Antimicrobial evaluation of the crude extract of symbiotic fungi from marine sponge Reniera japonica

Materials and Methods

Sponge collection and classification

The specimen of marine sponge (MNP-2016) was collected from the Xinghai Bay of Dalian city in China and classified as R. japonica by the Associate Professor Mingguang Mao at the Dalian Ocean University, China.

Fungal strain isolation and identification

A total of five fungal strains (coded as L1-2, L2-1, L4, L8-1 and L14) were isolated and purified from the R. japonica using the method previously reported (Miki et al., 1995). All these fungal strains were transferred into the potato dextrose agar slants separately followed by storing at 4°C. According to molecular phylogeny based on 18S rDNA sequence and morphological characterization by microscope, genus of bioactive strain was classified (Corsaro et al., 2017).

Preparation of fermentation broth and ethyl acetate extract

Each strain was cultured on the potato dextrose agar at 30°C for 7 days. Then a balanced amount of fungal colony was transferred to the culture broth in 500 mL Erlenmeyer flask which contained 300 mL sterilized potato dextrose broth, followed by shaking at 200 rpm for 7 days at 30°C. After that, the mycelium and the fermentation broth were separated with medical absorbent gauze. The filtrate was extracted twice with 450 mL ethyl acetate (Merck) each Erlenmeyer flask. The upper solvent was dried by a rotary evaporator at 25°C in vacuum to yield the ethyl acetate extract, which possibly had antimicrobial secondary metabolites. And according to the medium whether added with salt or not, these ethyl acetate extracts of fungal strains were numbered as L1-2, L1-2J, L2-1, L2-1J, L4, L4J, L8-1, L8-1J, L14 and L14J (J represents salting). Each afforded extract was kept in a vacuum drier for 3 days and dissolved in dimethyl sulfoxide (Merck) in 0.1, 1 and 10 mg/mL.

Antibacterial test

Anibacterial assay was carried out using the method developed by Wang et al. (2008). Two human pathogenic bacteria, Staphyloccocus aureus and Escherichia coli, were used in the screening process. Each strain was transferred into one 250 mL Erlenmeyer flask which contained 150 mL sterilized Luria-Bertani medium and incubated at 30°C on a rotary shaker at 180 rpm for 24 hours. About 10 mL of the melted water agar medium was poured into a petri dish (φ=9 cm) followed by lay with five Oxford cups equidistantly. 200 μL of cultured bacterium was thoroughly mixed with 150 mL of sterilized nutrition agar at 50°C and immediately put on the solidified water agar medium. After cooling, Oxford cups were removed and resulted in five holes (φ = 6 mm) in each petri dish. Three holes were respectively added with 100 μL of each sample. Two remaining holes were added with 100 μL of the positive control ampicillin sodium (0.2 mg/mL, Amresco) and 100 μL of negative control dimethyl sulfoxide. Then the plates were incubated at 30°C for 24 hours. All tests were performed in triplicate and the average value of diameters of inhibition zone (mm) were expressed as the antibacterial activity.

Antifungal test

The antifungal assay was carried out using our previous described method (Zhang et al., 2014). The human pathogenic fungus, Candida albicans, was transferred into a 250 mL Erlenmeyer flask which contained 150 mL sterilized potato dextrose broth and incubated at 30°C on a rotary shaker at 180 rpm for 48 hours. During the process of prepare the testing plate, potato dextrose agar was used as the upper medium layer. The positive control was amphotericin B (0.3 mg/mL, Sigma-Aldrich) and each plate was incubated at 30°C for 48 hours. All tests were carried out in triplicate and the inhibitory effect was expressed as the average value of diameters (mm).