Tunicates (Ascidians, sea squirts) are marine protochordates, which live sedentary or sessile in colonial or solitary forms. These invertebrates have to protect themselves against predators and invaders. A most successful strategy, to not being eaten by predators and prevent pathogenic microorganisms to settle, is the usage of chemical molecules for defence. To accomplish this, tunicates take advantage of the specialized metabolites produced by the bacteria associated with them. Therefore, the microbiome of the tunicates can be regarded as a promising bioresource for bacterial strains producing compounds with antibacterial activity. The aim of this study was to test this hypothesis by (i) isolation of tunicate-associated bacteria, (ii) analysis of the antibacterial activities of these strains, and (iii) purification and structure elucidation of an active compound derived from this bioresource. In total, 435 bacterial strains were isolated and thereof 71 (16%) showed antibacterial activity against multidrug resistant (MDR) bacteria. Therefrom, the ethyl acetate crude extracts from liquid fermentations of 25 strains showed activity against MDR Extended-Spectrum Beta-Lactamase (MDR-ESBL) Escherichia coli, MDR Bacillus cereus, Micrococcus luteus, and Bacillus megaterium. Phenotypic analysis based on 16S rDNA sequencing revealed the active strains belonging to different genera and phyla, like Bacillus, Pantoea, Pseudoalteromonas, Salinicola, Streptomyces, Vibrio and Virgibacillus. To obtain first insights into the molecules responsible for the antibacterial activities observed, strain Pseudoalteromonas rubra TKJD 22 was selected for large-scale fermentation and the active compound was isolated. This allowed the purification and structure elucidation of isatin, a compound known for its strong biological effects, thereunder inhibition of Gram-positive and Gram-negative pathogens.
Sponge-associated fungi are attractive targets for the isolation of bioactive natural products with different pharmaceutical purposes. In this investigation, 20 fungi were isolated from 10 different sponge specimens. One isolate, the fungus Penicillium citrinum strain WK-P9, showed activity against Bacillus subtilis JH642 when cultivated in malt extract medium. One new and three known citrinin derivatives were isolated from the extract of this fungus. The structures were elucidated by 1D and 2D NMR spectroscopy, as well as LC-HRMS. Their antibacterial activity against a set of common human pathogenic bacteria and fungi was tested. Compound 2 showed moderate activity against Mycobacterium smegmatis ATCC607 with a minimum inhibitory concentration (MIC) of 32 µg/mL. Compound 4 exhibited moderate growth inhibition against Bacillus subtilis JH642, B. megaterium DSM32, and M. smegmatis ATCC607 with MICs of 16, 16, and 32 µg/mL, respectively. Furthermore, weak activities of 64 µg/mL against B. subtilis DSM10 and S. aureus ATCC25923 were observed for compound 4.
The potential of sponge-associated bacteria for the biosynthesis of natural products with antibacterial activity was evaluated. In a preliminary screening 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, showing synergistic effects between C14-surfactin and macrolactin A against methicillin-resistant Staphylococcus aureus (MRSA).
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