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

摘要 在全球范围内，大约有280万人感染了被认为对临床相关药物具有耐药性的细菌。 抗生素每年。在这些感染中，据美国统计， 占死亡人数的5%（35，000）。在2019年的报告中，世界卫生组织指出，如果不采取行动， 到2050年，抗药性疾病每年可能导致1000万人死亡，到2030年，抗生素 抵抗可能迫使多达2 400万人陷入极端贫困。迫切需要 开发新的抗生素，以对抗抗生素耐药性细菌数量的急剧上升。在 特别是，几乎没有对多药耐药病原体具有广谱活性的分子。之一 阻碍新型抗生素进一步开发的瓶颈是缺乏可识别的基因， 集群产生它们，这可能导致优化的异源生产。这个项目的首要目标是 建议是确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，在粗提物中， 杀死耐多药病原体。这项拟议的研究是重要的，因为抗生素耐药性， 临床上相关的抗生素在不同类型的病原菌中不断增加，特别是， 治疗多药耐药菌株的广谱选择目前是有限的。PI和他的学生 近年来分离鉴定了几株具有广谱性的根际细菌， 抗菌活性这些最初的实验是使用基于培养的方法进行的， 对于在固体培养基上相互竞争的菌株，利用化学生态学概念， 来自同一环境样品的细菌物种之间的竞争。其中一种菌株， 铜绿假单胞菌属RIT 594产生抗生素化合物的混合物（响应于另一种分离物 不动杆菌属RIT 592）。抗生素活性在三种相关耐药菌株中产生> 6个对数减少， 临床分离株：（1）流行株MRSA USA300（FPR3757）;大肠杆菌（MCR1_NJ） 和（3）在24小时时间杀灭测定中的VIM-产生铜绿假单胞菌（AR-0266）。重要的是，初步数据 表明在时间杀灭试验中没有检测到体外抗性的出现。的后续分析 使用LC-MS的粗提物提供了证据，并鉴定了200多种新化合物， 在质谱数据库中未知。全基因组测序和注释，揭示了8个推定的 基因簇含有大量的未表征的蛋白质，这些蛋白质被预测会产生一种 具有抗生素特性的各种次级代谢产物。

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.