Oral microbial associations in HIV+subjects

描述（由申请人提供）：本提案的主要目的是检验HIV阳性受试者中更常见的牙周感染是由特定细菌或真菌物种引起的假设，这些细菌或真菌物种不一定是通常在HIV阴性受试者中发现的典型假定牙周病原体。我们的初步数据表明，HIV阳性受试者患有坏死性溃疡性牙周炎（NUP），这是这些个体特有的更严重的牙周病，确实缺乏许多经典的牙周病原体，但却具有“不寻常的”分类群，如外展弹子菌和Dialister肺炎菌，以及新的“不可培养”的Dialister属、Peptostreptococcus、Selenomonas和“不可培养”门TM7成员。我们的目标分为两个具体目标。目的1将确定与HIV+受试者牙周感染相关的主要可培养和未培养的细菌和真菌，如牙龈炎，低/中度和重度成人牙周炎，线性牙龈红斑（LGE）， HIV+受试者特有的另一种牙周感染。这些研究将通过分析菌斑文库中克隆插入物的细菌16S rRNA和真菌18S rRNA基因序列来进行。文库将通过对龈下菌斑中存在的rRNA基因进行PCR扩增，然后将扩增子连接到适当的载体上，并通过大肠杆菌的转化来产生。每个克隆的序列将用于确定物种身份，如果未知，则用于确定其系统发育位置。预计还将发现25至50个新的分类群。目的2将验证特定细菌和真菌种类与牙周感染与HIV+患者免疫状态恶化有关的假设。在每个牙周病类别中，将分析25名高病毒载量和低CD4水平的受试者和25名低病毒载量和高CD4水平的受试者。将比较这些HIV阳性组与HIV阴性对照组之间的龈下微生物和真菌种群。最多200种优势细菌将用于检测每个样品使用棋盘杂交，这允许同时分析多个样品与多个探针。多达30种真菌将分析每个样品在第二，单独的棋盘杂交。可能是特定的细菌复合物（例如，细菌/真菌的特定组合）与这些牙周病有关，并可能随着艾滋病毒感染的严重程度而改变。所提出的研究可能提供的见解，在可比的牙周感染的HIV阴性个体推定病原体的身份。

DESCRIPTION (provided by applicant): The broad objectives of this proposal are to examine the hypothesis that the more common periodontal infections in HIV+ subjects are caused by specific bacterial or fungal species, which are not necessarily the typical putative periodontal pathogens usually found in comparable periodontal infections in HIV negative subjects. Our preliminary data indicated that HIV+ subjects with necrotizing ulcerative periodontitis (NUP), a more severe periodontal disease unique to these individuals, indeed lacked many of the classical periodontal pathogens, but instead possessed "unusual" taxa, such as Bulleidia extructa and Dialister pneumosintes, and novel "uncultivable" phylotypes of the genera Dialister, Peptostreptococcus, Selenomonas, and members of the "uncultivable" phylum TM7. Our objectives are addressed in two Specific Aims. Aim 1 will identify the predominant cultivable and not-yet-cultivated bacteria and fungi that are associated with periodontal infections in HIV+ subjects, such as gingivitis, low/moderate and severe adult periodontitis, and Linear Gingival Erythema (LGE), another periodontal infection unique to HIV+ subjects. These studies will be performed by analyzing bacterial 16S rRNA and fungal 18S rRNA gene sequences of inserts in clones from plaque libraries. The libraries will be generated by PCR amplification of rRNA genes present in subgingival plaque followed by ligation of the amplicons into appropriate vectors and by transformation of E. coli. The sequence of each clone will be used to determine species identity, or if unknown, its phylogenetic position. It is anticipated that 25 to 50 additional new taxa will be identified. Aim 2 will test the hypothesis that specific bacterial and fungal species are associated with periodontal infections with the deterioration of immune status in HIV+ patients. In each periodontal disease category, 25 subjects with high viral loads and low CD4 levels and 25 subjects with low viral load and high CD4 levels will be analyzed. Comparisons of subgingival microbial and fungal populations will be made between these HIV+ groups vs. control groups of HIV negative subjects. Up to 200 predominant bacterial species will be assayed for each sample using Checkerboard hybridization, which allows for the simultaneous analysis of multiple samples with multiple probes. Up to 30 fungal species will be assayed for each sample in a second, separate checkerboard hybridization. It is likely that specific bacterial complexes (e.g., specific combinations of bacteria/fungi) are associated with these periodontal diseases and may change with the severity of HIV infection. The proposed studies may provide insight for the identity of putative pathogens in comparable periodontal infections in HIV negative individuals.