057 | Bioactive metabolites from antarctic sponge-associated bacteria: bioactivity and cytotoxic assessment

Claudia Gravagno^1|2, Stefano Putaggio², Teresa Romeo^2|3, Annamaria Russo², Carmen Rizzo^2|4, Ester Tellone², Angelina Lo Giudice⁴ | ¹Stazione Zoologica Anton Dohrn, Sicily Marine Centre, Messina, Italy; ²University of Messina, Italy; ³National Institute for Environmental Protection and Research, Milazzo [ME], Italy; ⁴National Research Council, Institute of Polar Sciences, Messina, Italy.

Marine sponges are among the oldest and most biologically complex invertebrates and host diverse symbiotic microorganisms capable of producing a wide range of bioactive secondary metabolites. Sponge symbionts from extreme environments, such as Antarctica, are of particular interest due to the potential evolution of unique metabolic adaptations. In this study, the cultivable bacterial fraction associated with specimens of Dendrilla antarctica Topsent, 1905 and Sphaerotylus antarcticus Kirkpatrick, 1907 was isolated, phylogenetically characterized and screened for its biotechnological potential, by assessing the production of extracellular bioactive metabolites, and simultaneously investigating its cytotoxic effect on red blood cells (RBCs). Following phylogenetic identification through Sanger sequencing, a preliminary screening of antagonistic activity was carried out via cross-streak testing. Extraction of metabolites was performed from cultures of the positive strains. The biological activity of the extracts was assessed by human erythrocyte hemolysis assays, antioxidant activity assays, and finally, plate diffusion assays for antimicrobial activity analysis. The isolation of symbiotic bacteria led to the production of 50 strains, mainly belonging to the phyla Proteobacteria, Firmicutes and Actinobacteria, with a predominance of the genera Psychrobacter, Planomicrobium and Kocuria. Molecular characterization, based on 16S rDNA gene amplification and sequence comparison with reference databases, highlighted similarity levels ranging from 97 to 100%. The cross-streak test involved approximately 2500 crosses, from which 23 strains showed antagonistic activity. Cytotoxic analysis, conducted on RBCs, showed that microbial extracts did not exhibit hemolytic activity up to 12 h of incubation (0.5-1.0 mg/mL), while an overall increase in the percentage of hemolysis was recorded after 24 h of incubation at both concentrations tested. In addition, all extracts resulted in a reduction in meta-Hb values compared to untreated RBCs after 24 h of incubation. A selection of extracts with lower or none haemolytic activity was subsequently selected to evaluate their antioxidant and antimicrobial actions. The results obtained show that the extracts exhibit a good antioxidant action against different radical species (DPPH, Superoxide Anion, Hydroxyl Radical, ABTS) and that they have the ability to chelate and reduce iron as shown by the results obtained from the iron chelation assay and FRAP, respectively. Antimicrobial activity assays conducted against potentially pathogenic target strains, such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Proteus mirabilis, showed clear inhibition of microbial growth, indicating the presence of metabolites with significant antimicrobial activity. Overall, the results indicate Antarctic sponge-associated bacteria as a source of novel bioactive compounds. The integration of hemolysis assays into the screening pipelines proved for both safety assessment and selection of strains with higher biotechnological potential, providing a foundation for future analysis including chemical characterization and heterologous production of promising compounds.