanisms of oral polymicrobial diseases.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Dewhirst, F E" uniqKey="Dewhirst F">F. E. Dewhirst</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Seymour, G J" uniqKey="Seymour G">G. J. Seymour</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhou, Y" uniqKey="Zhou Y">Y. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Sci Rep</journal-id><journal-id journal-id-type="iso-abbrev">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type="epub">2045-2322</issn><publisher><publisher-name>Nature Publishing Group</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">25308100</article-id><article-id pub-id-type="pmc">4194447</article-id><article-id pub-id-type="pii">srep06602</article-id><article-id pub-id-type="doi">10.1038/srep06602</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Intraindividual variation in core microbiota in peri-implantitis and periodontitis</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Maruyama</surname><given-names>Noriko</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Maruyama</surname><given-names>Fumito</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Takeuchi</surname><given-names>Yasuo</given-names></name><xref ref-type="corresp" rid="c2">b</xref><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Aikawa</surname><given-names>Chihiro</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Izumi</surname><given-names>Yuichi</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Nakagawa</surname><given-names>Ichiro</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><aff id="a1"><label>1</label><institution>Department of Periodontology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University</institution></aff><aff id="a2"><label>2</label><institution>Department of Microbiology, Graduate School of Medicine, Kyoto University</institution></aff></contrib-group><author-notes><corresp id="c1"><label>a</label><email>maruyama.fumito.5e@kyoto-u.ac.jp</email></corresp><corresp id="c2"><label>b</label><email>takeuchi.peri@tmd.ac.jp</email></corresp></author-notes><pub-date pub-type="epub"><day>13</day><month>10</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>4</volume><elocation-id>6602</elocation-id><history><date date-type="received"><day>24</day><month>04</month><year>2014</year></date><date date-type="accepted"><day>19</day><month>09</month><year>2014</year></date></history><permissions><copyright-statement>Copyright © 2014, Macmillan Publishers Limited. All rights reserved</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Macmillan Publishers Limited. All rights reserved</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by-nc-nd/4.0/"><pmc-comment>author-paid</pmc-comment>
<license-p>This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder in order to reproduce the material. To view a copy of this license, visit <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc-nd/4.0/">http://creativecommons.org/licenses/by-nc-nd/4.0/</ext-link></license-p></license></permissions><abstract><p>The oral microbiota change dramatically with each part of the oral cavity, even within the same mouth. Nevertheless, the microbiota associated with peri-implantitis and periodontitis have been considered the same. To improve our knowledge of the different communities of complex oral microbiota, we compared the microbial features between peri-implantitis and periodontitis in 20 patients with both diseases. Although the clinical symptoms of peri-implantitis were similar to those of periodontitis, the core microbiota of the diseases differed. Correlation analysis revealed the specific microbial co-occurrence patterns and found some of the species were associated with the clinical parameters in a disease-specific manner. The proportion of <italic>Prevotella nigrescens</italic> was significantly higher in peri-implantitis than in periodontitis, while the proportions of <italic>Peptostreptococcaceae</italic> sp. and <italic>Desulfomicrobium orale</italic> were significantly higher in periodontitis than in peri-implantitis. The severity of the peri-implantitis was also species-associated, including with an uncultured <italic>Treponema</italic> sp. that correlated to 4 clinical parameters. These results indicate that peri-implantitis and periodontitis are both polymicrobial infections with different causative pathogens. Our study provides a framework for the ecologically different bacterial communities between peri-implantitis and periodontitis, and it will be useful for further studies to understand the complex microbiota and pathogenic mechanisms of oral polymicrobial diseases.</p></abstract></article-meta></front><body><p>The oral microbiota comprises hundreds of prevalent taxa<xref ref-type="bibr" rid="b1">1</xref> that are also associated with oral and systematic diseases (e.g., diabetes and cardiovascular disease)<xref ref-type="bibr" rid="b2">2</xref>. Therefore, the oral microbiota are as important to human health as are the gut microbiota. The NIH launched the Human Microbiome Project (HMP), which aims to more fully characterize the human microbiota and to address their role in health and disease<xref ref-type="bibr" rid="b3">3</xref>. The HMP classifies oral specimens as coming from the saliva, buccal mucosa, hard palate, palatine tonsil, sub-gingiva, supra-gingiva, throat, or tongue dorsum, and their data indicate that healthy sites are dominated by genera such as <italic>Streptococcus</italic>, <italic>Prevotella</italic>, <italic>Haemophilus</italic>, <italic>Fusobacterium,</italic> and <italic>Veillonella</italic>. However, the microbial composition and abundances are different in each of these habitats. Of the body's habitats, the oral habitat has the most stable microbiota with the highest alpha diversity, suggesting that the oral environment is likely to be complex<xref ref-type="bibr" rid="b3">3</xref>. The ecology of the microbial communities of dental caries and periodontitis have been studied<xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref>. The oral disease peri-implantitis has become a concern recently because several studies have suggested that patients with a history of periodontitis are susceptible to peri-implant disease and are likely to undergo a bacterial shift from periodontitis to the peri-implant<xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref>. Although peri-implantitis is an oral microbial infection, information is limited, and the microbiota have been treated as the same as those of periodontitis<xref ref-type="bibr" rid="b9">9</xref>.</p><p>Implant-borne reconstructions are now preferred over conventional fixed or removable partial dentures, and marketing estimates indicate that over 2 million implants were installed per year in the past decade<xref ref-type="bibr" rid="b10">10</xref>. According to the American Academy of Implant Dentistry (AAID), 3 million Americans have implants and that number is growing by 500,000 a year. However, the number of problems associated with the implants has been increasing, and peri-implantitis has been identified in 28–56% of implant recipients and in 12–43% of their implant sites<xref ref-type="bibr" rid="b11">11</xref>. Peri-implantitis is considered an inflammatory disease that is strongly associated with the oral microbiota<xref ref-type="bibr" rid="b12">12</xref>, and its clinical symptoms are similar to those of periodontitis. However, because pain is infrequent in peri-implantitis, patients do not recognize that a problem exists until implant mobility occurs, which indicates the disease's final stage<xref ref-type="bibr" rid="b13">13</xref>. The progression of peri-implantitis can trigger a loss of the implants, maxillary sinusitis, mandibular fracture, or the infection of other implants or natural teeth<xref ref-type="bibr" rid="b14">14</xref>. While the progression of periodontitis can be suppressed by appropriate infection controls (e.g., surgical therapy and antibiotic therapy), the same results cannot be obtained for peri-implantitis because of the difficulty in decontaminating the roughened, threaded surfaces of the endosseous implants<xref ref-type="bibr" rid="b15">15</xref>.</p><p>Oral implants are exposed to the microbiota present in the oral cavity and easily develop a biofilm on the implant surfaces; therefore, a survey of the microbiota involved is necessary to our understanding of the disease. Recently, several studies have used high-throughput sequencing based on the 16S rRNA gene to establish the core microbiota by characterizing the human oral bacteria under healthy or diseased conditions, such as dental caries or periodontitis<xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b16">16</xref><xref ref-type="bibr" rid="b17">17</xref>. These studies have found that even in healthy people, the bacteria comprising the oral microbiota varied considerably between individuals. To further understand the bacterial ecology of peri-implantitis, and to determine the core microbiota for future prevention and treatment, we sought to clarify the microbial differences between peri-implantitis and the clinically similar disease of periodontitis by comparing the individual microbiota of each disease in patients with both conditions.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Patient's clinical characteristics and obtained sequences</title><p>Twenty patients with both peri-implantitis and periodontitis were recruited for this study, 7 men (35.0%) and 13 women (65.0%), with an age range of 40–76 (average = 60.1 ± 1.7) years. The clinical characteristics of the patients are shown in <xref ref-type="table" rid="t1">Table 1</xref>. The probing pocket depth (PPD) and number of sites with pus differed significantly between the peri-implantitis and periodontitis samples. After trimming the disqualified sequences, we obtained a total of 436,320 sequences for the 16S rRNA gene (average-read length = 294.4 bp) that were used for the analyses (<xref ref-type="supplementary-material" rid="s1">Supplementary Table S1</xref>). From these sequences, 19 phyla, 188 genera, and 235 species were identified (<xref ref-type="supplementary-material" rid="s1">Supplementary Figure S1</xref>).</p></sec><sec disp-level="2"><title>Overall bacterial community composition in peri-implantitis and periodontitis</title><p>The Ribosomal Database Project (RDP) classifier was initially used to assign each sequence to the taxonomy at the phylum or genus level. At the phylum level, the microbial compositions of the peri-implantitis and periodontitis sites were similar, but the abundance of some genera differed significantly (<xref ref-type="fig" rid="f1">Figure 1</xref> and <xref ref-type="supplementary-material" rid="s1">Supplementary Figure S2</xref>). Compared with periodontitis, peri-implantitis-associated bacterial communities had significantly higher levels of the genera <italic>Olsenella</italic>, <italic>Sphingomonas</italic>, <italic>Peptostreptococcus</italic>, and unclassified <italic>Neisseriaceae</italic>, and lower levels of the genus <italic>Desulfomicrobium</italic>. Furthermore, the <italic>Methanobrevibacter</italic> archaeal genus was detected with 100% RDP confidence using the prokaryotic universal primers; however, we did not detect it at the species level using the Human Oral Microbiome Database (HOMD) 16S rRNA Gene Database. This genus was more abundant in peri-implantitis than periodontitis, but the difference was not significant (<xref ref-type="fig" rid="f1">Figure 1</xref> and <xref ref-type="supplementary-material" rid="s1">Supplementary Figure S2a</xref>).</p></sec><sec disp-level="2"><title>Biodiversity in peri-implantitis and periodontitis</title><p>Next, we estimated the community diversity for all samples to compare the complexity between peri-implantitis and periodontitis. The Shannon index, number of operational taxonomic units (OTUs) based on a 3% genetic difference, and Chao1 estimates were not significantly different between peri-implantitis and periodontitis (<xref ref-type="supplementary-material" rid="s1">Supplementary Figure S3a–c</xref>), and the rarefaction curves indicated similar species richness for both diseases (<xref ref-type="supplementary-material" rid="s1">Supplementary Figure S3d</xref>). We also compared the characteristics of the constituent species of the peri-implantitis and periodontitis communities based on their oxygen requirements, Gram-staining statuses, and cultivation statuses, and found no significant differences (<xref ref-type="supplementary-material" rid="s1">Supplementary Figure S4a–c</xref>, and <xref ref-type="supplementary-material" rid="s1">Supplementary Table S2</xref>). The abundances of anaerobic and gram-negative bacteria were statistically higher than the abundances of the aerobic and gram-positive bacteria in both communities (<xref ref-type="supplementary-material" rid="s1">Supplementary Figure S4a–b</xref>).</p></sec><sec disp-level="2"><title>Comparison of the bacterial community structures between peri-implantitis and periodontitis</title><p>To compare the community structure of the samples, the overall bacterial community composition was calculated based on the unweighted UniFrac distance and visualized with a principal coordinate analysis (PCoA) plot. These plots did not reveal any distinct partitioning of the bacterial communities associated with peri-implantitis or periodontitis, and the similarities of the phylogenetic distances between peri-implantitis and periodontitis varied by patient. However, the analysis of similarities (ANOSIM) tests on the Unifrac distances showed that clustering within the same individual was significant (<italic>P</italic> = 0.001) relative to the disease status (peri-implantitis versus periodontitis; <italic>P</italic> = 0.212; <xref ref-type="fig" rid="f2">Figure 2a</xref>).</p></sec><sec disp-level="2"><title>Core microbiota of peri-implantitis</title><p>We tried to determine the core microbiota in peri-implantitis based on previous studies<xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref>. First, we investigated the species-level differences using the HOMD 16S rRNA Gene Database to further assess the differences in the microbial communities for peri-implantitis and periodontitis. Despite inter-individual variability, there were core microbiota representing a baseline oral community for peri-implantitis. We found that some species had significantly different abundance levels between peri-implantitis and periodontitis. <italic>Prevotella nigrescens</italic> was more abundant in peri-implantitis, while <italic>Peptostreptococcaceae</italic> [XI][G-4] sp. HOT369 and <italic>Desulfomicrobium orale</italic> were more abundant in periodontitis (<xref ref-type="fig" rid="f2">Figure 2b</xref>).</p><p>Second, considering that peri-implantitis was caused by intra-individual microbial infections, we considered that there were common species between peri-implantitis and periodontitis. We evaluated the species that were relatively abundant and prevalent in peri-implantitis and periodontitis (<xref ref-type="fig" rid="f3">Figure 3a</xref>). We found that <italic>Actinomyces johnsonii</italic>, <italic>Fusobacterium nucleatum</italic>, <italic>Porphyromonas gingivalis</italic>, <italic>Streptococcus oralis,</italic> TM7 [G-1] sp. HOT-346, <italic>Treponema denticola</italic>, and <italic>Treponema socranskii</italic> were highly abundant and prevalent in both diseases. <italic>Porphyromonas gingivalis</italic>, <italic>Treponema denticola,</italic> and <italic>Tannerella forsythia</italic>, which are the important periodontal pathogens of the “red complex”<xref ref-type="bibr" rid="b18">18</xref>, were abundant and prevalent in most samples in both diseases. However, other species (<italic>Achromobacter xylosoxidans,</italic> TM7 [G-5] sp. HOT-437, <italic>Actinomyces massiliensis</italic>, <italic>Porphyromonas</italic> sp. HOT-395, <italic>Prevotella nigrescens</italic>, and <italic>Prevotella oris</italic>) dominated in peri-implantitis samples when compared with periodontitis samples. Although <italic>Achromobacter xylosoxidans,</italic><italic>Actinomyces massiliensis</italic>, and <italic>Porphyromonas</italic> sp. HOT-395 were prevalent in both diseases, they were more abundant in peri-implantitis. Thus, some species were associated with either peri-implantitis or periodontitis, whereas other species were present in both diseases.</p></sec><sec disp-level="2"><title>Species associated with the clinical parameters</title><p>We hypothesized that some bacteria might be associated with the clinical data and therefore examined the relationship between the bacterial taxa and clinical parameters by calculating Spearman's ranked correlations. Although we did not find a significant correlation between clinical parameters and microbial characteristics (<xref ref-type="supplementary-material" rid="s1">Supplementary Table S3</xref>), smoking was positively correlated with <italic>Lachnospiraceae</italic> [G-1] sp. HOT-496 in peri-implantitis and <italic>Actinomyces</italic> sp. HOT848 in periodontitis (<xref ref-type="supplementary-material" rid="s1">Supplementary Table S4</xref>). Next, we tried to identify the species associated with the severity of peri-implantitis, as evidenced by the clinical examination parameters; PPD, clinical attachment loss (CAL), bleeding on probing (BOP), bone loss, and pus discharge, but no species could be associated with BOP because it was seen at all of the sampled sites. However, the significantly different aerobic/facultative bacteria were negatively correlated with the other parameters, indicating increased inflammation. Not all of the anaerobic bacteria correlated positively with the clinical parameters, and some were negatively correlated (<xref ref-type="supplementary-material" rid="s1">Supplementary Table S5 and S6</xref>). We also found that more species were associated with the severity of the clinical parameters for peri-implantitis than were for those for periodontitis (<xref ref-type="supplementary-material" rid="s1">Supplementary Table S5</xref>), and that some species were associated with ≥3 parameters. The bacterial species <italic>Treponema</italic> sp. HOT257, <italic>Eubacterium nodatum</italic>, <italic>Peptococcus</italic> sp. HOT168, <italic>Clostridiales</italic> [F-1][G-1] sp. HOT093, and <italic>Catonella morbi</italic> correlated positively with the clinical parameters (<xref ref-type="fig" rid="f3">Figure 3b</xref> and <xref ref-type="supplementary-material" rid="s1">Supplementary Table S5</xref>), while some species such as <italic>Eubacterium saburreum</italic> and <italic>Selenomonas noxia</italic> correlated negatively (<xref ref-type="supplementary-material" rid="s1">Supplementary Table S6</xref>). Among the species associated with the severity of peri-implantitis, some were uncultured bacteria, including <italic>Treponema</italic> sp. HOT257, which correlated with all 4 parameters.</p></sec><sec disp-level="2"><title>Co-occurrence of the bacterial taxa</title><p>To compare the ecologically significant competitive interactions of the bacteria in peri-implantitis with those in periodontitis, we performed a co-occurrence analysis of the abundant species (<xref ref-type="fig" rid="f4">Figure 4</xref>) and found several interesting microbial relationships. First, although 24 of the top 30 abundant species were shared between peri-implantitis and periodontitis, their correlations were not the same. For peri-implantitis, <italic>Synergistetes</italic> [G-3] sp. HOT-360 and <italic>Pseudoramibacter alactolyticus</italic> were the most positively correlated (ρ = 0.849), whereas <italic>Eubacterium nodatum</italic> and <italic>Streptococcus intermedius</italic> were the most negatively correlated (ρ = −0.692). For periodontitis, the most positively correlated species were <italic>Streptococcus oralis</italic> and <italic>Veillonella parvula</italic> (ρ = 0.826), whereas <italic>Synergistetes</italic> [G-3] sp. HOT-453 and <italic>Neisseria subflava</italic> were the most negatively correlated (ρ = −0.794). Second, associations between red complex bacteria were common in peri-implantitis and periodontitis. The abundance of <italic>Porphyromonas gingivalis</italic> was significantly and positively correlated with that of <italic>Tannerella forsythia</italic> in both diseases (ρ = 0.768 and 0.752).</p><p>Finally, the microbial correlations within the same genera changed in a manner dependent on the species. All of the species in the same genera of <italic>Streptococcu</italic>s and TM7 were positively correlated in both diseases (<xref ref-type="fig" rid="f4">Figure 4</xref>). By contrast, the abundance of some <italic>Synergistetes</italic> sp. was correlated negatively even within the same genus (e.g., <italic>Synergistetes</italic> [G-3] sp. HOT360 and <italic>Synergistetes</italic> [G-3] sp. HOT453 in periodontitis), although the former correlated positively with <italic>Synergistetes</italic> [G-3] sp. HOT363 in peri-implantitis.</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>Peri-implantitis is caused by a microbial infection that shifts from other oral sites to the peri-implant, affecting the tissues around and function of an osseointegrated implant. We recently reported that the microbiota of peri-implantitis were more complex than those of periodontitis using a 16S rRNA gene clone library technique<xref ref-type="bibr" rid="b19">19</xref><xref ref-type="bibr" rid="b20">20</xref>. However, the oral microbiome is comprised of hundreds of prevalent taxa, and we therefore believed it important to investigate the microbiota associated with peri-implantitis more precisely. Using high-throughput sequencing, we obtained enough sequence information to clarify the ecology of the microbial communities of peri-implantitis.</p><p>In this study, we showed that the unweighted Unifrac distances for the same individual were significantly similar in comparison to the disease status (peri-implantitis or periodontitis), and the PCoA revealed no distinct partitioning of the bacterial communities associated with peri-implantitis or periodontitis. These results differed from a previous study<xref ref-type="bibr" rid="b20">20</xref><xref ref-type="bibr" rid="b21">21</xref>, probably because we enrolled more patients and compared the microbiota in patients that had both diseases. The bacteria colonized around the implants were transmitted from the teeth<xref ref-type="bibr" rid="b12">12</xref> and the results indicated that the inflammatory peri-implantitis was caused by intra-individual microbial infections. However, the abundant bacteria in peri-implantitis differed from those in periodontitis in the same patients at the species level, which may suggest that the two environments are different. The soft and hard tissues surrounding the implant and the periodontium have several differences<xref ref-type="bibr" rid="b22">22</xref>. There are no periodontal ligaments in the peri-implant region because of osseointegration, and the collagen fibres surrounding the implant are arranged circumferentially with minimal vasculature. In addition, the connective tissue attachment of the peri-implant tissue is weaker than that of the periodontium. Severe inflammation with pus is therefore more likely to occur in the peri-implant tissue than in the periodontium, which may then result in a difference in core microbiota between peri-implantitis and periodontitis.</p><p>The species <italic>Prevotella nigrescens</italic>, a member of the orange complex of periodontal pathogens that are thought to be most important for the progression of periodontitis after those of the red complex<xref ref-type="bibr" rid="b18">18</xref>, was significantly abundant in peri-implantitis, which is consistent with a previous study<xref ref-type="bibr" rid="b23">23</xref>. In contrast, this result did not coincide with those of recent studies<xref ref-type="bibr" rid="b21">21</xref><xref ref-type="bibr" rid="b24">24</xref> (<xref ref-type="supplementary-material" rid="s1">Supplementary Table S7</xref>). We considered that this apparent discrepancy was due to differences in the study population; the previous studies also investigated peri-implant biofilm using pyrotag sequencing, while the samples were collected from not only peri-implantitis but also gingivitis/peri-mucotitis<xref ref-type="bibr" rid="b24">24</xref> or from different individuals<xref ref-type="bibr" rid="b21">21</xref>. Although inter-individual variability was observed, some species were abundant in all of the patients. Some abundant species common to peri-implantitis and periodontitis, i.e., <italic>Fusobacterium nucleatum</italic>, <italic>Neisseria subflava</italic>, and <italic>Streptococcus oralis</italic>, and red complex play important roles in biofilm formation<xref ref-type="bibr" rid="b25">25</xref>. In previous studies using high-throughput sequencing, <italic>Fusobacterium nucleatum, Treponema denticola</italic>, and TM7 sp. HOT346 were found to be core microbiota that were highly abundant and highly prevalent in periodontitis<xref ref-type="bibr" rid="b4">4</xref>. <italic>Porphyromonas gingivalis</italic> and <italic>Treponema denticola</italic> were two of the top three periodontitis-associated species<xref ref-type="bibr" rid="b5">5</xref>. We suggested microbial shifts in community structure from periodontitis to peri-implantitis, and some abundant species were common in both diseases as well as from the healthy condition to periodontitis<xref ref-type="bibr" rid="b4">4</xref>. Furthermore, we found other abundant species in peri-implantitis or periodontitis, suggesting that these species may be key players in these refractory pathogenic conditions. A previous study<xref ref-type="bibr" rid="b21">21</xref> demonstrated lower levels of the genus <italic>Prevotella</italic> in peri-implantitis than in periodontitis-associated biofilms (<xref ref-type="supplementary-material" rid="s1">Supplementary Table S7</xref>). In this study, <italic>Prevotella nigrescens</italic> and <italic>Prevotella oris</italic> were more abundant in peri-implantitis than in periodontitis, and the difference was significant for the former. They may therefore play important roles in the microbial community in peri-implantitis at the species level. <italic>Porphyromonas gingivalis</italic> was abundant in both diseases. In contrast, <italic>Porphyromonas</italic> sp. HOT395 was more abundant in peri-implantitis. <italic>Porphyromonas</italic><italic>endodontitis</italic> is considered to be the core species in periodontitis<xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref>, but was not abundant in our study. We considered that the rules of species in the same genus vary in different environments.</p><p>Although the biofilm formed at peri-implantitis and periodontitis sites appears to contain similar species until late colonizers such as the red complex are established, the colonization of the bacteria constituting the biofilm differs between the diseases. Thus, a species-level analysis may be important for understanding the prognosis of these diseases.</p><p>We also detected <italic>Methanobrevibacter</italic>, which belongs to the Archaea (<xref ref-type="fig" rid="f1">Figure 1</xref>), by using prokaryotic universal primers targeting 16S rRNA genes because the study aim was to further elucidate the community associated with peri-implantitis. Although <italic>Methanobrevibacter</italic><italic>oralis</italic> is included in the HOMD database, this species was not identified with the representative sequence by our method in this study. In previous studies, the <italic>Methanobrevibacter oralis</italic>-like phylotype increased in periodontitis<xref ref-type="bibr" rid="b26">26</xref>, and <italic>Methanobrevibacter oralis</italic> has been detected in peri-implantitis<xref ref-type="bibr" rid="b27">27</xref>. Although the role for the archaeal species in these diseases is not clear, further investigation of the association between oral diseases, including peri-implantitis, and archaeons is needed to better understand the ecology of the oral cavity.</p><p>We hypothesized that the microbiota in these habitats may be associated with the severity of disease. Some anaerobic species showed a positive correlation to the clinical parameters of peri-implantitis (<xref ref-type="supplementary-material" rid="s1">Supplementary Table S5</xref>), although the total number of anaerobic or aerobic bacteria was not significantly correlated with either disease (<xref ref-type="supplementary-material" rid="s1">Supplementary Table S3</xref>). In contrast, some aerobic/facultative bacteria such as <italic>Streptococcus oralis</italic> were negatively correlated to the clinical parameters, suggesting the proportion of these species might indicate the degree of severity of the disease. A higher number of species were significantly correlated with the peri-implant destruction than with the destruction from periodontitis. This suggests that for the samples used in this study, the diseased state of the peri-implant sites was greater than the sites of periodontitis, and the progression of disease was faster for peri-implantitis in comparison to periodontitis in terms of the structural differences, at least as measured by the same clinical parameters.</p><p>Regarding the clinical parameters, <italic>Treponema</italic> sp. HOT257, <italic>Eubacterium nodatum</italic>, <italic>Peptococcus</italic> sp. HOT-168, <italic>Clostridiales</italic> [F-1][G-1] sp. HOT-093, and <italic>Catonella morbi</italic> were positively correlated with ≥3 parameters in peri-implantitis, and are probably more associated with the increased inflammation of the disease than are other species, even though <italic>Eubacterium nodatum</italic> was more abundant in periodontitis than in peri-implantitis (<xref ref-type="fig" rid="f3">Figure 3a</xref>). These species were thought to be associated with the progression of disease as a group rather than individually because they have only been detected in oral disease. For example, <italic>Catonella morbi</italic> and <italic>Eubacterium</italic> spp. increased or persisted at a high frequency in refractory periodontitis, but were significantly reduced in treatable periodontitis, allowing good responders for periodontal therapy to be identified<xref ref-type="bibr" rid="b28">28</xref>. In our study, however, <italic>Eubacterium saburreum</italic> was negatively correlated to the increased inflammation of peri-implantitis (<xref ref-type="supplementary-material" rid="s1">Supplementary Table S6</xref>); thus, the correlation was different even within the same genus. Another species associated with peri-implantitis, <italic>Clostridiales</italic> sp. has also been detected in dental caries and periodontitis<xref ref-type="bibr" rid="b29">29</xref><xref ref-type="bibr" rid="b30">30</xref>, suggesting an association with oral disease. Smoking is a risk factor for both peri-implantitis and periodontitis<xref ref-type="bibr" rid="b12">12</xref>, but the disease severity was not associated with smoking in this study. Although 30% of patients were smokers, the species correlated with smoking were different in both diseases, which may reflect different environments. A previous study mentioned that levels of uncultivated <italic>Peptostreptococci, Parvimonas</italic> (e.g., <italic>P. micra</italic>)<italic>, Fusobacterium, Campylobacter</italic> (e.g., <italic>C. gracilis</italic>)<italic>, Bacteroides,</italic> and <italic>Treponema</italic> (e.g., <italic>T. socranskii</italic>) were elevated in smokers<xref ref-type="bibr" rid="b31">31</xref>. We included 6 smokers in this study; therefore we could not evaluate correlations between these taxa and smoking. As smoking influences the sub-gingival microbial composition<xref ref-type="bibr" rid="b31">31</xref>, further investigation of bacteria, including unclassified species, associated with smoking in peri-implantitis and periodontitis is necessary.</p><p>It has recently been reported that <italic>Treponema</italic> spp., including <italic>Treponema denticola</italic>, were core microbiota in periodontitis<xref ref-type="bibr" rid="b4">4</xref>. In our study, <italic>Treponema</italic> sp. was more associated with the increased inflammation of peri-implantitis. A previous study<xref ref-type="bibr" rid="b21">21</xref> demonstrated higher levels of the genera <italic>Eubacterium</italic>, <italic>Peptococcus</italic>, and <italic>Treponema</italic> associated with peri-implantitis in comparison to periodontitis-associated biofilms (<xref ref-type="supplementary-material" rid="s1">Supplementary Table S7</xref>). Although the abundance of these genera were not significantly different between the diseases, they might be important for the progression of peri-implantitis at the species level.</p><p>Our data confirmed that peri-implantitis was a polymicrobial infection and not associated with a specific pathogen, so we need to regard the microbiota of this disease as a complex microbial community. The co-occurrence analysis revealed that the strength of the bacterial correlations in peri-implantitis and periodontitis were different, and we believe that this would reflect environmental differences. Interesting bacterial interactions were also observed in both diseases. First, the associations between anaerobic bacteria and aerobic/facultative bacteria were different in the situation of biofilm formation. The early colonizers<xref ref-type="bibr" rid="b25">25</xref> were correlated positively with each other (e.g., <italic>Fusobacterium nucleatum</italic> and <italic>Streptococcus salivarius</italic> in both diseases). The early colonizers and late colonizers<xref ref-type="bibr" rid="b25">25</xref> were negatively correlated (e.g., <italic>Streptococcus intermedius</italic> and <italic>Eubacterium nodatum</italic> in peri-implantitis or <italic>Streptococcus oralis</italic> and <italic>Treponema denticola</italic> in periodontitis). Second, associations among the red-complex species were common. <italic>Porphyromonas gingivalis</italic> was correlated significantly with <italic>Tannerella forsythia</italic> at both sites.</p><p>Our data also corresponded with a previous study<xref ref-type="bibr" rid="b32">32</xref> that reported a positive relationship between members of the phyla Synergistetes and Spirochaetes in the sub-gingival biofilm; and we showed this correlation at the species level (<italic>Synergistetes</italic> sp. HOT453 and <italic>Treponema denticola</italic>) for peri-implantitis (ρ = 0.615).</p><p>In conclusion, this initial characterization furthers our understanding of the microbial community in the oral cavity by defining the microbiota associated with peri-implantitis. We have shown, by comparing the 2 diseases within the same patients using high-throughout sequencing and correlation analysis, that similar to other oral diseases, peri-implantitis is a polymicrobial disease. We showed that the core microbiota of peri-implantitis was different from that of periodontitis, even though the sources of bacteria around the implant were present elsewhere in the oral cavity, such as the remaining teeth. Although the red-complex species co-occurred as in past studies, other species were significantly abundant or associated with the progression of disease in peri-implantitis sites, suggesting that other periodontal pathogens and polymicrobial infections are associated with peri-implantitis, and therefore the target pathogens for its treatment or prevention may be different from those for periodontitis. Thus, our data could provide the basis for future molecular analyses of the bacteria suspected of being associated with peri-implantitis, including the uncultured species In addition, our finding that peri-implantitis results from the complexity of the oral microbiota suggests that understanding of the healthy oral microbiota will require more studies of the microbial ecology of other oral infectious diseases.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>Ethical statement</title><p>This study was carried out in accordance with the Ethical Guidelines for Clinical Studies (2008 Notification number 415 of the Ministry of Health, Labor, and Welfare) and approved by the Tokyo Medical and Dental University Institutional Review Board (No.661). Written informed consent was obtained from all patients.</p></sec><sec disp-level="2"><title>Patients and clinical examinations</title><p>Twenty patients with both peri-implantitis and periodontitis were recruited from the clinics of the Department of Periodontology, Tokyo Medical and Dental University Hospital. The patients were systemically healthy adults and had not received anti-inflammatory drugs, oral anti-microbial agents, or systemic antibiotics within the previous 3 months.</p><p>The following clinical examinations of each implant or tooth were performed at the mesio-buccal, buccal, disto-buccal, mesio-lingual, lingual, and disto-lingual sites: PPD, CAL, BOP, and noting the presence of pus. Intraoral periapical radiographs (Insight Dental Film; Eastman Kodak Company, SP, Tokyo, Japan) were obtained using the parallel technique, and the same examiner analysed the radiographs for bone loss from peri-implantitis at implants functioning at least 1 year and from periodontitis<xref ref-type="bibr" rid="b33">33</xref><xref ref-type="bibr" rid="b34">34</xref>. Based on the clinical and radiographic data, peri-implantitis and periodontitis sites were selected that exhibited PPD ≥ 4 mm, BOP and/or pus-discharge presence, and concomitant radiographic bone-loss presence.</p></sec><sec disp-level="2"><title>Sample collection and DNA extraction</title><p>Sub-mucosal and sub-gingival plaque samples were obtained from the deepest pockets at the peri-implantitis and periodontitis sites, respectively. The sampling sites were isolated with sterile cotton rolls and the supra-mucosal or supra-gingival plaque was removed. After drying the target sites, 3 paper points were inserted into the pocket for 30 s and then placed in a sterile tube for storage at −80°C until further analysis. To separate the bacteria from the paper points, 1 mL of sterile distilled water was added and they were vortexed for 1 min before removing the points. The tubes were then centrifuged at 12,000 × <italic>g</italic> for 5 min to pellet the bacterial samples, and the DNA was extracted and purified with the MORA-EXTRACT DNA extraction kit (Kyokuto Pharmaceuticals, Tokyo, Japan) in accordance with the manufacturer's instructions<xref ref-type="bibr" rid="b35">35</xref>. The total bacterial DNA was eluted with 200 μL of Tris-EDTA buffer and was stored at −20°C.</p></sec><sec disp-level="2"><title>Preparation of the bacterial 16S rRNA gene amplicons</title><p>The 16S rRNA genes from each sample were amplified with the following PCR primers<xref ref-type="bibr" rid="b36">36</xref>: 806R with the adaptor B sequences from 454 Life Sciences (Roche, Basel, Switzerland) (5′-CCTATCCCCTGTGTGCCTTGGCAGTC<bold>GGACTACVSGGGTATCTAAT</bold>-3′) and 515F with adaptor A and subject-specific ten-base barcode sequences (5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG -10-bp-barcode- <bold>GTGCCAGCMGCCGCGGT</bold>-3′); the small subunit ribosomal rRNA sequence is in bold.</p><p>The amplification of the V3–4 region of the 16S rRNA gene was performed in 50-μL reaction mixtures composed of 10× polymerase buffer, 2.5 mM dNTPs, 0.2 μM of each primer, 1.25 U Takara Ex Taq Hot Start (TaKaRa Biomedicals, Tokyo, Japan), and 1–2 μL of template DNA. After a denaturation step at 94°C for 1 min, the PCR cycling parameters were 35 cycles of 98°C for 10 s, 55°C for 30 s, and 72°C for 2 min, followed by a final extension at 72°C for 1 min. The amplicons were visualized by electrophoresis on 2% agarose gels, and the bands were purified using the NucleoSpin Extract II kit (Macherey-Nagel, Düren, Germany) followed by AMPure paramagnetic beads (Agencourt Bioscience Corporation, Beverly, MA, USA) according to the manufacturers' protocols.</p></sec><sec disp-level="2"><title>Amplicon quantitation, pooling, and pyrosequencing</title><p>The amplicons were quantified using a KAPA Library Quantification Kit optimized for the Roche 454 GS Titanium system (KAPA Biosystems, Woburn, MA) and pooled in equal amounts into a single tube, and a final amount of 1 × 10<sup>7</sup> molecules were analysed via pyrosequencing on a 454 Life Sciences Genome Sequencer Junior (GS-Junior; Roche Diagnostics, Basel, Switzerland). The emulsion PCR, bead enrichment, and 454 GS Junior sequencing were performed as indicated in the manufacturer's protocols, and the resulting flowgram files were used for the downstream analyses.</p></sec><sec disp-level="2"><title>Sequence analysis</title><p>The sequence data were processed and analysed with the software package Quantitative Insights into Microbial Ecology (QIIME) version 1.6.0<xref ref-type="bibr" rid="b37">37</xref>. The sequences were denoised<xref ref-type="bibr" rid="b38">38</xref> and removed if they had a length <200 bp or >800 bp, average quality score <25, ambiguous bases present, primer mismatches >1.5, homopolymer runs >6 bases, uncorrectable barcodes, or lacked the primer<xref ref-type="bibr" rid="b39">39</xref>. The remaining sequences were assigned as samples based on their barcodes, and the software program UCHIME<xref ref-type="bibr" rid="b40">40</xref> was used to identify putative chimeric sequences.</p><p>The similar sequences were binned into OTUs using UCLUST<xref ref-type="bibr" rid="b41">41</xref> with a minimum pairwise identity of 97%, and the most abundant sequence in each OTU was chosen to represent its OTU. The taxonomy of representative sequences at the phylum or genus level was determined using RDP classifier version 2.2<xref ref-type="bibr" rid="b42">42</xref> against the RDP 16S database in the QIIME with a minimum support threshold of 60%. Because all of the abundant genera were contained in the NCBI database, the phylogenetic tree was generated by PhyloT implemented in the Interactive Tree Of Life (iTOL) tool version 2.2.2<xref ref-type="bibr" rid="b43">43</xref>, which generates phylogenetic trees based on the NCBI taxonomy, and visualized using the iTOL. Because the pathogenicity often differs even among bacterial species of the same genus, refinement of the taxonomic assignment was performed at the species level based on HOMD database version 13.0<xref ref-type="bibr" rid="b1">1</xref>, a curated database of comprehensive information for oral 16S rRNA gene sequences. The species were assigned if a sequence had ≥98.5% identity for ≥250 bp of the sequence in a BLASTN search following the methods of previous studies<xref ref-type="bibr" rid="b44">44</xref><xref ref-type="bibr" rid="b45">45</xref>. The bacterial cultivation status of the bacteria was obtained from the HOMD, and the Gram-staining status or oxygen requirement for each taxon was obtained from the NCBI Entrez Genome Project database (<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi">http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi</ext-link>).</p><p>Alpha-diversity indexes were estimated from the number of observed OTUs, Chao1<xref ref-type="bibr" rid="b46">46</xref> species richness estimates, and the Shannon diversity index<xref ref-type="bibr" rid="b47">47</xref> that measures both species richness and evenness. Rarefaction curves were generated to calculate the species richness based on the bacterial OTUs at a 97% identity level and were compared between the peri-implantitis and periodontitis samples. To compare the beta-diversity between peri-implantitis and periodontitis, the representative sequences from OTUs were aligned against the Greengenes database<xref ref-type="bibr" rid="b48">48</xref> (core set aligned February 4, 2011) by PyNAST<xref ref-type="bibr" rid="b49">49</xref>, a Python-based implementation of the Nearest Alignment Space Termination (NAST) tool, implemented in the QIIME with a 200-bp minimum length and 75% minimum per cent identity. Subsequently, the hypervariable regions were masked with maskPH<xref ref-type="bibr" rid="b39">39</xref> and we used the unweighted UniFrac distance metrics<xref ref-type="bibr" rid="b50">50</xref> computed using the tree file generated by FastTree<xref ref-type="bibr" rid="b51">51</xref>; the resulting distance matrix was then visualized using PCoA. The co-occurrence of taxonomic groups across samples was explored by calculating the Spearman's ranked correlation (ρ), which was >0.6 and statistically significant (<italic>P</italic> < 0.05 and q <0.1)<xref ref-type="bibr" rid="b52">52</xref>, clustering the correlations using Cluster 3.0<xref ref-type="bibr" rid="b53">53</xref>, and visualizing the results with Java TreeView version 1.1.6r2<xref ref-type="bibr" rid="b54">54</xref>.</p></sec><sec disp-level="2"><title>Statistical analysis</title><p>Paired <italic>t-</italic>tests were performed to compare the clinical data and alpha diversity between the peri-implantitis and periodontitis samples<xref ref-type="bibr" rid="b4">4</xref>. ANOSIM tests were performed on the unweighted UniFrac distances, and the relative abundances of each bacterial taxon were compared by conducting Wilcoxon signed-rank tests<xref ref-type="bibr" rid="b5">5</xref>. Spearman's ranked correlations were used for the analyses of co-occurrence and the bacterial associations with the clinical data<xref ref-type="bibr" rid="b52">52</xref><xref ref-type="bibr" rid="b55">55</xref>. We corrected the obtained P-values for multiple tests using the Benjamini-Hochberg false discovery rate (q-value)<xref ref-type="bibr" rid="b56">56</xref>.</p></sec></sec><sec disp-level="1"><title>Author Contributions</title><p>This study was conceived by N.M. and F.M. Samples and clinical data were collected by Y.T. and Y.I. Laboratory work was done by N.M., F.M. and I.N. The manuscript was written by N.M., F.M. and C.A. All authors reviewed the manuscript.</p></sec><sec disp-level="1"><title>Additional information</title><p><bold>Accession codes:</bold> The data are available at the DNA Data Bank of Japan (DDBJ) under accession no. DRA000946 (<ext-link ext-link-type="uri" xlink:href="http://www.ddbj.nig.ac.jp/">http://www.ddbj.nig.ac.jp/</ext-link>).</p></sec><sec sec-type="supplementary-material" id="s1"><title>Supplementary Material</title><supplementary-material id="d33e23" content-type="local-data"><caption><title>Supplementary Information</title><p>Supplementary information</p></caption><media xlink:href="srep06602-s1.pdf"></media></supplementary-material></sec></body><back><ack><p>This research was supported by Grant-in-Aid for Japan Society for the Promotion of Science (JSPS) Fellows; the Japanese Ministry of Education, Global Center of Excellence program, International Research Center for Molecular Science in Tooth and Bone Diseases, Ministry of Education, Culture, Sports, Science and Technology Japan; Grant-in-Aid for Scientific Research 22592032 (F.M.), 21792110 (Y.T.), 24792321(Y.T.), 25670776 (F.M.), and 25713060 (F.M.); a Grant-in-Aid for Scientific Research on Innovative Areas 24117508 (F.M.); and the Funding Program for Next Generation World-Leading Researchers LS041 (I.N.), JSPS.</p></ack><ref-list><ref id="b1"><mixed-citation publication-type="journal"><name><surname>Dewhirst</surname><given-names>F. 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The outer bands show the relative abundance: red for peri-implantitis, blue for periodontitis, and green for overall relative abundance. The tree was constructed with the Interactive Tree of Life tool and the taxonomic names were based on results from the Ribosomal Database Project classifier. The statistical differences were calculated by Wilcoxon signed rank tests. *<italic>P</italic> <0.05 and q <0.1.</p></caption><graphic xlink:href="srep06602-f1"></graphic></fig><fig id="f2"><label>Figure 2</label><caption><title>Principal coordinate analysis (PCoA) and microbial differences at the species level.</title><p>(a) PCoA plots of the unweighted UniFrac distances for the samples by disease. (b) The most abundant species (>0.5% abundance) in the peri-implantitis and periodontitis samples. The species name or Human Oral Taxon (HOT) ID in the Human Oral Microbiome is shown. The taxonomy assignments were based on information in the Human Oral Microbiome Database, and the statistical differences were calculated by Wilcoxon signed rank tests. *<italic>P</italic> <0.05 and q <0.1.</p></caption><graphic xlink:href="srep06602-f2"></graphic></fig><fig id="f3"><label>Figure 3</label><caption><title>The microbiota associated with peri-implantitis.</title><p>(a) The core microbiota of peri-implantitis and periodontitis. The model includes the species detected at peri-implantitis (red), periodontitis (blue), and both sites (green), where the species were found in at least 50% of patients with a mean relative abundance of >1%, or were statistically different (outside of the square boxes; see <xref ref-type="fig" rid="f2">Figure 2b</xref>). The species detected in at least 80% of patients in both sites are indicated in bold. The inner box labelled with 1 indicates a mean relative abundance of ≥2% in periodontitis and <2% in periodontitis. The inner box labelled with 2 indicates a mean relative abundance of ≥2% in both sites. The inner box labelled with 3 indicates a mean relative abundance of <2% in periodontitis and ≥2% in periodontitis. The inner box labelled with 4 indicates a mean relative abundance of <2% in type of site. <italic>Peptostreptococcaceae</italic> [XI][G-4] sp. HOT369 is statistically abundant, although showed a mean relative abundance of <1% in periodontitis (see <xref ref-type="fig" rid="f2">Figure 2b</xref>). The species name or Human Oral Taxon (HOT) ID in the Human Oral Microbiome Database is shown. The statistical differences were calculated by Wilcoxon signed rank tests. *<italic>P</italic> <0.05 and q <0.1. (b) Bacterial taxa associated with the progression of peri-implantitis. The model represents all bacterial taxa associated with each of the four clinical parameters of peri-implantitis (<italic>P</italic> <0.05 and q <0.1). The species name or HOT ID is shown. The taxa correlated to four parameters (red box) and three parameters (black boxes) are shown. PPD, probing pocket depth; CAL, clinical attachment loss.</p></caption><graphic xlink:href="srep06602-f3"></graphic></fig><fig id="f4"><label>Figure 4</label><caption><title>Co-occurrence and co-exclusion analysis of the bacterial taxa.</title><p>Co-occurrence and co-exclusion were calculated by Spearman rank correlations between the abundant bacterial taxa. The co-occurrence of the top 30 species in the peri-implantitis and periodontitis samples is shown on the right and left, respectively. The species name or Human Oral Taxon ID in the Human Oral Microbiome Database is shown. The correlation values range from -1.00 (green) to 1.00 (red).</p></caption><graphic xlink:href="srep06602-f4"></graphic></fig><table-wrap position="float" id="t1"><label>Table 1</label><caption><title>Summary of meta-data on patients</title></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"></col><col align="center"></col><col align="center"></col><col align="center"></col></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50"> </th><th align="center" valign="top" charoff="50">Peri-implantitis sites</th><th align="center" valign="top" charoff="50">Periodontitis sites</th><th align="center" valign="top" charoff="50"><italic>P</italic> value<xref ref-type="fn" rid="t1-fn2">a</xref></th></tr></thead><tbody valign="top"><tr><td align="justify" valign="top" charoff="50">Age</td><td colspan="2" align="center" valign="top" charoff="50">60.1 ± 1.7</td><td align="left" valign="top" charoff="50"> </td></tr><tr><td align="justify" valign="top" charoff="50">Gender</td><td colspan="2" align="center" valign="top" charoff="50">7 males, 13 females</td><td align="left" valign="top" charoff="50"> </td></tr><tr><td align="justify" valign="top" charoff="50">Smoking</td><td colspan="2" align="center" valign="top" charoff="50">6</td><td align="left" valign="top" charoff="50"> </td></tr><tr><td align="justify" valign="top" charoff="50">Maxillary anterior</td><td align="center" valign="top" charoff="50">7</td><td align="center" valign="top" charoff="50">5</td><td align="char" valign="top" char="." charoff="50">0.49</td></tr><tr><td align="justify" valign="top" charoff="50">Maxillary posterior</td><td align="center" valign="top" charoff="50">5</td><td align="center" valign="top" charoff="50">8</td><td align="char" valign="top" char="." charoff="50">0.33</td></tr><tr><td align="justify" valign="top" charoff="50">Mandibular anterior</td><td align="center" valign="top" charoff="50">0</td><td align="center" valign="top" charoff="50">2</td><td align="char" valign="top" char="." charoff="50">0.16</td></tr><tr><td align="justify" valign="top" charoff="50">Mandibular posterior</td><td align="center" valign="top" charoff="50">8</td><td align="center" valign="top" charoff="50">5</td><td align="char" valign="top" char="." charoff="50">0.33</td></tr><tr><td align="justify" valign="top" charoff="50">Years in function</td><td align="char" valign="top" char=" " charoff="50">5.7 ± 0.7</td><td align="left" valign="top" charoff="50"> </td><td align="left" valign="top" charoff="50"> </td></tr><tr><td align="justify" valign="top" charoff="50">PPD (sampled sites)<xref ref-type="fn" rid="t1-fn2">b</xref></td><td align="char" valign="top" char=" " charoff="50">7.0 ± 0.6 mm</td><td align="char" valign="top" char=" " charoff="50">5.5 ± 0.4 mm</td><td align="char" valign="top" char="." charoff="50"><0.05</td></tr><tr><td align="justify" valign="top" charoff="50">CAL (sampled sites)<xref ref-type="fn" rid="t1-fn3">c</xref></td><td align="char" valign="top" char=" " charoff="50">7.5 ± 0.6 mm</td><td align="char" valign="top" char=" " charoff="50">6.4 ± 0.4 mm</td><td align="char" valign="top" char="." charoff="50">0.09</td></tr><tr><td align="justify" valign="top" charoff="50">BOP (sampled sites)<xref ref-type="fn" rid="t1-fn3">d</xref></td><td align="center" valign="top" charoff="50">100%</td><td align="center" valign="top" charoff="50">100%</td><td align="left" valign="top" charoff="50"> </td></tr><tr><td align="justify" valign="top" charoff="50">Number of sites with pus</td><td align="center" valign="top" charoff="50">9</td><td align="center" valign="top" charoff="50">0</td><td align="char" valign="top" char="." charoff="50"><0.05</td></tr><tr><td align="justify" valign="top" charoff="50">Radiographic bone loss</td><td align="char" valign="top" char=" " charoff="50">45.2 ± 7.2%</td><td align="char" valign="top" char=" " charoff="50">42.9 ± 5.2%</td><td align="char" valign="top" char="." charoff="50">0.78</td></tr></tbody></table><table-wrap-foot><fn id="t1-fn1"><p>Numbers shown are mean ± s.d.</p></fn><fn id="t1-fn2"><p><sup>a</sup>Statistical differences were calculated using paired t-tests; <sup>b</sup>Probing pocket depth.</p></fn><fn id="t1-fn3"><p><sup>c</sup>Clinical attachment loss; <sup>d</sup>Bleeding on probing.</p></fn></table-wrap-foot></table-wrap></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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