Abstract
Despite the wide availability of antibiotics, infectious diseases remain a leading cause of death worldwide1. In the absence of new therapies, mortality rates due to untreatable infections are predicted to rise more than tenfold by 2050. Natural products (NPs) made by cultured bacteria have been a major source of clinically useful antibiotics. In spite of decades of productivity, the use of bacteria in the search for new antibiotics was largely abandoned due to high rediscovery rates2,3. As only a fraction of bacterial diversity is regularly cultivated in the laboratory and just a fraction of the chemistries encoded by cultured bacteria are detected in fermentation experiments, most bacterial NPs remain hidden in the global microbiome. In an effort to access these hidden NPs, we have developed a culture-independent NP discovery platform that involves sequencing, bioinformatic analysis and heterologous expression of biosynthetic gene clusters captured on DNA extracted from environmental samples. Here, we describe the application of this platform to the discovery of the malacidins, a distinctive class of antibiotics that are commonly encoded in soil microbiomes but have never been reported in culture-based NP discovery efforts. The malacidins are active against multidrug-resistant pathogens, sterilize methicillin-resistant Staphylococcus aureus skin infections in an animal wound model and did not select for resistance under our laboratory conditions.
Highlights
Despite the wide availability of antibiotics, infectious diseases remain a leading cause of death worldwide[1]
We have developed sequencing strategies that rely on the barcoding of biosynthetic genes using degenerate polymerase chain reaction (PCR) primers to parse mixtures of biosynthetic gene clusters (BGCs) present in environmental samples[7,8]
Primers targeting non-ribosomal peptide synthetases (NRPS) adenylation domains (ADs) were used to screen environmental DNA (eDNA) isolated from small aliquots of each soil to identify environments predicted to contain gene clusters that encode for unidentified calciumdependent antibiotics
Summary
Following recovery, transfected cells were inoculated into 8 ml LB with selective antibiotic (12.5 μg ml−1 chloramphenicol) in 24-well plates at a density of ~25,000 clones per well and grown overnight. At 30 min post infection, rats were given single daily topical treatments of vehicle (25 mM CaCl2 in sterile water), or 0.5 mg malacidin A or daptomycin suspended in 25 mM CaCl2, and the wounds were covered in fresh Tegaderm dressing. In the cases where bacterial growth was observed in the 4×MIC overnight cultures, the resistant culture was plated in successively higher concentrations of antibiotic the following day This was repeated over the course of the experiment to assess fold change in MIC at day 0 to day 20. The intracellular accumulation of the cell wall precursor UDP-MurNAc-pentapeptide after treatment of MRSA USA300 with malacidin was assessed as previously described[16]. Received: 15 July 2017; Accepted: 3 January 2018; Published online: 12 February 2018
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