In vitro evaluation of antimicrobial activity of methanolic extract from selected species of Cephalopods on clinical isolates

Bioactive substances from marine biota have been found useful as special tools in pharmacological and biomedical research. In the present study, the in vitro antimicrobial activity of crude methanolic extracts of six species of cephalopods (Sepia kobiensis, Sepiella inermis, Sepioteuthis lessoniana, Octopus aegina, Octopus aerolatus, Octopus dollfusi) from Cuddalore (Southeast coast of India) was studied. The antimicrobial activity was screened against 10 species of clinically isolated human pathogenic bacteria namely Vibrio chlolerae, Pseudomonas aeruginosa, Klebsiella pnemoniae, Vibrio alginolyticus, Staphylococcus aureus, Vibrio parehaemolyticus, Streptococcus sp., Streptococcus pnemoniae, Salmonella sp. and Escherichia coli and five fungal strains such as Alternaria alternata, Candida tropicalis, Pencillium italicum, Fusarium equisetii and Candida albicans. Different concentrations such as 25, 50, 75 and 100% were prepared and tested against the microbial strains for their inhibitory activities, using the disc diffusion method. The minimum inhibitory concentration (MIC) of methanolic extract of cephalopods ranged from 60 to 100 mg/ml. The results were discussed in the light of positive and negative control apart from the concentrations tested.


INTRODUCTION
The Class: Cephalopoda includes nautilus, cuttlefishes, squids and octopods which are exclusively marine, varying in their form, size and nature (Voss and Williamson, 1971;Voss, 1973Voss, , 1977;;Worms, 1983).They occupy littoral and benthic to pelagic environments of oceans.Cephalopods are important as a food resource as well as animal models in scientific investigations (Ngoile, 1987) and they are the store-house of many biologically important substances.There is an everlasting need to discover new antimicrobial compounds with diverse chemical structures and novel mechanisms of action due to the alarming increase that has been *Corresponding author.E-mail: shanpappu48@gmail.com.Tel: +91-9443043597.
witnessed in the incidence of both new and re-emerging infectious diseases.A further big concern is the development of resistance to the antibiotics in current clinical use (Ilhan et al., 2007).
The first systematic search for antibiotics resulted in the discovery of actinomycetin from Actinomycete bacteria.In United States and Japan between 1953 and 1970 approximately 85% of the antibiotics were produced by Actinomycetes, 11% by fungi and 4% by bacteria (Reiner, 1982).Although antibiotics are life saving drugs but nowa-days, due to careless and promiscuous use of antibiotics, various pathogenic microbes are gaining resistance.Among marine invertebrates, cephalopods belong to a molluscan group comprising of 700 species in which bacterial associations have been known for a long time (Pierantoni, 1917;Bloodgood, 1977) which include the reproductive organs (accessory nidamental glands) of myopsids, sepiolids and sepiids (Kaufman et al., 1998;Grigioni et al., 2000;Pichon et al., 2005) and the light organ of sepiolids (McFall-Ngai and Ruby, 1991;Nishiguchi, 2002).
Marine invertebrates offer a good source of potential antimicrobial drugs (Bansemir et al., 2006;Mayer et al., 2007;Jayaraj et al., 2008).Discovered bioactive compounds in molluscs were identified essentially as peptide, depsipeptide, sterols, sesquiterpene, terpenes, polypropionates, nitrogenous compounds, macrolides, prostaglandins and fatty acid derivatives, miscellaneous compounds and alkaloids which presented specific types of activities (Maktoob and Ronald, 1997;Balcázar et al., 2006;Blunt et al., 2006).Studies on antimicrobial activity that lead to valuable information for new antibiotic discoveries and give new insights into bioactive compounds in aquacultured molluscs.In most of the publications concerning antimicrobial activity in molluscs, either single body compartments alone, like haemolymph and egg masses, or extracts of whole body have been tested for activity.Recently crustaceans (Haug et al., 2002a) and echinoderms (Haug et al., 2002b) are reported to possess antibacterial factors in different tissues.The antimicrobial activity of polysaccharides extracted from cephalopods such as Sepia aculeata and Sepia brevimana and heparin and heparin -like glycosaminoglycans (GAGs) from the cephalopod Euprymna berryi was reported against the human pathogenic microorganism (Shanmugam et al., 2008a;Shanmugam et al., 2008b).In the present investigation, an attempt has been made to screen the antibacterial and antifungal activity of the crude methanolic extract of selected cephalopods on some important human pathogens.

Sampling and identification
Cephalopods such as, S. kobiensis, S. inermis (Cuttlefishes), S. lessoniana (Squid), Octopus aegina, O. aerolatus and O. dollfusi (Octopods), used in this study were obtained from Cuddalore landing centre (Latitude 10° 42'N; Longitude 79° 46'E) which is situated in the Southeast coast of India.The studies carried out by Voss (1973), Voss and Williamson (1971), Roper et al. (1984), Jothinayagam (1987) and Shanmugam et al. (2002) have been of considerable help on developing the identification keys and description which in most cases have also been corroborated with examination of actual specimen.

Preparation of extracts
Cephalopods were brought to laboratory; body tissues were removed, cut into small pieces and homogenized (REMI, RQ-127 A) and extracted with MeOH at room temperature for 24 -48 h (Ely et al., 2004).Then the methanolic extract was centrifuged to collect the supernatant and concentrated under vacuum in a rotary evaporator (LARK, Model: VC-100A) at low temperature.The crude methanolic extract was assayed for antibacterial and antifungal activities using standard disc diffusion method.

Microbial cultures
Ten species of bacteria and five species of fungi were used as test organisms.

Inoculum preparation for bacteria
Nutrient broth was prepared and sterilized in an autoclave at 15 lbs pressure for 15 min.All the ten bacterial strains were individually inoculated in the sterilized nutrient broth and incubated at 37°C for 24 h.Mueller Hinton Agar (MHA, Himedia) was prepared, sterilized in an autoclave at 15lbs pressure for 15 min and poured into sterile petridishes and incubated at 37°C for 24 h.The 24 h old bacterial broth cultures were inoculated in the petridishes by using a sterile cotton swab.

Inoculum preparation for fungi
Czapek dox (Hi-media) broth was prepared and sterilized in autoclave at 15 lbs pressure for 15 min.Five fungal strains were inoculated in the broth and incubated at 37°C for 72 h.The sterilized Czapek dox agar was poured into sterile petridishes and incubated at 37°C for 3 days.The 72 h old fungal broth cultures were inoculated in the petridishes using a sterile cotton swab.

Disc diffusion method
Antibacterial and antifungal activity was determined following the method of (El-Masry et al., 2000).Briefly, a suspension of each tested microorganism was carefully mixed in the tube containing bacterial and fungal inoculums and media for bacterial and fungal were plated separately, respective strains were cotton swabbed on petridishes.Sterile antimicrobial disc (Hi-media) was impregnated with 50 µl of crude methanolic extract of the four concentrations tested.Positive control discs containing 50 µl of tetracycline (1 mg/ml) and negative control, 50 µl of methanol were used.The stocks for methanolic extracts were prepared in the concentration of 100 mg/ml.These impregnated discs were allowed to dry at laminar air flow chamber for 3 h, and were placed at the respective bacterial and fungal plates and incubated at 37°C for 24 h for bacteria and 72 h for fungi.The diameter (mm) of the growth inhibition halos produced by the methanolic extracts of cephalopods was examined.
Result was calculated by measuring the zone of inhibition in millimeters.All the tests were performed in triplicates.

Determination of the minimum inhibitory concentration (MIC)
The methanolic extract of cephalopods which showed significant antimicrobial activity was selected for the determination of MIC followed by the method of Rajendran and Ramakrishnan (2009).A stock solution of 100 mg/ml was prepared and was serially diluted to obtain various ranges of concentrations between 20 and 100 mg/ml.0.5 ml of each of the dilutions of different concentrations was transferred into sterile test tube containing 2.0 ml of nutrient broth.To the test tubes, 0.5 ml of test organism previously adjusted to a concentration of 10 5 cells/ml was then introduced.A set of test  tubes containing broth alone was used as control.All the test tubes and control were then incubated at 37°C for 24 h.After the period of incubation, the tube containing the least concentration of extract showing no visible sign of growth was taken as the minimum inhibitory concentration.

Statistical analysis
Data on the inhibitory effect of methanolic extracts of cephalopods was analyzed by one-way analysis of variance (ANOVA) using SPSS-16 version software followed by Duncun's multiple range test (DMRT).P values ≤0.05 were considered as significant.

RESULTS
The methanolic extract of cephalopods showed antibacterial and antifungal activity against all pathogenic strains which were concentrationdependent i.e., the activity was higher in 100% concentration and lower in 25% concentration but activity was absent in negative control ( In 25% concentration, maximum activity of 10 mm was recorded against Salmonella sp. in O.

MIC of the active extract against the test organisms
The MIC results are given in Table 2. MIC values of S. inermis against bacterial strains such as V. cholerae, K. pneumonia, S. aureus, Streptococcus sp., S. pneumonia and E. coli showed 100, 80, 80, 80, 100 and 100 mg/ml respectively.In S. lessoniana the MIC for V. cholerae, P. aeruginosa, K. pneumoniae, V. alginolyticus, S. aureus, V. parahaemolyticus and Streptococcus sp.

DISCUSSION
The overall objective of the current study is to compare the ability of antibacterial and antifungal activity of crude methanolic extract of six species of cephalopods collected from the same habitat.
In the present study the results clearly showed (Tables 1 and 2) that majority of extracts exhibited appreciable antimicrobial activity against most of the clinically isolated human pathogens.The effects of extracts were different for different bacterial strains.Interestingly the present study reported no antifungal activity for all methanolic extracts from the cephalopods studied.
In recent years, great attention has been paid to study the bioactivity of natural products due to their potential pharmacological utilization.The rationale of searching for drugs from marine environment stems from the fact that marine plants and animals have adapted to all sorts of habitats in the marine environment and these creatures are constantly under tremendous selection pressure including competition for space, predation, surface fouling and reproduction.Many of these organisms are showing antimicrobial properties.Although most of the antibacterial agents isolated from marine sources have not been active enough to compete with classicical anti-microbials obtained from microorganisms (Rinehart et al., 1981).However, majority of marine organisms are yet to be screened for discovering useful antibiotics.
Antibacterial activity has previously been described in a wide range of molluscan species such as oyster (C.virginica), mussel (Mytilus edulis and Geukensia demissa), muricid mollusks (Dicathais orbita) and sea hare (Dolabella auricularia) (Constantine et al., 1975;Gunthorpe and Cameron, 1987;Prem Anand et al., 1997;Anderson and Beaven, 2001;Benkendorff et al., 2001).In most of the species studied, the haemolymph, egg masses or the whole body have been tested for activity.Antimicrobial peptides have been isolated and characterized from the haemocytes of Mytilus edulis (Charlet et al., 1996;Mitta et al., 2000a) and M. galloprovincialis (Hubert et al., 1996;Mitta et al., 1999;Mitta et al., 2000b), and from the seahare Dolabella auricularia (Iijima et al., 2003).In the present investigation the crude methanolic extract from the whole body tissue of the cephalopods was used to study the antimicrobial activity against selected human pathogens.
Prem Anand and Patterson Edward (2002) reported moderate antibacterial and antifungal activity from the extracts of various bivalves molluscs.Patterson and Murugan (2000) reported broad spectrum of antibacterial activity for aqueous ink extract of the cephalopods L. duvaceli and S. pharaonis against nine human pathogens.
EDTA extract (polysaccharides) of D. sibogae gladius reported 10 mm inhibition zone against E. coli and K. pneumonia, 9 mm inhibition zone against S. aureus and 7 mm against S. typhii.Whereas the EDTA extract of L. duvauceli showed only low activity i.e., 5 mm against P. aeroginosa, 4mm against S. typhii and E. coli.At the same time, the gladius extract of both the species showed no activity against V. cholerae.The polysaccharide extract from the gladius of D. sibogae recorded potent antibacterial activity against all the bacterial strains mentioned above and at the same time the polysaccharide extract of the L. duvauceli gladius recorded only low activity.The polysaccharides extracted from the gladius of L. duvauceli showed activity against the fungi such as A. fumigatus, A. flavus and Rhizopus sp; whereas gladius extract of D. sibogae reported activity against A. fumigatus and Rhizopus sp.only.But at the same time both the species showed no activity against Candida sp. at all the concentrations tested (Barwin vino, 2003).The cuttlebone extract (using EDTA) of S. aculeata and S. brevimana showed antibacterial activity against almost all the 9 pathogenic bacterial stains tested viz., B. subtilis, E.coli, K. pnemoniae, S. aureus, V. parahaemolyticus, V. cholerae, S. typhii.P. aeroginosa and Shigella sp.The activity was recorded in almost all the concentrations except in negative control.The antifungal activity of cuttlebone extracts of S. aculeata and S. brevimana against four fungal stains such as A. fumigatus, A. flavus, Candida sp. and Rhizopus sp.showed the maximum activity of 100% and activity was found to be in an increasing order from the lower to higher concentration.On comparison the activity was higher in the cuttlebone extract of S. aculeata than S. brevimana (Shanmugam et al., 2008a).Shanmugam et al. (2008b) reported that the crude and purified sample of Glycosaminoglycans (GAGs) from E. berryi showed activity against five pathogenic bacteria and four fungal strains.The activity was higher in 100% concentration and lower in 25% concentration; but activity was absent in negative control.The maximum antibacterial activity was shown in Shigella sp.(5 mm) for crude sample and S. aureus for purified sample.The minimum antibacterial activity showed (1.5 mm) against E. coli in crude sample.The maximum antifungal activity (5.5 mm) was observed against C. albicans and A. fumigatus in crude and purified sample, respectively.The minimum antifungal activity (0.5 mm) was recorded against A. fumigatus and C. neofromans in crude sample.
Although different species and experimental procedures were used in the different studies, they indicated the high frequency of detectable antimicrobial activity in marine molluscs.These results enforce the idea that cephalopods are a source to be considered in the discovery of new substances for drug development to control microbial diseases.In the present investigation highest inhibition was recorded in Salmonella sp. and E. coli against O. dollfusi extract.Kagoo and Ayyakkannu (1992) reported a broad spectral activity in the hypobranchial gland extract of Chicoreus ramosus against 10 bacterial strains.In the present study a wide spectrum of antibacterial activity has been recorded in almost all the extracts, (Table 1).

Conclusion
Cephalopods collected from Cuddalore landing centre, southeast coast of India showed potential antibacterial activity against human pathogenic bacterial strains.In the present evaluation good antimicrobial activity was seen in the extracts of O. dollfusi, S. lessoniana and S. inermis species, which indicates the presence of potent antimicrobial compounds in them.Further research on purification and characterization of these potentially active compounds, may pave the way for the development of potent antibacterial drugs.

Table
Antibacterial activity of methanolic extract of cephalopods.

Table 2 .
MIC of methanolic extracts of cephalopods against clinically isolated human pathogens.
* MIC concentration, -No growth, + Cloudy solution (slight growth), ++ Turbid solution (strong growth), +++ Highly turbid solution (dense growth).dollfusiextract, 8mm against Streptococcus sp. in S. lessoniana extract, against V. parahaemolyticus in O. aegina extract and against K. pneumoniae in S. inermis extract.The lowest activity of 7 mm against V. alginolyticus in both O. aegina and O. dollfusi extract, against P. auruginosa in S. inermis extract and the same level of inhibition found V. parahaemolyticus in S. lessoniana extract.But at the same time, no activity was recorded against all the fungal strains studied.