Screening of Antibacterial Activity of Mucus Extract of Snakehead Fish, Channa Striatus (Bloch)

European Review for Medical and Pharmacological Sciences 2010; 14: 675-681 Screening of antibacterial activity of mucus extract of Snakehead fish, Channa striatus (Bloch) ONG YEONG WEI, R. XAVIER, K. MARIMUTHU Department of Biotechnology, Faculty of Applied Sciences, AIMST University Batu 31/2 Bukit Air Nasi, Jalan Bedong Semeling, Bedong, Kedah Darul Aman (Malaysia) Abstract. – Aim: The objective of this Introduction study is to gain a better understanding of the antimicrobial properties of the mucus extract Advanced improvements and new formulations of snakehead fish, Channa striatus against selected human and fish pathogenic microbes. in the modern chemotherapeutic techniques have Materials and Methods: The fish mucus been applied. However, infectious diseases are still samples were extracted with crude, acidic and an increasingly important public health issue in aqueous solvents to identify potential antimi- the world1. It has been reported that two million crobial agents including aqueous and acid people died in 2000 due to diarrhoeal disease soluble compounds. The study also deter- worldwide1. To combat this, recently research has mined the protein content of the three different mucus extracts. The highest protein con- been made to find out an effective method to pre- tent (0.589 mg/ml) was noticed in the crude ex- vent or cure diseases. Nowadays, the development tract followed by aqueous mucus extract of resistance to many of the commonly used an- (0.291 mg/ml) and acidic extract (0.267 mg/ml). tibiotics provides a further attempts to search for Preliminary screening for antimicrobial activi- new antimicrobial agents to combat infections and ty of all three mucus extracts were tested overcome problems of their resistance and side ef- against 5 human pathogens (Bacillus subtilis, fects. Several attempts have been made exploring Klebsiella pneumoniae, Salmonella enteritidis, Proteus vulgaris and Pseudomonas aeruginosa) new antimicrobial drugs from natural sources in- and fish pathogen (Aeromonas hydrophila) us- cluding plant and animal products. ing the British Society for Antimicrobial Approximately 20 million metric tones of fish Chemotherapy (BSAC) standardized disc sus- by-products are discarded annually from the ceptibility test method. The activity was mea- world fisheries. Fish by-products are rich in po- sured in terms of zone of inhibition in mm. tentially valuable proteins, minerals, enzymes, Results: The acidic mucus extracts exhibited a bactericidal activity and inhibited the growth of pigments or flavours. Off the fish by products Klebsiella pneumoniae, Pseudomonas aeruginosa fish mucus is considered more valuable and has and Bacillus subtilis while aqueous and crude ex- been reported that it contains antimicrobial pro- tract showed no bactericidal activity for any of teins. In fish the epidermal mucus is considered the human pathogens tested. Further test a key component of innate immunity and plays a against fish pathogen Aeromonas hydrophila role in the prevention of colonization by para- showed that the aqueous and crude extracts are sites, bacteria and fungi2-6. The epidermal mu- capable of inhibiting the growth of the pathogen, demonstrating the presence of antimicrobial cus, primarily produced by epidermal goblet or agents and the role of fish mucus in antimicro- mucus cells, is composed mainly of water and bial protection. gel-forming macromolecules including mucins Conclusions: The present results suggest and other glycoproteins7,8. The composition and that the mucus extracts of snakehead fish Chan- rate of mucus secretion has been observed to na striatus may be a potential source of antimi- change in response to microbial exposure or to crobial agents for human and fish pathogens. environmental fluctuations such as hyperosmo- 9-11 Key Words: larity and pH . The mucus layer on the fish surface performs a number of inevitable func- Snakehead fish Channa striatus, Epidermal mucus, Antibacterial activity. tions including disease resistance, respiration, ionic and osmotic regulation, locomotion, repro- Corresponding Author: Kasi Marimuthu, MSc, Ph.D; e-mail: [email protected] 675 Ong Yeong Wei, R. Xavier, K. Marimuthu duction, communication, feeding and nest build- prior to mucus collection. On the day of mucus ing3,7,12. It has been known that fish mucus con- collection fish was washed and transferred into a tains a variety of biologically active compounds sterile polyethylene bag for 10 to 20 minutes and such as lysozyme, lectins, proteolytic enzymes, moved front and back to slough off the fish mu- flavoenzymes, immunoglobulins, C-reactive pro- cus. Then, the fish was returned to recovery tein, apolipoprotein A-I and antimicrobial pep- tanks. Fish mucus samples obtained from five tides that are constitutively expressed to provide fish was then pooled and stored in refrigerator at immediate protection to fish from potential path- 4°C until further use. The pooled mucus sample ogenic microbes and parasites13-17. An increased was then divided into three parts, which were ex- mortality rate has been reported in several fish tracted separately with crude, acidic, and aque- species when removal of epidermal mucus and ous solvents. after challenging them with pathogenic microbe Listonella anguillarum18,19. The loss of epidermal mucus increased the rate of susceptibility to Mucus Extraction and bacterial infection in carp (Cyprinus carpio)5. Protein Quantification Antibacterial activity in fish mucus has been For crude extract, 50 ml of fish mucus was demonstrated in several fish species20; yet this activ- centrifuged at 5000 rpm for 10 minutes. The su- ity seems to vary from species to species such as pernatant obtained was then stored in refrigerator rockfish (Sebastes schlegelii)17, rainbow trout (On- at 4°C25. The aqueous extract of fish mucus was corhynchus mykiss)21 and tilapia (Tilapia prepared using a method as described by Hellio hornorum)22 and can be specific towards certain et al26. Fifty ml of fish mucus was mixed with 50 bacteria23. The exploration of the antimicrobial ml of distilled water and homogenized using a properties of epidermal mucus of Malaysian fresh- polytron homogenizer. The mixture was then water fish species is very limited and to date no centrifuged at 30,000 g for 30 minutes at 4°C studies have been made especially in snakehead (Beckman coulter, Avanti J-26 XPI, Brea, CA, fish, Channa striatus. Hence in the present study an USA). Supernatant was then collected and fil- attempt was made to find an antibacterial activity of tered with Whatman no.1 filter paper. The filtrate the mucus extracts of snakehead fish Channa stria- was then collected and stored in refrigerator at tus against selected human and fish pathogens. 4°C. The acidic extract of fish mucus was prepared by using a modified method of Subraman- ian et al24. Thirty mL of the fish mucus was mixed with 30 mL of 3% acetic acid and placed Materials and Methods in a boiling water bath for 5 minutes. The acid- mucus mixture was then cooled in ice and ho- Fish Collection and Maintenance mogenized using polytron homogenizer. The Mucus sample was collected from snakehead mixture was then centrifuged at 18,000 g for 35 fish, Channa striatus (body weight; 186.6 g ± minutes at 4°C. Then, the supernatant was col- 7.436 g) that were obtained from a local fish lected and purified using a syringe with 0.22 µm market in Sungai Petani, Kedah Darul Aman, filter. Elutes were then collected and stored in re- Malaysia. Then the fish were stocked into the frigerator at 4°C. Protein quantification was de- 500 L capacity circular plastic tanks. The fish ac- termined based on Bradford protein assay27 by climatized to laboratory conditions in a tap water using bovine serum albumin as standard. and they were maintained for one week. During this period the fish were fed with commercial feed once a day at ad libitum. Every day 50 % of Bacteria Culture Conditions the water was changed. After one week of ac- Antimicrobial activities of mucus extracts climatization the fish were used for mucus col- were tested against a range of human and fish lection. Only healthy fish were chosen for mucus pathogens including both Gram positive (Bacil- collection. Dead fish or fish with skin lesions lus subtilis) and Gram negative bacterium were removed from the tanks. (Aeromonas hydrophila, Klebsiella pneumoniae, Salmonella enteritidis, Proteus vulgaris and Mucus Collection Pseudomonas aeruginosa). All the microbes Mucus was collected by a modified method of were grown at 37°C in Luria-Bertani (LB) broth Subramanian et al24. Fish was starved for one day and maintained at 37°C in Luria-Bertani (LB) 676 Screening of antibacterial activity of mucus extract of Snakehead fish, Channa striatus (Bloch) agar except fish pathogen Aeromonas hydrophila. MIC test was carried out by using broth microdi- The fish pathogen was grown in Nutrient broth at lution method as described by Subramanian et 37°C. al24 with slight modification. Mucus extract was serially two-fold diluted with 100 µl with Screening for Antimicrobial Activity Mueller-Hinton broth (HImedia, Mumbai, India) Preliminary screening for antimicrobial activ- in order to determine the minimum concentra- ity of all three mucus extracts prepared from tion that can be used to inhibit the growth of mi- Channa striatus was carried out against human croorganism. Fifty µl of overnight inoculum was pathogens (Bacillus subtilis, Klebsiella pneumo- then added into each tube containing different niae, Salmonella enteritidis, Proteus vulgaris concentration of mucus extract and incubated at and Pseudomonas aeruginosa) and fish room temperature for 16 to 18 hours. Growth in- pathogen (Aeromonas hydrophila) using the hibition was observed by visual inspection of the British Society for Antimicrobial Chemotherapy turbidity of the mixture. (BSAC) standardized disc susceptibility testing method28. Briefly, 20 µl of mucus extract was Statistical Analysis impregnated onto a disc. The disc with mucus One way analysis of variance and Duncan’s extract was then transferred into the Mueller multiple-range tests were employed to analyze Hinton agar plate with bacterial culture.

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