口腔疾病防治 ›› 2021, Vol. 29 ›› Issue (3): 157-165.DOI: 10.12016/j.issn.2096-1456.2021.03.003

• 基础研究 • 上一篇    下一篇

牙周炎患者龈下菌斑菌群的高通量测序分析

杨万娟(),徐杰()   

  1. 昆明医科大学附属口腔医院牙周病科,云南 昆明(650106)
  • 收稿日期:2020-04-24 修回日期:2020-05-30 出版日期:2021-03-20 发布日期:2021-01-12
  • 通讯作者: 徐杰
  • 作者简介:杨万娟,住院医师,硕士研究生在读,Emai:2809688235@qq.com
  • 基金资助:
    云南省科技厅科技计划项目(2018FE001[-261]);云南省卫生科技计划项目(2017NS267)

High-throughput sequencing analysis of the microbiota of subgingival plaque in patients with periodontitis

YANG Wanjuan(),XU Jie()   

  1. Department of Periodontics, Stomatological Hospital of Kunming Medical University, Kunming 650106, China
  • Received:2020-04-24 Revised:2020-05-30 Online:2021-03-20 Published:2021-01-12
  • Contact: Jie XU
  • Supported by:
    Science and Technology Planning Project of Yunnan Science and Technology Department(2018FE001[-261]);Health Science and Technology Project of Yunnan Province(2017NS267)

摘要:

目的 利用高通量测序(high-throughput sequencing,HTS)分析广泛型侵袭性牙周炎(generalized aggressive periodontitis,GAgP)和重度慢性牙周炎(severe chronic periodontitis,SCP)龈下菌群的组成,利用生物信息学分析其多样性及功能,并观察牙周基础治疗前后龈下菌群的变化。方法 选择2018年9月至2019年5月在昆明医科大学附属口腔医院牙周病科就诊的11例GAgP患者和14例SCP患者作为研究对象。在基线及牙周基础治疗后第6周采集龈下菌斑样本并提取DNA,进行MiSeq测序,采用QIIME(quantitative insights in microbial ecology)、Mothur及SPSS等软件分析群落信息,并进行LEfSe差异分析(linear discriminant analysis Effect size,LEfSe)、Network网络分析,使用KEGG PATHWAY数据库(https://www.kegg.jp/kegg/pathway.html)对群落功能进行预测。结果 基线时,GAgP和SCP患者的优势菌群相似,均包括拟杆菌门、卟啉单胞菌属和牙髓卟啉单胞菌等。治疗后第6周,GAgP和SCP患者菌群变化相似,由于牙周袋变浅,拟杆菌门、卟啉单胞菌属和牙髓卟啉单胞菌等革兰阴性菌相对丰度降低,变形菌门、放线菌属和空间罗氏菌等革兰阳性菌相对丰度增加;其中放线菌是GAgP治疗后龈下菌群中显著增加的生物标志物;链球菌属是联系牙周炎相关菌属和牙周健康相关菌属的重要菌属。群落功能预测表明基础治疗可降低GAgP和SCP患者群落中的氨基酸代谢、甲烷代谢和肽酶等功能。 结论 GAgP和SCP患者的龈下菌群相似,链球菌属作为早期定植菌,在龈下菌群由健康向失调转变的过程中可能发挥着促使菌斑生物膜形成及成熟的重要作用;牙周基础治疗可以改变GAgP和SCP患者龈下菌群的组成和结构,降低群落多样性,降低氨基酸代谢、甲烷代谢和肽酶等群落功能。

关键词: 广泛型侵袭性牙周炎, 重度慢性牙周炎, 龈下菌斑, 龈下菌群, 牙周基础治疗, 高通量测序, 群落多样性, 群落功能预测

Abstract:

Objective To detect the composition of the subgingival microbiota in generalized aggressive periodontitis (GAgP) and severe chronic periodontitis (SCP) patients tested by high-throughput sequencing (HTS) technologies, analyze its diversity and function by using bioinformatics, and observe changes in the subgingival microbiota before and after periodontal initial therapy. Methods Eleven patients with GAgP and 14 patients with SCP who visited the Department of Periodontics in Stomatological Hospital of Kunming Medical University from September 2018 to May 2019 were recruited, and subgingival plaque samples were collected at baseline and 6 weeks after initial therapy. Then, the genomic DNA was distracted and sequenced by the Illumina MiSeq high-throughput sequencing platform. QIIME (quantitative insights in microbial ecology), Mothur, SPSS and other software were used to analyze community information. LEfSe difference analysis (linear discriminant analysis effect size), network analysis, and the KEGG PATHWAY database (https://www.kegg.jp/kegg/pathway.html) were used to predict community function. Results At baseline, the dominant microbiota of GAgP and SCP patients were similar, including Bacteroidetes, Porphyromonas and Porphyromonas endodontalis. Six weeks after initial therapy, as the periodontal pocket became shallower, the variation trend of the microbiota of GAgP and SCP patients was similar. The relative abundance of gram-negative bacteria, such as Bacteroidetes, Porphyromonas and Porphyromonas endodontalis, decreased, while the relative abundance of gram-positive bacteria, such as Proteobacteria, Actinomyces and Rothia aeria, increased. Actinobacteria were significantly increased biomarkers of the subgingival microbiota in GAgP after treatment. Streptococcus is an important genus that connects the microbiota related to periodontitis and the microbiota related to periodontal health. Community function prediction result showed that initial treatment can reduce the functions of amino acid metabolism, methane metabolism, and peptidase in GAgP and SCP patients. Conclusion The subgingival microbiota of GAgP and SCP patients are similar. Streptococcus, as an early colonizer, may play an important role in promoting plaque biofilm formation and maturation in the process of subgingival flora from health to imbalance. Initial therapy can change the composition and structure of the subgingival microbiota, reduce community diversity, and reduce the functions of amino acid metabolism, methane metabolism, and peptidase in GAgP and SCP patients.

Key words: generalized aggressive periodontitis, severe chronic periodontitis, subgingival plaque, subgingival microbiota, periodontal initial therapy, high-throughput sequencing, community diversity, community function prediction

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