Isolation, identification and characterization of potentially novel antibiotics from rhizospheric bacteria without detectable in vitro resistance

从根际细菌中分离、鉴定和表征潜在的新型抗生素，且体外未检测到耐药性

• 批准号: 10358855
• 负责人: ANDRE O HUDSON
• 金额: $ 44.36万
• 依托单位: ROCHESTER INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10358855
• 关键词: Accounting; Acinetobacter; Anabolism; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Aspartate; Bacteria; Bacterial Antibiotic Resistance; Biochemical; Biological Assay; Biotin; Carbohydrates; Centers for Disease Control and Prevention (U.S.); Cessation of life; Chemical Structure; Chemicals; Clinical; Cloning; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Complement; Crude Extracts; Crystallization; Data; Databases; Development; Disease Resistance; Drug resistance; Drug resistance in tuberculosis; Epidemic; Epithelial Cells; Erythrocytes; Escherichia coli; Event; Evolution; Exclusion; Foundations; Future; Gene Cluster; Gene Expression Profiling; Genes; Genome; Goals; Health; Horizontal Gene Transfer; Housekeeping; Human; Human Cell Line; In Vitro; Individual; Infection; Intermediate resistance; Lead; Link; Liquid Chromatography; Mass Spectrum Analysis; Mining; Molecular; Molecular Target; Morbidity - disease rate; Multi-Drug Resistance; Multiple Bacterial Drug Resistance; Ornithine; Pathway interactions; Patients; Poverty; Production; Property; Proteins; Proteomics; Pseudomonas aeruginosa; RNA methylation; Reporter; Reporting; Research; Resistance; Role; Sampling; Series; Signal Transduction; Solid; Staphylococcus aureus; Streptomyces; Structure; Students; Sulfhydryl Compounds; System; Thin Layer Chromatography; Time; Toxic effect; Vancomycin; Work; World Health Organization; Yeasts; bacterial resistance; base; clinically relevant; combat; comparative; experimental study; gene product; genome annotation; genome sequencing; homoserine lactone; human mortality; in vivo; intestinal epithelium; knock-down; knockout gene; liquid chromatography mass spectrometry; methicillin resistant Staphylococcus aureus; microorganism interaction; mortality; multi-drug resistant pathogen; mutant; novel; overexpression; pathogenic bacteria; public health relevance; quorum sensing; resistance gene; resistant strain; response; transcriptome sequencing; transcriptomics; whole genome

ABSTRACT Globally, approximately 2.8M people are infected with bacteria that are deemed resistant to clinically relevant antibiotics an annual basis. Of these infections, 700,000 individuals will die with the United Sates accounting for 5% (35,000) of these deaths. In the 2019 report, The World Health Organization states that if no action is take, drug resistant diseases could cause 10 million deaths each year by 2050 and that by 2030, antibiotic resistance could force up to 24 million people into extreme poverty. There is an urgent need for the development of novel antibiotics to combat the drastic rise in the number of antibiotic resistant bacteria. In particular, there are few molecules with broad spectrum activity against multidrug resistant pathogens. One of the bottlenecks that is impeding the further development of novel antibiotics is the lack of identified gene clusters producing them, which could lead to optimized heterologous production. The overarching goal of this proposal is to identify and characterize the biosynthetic gene clusters and products from these anabolic pathways that responsible for the production of antibiotics, which in crude extracts were shown to be active in killing multi-drug resistant pathogens. The proposed research is significant since antibiotic resistance to clinically relevant antibiotics is increasing across different types of pathogenic bacteria, and particularly, broad-spectrum options to treat multidrug resistant strains are currently limited. The PI and his students recently isolated and identified several strains of rhizospheric bacteria, which possess broad-spectrum antibiotic activity. These initial experiments were performed using a culture-based approach, which screened for strains competing against each other on solid media, leveraging the chemical ecological concept of competition between bacterial species from the same environmental sample. One of the strains, Exiguobacterium sp RIT 594 produces a cocktail of antibiotic compounds (in response to another isolate Acinetobacter sp RIT 592). The antibiotic activity produce a >6 log reductions in three relevant resistant clinical isolates: (1) the epidemic strain MRSA USA300 (FPR3757), (2) NDM-producing E. coli (MCR1_NJ) and (3) VIM-producing P. aeruginosa (AR-0266) in 24 h time kill assays. Importantly, the preliminary data show that the emergence of in vitro resistance was not detected in the time kill assays. Subsequent analysis of crude extracts using LC-MS provided evidence and identified more than 200 novel compounds that are unknown in the mass spectrometry databases. Whole genome sequencing and annotation, revealed 8 putative gene clusters that contained a large number of uncharacterized proteins that are predicated to produce a variety of secondary metabolites that are putatively endowed with antibiotic properties.

抽象的 全球范围内，大约有 280 万人感染了具有临床相关耐药性的细菌 每年使用抗生素。根据美国统计，在这些感染中，将有 70 万人死亡 占这些死亡人数的 5% (35,000)。世界卫生组织在 2019 年报告中指出，如果不采取行动 假设，到 2050 年，耐药性疾病每年可能导致 1000 万人死亡，到 2030 年，抗生素 抵抗可能使多达 2400 万人陷入赤贫。迫切需要 开发新型抗生素以对抗抗生素耐药细菌数量的急剧增加。在 特别是，很少有分子具有针对多重耐药病原体的广谱活性。之一 阻碍新型抗生素进一步开发的瓶颈是缺乏已鉴定的基因 生产它们的集群，这可能会导致优化的异源生产。本次活动的总体目标是 提议是鉴定和表征这些合成代谢的生物合成基因簇和产物 负责生产抗生素的途径，粗提物中的抗生素被证明具有活性 杀死多重耐药病原体。这项研究具有重要意义，因为抗生素耐药性 针对不同类型的病原菌，临床相关的抗生素正在增加，特别是 目前治疗多重耐药菌株的广谱选择有限。 PI和他的学生 最近分离并鉴定了几株根际细菌，它们具有广谱性 抗生素活性。这些最初的实验是使用基于培养的方法进行的，该方法筛选了 对于在固体培养基上相互竞争的菌株，利用化学生态概念 来自同一环境样本的细菌物种之间的竞争。其中一种菌株， Exiguobacter sp RIT 594 产生抗生素化合物混合物（响应另一种分离物 不动杆菌属 RIT 592）。抗生素活性使三种相关耐药性减少>6个对数 临床分离株：(1) 流行菌株 MRSA USA300 (FPR3757)，(2) 产 NDM 大肠杆菌 (MCR1_NJ) (3) 24 小时杀灭试验中产生 VIM 的铜绿假单胞菌 (AR-0266)。重要的是，初步数据 表明在时间杀灭测定中没有检测到体外耐药性的出现。后续分析 使用 LC-MS 的粗提物提供了证据并鉴定了 200 多种新化合物 质谱数据库中未知。全基因组测序和注释，揭示了 8 个假定的 包含大量未表征蛋白质的基因簇，这些蛋白质预计会产生 多种被认为具有抗生素特性的次级代谢产物。