Screening and Isolation of Antagonistic Actinobacteria Associated With Marine Sponges from Indian Coast

Sponges are host organisms for various symbiotic microorganisms such as Archea, Bacteria, Cyanobacteria and Microalgae. Sponges associated microorganisms are sources of wide variety of useful natural products like antibiotics, anti-inflammatory, antioxidant, antiviral, antifouling and cytotoxic compounds. Nearly 60 isolates of action bacteria were found to be associated with 6 species of marine sponges collected at various locations. The isolates were screened for antimicrobial activity against 6 pathogenic bacteria and 4 pathogenic fungi. Among 60 isolates, 15 isolates showed antibacterial activity and 6 isolates showed antifungal activity. Among active isolates, isolate no.42 showed highest antimicrobial activity against all the pathogenic bacteria and fungi studied and it was identified as streptomyces species. Screening and Isolation of Antagonistic Actinobacteria Associated With Marine Sponges from Indian Coast


Introduction
Marine sponges are a rich source of structurally unique natural compounds possessing a wide range of biological activities [1]. Recently several studies proved that bioactive natural products, initially isolated from marine sponges, are produced by microorganisms, which are associated commensally or symbiotically with marine invertebrates [2]. It is generally assumed that the interior of the sponge body is continuously oxygenated, due to the efficient pumping of water through the aquiferous system [2,3]. Hence sponges are not likely to harbour anaerobic microorganisms. However, the presence of facultative anaerobic bacteria in sponges has been demonstrated [4,5] and the recent discovery of sulfate reducing bacteria [6,7] and other symbiotic archea in sponges show that anaerobic microbial process may take place in sponges tissues. Actinomycetes have been traditionally a rich source for biologically active metabolites [8]. They have provided many important bioactive compounds of high commercial value and continue to be routinely screened for new bioactive compounds. Almost 80% of the world's antibiotics are known to come from Actinomycetes, mostly from the genera streptomyces [9]. Thus in the present study, an attempt was made on isolation of streptomyces strains associated with marine sponges along the Indian Coast.

Collection of sponges
Sponges were collected from the Indian coast (17° 43' N, 83° 18' E and 16.93° N, 82.22° E). Samples were collected in sterile polythene bags containing sterile sea water and kept on ice. Then the samples were transported to the laboratory with minimum possible time to avoid the external microbial contamination and stored at 4°C.

Isolation of actinobacteria from Marine sponges
Sponge specimens were rinsed in sterile sea water, cut into pieces of 1 cm 3 with a help of a sterile knife. Further, homogenized in a sterile mortar with 10 volumes of sterile sea water. The supernatant was diluted in ten-fold series and subsequently plated out on various media like Actinomycetes isolation agar, starch casein, Glycerol asparagine agar, M1 medium, ISP 2 medium and R2A agar for isolating actinobacteria [10,11]. All the media were supplemented with 0.2 µm pore size filtered cycloheximide (100 µg/ml), nystatin (25 µg/ml) and nalidixic acid (25 µg/ml) to facilitate the isolation of slow-growing actinobacteria and to inhibit fungal contamination. The inoculated plates were incubated in inverted position for 1-8 weeks at [28+2°C].

Enumeration and maintenance of cultures
In six selective media the number of Actinobacterial colonies found was sub-cultured on starch-casein agar. Later they were kept in refrigerator (4°C) till further analysis was to be carried out [12].

Characterization of the isolates
The isolates were characterized upto genus level by observing the spore bearing hyphae, structure of spore chain, color of the spore, aerial mass color and color of substrate mycelia as described by Bergey's manual [13] and International Streptomyces Project [13,14].

Screening of actinobacteria for antibiotic compounds
Initial screening of actinobacteria for antibiotic production was performed by cross streak method. The isolates having the activity were cultured in 100 ml of production medium (0.8 g NaCl, 1 g NH 4 Cl, 0.1 g KH 2 PO 4 , 0.2 g MgSO 4 . 7H 2 O, 0.04 g CaCl 2 , 2H 2 O, 2 g glucose, 3 g yeast extract in 1 l of distilled water, pH 7.3). These cultures were grown in a rotary shaker at 200 rpm, 28°C for 120 hrs under the standard conditions of aeration and agitation. The resultant cultures were centrifuged for 15 min. activities by agar well diffusion method [15]. The Muller Hinton agar plates were seeded with S. aureus MTCC 7443, B. sublitis MTCC 8141, P. vulgaris NCIM 2813, E. coli MTCC 6365 for antibacterial activity. Potato dextrose agar plates were seeded with S. cerevisiae MTCC 463, C. albicans MTCC 1346, A. niger MTCC 6484 for antifungal activity. Wells of 6 mm diameter were prepared in the plates, filled with 50 µl of crude culture supernatant samples and the diameter of inhibition zones were measured after incubation at 37°C for 24 hours for antibacterial activity and 28°C for 72 hours for antifungal activity. Ciprofloxacin (for bacteria) and Griseofulvin (for fungi) were used as positive control (10 µg).

Isolation of actinobacteria from sponges
In the present study a total of six species of sponges were collected along the Indian Coast. Six species of sponges were identified as:

vi. unknown Demospongiae
A total of 60 actinomycetes were isolated from these sponges. Out of the 60 isolates of actinomycetes, 4 isolates were identified as genus streptomyces (spore chain was coiling spiral and looped), 10 as Micromonospora, (clusters of single conidia on substrate mycelium), 8 as Nocardia (conidia on powdery appearance aerial hyphae, carotenoid like pigments) and 2 as Streptoverticillium (whorls of straight chain of conidia formed) ( Table 1).

Antimicrobial activity of selective isolates
The mean diameter of inhibition zones was taken as the degree of antimicrobial activity of the isolates. In the present study, the zone of inhibitions is the mean of triplicates. As shown in Table 3, out of the 60 isolates 9 isolates were active against the pathogenic bacteria. Out of 9 isolates, 4 isolates [MB 31, MB 33, MB 42 and MB 60] showed excellent activity against the pathogenic bacteria. Whereas 2 isolates [MB 15 and MB 54] inhibited the growth of only gram positive bacteria and did not exhibit any activity against gram negative bacteria. Table 4, out of the 60 isolates 6 isolates were active against pathogenic fungi. Out of the 6 isolates, 2 isolates [MB 42 and MB 60] showed excellent activity. Whereas, the remaining 4 isolates were active against only three fungal species. The antimicrobial studies revealed that isolate MB 42 and MB 60, showed excellent antagonistic activities against both the bacterial and fungal species under study.

Conclusion
The main focus of the study reveals that the marine sponges act as the potential source for the development of new active compounds in the development of drugs. However, further studies are needed in the characterization of the isolates, to identify the chemical nature of the active compound.