Extreme Halophiles: A Potential Reservoir of New Antibiotics

描述（由申请人提供）：病原微生物正在迅速对我们几十年来用于治疗疾病的许多抗生素产生耐药性。这对于引起医院获得性（医院内）感染的病原体尤其如此。需要新的抗生素，但在哪里寻找？一个未开发的来源是来自极端环境的微生物。该提案的长期目标是从高盐环境中分离微生物，并确定它们是否能产生适合对抗疾病的新型抗菌剂。这些目标涉及的具体目标包括确定这些分离株是否是新的，它们与现有生物的关系有多密切，它们是否产生新的抗生素，纯化和表征这些新抗生素，确定它们抑制哪些细菌生物，以及克隆产生这些新抗菌剂的基因。实现这些目标需要采取以下方法。首先，必须制定一项有效的方案，从各种高盐环境（表面盐层、盐壳内部和盐水）中回收数百种极端嗜盐生物。第二，采用结构域特异性引物对的聚合酶链反应（PCR）将用于扩增来自新纯化的生物体的16 S或18 S基因。这些基因的DNA序列将在生物分类学上与现有的分离株相比较。第三，通过拮抗作用研究发现产生抑制产物的微生物。这涉及将少量培养物置于所有其他极端嗜盐分离物的“菌苔”（一个含有薄层生物体的生长培养基平板）上，并在孵育后寻找抑制区的存在。此外，将通过挑战新分离株对各种革兰氏阳性和革兰氏阴性微生物的菌苔进行相同的试验。第四，将使用一系列生物化学方法纯化产生抑制区的材料，包括通过越来越小孔径的过滤器浓缩培养上清液、凝胶过滤柱色谱、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）以及反相和离子交换高效液相色谱（HPLC）。最后，编码这些新抗生素的基因将通过基于纯化抗菌剂的氨基酸序列合成简并寡核苷酸探针来克隆。探针将被赋予放射性，并用于搜索与探针杂交并含有抗生素的限制性片段（该过程称为Southern印迹）。将对含有新基因的片段进行克隆和测序，并对新基因进行遗传和生理分析。致病微生物正在以非常快的速度对目前使用的抗生素产生耐药性。这一提议与公共卫生的相关性在于开发一个新的微生物库，以产生新的抗生素。新的水库是微生物，在非常高盐（高盐）的环境，如大盐湖，UT。

DESCRIPTION (provided by applicant): Pathogenic microorganisms are rapidly becoming resistant to many of the antibiotics that we have used for decades to treat disease. This is especially true for pathogens that cause hospital-acquired (nosocomial) infections. New antibiotics are needed, but where does one look? One untapped source is microorganisms from extreme environments. The long-term objectives of this proposal are to isolate microorganisms from hypersaline environments and determine if they produce new classes of antimicrobials suitable for fighting disease. The specific aims involved in these objectives include determining if these isolates are new, how closely related to existing organisms they are, if they produce new antibiotics, purifying and characterizing these new antibiotics, determining which bacterial organisms they inhibit, and cloning the genes that produce these new antimicrobials. The achievement of these goals will require the following methods. First, an efficient protocol for recovering hundreds of extreme halophiles from various hypersaline environments (surface salt deposits, inside salt crust and from brine) must be in place. Second, the Polymerase Chain Reaction (PCR) employing domain-specific primer pairs will be used to amplify either the 16S or 18S genes from the newly purified organisms. The DNA sequence of these genes will place the organisms taxonomically with respect to existing isolates. Third, organisms that produce inhibitory products will be found by an antagonism study. This involves placing a small amount of culture onto "lawns" (a plate of growth medium containing a thin layer of organism spread onto the surface) of all of the other extremely halophilic isolates and looking for the presence of zones of inhibition after incubation. In addition, the same test will be done by challenging the new isolates against lawns of various gram positive and gram negative organisms. Fourth, the material producing the zone of inhibition will be purified using a series of biochemical methods including concentration of culture supernatants by filters of smaller and smaller pore sizes, gel filtration column chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and reversed-phase and ion exchange high performance liquid chromatography (HPLC). Finally, genes that encode these new antibiotics will be cloned by synthesizing degenerate oligonucleotide probes based upon the amino acid sequence of the purified antimicrobial. The probes will be rendered radioactive and used to search for restriction fragments that hybridize to the probe and contain the antibiotic (this process is called Southern blotting). The fragments containing the new genes will be cloned and sequenced and the new genes subjected to genetic and physiologic analysis. At a very rapid rate, disease-causing microorganisms are becoming resistant to the antibiotics currently in use. The relevance of this proposal to public health is tapping a new reservoir of microorganisms that produce new antibiotics. The new reservoir is microorganisms that thrive in very high salt (hypersaline) environments like the Great Salt Lake, UT.