Antimicrobial Potency Evaluation of Skin Mucus of Three Major Indian Carp Species

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       Evaluation of Antimicrobial Potency on Skin Mucus of Three Major Indian Carp Species

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Evaluation of antimicrobial potency on Skin Mucus of Three Major Indian Carp Species


Zoonotic infections caused by bacterial pathogens threaten both humans and the aquaculture industry. This problem probed search for different epidermal mucus as natural products from three Indian Major Carp species to evaluate the potency of the fish skin mucus as an effective antimicrobial against human and fish pathogenic bacteria. Here, the skin mucus of the three indigenous Indian Major Carp species got collected against pathogenic fish bacteria and human viz. Catla catla, Labeo rohita, and Cirrinus mrigala. The activities of the microbes got measured based on Zone of Inhibition (ZOI) in millimeters and comparison made with two antibiotics. The pooled mucus of fish got dug out with centrifugation and filtration to assess the MICs of various mucus extracts against a variety of bacteria by methods of agar-overlay diffusion. Comparison of the research involved the use of chloramphenicol with standard antibiotics amikacin (antibiotics) to determine fish skin mucus antimicrobial potency.

Moreover, the three selected fish species were also challenged with A. Hydrophila to evaluate the presence of any change in their mucus and antibacterial influence after exposure of bacterial. Afterward, the agar plate dilution test was used to analyze the minimum inhibitory concentration of HFM and CFM from every fish species against the selected microbial strains. The results showed variations in antibacterial effect with both the same (healthy and challenged) different three carp species. Generally, antibiotic chloramphenicol and aqueous extracts showed less bacterial action than crude mucus of all species. Therefore the results of the study were useful in identifying the possibility of using the fish skin mucus as a potential alternative of more effective and newer antibacterial components in fish and human health associated complications.

Keywords: Zone of Inhibition (ZOI), fish, antimicrobial activity, skin mucus, major Indian carps (IMCs), Healthy Fish Mucus (HFM), Challenged Fish Mucus (CFM)























The role of fish in an aquatic system is broad. Their diversity forms the largest class of marine environment, especially cold-blooded vertebrate species.  Therefore fishes are essential in the interconnection between food webs of the marine ecosystems, which harbors a variety of pathogenic microorganisms (Jayanthi et al., 2018). The production of fish globally for the consumption of human has been gradually rising. This increase has led to the growth of the aquaculture industry. However, fishes are susceptible to maladies caused by pathogenic organisms. According to Alvarez-Pellitero (2008), infectious diseases get widely spread when fish get reared in intensive and high densities aquaculture. Increased population density and limited resources provide a favorable environment for the thriving of microorganisms.

Fish get stressed in a crowded and unnatural environment characterized by a limited flow of water hence making the fish family more susceptible to diseases. Consequently, through the consumption of fish, the transmission of the bacterial pathogens from infected fish poses a severe danger to the aquaculture industry, and human health. Increased density of bacteria, viruses, and parasitic microorganisms are, therefore, critical for the development of aquaculture (Cooper and Shlaes, 2011). Infectious diseases cause complications such as edwardsiellosis, columnaris, and microbial hemorrhagic ascites in fish. Interestingly, fishes pose mucus substances on their surfaces, which aides them in locomotion, osmoregulation, body lubrication that wades off pathogens and also immunological functions. 

According to Lirio et al. (2018), the mucus layer defends the fish against the microbe invasion. Moreover, Angeles Esteban (2012) asserts that aquatic life, such as fish gets covered with an innate immunological subsystem. This subsystem offers the primary defense line in epidermal mucus form consisting of components of immune through goblet cells secretion. Furthermore, According to Nigam et al. (2017), three carp species examined showed a relatively large amount of mucus was produced by the species. However, the secretion varied among the species. These wide ranges in mucus could be due to the diversity in ecological and physiological state. Besides, there are various mucus-secreting cells in different fishes.

 The use of antibiotics to treat and prevent dynamic illness caused by the microbes in the aquaculture has intensified over the years; however, its effectiveness has gotten compromised. This cause is primarily attributed to the increased resistance of bacteria. Therefore uncontrolled use of the antimicrobial agents has immensely contributed to antimicrobial resistance of the fish microbes. Spread of the opposition from the aquatic environment to the terrestrial leads to massive treatment failure and more human being diseases. Bacterial infections proliferation among fishes can, therefore, get acquired unintentionally by a human being through ingestion (Mukherjee et al., 2016). The Indians occasionally experience emerging outbreaks of bacterial infections, for example, salmonellosis and typhoid. Hence, resulting in individuals becoming victims of nosocomial diseases contracted from medical equipment and health facilities.

Other consequences of the disorders may include a prolonged usage of antibiotics, increased hospital bills, as well as economic losses. According to Mukherjee et al. (2016), drug resistance pervasiveness is currently a threat to the efficacy of the antibiotics used in the treatment of infections. Increased antibiotics dosage functions faster on pathogenic viruses, thus, leasing to an increased risk for neurotoxicity, hepatoxicity plus, several adverse effects to humans. As a way out, the development and discovery of new-fangled alternatives for available antibiotics are presently commercially promoted worldwide. New options of drugs from compounds extracted from animals, plants, and microorganisms get considered as promising products.

 Substances produced naturally by organisms are relatively reasonable, cost-efficient, and environmentally friendly and have less serious side effects as compared to the commercially present antibiotics (Dash, Samal, and Thatoi, 2014). As a result, natural products antibacterial activities of organisms, particularly of wide-range of fish species has drawn interests as a promising source of compounds of antibiotics against fish and humans pathogens.

Mucus produced by fish species is a naturally secreted product that carries several functions for the organisms to survive. For example, it is the first line of the innate mechanism of innate defense in fishes that protects them from pathogen by preventing or killing pathogens.

            India currently is the second-largest fish producer worldwide consisting of 6.3 percent of the total world fish production. It is a vital production mainly in its coastal states, making employment to over 14 million individuals. For example, during the 2017and 2018 season, the total output of fish got projected to be over 12.61 million metric tons and, inland fishing constituting 65 percent. On the other hand, culture fisheries represented about 50 percent of the total production (Jayanthi et al., 2018). Therefore, there might be a potency of antibiotic properties in the diverse fish species produced in India aquatic ecosystem.

           The primary goal of this research was to contribute to the general awareness of the search for novel antimicrobials. Therefore, this research explored extensively on the assessment of the potency of three major Indian fish of indigenous origin. The study examined the dominant skin mucus of Carp species, including Catla catla, Labeo rohita, and Cirrinus mrigala as an effective antimicrobial against human and fish pathogenic bacteria. The study particularly sought to test the effects of antimicrobial of the mucous against human and fish pathogenic bacteria to expound on possible agents in the mucus of fish for drug discovery.

The three types of fish species are the leading Indian Carps (IMCs) which are essential for aquaculture and mostly used in polyculture. According to Mukherjee et al. (2016), all three species have distinct food habitats, thereby making it easier to get grown together. Subsequently, the difference in food habitats among the three species presents an ideal discovery of different mucus components, hence difference in action against pathogenic bacteria. For example, Catla catla is a surface feeder, Labeo rohita is adapted as a mid-water feeder whereas Cirrinus mrigala thrives best as a bottom feeder. 

Recent related studies have also indicated that skin mucus of fish plays essential roles in fish immune defense. For example, according to Lirio, De Leon, and Villafuerte (2019), fish skin mucus lies at the interface between aqueous surrounding and fish. It is a vital component of the innate immune system of fish that fights pathogens immediately they get in contact with fish skin. Angeles Esteban (2012) established that the skin mucus of fish is continuously replaced and secreted. Thus the microbes and other substances get washed when entrapped, this aid in the prevention of the stable colonization of probable infectious bacteria. Alvarez-Pellitero (2008), reported that mucus act as a storeroom for components of innate immune such as proteolytic enzymes, pentraxins, alkaline phosphatase, and immunoglobulin. The parts offer strong antimicrobial point to skin mucus.

Al-Arifa, Batool, and Hanif (2013) asserted that, the fish skin mucus layer consists of a complex fluid with a varying composition throughout the fish epithelial surface. However, extensive evidence on the epidermal mucus antimicrobial activity in freshwaters of various habitats and habits still lacks. Every fish species possess its habit and habitat, hence, surviving in the different aquatic surrounding, consumes different food types. This distinct adaptation of each species may influence the quantity of secreted mucus and its components between or within species and can be significant in offering different immune parts and response. This study presents the variation in fish mucus secretion by the three Indian carp species concerning their habits and habitats.

The objectives of the study were, therefore, guide in the evaluation of the effectiveness of fish skin mucus that could constitute compounds of antibacterial. Hence, the findings could get utilized as an alternative to antibiotics that can get employed in both aquaculture and for humans. The study, therefore, presents the following research objectives; to evaluate the effect of antibacterial of skin mucus among the three IMCs viz. Catla catla, Labeo rohita, and Cirrinus mrigala, secondly, to determine any changes in secretion of mucus and its antibacterial influence after bacterial challenge, and to examine both challenged and healthy fish skin mucus among the three species under study and assess the MICs against all chosen microbial strain for investigations.          

Materials and Methods

Fish collection

Experimental fish constituted healthy yearlings from the three species which will get obtained from the fish farms and transported to the Department of Zoology, University of Kurukshetra. The daily recording of fish health got conducted as they fed. Again, observations on dead fish also noted. Measurements of changes in average length and weight for each species were recorded daily during the acclimation period.

Collection of Mucus: 

The fish species got acclimated for seven days. Mucus was then collected at regular intervals by careful scraping from the dorsal body surface using a sterile spatula. Mucus collection in the fish ventral area was avoided to eliminate urogenital and intestinal contamination (Al-Arifa, Batool, and Hanif, 2013). After that, the samples of mucus got frozen in ice at 0 degrees Celsius. This low temperature prevents bacterial growth (Al-Arifa, Batool, and Hanif, 2013).

Test Microorganisms

Both gram-negative and positive bacterial strains included human pathogens such as Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, which are gram-negative. The gram-positive strains of bacteria consisted of Bacillus cereus, Staphylococcus epidermidis, Staphylococcus aureus plus one Gram-negative bacterial strain of the fish pathogen, Aeromonas hydrophila. The bacterial strains got procured from a recognized institution of microbial technology in India. The microbial strains were cultured regarding microbiological safety conditions and procedures (Eder et al., 2009).  

Challenge Test

Ten fishes from every species got challenged with pathogenic bacteria of fish using A. Hydrophila maintained and cultured in the media of agar. For the first immersion, the mucus got collected on third and seventh days in a suspension of approximated107 CFU ml-1, and significant changes recorded regarding the amount of mucus secreted both by challenged and healthy fish. After that, second immersion got performed in an immersion of about109 CFU ml-1 for fifteen days and mucus collection conducted after a one-week interval through the method of a skin scraping. 


Preparations of Mucus Extracts

Extract of thick mucus got prepared for both challenged and healthy fish species through centrifuging raw mucus at a revolution of 5000 per minute for 300 seconds. On the other hand, aqueous mucus, a similar amount of sterilized physiological saline was mixed with thick mucus and centrifuged for the same revolutions and time.  

Data Analysis

All the samples were analyzed in triplicate using statistical analysis performed by T-test as well as one-way ANOVA at significant level 0.05. The test used SPSS Version 11.5 for Windows among all the selected species and antibiotics. Besides, the student’s test was used to evaluate the considerable difference between ZOIs gotten from extracts of CFM and HFM of each selected species of fish against various pathogenic strains performed under research. Finally, Statistical significance got settled at P\0.05 probability value.