Oral microbial associations in HIV+subjects

描述(由申请人提供)：这项建议的广泛目标是检验一种假设，即HIV+受试者中更常见的牙周感染是由特定的细菌或真菌引起的，这些细菌或真菌不一定是通常在HIV阴性受试者的可比牙周感染中发现的典型假定牙周病原体。我们的初步数据表明，HIV+患者患有坏死性溃疡性牙周炎(NUP)，这是一种更严重的牙周疾病，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，如大泡杆菌和Dialister气球菌属，以及新的“不可培养”的Dialister属、消化链球菌和Selenomonas属，以及“不可培养”门TM7的成员。我们的目标有两个具体目标。目的1鉴定与HIV阳性受试者牙周感染相关的主要可培养和尚未培养的细菌和真菌，如牙周炎、低/中和重度成人牙周炎，以及另一种HIV阳性受试者特有的牙周感染--线性牙龈红斑(LGE)。这些研究将通过分析菌斑文库中克隆的细菌16S rRNA和真菌18S rRNA基因序列来进行。文库将通过聚合酶链式反应扩增龈下菌斑中的rRNA基因，然后将扩增产物连接到适当的载体上，并转化大肠杆菌来生成。每个克隆的序列将被用来确定物种身份，或者如果未知，则用于确定其系统发育位置。预计将确定另外25至50个新的分类群。目的2验证随着HIV+患者免疫状态的恶化，特定细菌和真菌与牙周感染有关的假说。在每个牙周疾病类别中，将分析25名病毒载量高、CD4水平低的受试者和25名病毒载量低、CD4水平高的受试者。将在这些HIV+组和HIV阴性受试者的对照组之间进行牙龈下微生物和真菌种群的比较。将使用棋盘杂交技术对每个样本进行多达200种优势细菌的分析，这允许使用多个探针同时分析多个样本。在第二次单独的棋盘杂交中，将对每个样本进行多达30种真菌的检测。可能是特定的细菌复合体(例如细菌/真菌的特定组合)与这些牙周疾病有关，并可能随着艾滋病毒感染的严重程度而改变。拟议的研究可能为HIV阴性个体中可比的牙周感染中假定病原体的身份提供洞察力。

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.