ISOLATION OF ANTIBACTERIAL COMPOUND FROM MARINE SOIL ACTINOMYCETES

The antibacterial study revealed that the isolated marine soil actinomycetes by crowded plate method and was identified by MIDI Advance technology (gas chromatographic) method and Thus, the microbes were identified according to their fatty acid sequences. The actinomycetes species were identified as Streptomyces rimosus, Streptomyces fradiae, and Streptomyces griseoflavus and the antibacterial assay was carried out by well diffusion method. Out of these only Streptomyces rimosus having high antimicrobial potential against Gram positive as well as Gram negative bacteria. In Gram positive Streptomyces aureus exhibits higher sensitivity followed by Bacillus subtilis where as gram negative bacteria E.Coli was extremely sensitive to the metabolites followed by Pseudomonas aeruginosa.


Introduction
Actinomycetes are aerobic, gram positive bacteria that form branching filaments or hyphae and asexual spores and frequently filamentous and sporulating with DNA rich in G+C from 57-75%. The name Actinomycetes are derived from Greek word "aktis" means a ray and "mykes" means fungus. Immediately after the first shower the earth's smell of the musty odour is due to the presence of Actinomycetes. The Streptomycetes metabolites known as the 'Geosmin' is responsible for the earthy odour. However volatile product secreted by Streptomyces may also be responsible for the characteristic smell. 1 After the revolution in the 'golden era", in forties and early fifties, when almost all groups of important antibacterial antibiotic (Tetracycline, Cephalosporines, Amino glycosides, Macrolides) were discovered, the success story had continued. It seemed in the fifties and sixties that the main problems of chemotherapy had been solved. Antibiotic discovered in this period were mainly isolated from Streptomyces species representing some 70-80 % of the all isolated compounds. During this period the discovery of antitumour, antiviral and non antibiotic-enzyme inhibitory-metabolites had just started . In the next period, between the seventies and nineties the efficiency of research had decreased. In this period, besides the leading role of actinomycetes products (65-70%), the considerable increase of the discovery of "rare Actinomycetes" products (up to 30%) were also noticeable. The chemical structures of almost all discovered compounds had been elucidated. 2 2. Materials and Methods 2.1Collection of soil sample: Soil samples were collected from three locations, Chilka lake, Puri beach and Bhuvneshwar. In the month of May the soil samples were taken from the 20 cm depth after removing approximately 3 cm of upper soil surface. The samples were placed in the polythene bags, closed tightly and stored in a refrigerator. The processed samples were given proper identification code (ID). 8

Isolation of the Actinomycetes organism:
Collected soil samples were treated with 2% calcium carbonate and incubated at 37 0 C for 15 min. Isolation of the Actinomycetes was carried out by the serial dilution method and followed by the crowded plate technique. 3

Primary screening of the Isolates:
Few colonies were selected which showed the antimicrobial activity and grown by streaking horizontally on the nutrient agar medium plate and incubated for 24 hours at 28 0 C. After incubation test organisms were streaked perpendicular on it with sterile inoculating loop and then incubated for 24 hours at 28 0 C. The zones of inhibition near to the test organism were observed. 4

Primary screening for isolated marine
Actinomycetes : Primary screening of the isolated Actinomycetes were carried out against the test organism Staphylococcus aureus, Bacillus subtilis, Escherichia coli. Out of 12 isolates only 3 isolates were shown the antibacterial activity in Table 1.

Microbial Identification of Isolated marine soil Actinomycetes by MIDI (GC):
The identification of isolates by midi was carried out and the results were tabulated in table 2. From the result of MIDI it was found that Isolate No.3, 9, and 11 were identified as Streptomyces species and found to be Streptomyces rimosus, Streptomyces fradiae, and Streptomyces griseoflavus respectively.

Thin layer chromatography of Extracts:
The extracts were separated by TLC and observed on Iodine vapor and UV spectrophotometer and the R f values were tabulated in Table 3.

Antibacterial activity of extracted crude antibiotic
Antibacterial activity was carried out by well diffusion method, zone of inhibition were observed and tabulated in Table 4.

Summary and Conclusion
The collected soil samples from the different locations were brought to the laboratory under aseptic condition and used for isolation of antibacterial compounds. Serial dilutions of sample were made and isolation of the antibiotic MIC studies were carried out to found that the exact concentration of drug required to inhibiting the growth of test organisms. Extracts were further studied for their spectral analysis by UV and FTIR Spectroscopic analysis. The UV spectrum of all crude antibiotics showed λ max between 268 to 277 nm. FTIR showed the antibacterial groups but the graphs didn't match with the previous antibiotic produced by Streptomyces rimosus Streptomyces fradiae and Streptomyces griseoflavus. Hence the produced antibiotics might be newer antibiotics. To conclude, all the isolated Actinomycetes had good antibacterial activity except few had some inhibitory effect against test organisms. Among these isolate 3 had got more potent activity as compared to isolate 9 and 11. The identification and elucidation of the structure of these antibiotics required C 13 NMR, MS spectral analysis and can be carried out in future studies.