INTRODUCTION:

In terms of evolution and biodiversity, the sea appears to be superior to the terrestrial ecosystem. One has to consider that the most important biological explosion took place in the marine ecosystem during the Cambrian period 600 million years ago and marine species comprise approximately a half of the total diversity thus offering a vast source from which to discover useful therapeutics (Cragg, 2005).The sponge or poriferans (from Latin pores “pore” and ferre “to bear”) are animals of the phylum porifera. Porifera translated to “pore-bearer”. Pores in their body are used for their respiration. It is generally accepted that sessile, soft bodied marine invertebrates such as sponge, corals, tunicates etc., which lack physical defenses, produce toxic chemicals have been developed in the oceans for thousands of year (Kahthiresan et al., 2008). Sponges exclusively aquatic and mostly marine are found from the deepest oceans to the edge of the sea.

There are approximately 15,000 species of sponge in the world, of which 150 occur in freshwater, but only about 17 are of commercial value (Brusca, 1990).

Sigmadocia medussa is thickly encrusting or massive with irregular, solid form. Raised thick – walled volcano shaped oscules reaching several centimeters in height. Exterior is pale blue green, while interior is dirty yellow in color, soft and easily torn, even, slightly smooth surface. An endosymbionts is any organism that lives within the body or cells of another organism. Many instances of endosymbionts are obligate, that is either the endosymboint or the host cannot survive without the other. Sponges gain 48% to 80% of their energy supply from these microorganisms (Barnes, 2004). The wider biosynthetic capability of sponges could be attributed to their biological association with other symbionts (Vacelet, 1971). Many different bacterial species permanently inhabit sponges and considerably to the total sponge biomass. (Wilkinson, 1978). It has been suggested that the growth of useful microorganism may be under control of the sponge host and serve as source of food or supply other metabolic product (Muller et al., 1981). The sponges contain the bioactive compounds that are potential medical importance. (Richter,1907).The dry powder of this sponge is rubbed on the chest or back of patients with lung disease or the sore places in case of foot and leg aches such as rheumatism.(Schroder,1942). Sponge derived antifouling molecules have been found to inhibit the settlement of barnacle larvae. (Okino et al., 1955).Most bioactive compounds from sponges can be classified as anti-inflammatory, antitumors, immuno or neurosuppressive, antiviral, antimalarial, antibiotic or antifouling. The presence of bioactive terpenes, sterols, cyclic peptides, alkaloids, fatty acids, peroxides and amino acid derivatives have been described from sponges. (Tramper, 2000)

The first report of antimicrobial activity of sponge extract was reported by (Nigrelli et al., 1959). Since then, there has been growing number of antimicrobial extracts reported from marine sponges, but these studies have generally used terrestrial microorganism and human pathogens to assess antimicrobial activity. More recent reports showed that the sponge aquaculture could be an alternative approach for the production of bioactive metabolites (Duckworth et al., 2003).The role of sponge associated bacteria in the host ebibiotic defense is also highlighted in a recent investigation (Thakur et al .,2) In this background, the present study was initiated to isolate the antimicrobial fraction from Sigmadocia medussa which showed potential antimicrobial activity.

MATERIALS AND METHODS

Collection of sponge

Collection of sample was carried out at Nagapattinum coast, TamilNadu,India. After washing, their color pattern was recorded for identification then the sponge species were separately preserved in methanol for the isolation of associated microbes.

Preparation of crude extract from sponge

The sponge Sigmadocia medussa was cut into small pieces (2cmx2cm) and squeezed to prepare the crude extract in a mortar and pestle, using methanol as solvent. They were extracted thrice and the extract was concentrated in a rotary vacuum evaporator at room temperature. The concentrated crude extract was collected in the air tight plastic containers and kept in the refrigerator.

Anti-microbial activity of selected marine sponge Sigmadocia medussa

The principles of antibiotic diffusion assays and specific solid and liquid medium were prepared according to Grove and Randall (1955). Initially nutrient agar spread plates were prepared using 0.1 ml of inoculum containing appropriate bacteria of 18 hours culture. The plates were kept as such for 15 minutes for the adhesion of medium. Filter paper discs were prepared using size ranging 7 mm diameter. The paper discs were then dipped in the methanol extracts of the sponge and placed over the bacterial culture. Control discs were also placed in each culture plates. After 24 hours of incubation at 30± 20°C in a B.O.D incubator, the diameters of inhibition zones were measured.

The area of inhibition zone was calculated as follows.

Cross diameter of the inhibition zone = m

Net diameter of the disc = n

Net diameter of the inhibition zone = m-n

Net radius of the inhibition zone = x/2

Area of the inhibition zone = pr2 (p=3.14)

Isolation and Enrichment of the endosymbiont from Sigmadocia medussa

Frozen sponge tissue was cut in to small pieces (<1 cm3) and placed in the sterile PBSE buffer (1x phosphate- buffered saline plus 10mM EDTA at 10ml/g of sponge). Collagenase was added at a final concentration of 500µg/ml, and the mixture was shaken on ice for 30minutes. For further disruption, the sponge suspension was blended briefly. The suspension was filtered (45-µm pore size) and centrifuged at 500xg for 6min at4°C. The filtrate was collected and transferred in to a petriplate containing marine agar medium (Himedia). The inoculated plates were incubated at 37°C for 24-48 hours. An endosymbiotic bacterium needs nutrient media supplemented with sponge extract for growth. So the individual colonies obtained after incubation were streaked across the medium containing sponge tissue extracts (autoclaved) and incubated at 37°C for 24-48 hours. For better results the above step was repeated several times. Then the pure culture was transferred to the nutrient broth, supplemented with autoclaved sponge extract and incubated at 37°C in a shaker for further analysis.

Morphological characterization

The colony morphology was observed from 18 hours colonies by analyzing the morphology as well as staining methods.

Biochemical Characterization

Biochemical characteristics of identified bacterial colonies were studied by different biochemical tests viz. Hydrogen sulphide production test, Indole production, Methyl red test, Voges proskauer, Citrate utilization, triple sugar Iron test, Urease test, (Dubey and Maheshwari, 2006) and the results are tabulated in Table 3.

PURIFICATION OF ANTIBACTERIAL FRACTION

Ammonium sulphate precipitation

During ammonium sulphate precipitation, the salt has to be added small portions under constant stirring to prevent increase of high local concentration. Ammonium sulphate was used for precipitation of total proteins at-90% saturation or for differential precipitation the proteins from the culture. After complete dissolution of the salt, the solution was equilibrated for approximately one day in cold condition to ensure complete precipitation and then the precipitation was collected by centrifugation.

Dialysis

The protein obtained by ammonium sulphate precipitation was suspended in a buffer (pH 6.8) and dialysis was done.

Procedure

For the biological work, the membranes are pretreated to remove some undesirable impurities such as glycerol, heavy metals, sulphides etc, which are associated during manufacturing process.

To remove glycerol, heavy metals, sulphur and also to inactivate and enzyme that may be present in the dialysis tube, the tube is cut into pieces of about 5=50 cm and place inside a beaker containing 500ml of 2% NaHCO, 10ml EDTA and boiled for 20minutes.After 20 minutes boiling, the inside and outside of the tubes are washed with distilled water using a squeeze bottle. The tubes are again boiled for 10 minutes in 1Mm EDTA to remove excess NaCo3. The inside and outside of the tubes are again washed thoroughly as above and stored in 10% ethanol at 4°C.

The bag is placed in an appropriate buffer solution (about 50 to 100 fold buffer volume with respect to sample volume can be used) and dialysed for 3 to 4 hours at required temperature. During this time the small molecules will be removed from the bag.

RESULTS AND DISCUSSION

Collection of sponge

Sigmadocia medussa (fig.1) species of sponges were collected, identified and taken up for isolation and bioactivity screening of secondary metabolites

Fig.1

Antimicrobial activity

The antibacterial activity of the sponge extract was determined for 5 species of gram positive bacteria(Acetobacter pasteuriances, Bacillus subtilis, Lactobacillus acidophilus, Klebsiella species, Lactococcus lactis) and 2 species of gram negative bacteria (Proteus vulgaris, Pseudomonas fluorescence) obtained from MTCC. The extract released only on the active principles but it may change due to the assay method, incubation temperature and culture media. The cylinder plate double layer method found effective to determine antibiogram.

The antibacterial activity of Sigmadocia medussa against chosen gram positive and negative bacteria presented in the table.1

Table.1

Sl.No	Organism	Zone of inhibition cm
Gram positive
1.	Acetobacter pasteuriances	0.75
2.	Bacillus subtilis	0.92
3.	Lactobacillus acidophilus	1.10
4.	Klebsiella	0.81
5.	Lactococcus lactis	0.98
Gram negative
6.	Proteus vulgaris	0.36
7.	Pseudomonas fluorescense	2.30

Fig. 1 Inhibition zone is 0.75

Fig. 2 Inhibition zone is 0.92

Fig. 3 Inhibition zone is 1.10

Fig. 4 Inhibition zone is 0.81

Fig. 5 Inhibition zone is 0.98

Fig. 6 Inhibition zone is 0.36

Fig. 7 Inhibition zone is 2.30

It was found that the extract prepared from Sigmadocia medussa successfully prevent the growth of gram positive bacteria as well as gram negative bacteria. The maximum inhibition zone was produced by the extract against gram positive bacteria, Lactobacillus acidophilus was found to be 1.10cm and gram negative bacteria, Pseudomonas fluorescence was found to be 2.30cm.

Based on the results, it was found that the extract contain antimicrobial agent which was successfully prevent the growth of microbes. The isolates showed inhibitory interactions with various gram positive and negative bacteria. This property clearly indicates that the isolates might have a defense function in the host sponge. The host sponge Sigmadocia was reported as a moderate antibacterial agent. The biosynthetic origin of most sponge secondary metabolites were unknown, although several recent studies have shown that attend some metabolites were synthesized by the symbolist and not the sponge (Brantley et al., 1995).

The antibiogram of extract against specific bacteria was presented in the chart1.

Chart 1-Isolation and characterization of Sigmadocia medusa associated endosymbiotic bacteria

Table 2

Test/Characteristic features	SES1	SES2	SES3	SES4	SES5	SES6	SES7	SES8	SES9	SES10
Simple staining	Rod	Rod	Cocci	Rod	Cocci	Rod	Rod	Rod	Cocci	Rod
Gram staining	+	+	-	-	-	+	-	+	-	-

Table3

Test	SES1	SES2	SES3	SES4	SES5	SES6	SES7	SES8	SES 9	SES10
H₂S Production	+	-	-	+	-	-	-	+	-	+
Indole test	+	+	+	-	-	-	+	+	-	-
Methyl red	+	+	+	+	+	+	+	+	+	+
Citrate utilization	-	-	-	-	+	+	-	-	+	-
Urease test	-	+	+	+	+	+	+	+	+	+

Screening of isolates for inhibitory interactions

From the agar medium supplemented with organic or aqueous extract of the sample, 10 species of bacterial endosymbionts were isolated. The isolates were classified according to the colony morphology and denoted as SES1-SES10. There is ample evidence documenting the existence of bacteria associated with sponges that produce antibacterial metabolites (Kobayashi and Ishibashi, 1993; Thakur and Anil, 2000). In the present study the extracts obtained from sponge-associated bacteria was showed antimicrobial activity (Table 1). Microorganisms play a central role in sponge biology: they serve as food particles and are found to live associate with many sponges inter- and intracellularly (Friedrich et al., 1999). Several observations support the idea that bacteria synthesize sponge-specific compounds either completely or in the form of precursors completed subsequently by sponge metabolism (Bewley et al., 1998& Jensen et al., 1994) Antimicrobial and other biological activities of associated bacteria may play a significant ecological role in sponge– bacteria associations.

Morphological and Biochemical characterization

Morphological characterization of endosymbionts was identified through the colony morphology and different staining methods were presented in the table (2) and determined biochemical characterization was presented in the table (3)

Antimicrobial and other biological activities of associated bacteria may play a significant ecological role in sponge– bacteria associations. Furthermore, the isolation of sponge-associated bacteria producing bioactive metabolites, which were originally isolated from sponges, strongly supports the hypothesis of the microbial origin of the compounds formerly ascribed to sponges (Oclarit et al., 1994& Stierle et al., 1988). Sponge associated microorganisms are responsible for the bioactivity than the host sponges. The true bacterial endo- symbionts from SES1-SES10 influenced the synthesis of secondary metabolites of the host sigmadocia medusa. This study concluded that the discovery of new classes of antibiotics is highly necessary due to the increased incidence of resistant pathogens to drugs that are currently in use. Antibiotics developed from marine microbes are particularly important because they have high potency when compared with terrestrial counterparts.

REFERENCES:

1. Brantley, Uriz M.J, Turon X, 1995. Antimalarial antiviral and antitoxoplasmosis norsester peneperoxides acids from the Red sea sponge. J. N. At. product. 64:522 – 4

2. Bewley, C.A. and D.J. Faulkner, 1998. Lithistid sponges: star performers or hosts to the stars. Chem. Int. Ed., 37: 2 Unson M.D., N.D. Holland and D.J. Faulkner, 1994.

3. Brusca, 1990. Antimicrobial activity of sum marine sponges. Nature; 222; 983 – 984

4. Cragg, 2005. Antimicrobiol activity of untenospongin B, a metabolite from the marine sponge. 144; 19 – 29.

5. Dubey R C and Maheshwari D K (2006),“Practical Microbiology” (2nd Edition), S. Chand, New Delhi.

6. Duckworth F., Heilmann, K.P., Rhomberg P, 2003. Antimicrobial activity of trophical and subtrophical sponges. California; 92093 – 0236, USA.

7. Friedrich, A.B., H. Merkert, T. Fendert, J. Hacker, P., Proksch and U. Hentschel, 1999. Microbial diversity.in the marine sponge Aplysina cavernicola (formerly Verongia cavernicola) analyzed by Xuorescence in situ hybridization (FISH). Mar Biol., 134: 461-470.

8. Jensen, P.R. and W. Fenical, 1994. Strategies for the A brominated secondary metabolite synthesized by cyanobacterial symbiont of a marine sponge and bacteria and accumulation of the crystalline metabolite in the. sponge tissue. Mar Biol., 119: 1-11.162-2178.

9. Kahtiresan H., Kanagasabhapathy M., Nagata M., 2008. Conservation of the positions of metazoan introns from sponge to humans gene 295; 299-309

10. Kobayashi J, Ishibashi M (1993) Bioactive metabolites from symbiotic marine microorganisms. Chem Rev. , 1753–1769

11. Muller W., Bohm M.,Grebenjuk V.,1981.Molecular chemical cology in sponges; Evidence for an adaptive antibacterial response in sponge.Mar.Biol:144:19-29.

12. Nigrelli, Carte B.K, Mokashe S,1959.Bio medical potential of marine Natural Products. Biosciences,April,271-286.

13. Oclarit, J.M., H. Okada, S. Ohta, K. Kaminura,Y. Yamaoka, T. Iizuka, S. Miyashiro and S. Ikegami,1994. Anti-bacillus substance in the marine sponge, species produced by an associated Vibriospecies bacterium. Microbios, 78: 7-16.

14. Okino, Corley G, Moore R.E., 1955. The Spongistatin, potently cytotoxic inhibitors of tubulin Polymerization. Biochemistry 34;9714-9721.

15. Richter, 1907, Marine metabolites as drug leads-Retrospect and prospect Colorado;1-12

16. Schroder., 1942. New perspectives in sponge Biology, Washington. 455-466.

17. Stierle, A.C., J.H. Cardellina and F.L. Singleton, 1988. A marine Micrococcus produces metabolites ascribed to the sponge Tedania ignis. Experientia, 44: 1021.

18. Thakur NL, Anil AC (2000) Antibacterial activity of the sponge Ircinia ramosa: importance of its surface associated bacteria. J Chem Ecol 26, 57–71. 26.

19. Tramper, 2000. Cell and tissue Research Springer, 38;455-457.

20. Vacelet, 1971. Electron microscope study of the association between some sponges and bacteria J. Exp. 30, 301-314.

Received on 08.11.2012 Accepted on 21.11.2012

Asian J. Pharm. Tech. 2(4): Oct. - Dec. 2012; Page 148-153