Cytotoxic and Antimicrobial Potential of Actinomycete Species Saccharopolyspora salina VITSDK4 Isolated from the Bay of Bengal Coast of India K. Suthindhiran and K. Kannabiran

Problem statement: Although the diversity of marine Actinobacteria ha ve been studied and biotechnologically exploited throughout the world, the studies on marine actinobacteria in Indian peninsula are largely unexplored. Of 9 maritime sta te in India, only 4 states have been extensively studied for the diversity of actinobacteria. Furthe , the studies on bioactive actinomycetes from sali ne soil are very scanty. In the present study, we had taken an initiative to isolate culturable halophili c actinomycetes and to screen the bioactive potential . Approach: The marine sediment sample was collected at a depth of 400 cm at Marakkanam. The s train was isolated using ISP No. 2 medium supplemented with 25% sea water. The polyphasic tax onomy of the strain was evaluated by phenotypic, chemotaxonomic and phylogenetic analysi s. The 16S rRNA was sequenced and phylogenetic relationship with the closest related species were studied. The growth conditions and the medium had been optimized under shake-flask conditi ons by measuring the dry weight of the mycelium. The isolate was subjected to fermentation and the crude extract was screened for cytotoxic, hemolytic and antimicrobial activity. The cytotoxic ity was evaluated on HeLa cells by MTT assay, hemolytic assay on mouse erythrocytes and the antim icrobial activity was determined by agar diffusion assay. Results: Based on polyphasic taxonomy the species was ident ified as Saccharopolyspora salina and belongs to the genus Saccharopolyspora. The 16S rRNA sequence of the isolate showed 100% similarity with Saccharopolyspora salina. Based on the phylogenetic and phenotypic evaluatio n the isolate was designated as Saccharopolyspora salina VITSDK4. The growth was maximal in the designed production medium with the incubation temp rature of 28°C and pH of 7.4. It is a moderately halophilic species requires 9% NaCl concentration f or optimal growth. The cytotoxicity on HeLa cells showed the IC50 value of 26.2 μg mL −1 by MTT assay. The hemolytic activity on mouse eryt hrocytes showed the EC 50 value of 266 μg mL . The crude extract also exhibited significant anta gonistic activity against fungal pathogens Aspergillus niger, Aspergillus fumigatus, Candida albicans and the Gram negative bacteria Escherichia coli and Klebsiella pneumoniae. Only moderate activity was seen against Gram positive bacteria Staphylococcus aureus and Bacillus subtilis. Conclusion/Recommendations: Based on the results of our investigation, the isol ated strain was identified as Saccharopolyspora salina VITSDK4, which was moderately halophilic, produces an extracellular bioactive metabolite, which inhibits the proliferation of HeLa cells as well as antagoni stic to fungal and bacterial pathogens. Further studies on purification and characterization of the pure compound from the strain were ongoing. The results of this study suggested that t e marine actinomycetes from the unexplored Indian coast coul d provide lead compounds of therapeutic value.


INTRODUCTION
Marine microorganisms continue to provide pharmacologically important secondary metabolites which are unique and novel chemical compounds.
Marine microbes are continuously explored for drug discovery. Apart from microbes all other marine sources have also provided valuable chemical diversity [1] . Marine actinomycetes have been traditionally a rich source for biologically active metabolites. Although heavily studied over the past three decades, actinomycetes continue to prove themselves as reliable sources of novel bioactive compounds.
Among the well-characterized pharmaceutically relevant microorganisms, actinomycetes remain major sources of novel, therapeutically relevant natural products [2] . The majority of these compounds demonstrate one or more bioactivities many of them developed into drugs for treatment of wide range of diseases in human, veterinary and agriculture sectors [3] . Searching for novel actinomycetes constitutes an essential component in natural product-based drug discovery. Analytical methods continue to improve to allow the rapid elucidation of structures and make natural products valuable components of modern drug discovery.
The isolated compounds from marine actinomycetes has a broad spectrum of biological activities such as antibiotic, antifungal, toxic, cytotoxic, neurotoxic, antimitotic, antiviral and antineoplastic activities [4] . Recently, new targets have been added to the general screening like AIDS, immunosuppression, anti-inflammation, Alzheimer disease, ageing processes, some tropical diseases and resulted in discovery of several drugs [5] Studies on marine actinomycetes are facing some unexpected problems. For many of the marine actinomycetes the taxonomy of the strain is very poorly defined, so that binomial identifications are frequently uneasy to be carried out.
The genera Saccharopolyspora belongs to the class actinobacteria and the order Actinomycetales. The genus Saccharopolyspora belongs to the family Pseudonocardiaceae, is known for producing antibiotics such as vancomycin, erythromycin and rifamycins [6] . The genus Saccharopolyspora was proposed by [7] originally isolated from spontaneously heated sugarcane bagasse. Representatives of these species form a distinct phyletic line within the evolutionary radiation encompassed by the family Pseudonocardiaceae can be distinguished by using phenotypic properties [8] .
Members of the genus Saccharopolyspora are characterized by aerobic, extensively branched substrate hyphae that fragment into rod-shaped elements and aerial hyphae that segment into bead-like chains of spores. The colonies are thin, raised, slightly wrinkled and mucoid in appearance. The cell wall envelope is rich in iso-and anteiso branched chain fatty acids, wall chemotype IV; do not contain mycolic acids, MK-9(H 4 ) as predominant menaquinone and DNA G+C contents in the range of 66-77 mol% [9] . Members of the genus Saccharopolyspora are a potentially rich source of natural products, but only erythromycin, produced by Saccharopolyspora erythraea, is of commercial importance [10] . The isolation of the genus Saccharopolyspora is complicated because of the poor knowledge of its habitation and slow growth. In this study, we report a polyphasic taxonomic study of Saccharopolyspora salina VITSDK4 isolated from a saltpan marine soil sample collected at the Marakkanam coast and its cytotoxic, hemolytic and antimicrobial activity.

MATERIALS AND METHODS
Sampling and isolation of actinomycetes: The marine sediment samples collected at the depth of 400 cm in the Marakkanam, [Latitude (N) 12°20′; Longitude (E) 79°95′)] coast of the Bay of Bengal, India. The actinobacteria was isolated using the ISP no. 2 media supplemented with 25% sea water, 25% soil extract, cycloheximide (25 mg mL −1 ) and nalidixic acid (25 mg mL −1 ) (Himedia, Mumbai, India) with 9% NaCl concentration. Plates were incubated at 28±2°C for 15 days. The isolate was subcultured and maintained in slant culture at 4°C as well as at 20% (v/v) glycerol stock at -80°C.

Optimization of media and cultural conditions:
To determine the optimal nutritional and cultural conditions and to identify the suitable media for growth, the isolate was inoculated in different culture medias (SCA, ISP 2, ISP 5, ISP 6, ISP 7, Czapek's agar, modified Bennett's agar, glucose/peptone, glycerol/calcium malate agar (Waksman medium No.7) and nutrient agar) and the growth was investigated. The effect of cultural conditions like different incubation temperatures (15,28,37 and 50°C), different pH (5.0, 6.0, 7.6 and 9.0) and NaCl concentrations (1,3,6,12,15,18,22,24 and 26%) on the growth of the isolate was also studied. The characteristics of physiological, biochemical and concentration of carbon and nitrogen sources were determined as described by [11] . After incubation the dry weight of the mycelium was measured and correlated with the growth of the isolate. Based on the growth of the isolate in different media the cultural conditions were optimized.

Taxonomy:
The morphological, cultural, physiological and biochemical characterization of the isolate was carried out as described in International Streptomyces Project (ISP) [12] . The morphological properties of the isolate were examined by using light microscope as well as scanning electron microscope (Hitachi, S-3400N). The cell wall fraction and sugar composition were analyzed as per the procedures described earlier [13,14] . The whole-cell sugar composition was determined as reported by Becker et al. [15] and Lechevalier and Lechevalier [16] . Polar lipids were examined and identified using the method of Minnikin et al. [17] . Menaquinones were determined as described by Collins [18] . The isolate was inoculated in TSB agar plates [trypticase soy broth (BBL), 3% (w/v); Bacto agar (Difco), 1.5% (w/v)], incubated for 7 days at 28°C and used for fatty acid analysis. The fatty acids were extracted, methylated and analysed using the standard MIDI (Microbial Identification) system [19,20] .
The DNA was isolated by HiPurA bacterial DNA isolation and purification kit (Himedia, India) and amplified by PCR using a master mix kit, Medoxmix (Medox, India) as per user manual. The primers and the PCR conditions were adapted from Rainey et al. [21] . The design of the sequencing primers and the methodology for the sequencing were adapted from previous reports [21][22][23] . The 1273 bp fragment of the 16S rRNA gene of the strain was sequenced in both the sense and antisense directions. The 16S rRNA sequence was analyzed for the similarity and homology with the existing sequences available in the data bank of National Center for Biotechnology Information (NCBI) using BLAST search. The DNA sequences were aligned and phylogenetic tree was constructed by neighbor joining method using ClustalW software [24] . A bootstrap analysis of 1000 replicates was carried out. The phylogenetic tree based on Maximum-parsimony method was also carried out using MEGA version 3.1 [25] . To determine the G+C content, the DNA was isolated by the method of Marmur [26] and the G+C content was determined using the thermal denaturation method of Marmur and Doty [27] .
Fermentation and extraction of secondary metabolites: Well grown slant cultures of the isolate optimized in production medium were inoculated into 50 mL medium in 250 mL Erlenmeyer flasks containing the optimized production medium with sea water 25%, distilled water 75%, pH 7.2 and incubated for 2 days in rotary shaker (200 rpm) at 28°C. The inoculums (10%) were transferred into 200 mL production medium in 1L Erlenmeyer flasks. The inoculated cultures in the production medium were incubated for 72 h on a rotary shaker (200 rpm) at 28°C. After fermentation the broth was centrifuged at 4000 rpm for 10 min at 10°C and the filtrate was separated. The supernatant was extracted twice with ethyl acetate (400 mL) and washed with 500 mL water. The extract was then concentrated in rotary vacuum and lyophilized using a freeze drier (Thermo, USA) at 5°C for 5 h.

Preparation of stock solution:
The lyophilized ethyl acetate extract was used to prepare stock using distilled water (1 mg mL −1 ) and filtered through 0.2 m filter (Sartorius, India) to avoid contamination. The appropriate concentration of the extract was made by serial dilution. In vitro antibacterial assay: The antibacterial activity of crude extract (25 mg mL −1 ) was tested by agar diffusion assay [28] . The plates were incubated at 37°C for 24 h during which activity was evidenced by the presence of a zone of inhibition surrounding the well. Each test was repeated three times and the antibacterial activity was expressed as the mean of diameter of the inhibition zones (mm) produced by the secondary metabolite when compared to the controls. Chloramphenicol was used as positive control.
In vitro antifungal assay: Antifungal activity of the crude extract was determined by using the standard method CLSI M38-A (formerly NCCLS) [29] . The fungal cultures were maintained in 0.2% dextrose medium and the optical density 0.10 at 530 nm was adjusted using spectrophotometer. Each fungal inoculums were applied on plate and evenly spread on Sabouraud's Dextrose agar (HiMedia, India) using a sterile swab. Agar diffusion assay was followed to evaluate the antimicrobial activity along with amphotericin B. The Petri plates were incubated at 30°C for 2 days. At the end of the 48 h, inhibition zones formed in the medium were measured in millimeters (mm). All experiments were done in three replicates.

Minimum Inhibitory Concentration (MIC):
MIC was determined by the broth 2-fold macro dilution method [30] . The crude extract was serially diluted in Mueller Hinton broth for bacteria and Sabouraud's dextrose broth for fungi. Varying concentrations of the extracts 1,500-5 mg L −1 for bacteria and 2,800-5 mg L −1 for fungi were prepared from the stock solution. 0.1 mL of the culture was added to the broth each containing the crude extracts. The tubes were incubated aerobically at 37°C for 24 h for bacteria and 30°C for 48 h for fungi. Positive controls were prepared separately for both bacteria and fungi with respective organisms in the same culture media without the extract. After incubation, the tube with least concentration of extract shows no growth was taken as the MIC value for the respective organism.
In vitro hemolytic assay: The hemolytic activity of the crude extract on erythrocytes was tested by using the washed erythrocytes (RBCs) from mouse [31] under in vitro conditions in 96-well plates. Each well received 100 mL of 0.85% NaCl solution containing 10 mM CaCl 2 . The first well served as negative control contained only water and in the second well, 100 mL of crude extract of various concentrations (5-500 mg mL −1 ) were added. The last well served as positive control containing 20 mL of 0.1% Triton X-100 in 0.85% saline. Then, each well received 100 mL of a 2% suspension of mouse erythrocytes in 0.85% saline containing 10mM CaCl 2 . After 30 min incubation at room temperature, centrifuged and the supernatant was used to measure the absorbance of the liberated hemoglobin at 540 nm. The average value was calculated from triplicate assay.
Cell culture: HeLa cell lines were obtained from ATCC and maintained in DMEM (Himedia, Mumbai, India) medium supplemented with 10% FBS (v/v) and 100 mg L −1 streptomycin and 100 IU mL −1 penicillin (Himedia, India) at 37°C in a CO 2 incubator with 5% carbon dioxide.
MTT cell proliferation assay: The cytotoxic activity of the crude extract (diluted in DMSO 0 to100 µg mL −1 ) on HeLa cells (1×10 5 cells/well) were tested by using the CellQuanti-MTT cell viability assay kit (Bioassay Systems). The wells with only culture medium or cells treated with 0.1% of DMSO served as control. The graph was plotted with cell viability against the time period in hours at increasing concentrations of secondary metabolite. The mean and the IC 50 value were calculated by non-linear regression analysis using the data analysis software (prism) from three independent experiments.

RESULTS
The isolated strain is Gram +ve, non-motile and aerobic bacteria. The aerial mycelium is straight, segmented, 0.6-1.0 mm, white in color. The diameter of cells is 1.2×0.5 mm. The spores are rough arranged in straight to loosely spiral shape and non-motile (Fig. 1). Extensively fragmented substrate mycelia were observed under light and scanning electron microscopy (Fig. 2). Sporangia, flagellated spores and sclerotia were not observed. The cultural, biochemical and physiological characteristics of the isolate is given in Table 1.   The typical polar lipids are diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol and phosphatidylinositol. (phospholipids type III sensu Lechevalier et al. [16] ). The fatty acid profile comprised mainly of saturated straight-chain and iso-and anteiso-branched-chain fatty acids. The major fatty acids are iso-C 15:0 , iso-C 16:0 , iso-C 17:0 , anteiso-C 16:0 and anteiso-C 17:0 . The G+C content of the DNA isolated from the species is 68.2 mol%. The chemical and morphological properties of the isolate resembles with the genus Saccharopolyspora. A BLAST search of the 1273 bp 16S-rRNA gene sequence of the isolate showed 100% homology to Saccharopolyspora salina. The phylogenetic tree was constructed based on neighbor joining method shows that the isolate is most closely related to Saccharopolyspora salina which is also supported by the high boot strap value. Based on the molecular taxonomy and phylogeny the isolate was identified as Saccharopolyspora salina and designated as Saccharopolyspora salina VITSDK4. The 16S rRNA sequence of Saccharopolyspora salina VITSDK4 has been deposited in the GenBank (NCBI, USA) under the accession number EU551240. A neighbor-joining tree based on 16S rRNA gene sequences showed that the isolate shares a same clade with Saccharopolyspora salina and occupies a distinct phylogenetic position within the radiation including representatives of the family Saccharopolyspora (Fig. 3).
The optimal growth of the isolate was attained in the production medium when cultivated at temperature 28°C; pH 7.6. The growth was also analyzed with different organic and inorganic nitrogen sources. Both organic and inorganic sources support the growth but organic nitrogen sources produce abundant colonies and sporulation. Among carbon sources, Dglucose, starch, sucrose, D-xylose, D-galactose, maltose, L-arabinose, lactose, inositol were utilized for growth, but glycerol was not utilized. The isolate grows well between 6-18% NaCl concentrations with the optimal concentration of 9% and no growth was observed at 0 and 22% salt concentration. 2) The zone of inhibition and MIC for the standard antibiotics is given within parenthesis. Chloramphenicol and Amphotericin B are the standards for bacteria and fungi respectively Based on the results of physiological test the medium has been designed for the optimum growth which contains glucose 0.5%, soluble starch 2%, meet extract 0.4%, yeast extract 0.5%, peptone 0.5%, calcium carbonate 0.4%, sodium sulfate 0.1%, potassium chloride 0.05%, magnesium chloride 0.2%, dipotassium phosphate 0.05%, sodium chloride 12%, pH 7.6.
The antimicrobial activity of ethyl acetate extract of the isolate is shown in Table 2. The maximal activity was observed against Klebsiella pneumoniae with the MIC value of 126 mg mL −1 when compared to chloramphenicol (26 mg mL −1 ). The other bacterial pathogens susceptible to the crude extract are E. coli (MIC of 174 mg mL −1 and 13.6 mm zone of inhibition), S.aureus (8.4 mm) and B.subtilis (5.1 mm). It crude extract also exhibits a good antagonistic activity against A.niger, A.flavus and C.albicans. The zone of inhibition is 15.9, 13.3 and 8.5 mm and the MIC is 96.5, 108.5 and 52 mg mL −1 respectively. Among the fungal pathogens C.albicans was more susceptible to the extract when compared amphotericin B. The effect of crude extract on cytotoxicity on HeLa cells showed a concentration and time dependent activity. The IC 50 value against HeLa cells was found to be 26.2 mg mL −1 . At 40 mg mL −1 concentration more than 70% cell death was observed. The hemolytic activity on erythrocytes showed IC 50 value of 266 mg mL −1 . The results suggest that the crude extract is less toxic to normal cells and toxic to He La cells.

DISCUSSION
The marine microorganisms are known to be rich sources of novel compounds; to date about 1000 natural products were derived from marine microbes. Interestingly marine actinomycetes produce many pharmacologically potential compounds with antibiotic and antitumor properties. Actinobacteria have been proven as a potential source of bioactive compounds and richest source of secondary metabolites. They are the most economically and biotechnologically valuable prokaryotes. However, the research on marine Actinobacteria from Indian peninsula is very scanty. In the present study we have demonstrated that our isolated halophilic Saccharopolyspora salina VITSDK4 produces a extracellular compound and inhibit the growth of tumor cells as well as microbial cells.
Our sampling site Marakkanam is situated in southern coast of India within Tamil Nadu province. It is a narrow sandy coastal belt exhibits tidal flats and marsh zones. It is perched at a height of 14 meters (45 feet) above mean sea level and has large areas of salt pans near wide backwater with salinity of 32-38 parts per thousand. In the course of our systematic screening for bioactive marine actinobacteria, 116 strains were isolated and only 7 isolates showed broad spectrum activity. The majority of the isolated strains were belongs to Streptomyces, some were belongs to Micromonospora, Actinopolyspora and few belongs to other types. The strain VITSDK4 with significant antitumor and antimicrobial activity was further identified as Saccharopolyspora salina. The cultural, morphological, biochemical, physiological characteristics indicates that the strain belongs to the genus Saccharopolyspora. Chemotaxonomic properties are typical to the genus Saccharopolyspora. It is evident from the phylogenetic analysis that the strain VITSDK4 closely matches with the strain Saccharopolyspora salina. The strain shows 100% similarity with Saccharopolyspora salina. A tree constructed by the neighbor joining method shows the distinct phyletic line which is closesly related with Saccharopolyspora salina. The strain is sensitive to, rifampicin and lincomycin, but resistant to neomycin and penicillin G.
Optimization of media and cultural conditions were carried out by a systematic study. Various carbon and nitrogen sources were supplemented and the growth was measured as dry weight of the mycelium. Glucose, xylose and starch serves as sole carbon source and better growth was seen if yeast extract is used as a sole nitrogen source and poor growth was seen when inorganic nitrogen source was used. As the samples were collected from the ocean near-salt pans, the salt concentration in the media was increased and the growth was evaluated. The organism requires salt concentration of 9% for growth and at low concentrations no growth was observed. At the concentrations of less than 3% (w/v) the colonies appear as plaque like holes. The salt requirement suggests that the organism is moderately halophilic. The sediment sample collected for the isolation of this organism was from the shore where many salt pans exist. The organism tends to grow and sporulate even at 12% salt concentration, but it exhibits very slow growth which takes 10-15 days. This suggests that the organisms ability to grow and multiply in the marine environment at various salt concentrations.
Halophilic microorganisms can be conveniently grouped according to NaCl requirements for growth [32] . Extreme halophiles are able to grow in saturated NaCl and unable to grow in the presence of NaCl concentrations less than 12%. Larsen [33] defined moderate halophiles as organisms growing optimally between 5 and 20% NaCl. Since our isolate also showed similar NaCl requirements for their growth; the strain is considered as halophiles and absence of NaCl in the medium inhibits their growth. Thus VITSDK4 phylogenetically distinct and exhibited different physiological characteristics from other Streptomyces described. Apart from sodium chloride, addition of potassium chloride 6-20%) and magnesium chloride (6-22%) also enhances the growth with maximal growth at 16% for KCl and 18% for MgCl 2 .6H 2 O.
Five liter of the fermentation broth yield 786 mg of crude extract. The extract was dissolved in DMSO (1 mg mL −1 ) and made as the stock for the assessment of cytotoxic properties. The DMSO control was maintained for the assays and results were calculated. The extract showed a strong growth inhibition against HeLa cells with an IC 50 of 26.2 mg mL −1 , which is significant with control. The IC 50 against mouse erythrocytes was found to be 266 mg mL −1 which shows that the cytotoxicity on HeLa cells was not related to membrane integrity. Also, notable activity was seen against fungal and Gram positive pathogens, but the activity against Gram negative microbes was less. Previously we have reported a Streptomyces strain with moderate hemolytic and antimicrobial activity isolated from Marakkanam coast [34] . The cytotoxic activity of VITSDK4 on HeLa cells suggests that the strain could be clinically important and need to be investigated further for the anticancer properties.

CONCLUSION
Our results indicate that the isolated Saccharopolyspora salina is a moderately halophilic strain which exhibits significant cytotoxic and strong antibiosis against fungal and Gram negative pathogens. Further studies on the purification and characterization of bioactive compounds from the strain are underway. Most of the studies conducted in Indian peninsula have been restricted to isolation, identification and studied for their antagonistic properties against different microbial pathogens. But the detailed studies on the bioactive compounds from the marine actinomycetes are lacking. It is suggested that the frequent and systematic screening of marine actinomycetes in the Indian peninsula could provide novel species as well as novel bioactive compounds.

ACKNOWLEDGEMENT
The researchers wish to thank the management of VIT University for providing necessary facilities for the completion of this study and KS thankful to the VIT management for the award of TRA. Researchers are also thankful to Mr.Elumalai, Pondicherry University, for SEM photographs.