Antibacterial Attributes of Marine Gastropod Hemifusus pugilinus (Born, 1778) against Human Pathogenic Bacteria  

J. Mohan Raj1 , G. Chelladurai1 , P. Ramasamy1,2 , P.K Deepa1 , M. Sasirekhamani3
1. Department of Zoology, Kamaraj College, Tuticorin, Tamilnadu, India
2. Post- Doctoral Fellow, Institute of Marine Microbes and Ecospheres, Xiamen University, Xiamen, China
3. Centre of Advanced Studies in Botany, University of Madras, Gundy campus, Chennai ,Tamil Nadu, India
Author    Correspondence author
International Journal of Marine Science, 2014, Vol. 4, No. 57   doi: 10.5376/ijms.2014.04.0057
Received: 02 Sep., 2014    Accepted: 13 Oct., 2014    Published: 21 Oct., 2014
© 2014 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Raj et al., 2014, Antibacterial Attributes of Marine Gastropod Hemifusus pugilinus (Born, 1778) against Human Pathogenic Bacteria, International Journal of Marine Science, Vol.4, No.57 1-4 (doi: 10.5376/ijms.2014.04.0057)

Abstract

To study the antibacterial activity of marine gastropods Hemifusus puglinius with different solvent extracts against clinically isolated human pathogenic bacteria. Different solvent extracts of Hemifusus pugilinus was screened for their activity against Vibrio parehaemolyticus (J13300), Aeromonas hydrophilla (IDH1585), Salmonella typhi (C6953), Salmonella paratyphi A (C6915), Vibrio cholerae (IDH5439) and Escherichia coli (H10407) using standard well diffusion method and its minimum inhibitory concentration. The study revealed that the ethyl acetate extract of the tissues inhibited the growth of the tested pathogenic bacterial strains. The minimum inhibitory of the tissue extract ranged from 05 to 20 mg/mL. These above observed results showed the marine gastropods H. pugilinus extracts were may use as additional antibacterial compounds to inhibit the growth of harmful human bacteria.

Keywords
Hemifusus pugilinus; Antibacterial activity; Solvent extracts; Minimum inhibitory concentration

Molluscs are considered as one of the most important sources to derive bioactive compounds that exhibit antitumor, antimicrobial, anti-inflammatory and antioxidant activities (Anbuselvi et al., 2009).The compounds isolated from molluscs were also used in the treatment of rheumatoid arthritis and osteoarthritis (Chellaram and Edward, 2009).The marine environment is a huge source for discovering many antibacterial drugs. Apart from the food that is derived from the marine environment, wide varieties of antibacterial drugs are being isolated and characterized with great promise for the treatment of human diseases. The mollusc extracts also exhibited antiviral and antibacterial activity against fish pathogenic bacteria (Defer et al., 2009). Hemifusus pugilinus is an herbivorous gastropod that feeds on algae and other flora present abundantly in the Gulf of Mannar (Edward et al., 2008). The first attempt to the screening of antimicrobial activity in marine organisms was initiated around 1950’s. Since this time large number of marine organisms from a wide range from 1960’s to 1990’s approximately 300 bioactive marine natural products were fields of patent (Dhinakaran et al., 2011). Approximately 6500 bioactive compounds were isolated from marine organisms. Among the invertebrates, the molluscs are highly delicious seafood because of their nutritive value next to fin fish and crustaceans. They are also a very good source for biometrically important products (Kamboj, 1999). Literature survey revealed the urgent need to explore marine molluscs and not much work has been carried out in H. pugilinus. In the present investigation, we aimed to study the antibacterial activity of tissue extracts from marine gastropod H.pugilinus against clinically isolated human pathogenicbacteria.
1 Material and Methods
1.1 Collection of species
Fresh and live animals of H. pugilinus were collected from the Mandapam coastal area (Lat. 09º17΄11.3˝ Nand Long. 79º 09΄17.1˝ E), Ramanathapuram District, Tamil Nadu, India. The collected tissues were washed with tap water until the sand and mud were removed from the shells. After that, the shells were broken using a hammer to remove the soft body tissue. The removed tissues were rinsed with sterile distilled water, cut into small pieces and kept in Petri dishes and dried at a constant temperature of 50 ℃ for 24 h in a hot air oven. The dried material was powdered thoroughly for solvent extraction (Vimala and Thilaga, 2012).
1.2 Solvent extraction
The powdered tissues of H. pugilinus were extracted with eight different solvents like methanol, ethanol, acetone, acetonitrile, dichloro methane, chloroform, ethylacetate and distilled water with the help of soxlet apparatus then the solvents were concentrated with the help rotary evaporator (VC100ALark Rotavapor® at 30℃) with reduced pressure to give a dark brown gummy mass. The resultant residues were stored at 4 ℃ for further antibacterial activity.
1.3 Antibacterial Attributes of H. pugilinus
Six species of bacteria were used for the antibacterial activity, including V.parehaemolyticus (J13300), A.hydrophilla (IDH1585), S.typhi (C6953), S.paratyphi A (C6915), V.cholera (IDH5439) and E.coli (H10407). All the bacterial strains were clinical isolates, obtained from the Microbial Type Culture Collection & the Gene Bank, Institute of microbial technology, Chandigarh, India. Pathogenic bacterial strains were inoculated in sterile nutrient broth and incubated at 37° C for 24h. Pathogens were swabbed on the surface of the Muller Hinton agar plates and. wells of 5 mm in diameter were made aseptically using well cutter, and50 μL of eight different solvent extracts of tissues and eggs were inoculated. The stock solutions were prepared at a concentration of 20 mg/mL. Positive control well containing 50 μL of tetracycline (1mg/mL) and negative control containing 50 μL of appropriate solvents were used. The result was calculated by measuring the zone of inhibition in millimetres. For each concentration tested, triplicates were maintained for the confirmation of activity.
1.4 Determination of the minimum inhibitory concentration (MIC)
The solvent extracts of marine gastropods H.pugilinus which showed significant antibacterial activity were selected for the determination of MIC. A stock solution of 20 mg/mL was prepared and serially diluted to obtain various concentrations between 4 mg/mL and 20 mg/mL. About 0.5 mL of each dilution of different concentrations was transferred into a sterile test tube containing 2 mL of nutrient broth. To the test tubes, 0.5 mL of test organism previously adjusted to a concentration of 105cells/mL was then introduced. A set of test tubes containing broth alone was used as a control. All the test tubes and control were then incubated at 37℃ for 24 h. After the period of incubation, the tube containing the least concentration of extracts showing no visible sign of growth was taken as the minimum inhibitory concentration (Pasiyappazham Ramasamy et al., 2013).
1.5 Statistical analysis
Data on the inhibitory effects of solvent extracts of H.pugilinuswas analyzed by one-way analysis of variance (ANOVA) usingSPSS-16 version software followed by Duncun’s multiple range tests and standard deviation. P<0.05 were considered for describing the significant levels.
2 Results
2.1 Antibacterial Attributes
The inhibition zone in different solvent tissue extracts of H.pugilinusagainst clinical isolate human pathogenic bacteria was showed in Table 1. Among H. pugilinus the various strains, the maximum zone of inhibition (21.00±.0.74 mm) was recorded in Ethyl acetate extracts against S.paratyphi A, followed by (18.00± 0.84 mm) was recorded in Aceton nitrile against V.cholerae and the minimum zone of inhibition(4.00±0.51mm) was noticed in Acetone, methanol, chloroform and water extract (Figure 1).The positive control(tetracycline) was active against all the bacterial strains tested.


Figure 1 Antibacterial activity of H. pugilinus against Human pathogenic bacteria



Table 1 Antibacterial activity of tissue extract of H. pugilinus against clinically isolated human pathogen (Mean±SD)


2.2 MIC of the active extract against isolated pathogens
MIC values of ethyl acetate and acetonitrile tissue extracts against bacterial strains such as A. hydrophilla, S. typhi, S. paratyphi A, V.cholerae and E. coli were reported as 20, 15, 10, 5, mg/mL respectively (Table 2).


Table 2 MIC of tissue and egg extracts of H. pugilinus against clinically isolated human pathogens


3 Discussions
Recently, sea creatures constitute a large reservoir for pharmacologically active drug recently reviewed (Mayer et al., 2007). Molluscs are widely used in world research institution for various studies, but only recently they have been recognized as potential sources of antibacterial and antifungal metabolites.Some of the molecules responsible for antimicrobial activities have been identified and characterized. According to (Suresh et al., 2012), some of the molecules were responsible for significant antimicrobial activities and yet have to be identified and characterized. In result of the present study clearly showed that the antibacterial activities maximum inhibition zone was observed in Ethyl acetate extract of H. pugilinus against S. paratyphi A and minimum zone of inhibition (7 mm) was notice in Acetone, methanol, chloroform and water extract. These results encourage the idea that marine molluscs are potent sources for drug development. The maximum inhibition zone was observed against Klebsiella pneumonia in the ethanol extract of Babylonia zeylanica and the minimum inhibition zone mm) was observed against V. cholera (Suresh et al., 2012). It’s also reported that the acetone extract of the winged oyster Peteria chinensis was found to have a board spectral activity in habiting all the fish pathogenic strains tested and the extract of chloroform inhibited eight pathogens, these also support the present study on antimicrobial activity of gastropod extracts (Chellaram et al., 2004). MIC methods are widely used in the comparative testing of new drugs. In clinical laboratories they are used to establish the susceptibility of organisms that give equivocal results in disc or well tests, for tests on organisms where disk or well tests may be unreliable and when a more accurate result is required for clinical management.
The present study MIC values of acetone tissue and egg extracts against bacterial strains such as A.hydrophilla, S. typhi, S. paratyphi A, V. cholera and E.coli were reported 20,15,10,5 mg/mL respectively. The isolation of compounds from marine molluscs were also used in the treatment of rheumatoid arthritis and osteoarthritis. Marine mollusc extracts also exhibited antibacterial and antiviral activity against fish pathogen bacteria and the extract also may be applied in aquaculture (Rajaganapathi, 1996). The methanolic extract of C.virgineus and C. ramous experimentally analyzed and observed the broad spectrum antimicrobial activities of body tissue extract (Santhana Ramasamy and Murugan, 2005). The different antibacterial activity found in gastropod extract may depend on extracting capacity of the solvents and the compound extracted (Periyasamy et al., 2012).Commercial antibiotics are highly effective to kill the bacterial and fungal pathogens involved in common infection. This study revealed that the ethyl acetate extract of the tissues inhibited the growth of the tested pathogenic bacterial strains. Further investigations intending to purify these active compounds should be considered to clarify their chemical nature.
Acknowledgements
Authors are thankful to Dr. T. Ramamurthy, Scientist F, National Institute of Cholera & Enteric Diseases, Kolkata with necessary facilities. The authors are also thankful to the Centre for Marine Living Resources and Ecology, Cochin, Ministry of Earth Sciences, New Delhi, India for providing the financial support (Grant No. MoES/10-MLR/01/12).
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