Quantitative analysis of subgingival bacteria in smokers

描述（由申请人提供）：在美国，41.9%的牙周炎可归因于吸烟，吸烟者比非吸烟者患更严重和更广泛疾病的风险更高。虽然菌斑和吸烟都被报道在牙周炎中起重要作用，但单个细菌物种或聚生体与吸烟之间的关联尚未得到很好的阐明。最近对龈下微生物群落的非培养性探索表明，大多数物种是未培养的，因此，在使用传统的基于培养的方法时无法识别。因此，重要的是要检查龈下微生物的配置文件，这一高风险的人口使用的分子方法，规避需要培养。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基因的珠探针，同时用流式细胞仪测量多个物种的水平。具体目标1提出，通过比较牙周健康吸烟者和牙周炎吸烟者之间的种植物种和尚未种植的牙周类型的水平，调查吸烟是否与龈下生态系统的全球扰动有关。这些菌种是基于我们早期的工作，使用开放式分子方法进行细菌鉴定而选择的。具体目标2将调查吸烟者微生物谱的改变是否可能归因于该队列疾病严重程度的增加。我们将比较牙周炎严重程度相似的当前吸烟者和从不吸烟者之间所选微生物的水平。这些研究代表了第一个全面的方法来检查吸烟和龈下微生物群落的变化之间的关联，并将提供重要的初步数据，更广泛的研究建议，以评估与牙周炎的发病和进展，在这个高危人群的细菌变化。在本研究过程中开发的灵敏、高通量定量分析方法可用于任何微生物生态系统的表征，并将填补急需的方法学空白。在这一高危人群中确定假定的病原体和健康相关的细菌将导致更好地了解吸烟在牙周病病因学中的作用。这是开发临床有用的疾病标志物和靶向生物干预的重要一步。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龈下微生物群落变化之间的关联。在这一高危人群中鉴定推定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，并可能导致更好地了解吸烟在牙周病病因学中的作用。这可能导致临床上有用的疾病标志物和预测因子以及用于预防疾病的生物干预的发展。

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.