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Antibacterial Properties of the Skin Mucus of the Freshwater Fishes, Rita Rita and Channa Punctatus

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Antibacterial Properties of the Skin Mucus of the Freshwater Fishes, Rita Rita and Channa Punctatus European Review for Medical and Pharmacological Sciences 2011; 15: 781-786 Antibacterial properties of the skin mucus of the freshwater fishes, Rita rita and Channa punctatus U. KUMARI, A.K. NIGAM, S. MITTAL*, A.K. MITTAL Skin Physiology Laboratory, Centre of Advanced Study, Department of Zoology, Banaras Hindu University, Varanasi (India) Abstract. – Objectives: The skin mucus unfavorable environmental conditions e.g. os- of Rita rita and Channa punctatus was investigat- moregulation1, reduces body friction in water and ed to explore the possibilities of its antibacterial assists in swimming2, protects from abrasion dur- properties. ing burrowing3, precipitates mud held in suspen- Materials and Methods: Skin mucus was ex- sion in water4, chelates cations especially toxic tracted in acidic solvents (0.1% trifluoroacetic 5 6 acid and 3% acetic acid) and in triple distilled metal ions , traps nanoparticles . Trapping water (aqueous medium). The antibacterial activ- nanoparticles and chemicals in the mucus, with ity of the mucus extracts was analyzed, using subsequent sloughing of the contaminated mucus disc diffusion method, against five strains of protects the sensitive epithelial cells6. bacteria – the Gram-positive Staphylococcus au- The aqueous environment, inhabited by fish reus and Micrococcus luteus; and the Gram nega- often, contains different types of pathogens. Fish, tive Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi. however, maintains a healthy state by defending Results: In both Rita rita and Channa puncta- against pathogens by a complex system of innate tus, the skin mucus extracted in acidic solvents defense mechanisms. Mucus layer provides as well as in aqueous medium show antibacteri- physical barrier which acts as first line of defense al activity against Staphylococcus aureus and Mi- against attachment and penetration by potentially crococcus luteus. Nevertheless, the activity is harmful agents and thus plays an important role higher in acidic solvents than that in aqueous 7-10 medium. The acidic mucus extracts of Rita rita, in the protection of the fish . show antibacterial activity against Salmonella Antibacterial agents are widely expressed by a typhi as well. diverse range of organisms such as invertebrates, Conclusions: The results suggest that fish fishes, amphibians, and mammals11-13. In recent skin mucus have bactericidal properties and years, attention of researchers in the field has thus play important role in the protection of fish been drawn to explore antibacterial properties of against the invasion of pathogens. Fish skin mu- fish skin mucus. Austin and McIntosh14 demon- cus could thus be regarded as a potential source of novel antibacterial components. strated the antibacterial compounds on the sur- face of rainbow trout, Salmo gairdneri. Na- Key Words: gashima et al15 found antibacterial factors in skin Fish, Skin, Mucus, Antibacterial properties. mucus of two species of rabbitfishes, Siganus fuscescens and Siganus guttatus. Hellio et al16 and Subramanian et al17 compared antibacterial activities in epidermal mucus extracts of several fish species of North Atlantic. Exploiting indigenous species for potential an- Introduction tibacterial agents is of substantial interest. How- ever, a review of literature reveals that studies on In fishes, in general, the external body surface the antibacterial properties of skin mucus secre- is covered by a mucus coat, elaborated by skin tions of food fishes in Indian subcontinent are gland cells. The mucus coat varies considerably limited. Kuppulakshmi et al18 in Channa puncta- in its thickness and viscosity in different fish tus and Cirrhinus mrigala, reported antibacterial species, and is important to the fishes in various properties of the mucus based on their studies on ways. Fish skin mucus plays a significant role in only the aqueous mucus samples from skin. Ra- mechanical and physiological protection against jansamantaray et al19 described antibacterial Corresponding Author: Swati Mittal, Ph.D.; e-mail: [email protected] 781 U. Kumari, A.K. Nigam, S. Mittal, A.K. Mittal properties of skin mucus of the fish Ompok pab- Collection of Fish Skin Mucus Samples da based on their studies on mucus collected by Fishes were cold anaesthetized20 and then dipping the fish in methanol. washed with phosphate buffered saline (PBS, Multidrug resistance and/or low antibiotic sus- pH7.4) to clean the surface from contaminants. ceptibility by bacterial strains necessitate search Mucus of the two fish species was collected sep- for development of new antibacterial agents arately from the dorso-lateral region of the skin which can control infectious disease effectively using blunt edge of a clean steel scalpel. Ventral without inducing resistance. Search of natural an- skin mucus was not collected to avoid contami- tibacterial agents is gaining importance over con- nation from urinogenital and intestinal excreta. ventional antibiotics, because of a broad range of Mucus thus collected from ten fishes of each fish rapid acting and potent bactericidal activity. In species was pooled separately. Pooled mucus of view of this, the focus of the present study has each fish species was homogenized in PBS, for been to explore antibacterial properties of the skin 10 minutes at 4°C and then lyophilized and mucus secretions of the catfish, Rita rita (Order, stored at –20°C for further analysis. Siluriformes; Family, Bagridae) and the murrel, Channa punctatus (Order, Channiformes; Family, Screening of Mucus Extracts for Channidae). The two fish species are considered Antibacterial Activities as valuable food fishes in India both from the The lyophilized mucus was divided into three consumers taste and preference. Rita rita is a parts. One part (10 mg ml-1) was dissolved in 3% sluggish, bottom-dwelling carnivorous catfish and (v/v) acetic acid, the second part (10 mg ml-1) in inhabits the regions of river with accumulations 0.1% (v/v) trifluoroacetic acid, and the third part of dirty water. Channa punctatus is an active (10 mg ml-1) in triple distilled water. The three predator, inhabits rivers and ponds, and is able to aliquots were then separately homogenized at survive droughts and to live in semi-fluid mud. 4°C and centrifuged at 10,000 × g for 15 minutes The aquatic environment inhabited by the two at 4°C. Supernatants were collected separately fish species is thus likely to get infested, predomi- and stored at –20°C. The mucus extracts, thus nantly with potentially harmful microorganisms obtained from each fish species were evaluated in and the fishes thriving in such conditions in turn vitro for antibacterial activity. are prone to get exposed to different types of bac- Antibacterial properties of the skin mucus of terial pathogens. In fishes, mucus elaborated at the fish species was assessed using two Gram- the surface from the skin, which is in intimate positive bacteria – Staphylococcus aureus ATCC contact with the surrounding hostile environment, 25923, Micrococcus luteus MTCC 3911, and is liable to possess antibacterial activity, an adap- three Gram-negative bacteria – Escherichia coli tation to play significant role in protecting the fish ATCC 25922, Pseudomonas aeruginosa ATCC from pathogenic infections. An understanding on 27853, Salmonella typhi MTCC 3216. Each the antibacterial properties of skin mucus could strain of bacteria was grown to mid-logarithmic be useful to gain insight in the involvement of phase in Mueller-Hinton broth and then diluted mucus in innate host defense mechanism at or- to achieve cell density to an OD600 = 0.1 ab- ganism-environment interfaces and could aid in sorbance to create a working bacterial stock. The the development of antibacterial agents for thera- antibacterial properties of mucus extracts were peutical applications. studied by standard Kirby-Bauer antibacterial sensitivity test (Disc diffusion method). Mueller-Hinton agar (M-H agar) Petri-plates prepared in laboratory were inoculated with 50 Materials and Methods µl of diluted bacterial culture by spreading it over agar surface with the help of sterile cotton Live specimens of the fishes Rita rita (standard swabs. Filter paper (Whatman Filter Paper No. length, 105 ± 6 mm; n = 30) were collected from 1) discs of 4.5 mm diameter were impregnated the river Ganges and Channa punctatus (standard with 50 µl of mucus extracts and then were length, 138 ± 8 mm; n = 30) were collected from placed on the inoculated agar surface using a local ponds at Varanasi and were kept in a labora- sterilized forceps. The Petri-plates were pre- tory aquarium at controlled room temperature (25 pared, in triplicate, for each mucus extract and ± 2°C). The fishes were fed on alternate days with incubated at 4°C for 1 hour, and then at 37°C in minced goat obtained from market. an incubator for 16-18 hours. The controls for 782 Antibacterial properties of the skin mucus of the freshwater fishes, Rita rita and Channa punctatus each solvent (0.1% trifluoroacetic acid, 3% (aqueous extract), the zone of inhibition was acetic acid and triple distilled water) without the minimum (average diameter, 9.75 ± 1.70 mm), mucous samples were assayed simultaneously. demonstrating that the inhibition of the growth of The diameter of inhibition zone i.e., clear zone the bacteria with this extract was slight. Average around the disc resulting from the inhibition of diameter of the inhibition zone obtained by the bacterial growth by mucus were recorded. Each three extracts using agar disc diffusion test
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