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Metagenomic analysis of the microbial changes following non-surgical periodontal therapy in aggressive periodontitis.

机译：侵袭性牙周炎非手术牙周治疗后微生物变化的元基因组学分析。

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摘要

Background. Metagenomics is the sequencing of DNA from whole communities of organisms taken directly from the environment. As opposed to culturing methods which focus on individual species, metagenomics allows for the characterization of intact community genomes, including the identification of noncultivable bacterial species. The objective of this pilot study was to test the feasibility of a metagenomic approach in combination with a novel design of primers to analyze the pre- and post-treatment of subgingival plaque in two aggressive periodontitis patients.;Methods. Periodontal examinations and microbial sampling were performed before and after non-surgical initial phase therapy. DNA was extracted from the subgingival plaque samples and subjected to PCR amplification using primers that amplified the c2-c4 regions of the 16S rDNA. Specific bar codes were included in the primers to identify individual samples. The PCR products were pooled and sequenced for the v4 region of the 16S rDNA using the pyrosequencing 454 FLX standard platform. The results were analyzed for species identification in the Human Oral Microbiome Database (HOMD) and Ribosomal Database Project (RDP) database. The observed proportional changes of the subgingival species after therapy were verified by testing the pre- and post-therapy levels of Porphyromonas gingivalis by real-time PCR.;Results. The sequencing of the amplicons resulted in 24,673 reads and identified 208 species/phylotypes. Of those, 129 species/phylotypes were identified in both patients but their proportions varied. While >120 species/phylotypes were identified in all samples, 85--90% of the species/phylotypes were represented by ≤100 sequencing reads (≤0.2% of the total reads). Similar levels of subgingival P. gingivalis were found based on the numbers of sequencing reads or quantitative Real-time PCR analyses. Pre- and post-therapy samples showed significant quantitative and distributive species changes along with clinical improvements. Quantitatively, many known pathogenic species went from easily detectable to zero counts while the opposite occurred for many known commensal bacteria. Additionally, several novel, pathogenic and health-associated species were found.;Conclusion. High throughput metagenomic analysis is applicable to assess the changes in subgingival microbiota after nonsurgical periodontal therapy in aggressive periodontitis. The examined subgingival microbiota are characterized by high species richness (>120 species/phylotypes) dominated by a few species/phylotypes. The composition of the subgingival microbiota appears to be distinct between individuals before and after the treatment. The considerable variety of known and novel species/phylotypes detected supports further research to better understand the microbial etiopathogenesis of periodontitis.
机译：背景。元基因组学是对直接来自环境的整个生物群落中的DNA进行测序。与专注于单个物种的培养方法相反，宏基因组学可以表征完整的群落基因组，包括鉴定不可培养的细菌物种。这项初步研究的目的是测试一种宏基因组学方法与新颖的引物设计相结合的可行性，以分析两名侵略性牙周炎患者的龈下斑块的治疗前后。在非手术初期治疗之前和之后进行牙周检查和微生物取样。从牙龈下菌斑样品中提取DNA，并使用能扩增16S rDNA c2-c4区的引物进行PCR扩增。引物中包括特定的条形码，以识别单个样品。汇集PCR产物，并使用焦磷酸测序454 FLX标准平台对16S rDNA的v4区域进行测序。在人类口腔微生物组数据库（HOMD）和核糖体数据库计划（RDP）数据库中对结果进行了分析，以进行物种鉴定。通过实时PCR检测牙龈卟啉单胞菌治疗前后的水平，验证了治疗后观察到的龈下种类的比例变化。扩增子的测序导致24,673个读数，并鉴定了208种/系统型。其中，在两名患者中鉴定出129种/系统型，但比例不同。尽管在所有样品中鉴定出> 120种/系统型，但≤100个测序读数代表了85--90％的物种/系统型（占总读数的0.2％）。根据测序读数或实时荧光定量PCR分析的数量，发现牙龈下龈沟假单胞菌的水平相似。治疗前和治疗后样品显示出明显的数量和分布物种变化以及临床改善。从数量上讲，许多已知的致病菌种从易于检测到零计数，而许多已知的共生细菌则相反。此外，还发现了几种新颖的，致病的和与健康有关的物种。高通量宏基因组学分析适用于评估侵袭性牙周炎非手术牙周治疗后龈下微生物群的变化。被检查的龈下微生物群的特征在于高物种丰富度（> 120个物种/系统型），而少数物种/系统型占优势。在治疗前后，个体之间的龈下微生物群的组成似乎是不同的。检测到的相当多的已知和新物种/系统型支持进一步研究，以更好地了解牙周炎的微生物病因。

著录项

  • 作者

    Laksmana, Theresia.;

  • 作者单位

    University of Southern California.;

  • 授予单位 University of Southern California.;
  • 学科 Biology Microbiology.;Health Sciences Dentistry.
  • 学位 M.S.
  • 年度 2011
  • 页码 60 p.
  • 总页数 60
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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