H. influenzae Genes Associated with Acute Otitis Media

描述（由申请人提供）：急性中耳炎是由宿主防御、环境因素和细菌毒力特性的复杂相互作用引起的。以往的研究表明，流感嗜血杆菌（Hi），在儿童急性中耳炎的一个重要原因，具有独特的基因在这种感染的发病机制中的重要性。在这个项目中，我们将确定潜在的毒力因子的具体Hi中耳炎。使用消减杂交技术，我们将确定存在于Hi中耳炎分离株和Hi咽喉分离株中缺失的DNA片段。将对减去的DNA片段进行克隆、测序，并用于确定其在按各种流行病学参数分组的大组Hi菌株中的流行率。我们将使用基于基因检测的微阵列对大量Hi菌株进行中耳炎相关基因的筛选，所述微阵列由Hi菌株的基因组和这些基因之间的关联以及这些基因与建立的流行病学因素之间的关联组成。该项目的具体目标是： 1.对急性中耳炎患儿中耳积液和鼻咽咽部标本中分离的Hi进行生物型、血清型、产β-内酰胺酶和脉冲场凝胶电泳基因型鉴定。 2.利用消减杂交技术筛选Hi中耳炎相关基因。 3.描述候选Hi中耳炎相关基因之间的关联，Hi中耳炎相关基因和流行病学参数之间的关联，包括分离部位、地理位置、鼻咽定植的持续性和儿童间传播的能力，使用我们收集的每个菌株的Hi基因组DNA微阵列。 4.通过大小、预测功能和Hi染色体中的位置来表征Hi中耳炎相关基因。 鉴定独特的Hi中耳炎毒力因子将有助于开发预防和治疗Hi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.