根管内細菌叢パンゲノムの特性評価に基づく新しい歯内治療ストラテジー

2015 Fiscal Year Annual Research Report

• Project/Area Number: 25462945
• Research Institution: Tohoku University
• Principal Investigator: 八巻 惠子 東北大学, 歯学研究科(研究院), 助教 (90182419)
• Co-Investigator(Kenkyū-buntansha): 佐藤 拓一 東北大学, 歯学研究科(研究院), 講師 (10303132) ; 真柳 弦 東北大学, 歯学研究科(研究院), 助教 (10451600) ; 島内 英俊 東北大学, 歯学研究科(研究院), 名誉教授 (70187425)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Keywords: 歯内治療学 / 根管内細菌叢 / 16s ribosomal RNA / メタゲノム解析 / PCR / 感染根管 / 根尖性歯周炎

Outline of Annual Research Achievements

根尖性歯周炎の原因は根管内の細菌感染である。しかし感染根管から検出される細菌は多種多様で、根尖歯周組織に対する病原性が特に強い細菌種はまだ特定されていない。根管内に棲息する細菌の総量や、細菌集合体であるフローラ自体が病原性を発揮している可能性もある。本研究では根管内フローラをパンゲノムとして捉え、その構造や機能特性を解明し、歯内治療ストラテジーへの応用を模索した。
インフォームドコンセントの得られた根尖性歯周炎症例において、①根管治療前、②根管の拡大形成後　③根管充填直前の計3回、根管壁を手用ファイルで切削し象牙質を採取した。試料は実験室に移送し生理食塩水を加えて均一分散化後、専用機器を用いて細菌数を計測した。さらに嫌気ボックス内で10倍連続希釈し血液寒天培地に接種、37℃で1週間嫌気培養した。残った試料は培養を経ずにPCR増幅しpyrosequencingを行い、メタゲノム解析した。嫌気培養後、CFUを求め、生育したコロニーを継代純培養しDNAを抽出、PCR増幅しシークエンスを決定、Blast search解析により菌種を同定した。
迅速計数は嫌気培養で求めたCFUよりつねに約4～100倍近く大きい値を示し、培養困難菌の存在によりCFUが低下するだけでなく、象牙質削片試料を用いると装置の測定誤差（過大に計数）をもたらす可能性が窺われた。本年度解析した6症例から嫌気培養を通じて123コロニーが得られた。Propionibacterium属が67コロニーと最も多く、5症例から検出された。P. acidipropionici、P. propionicum、Enterococcus faecalisの単一感染が各1例認められた。術前の臨床所見から感染菌種数の多寡を推測することは困難であったが、単一感染でも重複感染でも通法の機械的化学的根管の拡大形成により感染制御可能であった。

Research Products

• [Journal Article] Profiling subgingival microbiota of plaque biofilms in the elderly. (2016)
  • Author(s): Abiko Y, Sato T, Sakashita R, Tomida J, Kawamura Y, Takahashi N:
  • Journal Title: J Oral Biosci Volume: 58 Pages: in press
  • DOI: 10.1016/j.job.2015.12.002
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] PCR-dipstick DNA chromatography for profiling of a subgroup of caries-associated bacterial species in plaque from healthy coronal surfaces and periodontal pockets. (2016)
  • Author(s): Tian L, Sato T, Niwa K, Kawase M, Mayanagi G, Washio J, Takahashi N:
  • Journal Title: Biomed Res Volume: 37 Pages: 29-36
  • DOI: https://www.jstage.jst.go.jp/browse/biomedres/37/1/_contents
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Oral malodorous gases and oral microbiota: From halitosis to carcinogenesis. (2015)
  • Author(s): Tanda N, Hoshikawa Y, Ishida N, Sato T, Takahashi N, Hosokawa R, Koseki T:
  • Journal Title: J Oral Biosci Volume: 57 Pages: 175-178
  • DOI: 10.1016/j.job.2015.05.004
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Porphyromonas bronchialis sp. nov., isolated from intraoperative bronchial fluids of a patient with non-small cell lung cancer. (2015)
  • Author(s): Sato T, Tomida J, Naka T, Fujiwara N, Hasegawa A, Hoshikawa Y, Matsuyama J, Ishida N, Kondo T, Tanaka K, Takahashi N, Kawamura Y:
  • Journal Title: Tohoku J Exp Med Volume: 237 Pages: 31-37
  • DOI: 10.1620/tjem.237.31
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Responses of infected dental pulp to αTCP-containing antimicrobials in rat molars. (2015)
  • Author(s): Sato T, Kenmotsu S, Nakakura-Ohshima K, Takahashi N, Ohshima H:
  • Journal Title: Arch Histol Cytol Volume: 73 Pages: 165-175
  • DOI: http://doi.org/10.1679/aohc.73.165
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Microbiota profiling of bronchial fluids of patients with pulmonary carcinoma. (2015)
  • Author(s): Ishida N, Sato T, Hoshikawa Y, Tanda N, Sasaki K, Kondo T, Takahashi N:
  • Journal Title: J Oral Biosci Volume: 57 Pages: 110-117
  • DOI: 10.1016/j.job.2014.11.001
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Porphyromonas pogonae sp. nov., an anaerobic but low concentration oxygen adapted coccobacillus isolated from lizards (Pogona vitticeps) or human clinical specimens, and emended description of the genus Porphyromonas Shah and Collins 1988. (2015)
  • Author(s): Kawamura Y, Kuwabara S, Kania SA, Kato H, Hamagishi M, Fujiwara N, Sato T, Tomida J, Tanaka K, Bemis DA:
  • Journal Title: Syst Appl Microbiol Volume: 38 Pages: 104-109
  • DOI: 10.1016/j.syapm.2014.11.004
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] PCR-dipstick DNA chromatography for multiplex and semi-quantitative analysis of plaque biofilm microbiota. (2016)
  • Author(s): Tian L, Sato T, Niwa K, Kawase M, Mayanagi G, Abiko Y, Washio J, Takahashi N:
  • Organizer: Innovative Research for Biosis-Abiosis Intelligent Interface Symposium: The 6th International Symposium for Interface Oral Health Science
  • Place of Presentation: Sendai, Japan
  • Year and Date: 2016-01-18 – 2016-01-19
  • Int'l Joint Research
• [Presentation] メタボローム解析：口腔疾患の発生機序解明への新たなアプローチ (2015)
  • Author(s): 鷲尾純平、小川珠生、北村淳、森島浩允、田玲錫、石黒和子、真柳弦、安彦友希、佐藤拓一、高橋信博：
  • Organizer: 第68回東北大学歯学会
  • Place of Presentation: 仙台
  • Year and Date: 2015-12-11
  • Invited
• [Presentation] Malodorous gases from saliva of perioperative patients of lung resection. (2015)
  • Author(s): Tanda N, Ishida N, Takahashi N, Sato T, Hoshikawa Y, Hosokawa R, Koseki T:
  • Organizer: The 63rd Annual Meeting of the Japanese Association for Dental Research
  • Place of Presentation: Fukuoka, Japan
  • Year and Date: 2015-10-31
  • Int'l Joint Research
• [Presentation] 嫌気性無芽胞グラム陽性桿菌Olsenella spp.の薬剤感受性 (2015)
  • Author(s): 田中香お里、佐藤拓一、八巻恵子、林将大、河村好章：
  • Organizer: 第63回日本化学療法学会西日本支部総会
  • Place of Presentation: 奈良
  • Year and Date: 2015-10-15 – 2015-10-17
• [Presentation] Profiling of microbiota from infected root canals with and without marginal leakage using anaerobic culture and molecular techniques (16S rRNA gene sequencing) (2015)
  • Author(s): Sato T, Yamaki K, Kawamura Y, Tomida J, Tian L, Ishida N, Takeuchi Y, Hashimoto K, Abiko Y, Matsuyama J, Takahashi N:
  • Organizer: The 25th European Congress of Clinical Microbiology and Infectious Diseases
  • Place of Presentation: Copenhagen, Denmark
  • Year and Date: 2015-04-28
  • Int'l Joint Research
• [Presentation] Antimicrobial susceptibility profiles of Helicobacter cinaedi strains isolated from patients in Japan. (2015)
  • Author(s): Tomida J, Morita Y, Sato T, Kawamura Y:
  • Organizer: The 25th European Congress of Clinical Microbiology and Infectious Diseases
  • Place of Presentation: Copenhagen, Denmark
  • Year and Date: 2015-04-25
  • Int'l Joint Research
• [Book] Oral microbiota in crevices around dental implants: profiling of oral biofilm. In: Interface Oral Health Science 2014: Innovative Research on Biosis-Abiosis Intelligent Interface. (2015)
  • Author(s): Sato T, Kawamura Y, Yamaki K, Ishida N, Tian L, Takeuchi Y, Hashimoto K, Abiko Y, Mayanagi G, Washio J, Matusyama J, Takahashi N:
  • Total Pages: 351 (45-50)
  • Publisher: Springer
• [Book] Preventing aspiration pneumonia for the elderly: a review focused on the impact of the consistency of food substances. In: Interface Oral Health Science 2014: Innovative Research on Biosis-Abiosis Intelligent Interface. (2015)
  • Author(s): Sakashita R, Takami M, Ono H, Nishihira T, Sato T, Hamada M:
  • Total Pages: 351 (335-351)
  • Publisher: Springer