World J Microbiol Biotechnol (2008) 24:19891992 DOI 10.

1007/s11274-008-9695-5

SHORT COMMUNICATION

Antibacterial activity of three South Indian seagrasses, Cymodocea serrulata, Halophila ovalis and Zostera capensis
Chinnadurai Sreenath Kumar Dronamraju V. L. Sarada Thomas Paul Gideon Ramasamy Rengasamy

Received: 14 September 2007 / Accepted: 7 February 2008 / Published online: 22 February 2008 Springer Science+Business Media B.V. 2008

Abstract The antibacterial properties of the three seagrasses namely Cymodocea serrulata, Halophila ovalis and Zostera capensis were tested against the human pathogens Staphylococcus aureus, Bacillus cereus, B. subtilis, Escherichia coli, Salmonella paratyphi, Salmonella typhimurium and Micrococcus luteus, using six different solvents namely, petroleum ether, chloroform, ethyl acetate, acetone, methanol and water. Ethyl acetate and methanol extracts showed maximum activity against most of the pathogens when compared to other solvents. Experiments are underway to isolate active compound(s) implicated in controlling the growth of the pathogens in vitro. Keywords Antibacterial activity Seagrass Human pathogens MIC

Introduction It has long been recognized that tropical coastal rural economies are to a large extent dependent on their marine resources as a source of food and income (Coughanowr et al. 1995; Moffat et al. 1998). Most studies have focused on the importance of coral reefs and mangroves, while seagrasses have generally been neglected. However, recent studies have addressed the need to focus on seagrass research in relation to biodiversity and ecosystem services
C. S. Kumar T. P. Gideon R. Rengasamy (&) Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai 600 025, India e-mail: rengasamy50@yahoo.co.in D. V. L. Sarada Department of Biotechnology, SRM University, Chennai 600 089, India

(Duarte 2000) importance for the local populations (Gullstrom et al. 2002), food webs dynamics (Valentine nnba ck et al. 2002), seascape interactions (Moberg and Ro 2003) and ecological resilience (Gunderson 2001) beside fertilizers and traditional medicine (Torre-Castro and nnba ck 2004). The use of the roots of Enhalus acoroides Ro as a remedy against stings of different kinds of rays and scorpion is very popular. Cymodocea spp. is used as a tranquillizer for babies, as soothing help during pregnancy and against cough and malaria. Halophila spp. is a strong medicine against malaria and skin diseases and found to be very effective in early stages of leprosy. Interest in the importance of marine organisms as a source of new substances is growing. With marine species comprising approximately half of the total global biodiversity, the sea offers an enormous resource for novel compounds (de Vries and Beart 1995), and it has been classied as the largest remaining reservoir of natural molecules to be evaluated for drug activity (Gerwick and Bernart 1993). Marine and estuarine submersed aquatic angiosperms, or seagrasses, produce antimicrobial compounds that may act to reduce or control microbial growth. Workers have described antibacterial (Harrison and Chan 1980; Bernard and Pesando 1989; Devi et al. 1997; Bhosale et al. 2002), antialgal (Harrison 1982), antifungal (Ballesteros et al. 1992; Jensen et al. 1998) and antiviral (Premnathan et al. 1992) activities among this group of organisms. The objective of the present article is to examine antibacterial activity and Minimum Inhibitory Concentration of three seagrasses Cymodocea serrulata (R.Br.) Ascherson, Halophila ovalis R.Br.Hooke and Magnus and Zostera capensis Setchell, collected along the coast of Tamil Nadu, India, against the bacteria Staphylococcus aureus, Bacillus cereus, B. subtilis, Escherichia coli, Salmonella paratyphi, S. typhimurium and Micrococcus luteus, using six different

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World J Microbiol Biotechnol (2008) 24:19891992

solvents: petroleum ether, chloroform, ethyl acetate, acetone, methanol and water.

which no growth was observed is the Minimum Bactericidal Concentration. The data were statistically analysed by applying One-Sample T Test.

Materials and methods Results Fresh seagrass samples of Cymodocea serrulata (R.Br.) Ascherson, Halophila ovalis R.Br.Hooke and Magnus and Zostera capensis Setchell were collected from the Coast of Tamil Nadu during the year 2006. They were brought to the laboratory washed in seawater to remove the macroscopic epiphytes and extraneous matter, and then rinsed in distilled water, and shade dried for 10 days. The herbarium specimens were submitted to the algal herbarium in Centre for Advanced Studies in Botany, University of Madras. The samples were powdered and stored in a refrigerator. Fifty grams of shade-dried seagrass powder were soaked in the organic solvents petroleum ether, chloroform, ethyl acetate, acetone, methanol and water (1:4 w/v), and extracted for 10 days in shaker (30 rev/min), the extracts were collected and concentrated. 5% DMSO was added for concentrated extracts and used for antibacterial assay (Bauer et al. 1966). The human pathogens Staphylococcus aureus MTCC 96, Bacillus cereus MTCC 430, B. subtilis MTCC 441, Escherichia coli MTCC 443, Salmonella paratyphi MTCC 735, S. typhimurium MTCC 98 and Micrococcus luteus MTCC 1541 were obtained from the Institute of Microbial Technology, Chandigarh, India. Antibacterial activity of seagrass extract was evaluated using an agar diffusion method. 5% DMSO served as a control and ampicillin was used as a positive control. A broth microdilution method was used to determine the Minimum Inhibitory Concentration (NCCLS 2003; Mazzanti et al. 2000; Devienne and Raddi 2002). All tests were performed in Mueller-Hinton medium (Himedia). Serial double dilutions were prepared with a mixture of maximum active seagrass extracts: Dimethylsulfoxide 95:5 in a 96well microtiter plate over the range of 73,125 ll/l. Overnight broth cultures of each strain were prepared and the nal concentration of the microbe in each well was adjusted to 2 9 103 c.f.u./ml. Plates were incubated at 37C for 24 h. The MIC dened as the lowest concentration of the seagrass extract at which the microorganism does not demonstrate visible growth and the absorbance of each well were determined using an automatic ELISA tray reader adjusted at 630 nm (SLT Spectra). The samples were analysed in duplicate and the assay was repeated twice. The antibiotic ampicillin was employed as positive control. The wells showing complete absence of growth were identied and 10 ll of each well were transferred to Mueller Hinton agar plates and incubated at previously mentioned times and temperatures. The concentration in Table 1 shows the effect of Cymodocea serrulata, Halophila ovalis and Zostera capensis extracts on seven bacterial pathogens. Control disks (DMSO) always showed no inhibition, with bacterial lawns growing in contact with the disk edges. The antibiotic ampicillin inhibited the pathogens Bacillus cereus, B. subtilis, Escherichia coli and Micrococcus luteus. Of the three different seagrasses examined in the present study Halophila and Zostera were more effective than Cymodocea in controlling the growth of all pathogens except Micrococcus luteus. Fifty percent of activity was recorded in Halophila ovalis followed by Zostera capensis (45.2%) and Cymodocea serrulata (44.1%). Ethyl acetate extracts of all the seagrasses were active against the all gram-positive and most of the gram-negative pathogens tested namely, Staphylococcus aureus, Bacillus cereus, B. subtilis, Salmonella typhimurium and Micrococcus luteus and methanolic extracts active against Staphylococcus aureus, Salmonella paratyphi, S. typhimurium and Micrococcus luteus. Ethyl acetate proved to be the best solvent for isolation of bioactive substances from seagrasses (81.9%) followed by methanol (76.2%). Maximum activity was recorded in ethyl acetate extract of Halophila ovalis and followed by methanolic extract of Zostera capensis. Acetone, petroleum ether and chloroform showed less activity. Minimum Inhibitory Concentrations of the methanol and ethyl acetate extracts of the three seagrasses which showed maximum activity against all the six pathogens are depicted in Table 2. Of the three seagrasses studied, minimum concentrations of Halophila extracts were effective in controlling the growth of Micrococcus luteus Escherichia coli and Salmonella typhimurium.

Discussion and conclusion Natural products are considered an important source of new antibacterial agents. Drugs derived from unmodied natural products or drugs semi-synthetically obtained from natural sources corresponded to 78% of the new drugs approved by the FDA between 1983 and 1994 (Cragg et al. 1997). With marine species comprising approximately a half of the total global biodiversity, large-scale screening will continue to play an important role in development of new drugs (Xu et al. 2004). Marine organisms are a rich source of structurally novel and biologically active metabolites. Many chemically

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World J Microbiol Biotechnol (2008) 24:19891992 Table 1 Antimicrobial activity of seagrasses Pathogens Sea Inhibition zone (mm) 50 lg concentration per disc grasses Organic solvents PE Staphylococcus aureus S-1 S-2 Bacillus cereus S-3 S-1 S-2 S-3 B. subtilis S-1 S-2 S-3 Escherichia coli S-1 S-2 S-3 Salmonella paratyphi S-1 S-2 S-3 S. typhimurium S-1 S-2 Micrococcus luteus S-3 S-1 S-2 S-3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 9.00 0.58 0.00 0.00 8.00 0.00 12.33 0.33 9.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 9.00 0.58 0.00 0.00 0.00 0.00 C 0.00 0.00 EA 9.33 0.33 A M W 0.00 0.00 9.00 0.58

1991

Ampicillin (50 lg/ml)

8.33 0.67 15.67 0.33 0.00 0.00 14.33 0.33 0.00 0.00 10.00 0.58 0.00 0.00 9.00 0.58 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 8.00 0.58 0.00 0.00 0.00 0.00 8.00 0.00 0.00 0.00

0.00 0.00

9.33 0.33 10.33 0.33 0.00 0.00 10.00 0.58 0.00 0.00 8.00 0.00 8.67 0.67 12.67 0.67 0.00 0.00 18.00 0.00 0.00 0.00 8.67 0.33

0.00 0.00 0.00 0.00 10.00 0.58 0.00 0.00 9.00 0.00 0.00 0.00 12.33 0.33 0.00 0.00 7.33 0.33

0.00 0.00 10.33 0.33 17.00 0.00 9.33 0.33 15.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 14.67 0.33 0.00 0.00 0.00 0.00

0.00 0.00 12.33 0.33

0.00 0.00 11.00 0.58 13.00 0.58 0.00 0.00 14.67 0.33 0.00 0.00 9.00 0.58 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 19.67 0.33 12.00 0.00 0.00 0.00 12.33 0.33 0.00 0.00 14.33 0.33 0.00 0.00 16.67 0.67 0.00 0.00 12.33 0.33 0.00 0.00 12.00 0.58

8.00 0.00 10.67 0.33

0.00 0.00 10.00 0.00 0.00 0.00 10.67 0.67 0.00 0.00 18.33 0.67

0.00 0.00 12.33 0.33 0.00 0.00 16.00 0.00 0.00 0.00 0.00 0.00 17.33 0.33 10.00 0.58 10.00 0.58 10.00 0.00 12.00 0.58 7.33 0.33 11.00 0.58 0.00 0.00 10.00 0.58 8.00 0.00 20.00 0.00 0.00 0.00 17.00 0.58 0.00 0.00 0.00 0.00

S-1: Cymodocea serrulata, S-2: Halophila ovalis and S-3: Zostera capensis PEPetroleum ether; CChloroform; EAEthyl acetate; AAcetone; MMethanol; WWater; ConControl Mean Standard error; Standard deviation P B 0.05 Table 2 MIC of maximum active seagrass extracts Pathogens Staphylococcus aureus Bacillus cereus B. subtilis Escherichia coli Salmonella paratyphi S. typhimurium Micrococcus luteus
a

Seagrass extracts Methanolic extract of Cymodocea serrulata Ethyl acetate extract of Zostera capensis Ethyl acetate extract of Halophila ovalis Methanolic extract of Halophila ovalis Methanolic extract of Zostera capensis Ethyl acetate extract of Halophila ovalis Methanolic extract of Halophila ovalis
3

MIC (lg/ml)a 150 100 100 75 125 75 50

Ampicillin MIC (lg/ml)a 170 160 160 160

Values are the mean of three replicates using 1 9 10 cells of each culture cells of each culture

unique compounds of marine origin with different biologically activity have been isolated and a number of them are under investigation and/ or are being developed as new pharmaceuticals (Faulkner 2000a, b; da Rocha et al. 2001; Schwartsmann et al. 2001). Several species of seagrass produce antimicrobial compounds that may act to reduce or control microbial growth. Marine organisms collected from the Southeast coast of India have been shown to possess a number of biological activities (Ely et al. 2004). The present

study demonstrates that antibacterial agents exist in three species of seagrass Cymodocea serrulata, Halophila ovalis and Zostera capensis which are effective against grampositive bacteria, inhibiting growth of species in the genera Staphylococcus aureus, Bacillus cereus, B. subtilis, and Micrococcus luteus and gram-negative Escherichia coli, Salmonella paratyphii, and S. typhimurium. The results presented here are consistent with some previous studies of seagrass antibacterial activity. Harrison and Chan (1980)

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World J Microbiol Biotechnol (2008) 24:19891992 Dumay O, Costa J, Desjobert J, Pergent G (2004) Variations in the concentration of phenolic compounds in the seagrass Posidonia oceanica under conditions of competition. Phytochemistry 65:32113220 Ely R, Supriya T, Naik CG (2004) Antimicrobial activity of marine organisms collected off the coast of South East India. J Exp Mar Bio Ecol 309:121127 Faulkner DJ (2000a) Marine natural products. Nat Prod Rep 17:755 Faulkner DJ (2000b) Marine pharmacology. Antonie Van Leeuwenhoek 77:13545 Francois R, Weber JH (1988) Speciation of methyltin and butyltin compounds in eelgrass (Zostera marina L.) leaf tissue from the great bay estuary (NH). Mar Chem 25:279289 Gerwick WH, Bernart MW (1993) Eicosanoids and related compounds from marine algae. In: Attaway DH, Zaborsky OR (eds) Marine biotechnology. Pharmaceutical and bioactive natural products, vol 1. Plenum Press, NY, pp 101152 Gullstrom M, Torre-Castro M, Bandeira SO, Bjork M, Dahlberg M, Kautsky N, Ronnback P, Ohman MC (2002) Seagrass ecosystems in the Western Indian Ocean. Ambio 31:588596 Gunderson LH (2001) Managing surprising ecosystems in southern Florida. Ecol Econ 37:371378 Harrison PG (1982) Control of microbial growth and of amphipod grazing by water-soluble compounds from leaves of Zostera marina. Mar Biol 67:225230 Harrison PG, Chan AT (1980) Inhibition of the growth of microalgae and bacteria by extracts of eelgrass (Zostera marina) leaves. Mar Biol 61:2126 Jensen PR, Kensin KM, Porter D, Fenical W (1998) Evidence that a new antibiotic avone glycoside chemically defends the sea grass Thalassia testudinum against zoosporic fungi. Appl Environ Microbiol 64:14901496 Kawasaki W, Matsui K, Akakabe Y, Itai Kajiwara N (1998) Volatiles from Zostera marina. Photochemistry 45:2729 Mazzanti G, Mascellino MT, Battinelli L, Coluccia D, Manganaro M, Saso L (2000) Antimicrobial investigation of semipuried fractions of Ginkgo biloba leaves. J Ethnopharmacol 71:8388 McMillan C (1986) Sulfated avonoids and leaf morphology in the Halophila ovalis - minor complex (Hydrocharitaceae) of the Indo-Pacic Ocean. Aquat Bot 25:6772 McMillan C, Zapata O, Escobar L (1980) Sulphated phenolic compounds in seagrasses. Aquat Bot 8:267278 nnba ck P (2003) Ecosystem services of the tropical Moberg F, Ro seascape: interactions, substitutions and restoration. Ocean Coast Manage 46:2746 n O, Francis J (1998) The reality of the Moffat D, Ngoile M, Linde stomach: coastal management at the local level in eastern Africa. Ambio 27:590598 NCCLS National Committee for Clinical Laboratory Standards (2003) Methods for dilution antimicrobial susceptibility test for bacteria that grow aerobically; approved standard M7-A6, 6th edn. National Committee for Clinical Laboratory Standards, Wayne, Pa Premnathan M, Chandra K, Bajpai SK, Kathiresan K (1992) A survey of some Indian marine plants for antiviral activity. Bot Mar 35:321324 Schwartsmann G (2001) Breast cancer in South America: Challenges to improve early detection and medical management of a public health problem. J Clin Oncol 19:118s124s nnba ck P (2004) Links between humans and Torre-Castro M, Ro seagrasses an example from tropical East Africa. Ocean Coast Manage 47:361387 Valentine JF, Kenneth L, Heck KL Jr, Cinkovish AM (2002) Impacts of seagrass food webs on marine ecosystems: a need for a broader perspective. Bull Mar Sci 7:13611368 Xu N, Fan X, Yan X, Tseng CK (2004) Screening marine algae from China for their antitumor activities. J App Phycol 16:451456

found Zostera marina effective against S. aureus, which was also sensitive in this study. Our nding of Bacillus sensitivity agrees with Bhosale et al. (2002), who reported extracts from Cymodocea rotundata as being effective against several Bacillus species (Bernard and Pesando 1989). The estuarine submerged aquatic plants Potamogeton pectinatus, P. perfoliatus and Ruppia maritima inhibited growth of gram-positive bacteria Bacillus, Mycobacterium, Staphylococcus and Streptococcus and gram-negative species like Vibrio, Listonella and Pasteurella (Bushmann and Ailstock 2006). In the present study the three seagrasses Cymodocea serrulata, Halophila ovalis and Zostera capensis inhibited gram-positive and gram-negative bacterial pathogens. This report demonstrating the antimicrobial activity of the seagrasses is an encouraging trend unraveling the potential of the Indian coastline as a source of marine organisms worthy of further investigation. These organisms are currently being investigated in detail with the objective of isolating biologically active molecules which would prove to be lead chemicals for drug discovery.
Acknowledgements The authors are thankful to the Director, CAS in Botany, University of Madras, for extending laboratory facilities.

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