Marine sponges and their associated bacteria as bioresource for the production of antimicrobial compounds
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The discovery of novel natural products (NPs) that can serve as lead structures has to be an ongoing effort to fill the respective development pipelines. However, identification of NPs, which possess a potential for application in e.g., the pharma or agro sector, must be as cost effective and fast as possible. Furthermore, the amount of sample ... available for initial testing is usually very limited, not least because of the fact that the impact on the environment, i.e., the sampled biosystem, should be kept minimal. Here, the pipeline SeaPEPR was implemented, in which a primary bioactivity screening of crude extracts is combined with the analysis of the corresponding metabolic fingerprint. This enables prioritization of samples for subsequent microfractionation and dereplication of the active compounds early in the workflow. As a case study, 76 marine sponge-derived extracts were screened against a microbial screening panel. Thereunder, human pathogenic bacteria (Escherichia coli ATCC35218 and Staphylococcus aureus ATCC33592) and yeast (Candida albicans FH2173), as well as the phytopathogenic fungus Septoria tritici MUCL45407. Overall, nine extracts revealed activity against at least one test organism. Metabolic fingerprinting enabled assigning four active extracts into one metabolic group; therefore, one representative was selected for subsequent microfractionation. Dereplication of the active fractions showed a new dibrominated aplysinopsin and a hypothetical chromazonarol stereoisomer derivative. Furthermore, inhibitory activity against the common plant pest Septoria tritici was discovered for NPs of marine origin. The pipeline represents a valuable tool for further bioprospecting projects, since only low sample volumes are needed that in turn renders an extensive collection of limited bioresources for screening purposes (e.g., slow-growing macroorganisms like sponges) obsolete. The potential of sponge-associated bacteria for the biosynthesis of natural products with antibacterial activity was evaluated. Out of all 396 marine bacteria from campaign 1, which were isolated from five sponges, 6.1% showed antibacterial activity against at least one of the test organisms. Most of the active isolates belonged to the genus Bacillus. Further antimicrobial activities were observed for Salinispora and Microbacterium species. The automatic and manual analysis of LC/MS2 data allowed dereplication of ten compounds, i.e., saliniketal A, rifamycin S, staurosporine, staurosporine M1, and staurosporine-7-OH, derivatives of staurosporine-7-OH, desf-05, desferrioxamine, desferrioxamine E, and ferrioxamine D1. In a preliminary screening, bacteria isolation campaign 2 revealed that 108 of 835 axenic isolates showed antibacterial activity. Active isolates were identified by 16S rRNA gene sequencing and selection of the most promising strains was done in a championship like approach, which can be done in every lab and field station without expensive equipment. In a competition assay, strains that inhibited most of the other strains were selected. In a second round, the strongest competitors from each host sponge competed against each other. To rule out that the best competitors selected in that way represent similar strains with the same metabolic profile, BOX PCR experiments were performed, and extracts of these strains were analysed using metabolic fingerprinting. This proved that the strains are different and have various metabolic profiles, even though belonging to the same genus, i.e., Bacillus. Furthermore, it was shown that co-culture experiments triggered the production of compounds with antibiotic activity, i.e., surfactins and macrolactin A. Since many members of the genus Bacillus possess the genetic equipment for the biosynthesis of these compounds, a potential synergism was analysed. It was shown that molecules with assumed antibacterial effects, e.g., surfactins, are inactive alone (at the concentrations tested), but can have synergistic effects combined with other molecules produced by Bacillus species. From the third isolation campaign, one bacterium belonging to the genus Flammeovirga was selected for further investigation. The strain was isolated from an unidentified sponge collected at the Cenderawasih Bay National Park, Papua, Indonesia. However, despite the observed activity in the initial screening, the bioassay-guided isolation approach was not successful to isolate compounds with antibacterial activity produced by Flammeovirga. Putatively active compounds were isolated; however, in very less amount and the compounds decomposed during further analysis. Furthermore, compounds with antibacterial activity which were isolated are predicted to be medium-derived. Hence, further optimization of fermentation and purification conditions is needed. In addition, genome mining could be a way forward for the further investigation of this bacterial group.