The aim of our study was to screen Moroccan actinomycete isolates able to produce nonpolyenic antifungal metabolites in order to contribute to limiting the problem of fungal infections emergence in particular mycotic diseases and to discover known antifungal families, especially nonpolyenic drugs. 480 isolates were tested against 5clinically pathogenic Candida species. Several methods have been used to study the polyenic or non-polyenic nature of the antifungal molecules produced by Actinobacteria (i) The study of the antibacterial activity (the bacterial plasma membrane is devoid of sterols); (ii) The screening of the antimicrobial activity of resistant strains to polyenic antifungals essentially Candida tropicalis R2 and Pythium irregular; (iii) The inhibition of antifungal activity by exogenous ergosterol addition in the culture medium and (iv) The UV-Visible light spectrophotometric analysis of the crude extracts of the actinomycete isolates. Among all isolate tested, only 60 (29 %) showed an antifungal activity against all test microorganisms used. Six active isolates meet all the selection criteria and produced nonpolyenic antifungal metabolites. The taxonomic study of the promising isolate Z26, using morphological, physiological and molecular characters, showed that it has 99,43% of similarity with Streptomyces phytohabitans but there were some differences in morphological characteristics. In addition, the chemical study, using chromatographic and spectroscopic techniques, of the bioactive substances produced in the Z26 isolate fermentation broth in NL300 culture medium allowed the determination of two macrolide derivatives with chemical structures C 62 H 100 O 24 (m/z 1251.6504 [M+Na] +) and C 68 H 110 O 26 (m/z 1365.7174 [M+Na] +). NMR experiments revealed that the active compounds were Novonestmycin A and B. The Novonestmycins A and B are for the first time produced by an actinomycete strain purely isolated from the Moroccan ecosystems.
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.
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