Antimicrobial and cytotoxic activity of Marine Bacteria Isolated from the Karwar Coast, West coast of India

INTRODUCTION

INTRODUCTION

The Ocean, which is called the Mother of origin of life', is also the source of structurally unique natural products that are mainly accumulated in living organisms. Several of these compound show pharmacological activities and are helpful for the invention and discovery of bioactive compound, primarily for deadly diseases like cancer, acquire immuno-deficiency syndrome, etc. The lives saving drugs are mainly found abundantly in microorganisms, algae and invertebrates and vertebrates. Modern technologies have opened vast areas of research for the extraction of biomedical compounds from ocean and seas to treat the deadly diseases.

The number of natural products isolated from marine organisms increases rapidly, and now exceeds 18,000, with hundreds of new compounds being discovered every year. A large proportion of these natural compounds have been extracted from marine invertebrates, especially sponges, ascidians, bryozoans and molluscs, and some of them are currently in clinical trials. Most marine invertebrates are sessile soft bodies that inhabit benthic rock environments. In the sea, rock substrate is limited, and benthic organisms have to compete for the space to live and develop. Invertebrate organisms have evolved defence strategies based on the synthesis of cytotoxic compounds in order to avoid predation and epibiosis. Many invertebrate animals, like sponges, tunicates, bryozoans, molluscs and oligochaetes are symbiotically associated with microorganisms belonging to the Bacteria and Archaea domains. In some cases, the source of the cytotoxic compounds isolated from marine invertebrates are the symbiotic bacteria. For instance, the tunicate Lissoclinum patella is simbiotically associated with the cyanobacteria Prochloron sp., which produces the cytotoxic compounds patellamides A and C, each with clinical potential. Davidson et al. provided evidence in the bryozoan Bugula neritina that its symbiont "Candidatus Endobugula sertula" is the source of bryostatins, which show excellent potential as therapeutic agents against leukemias, lymphomas, melanomas and solid tumors.

Screening of marine bacteria isolated from the surface of marine algae and invertebrates has shown that a high percentage produce Bioactive compounds. In addition, bacteria in biofilms formed on the surface of marine organisms have been documented to contain a high proportion of antibiotic producing bacteria than some other marine environment. The aim of this study was to investigate the antimicrobial and cytotoxic activity of Pseudomonas species isolates from marine environment of Karwar coast, West coast of India.

MATERIALS AND METHODS

Sample collection: The deep sea sedimentand water sample were collected from the Karwar region, west coast of India at a depth of 20 meter. (Lat. 14o 45.46'N. Long. 074o 02.84'E). The samples were brought to the laboratory in aseptic condition. Than the microorganisms were cultivated on Zobell Marine Agar 2216, than it was sub cultured on Modified Nutrient agar (MNA).

Screening of Isolates for antimicrobial activity: In preliminary screening, determination of the antimicrobial activity of pure isolates was done by zone of inhibition method on Nutrient agar (NA) using Salmonella typhi as pathogen. Further screening was performed by zone of inhibition method against the standard test organisms. Active microbes were cultured for the screening of antibiotic substances in nutrient agar at 28oC for overnight. The test organisms used were: Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Proteus vulgaris , Klebsiella spp., S. typhi and Enterococcus faecalis.

Characterization of Isolates: The potent isolates selected from the secondary screening were characterized by morphological and biochemical methods. The results of microscopic examination were compared with Bergey's manual of Determinative Bacteriology, Ninth edition (2000) and the organism was identified. Various biochemical tests were performed for the identification of the potent isolates are as follows; Fermentation of sugars, Hydrolysis of starch, Indole production, Methyl red, Vogues Prauskauer, Citrate utilization, Urease test, 2%Peptone water, Nitrate reduction test, Gelatin liquefaction, Catalase test, Oxidase test.

Isolation and purification of antibacterial metabolites: Samples from Pseudomonas Culture No.7 grown in 1 L nutrient broth at 28oC and 220 rpm were taken and extraction was carried out using ethyl acetate solvent extraction [ethyl acetate : filtrate 1:1 (v/v)] on shaker at 220 rpm for 1 hr. The ethyl acetate phase that contains antibiotic was separated from the aqueous phase and by evaporation in water bath at 80-90C the residue was obtained and weighed. The compound thus obtained was used to determine antimicrobial activity and minimum inhibitory concentration. The antimicrobial activity was determined by spectrophotometric method. The residue obtained was dissolved in 1 ml 0.2 M phosphate buffer (pH 7.0). 100 l of preparation was loaded into a well in the 24 well plates, containing pathogenic cultures and were incubated at 37C for 18 - 48 hr and optical densities measured at 540 nm using ELISA plate reader (Figure 1). For thin layer chromatography (Becker and Lechavier, 1964), silica gel plates (Merck) 5 X 20 cm, 1 mm thick were used. 10 l of the ethyl acetate fractions and reference antibiotics were spotted on the plates and the chromatogram was developed using ethylacetate : iso-propanol : acetonitrile (1:4:5) as solvent system.

Cytotoxicity assay:Human Lung carcinoma cell lines were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS). For cytotoxicity assays, cells were seeded into 24-well plates at a density of 2.1 105 cells/well and the dilutions (1:500 and 1:1000) of the extract made in DMEM in the volumes of 10 l and 25 l were added. After incubation period (24, 48 h), the growth media was removed and the cells were trypsinized and counted in a Hemocytometer (Freshney, 2006).

RESULTS AND DISCUSSION

Out of 38 isolates were subjected for primary screening process, only 21 isolates showed activity against test organisms. Of which, only 3 were active against gram negative organism, 10 against gram positive organisms and 12 against both gram positive and gram negative organisms. Among these isolates, 29 of them showed positive inhibitory effect against B. subtilis, 27 against S. aureus, 17 against E. coli, 15 against S. typhi and 14 against Proteus species. Out of the 21 isolates those were subjected for the secondary screening, 10 isolates were active against B. subtilis, 12 against S. aureus, 6 against E. coli, 3 against P. vulgaris and 4 against S. typhi. It can be seen that the Pseudomonas culture no.7 shows maximum zone of inhibition against pathogenic culture. Culture no 7 which showed activity against all pathogenic strains (Figure 1). Figure 2 extrapolates the effect of extract of 48 h grown culture of Pseudomonas spp. colony 7 on various pathogenic cultures in the form of optical densities obtained. Identification of the potent antibiotic producing strains reveals that most of the specimens belong to the genus Pseudomonas. The minimum inhibitory concentration of the extract from Pseudomonas spp. no.7 was 0.73 mg.mL-1. Also the results from thin layer chromatography showed the spot given by the extract of Pseudomonas spp. was with Rf value 0.78.

We examined the effect of Pseudomonas spp. and its metabolites on the growth of eukaryotic cells. For the cytotoxicity studies human lung carcinoma cell were chosen. Monolayer cultures of human lung carcinoma cells were exposed to various concentrations of the extracts and cell viability evaluated by counting live cells after 24 and 48 h of exposure (Figures 3). The extract caused cell death at dilutions of 1/500 (10 l). At dilution of 1/1000 (25 l) cell viability greatly reduced after 48 h of exposure.

Although the antimicrobial and cytotoxic agents obtained in this study can't be declared as new bioactive compound, there is the probability of finding new bioactive compound in Karwar because of its wide biodiversity. For proper identification of the extracts of bioactive compound, it is necessary to obtain in pure form, which requires a series of purification process and different chemical analysis. As we know the land of Karwar is virgin in this field, so lots of works should be done to explore the new bioactive compound because any new bioactive compound and its producing organism have been a great demand.

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Antimicrobial and cytotoxic activity of Marine Bacteria Isolated from the Karwar Coast, West coast of India

By: pradeep

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