H. influenzae Genes Associated with Acute Otitis Media

描述(由申请人提供)：急性中耳炎是宿主防御、环境因素和细菌毒力特征的复杂相互作用的结果。以前的研究表明，流感嗜血杆菌(Hi)是儿童急性中耳炎的重要原因，具有独特的基因，在这种感染的发病机制中发挥着重要作用。在这个项目中，我们将确定针对Hi中耳炎的潜在毒力因子。使用消减杂交技术，我们将鉴定在Hi中耳炎分离株中存在的DNA片段和在Hi咽喉分离株中不存在的DNA片段。减去的DNA片段将被克隆、测序，并用于确定它们在按不同流行病学参数分组的大量Hi毒株中的流行率。我们的大量Hi菌株将使用基于基因检测的微阵列来筛选与中耳炎相关的基因，该微阵列由Hi菌株的基因组以及这些基因之间的关联以及这些基因与流行病学因素之间的关系组成。该项目的具体目标是： 1.用脉冲场凝胶电泳法对急性中耳炎患儿中耳积液和儿童鼻咽部标本中分离的Hi进行了生物型、血清型、产β-内酰胺酶和基因分型鉴定。 2.利用消减杂交技术筛选Hi中耳炎相关候选基因。 3.使用我们收集的每个菌株的Hi基因组DNA微阵列，描述候选Hi中耳炎相关基因之间的关系，以及Hi中耳炎相关基因与流行病学参数之间的关系，包括隔离地点、地理位置、鼻咽定植的持久性和儿童传播的能力。 4.确定Hi中耳炎相关基因的大小、预测功能和在Hi染色体中的位置。 识别独特的高中耳炎毒力因子将有助于开发预防和治疗高中耳炎的新方法。

DESCRIPTION (provided by applicant): Acute otitis media results from a complex interplay of host defenses, environmental factors, and virulence characteristics of bacteria. Previous studies suggest that Haemophilus influenzae (Hi), an important cause of acute otitis media in children, possess unique genes important in the pathogenesis of this infection. In this project, we will identify potential virulence factors specific for Hi otitis media. Using a subtractive hybridization technique, we will identify DNA fragments present in Hi otitis media isolates and absent in Hi throat isolates. The subtracted DNA fragments will be cloned, sequenced and used to determine their prevalence among large panels of Hi strains grouped by various epidemiologic parameters. Our large collection of Hi strains will be screened for the otitis media associated genes using a gene detection-based microarray consisting of genomes of the Hi strains and associations among these genes as well as between these genes and epidemiologic factors established. The Specific Aims of this project are: 1. Characterize Hi isolated from middle ear effusions of children with acute otitis media and from nasopharyngeal throat specimens of children by biotype, serotype, b-lactamase production and genotype by pulsed field gel electrophoresis. 2. Identify candidate Hi otitis media associated genes through subtractive hybridization. 3. Describe associations among candidate Hi otitis media associated genes and between Hi otitis media associated genes and epidemiologic parameters, including site of isolation, geographic location, persistence of nasopharyngeal colonization and ability to be transmitted child to child, using a microarray of Hi genomic DNA from each strain in our collections. 4. Characterize the Hi otitis media associated genes by size, predicted function, and location in the Hi chromosome. Identification of unique Hi otitis media virulence factors will facilitate the development of novel approaches to the prevention and treatment of Hi otitis media.