View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Archivio della ricerca - Università degli studi di Napoli Federico II Marine Biotechnology (2018) 20:502–511 https://doi.org/10.1007/s10126-018-9821-9 ORIGINAL ARTICLE Identification of a Sorbicillinoid-Producing Aspergillus Strain with Antimicrobial Activity Against Staphylococcus aureus:aNew Polyextremophilic Marine Fungus from Barents Sea Paulina Corral1,2 & Fortunato Palma Esposito1 & Pietro Tedesco1 & Angela Falco1 & Emiliana Tortorella1 & Luciana Tartaglione3 & Carmen Festa3 & Maria Valeria D’Auria3 & Giorgio Gnavi4 & Giovanna Cristina Varese4 & Donatella de Pascale1 Received: 18 October 2017 /Accepted: 26 March 2018 /Published online: 12 April 2018 # Springer Science+Business Media, LLC, part of Springer Nature 2018 Abstract The exploration of poorly studied areas of Earth can highly increase the possibility to discover novel bioactive compounds. In this study, the cultivable fraction of fungi and bacteria from Barents Sea sediments has been studied to mine new bioactive molecules with antibacterial activity against a panel of human pathogens. We isolated diverse strains of psychrophilic and halophilic bacteria and fungi from a collection of nine samples from sea sediment. Following a full bioassay-guided approach, we isolated a new promising polyextremophilic marine fungus strain 8Na, identified as Aspergillus protuberus MUT 3638, possessing the potential to produce antimicrobial agents. This fungus, isolated from cold seawater, was able to grow in a wide range of salinity, pH and temperatures. The growth conditions were optimised and scaled to fermentation, and its produced extract was subjected to chemical analysis. The active component was identified as bisvertinolone, a member of sorbicillonoid family that was found to display significant activity against Staphylococcus aureus with a minimum inhibitory concentration (MIC) of 30 μg/mL. Keywords Sediments . Marine fungi . Aspergillus protuberus . Bisvertinolone . Antimicrobial activity . MDR Introduction use of antimicrobial drugs has affected the development of a new group of microorganisms, the multidrug-resistant (MDR) Antimicrobial resistance has spread dramatically in the last bacteria, which show resistance towards the most common 30 years, leading to an increase in the number of deaths due antibiotics (Muller et al. 2017). Currently, there is a pressing to infectious diseases. The excessive and often inappropriate need to discover novel and effective antimicrobial drugs to counteract this dramatic emergence of MDR infections. Paulina Corral, Fortunato Palma Esposito and Pietro Tedesco contributed Nature has always been the main source of new molecular equally to this work. scaffolds; a prime example was the discovery of penicillin. Electronic supplementary material The online version of this article Henceforth, bioprospecting of marine macro- and microor- (https://doi.org/10.1007/s10126-018-9821-9) contains supplementary ganisms for new natural drug candidates still represents the material, which is available to authorized users. best opportunity for the discovery of new bioactive com- pounds. In particular, extreme environments constitute an un- * Donatella de Pascale [email protected] explored reservoir of biodiversity (Poli et al. 2017). The use of different strategies of adaptation by organisms and microor- 1 National Research Council of Italy (CNR)-Institute of Protein ganisms allowed the colonisation of extreme habitats, which Biochemistry (IBP), Naples, Italy are characterised by low nutrient concentration, extremes of 2 Burnett School of Biomedical Sciences, College of Medicine, pH, low temperature, high pressure and salinity. Some of the University of Central Florida, Orlando, FL, USA most extreme marine habitats known like Mediterranean deep 3 Department of Pharmacy, University of Naples Federico II, hypersaline anoxic basins (DHABs; water depth ∼ 3500 m) Naples, Italy are nearly saturated with salt, from these sediments were re- 4 Department of Life Science and Systems Biology, University of trieved different halotolerant/halophilic fungal groups that Torino,Turin,Italy might have adapted to different local environments Mar Biotechnol (2018) 20:502–511 503 (Bernhard et al. 2014). Currently, particular attention has been the isolation of bisvertinolone as an active antimicrobial focused on marine fungi (Zhao et al. 2016; Saleem et al. component. 2007), since they are considered an interesting untapped re- source of biodiversity and biotechnological potential for pro- duction of secondary metabolites (Ebada and Proksch 2015). Materials and Methods The ability to grow in different ranges of salt concen- tration is probably one of the most remarkable features Sediment Collection of some species of marine fungi that could confer the potential to synthesise new bioactive metabolites The strains used in this work were isolated from sub-sea sed- (Gostincar et al. 2010). Particularly, under hypersaline iments collected from the Barents Sea (Fig. 1), during the conditions, the cell keeps its stability and integrity accu- Barkut expedition in June 2015, in collaboration with Prof. mulating compatible solutes in its interior to overcome Bjarne Landfald of the University of Tromsø. Sediments were osmotic stress and to prevent lysis (Delgado-Jarana et al. collected from nine different sampling sites and depths 2006). This condition induces changes in the membrane (Table 1) using a multicorer drill device. Several layers were composition that effectively increases the sterol to phos- cut and then aseptically placed into 50-mL conical tubes and pholipid ratio, and the fatty acid unsaturation in turn kept at 4 °C during the shipments and stored at − 80 °C after increases the membrane fluidity (Turk et al. 2004). their arrival to the laboratory. Among all marine fungi, Penicillium and Aspergillus are a very powerful source of new bioactive compounds (Blunt et al. 2015). The genus Aspergillus is widespread Culture Media all over the marine ecosystem. Aspergillus sp. fungi are heterotroph and polyextremophiles. This significant nu- Specific culture media were designed for the isolation of dif- tritional and physiological versatility is complemented by ferent groups of psychrophilic and halotolerant/halophilic mi- the metabolic capacity to produce numerous secondary croorganisms present in extreme marine environments. Each metabolites, which is believed to be important in ecolog- medium was prepared with natural seawater and artificial sea- ical signalling (Baker and Bennett 2007). Genomic stud- water in a range of six salinities [3, 5, 10, 15, 20 and 25% (w/v) ies are revealing putative biosynthetic genes of of total salts] and three different pH conditions. Natural sea- Aspergillus strains; however, the biocomposites described water was used to prepare media with a basal salt concentra- in the literature are still limited. The prediction of genes tion in order to mimic the marine environment (3–3.5% of exceeds the number of new molecules obtained so far, total salinity) while a stock solution of artificial seawater leaving a large number of compounds yet to be discov- SW30 (Subov’ssaltsolution30%(w/v)) was used to prepare ered (Rateb and Ebel 2011). other media with higher salt concentrations. Despite the fact that natural products from some fungal All media except LB were adjusted to three different pH genera have been often and intensively studied, in particular, values (4.0, 7.0 and 9.0) and sterilised by autoclaving at Aspergillus and Penicillium, there is still a great potential of 121 °C for 15 min. The names and formula of each medium secondary metabolites produced by these fungi, which have are given in grams per litre, and for solid media, 20 g/L of agar not as yet been fully and adequately explored. was added. Barents Sea is part of Arctic Continental Shelf, and it is also Artificial seawater (SW30): 234.0 g NaCl, 39.0 g MgCl2· * the deepest of the Arctic shelf seas. The most distinctive 6H2O, 61.0 g MgSO4·7H2O, 1.0 g CaCl2;6.0gKCl,0.2g oceanographic feature of the Barents Sea is the influx of the NaHCO3, 0.7 g NaBr. This medium was prepared with dis- * salty Atlantic waters from the southwest that meets and mixes tilled water up to 1000 mL. CaCl2 was dissolved separately in with Arctic water masses along the Polar Front (Loeng 1991). 20 mL of distilled water to prevent the formation of insoluble This condition makes the Barents Sea warmer in comparison complexes of CaCO3. with other areas of the same latitude, but the oceanographic The following media were prepared dissolving the com- conditions are highly variable. Barents Sea is a poorly ex- pounds in natural seawater. For media with higher salinities, plored environment, and therefore it is an appealing prospect the artificial seawater SW30 diluted in distilled water was within the biodiscovery pipeline. used to reach the salt concentration required. Herein, we report the isolation of 14 fungi from marine sediments collected in the Barents Sea and their taxonomic Seawater minimal (SWM): 1.0 g casamino acids, 1.0 g characterisation. Furthermore, we also report the identification yeast extract, 1.0 g peptone, 1.0 g glucose. of a sorbicillonoid-producing strain of Aspergillus protuberus Casein yeast chitin (CYC): 5.0 g casein, 0.5 g yeast ex- MUT 3638, a species recently described belonging to the tract, 5.0 g chitin, 0.5 g K2HPO4, 5.0gmaltextract,0.1g Aspergillus section Versicolores (Jurjevic et al. 2012),