International Journal of Advanced Life Sciences (IJALS)

Ramamurthy et al., IJALS, Vol.1. Jan. 2012

ISSN 2277-758X RESEARCH ARTICLE

Antimicrobial activity of Heterocytic Cyanobacteria

V. Ramamurthy*, S. Raveendran**, S. Thirumeni** and S. Krishnaveni***
*Post Graduate and Research Department of Biochemistry, Marudupandiyar College, Vallam, Thanjavur 613 403, ** Post Graduate and Research Department of Zoology, Khadir Mohideen College, Adirampattinam - 614 701, Tamil Nadu, ***Department of Zoology, ADM Women`s College, Nagappattinam, Tamil Nadu, India Email address : v.ramamoorthy07@gmail.com

Abstract
Invitro screening of antimicrobial activities of some heterocystous cyanobacteria from agricultural fields in the Pattukkottai, Thanjavur District were studied. The collected cyanobacterial supernatants, ethanolic extract from biomass of 80 strains of cyanobacteria were isolated and screened against five bacteria and five fungi. Ethanolic extracts and culture supernatants of 10 strains of cyanobacteria significant antimicrobial effect. According to these results, it is concluded that strains of Anabaena species, seem to be more potential for producing antimicrobial substances than other strains. This is interesting in that the traditional method of treating a microbial infection was by administering a decoction of the cyanobacteria, whereas according to our results a bacterium is better than fungi; hence this may be more beneficial.

Corresponding Author
V. Ramamurthy Post Graduate and Research Department of Zoology, Khadir Mohideen College, Thanjavur District, v.ramamoorthy07@gmail.com

Keywords: Antimicrobial active compound, Blue-green algae, cyanobacteria

and heterocystous

Introduction Cyanobacteria are a very old group of organisms and represent relics of the oldest photoautotrophic vegetation in the world that occur in freshwater, marine and terrestrial habitats (Mundt and Teuscher 1986). Cyanobacteria have drawn much attention as prospective and rich sources of biologically active constituents and have been identified as one of the most promising groups of organisms to be able of producing bioactive compounds (Schlegel et al., 1999). Cyanobacteria are known to produce metabolites with diverse biological activities such as antibacterial (Jaki et al., 2000), antifungal (Kajiyama et al., 1998), antiviral (Patterson et al., 1994), anticancer (Luesch et al., 2000), antiplasmodial (Papendorf et al., 1998), algicide (Papke et al., 1997), antiplatelet aggregation (Rho et al., 1996) and immunosuppressive (Koehn et al., 1992) activities. Cyanobacteria from local habitats seem to be a source of potential new active substances that could contribute to reduction of the number of bacteria, fungi, viruses and other microorganisms (Mundt et al., 2001). Cyanobacteria have not yet been studied for antimicrobial activity and little work has been done to screen cyanobacteria isolated from soils with regard to their production of bioactive compounds. In order to find the potential of cyanobacteria for production

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Ramamurthy et al., IJALS, Vol.1. Jan. 2012 of antimicrobial compounds in agricultural field. In this study, some heterocystous microalgae were tested wherein we report their efficacy against ten pathogenic microorganisms. Materials and Methods Isolation and identification of cyanobacteria Soil samples were collected from different agricultural fields in Pattukkottai, Thanjavur District, Tamil Nadu, India. Soil samples in laboratory were cultured directly in Nitrogen free BG11 media (Browitzka and Browitzka, 1988), after colonization, cyanobacteria were transferred to the same medium. Each isolated cyanobacterium was cultured in a 250 ml flask containing 100 ml of BG11 medium without shaking, for 30 days. The incubation temperature was 28C 2 and illumination at 3000 lux with a white continuous light. The isolated cyanobacteria and identification was done using morphological variation studies and taxonomical approaches (Anagnostidis and Komarek, 1988; Desikachary, 1959; Prescott, 1962 and Anand et al., 1990). Preparation of extracts for antimicrobial activity The cultures were harvested after 30 days by centrifugation at 10000 rpm for 10 min. The aqueous supernatant was collected and the algal pellet was extracted with 15 ml of ethanol followed by 15 ml hexane, with shaking for 20 min. The culture supernatants and solvent extracts were dried under reduced pressure at 40C and were stored at 10C for further studies. The following bacteria and fungi were used as test organisms: Staphylococcus aureus,

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Staphylococcus epidermidis, Staphylococcus haemolyticus, Shigella sonnei, Proteus vulgaris, Candida krusei, Candida albicans, Cryptococcus neoformans, Aspergillus niger and Aspergillus fumigatus. Dried extracts and supernatants were dissolved in 4 ml of their extraction solvent and antimicrobial activity was determined by the disc method. Filter paper discs (6.0mm) were saturated with 50 l of the test solution, dried under laminar air flow and placed on the MullerHinton agar plate for bacteria and potato dextrose agar plate for fungi, which had been inoculated with a lawn of the test microorganisms. Plates were incubated at 37C, for a period of 18-24 hour for bacteria and at 25C, for 24-48 hours for fungi. Discs treated with 50l ethanol was used as negative controls and gentamycine discs were used (10 g) as positive controls. The extracts and supernatants containing antimicrobial components produced distinct, clear, circular zones of inhibition around the discs and the diameters of clear zones were determined and used as an indication of antimicrobial activity. Results and Discussion The results of culture supernatants and ethanolic extracts of the isolated cyanobacteria that demonstrated antimicrobial activity are shown in Tables 1 and 2. Supernatant and ethanolic extract of 10 strains from the 80 cyanobacteria strains, showed significant antimicrobial activity against bacteria and fungi. Three of them were identified as Nostoc species, where 2 Anabena species, two Haplosiphan species and each one Westielliopsis, Cylindrospermum plectonema species were also among those showing antimicrobial activity.

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International Journal of Advanced Life Sciences (IJALS)

Ramamurthy et al., IJALS, Vol.1. Jan. 2012

ISSN 2277-758X RESEARCH ARTICLE

Table - 1. Antibacterial activity of heterocytic cyanobacteria as presented by inhibition zone diameter (in mm) S. No 1 2 3 4 5 6 7 8 9 10 Name of cyanobacteria Nostoc muscorum N. pauludosum N. spongiaeforme Anabaena sphaerica A. cylindrica Haplosiphon welwitchii H. fontinalis Plectonema sp Westielliopsis prolifira Cylindrospermum sp. S. epidermidis 22 19 21 16 19 20 18 12 15 19 S. aureus 24 22 22 17 19 21 19 15 19 22 S. haemolyticus 16 13 15 14 16 17 13 09 15 16 P. vulgaris 18 15 17 15 17 19 16 10 16 18 S. sonnei 15 16 19 16 18 18 14 10 17 17

Table - 2. Antifungal activity of heterocytic cyanobacteria as presented by inhibition zone diameter (in mm) S. No 1 2 3 4 5 6 7 8 9 10 Name of cyanobacteria Nostoc muscorum N. pauludosum N. spongiaeforme Anabaena sphaerica A. cylindrica Haplosiphon welwitchii H. fontinalis Plectonema sp Westielliopsis prolifira Cylindrospermum sp C. krusei 9 10 9 8 9 10 9 8 6 9 C. albicans 8 9 10 7 9 9 8 7 6 10 A. niger 9 8 8 7 6 6 6 5 3 7 A. fumigatus 7 8 6 6 5 4 7 6 4 6 C. neoformans 10 11 9 9 8 9 7 7 7 11

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International Journal of Advanced Life Sciences (IJALS)

Ramamurthy et al., IJALS, Vol.1. Jan. 2012

ISSN 2277-758X RESEARCH ARTICLE

In the present pilot screening of cyanobacterial extracts of 10 species were found to show species specific activity against the 10 human pathogens. The details of activity of aqueous and ethanol extracts of algae along with activity profile with standard commercial antibiotics (gentamycine) was presented in Table 1 and 2. The antibacterial activity of cyanobacteria aqueous and ethanolic extracts against Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Shigella sonnei and Proteus vulgaris shown in Table 1. Maximum antibacterial activity was shown against Staphylococcus aureus followed by Staphylococcus epidermidis and Proteus vulgaris. Neither of the extracts was able to inhibit Shigella sonnei and Staphylococcus haemolyticus. Generally fungal pathogens have less inhibition than bacterial pathogens. This is interesting in that the traditional method of treating a microbial infection was by administering a decoction of the cyanobacteria, whereas according to our results an organic solvent is better; hence this may be more beneficial. Amongst the 5 bacterial and 5 fungal strains investigated Staphylococcus aureus and Staphylococcus epidermidis the most resistant and Aspergillus less resistant. A large number of microalgal extracts and extracellular products have been found to have antibacterial activity. However, pH of the medium, incubation period and temperature of incubation were very important for the biosynthesis of antimicrobial agent products as secondary metabolites. Previously (Noaman et al., 2004) reported that temperature 35 C, pH 8 and days

of incubation were the best for growth and antimicrobial agent production. In this study, these were chosen as an optimum pH, incubation periods and temperature. The cyanobacteria such as Nostoc commune (Jaki et al., 2000), Scytonema hofmanni (Pignatello et al., 1983), Hapalosiphon fontinalis (Moore et al., 1987), Anabaena spp (Frankmolle et al., 1992), Nostoc spongiaeforme (Hirata et al., 1996), Microcystis aeruginosa (Ishida et al., 1997) Spirulina platensis and Lyngbya majuscula (Ramamurthy and Raveendran, 2009) have been reported as the main cyanobacteria to produce antimicrobial substances. Screening efforts aimed to identify antimicrobial agents in cyanobacteria have revealed several promising lead compounds. Some of these substances identified including Nostocyclyne A (Ploutno et al., 1997), Nosto fungicidine (Kajiyama et al., 1998), Kawaguchipeptin B (Ishida et al., 1997), Nostocin A (Hirata et al. 1996), Ambigol A and B (Falch et al., 1995), Hapalindoles (Moore et al., 1987) and Scytophycins (Ishibashi et al., 1986). Most of the studies have only done as in vitro assays and, it is likely that most of these compounds will have little or no clinical application, as they are either too toxic or inactive in-vivo (Browitzka, 1995). They may however serve as useful lead compounds for synthesis of antibiotics or may find application in agriculture. For example Tjipanazoles isolated from the cyanobacterium, Tolypothrix tjipanensis, showed appreciable fungicidal activity against rice blast and leaf rust wheat infections.

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Ramamurthy et al., IJALS, Vol.1. Jan. 2012 A few studies have been done to screen cyanobacteria for production of antimicrobial substances from agricultural fields. Possibly the synthesis of highly active toxin is a defense option of cyanobacteria in these environments against other organisms like bacteria, fungi, viruses and eukaryotic microalgae. In one study, the culture media of cyanobacteria belonging to Nostaceae, Microchaetaceae and Scytonematacaea isolated from the Argentinian agricultural fields were found to be active against S. aureus and Candida albicans (De Caire et al., 1993). In another study, it was shown that cyanobacteria from the agricultural fields of northern Thailand produce bioactive substances with antibiotic activity against Bacillus subtilis (Chetsumon et al., 1993). Cyanobacteria of Pattukkottai have not yet been studied for antimicrobial activity and this screening program is among the first studies done for assessment of antibacterial and antifungal activity of Pattukkottai agricultural field cyanobacteria. In this investigation, out of 80 strains of cyanobacterial isolates, 10 showed significant in vitro antimicrobial effect. The proportion of the isolates with antibacterial and antifungal activities were approximately 13% and 12.5%, respectively, which is comparable with those published earlier in other screening programs 11% (Flores and Wolk, 1986), 7% (Patterson et al., 1993) and 10% (Schlegel et al., 1999). As depicted in Table1 and 2 Nostoc, Anabena and Hapalosiphon species produce bioactive substances, which may have potential

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for antibacterial and antifungal activity. Although some of the cyanobacteria produce active compounds against Gram-negative bacteria such as S. sonnei and P. vulgaris but less activity was found against S. haemolyticus. Different results have been reported by other authors in this case. Falch et al. (1995) and Hirata et al. (1996) reported active compounds against E. coli in the petroleum ether fraction of Fischerella ambigua and supernatant of Nostoc spongiaeforme, respectively. Antifungal activity assays showed a good activity against C. krusei, C. albicans and C.neoformans and the minimum against Aspergillus sp. Among the isolated cyanobacteria, Westielliopsis species had the minimum activity against the test organisms. Nostoc and Anabaena species from chemical components had the greatest frequency among the species that showing antimicrobial activity and exhibited the most prominent effect. The effect of antimicrobial activity of Stigonemataceae has been reported in other studies (Patterson et al., 1994; Falch et al., 1995 and Smitka et al., 1992). Among all of the species studied in this investigation for antibacterial and antifungal activity, it seems Plectonema strains are being reported for the first time as producer of antibacterial substances. The results of this work indicate that this group of organisms displays a potential that warrants further investigations. Acknowledgements The authors are thankful to the Director, PRILS Research Institute, Pattukkottai for providing necessary facilities to carryout this work.

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Ramamurthy et al., IJALS, Vol.1. Jan. 2012 References Anagnostidis, K. and Komarek, G. 1988. Modern approach to the classification system of cyanobacteria, Arch Hydrobiol. Suppl., 80: 372 - 470. Anand, N.L., Radha, R.S., Hopper, S., Ravati, G. and Subramianan, T.D. 1990. Perspectives in Phycology. Today and Tomorrows Printers and Publishers, New Delhi, pp. 383- 391. Browitzka, M.A. 1995. Microalgae as sources of pharmaceuticals and other biologically active compounds. J. Appl. Phycol., 7: 3-15. Browitzka, M.A. and Browitzka, L.J. 1988. Microalgal Biotechnology. Cambridge University Press, Cambridge, pp. 456-458. Chetsumon, A., Miyamoto, K., Hirata, K., Miura, Y., Ikuta, Y. and Hamsaki, A. 1993. Factors affecting antibiotic production in bioreactors with immobilized algal cells. Appl. Biochem. Biotech., 37: 573-586. De Caire, G.Z., De Cano, M.M.S., De Mule, M.C.Z. and De Halperin, D.R. 1993. Screening of cyanobacterial bioactive compounds against human pathogens. Phyton., 54: 59-65. Desikachary, T.V.1959. Cyanophyta, Indian Council of Agricultural Research New Delhi. Falch, B.S., Konig, G.M., Wright, A.D., Sticher, O., Angerhofer, C.K., Pezzuto, J.M. and Bachmann, H. 1995. Biological activities of cyanobacteria: evaluation of extracts and pure compounds. Planta Med., 61: 321-328.

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Flores, E. and Wolk, C.P. 1986. Production, by filamentous, nitrogen- fixing cyanobacteria, of a bacteriocin and of other antibiotics that kill related strains. Arch. Microbiol., 145: 215-219. Frankmolle, W.P., Larsen, L.K., Caplan, F.R., Patterson, G.M.L. and Knubel, G. 1992. Antifungal cyclic peptides from the terrestrial blue-green alga Anabaena laxa. Isolation and biological properties. J. Antibiot., 45: 14511457. Hirata, K., Takashina, J., Nakagami, H., Ueyama, S., Murakami, K., Kanamori, T. and Miyamoto, K. 1996. Growth inhibition of various organisms by a violet pigment, Nostocin A, produced by Nostoc spongiaeforme. Biosci. Biotech. Biochem., 60: 1905-1906. Ishibashi, M., Moore, R.E. and Patterson, G.M.L. 1986. Scytophycins, cytotoxic and antimycotic agents from the cyanophyte Scytonema pseudohofmanni. J. Org. Chem., 51: 5300-5306. Ishida, K., Matsuda, H., Murakami, M. and Yamaguchi, K. 1997. Kawaguchipeptin B, an antibacterial cyclic undecapeptide from the cyanobacterium Microcystis aeruginosa. J. Nat. Prod., 60: 724-726. Jaki, B., Heilmann, J., Linden, A., Volger, B and Sticher, O. 2000. Novel extra cellular diterpenoids with biological activity from the cyanobacterium Nostoc commune. J. Nat. Prod., 63: 339-343.

Int. j. Adv. Lif. Sci., Available online on at www. Int. J. Adv. Lif. Sci., Available online on at www. ijals.com

Page 37

International Journal of Advanced Life Sciences (IJALS)

Ramamurthy et al., IJALS, Vol.1. Jan. 2012 Kajiyama, S., Kanzaki, H., Kawazu, K. and Kobayashi, A. 1998. Nostifungicidine, an antifungal lipopeptide from the fieldgrown terrestrial blue-green alga Nostoc commune. Tetrahedron Lett., 39: 3737-3740. Koehn, F.E., Longley, R.E. and Reed, J.K. 1992. Microcolins A and B, new immunosuppressive peptide from the bluegreen alga Lyngbya majuscule. J. Nat. Prod., 55: 613-619. Lewin, R.A. 1993. Antiviral activity of cultured bluegreen algae. J. Phycol., 29: 125-130. Luesch, H., Yoshida, W.Y., Moore, R.E., Paul, V.J. and Mooberry, S.L. 2000. Isolation, structure determination, and biological activity of Lyngbyabellin A from the marine cyanobacterium Lyngbya majuscule. Ibid., 63: 611 - 615. Moore, R.E., Cheuk, C., Yang, X.G. and Patterson, G.M.L. 1987. Hapalindoles, antibacterial and antimycotic alkaloids from the cyanophyte Hapalosiphon fontinalis. J. Org. Chem., 52: 1036 - 1043. Mundt, S. and Teuscher, E. 1988. Blue-green algae as a source of pharmacologically-active compound. Pharmazie, 43: 809-815. Mundt. S., Kreitlow, S., Nowotny, A. and Effmert, U. 2001. Biological and pharmacological investigation of selected cyanobacteria. Int. J. Hyg. Environ. Health, 203: 327- 334. Noaman, N.H., Fattah, A., Khaleafa, M. and Zaky, S.H. 2004. Factors affecting antimicrobial activity of Synechococcus leopoliensis. Microbiological. Res., 159: 395-402.

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Papendorf, O., Knig, G.M. and Wright, A.D. 1998. Hirridin B and 2,4-dimethoxy-6heptadecylphenol, secondary metabolites from the cyanobacterium Phormidium ectocarpi with antiplasmodial 2383 - 2386. Papke, U., Gross, E.M. and Francke, W. 1997. isolation, identification and determination of the absolute configuration of Fischerellin B. A new algicide from the freshwater cyanobacterium Fischerella muscicola (Thuret), Tetrahedron Lett., 38: 379-382. Patterson, G.M.L., Baker, K.K., Baldwin, C.L., Bolis, C.M., Caplan, F.R., Larsen, L.K. and Patterson, G.M.L., Larsen, L.K. and Moore, R.E. 1994. Bioactive natural products from bluegreen algae. J. Appl. Phycol., 6: 151-157. Pignatello, J.J., Porwoll, J., Carlson, R.E., Xavier, A., Gleason, F.K. and Wood, J.M. 1983. Structure of the antibiotic cyanobacterin, a chlorine-containing freshwater -lactone from the cyanobacterium Scytonema activity. Phytochem., 49:

hofmanni. J. Org. Chem., 48: 4035-38. Ploutno, A. and Carmeli, S. 1997. Nostocyclyne A, a novel antimicrobial cyclophan from the cyanobacterium Nostoc sp. J. Nat. Prod., 63: 1524-1526. Prescott, G.W. 1962. Algae of the Western Great Lake Areas. W.M.C. Brown Company Publisher, Dubuqe. Iowa.

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Ramamurthy et al., IJALS, Vol.1. Jan. 2012

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Ramamurthy, V.

and

Raveendran,

S.

2009.

Antibacterial and antifungal activity of Spirulina platensis and Lyngbya majuscula. J. Ecobiol., 24 (1): 47 52. Rho, M., Matsunaga, K., Yasuda, K. and Ohizumi, Y.A.1996. Novel monogalactosylacylglycerol with inhibitory effect on platelet aggregation from the cyanophyceae Oscillatoria rosea. J. Nat. Prod., 59: 308-309. Schlegel, I., Doan, N.T., De Chazol, N. and Smith, G.D. 1999. Antibiotic activity of new cyanobacterial isolates from Australia and Asia against green algae and cyanobacteria. J. Appl. Phycol., 10: 471 - 479.

Smitka, T.A., Bonjouklian, R., Doolin, L., Jones, N.D., Deeter, J.B., Yoshida, W.Y., Prinsep, M.R., Moore, R.E. and Patterson, G.M.L. 1992. Ambiguine isonitriles, fungicidal hapalindole-type alkaloids from three genera of bluegreen algae belonging to Stigonemataceae. J. Org. Chem., 57: 857-861.

Corresponding to Author: V. Ramamurthy, Post Graduate and Research Department of Biochemistry, Marudupandiyar College, Vallam, Thanjavur 613 403, E - Mail : v.ramamoorthy 07 @ gmail.com

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