Biofilm Population Structure in the Gangetic Delta

描述（由申请人提供） 下面总结的研究项目是Schoolnik博士和Md. Sirajul Islam，将主要在孟加拉国国际腹泻病研究中心（ICDDR，B）进行，作为NIH资助RO 1 AI 43422的扩展。在孟加拉国和其他地方进行的实地观察研究表明，亚洲霍乱的病原体霍乱弧菌是自然水生生境的正常成员。在构成恒河三角洲的河流和河口，霍乱弧菌被认为与浮游植物和浮游动物有关，并居住在各种表面上的混合物种生物膜中。一个这样的表面，桡足类的几丁质外骨骼，可以被霍乱弧菌几丁质酶降解，从而提供氮，碳和能量的来源。其他表面，包括河口底栖层的沙粒，营养贫乏。这种非营养表面上的生物膜细菌必须从水柱中获得营养。反过来，水体的营养成分和化学特征也受到季风季节循环的显著影响。因此，认为霍乱弧菌在其水生水库和人类宿主中的这些地点之间的移动部分取决于季风气候对该栖息地理化特征的影响。为了开始了解这些季节变化如何影响恒河三角洲含有霍乱弧菌的混合物种生物膜，Islam和Schoolnik博士最近完成了一项为期一年的试点研究（作为母基金的补充），显示了以下内容：（1）霍乱弧菌存在于孟加拉国Matlab附近恒河三角洲淹没的非营养塑料表面上形成的混合物种生物膜中;（2）生物膜的物种组成在一年中发生变化。这些观察结果以及甲壳素作为营养表面的重要性和丰富性导致了以下假设：含有霍乱弧菌的混合物种生物膜的种群结构随着表面基质（甲壳素与非营养表面相比）和季节（季风前，季风或季风后）而变化。为了验证这一假设，本文提出的工作将使用一种不依赖于培养的、基于16 S rRNA序列的方法来确定在前季风、季风和后季风期间淹没在Matlab采样点的甲壳素或聚碳酸酯表面上的生物膜聚生体的种群结构。然后，将使用所确定的物种特异性序列来设计标记的pliglitazide探针，用于荧光原位杂交（FISH）检测;这些探针将用于该项目的最后两年，以确定每月从几丁质和聚碳酸酯表面的生物膜样品的种群结构。这些结果将与水化学和霍乱病例率的变化相关联。这些关键的分子生态学方法的每一个组成部分都转移到国际复员方案中心，B-通过在国际复员方案中心，B和斯坦福大学为孟加拉国科学家举办的培训方案实现-将大大提高这一重要中心的研究能力。

DESCRIPTION (provided by applicant) The research project summarized below, a collaboration between Drs. Schoolnik and Md. Sirajul Islam, will be performed mainly in Bangladesh at the International Center for Diarrhoeal Diseases Research (ICDDR,B) as an extension of NIH grant RO1 AI43422. Observational field studies performed in Bangladesh and elsewhere indicate that Vibrio cholerae, the agent of Asiatic cholera, is a normal member of natural aquatic habitats. In the rivers and estuaries that compose the Gangetic Delta, V. cholerae is believed to be associated with phyto- and zoo- plankton and to reside in mixed-species biofilms on a variety of surfaces. One such surface, the chitinous exoskeletons of copepods, can be degraded by V. cholerae chitinases, thus providing a source of nitrogen, carbon and energy. Other surfaces, including sand particles in the benthic layer of the estuary, are nutrient-poor. Biofilm bacteria on non-nutritive surfaces of this kind must obtain nutrients from the water column. In turn, the nutrient content and chemical features of the water column are dramatically affected by the monsoon seasonal cycle. As a result, the movement of V. cholerae between these sites in its aquatic reservoir and the human host is believed to be determined, in part, by the effects of the monsoon climate on the physicochemical features of this habitat. To begin to understand how these seasonal changes affect V. cholerae- containing mixed-species biofilms in the Ganges Delta, Drs. Islam and Schoolnik recently completed a one year pilot study (supported as a supplement to the parent grant) that showed the following: (1) V. cholerae resides in mixed species biofilms that form on non-nutritive plastic surfaces submerged in the Ganges Delta near Matlab, Bangladesh; and (2), the species composition of the biofilm changes during the year. These observations and the importance and abundance of chitin as a nutritive surface led to the following hypothesis: the population structure of V. cholerae-containing mixed-species biofilms varies as a function of the surface substratum (chitin compared to non-nutritive surfaces) and season (pre-monsoon, monsoon or post-monsoon). To test this hypothesis, the work proposed here will .use a culture-independent, 16S rRNA sequence-based method to determine the population structure of biofilm consortia on chitin or polycarbonate surfaces submerged at the Matlab sampling site during the pre-monsoon, monsoon .and post-monsoon periods. The identified species-specific sequences will then be used to design labeled pligonucleotide probes for fluorescence in situ hybridization (FISH) assays; these probes will be used in the last two years of the project to determine the population structure of monthly biofilm samples from chitin and polycarbonate surfaces. These results will be correlated with changes in water chemistry and cholera case rates. The transfer of each component of these key molecular ecology methods to the ICDDR,B--achieved by a training program for Bangladeshi scientists at the ICDDR,B and at Stanford--will significantly enhance the research capacity of this important Center.