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

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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.