Anita Bhatnagar, Sunil Kumari, Anil Kumar Tyor
Department of Zoology, Kurukshetra University, Kurukshetra, Haryana, 136119, India
Keywords:
Catfish
Epidermal mucus
Pathogen
Zone of inhibition
Bactericidal effect
A B S T R A C T
This research was directed to understand the bactericidal effect of epidermal mucus of two Asian catfish species viz.Clarias batrachus and Heteropneustes fossilis.Epidermal mucus extracts (raw and diluted) of both catfish species were tested against several Gram negative (Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumonia,A.hydrophila) and Gram positive bacterial strains (Bacillius cereus, Staphylococcus aureus, S.epidermidis) and antibacterial results were also compared with two standard antibiotics viz.amikacin and chloramphenicol used as positive control.An A.hydrophila challenge experiment was also performed on all selected test fish species to examine the change in the amount of mucus production and its bactericidal impact..Both epidermal mucus extracts (raw and diluted) of all selected normal and bacterial challenged test objects showed potent bactericidal effect against all pathogenic bacterial strains taken under study.However, former was more effective than later.Also raw epidermal mucus extracts of both normal and bacterial challenged catfish species exhibited signi ficantly higher ZOI values against all selected microbial strains than diluted mucus extracts and antibiotic chloramphenicol.Hence, these outcomes have clearly revealed that this cost effective natural product acquired from fishes is the key component of their defensive system.Therefore, it could be utilized as a novel ‘antimicrobial’ in human as well as veterinary sector for combating against several bacterial diseases.
Many infectious diseases can be spread directly/indirectly by zoonosis.Term zoonosis is used for transmission of diseases from animals to humans and vice versa through various routes (Stephen et al.,2004).Pathogenic bacteria are the main causative agents of infectious diseases in wide host range including humans and fish species.In aquaculture industry, several bacterial diseases in fish species like edwardsiellosis (Park et al., 2012), columnaris (Declercq et al., 2013),aeromonas septicemia (Igbinosa et al., 2018) and many others, not only affect the animal health but also affect the economics of aquaculture.Fish consumption as food by humans increasing day by day which unfortunately, may also increases the chance of zoonotic diseases.Most of fish species are the long term carrier of infectious pathogens likeAeromonas hydrophila,Vibrio cholerae,Erysipelotrix rhusiopathiae,Campylobacter jejuni,C.coli,C.upsaliensis, Mycobacterium marinum,Streptococcus iniaeetc.before clinical diseases is detected (Crane & Hyatt, 2011).Consumption of such fishes might cause the serious health problems in human.Studies had revealed thatStreptococcus iniaecarried by many fishes might cause arthritis, endocarditis in infected person (Brusaferro et al., 2015).Moreover,Aeromonas hydrophila, Vibrio cholerae,Campylobactersp.infections may be responsible for vomiting and diarrhea (Gauthier, 2015).Such diseases mainly target the immunocomprimsed people and results in serious diseases outbreaks.India is seeing flare-ups of rising bacterial contaminations, for example, typhoid,salmonellosis, pneumonia and many more (Chatterjee et al., 2017;Yadav & Awasthi, 2016).Diseases by bacterial pathogens brought about the expanded number of hospitalized natives who become disastrous casualties of nosocomial contaminations through medical equipments and surgical procedures used in hospitals which further lead to prolonged use of antibiotic and economic losses (Peleg & Hooper, 2010).
Inappropriate use of antibiotics in human and animals and extensive use of these as growth promoter in animal feed etc.leads to acquired resistance in microorganism against commercially available antibiotics(Paterson, 2006).The growing resistance in microorganisms is responsible for the emergence and re-emergences of several dangerous diseases and becomes major threat worldwide (Botreau et al., 2007; Fauci &Morens, 2012).Such critical situations strained new antibiotic discovery efforts from natural and synthetic resources.Natural products become the keen interest area for the development of new antibiotic research because of their least toxicity and cost effectiveness over synthetic drugs.Among animals based products, a fish by product, skin mucus emerges out as an excellent resource for antimicrobial components(Kuppulakshmi et al., 2008).Many previous reports (Alvarez-Pellitero,2008; Ellis, 2001; Hedmon, 2018) suggest that fish epidermal mucus is a dynamic layer which continuously secreted and sloughing off; carrying numerous functions such as reduces body friction against water current,help in osmo-regulion, respiration, feeding, excretion, nest building and most importantly providing protection against infectious pathogens.It is secreted by goblet cells, acts as barrier between fish and infectious microorganisms in their surroundings and protects fishes from pathogens by preventing their attachment to the surface of fishes or direct killing them with the help of antimicrobials (Dash et al., 2018).To this regard,Lemaitre et al.(1996) revealed thatC.carpiobecame more susceptible to diseases after removal of skin mucus coat.Also, loss of mucus leads to high mortality rate in bacterial challenged fishes (Guardiola et al.,2014).Mucus is the reservoir of biochemically diverse mixtures such as antimicrobial peptides (AMPs), fatty acids, carbohydrates, lysozymes,proteolytic enzymes, alkaline phosphatase, immunoglobulin etc.All these compounds collectively provide high antibacterial properties to the skin mucus (Alvarez-Pellitero, 2008).Consistently, several other researchers (Fuochi et al., 2017; Kumari et al., 2011; Lirio et al., 2018;Tyor & Kumari, 2016) had been also uncovered the presences of bactericidal effect in skin mucus of many fishes.Although many studies revealed the antimicrobial potential of fish skin mucus but very limited experimental evidence is available regarding these phenomena in catfish species.Catfish species are scale less and line in muddy environment; hence secretion of mucus could to be higher than other scaly fishes to combat adverse environment conditions (Nigam et al., 2012).Hence,there is possibility of getting variety of immunological response and related antimicrobial components which help them to survive in adverse environment.Therefore, present studies were intended to find out the bactericidal effect of skin mucus of Asian catfish species viz.Clarias batrachusandHeteropneustes fossilisvariation in its mucus secretion and bactericidal effect afterA.hydrophilaexposure and MICs of epidermal mucus extracts of both normal andA.hydrophilachallengedC.batrachusandH.fossilisagainst each experimentally selected bacterial strains.
2.1.Specimen collection and maintenance
Live adult specimen of Asian catfish speciesC.batrachus(average length (total length) 24.30 ± 0.48 cm and average weight 523 ± 66.10 g) andH.fossilis(average length (total length) 27.50 ±1.19 cm and average weight 570 ±50.38 g) were obtained from fish ponds situated in Karnal, Kurukshetra and Sonipat districts of Haryana.Captured fishes were transferred to Aquaculture Research Unit of Kurukshetra University, Kurukshetra.Prior to mucus collection, fishes were acclimatized to laboratory conditions for 10 days in glass fibre reinforced Plastics (F.R.P) tank (1500 L capacity), equipped with low pressure aerators and pumps.Almost one third water in the storage chamber/F.R.P tank was refilled on third day to maintain the water quality and hygiene conditions.Fishes were fed daily with freezed-dried blood worms and tubifex worms at the rate of 4% of their body weight, two times a day.
2.2.Mucus extraction
After 24 h starvation period, prior to mucus extraction, test organisms were cleaned with 4% potassium permagnate (KMnO4) solution to remove contaminants and debris associated with epidermis.Mucus was pooled from 10 specimens of each experimental animal.Scrapping of mucus was done by moving aseptic plastic spatula head towards tail, on dorsal-lateral surface of fishes.Mucus scrapping was avoided from ventral surface of fishes as this might be contaminated with anal and intestinal excreta.Mucus collected till it was discharged by fishes and stored in sterile plastic tubes.Then mucus samples were stored in refrigerator at 4 ℃ for further use.After collection of mucus test subjects were resealed to recovery tank.
2.3.Microbial strains
All selected Gram negative and Gram positive bacterial strains i.e.Pseudomonas aeruginosa, Escherichia coli,Klebsiella pneumonia, Bacillus cereus,Staphylococcus.Aureus, S.epidermidis(Human pathogenic bactrerial strains) andAeromonas hydrophila( fish pathogenic bacteria) were obtained from Institute of Microbial technology (IMTECH), Chandigarh,India.
Liquid nutrient broth media composed of 0.3% beef extract, 0.5% peptone, 0.5% NaCl, distilled water, pH 6.8 at 28 ℃ were used to develop the selected microbial strains.Then, Colony Forming Unit(CFU) method was used to calculate the CFU of each bacterial strain(Goldman & Green, 2008).
2.4.Mucus extraction preparations
Obtained raw mucus from both normal and bacterial challenged fish species was centrifuged at 5000 RPM (revolutions per minute) for 10 min and named as raw mucus extract.Diluted mucus extracts were prepared by centrifuging equal amount of physiological saline solution(0.85% NaCl) and raw mucus at 5000 RPM for 10 min.Supernatants of both raw and diluted extracts were filtered through whatman filter paper separately and stored at 0 ℃ until use.
2.5.Bacterial challenge test
In test analyze, 15 representatives of each fish were challenged withA.hydrophila.First drenching of all test subjects were done in approximately 107CFU ml-1A.hydrophilasuspension.Extraction of mucus was done after one week but no huge change was reported in amount of mucus secretion by A.hydrophilla exposed fishes.For that reason, second immersion of approximately 109CFU ml-1 was accomplished for 10 days and extraction of mucus was done on the fifth day.Obtained bacterial challenged fish mucus (BCFM) samples were analyzed for protein content and antibacterial activity and compared with normal fish mucus(NFM) samples of both fish species to check the difference, if any, in the constituents of mucus after inoculation of A.hydrophilla.
2.6.Protein estimation assay
Biuret test (Smith et al., 1985) and Lowry assay (Lowry et al., 1951)was adopted for qualitative and quantitative analysis of protein content in mucus of both normal and bacterial challenged Asian catfishes.
2.7.Bactericidal activity assay
Agar well diffusion method (Valgas et al., 2007) was employed to evaluate the bactericidal effect of NFM and BCFM extracts of every test fish species on the chosen bacterial strains.The standardized diluted inoculum of each microbial strain (approximately 107CFU/mL) was spread on nutrient agar plates with sterile cotton swabs.1liter (l) of nutrient agar media composed of 0.5% peptone and 0.3% beef extract,0.5% NaCl, 1.5% agar with of pH 7.3.After 1 h, around 7–8 mm diameter, wells were punctured with the help of a sterile cork borer in each of these plates and labeled.Then, labeled wells were filled with 100 μL/mL of mucus extracts (raw and diluted) from all selected catfish species and antibiotics.Two standard antibiotics viz.amikacin and chloramphenicol were used as positive control in the concentration of 40 μg ml-1(raw mucus extracts) and 20 μg ml-1(diluted mucus extracts).0.85% NaCl (physiological saline solution) was utilized as negative control during diluted mucus antibacterial measure.For proper diffusion of mucus extracts and antibiotics into agar media, plates were kept at room temperature for 2 h and then were incubated for 20–24 h at 37 ℃, under aerobic conditions.Assessment of bactericidal effect was done by measuring the ZOI (mm) on every side of the well as described by Jorgensen and Turnidge (2015).ZOI values shown by mucus extracts against all pathogenic strains were compared with standard antibiotics.
2.8.Minimum inhibitory concentration (MIC) assay
MICs of NFM and BCFM extracts of bothC.batrachusandH.fossilisagainst all studied pathogens were determined by agar plate dilution assay (National Committee for Clinical Laboratory Standards (NCCLS),2000).
Different concentrations viz.100 μL/mL, 50 μL/mL, 25 μL/mL, 12.5 μL/mL and 6.5 μL/mL dilutions of individual fish skin mucus extracts were made by adding sterile distilled water into raw mucus extracts.ZOI was calculated for each concentration.Distinct minimum concentrations ZOIs determine the MICs of different mucus extracts against all selected microbial strains used for studies.
2.9.Data analysis
All the samples were analyzed in triplets.Mean ±S.E was used for each parameter.Data generated from bactericidal assay was evaluated using One way - analysis of variance (ANOVA) followed by Duncan’s multiple range test (Duncan, 1955) to determine the significant variation among the mucus extracts (raw and diluted) of all experimental fishes and antibiotics against all selected pathogens.Difference between antibacterial effect obtained from NFM and BCFM extracts of bothH.fossilisandC.batrachusagainst all selected microbial strains was determined by using Student’t’ test.All statistics were done by utilizing SPSS Version 11.5 for Windows.Probability value ofP ＜0.05 was considered statistically significant.
Both selected catfish species i.e.C.batrachusandH.fossilissecreted variable amount of mucus which was also varied within and in between the fish species.As depicted in Table 1 mucus secretion was higher in amount in both healthy and challengedC.batrachusthanH.fossilis.Beside this, we had also noticed that after challenge experiment, mucus secretion increased in selected catfish species.
3.1.Protein assay
Qualitative assay (Biuret test) had confirmed the presence of protein in all selected experimental fishes.Mean ±S.E values of protein concentrations in Normal fish mucus (NFM) and bacterial challenged fish mucus (BCFM) of all selected Asian catfishes presented in Table 1.Protein content was found higher inC.batrachusthanH.fossilisin both states (normal and bacterial challenged).Moreover, our studies also revealed that mucus of all chosen bacterial challenged catfish speciesexhibited significantly (P ＜0.05) higher protein concentration than the mucus of all selected normal/healthy catfish species.However, the increase in protein content was 99 mg/mL inC.batrachusand 91 mg/mL inH.fossilis.
Table 1Amount of mucus (ml) and protein content (mg/mL) in all selected normal and bacterial challenged catfish species.
3.2.Bactericidal assay
Results of agar well diffusion assay demonstrated that, all eight extracts (one raw and one diluted of NFM and BCFM of individual fish species) and two antibiotics viz.amikacin and chloramphenicol effectively able to inhibits the growth of all selected microbial strains.Variation in zone of inhibition (ZOI) values, within and between all the selected fishes against all selected bacteria was also observed in present studies.Raw extracts of NFM (Table 2) and BCFM (Table 4) of both selected catfish species showed higher antibacterial effect than diluted extracts of NFM (Table 3) and BCFM (Table 5).Also difference in mean±S.E values of bactericidal effect of all extracts (raw NFM and BCFM) of all test subjects against all microbial strains were considerably greater(P ＜0.05) than antibiotic chloramphenicol and substantially (P ＜0.05)lower than antibiotic amikacin except raw BCFM ofC.batrachuswhich exhibited higher ZOI (35.33 ± 0.54 mm) than amikacin (34.90 ± 0.10 mm) as shown in Table 4.However, diluted extracts of NFM and BCFM of both selected catfish species were also found much higher (P ＜0.05)when contrasted with antibiotic chloramphnenicol except few extracts such as diluted extracts of NFM ofH.fossilis(7.66 ±0.27 mm) &C.batrachus(8.26 ±0.32 mm) and BCFM ofC.BatrachusagainstS.aureusshowed lower bactericidal effect than chloramphenicol (10.07±0.06 mm in NFM containing agar nutrient plates and 10.28 ±0.20 mm in BCFM containing agar nutrient plates) as depicted in Tables 3 and 5 respectively.All diluted mucus extracts of all experimental healthy and challenged fish species against all bacterial pathogens demonstrated lower inhibitory impact when compared with antibiotic amikacin.However, lower ZOIs shown by diluted mucus extracts than raw mucus extracts in all healthy and challenged test objects were expected as the volume of mucus in diluted extracts was half than that of raw extracts.But surprisingly, the pattern of lower antibacterial activity against all selected microbial strains was not similar to raw extracts i.e.raw NFM ofH.fossilisexhibited maximum bactericidal effect againstB.cereus(Table 2) but diluted NFM of same fish was most effective againstK.pneumonia(Table 3), raw mucus of challengedH.fossilisdisplayed least bactericidal impact againstK.pneumoniae(Table 4), but diluted mucus extract of the same was least effective againstS.aureus(Table 5).
Table 2Bactericidal effect in term of ZOIs (mm) exhibited by raw NFM extracts of all selected catfish species, and positive control (antibiotics) against all selected pathogenic bacterial strains.
Table 3Bactericidal effect in term of ZOIs (mm) exhibited by diluted NFM extracts of all selected catfish species, and positive control (antibiotics) against all selected pathogenic microbial strain.
Table 4Bactericidal effect in term of ZOIs (mm) exhibited raw BCFM extracts of all selected fishes, and positive control (two antibiotics) against selected human and fish pathogenic microbial strain.
Table 5Bactericidal effect in term of ZOIs (mm) exhibited by diluted BCFM extracts of all selected catfish species, and positive control (two antibiotics) against selected microbial strains.
3.3.Bactericidal effect of raw and diluted NFM extracts of all experimental catfishes
Photographic presentation of nutrient agar plates depicting ZOIs of raw and diluted mucus extracts of all experimental catfish species and positive control (amikacin and chloramphenicol) against all selected pathogenic microbes have been given in Fig.1(a–g) and Fig.2(a –g)respectively.Antibacterial activity estimation of raw and diluted mucus extracts were in the scope of 24.33 ± 0.27 to 34.00 ± 0.40 mm forC.batrachus& 24.33 ± 0.27 for mm to 31.33 ± 0.21 mm forH.fossilis(Table 2) and 08.26 ± 0.32 mm to 15.03 ± 0.07 mm forC.batrachus&07.66 ± to 15.00 ± 0.23 mm forH.fossilis(Table 3) individually.
Fig.1.Photographic agar plates images showing the ZOIs exhibited by raw NFM extracts of all experimental fishes against all selected pathogenic bacterial strains,(a) P.aeruginosa, (b) E.coli, (c) K.pneumoniae, (d) B.cereus, (e) S.aureus, (f) S.epidermidis and (g) A.hydrophila.
Fig.2.Photographic agar plates images showing the ZOIs exhibited by diluted NFM extracts of all experimental fishes against all selected pathogenic bacterial strains, (a) P.aeruginosa, (b) E.coli, (c)K.pneumoniae, (d) B.cereus, (e) S.aureus, (f) S.epidermidis and (g) A.hydrophila.
As shown in Table 2 and Fig.1 (a –g), for raw extracts, among all pathogenic bacteria studied, NFM ofC.batrachusandH.fossilisexhibited higher inhibitory effect againstB.cereusby showing ZOIs of 34.00 ±0.40 mm and 31.33 ±0.21 mm respectively.These ZOIs values were found significantly (P ＜0.05) higher when compared with antibiotic chloramphenicol (25.43 ±0.43 mm).On the other hand, as shown in Fig.1(f) and (e), and mucus ofC.batrachusandH.fossilisexhibited lesser inhibitory effect againstS.epidermidisandS.aureusseparately.
In case of diluted mucus extracts, as depicted in Table 3, Mucus ofC.batrachus(15.03 ±0.07 mm) exhibited maximum inhibitory effect againstE.colias depicted in Fig.2 (b).NFM ofH.fossilisshowed highest ZOI of 15.00 ±0.23 mm againstK.pneumoniaeamong all selected microbial strains as shown in Fig.2 (c).At the same time, both catfish species showed minimum bactericidal effect againstS.aureusas shown in Fig.2 (e).
3.4.Bactericidal effect of raw and diluted BCFM extracts of all experimental catfishes
Photographic pictures of agar nutrient plates demonstrating the bactericidal effect (ZOIs) of raw and diluted BCFM extracts of all chosen test fish species and positive control (amikacin and chloramphenicol)against all bacterial strains studied have been portrayed in Fig.3(a–g)and Fig.4(a–g) respectively.
Antibacterial impact of raw and diluted BCFM of all selected catfish species and two antibiotics against every single microbial strain were ranges from 17.22 ± 0.17 mm to 34.90 ± 0.10 mm (Table 4) and 07.85±0.12 to 25.60 ±0.05 mm (Table 5) individually.
As depicted in Table 4, for raw extracts, mucus ofH.fossilis(30.66 ±0.16 mm) was found to be more effective againstA.hydrophila, also depicted in Fig.3 (g).Among all bacterial strains, As depicted in Fig.3(d), mucus obtained fromC.batrachus(35.33 ±0.54 mm) displayed most extreme antibacterial impact againstB.cereuswhich was also found additionally essentially higher (P ＜0.05), when compared to antibiotic amikacin (34.90 ±0.10 mm).As shown in Fig.3(e), both catfish species exhibited least bactericidal effect againstS.aureus.
Fig.3.Photographic agar plates images showing the ZOIs by raw BCFM extracts of all experimental fishes against all selected pathogenic bacterial strains, a)P.aeruginosa, (b) E.coli, (c)K.pneumoniae, (d) B.cereus, (e) S.aureus, (f) S.epidermidis and (g) A.hydrophila.
In case of diluted mucus extracts (Table 5) among all microbial strains studied, like raw BCFM, as depicted in Fig.4 (g), diluted mucus ofC.batrachus(16.40 ±0.24 mm) was observed to be best againstA.hydrophilaamong all microbial strains taken under study.Whereas,BCFM ofH.fossilismost successfully repressed theB.cereusgrowth by showing ZOI of 15.13 ±0.72 mm as seen in Fig.4 (d).As shown in Fig.4(e), both catfish species indicated least ZOIs againstS.aureuswhich were also significantly (P ＜0.05) lower than positive controls.
3.5.Comparison between raw NFM and BCFM extracts of all selected test subjects against all microbes studied
ForC.batrachus, as depicted in Fig.5 (a), bactericidal effect shown by BCFM extract was found to be more effective than NFM against all bacteria taken under study.But the difference in ZOI (mean ±S.E)values of NFM and BCFM extracts were found to be statistically consequential (P ＜0.5) against five microbial strains viz.P.areuginosa, E.coli,K.pneumoniae,S.epidermidisandA.hydrophila.ForB.cereusandS.aureus, no significant difference was seen in mean ±S.E values of NFM and BCFM extracts.In case ofH.fossilisas appeared in Fig.5 (b), Mean ±S.E.values of antibacterial activity of NFM was found to be higher than BCFM againstP.aeruginosa,E.coli,B.cereusandS.epidermidis.Reverse was true in remaining selected pathogenic microbes.But the remarkable(P ＜0.05) difference have been seen among the ZOI values of NFM and BCFM extracts againstK.pneumoniae,S.aureusandA.hydrophila.Whereas, there were no significant difference among NFM and BCFM extracts for remaining microbial strains.
Fig.5.Comparison of bactericidal effect of raw NFM and BCFM extracts of experimental fishes against all selected bacterial strains, (a) C.batrachus, (b) H.fossilis.
3.6.Comparison between inhibitory effect displayed by diluted NFM and BCFM extracts of chosen test subjects against all selected microbial strains
In case ofC.batrachusas given in Fig.6 (a), BCFM exhibited higher bactericidal effect than NFM.The difference in mean ±S.E values of ZOI of NFM and BCFM extracts were found to be notably higher (P ＜0.05)against all selected microbial strains.As Fig.6 (b) showed, NFM extract ofH.fossiliswas higher than BCFM extracts againstK.pneumoniaeandA.hydrophila.Opposite to this, BCFM showed higher inhibitory effect than NFM extracts against remaining bacteria.But the difference in antibacterial effect of NFM and BCFM extract was found to be considerably high (P ＜0.05), only againstB.cereus.In case of other selected pathogens, this difference was statistically insignificant.
Fig.6.Comparison of bactericidal effect of diluted NFM and BCFM extracts of all experimental fishes against all selected bacterial strains, (a) C.batrachus,(b) H.fossilis.
3.7.MIC assay
Different MIC values have been exhibited by different mucus extracts of all chosen fishes against different pathogenic strains taken under study.As shown in Table 6, NFM and BCFM extracts of both catfish species demonstrated MICs at 50 μL/mL concentration againstP.aeruginosa,A.hydrophila, S.aureusandS.epidermidis.Whereas, 25 μL/mL concentration was sufficient to stop the visible growth ofE.coli, K.pneumoniae, andB.cereus.
Table 6MIC and ZOI shown by NFM and BCFM of selected catfish species against all microbial strain.
Results of current studies have confirmed that both selected Asian catfishes i.e.C.batrachusandH.fossilissecreted enormous mucus and amount of its secretion changed within and between the fish species.These outcomes are like the investigations conducted for various catfishes likeRita ritaandChanna punctata(Nigam et al., 2012).Scale less fish species secreted more substantial amount of mucus than scaled fish(Dash et al., 2018).Diverse environmental conditions and furthermore presence of different mucus secreting cells in epidermis of different fishes might be responsible for variations in mucus secretion.Present studies had uncovered the progressively increase in the volume of mucus secretion in all selected fish species, afterA.hydrophilaexposure (challenge test).Undesirable pH/temperature, increased level of ammonia,nitrates, carbon dioxide, bacterial load etc., credited to influence the volume of mucus discharge in fish species (Jung et al., 2012).Our results are in significant concurrence with these examinations.Increased mucus secretion might be a marker high stress level and initiation of natural resistant framework against bacterial assault.Bacterial contaminated Hag fish secreted higher volume of mucus than normal fish which additionally bolsters our finding that increased bacterial load upgraded mucus production in bacterial challenged fish species (Jones, 2001;Subramanian et al., 2008).Similar results were observed by Holm et al.(2015) inSalmo.salarafter Sea lice infection, which supporting our results.According to previous studies by number of authors (Kumari et al., 2011; Lirio et al., 2018; Reverter et al., 2018) demonstrated that,being at the interface between fish and its surrounding opportunistic pathogens, skin mucus acts as primary immunological defensive barrier against the entry of pathogens.Slippery nature of mucus helps in preventing the adherence and stable colonization of pathogen (Rakers et al.,2013).Alongside this, mucus is also the repository of bactericidal agents which carries inborn antibacterial capacity and works via different mechanisms to kill pathogenic microbes (Esteban, 2012).Many previous reports (Arulvasu et al., 2012; Dhotre et al., 2013; Wei et al., 2010) had concurred the proteinaceous nature of epidermal mucus in many fish species, for e.g.Channa straitus, C.punctatus, C.gachua, Cypernius carpioandArius dussmieri.Protein as major component in epidermal mucus ofC.batrachusandOreochromis.mossambicushaving concentrations of 0.022 mg/mL and 0.034 mg/mL respectively (Elavarasi et al., 2013).Similarly, protein concentration in raw mucus extracts of different fish species likeC.micropeletes,C.striatus, Oreochromisnitolituswas ranging from 432.90 ± 28.20 μg/mL to 579.90 ± 32.30 μg/mL (Rao et al., 2015).Furthermore, protein also reported as major component with concentration of 0.66 ±0.04 mg/mL in the mucus extract ofPeriophthalmodon schlosseri(Mahadevan et al., 2019).Our outcomes are likewise in concurrence with above investigations.In present studies, total protein estimation of all selected fish species was found to be in the range of 178.91 ± 1.20 mg/mL to 263.00 ± 2.35 mg/mL.Beside different selected fish species, different experimental set up high protein concentration could be also related with mucosal intrinsic resistance in light of the fact that the majority of bactericidal constituents found in skin mucus, are likewise proteinous in nature.Our outcomes additionally uncovered that protein content not just change within the species(among NFM & BCFM extracts) but also between the fish species in both extracts (NFM & BCFM).Moreover, protein concentration of BCFM extracts of all chosen test subjects was higher than NFM.Similarly, mucus obtained from hag fish (during bacterial stress) showed higher protein content than healthy skin mucus Subramanian et al.(2008).This enhanced mucus secretion and protein concentration after bacterial stress might be due to activation of bioactive components in mucus for providing protection against problematic conditions.Present studies on antibacterial activity exhibited by selected catfish species also suggest the existence of bactericidal components in fish epidermal mucus.Many previous reports by numerous researchers also indicated toward the presence of potent bactericidal activity in epidermal mucus of wide range of fish species (Balasubramanian et al., 2012; Mahadevan et al.,2019; Nurtamin et al., 2016).Raw mucus ofC.straitusexhibited inhibitory effect against fish pathogenA.hydrophila(8 mm) and no antibacterial impact was observed against human pathogens viz.E.coliandK.pneumonia(Wei et al., 2010).However, in present findings, raw as well as diluted extracts (NFM and BCFM) of both selected catfish species showed strong microcidal effect (ZOIs) against all microbial strains studied by Wei et al.(2010).Raw mucus ofCynoglossus arelandArius caelatusexhibited ZOIs of 5 mm and 4 mm againstS.aureusrespectively.But no antibacterial activity was reported in their diluted mucus extract (Bragadeeswaran & Thangaraj, 2011).In our results, also lesser ZOIs were observed in raw and diluted extracts (NFM and BCFM)of all selected fish species againstS.aureusbut found higher when compared with above said studies.Mucus extracts of male and femaleRutilus frisiishowed mean ± S.E ZOIs of 23.2 ± 1.4 mm & 24.0 ± 1.8 mm againstP.aureginosaand 24.3 ± 1.9 & 25.4 ± 1.5 mm againstE.colirespectively (Adel et al., 2018).Our outcomes are in agreement with above said studies.However, our results underline that ZOI values shown by raw and diluted extracts of NFM and BCFM of all selected catfish species vary among different fish species even for same bacteria.Different environmental conditions, different niches, different feeding habits, different stress level, different developments stages, different susceptibility of different fish species against different/same pathogen/s, different expression of antimicrobial constituents may be the cause of variations in ZOI values.Furthermore, different type of solvent used in mucus extracts preparation could also influence the antibacterial effect of mucus extracts.
Bactericidal activity in diluted mucus extracts of wide range of fish species, but in their further studies, they didn’t notice inhibitory effect in diluted mucus extracts of several fish species, for example,Salvelinus Peaks,S.fontinalis, C.carpio, Menticirrhus aeglefinus,M.Glutinosa, andM.saxatalis(Subramanian et al., 2007).Different incubation temperature or pH conditions during bactericidal test may be the explanations for the nonappearance of antibacterial action.However, in present investigations, diluted extracts of NFM and BCFM of all chosen fish species showed bactericidal impact against all the selected pathogenic strains taken under study.Investigations on antibacterial impact of diluted mucus extracts of two catfish species i.e.Arius maculatusandMystus guliorevealed that,out of ten selected pathogenic bacterial strainsA.maculatusshowed effective inhibitory effect againstE.coli(8 mm) andK.pneumonia(6.5 nm) whereasM.gulio(11 mm) able to inhibit the growth of onlyP.areuginosa.Very low antibacterial activity up to 2–3 mm was observed againstS.aureusby both of these fish species(Anbuchezhian et al., 2011).However, in present outcomes, ZOIs shown by diluted extracts of NFM and BCFM of both catfish species was found higher against all these microbial strains.Antibacterial activity of diluted mucus ofC.batrachusagainstE.coli(18 mm),A.hydrophila(22 mm) andP.aeruginosa(25 mm) had been studied previously by Loganathan et al.(2011).Whereas, in present study raw and diluted mucus extracts of both (NFM and BCFM) ofC.batrachusexhibited lower ZOIs against these bacteria.All these studies strongly support our results.
Contradictory to our results, no antibacterial activity was noticed in raw and diluted mucus extracts of several fish species such asConger conger,Trisoptereus luscus,Pollachius pollachius,P.viren,Gadus morthua,T.trachurus, Scomber scomber, Labrus bergylta, L.piscatorius, Scophthalmus rhombus, P.flesus, P.platessa,Solea solea, O.niloticusand bigrid catfish against human pathogenic bacteria and fish pathogenic bacterial strains (Hellio et al., 2002; Rao et al., 2015).
In present findings, after bacterial exposure, changes in bactericidal impact (either increased or decreased) had been observed.Raj et al.(2011) also noticed the increased bactericidal activity with differential protein expression in mucus of codfish after bacterial challenge withV.anguillarum.Present studies are in substantial agreement with these reports.Moreover, this change in inhibitory impact in fish species may be because of various chemicals level or distinctive protein discharge in same or diverse fishes, during unfriendly environmental situations.
Presence of broad spectrum of bactericidal activities in NFM and BCFM of selected test subjects against all tested pathogenic strains might be supporting the existence of antibacterial constituents fish skin mucus.Antibacterial components present in fish mucus employed different strategies to kill pathogens.These include the pores formation in cell membrane of bacteria followed by subsequent leakage of the cell contents or the damaging of critical intracellular targets after internalization of the peptide (Hedmon, 2018).
Studies of Hellio et al.(2002) had revealed that dichromate and ethanol extracts of skin mucus of several fish against several pathogenic microbial strains viz.B.cereus,S.aureus,E.coli,K.pneumoniaandP.aeruginosaexhibited MICs in the range of 24 μg/mL to 48 μg/mL.Although in present investigations ethanol and dichromate extracts were not taken, yet the ranges of MICs were similar to these results.Similarly, MIC for mucus extracts ofDesyatis sephenandHimanturagerradi against many pathogenic bacterial strains ranging from 10.36 μg/mL to 25.48 μg/mL(Vennila et al., 2011), supporting our finding.
Contradictory to our results, MICs values ranging from 1 μg/mL to 5 μg/mL had been reported in hydrophobic friction of epidermal mucus of eel, tench and trout (Ebran et al., 2000).Bactericidal protein in the skin mucus ofSyngnathus schlegeliexhibited MIC at 25 μg/mL againstA.hydrophillawhereas＞200 μg/mL againstE.coliandS.aureus(Nagashima et al., 2003).Similarly, crude mucus ofC.straitusdisplayed the MIC at 0.29 μg/mL againstA.hydrophilla(Wei et al., 2010).This large variation in MICs values could be due to different susceptibility of fishes toward different pathogen or due to different experiment set up.However, the high value of MICs in present findings might be due to testing of whole mucus against different microbial strains instead of taking a purified particular component responsible for microbial effect shown by mucus.
The results obtained from present studies evidenced the fact that fish epidermal mucus is a prime component in innate defense system of fishes.NFM and BCFM of both selected Asian catfish species i.e.H.fossilisandC.batrachusexhibited broad range of antibacterial activities against all tested human and fish pathogenic bacteria.Increase in protein concentration after challenge experiment could reveal the proteinous nature of antibacterial components present in mucus which might be responsible for antibacterial effect.Moreover, the increase in the amount of mucus secretion after the bacterial attack might be utilized as a marker of increased stress level in the organisms.Therefore,fish mucus could be opted as an alternative natural source for available antibiotics in human and animal sector which also help in reducing antibacterial resistance.
CRediT authorship contribution statement
Anita Bhatnagar: interpreted the results, Writing – review & editing.Sunil Kumari: experimental work, Writing – original draft.Anil Kumar Tyor: Conceptualization.
Declaration of competing interest
There is no conflict of Interests among Authors.
Acknowledgements
We would like to thank Chairman, Department of Zoology, Kurukshetra University, Kurukshetra and Administration, Kurukshetra University, Kurukshetra for providing the necessary facilities.
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