Quantitative analysis of subgingival bacteria in smokers

描述(申请人提供)：在美国，41.9%的牙周炎是由吸烟引起的，吸烟者比不吸烟者患更严重和更广泛的疾病的风险更高。虽然细菌菌斑和吸烟都被报道在牙周炎中起重要作用，但个别细菌种类或菌群与吸烟之间的关系尚未很好地阐明。最近对龈下微生物群落的独立于培养的探索表明，大多数物种是未培养的，因此在使用传统的以培养为基础的方法时没有被识别。因此，使用避开培养需要的分子方法来检查这一高危人群的牙龈下微生物谱是很重要的。我们假设吸烟与龈下微生物谱的改变有关，并且这种改变与疾病的严重程度无关。我们建议使用针对16S rRNA基因的珠状探针，用流式细胞仪同时测量多个物种的水平。具体目标1建议通过比较牙周健康吸烟者和牙周炎吸烟者之间的培养物种和尚未培养的种型水平，来调查吸烟是否与全球牙龈下生态系统的扰动有关。这些物种是在我们早期工作的基础上，使用开放式分子方法进行细菌鉴定的。特定目标2将调查吸烟者的微生物谱变化是否可归因于该队列中疾病严重程度的增加。我们将比较目前吸烟者和从不吸烟者的牙周炎严重程度相似的选定微生物的水平。这些研究是研究吸烟与龈下微生物群落变化之间关系的第一个综合方法，并将为更广泛的研究建议提供重要的初步数据，以评估与牙周炎高危人群的牙周炎发生和发展相关的细菌变化。在本研究过程中开发的灵敏、高通量的定量分析可用于任何微生物生态系统的表征，并将填补亟需的方法学空白。在这一高危人群中鉴定可能的病原体和与健康相关的细菌将有助于更好地理解吸烟在牙周病病因中的作用。这是开发临床有用的疾病标记物和有针对性的生物干预措施的重要一步。关键词：牙周炎，吸烟，细菌，龈下，16S，分子微生物学，流式细胞术。这些研究代表了第一个全面的方法来检查吸烟和牙龈下微生物群落的变化之间的联系。在这一高危人群中鉴定可能的病原体和健康相关细菌将是研究它们与人类宿主相互作用的重要一步，并可能有助于更好地理解吸烟在牙周病病因中的作用。这可能导致开发临床上有用的疾病标记物和预报器以及预防疾病的生物干预措施。

DESCRIPTION (provided by applicant): 41.9% of periodontitis in the USA is attributable to smoking, and smokers are at a higher risk for more severe and extensive disease than non-smokers. Although both bacterial plaque and smoking are reported to play important roles in periodontitis, associations between individual bacterial species or consortia and smoking have not been well elucidated. Recent cultivation-independent explorations of the subgingival microbial community have revealed that the majority of species are uncultivated and hence, unrecognized when using traditional, cultivation-based approaches. Therefore, it is important to examine the subgingival microbial profile of this high-risk population using molecular approaches that circumvent the need for cultivation. We hypothesize that smoking is associated with an alteration in the subgingival microbial profile and that this alteration is independent of disease severity. We propose to use bead-probes directed to the 16S rRNA genes to simultaneously measure the levels of multiple species with a flow cytometer. Specific aim 1 proposes to investigate if smoking is associated with a global perturbation of the subgingival ecosystem by comparing levels of cultivated species and as-yet-uncultivated phylotypes between periodontally healthy smokers and those with periodontitis. These species have been selected based on our earlier work using an open-ended molecular approach for bacterial identification. Specific aim 2 will investigate if altered microbial profiles in smokers could be attributed to increased severity of disease in this cohort. We will compare the levels of selected organisms between current and never smokers with similar severity of periodontitis. These studies represent the first comprehensive approach to examine the association between smoking and changes in the subgingival microbial community and will provide important preliminary data for much broader research proposals to evaluate the bacterial changes associated with the onset and progression of periodontitis in this high-risk population. The development of a sensitive, high-throughput quantitative assay during the course of this study can be used in the characterization of any microbial ecosystem and will fill a much-needed methodological niche. Identification of putative pathogens and health-associated bacteria in this high-risk population will lead to a better understanding of the role of smoking in the etiology of periodontal diseases. This is an important step in developing clinically useful disease markers and targeted biological interventions. Keywords: periodontitis, smoking, bacteria, subgingival, 16S, molecular microbiology, flow cytometry. These studies represent the first comprehensive approach to examine the association between smoking and changes in the subgingival microbial community. Identification of putative pathogens and health-associated bacteria in this high-risk population will be an important step in studying their interactions with the human host and could lead to a better understanding of the role of smoking in the etiology of periodontal diseases. This could lead to the development of clinically useful disease markers and predictors as well as biological interventions for prevention of disease.