Abstract
Natural products are an important source of lead compounds for the development of drug substances. Actinomycetes have been valuable especially for the discovery of antibiotics. Increasing occurrence of antibiotic resistance among bacterial pathogens has revived the interest in actinomycete natural product research. Actinobacteria produce a different set of natural products when cultivated on solid growth media compared with submersed culture. Bioactivity assays involving solid media (e.g. agar-plug assays) require manual manipulation of the strains and agar plugs. This is less convenient for the screening of larger strain collections of several hundred or thousand strains. Thus, the aim of this study was to develop a 96-well microplate-based system suitable for the screening of actinomycete strain collections in agar-plug assays. We developed a medium-throughput cultivation and agar-plug assay workflow that allows the convenient inoculation of solid agar plugs with actinomycete spore suspensions from a strain collection, and the transfer of the agar plugs to petri dishes to conduct agar-plug bioactivity assays. The development steps as well as the challenges that were overcome during the development (e.g. system sterility, handling of the agar plugs) are described. We present the results from one exemplary screening campaign targeted to identify compounds inhibiting Agr-based quorum sensing where the workflow was used successfully. We present a novel and convenient workflow to combine agar diffusion assays with microtiter-plate-based cultivation systems in which strains can grow on a solid surface. This workflow facilitates and speeds up the initial medium throughput screening of natural product-producing actinomycete strain collections against monitor strains in agar-plug assays.
Highlights
Natural products are an indispensable source of lead compounds for the development of drug substances [1, 2]
About 60% of the known natural products with biological activity are derived from plants, about 40% are produced by microorganisms such as fungi and bacteria [3]
While actinomycetes can grow and produce secondary metabolites in submersed cultures, they naturally grow attached to solid surfaces
Summary
Natural products are an indispensable source of lead compounds for the development of drug substances [1, 2]. Of the bacterial natural product producers, especially in the field of antibiotics discovery, the most famous are undoubtedly the Gram-positive, filamentous actinomycetes [3,4,5,6], other bacteria such as myxobacteria and cyanobacteria have received increasing attention over the last years [7,8,9]. Both major pharmaceutical companies and academic groups have created impressive actinomycete strain collections, sometimes comprising of tens of thousands of strains [10,11,12]. Important compounds such as deferoxamine [16], a compound used to treat iron intoxication that is listed in the WHO Model List of Essential Medicines [17], or the herbicide glufosinate [18] resulted from research on this strain collection
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