Evaluation of Antimicrobial Potency on Skin Mucus of Three Major Indian Carp Species
I thank the Almighty God for his guidance and protection throughout this study. I extend my gratitude to my family and friends for the financial and moral support. I would also like to acknowledge my supervisor for the guidance and support throughout the study.
Zoonotic infections resulted from bacterial pathogens threatens both humans and the aquaculture industry. This outbreak 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. 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 were measured based on Zone of Inhibition (ZOI) in millimeters to assess the MICs of various mucus extracts against a variety of bacteria. The results were compared with standard antibiotics amikacin and chloramphenicol 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. Therefore the results of the study were useful in identifying the possibility of using the fish skin mucus as an alternative to antibiotics 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), Colony Forming Units (CFU)
Evaluation of antimicrobial potency on Skin Mucus of Three Major Indian Carp Species
Fishes play a significant role in the aquatic environment. Their diversity forms the largest class of water habitat, especially cold-blooded vertebrate species. They provide interconnection between food webs of the water ecosystems, which harbors a variety of pathogenic micro-organisms (Ellis 2001). Moreover, According to Alvarez-Pellitero (2008), infectious diseases are 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 pathogens.
Aquatic organisms like 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 parasites 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. 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 get adapted as a mid-water feeder, and 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.
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 is compromised. This nature gets 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 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 micro-organisms 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.
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 an 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.
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
Experimental fish constituted healthy yearlings from the three species which were obtained from the fish farms and transported to the Department of Zoology, University of Kurukshetra. Hygienic environment and the water quality were observed to ensure sustainability.
The daily recording of the fish health got conducted, and observations on dead fish noted.
Maintenance of fish involved checking of the health of fish, the quality of water as well as feeding the fish with proper food during acclimation.
Collection of Mucus:
The fish species then 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 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 of Micro-organisms
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, Institute of Microbial Technology (IMTECH), Chandigarh. The microbial strains were cultured regarding microbiological safety conditions and procedures (Eder et al., 2009). The growth of all microbial strains took place at 37°C in nutrient broth. Afterward, CFU of every strain of bacteria got calculated by the method of colony-forming units (CFU).
Ten fishes from every species then 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 performed by 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 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. The levels of protein in mucus got determined by the use of Lowry et al. (1951) for different fish species.
Agar Well Diffusion Assay
Agar well diffusion method was used to assay extracts of every tested fish species on the chosen bacterial strains of both HFM and CFM. Seeding involved 15ml nutrient agar medium contained in Petri-plates with a one day culture of averagely 107 CFU ml-1 strains of bacteria. The diameter of six to seven (6 to 7) mm of well was then pierced aseptically with a cork borer. The cork borer got sterilized at first, and extracts of antibiotics and mucus added. Amikacin and chloramphenicol (positive extracts) got used for extracts of aqueous and crude in a concentration of 20µgml-1 and 40µgml-1, respectively. Incubation period took one day (24 hours) at a temperature of 37°C. The bacterial effect evaluation got measured by using the ZOI diameter (in mm) established around the well (Eder et al., 2015). The results of the antibacterial activity got compared with the two standard positive control drugs. The negative control comprised of 0.85% NaCl, which got applied in the assay of aqueous mucus antibacterial.
Agar Dilutions Assay
The CFM and MIC of HFM of each species of fish got determined using agar plate dilution assay against every chosen microbial strain. All skin mucus of fish made by dilution of the mucous extract in sterile distilled water. The extract of desired dilutions of fish mucus got prepared and transferred in various wells. The different fish MICs get determined by the measurement of ZOIs to varying levels against different bacteria.
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.