Comprehensive detection of periodontal pocket bacterial flora and quantitation of disease-specific bacterial species

背景：在人类牙周袋中发现了近400种细菌。其中一半被认为至今仍未被培养。为了建立牙周感染的微生物诊断，应进一步确定尚未培养的微生物群落的生态学特征。本研究旨在通过序列法了解慢性牙周炎患者牙龈下菌群的生态学，为人类牙周微生物生态学诊断提供依据。方法：从4例慢性牙周炎患者所有病变部位的龈下菌斑样本中构建4个16S rRNA基因文库。对每个文库的480个克隆进行了系统分型分析。结果：物种丰富度包括一组丰富的共有物种（17个种型）和一组单独多样的稀有物种（58 ~ 83个种型）。两组由~ 25%的描述良好的和~ 75%的未培养的种型组成。分类为拟杆菌门、梭杆菌门、变形杆菌门、厚壁菌门、螺旋体门、放线菌门和TM7门。以前报道的牙周相关物种和种型在一些样本中发现，但并非所有样本都有。序列相似度在99%以上的不同亚种表现为个别偏态分布。结论：龈下菌群的物种丰富度和未培养菌群的丰度分别为70-80%和70-80%。然而，大多数这些系统型在系统发育上接近于描述良好的牙周相关细菌。在尚未培养的物种和描述良好的微生物区系物种的物种斑块中发现了个体差异。这些单独不同的斑块分布有望为“量身定制”牙周治疗提供微生物诊断。

Background : Nearly 400 species of bacteria have been found in the human periodontal pockets. Half of those are considered to remain not-yet-cultured to this date. Further characterization of the ecology of those not-yet-cultured species in the microflora should be done for establishment of microbiological diagnosis of periodontal infection. The purpose of this study is to know ecology of subgingival microflora of chronic periodontitis patients by sequence-based method for an approach to diagnosis in human periodontal microbial ecology.Methods : Four 16S rRNA gene libraries were constructed from subgingival plaque samples taken from all of diseased sites of 4 chronic periodontitis patients. The 480 clones from each libraries were analyzed by phylotyping.Results : The species richness contained a group of abundant shared species (17 phylotypes) and a group of individually diverse rare species (58〜83 phylotypes). Both groups consisted of〜25% of well-described and〜75% of not-yet-cultured phylotypes. The phylotypes were assigned to the Bacteroidetes, Fusobacteria, Proteobacteria, Firmicutes, Spirochetes, Actinobacteria, and TM7 phyla. Previously reported periodontally associated species and phylotypes were found in some samples but not to be shared by all. Diverse sub-phylotypes showing more than 99% of sequence similarity showed individually skewed distribution.Conclusions : The species richness and abundance of the not-yet-cultured species seemed to be 70-80% and 70-80% respectively in subgingival microflora. However most of those phylotypes were phylogenetically close to well-described periodontally associated bacteria. Individual differences were found in patchness of the species within not-yet-cultured as well as well-described species of the microflora. These individually distinct patchy distributions are expectable to serve microbial diagnosis for 'tailor-made' periodontal treatment.