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N. Yuvaraj, P. Kanmani, R. Satishkumar, K.A. Paari, V. Pattukumar and V. Arul. Department of ..... S.P. Singh, P. Shashikala, R. Kanungo and. S.Jayachandran ...
World Journal of Fish and Marine Sciences 3 (1): 51-57, 2011 ISSN 2078-4589 © IDOSI Publications, 2011

Extraction, Purification and Partial Characterization of Cladophora glomerata Against Multidrug Resistant Human Pathogen Acinetobacter baumannii and Fish Pathogens N. Yuvaraj, P. Kanmani, R. Satishkumar, K.A. Paari, V. Pattukumar and V. Arul Department of Biotechnology, Pondicherry University, Puducherry-605014, India Abstract: The antibacterial effect of the crude methanol extracts and purified fractions of Cladophora glomerata (Linnaeus) Kützing (Cladophoraceae) against multidrug resistant human pathogen Acinetobacter baumannii and fish pathogens were investigated. Cladophora glomerata demonstrated appreciable activity against the human pathogen A. baumannii and fish pathogens Vibrio fischeri, V. vulnificus, V. anguillarum, V. parahaemolyticus, E. coli and B. cereus. TLC Purified fractions III and V of green seaweed C. glomerata inhibited the human pathogen A. baumannii and fish pathogens V. fischeri and V. vulnificus. Purified fraction II of the same seaweed inhibited only V. fischeri and V. vulnificus. Methanol extract of C. glomerata inhibited E. coli and B. cereus growth at a minimum inhibitory concentration of 75 µg/ml and other species at 100 µg/ml. Whereas, V. vulnificus growth was inhibited at a minimum concentration of 125 µg/ml. GC-MS analysis revealed the presence of hydrocarbon compounds in active fractions II, III and V of C. glomerata. These findings demonstrate that the methanol extract and their purified fractions of C. glomerata exhibited appreciable antimicrobial activity and thus have great potential as a source for natural health products. Key words: Seaweeds % Methanol extracts % Antibacterial activity and GC-MS INTRODUCTION

septicaemia and urinary tract infections. Acinetobacter infections ranked second after Pseudomonas aeruginosa among the nosocomial, aerobic, non-fermentative, Gram negative bacilli pathogens [14, 15]. Clinical impact of A. baumannii infection has been a matter of continuing debate. Many studies reported high overall mortality rates in nosocomial patients susceptible to A. baumannii bacteraemia or pneumonia [16, 17]. A. baumannii is attracting much attention owing to the increase in antibacterial resistance and occurrence of strains that are resistant to virtually all available drugs [18]. Further Vibrio spp., especially luminous Vibrio harveyi and V. parahaemolyticus have been implicated as the main bacterial pathogens of shrimp farms [19]. These Vibrio species are resistant to every antibiotic used, including chloramphenicol, oxytetracycline and streptomycin, (and are more virulent than in previous years). Consequently, the problem of giving treatment against resistant pathogenic bacteria is becoming increasingly difficult [20]. Since pathogens gaining resistance to drugs, is common due to indiscriminate use of antibiotics, much attention is needed to kill or control the pathogens using bioactive substances. Though literature speaks diverse studies of bioactivity of marine flora against several pathogens, our

Seaweeds are one of the important renewable resources in the marine environment and have been a part of human civilization from time immemorial. They offer a wide range of therapeutic possibilities both for internal and external applications. Although terrestrial biodiversity is the foundation of pharmaceutical industry, the oceans have enormous biodiversity and potential to provide novel compounds with commercial value [1, 2]. It has been reported that seaweeds serve as an important source of bioactive natural substances [2]. Many marine macro algae produce a variety of secondary metabolites [3]. These metabolites are mainly terpenes, acetogenins, alkaloids and polyphenolics, with many of these compounds being halogenated [4]. Specific studies on seaweeds, carried out in the Atlantic, Pacific and Indian oceans, have demonstrated antibacterial, antifungal and antiviral activities [5-10]. Extracts of marine algae were reported to exhibit antibacterial activity and has been investigated most widely [11-13]. Acinetobacter baumannii has emerged as an important and problematic human pathogen as it is the causative agent of several types of infections including pneumonia, meningitis,

Corresponding Author: V. Arul, Department of Biotechnology, Pondicherry University, Puducherry-605014, India. Tel: +91-413-2655994 Extn. 429, Fax: +91-413-2655265, E-mail : [email protected].

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work on testing the antibacterial efficacies of seaweeds, mainly on human multidrug resistant pathogen A. baumannii and also on fish pathogens V. parahaemolyticus, V. fischeri, V. anguillarum, V. vulnificus, Bacillus cereus and Escherichia coli is relatively on a new concept as few attempts had been made earlier in this line. In this study, the crude extracts of Cladophora glomerata and their purified fractions were employed and their potential was reported against multidrug resistant A. baumannii and fish pathogens.

MTCC 1687 were procured from Microbial type culture collection, IMTECH, Chandigarh, India. V. anguillarum was procured from Central Institute of Brackish-water Aquaculture (CIBA), Chennai. The multidrug resistant bacteria Acinetobacter baumannii obtained from Pondicherry Institute of Medical Sciences (PIMS), Pondicherry, India and the strain was biochemically characterized [23]. The pathogenic bacteria were cultured individually on Tryptic soy broth at 37°C for 18 h, before inoculation for assay. Broth culture (100 µl), which contained 107-108 number of bacteria per ml was added to tryptic soy agar medium (Hi-media, Mumbai), poured into sterile petri dishes and allowed to solidify.

MATERIALS AND METHODS Collection of Plant Materials: A total of eight seaweeds was collected from Mandapam, Rameshwaram in Tamilnadu and Pondicherry coastal line, India during August 2008. Seaweeds Sargassum wightii Greville and Sargassum myriocystum J. Agardh (Sargassaceae), Kappaphycus alvarezzi Doty (Solieriaceae), Gelidiella acerosa F. Minima and Srinivasa rao (Gelidiellaceae), Grateloupia filicina Lamouroux and C. Agardh (Halymeniaceae), Ulva lactuca Linnaeus (Ulvaceae), Cladophora glomerata (Linnaeus) Kützing (Cladophoraceae) and Dictyota dichotoma (Hudson) Lamouroux (Dictyotaceae) was identified at Salim Ali School of Ecology, Pondicherry University, in comparison with data provided in a seaweed field manual by National Institute of Oceanography, Goa [21] and University Herbarium, University of California, Berkley, compiled by Silva and Moe [22]. Voucher specimens were preserved in 5% formaline solution.

Antibacterial Assay: Growth inhibition of pathogens by seaweeds extracts was assessed using the disc diffusion assay [24]. Briefly, crude extract impregnated discs (200 µg/ml), positive control (Ampicillin 50 µg ml-1) and negative control (methanol) disc was allowed to air dry and were subsequently placed equidistantly onto the surface of the pathogen seeded TS agar plates. The plates were kept in an inverted position and incubated at 37 °C for 18 h. The growth inhibition was assessed as the diameter (in mm) of the zone of inhibited microbial growth. The experiment was carried out in triplicate. Experimental data represent mean ± SD of each sample, unless otherwise stated. Minimum Inhibitory Concentration (MIC) Assay: A broth microdilution method was used to determine the Minimum Inhibitory Concentration [25-27]. All tests were performed in Mueller-Hinton agar medium (Himedia).

Preparation of Extracts: The seaweeds were washed with sea water three times and then successively with tap water and distilled water to remove the epiphytes and other wastes. Finally, they were air dried under the shade for two weeks. The dried plant material (10 g dry weight) was ground to fine powder and extracted with 100 ml of methanol for 24 h and the extract was filtered through a Buchner funnel with Whatman number 1 filter paper. This was repeated three times for the complete extraction of methanol-soluble compounds and all the three methanol extracts were pooled. The solvent was evaporated from crude extract by rotatory evaporator. The dried extracts (1 g) were dissolved in 2 ml of methanol and stored at 4°C until use.

Purification of Compound by Thin Layer Chromatography: Concentrated crude methanol extracts of Cladophora glomerata was purified by thin layer chromatography using hexane-ethyl acetate as solvent systems. The crude extract was separated in a pre coated aluminium TLC sheet with silica gel G 60 as stationary phase and ethyl acetate: hexane mixture in the ratio of 9.5:0.5 as mobile phase. The eluted spots, representing various compounds, were visualized under UV transilluminator at 254 nm and also in the iodine chamber. TLC resolved spots of methanol extract at various Rf values were scrapped from the TLC plate and the scrapped spots were dissolved in methanol, mixed well and centrifuged at 12,000 x g for 5 min. The supernatant (40 µl) of each fraction was used to check the antibacterial activity against pathogens using the disc diffusion method in triplicate. Ampicillin was used as positive

Test Organisms: Bacteria, Vibrio parahaemolyticus MTCC 451, V. fischeri MTCC 1738, V. vulnificus MTCC 1145, Bacillus cereus MTCC 430 and Escherichia coli

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control. Experimental data represent mean ± SD of each sample. Active fractions were subjected to Gas chromatography Mass spectrometry (GC-MS) for chemical constituent analysis.

lead compounds that could be used against infectious diseases and parasites [28]. In this study, crude methanol extracts of eight different seaweeds were screened for antibacterial activity. Among the eight crude methanol extracts, the green seaweed Cladophora glomerata showed high inhibiting activity against Acinetobacter baumannii and fish pathogens (Table 1). Similar observation was made in a methanol extract of green seaweed Ulva lactuca (200 µg/ml) which showed high inhibiting activity against Staphylococcus aureus [29]. In the present study, the minimum inhibitory concentration of methanol extract of C. glomerata was found to be 75 µg/ml against Bacillus cereus, E. coli and 100 µg/ml against Gram-negative pathogens A. baumannii and Vibrio spp (Table 1). Earlier studies showed that methanol extracts of seaweeds Enteromorpha intestinalis and Gracilaria corticata were active against Grampositive bacteria [30]. In the present study, we observed that methanol extracts of green seaweed C. glomerata was active against Gram-negative bacteria. Many species of seaweeds were screened and found that members of red algae exhibited high antibacterial activity [31, 32]. However, the present study revealed that green seaweed exhibited high antibacterial activity than red and brown seaweeds. The variation of antibacterial activity of our extracts might be due to the presence of antibacterial substances, which varied from species to species [33]. Previous reports showed that Gram-positive bacteria were more effectively controlled by the extracts of algae used in their study in comparison to Gram-negative bacteria [34, 35]. This may be probably due to the presence of more complex cell wall structure in Gramnegative bacteria [36-39]. In addition to that, the resistance displayed by the pathogens might be due to masking of antibacterial activity by the presence of some inhibitory compounds or factors in the extract [40]. Interestingly, in the present study, it was observed that the seaweeds which were used in this work exhibited

Gas Chromatography-mass Spectrometry: After the initial thin layer chromatography, the purified fractions were analyzed using an Agilent 6890 series hightemperature gas chromatography-mass spectrometer (GCMS), fitted with an autoinjector. For GC-MS analysis, a high-temperature column (DB-5ht; 30 m x 0.25 mm id x 0.25 µm film thickness) was purchased from Agilent Technologies (Agilent, USA). By employing a hightemperature column, we eliminated the need for derivatization of each sample. The injector and detector temperatures were set at 350°C while the initial column temperature was set at 80°C. A 2 µl sample volume was injected into the column and ran using split less mode. After 2 min, the oven temperature was raised to 150°C at a ramp rate of 10°C/min. The oven temperature was then raised to 250°C at a ramp rate of 5°C/min and finally the oven temperature was raised to 280°C at a ramp rate of 20°C/min and maintained at this temperature for 40 min. The helium carrier gas was programmed to maintain a constant flow rate of 1 ml/min and the mass spectra were acquired and processed using both Agilent ChemStation (Agilent, USA) and AMDIS32 software. The compounds were identified by comparison of their mass with library search and authentic standards. Statistical Analysis: The experiments were conducted three times or as indicated and all data are expressed as means± SD using SPSS version 7.5. RESULTS AND DISCUSSION Antibacterial Activity of Crude Extracts: The marine environment has a great potential for the discovery of Table 1:

Antibacterial activity of crude extracts and minimum inhibitory concentration of seaweed Cladophora glomerata against pathogens (inhibition zone was measured to the nearest millimeter) Cladophora glomerata methanol extracts --------------------------------------------------------------------------------------------------------------------------------------------------------------

Test organism

Crude extract (200 µg/ml)

MIC (µg/ml)

Positive control Ampicillin

Negative control Methanol

V. parahaemolyticus

09.00±1.53

100

12.00±0.50

0.00±0.00

V. anguillarum V. fischeri

12.00±1.00 09.00±2.52

100 100

10.00±0.00 13.00±0.50

0.00±0.00 0.00±0.00

V. vulnificus E. coli

11.00±2.00 09.00±0.58

125 75

12.16±0.28 12.00±0.50

0.00±0.00 0.00±0.00

B. cereus

09.00±0.58

75

10.16±0.28

0.00±0.00

A. baumannii

15.00±1.58

100

10.16±0.28

0.00±0.00

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World J. Fish & Marine Sci., 3 (1): 51-57, 2011 Table 2: Antibacterial screening of Cladophora glomerata purified fractions against pathogens (inhibition zone was measured to the nearest millimeter)

Test organism

Cladophora glomerata methanol extracts Purified fractions ------------------------------------------------------------------------------------------------------------------------Cg 1 Cg 2 Cg 3 Cg 4 Cg 5

Positive control ---------------------------Ampicillin (50µg/ml)

V. parahaemolyticus V. anguillarum V. fischeri V. vulnificus E. coli B. cereus A. baumannii

7±0.29 0±0.00 8±0.58 6±0.58 0±0.00 8±0.58 8±0.58

12.00±0.50 10.00±0.00 13.00±0.50 12.16±0.28 12.00±0.50 10.16±0.28 10.16±0.28

7±0.25 9±0.62 11±0.42 10±0.26 5±0.40 8±0.55 8±0.30

0±0.00 9±0.15 10±0.60 7±0.26 0±0.00 0±0.00 12±0.15

Table 3: GC-MS analysis of major compounds of Cladophora glomerata

8±0.25 8±0.40 6±0.92 6±0.60 0±0.00 0±0.00 9±0.15

7±0.66 9±0.92 6±0.66 10±0.15 5±0.40 0±0.00 10±0.15

Table 4: GC-MS analysis of major compounds of Cladophora glomerata purified fraction III

purified fraction II Rt (min)

Compound

Area (%)

Rt (min)

Compound

Area (%)

27.32

Pentadecane, 8-hexyl-

7.02

25.77

Tridecane, 7-hexyl-

3.43

28.41

Tridecane, 8-hexyl-

4.93

29.33

Heptadecane, 9-hexyl-

7.02

27.77 28.41

Heptadecane, 3-methylTridecane, 8-hexyl-

3.18 8.76

29.66

Triacontane

4.34

0.35

Octacosane

4.38

29.34 30.35

Heptadecane, 9-hexylOctacosane

6.48 4.07

31.53

Heptadecane, 9-octyl-

3.21

good antibacterial activity to all Gram-negative pathogens tested except a few. Conflicting reports were observed on the presence of bioactive compounds in the seaweeds related to the seasonal variation, as well as the method of extraction and organic solvents used for extraction of bioactive compounds and differences in assay methods.

Table 5: GC-MS analysis of major compounds of Cladophora glomerata purified fraction V

Purification of Antibacterial Compound: The crude extracts of Cladophora glomerata were purified by thin layer chromatography using hexane:ethyl acetate as solvent systems. A total of five different fractions were obtained. All the TLC purified fractions were assayed for antibacterial activity against an array of pathogens and summarized (Table 2). In the present study, the second fraction of green seaweed C. glomerata showed good antibacterial activity against fish pathogens Vibrio fischeri and V. vulnificus. Similarly, the third and fifth fractions of C. glomerata exhibited good antibacterial activity against fish pathogens V. fischeri, V. vulnificus and human pathogen A. baumannii respectively. The antibacterial activity of marine algae and mangrove plants were screened against fish pathogens and reported that fractions of methanol extract of red seaweed Gracilaria corticata showed good activity against fish pathogens Pseudomonas aeruginosa and V. alginolyticus [41]. In contrast, our study showed that fractions of green seaweed C. glomerata showed good activity against fish pathogens. Similarly, purified fractions of selected South African seaweeds showed broad spectrum activity against Gram-positive as well as Gram-negative pathogens than crude extracts of the same seaweeds [39].

Rt (min)

Compound

Area (%)

27.32

Pentadecane, 8-hexyl

10.01

27.77 28.41

Heptadecane, 3-methylTridecane, 8-hexyl-

3.44 7.27

29.34 30.36

Heptadecane, 9-hexylOctacosane

6.93 11.66

GC-MS Analysis: The Thin layered chromatography purified fractions of green seaweed Cladophora glomerata were dissolved in hexane and subjected to GCMS to analyze the chemical constituents. Characteristic Gas Chromatograph-Mass Spectrometry analysis of hydrocarbons has been summarized. In the second active fraction, Pentadecane, 8-hexyl-was found to be a major compound (7.02%) followed by heptadecane, 9-hexyl(7.02%) and tridecane, 8-hxyl-(4.93) (Table 3). Similarly, pentadecane, 8-hexyl-(8.76%) was also found as major compound followed by the hydrocarbon compounds heptadecane, 9-hexyl-(6.48%) and octacosane (4.07%) in third fraction (Table 4). Octacosane (11.66%) was found to be major compound followed by pentadecane, 8-hexyl(10.01%), Tridecane, 7-hexyl-(7.27%) and heptadecane, 9hexyl-(6.93%) in fifth fraction (Table 5). Our results are in accordance with the reported investigations [42, 43]. Straight chain paraffins (n-alkanes), branched chain paraffins (alkyl-alkanes) and unsaturated hydrocarbons (alkenes) were already reported from many marine algae. [44, 45]. Hydrocarbon distribution pattern mainly in C. glomerata is closely similar to prokaryotic Anacystis 54

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montane and Btryococcus braunii belonging to Cyanophycophyta and Chrysophycopgyta respectively. Similar group of hydrocarbons heptadecane and hexadecane have been reported as common major volatile components in many other algae [46]. Distribution of hydrocarbons among the studied algae showed a very interesting pattern in respect to geographical variations. The presence of methyl and hexyl groups could be a result of alkylation of hydrocarbons with methanol and hexane which was used in extraction and purification process in the present study. Use of antibiotics to control pathogens in shrimp and fish culture is banned. There is a need for alternative methods or substances for controlling fish and shrimp diseases. Vibrio fischeri and V. vulnificus are resistant to antibiotic treatment [47] and hence the control of these fish pathogens is possible by using probiotic bacteria or any other bioactive compounds [48]. The results of this study prove that methanol fraction of green seaweed C. glomerata can control V. fischeri and V. vulnificus. Further study is needed in the fish to confirm these results. In conclusion, the present study provides data to show the appreciable antibacterial activity of seaweed Cladophora glomerata crude extracts and purified fractions against Gram-negative human and fish pathogens. The result presumes that the long chain hydrocarbons may act as potential bioactive substance and can be exploited in pharmaceutical preparations. The cultivable nature of seaweeds is an added advantage for mass production of potential antibacterial products. Further study is in progress to find out the mechanism of inhibition of Gram-negative pathogens by the purified compounds and to study the antioxidant and antiinflammatory properties of C. glomerata.

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10. ACKNOWLEDGEMENTS This work was supported by grant from Department of Biotechnology (DBT), New Delhi, India. The authors also thank Dr. Baburajendran, Dept. of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli, India for the use of GC-MS facility.

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REFERENCES 1.

2.

13.

Hay, M.E., 1996. Marine chemical ecology: what’s known and what next? J. Exp. Mar. Biol. Ecol., 200: 103-34. Smit, A.J., 2004. Medicinal and pharmaceutical uses of seaweed natural products: A review. J. Appl. Phycol., 16: 245-62. 55

Van Alstyne, K.L., G.V. Wolfe, T.L. Freidenburg, A. Neill and C. Hicken, 2001. Activated defense systems in marine macro algae: evidence for an ecological role for DMSP cleavage. Mar. Ecol. Prog. Ser., 213: 53-65. Watson, S.B. and E. Cruz-Rivera, 2003. Algal chemical ecology: an introduction to the special issue. Phycologia, 42: 319-23. Burkholder, P.R., L.M. Burkholder and L.R. Almodovar, 1960. Antibiotic activity of some marine algae of Puerto Rico. Bot. Mar., 2: 149-56. Hornsey, I.S. and D. Hide, 1974. The production of antimicrobial compounds by British marine algae. I. Antibiotic producing marine algae. Br. Phycol. J., 9: 353-61. Naqvi, S.W., A. Solimabi, S.Y. Kamat, L. Femandes, C.V.G. Reddy, D.S. Bhakuni and B.N. Dhawan, 1980. Screening of some marine plants from the Indian coast for biological activity. Bot. Mar., 24: 51-55. Rinehart, Jr, K.L., P.D. Shaw, L.S. Shield, J.B. Gloer, G.C. Harbour, M.E.S. Koker, D. Samain, R.E. Schwartz, A.A. Tymiak, D.L. Weller, G.T. Carter, M.H.G. Munro, R.G. Hughes Jr, H.E. Renis, E.G. Swynenberg, D.A. Stringfellow, J.J. Vavra, J.H. Coats, G.E. Zurenko, S.L. Kuentzel, L.H. Li, G.J. Bakus, R.C. Brusca, L.L. Craft, D.N. Young and J.L. Connor, 1981. Marine natural products as sources of antivirus, antimicrobial and antineoplastic agents. Pure Appl. Chem., 53: 795-17. Reichelt, J.L. and M.A. Borowitzka, 1983. Antimicrobial activity from marine algae: results of a large-screening programme. Proc. Int. Seaweed Symp., 11: 158-68. Hodgson, L.M., 1984. Antimicrobial and antineoplastic activity in some South Florida seaweeds. Bot. Mar., 27: 387-90. Sachithananthan, K. and A. Sivapalan, 1975. Antibacterial properties of some marine algae of Sri Lanka. Bull. Fish. Res. Stn. Sri Lanka, 26: 1-2. Mahasneh, I., M. Jamal, M. Kashasneh and M. Zibdeh, 1995. Antibiotic activity of marine algae against multiantibiotic resistant bacteria. Microbiol., 83: 23-26. Siddhanta, A.K., K.H. Mody, B.K. Ramavat, V.D. Chauhan, H.S. Garg, A.K. Goel, M.J. Doss, M.N. Srivastava, G.K. Patnaik and V.P. Kamboj, 1997. Bioactivity of marine organisms: Part VIII-Screening of some marine flora of Western coast of India. Ind. J. Exp. Biol., 35: 638-43.

World J. Fish & Marine Sci., 3 (1): 51-57, 2011

14. Schreckenberger, P.C. and A. Von Graevenitz, 1999. Acinetobacter, Achromobacter, Alcaligenes, Moraxella, Methylobacterium and other nonfermentative Gram-negative rods, pp: 539-560. In P.R. Murray, E.J. Baron, M.A. Pfaller, F.C Tenover and R.H. Yolken (eds.), Man. Clini. Microbiol., 7th Ed. Ameri. Soci. Microbiol. Washington, D.C., USA. 15. Simor, A.E., S.F. Bradley, L.J. Strausbaugh, K. Crossley and L.E. Nicolle, 2002. An outbreak due to multiresistant Acinetobacter baumannii in a burn unit: risk factors for acquisition and management. Infect. Cont. Hosp. Ep., 23: 261-67. 16. Seifert, H., A. Strate and G. Pulverer, 1995. Nosocomial bacteremia due to Acinetobacter baumannii. Clinical features, epidemiology and predictors of mortality. Medicine, 74: 340-49. 17. Cisneros, J.M., 1996. Bacteremia due to Acinetobacter baumannii; epidemiology, clinical findings and prognostic features. Clin. Infec. Dis., 22: 1026-32. 18. Perez, F., 2007. Global challenge of multidrug-resistant Acinetobacter baumannii. Antimicrob. Age. Chemothera, 51: 3471-84. 19. Sandip, M.S., C. Singh and V. Arul, 2009. Inhibitory activity of Streptococcus phocae PI80 and Enterococcus faecium MC13 against vibriosis in shrimp Penaeus monodon. World J. Microbiol. Biotechnol., 25: 697-03. 20. Sieradzki, K., R.B. Robert, S.W. Haber and A. Tomasz, 1999. The development of vanomycin resistance in patient with methicillin resistant Staphylococcus aureus. New Eng. J. Med., 340: 517-23. 21. Dhargalkar, V.K. and D. Kavlekar, 2004. Seaweeds-A Field Manual. National Institute of Oceanography, Goa. 22. Silva, P.C. and L.R. Moe, 1999. The Index Nominum Algarum. Int associa plan taxo Berlin, 48(2): 351-53. 23. Prashanth, K., R. Srinivasa Rao, R. Uma Karthika, S.P. Singh, P. Shashikala, R. Kanungo and S.Jayachandran, 2008. Correlation between biofilm production and multiple drug resistance in imipenem resistant clinical isolates of Acinetobacter baumannii. Ind. J. Med. Microbiol., 26: 333-37. 24. Engel, S., M.P. Puglisi, P.R. Jensen and W. Fenical, 2006. Antimicrobial activities of extracts from tropical Atlantic marine plants against marine pathogens and saprophytes. Mar. Biol., 149: 991-02.

25. NCCLS., 2008. Performance Standards for Antimicrobial Susceptibility testing; Ninth Informational Supplement. NCCLS document M100-S9. National Committee for Clinical Laboratory Standards, Wayne, PA, pp: 120-126. 26. Mazzanti, G., M.T. Mascellino, L. Battinelli, D. Coluccia, M. Manganaro and L. Saso, 2000. Antimicrobial investigation of semipurified fractions of Ginkgo biloba leaves. J. Ethnopharmacol., 71: 83-88. 27. Devienne, K.F. and M.S.G. Raddi, 2002. Screening for antimicrobial activity of natural products using a microplate photometer. Braz. J. Microbiol., 33: 166-68. 28. Sultana, V.S., E. Haque, J. Ara and M. Athar, 2005. Comparative efficacy of brown, green and red seaweeds in the control of root infecting fungi and okra. Int. J. Environ. Sci. Tech., 2: 129-32. 29. Kandhasamy, M. and K.D. Arunachalam, 2008. Evaluation of in vitro antibacterial property of seaweeds of southeast coast of India. Afr. J. Biotechnol., 7: 1958-61. 30. Rao, P.S. and K.S. Parekh, 1981. Antibacterial activity of Indian Seaweed extracts. Bot. Mar., 24: 577-82. 31. Reichelt, J.L. and M.A. Borowitzka, 1984. Antimicrobial activity from marine algae: Results of a large scale screening programme. Hydrobiol., 116/117: 158-68. 32. Salvador, N., A. Gomez-Garreta, L. Lavelli and L. Ribera, 2007. Antimicrobial activity of Iberian macroalgae. Sci. Mar., 71: 101-13. 33. Lustigman, B. and C. Brown, 1991. Antibiotic production by marine algae isolated from the New York/New Jersey Coast. Bull. Environ. Contam. Toxicol., 46: 329-35. 34. Taskin, E., M. Ozturk and O. Kurt, 2001. Antibacterial activities of some marine algae from the Aegean Sea (Turkey). Afr. J. Biotechnol., 6: 2746-51. 35. Tuney, I., B.H. Cadirci, D. Unal and A. Sukatar, 2006. Antimicrobial activities of the extracts of marine algae from the Coast of Urla (zmir, Turkey). Turk. J. Biol., 30: 1-5. 36. Caccamese, S. and R. Azzolina, 1979. Screening for antimicrobial activities in marine algae from Eastern Sicily. Planta. Med., 37: 333-39. 37. Pesando, D. and B. Caram, 1984. Screening of marine algae from the French Mediterranean Coast for antibacterial and antifungal activity. Bot. Mar., 27: 381-86.

56

World J. Fish & Marine Sci., 3 (1): 51-57, 2011

38. Paz, E.A., R.N. Lacy and M. Bakhtian, 1995. The $-Lactum antibiotics penicillin and cephalosporin in perspective. Hodder strong, London, pp: 324. 39. Vlachos, V., A.T. Critchley and A. Von Holy, 1997. Antimicrobial activity of extracts from selected Southern African marine macroalgae. S. Afr. J. Sci., 93: 328-32. 40. Sastry, V.M.V.S. and G.R.K. Rao, 1994. Antibacterial substances from marine algae: Successive extraction using benzene, chloroform and methanol. Bot. Mar., 37: 357-60. 41. Sree, A., S. Choudhury, S.C. Mukherjee, P. Pattnaik and M. Bapuji, 2005. In vitro antibacterial activity of extracts of selected marine algae and mangroves against fish pathogens. Asian Fis. Sci., 18: 285-94. 42. Blumer, M., R.R.L. Guillard and T. Chase, 1971. Hydrocarbons of marine phytoplankton. Mar. Biol., 8: 183-189. 43. Youngsblood, W.W., M. Blumer, R.R.L. Guillard and F. Flore, 1971. Saturated and unsaturated hydrocarbons in marine benthic algae. Mar. Biol., 8: 190-201.

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44. Gelpi, E., H. Schneider, J. Mann and J. Oro, 1970. Hydrocarbons of geochemical significance in microscopic algae. Phytochem., 9: 1317-1324. 45. Weber, P.C. and A. Leaf, 1991. Cardiovascular effects of O-3 fatty acid. World. Rev. Nutr. Diet., 66: 2318-232. 46. Tellez, M.R., K.K. Schrader and M. Kobaisy, 2001. Volatile components of the cyanobacterium Oscillatoria perornata (Skuja). J. Agric. Food Chem., 49: 5989-5992. 47. Karunasagar, I., R. Pai, G.R. Malathi and I. Karunasagar, 1994. Mass mortality of Penaeus monodon larvae due to antibiotic resistant Vibrio harveyi infection. Aquaculture, 128: 203-209. 48. Gopalakannan, A., V. Atulkumar and V. Arul, 2004. Effect of gut probiotic lactic acid bacteria isolated from marine fish on Penaeus monodon post larvae to control the Vibrio anguillarum infection. Proc. Nat. Sem. New Front Ma.r Biosci. Res., pp: 169-76.