Extreme Halophiles: A Potential Reservoir of New Antibiotics

极端嗜盐菌：新型抗生素的潜在库

• 批准号: 7305462
• 负责人: Stephen M Beckstrom-Sternberg
• 金额: $ 22.2万
• 依托单位: NORTHERN ARIZONA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7305462
• 关键词: Achievement; Amino Acid Sequence; Antibiotic Resistance; Antibiotics; Archaea; Bacillus anthracis; Bacillus cereus; Bacteria; Biochemical; Biological Factors; Class; Clinical; Cloning; Column Chromatography; Culture Media; DNA Sequence; Deposition; Disease; Drug Industry; Environment; Gel Chromatography; Genes; Genetic; Goals; Head; Health; High Pressure Liquid Chromatography; Hospitals; Hot Springs; Incubated; Institutes; Ion Exchange; Laboratories; Liquid Chromatography; Methods; Multi-Drug Resistance; Nosocomial Infections; Oceanography; Oligonucleotide Probes; Organism; Peptides; Phase; Phylogenetic Analysis; Physiological; Placement; Polyacrylamide Gel Electrophoresis; Polymerase Chain Reaction; Price; Process; Production; Proteins; Protocols documentation; Public Health; Radioactive; Rate; Recombinant DNA; Resistance; Ribosomal DNA; Score; Series; Sodium Chloride; Sodium Dodecyl Sulfate; Source; Southern Blotting; Spottings; Surface; Testing; antimicrobial; base; brine; design; extreme halophile; fighting; gene cloning; interest; microorganism; pathogen; size; success

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.

描述（由申请人提供）：致病微生物正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。对于引起医院获得性（院内）感染的病原体尤其如此。我们需要新的抗生素，但从哪里找呢？一个未开发的来源是来自极端环境的微生物。这项建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对抗疾病的新型抗菌剂。这些目标所涉及的具体目标包括确定这些分离物是否是新的，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，纯化和表征这些新抗生素，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。实现这些目标需要下列方法。首先，必须制定一套有效的方案，从各种高盐环境（地表盐沉积、盐壳内部和盐水）中回收数百种极端嗜盐微生物。其次，采用区域特异性引物对的聚合酶链反应（PCR）将从新纯化的生物体中扩增16S或18S基因。这些基因的DNA序列将把生物体与现有的分离物进行分类。第三，产生抑制产物的生物体将通过拮抗研究发现。这包括将少量培养物放置在所有其他极端嗜盐分离物的“草坪”上（一个含有薄薄的生物体层的生长培养基板），并在孵育后寻找抑制区域的存在。此外，将通过挑战针对各种革兰氏阳性和革兰氏阴性生物的新分离物来进行相同的测试。第四，产生抑制区的物质将使用一系列生化方法进行纯化，包括用孔径越来越小的过滤器、凝胶过滤柱层析、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和反相和离子交换高效液相色谱（HPLC）对培养上清进行浓缩。最后，根据纯化的抗菌药物的氨基酸序列，通过合成简并寡核苷酸探针克隆这些新抗生素的编码基因。探针将呈现放射性，用于寻找与探针杂交并含有抗生素的限制性片段（该过程称为Southern blotting）。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。引起疾病的微生物正以非常快的速度对目前使用的抗生素产生耐药性。这一建议与公共卫生的相关性在于开发产生新抗生素的新微生物库。新的储层是微生物，它们在非常高盐（高盐）的环境中茁壮成长，比如德克萨斯州的大盐湖。