Extreme Halophiles: A Potential Reservoir of New Antibiotics

描述(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抗药性。对于导致医院获得性(医院内)感染的病原体来说尤其如此。需要新的抗生素，但从哪里看呢？一个尚未开发的来源是来自极端环境的微生物。这项提议的长期目标是从高盐环境中分离微生物，并确定它们是否产生适合抗击疾病的新类别的抗菌剂。这些目标涉及的具体目标包括确定这些分离株是否为新菌株，它们与现有生物体的关系有多密切，它们是否产生新的抗生素，提纯和鉴定这些新抗生素，确定它们抑制哪些细菌生物体，以及克隆产生这些新抗菌素的基因。要实现这些目标，需要采取以下方法。首先，必须制定一项有效的方案，从各种高盐度环境(表层盐层、盐壳内部和卤水)中恢复数百种极端嗜盐微生物。其次，使用结构域特异的引物对的聚合酶链式反应(PCR)将被用于从新纯化的生物体中扩增16S或18S基因。这些基因的DNA序列将把这些生物体与现有的分离物进行分类。第三，通过拮抗研究将发现产生抑制产物的生物体。这包括将少量的培养物放在所有其他极端嗜盐分离物的“草坪”(一块生长介质的平板上，表面覆盖着一层薄薄的有机体)上，并在培养后寻找是否存在抑制区。此外，还将通过挑战各种革兰氏阳性和革兰氏阴性生物的草坪来挑战新分离株，进行同样的测试。第四，对产生抑制区的物质将采用一系列生化方法进行纯化，包括用越来越小的孔径过滤器浓缩培养上清液、凝胶过滤柱层析、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法(SDS-PAGE)、反相离子交换高效液相色谱(HPLC)。最后，根据纯化的抗菌素的氨基酸序列，通过合成简并的寡核苷酸探针来克隆编码这些新抗生素的基因。这些探针将被赋予放射性，并用于寻找与探针杂交并含有抗生素的限制性片段(这一过程被称为Southern blotting)。含有新基因的片段将被克隆和测序，并对新基因进行遗传和生理分析。致病微生物正以非常快的速度对目前使用的抗生素产生抗药性。这项提议与公共卫生的相关性正在挖掘产生新抗生素的微生物的新储备库。新的水库是微生物，它们在非常高的盐分(高盐分)环境中茁壮成长，比如犹他州的大盐湖。

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.