African Catfish (Clarias gariepinus) skin mucus as an antibacterial agent against pathogenic bacteriaResearcher:Chella Elin R. CruzNabunturan National Comprehensive High School, Nabunturan, Compostela Valley, Philippines
______________________________________________________________________________ABSTRACTMost freshwater fishes have been proven effective in treating many kinds of disease mostly through antibiotics because of its medicinal contents. However, the mucus secreted by the skin of fish showed more antimicrobial properties than the rest of its body (Balasubramanian et. al., 2011). In the present study, the skin mucus of African Catfish (Clarias gariepinus) was tested against Klebsiella pneumoniae and Escherichia coli and was measured in terms of zone of inhibition in mm through aseptic technique. A galactose-specific lectin purified from the skin mucus of African Catfish made it inhibit growth of bacteria (Ayinde, 2015).Results showed strong growth inhibition treated than the negative control.Key Words:African Catfish, skin mucus, E.coli, K.pneumoniae______________________________________________________________________________INTRODUCTION
In recent years, the need for natural antibacterial agents is gaining importance in relation to the widespread occurrence of antibacterial resistance (Fair & Tor, 2014). The antibacterial resistance crisis has endangered the efficacy of antibiotics due to the lack of new drug development (Ventola, 2015). A post-antibiotic apocalypse, in which antibiotics can no longer be effective because of the ability of bacteria to resist, has also been warned unless new organic drugs are developed (Gabbatis, 2017).

The proportion that had bacteria resistant to at least one of the most commonly used antibiotics ranged from zero to 82 percent (World Health Organization,2018). WHO also said that 8% to 65 percent of E. coli associated with Urinary Tract Infections presented resistance. A 2013 report from the Center for Disease Control and Prevention (CDCP) also revealed that more than 2 million people in the USA alone become ill every year as a result of antibiotic-resistant infections, and 23,000 die from such infections (Whiteman, 2014).In the Philippines, particularly Region XI, there has been a significant increase in resistance rates by 51%-75% (Antimicrobial Resistance Surveillance Program, 2016).
Since then, the attempt to kill the bacteria has been an alternative approach to the antibacterial resistance problem, but bacteria were able to resist (Alliance for the Prudent Use of Antibiotics, 2014). Hence, development of new antibiotics is highly encouraged. Thus, urging the researcher to search for a new, organic, alternative and cost-effective antibacterial agent which has a stronger effect on bacterial resistance. Organic antibiotics were already produced to fight resistance however, bacteria have the ability to pass their resistance genes to other bacteria which urges continuous antibiotic development. African Catfish, which has a potential to inhibit bacterial growth can be found anywhere in the locality which makes it easier to develop.

The potential of the epidermal layer of the fish in inhibiting growth of pathogenic bacteria has been explored due to its ability to provide mucosal body which has a stronger effect on bacterial resistance.

In this present study, the evaluation of the antibacterial effect for the skin mucus of African Catfish (Clarias gariepinus) is conducted to pave way for a new antibacterial resource that is readily available. These will be tested against the strains of K. pneumoniae and E.coli.

Materials and Methods
Collection of fish specimen
Seven live African Catfish (Clarias gariepinus) approximately 6 months old were collected at KM 92, Magsaysay, Nabunturan, Compostela Valley Province. The specimen were acclimatized in an oxygenized aquarium for 3 days (Kuppulakshmi et al., 2008). Then they werefed with 23 g of floaters and growers every four hours a day (Alfetche, 2018).Collection of skin mucusFishes were cold anaesthesized. Then they were washed with distilled water to clean the surface from contaminants (Kumari et al., 2011). Mucus of the African Catfishes were gently scraped using a sterile spatula and forceps. The left hand with gloves was used to hold the African Catfish’ head and the right hand without gloves held the spatula. Mucus was collected in the ventral side to avoid intestinal and sperm contamination (Kuppulakshmi et al., 2008).

Extraction of fish mucus
The mucus that was collected was placed in a test tube and added with equal amount of saline (5 mL). Then, it was centrifuged for 5 minutes in 5000 µL to separate the protein particles from the mucus. To decontaminate the mucus, it was washed with equal amount of ethanol then washed with distilled water. Afterwards, it was centrifuged again to get the pure protein components of the mucus then, it was labeled refrigerated under 4°C until use to avoid contamination.

Preparation of Test Organism
The cultures of E. coli and K. pneumoniae were obtained from the Microbiological Section, University of Immaculate Concepcion, Bankerohan, Davao City.

Testing of Antibacterial Activity of fish mucus
Preparation of Materials
All the glass wares that will be needed will be washed before the testing. About one hundred 4 mm Whattman Filter paper discs were be prepared using a puncher, five replicates three trials. alcohol lamp, distilled water and Nutrient Agar will be prepared as well.Sterilization of Materials
The glass wares and filter paper discs will be wrapped with paper and will be placed inside an autoclavable bag. They will then be sterilized using the autoclave of the Microbiology Laboratory Section of University of Immaculate Concepcion, Bankerohan, Davao City at 121°C, 15 psi for 15 minutes.Laminar Flow Hood and working tables will be sterilized by 70% Ethyl alcohol before performing any tests.Preparation of MediaRapid Screening will be done in order to find out if the test organism will exhibit resistance against antibiotic that is available in the market
Twenty-three grams of Nutrient agar will be prepared and mixed in 1 L of distilled water. Mixture will be boiled and stirred constantly using the magnetic stirrer for 10 minutes. It will then be autoclaved for 15 minutes at 121°C, 15 psi. Afterwards, 6 petri dishes will be filled with four mm of prepared agar. The petri dishes will be half-covered to solidify for 3 hours.Inoculation of Test OrganismThe place of the experimentation will be kept sterile again using 70% Ethyl Alcohol. Materials such as petri dishes with solidified agar, inoculating loop, cotton swab, alcohol lamp, 4 mm filter discs, powdered antibiotic streptomycin and the cultures of pathogenic microorganisms will be prepared.Through aseptic technique, the inoculating loop will be heated in direct flame to avoid contamination. The micropipette was used to scrape bacteria, was checked with equal turbidity and inoculated in the solidified agar directly in zigzag motion. Only two scrapings were done. Same process will be done in the cultured E.coli.
Treatment of fish mucus and controlTen filter paper discs were impregnated separately. 3 filter paper discs each will be soaked in the 5 mL fish mucus of African Catfish. Three filter paper discs will be soaked in each of the cultured bacteria, including the negative and positive control.Each sector was labeled with sample, was placed with three soaked filter paper discs with fish mucus each and one soaked filter paper discs with the positive control (saline) and negative control mixture were placed in the center of the sector that will be labeled with control as well. They will undergo three replicates with three trials for each of the bacteria that will be used in the experiment.

Incubation of agar plates treated with sample
The agar plates were then covered andwill be wrapped with paper. Afterwards, they were incubated for 24 hours at 37°C.

Data Gathering ProcedureAfter 24 hours, the diameter of the zone of inhibition were measured using Orion steel ruler and the unit used was millimeter (mm). Zone of inhibition of each filter paper of the control and the experimental set-up were measured.Decontamination of agar plates and cleaning of materials
After the Antibacterial Assay, all the disposable petri dishes will be wrapped in a garbage bag and will be disposed properly. Glass wares will be sterilized in the autoclave for 15 minutes 121°C at 15 psi. It will be washed and dried thoroughly. The tables will be wiped with 80% Ethyl Alcohol and the room will be sprayed with disinfectant.
Data AnalysisThe results in each treatment will be recorded by measuring the zones of growth inhibition surrounding the disc using an Orion steel ruler. Clear inhibition zones around the discs will be expressed in terms of diameter of zone of inhibition and will be measured in mm using cm scale, recorded and the average were tabulated. All data obtained will be subjected to One-way Analysis of Variance (ANOVA).

Treatment Trials Average Mean Zones of Inhibition (mm)
K.pneumoniaeE.coliDisk 1 Disk 2 Disk 3 Disk 1 Disk 2 Disk 3
African Catfish T1 11 12 12 14 14 13
(Clarias gariepinus) T2 10 11 11 14 15 14
skin mucus T3 11 10 11 14 15 15
The average zones of inhibition of K.pneumoniae and E.coli treated with the fish mucus of African Catfish (Clarias gariepinus) ranged from… These are higher than that of the negative control which ranged from … and the positive control…
ACKNOWLEDGEMENT The researcher would like to take this opportunity to recognize the time and efforts of the people who extended their kindness, guidance, assistance and help to make this project possible.

To the researcher’s parents, relatives, friends, and teachers for giving unconditional love and support morally and financially.
To the researcher’s classmates who willingly extended help in one another and for being there during each hardship.

To Ma’am Leah R. Guirigay, Research Adviser for offering her time and knowledge in checking the researcher’s work.

To the UIC, Science Resource Center staff especially Ma’am Nelyn R. Cadotdot, Registered Medical Technologist, for her assistance and for providing all the equipment and materials necessary in the conduct of the study.

Above all, the researcher with all her heart, highly-acknowledge the Almighty God for giving the researcher wisdom and safety to finish her work and for bringing this Research Project into reality.