Introduction

Marine sponges are one of the rich sources of highly diverse microbial communities, including more than ten bacterial phyla (such as Proteobacteria, Actinobacteria, Nitrospira, Chloroflexi, lanctomycetes, Cyanobacteria, Acidobacteria), major lineages of Archaea and a range of unicellular eukaryotes like diatoms and dinoflagellates. These organisms as a whole are potentially useful because of their extensive metabolic diversity, including nitrification, photosynthesis, anaerobic metabolism and secondary metabolite production. However, the exact nature of the interactions between sponges and microbes is still an enigma to the scientists if the interaction is predation or parasitism or other types of symbiosis (V. Vasanthabharathi, 2012).

Novel marine natural products are isolated from marine bacteria, fungi, sponges, worms, fishes and mostly from plants (Mayer & Hamann, 2002). They are classified into six major chemical classes, namely, polyketides, terpenes, peptides, alkaloids, shikimates and sugars and have a wide variety of biological activities, such as antibacterial, anticoagulant, antimalarial, anti-inflammatory, antiprotozoal, antituberculotic and antiviral effects (Abad, Bedoya, & Bermejo, 2008; Carballeira, 2008; Soltani, Saadatmand, Khavarinejad, & Nejadsattari, 2011).

Marine fungi are one of potential sources of secondary metabolites having various biological activities. Penicillium brocae, obtained from a tissue sample of the Fijian sponge Zyzzya sp., produced three novel cytotoxic polyketides, Brocaenols A-C which showed cytotoxicity when tested against HCT-116 cell line. Bioactive extracts of a-Proteobacterial strains from the sponge surface as well as Pseudomonas sp. associated with primmorph exhibited antiangiogenic, antimicrobial, hemolytic and cytotoxic properties (Thomas, Kavlekar, & LokaBharathi, 2010).

Hemolytic power is considered as an important virulence factor for numerous bacterial pathogens.  It is due to various factors such as pore-forming toxins, thiol-dependent cytolysins, enzymes like phospholipases, biosurfactants or to a concomitant action of these substances. Antioxidants are the molecules, which prevent cellular damage by reducing the oxidative stress and therefore have a beneficial effect on human health. One of the major causes of mortality and morbidity world-wide is atherosclerosis, the accumulation of oxysterol, cholesterol, and peroxide lipids in arteries, generated by free radicals which lead to heart attack. Hence, there has been an increased interest in the application of antioxidants (Arora & Chandra, 2010; Rodrigues, Costa, Carvalho, & Epifanio, 2005).

Materials and methods

Isolation of bacteria and fungi from Callyspongia diffusa

Isolation of bacteria

 The sponge sample was collected from Mandapam Coast (Tamil Nadu, India), transferred to a sterile polyethylene bag and transported at 4 ºC to the laboratory for the isolation of associated micro-organisms. On reaching the laboratory, the invertebrate was brought to room temperature and cut aseptically into small pieces (2 × 2 cm) using a sterile scissors and washed twice with 2 ml of sterile seawater and vortexing for 20 s in order to remove adhering particles. Finally, the sample in sterile seawater was homogenized aseptically and the homogenate was serially diluted up to 10^-6 dilutions and then spread plated on Zobell marine agar plates (Hi-Media, Mumbai) and incubated at room temperature for 24-48 hrs.

Isolation of fungi

1g of sponge sample was mixed in sterile water and was serially diluted up to 10^-4. 0.1 ml of the diluted sample was taken from 10^-3 and 10^-4 dilutions and was pour plated using 15-20 ml Potato dextrose agar (PDA) (Hi-Media, Mumbai) prepared in 50% sea water (to eliminate the bacterial contamination 8 ml of 1% Streptomycin was added to 1 L of the sterilized medium) and incubated at 30 °C for 5 days. 

Identification of potential strain

The potential bacterial strain was identified by the conventional biochemical tests (Asha Devi, Rajendran, & Karthik Sundaram, 2011). Cell morphology was observed under a phase contrast microscope and confirmed through 16S rRNA gene sequencing. The tree Topologies were evaluated by bootstrap analyses based on 1,000 replicates and phylogenetic trees were inferred using the neighbour-joining method and submitted to NCBI GenBank (accession number: 1428145 HQ907732). The sponge associated potential fungi were identified by following the method of Thomas et al. (Richards, Jones, Leonard, & Bass, 2012) and Hend et al. (Hend A. Alwathnani, 2012).

Hemolytic activity of potential strains

Hemolytic activity was determined using a blood agar plate. Blood agar base (Meat extract 10.0 g, Peptone 10.0g, Sodium chloride 5.0 g, Agar 15.0 g, pH 7.3±0.2 and distilled water 1000 ml) was prepared by autoclaving at 121 °C for 15 min. and allowed to cool at 45-50 °C and aseptically 5% (v/v) sterile defibrinated sheep blood was added. Blood agar was poured into petri plates and wells were made.     

50 µl of 24 h cell free extract of P. fluorescens (5.0 x 10⁶ cfu/ml) and 96 h cell free extract of P. citrinum (2.5 X10⁶ cfu/ml) were inoculated on blood agar plate wells. Plates were examined for hemolysis after incubation at 37 °C for 24 hrs. The plates were observed for zone of clearance as hemolysis was determined by a clear zone around the colony.

Antioxidant activity

The antioxidant activity was evaluated by the Phospho-molybdenum method according to the procedure of Vijayabaskar et al., 2012 (Vijayabaskar & Shiyamala, 2012). This assay is based on the reduction of Mo (VI) – Mo (V) by the extract and subsequent formation of a green phosphate / Mo(V) complex at acidic pH.

0.6 M sulfuric acid, 28 mM sodium sulfate and 4 mM ammonium molybdate were mixed together in 250ml distilled water and labeled as Total Antioxidant Capacity (TAC) reagent. Different concentration of lyophilized culture filtrate (0.2, 0.4, 0.6, 0.8, 1.0 mg/ml) of P. fluorescens and P. citrinum were taken in separate test tubes. About 1 ml of TAC reagent was added to all tubes. Blank was prepared with distilled water replacing the TAC reagent. Absorbance was measured at 695 nm in a spectrophotometer where Gallic acid was used as standard. The total antioxidant activity was measured as follows:

Percentage of total antioxidant activity = (Control OD - Sample OD/ Control OD) ×100

Cytotoxicity assay

Cytotoxic property of the bacterial strain was carried out by MTT (3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay against HEp-2 cell line. HEp-2 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) which was supplemented with 10% fetal bovine serum (FBS) and 100 µg/ml streptomycin. 100 µl of cell suspension was seeded into 96-well plates (5 × 10³ cells/well) and incubated at 37 °C for 24 h. After 24 h, lyophilized cell free extract of P. fluorescens and P. citrinum at various concentrations (ranging from 1 mg/ml to 1.95 µg/ ml) were added and incubated at 37 °C + 5% CO₂ for 48 h. After 48 h, media was removed from the wells carefully for MTT assay. Wells were washed with MEM (w/o) FCS for 2-3 times and 200 µl of MTT (5 mg/ml) was added and incubated again for 6-7 h. Then 1ml of DMSO was added to each well and mixed by a pipette and left for 45 sec. The suspension was transferred into the cuvette of spectrophotometer and absorbance was taken at 595 nm where DMSO was used as blank. The % of cell viability was measured by the following formula:

 Cell viability (%)   = (Mean OD/Control OD) x 100

Results

Isolation and identification of potential strains

After biochemical analysis, phase-contrast microscopy and 16s rRNA gene sequencing, highly potential bacterium P. fluorescens and fungus P. citrinum were identified and selected for further characterization.

Hemolytic activity of potential strains

The present study showed alpha (a) type of hemolysis in culture filtrate of P. fluorescens and P.citrinum. Alpha hemolysis refers to the partial lysis of red blood cells and hemoglobin. This resulted in a greenish-grey discoloration of the blood around the well (Figure 1).

Figure 1. Assessment of hemolytic property. A) Hemolytic activity in culture filtrate of P. fluorescens, B) Hemolytic activity in culture filtrate of P. citrinum, C) Control (uninoculated)

Antioxidant activity

In the present study, lyophilized culture filtrate of P. fluorescens and P. citrinum showed concentration dependent antioxidant activity and it was incereasing linearly with gradual increase in concentration and exhibited 85% and 74% antioxidant activity in 1 mg/ml of the sample, respectively. (Figure 2 and Figure 3).

Figure 2. Antioxidant activity of P. fluorescens

Figure 3. Antioxidant activity of P. citrinum

Cytotoxicity assay

Toxicity was increased with increasing concentration of lyophilized cell free extract of P. fluorescens ranged from 1 mg/ml to 7.8125 µg/ml. Maximum viability was observed at 7.815 µg/ml for the cell free extract of P. fluorescens (95.68%) where at 1 mg/ml of cell free extract of P. fluorescens viability count was 12.28%. In control (without lyophilized cell free extract of P. fluorescens) viability was 100% (Figure 4 and Figure 5).

In case of P. citrinum toxicity was found to increase with increasing concentration of cell free extract of this fungus ranged from 1 mg/ml to 1.95 µg/ml. Maximum viability was observed at 1.95 µg/ml of cell free extract of P. citrinum (96.61%) and minimum at 1000 µg/ml which was  20.33%. Cell viability was 100% in control (only medium) (Fig 6 and Fig 7)

Figure 4. Cytotoxicity assay in HEp-2 cells for lyophilized extract of P. fluorescens BCPBMS-1

Figure 5. MTT assay for P. fluorescens

Figure 6. Cytotoxicity assay HEp-2 cells for lyophilized extract P. citrinum

Figure 7 . MTT assay for P. citrinum

Discussion

The present study showed alpha (a) type of hemolysis in culture filtrate of P. fluorescens BCPBMS-1 and P. citrinum. Most hemolysis-positive strains belonged to the genera Pseudoalteromonas, Aeromonas spp. and Bacillus spp (Romanenko, Uchino, Kalinovskaya, & Mikhailov, 2008). The extract of Pseudomonas spp. PB2 associated with a sponge, Suberites domuncula, exhibited anti-angiogenic, hemolytic, antimicrobial and cytotoxic activities (Thakur et al., 2005). Atagazli (Atagazli, Greenhill, Melrose, Pue, & Warner, 2010) observed hemolytic activity in culture filtrate of P. citrinum that yielded 80- 100% hemolysis in human erythrocytes. Hemolytic activity was observed in other Penicillium spp. as well (Taira, Marcondes, Mota, & Svidzinski, 2011). Bonassoli et al., (Bonassoli, Bertoli, & Svidzinski, 2005) reported hemolytic activity in Candida arapsilosis.

Antioxidant compounds scavenge free radicals such as peroxide, hydro peroxide or lipid peroxyl and thus reduce the level of oxidative stress slowing down or preventing the development of complications associated with oxidative stress related   diseases.  Many synthetic antioxidants have shown toxic and mutagenic effects, which have shifted attention towards naturally occurring antioxidants. In the present observation, lyophilized culture filtrate of P. fluorescens and P. citrinum showed concentration dependent antioxidant activity which incereased linearly with gradual increase in concentration and exhibited 85% and 74% antioxidant activity in 1 mg/ ml of the sample, respectively.

Endophytic Paenibacillus polymyxa isolated from the root tissue of Stemona japonica, produced exopolysaccharides (EPS) which had strong scavenging activities on superoxide and hydroxyl radicals (Liu et al., 2009). Graphislactone-A, a phenolic metabolite isolated from the endophytic fungus Cephalosporium spp. IFB-E001, had free radical-scavenging and antioxidant activities in  in vitro study  (Song, Huang, Sun, Wang, & Tan, 2005). Guo et al. (Guo et al., 2010) reported that extracellular polysaccharides ETW1 and ETW2 produced by marine bacterium Edwardsiella tard, exhibited strong antioxidant activities. Antioxidant activity was observed in intra-cellular and extra-cellular metabolites of marine Streptomyces species VITTK3 (Thenmozhi, Sindhura, & Kannabiran, 2010). Sun, et al., (Sun et al., 2009) isolated three different exopolysaccharides from marine fungus Penicillium sp. F23-2 and evaluated their antioxidant activity by assays in in vitro systems which revealed that those three polysaccharides possessed good antioxidant properties, especially scavenging abilities on superoxide radicals and hydroxyl radicals.  Srinivasan et al. (Srinivasan et al., 2010) observed it in fungal extract of endophytic Phyllosticta spp. Sadananda et al. (Sadananda et al., 2011) reported total antioxidant capacity of the endophytic fungus A. niger and A. alternata. In the present observation also endorsed the same.

The cytotoxicity of lyophilized cell free extracts of sponge-associated bacteria and fungi against HEp-2 cell line indicated that the presence of potent cytotoxic and probably anticancer components of these extracts. Cytotoxicity was increased with increasing concentration of lyophilized cell free extract of P. fluorescens and P. citrinum ranged from 1 mg/ml to 7.8125 µg/ml where highest cytotoxicity (87.72%) was observed in cell free extract of P. fluorescens with a concentration of 1 mg/ml. Cytotoxicity against normal cell lines is needed to be assessed to further characterize these highly potent anticancer cell free extracts.

There were some reports on  P. aeruginosa  and  Bacillus sp. in producing some biologically active compounds against cancer cell lines (Ohba, Mizuki, & Uemori, 2009). The cytotoxic activity of the Candida tropicalis, Acinetobacter baumannii, Pseudomonas aeruginosa and Bacillus sp., crude extracts were determined against four established cancer cell lines; MCF-7, HepG2, HeLa and U937 cells, and Vero cell line as a representative of normal cell line (Kantachote et al., 2010). Alkaloid Lodopyridone from a marine Saccharomonospora spp. found to be cytotoxic (IC₅₀ = 3.6 µM) to HCT-116 human colon cancer cells (Maloney et al., 2009). Sivonen et al. (Sivonen, Leikoski, Fewer, & Jokela, 2010) observed potent antitumor activitiy in ulithiacyclamide and patellamide-A belong to cyanobactins, produced by cyanobacteria. Phonnok et al. (Phonnok, Tanechpongtamb, & Wongsatayanon, 2010) reported cytotoxic activity of the microbial crude extracts against four established cancer cell lines, viz., MCF-7, HepG2, HeLa and U937 cells and Vero cell line. Yoghiapiscessa et al. (Yoghiapiscessa, Batubara, & Wahyudi, 2016) observed cytotoxic activity of (sponge) Stylotella sp.  associated Pseudoalteromonas flavipulchra. Marine derived fungus A. nomius (NC06) from sponge N. chaliniformis AR-01 showed the most selective cytotoxicity against WiDr cell line (Artasasta, Yanwirasti, Djamaan, & Handayani, 2017). Xiaoling et al., (Xiaoling et al., 2010) observed that mangrove associated endophytic fungi isolated from Zhuhai,China had cytotoxicity activity in KBV and KBV 200 cell lines. Almeida et al. (Kijjoa et al., 2010) observed anticancer activity in extract of E.cristatum, a fungi isolated from sponges. In vitro study proved its inhibitory activity against MCF-7 (breast adenocarcinoma), NCI-H460 (non-small lung cancer) and A375-15 (melanoma) cell lines.

However, the study proved that both P. fluorescens BCPBMS-1 and P. citrinum possess good antioxidant activity as well as potent anticancer property. The active components responsible for these activities need to be evaluated. The data may contribute to a rational basis for the use of antioxidant rich marine P. fluorescens and P. citrinum.

Conflict of interest statement

We declare that we have no conflict of interest.

Authors’ contributions

V. V. designed the whole research and finalized the manuscript and J. S. performed the experiments and drafted the manuscript.

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