Bactericidal activities and biochemical analysis of skin mucus of Cyprinid fish

Ethical approval

All methods used in this experiment were concented by the Research Ethical Committee of KUST1447, Kohat.

2.2 Isolation and characterization of bacteria

We collected fish from various fish farms in Khyber Pakhtunkhwa, Pakistan. We used the infected parts of the diseased fish for bacterial isolation, performed under aseptic conditions by serial dilution method. We determined the morphology and shape of bacterial colonies on the nutrient agar plates. Selective media such as MacConkey agar (MA) (Difco™, Becton, Dickinson and Company, NJ, USA), tryptic soy agar (TSA), eosin methylene blue (EMB), mannitol salt agar (MSA), and cetrimide agar were used for identification of bacterial species. Biochemical tests such as triple sugar iron (TSI), catalase, oxidase, motility indole urea (MIU), and sulfur indole motility (SIM) were also performed for the identification of bacteria as described earlier (Tonguthai et al., 1999). Luria Bertani (LB) broth was used to grown the pure culture of potential bacteria which were preserved in glycerol stock at −80 °C for further analysis.

Furthermore, DNA was extracted from purified bacterial samples, and universal primers (5′-ACGCGCGTGTGTAC-3′ Forward and 5′-CAGCCGCGGTMTA--3′ Reverse) were used for the amplification of bacterial DNA. PCR products were verified using 2% agarose gel electrophoresis in TBE buffer. Sequences acquired for gene 16sRNA were modified with Bio edit (created in MEGA X) and were submitted to GenBank. Sequences obtained were put in BLAST for searching the nearest neighbor species.

2.3 Skin mucus collection

Healthful alive fish (C. idella, L. rohita, G. catla, C. mrigala and H. molitrix) were kept in glass aquaria in the Laboratory of Fisheries and Aquaculture, Department of Zoology, Kohat University of Science and Technology (KUST), Kohat. Fish were kept starved for 24 h after seven days of acclimatization to maximize mucus secretion and avoid defecation during the mucus collection process. Fish was put on a sterile tray, washed with phosphate buffer saline, and gently scraped with a sterile slide from lateral sides. Skin mucus was collected from each 20 representative species and mixed. The collected mucus was put into falcon tubes (15 mL) and lyophilized using Labconco’s Freeze dryer and stored at −20 °C for Analysis of bactericidal activity (Nigam et al., 2017; Subramanian et al., 2008).

2.4 Preparations of skin mucus extracts

The extracted mucus from five fish was then partitioned into 3-portions, and isolated individually with acidic, aqueous, and organic (ethanol and methanol) solvents. The acidic mucus extracts were made with little modifications in Subramanian et al. (2008) protocols. Extracted mucus (15 mL) was homogenized with 80 mL of 5% (v/v) acetic acid and put in the water bath for 5 min. The acidic mucus was centrifuged at 18,000 rpm for 35 min at 4 °C after being properly vortexed and cooled to 4 °C. A reverse-phase Sep-Pak Vac 5 g C18 cartridge (125, 55–105 m; Waters Corporation, Milford, MA, USA) was used to collect and partially purify the supernatant. Before adding the supernatant, the cartridge was first activated with 15 mL of methanol and then equilibrated with 5 mL of 10% (v/v) acetic acid. Supernatant was placed into the cartridge, which was then washed once with 20 mL of an acetonitrile, water, and TFA mixture, persuaded by 5 mL of 0.1% (v/v) trifluoroacetic acid (TFA), before being eluted. The mucus samples were kept at −20 °C, then submerged in distilled water and used for bactericidal activity.

Organic extracts (ethanol and methanol) were used to activate skin mucus. The extracted mucus (15 mL) from all fish was immediately lyophilized and kept at −20 °C. Absolute ethanol was added to the dried mucus, then centrifuged at 11,000 rpm for thirty minutes at 4 °C. Ethanolic extract was vortex thoroughly and was kept under liquid nitrogen for 24 h. Then, the ethanolic extract was re-suspended in 15 mL distilled water, followed by adding 5% (v/v) DMSO (dimethyl sulphoxide), thus finally used to evaluate the bactericidal activity. Methanol was added to dried mucus and centrifuged at 11,000 rpm for thirty minutes at 4 °C. The methanolic extracts were mixed well and evaporated under liquid nitrogen for 24 h. To resuspend the dry pellet, 15 mL of distal water was added and extracted two more times with 50 mL of DCM (dichloromethane) then the mucus was analyzed for bactericidal activity. For aqueous extracts, 15 mL of the extracted mucus was re-suspended in 50 mM (w/v) ammonium bicarbonate under cold conditions and centrifuged at 10,000 rpm for ten minutes at 4 °C. The upper layer was amassed, immediately freeze-dried, and stored for further Analysis (Hellio et al., 2002).

2.5 Determination of the bactericidal activity of mucus

Bacterial species of 10⁸ CFU/mL were cultured on petri plates containing 25 mL muller hinton agar (MHA). Different extracts of skin mucus were prepared with four concentrations (1–4 mg/mL). Each MHA petri plate was bored with three distinct wells and named AQ) aqueous, A) acidic, and O) organic with the positive control (Fosfomycin). Then, wells were punched aseptically with a sterile blue tip with a diameter of 6–8 mm, and 100 µL of each mucus extract per well was added. Alongside each mucus concentration, positive controls (Fosfomycin) and ethanol, methanol, acetic acid, and ammonium bicarbonate were used as negative control during bactericidal activity. Bactericidal activity was evaluated by quantifying the diameter of the ZOI produced across the well in a millimeter (mm) after 24 h.

2.6 Determination of minimum inhibitory concentration (MIC) for mucus extracts

The MIC is the least concentration of an analyzed bactericidal component that prevents the apparent growth of bacteria examined after 24 h incubation. Microdilution procedures was used to determine the MIC of acidic mucus extract using Muller Hinton Broth (MHB) in accordance with the Clinical and Laboratory Standards Institute (CLSI) (Wang et al., 2014) with minor changes. The acidic mucus extracts were 2-fold diluted, ranged in concentration from 256 to 2 µg/mL (100 µL mucus/per well). Evaluation of bacterial growth control (MHB + bacteria + mucus extract) was carried out immediately with one column each for negative control (MHB) and for the positive control (MHB + bacteria) used (Silveira et al., 2009). Microtiter plates were then incubated at 37 °C for 24 h. Each well absorbance was calculated by using a biometra microplate spectrophotometer reader at 630 nm.

2.7 Minimum bactericidal concentration (MBC)

MBC was performed according to (Pillai Jr, 2005)) with slight modification. MBC was performed after the MIC test by dispersing 5 µL of mucus sample from the microtiter plates on MHA plates that showed no apparent growth. MBC was recorded after 18–24 h incubation at the least concentration that produced 3–4 colonies, i.e., 99.9% of bacteria was inhibited.

2.8 Biochemical characterization of fish skin mucus

Skin mucus extracts were prepared from the preserved mucus, thawed, and centrifuged at 5000 rpm for 5 min. For identification of biochemical constituents, 3 g of copper sulfate (CuSO₄·5H₂O), dissolve in 500 mL of 0.2 mol/liter sodium hydroxide, then 9 g of sodium potassium tartrate and 5 g of potassium iodide were added. Further, 1 mL of mucus of each species was procured in a distinct test tube, and added 1 mL of distilled water to a separate test tube that served as the blank. Furthermore, 3 mL of the biuret reagent was added to all the test tubes, involving the blank tube. The biuret reagent was mixed with mucus and warmed at 37 °C for 10 min, and the absorbance was recorded through spectrophotometry at 595 nm against blank tubes. The standard curve was drawn, with the concentration of proteins along the X-axis and the absorbance along the Y-axis, to determine the protein concentration in each sample. The same process was adopted for protein analysis in the mucus of each species in triplicate.

The anthrone test estimated carbohydrate content. Briefly, 0.2 g of anthrone was dispelled in 100 mL of diluted chilled sulfuric acid. 3 mL of anthrone reagent and 1 mL of mucus extract were incorporated in a test tube, and the mixture was then cooled in iced water. The reaction mixture was measured at 630 nm.

Lipid analysis was executed by a free fatty acid test. 5 g of mucus sample in a conical flask was mixed with 50 mL of ethanol. The burette was filled with a standardized solution of 0.1% NaOH, and a 2 mL phenolphthalein indicator was added. The solution was heated up to 40 °C, and the alkali solution (NaOH) was added to the mixture and gently shacked till a pale pink colour appeared that was shown the end point of titration, and absorbance was recorded.

2.9 Statistical analysis

The bactericidal activities of each fish skin mucus were analyzed in triplicate. Data were shown as mean ± SE from 3 replicate experiments. Significance was established using a one-way analysis of variance (ANOVA) where P < 0.05 were considered significant.

3.1 Identification and characterization of isolated bacteria

This study observed the morphological and biochemical characteristics of different bacteria (Table S1) isolated from diseased fish (G. catla, C. mrigala, and C. Idella) (Fig. S1A). Standard reference organisms were used to identified bacteria based on their cultural, morphological, and biochemical characteristics. Each bacterium produced round, rod-shaped, smooth, colorless, dew drop-like colonies on the petri dishes of nutrient agar and showed specific colour on respective media after incubation of 24 h (Table S2) (Fig. S1B). The isolated bacteria were identified as Edwardsiella spp, Aeromonas spp, Serratia spp, Enterobacter spp, Pseudomonas spp, Salmonella spp, Staphylococcus aureus, Escherichia coli, Klebsiella spp, and Bacillus spp.

The ten identified bacterial species belonged to three families Enterobacteriaceae (Staphylococcus aureus (ON915526), Salmonella enterica (ON920836), Enterobacter cloacae (ON920869), Escherichia coli (ON935728), Klebsiella pneumonia (ON935750), Bacillus wiedmannii (ON920835), Edwardsiella tarda (ON935051), and Serratia marcescens (ON920834), Pseudomonadaceae (Pseudomonas aeruginosa, ON935772) and Aeromonadaceae (Aeromonas hydrophila, ON920871). Amplified PCR product of genomic DNA of ten species using 16S rRNA bacterial universal primers generated 714–1251 bp amplicons (Fig. 1). The 16S rRNA gene sequencing results of all isolated bacterial DNA revealed 96–99% similarity with reference reported sequences. The relationship among sequences of 16S rRNA gene was clustered to each other in the phylogenetic tree (Fig. 2). These clusters were also intensely upheld by their high bootstrap values.

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Fig. 1

Amplified PCR product of 16 s RNA gene. A) S1, S2, S3, S4, S5, S6, and S7 are the bacterial DNA positive samples (∼850 bp) relative to L (1500 bp) ladder. B) S1 and S2 are the bacterial DNA positive samples (∼1251 bp) relative to L (1000 bp) ladder.

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Fig. 2

Phylogenetic tree based on 16S rRNA gene. The study identified 10 different bacteria belonging to three families. (A) Family Enterobacteriaceae (red colour shows gram (-ve) bacteria, the green colour shows the family Pseudomonadaceae while the dark blue colour is for the family Aeromonadaceae. (B) The light green colour shows the gram (+ve) and the dark blue colour shows the gram (-ve) bacteria. All the stains were compared with closely related species reported from other countries. Percentage bootstrap values (1000 replicates) are shown at each branch point.

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3.2 Mucus secretion

Secretion of skin mucus was different in quantity and appearance among each species. Secreted mucus of H. molitrix showed less viscosity and soon became watery. While the mucus of C. idella and L. rohita was more viscous and secrete mucus in equal quantity in both winter and summer whereas G. catla secreted pale-yellow mucus with suffocating odor, and C. mrigala secreted less mucus as compared to the other species. Moreover, the secretion of skin mucus in all the species was more in winter than in the summer. Furthermore, the average length (cm) and weight (g) of all five selected fish were recorded as (C. idella 38 ± 1.15 cm; 949 ± 1 g, L. rohita 37 ± 0.577 cm; 799.6 ± 0.577 g, G. catla 34 ± 0.57 cm; 701 ± 0.577 g, C. mirigala 28.66 ± 0.577 cm; 499 ± 1 g, H. molitrix 30 ± 1 cm; 501 ± 0.577 g.

3.3 Bactericidal activity of mucus extracts

Among the four different extracts (aqueous, acidic, ethanol, and methanol), the acidic extracts have shown strong bactericidal activity than aqueous and organic mucus extracts (Table 1–5). All four concentrations (1–4 mg/mL) of fish species showed significant activity against identified bacterial species, which was comparable to the standard antibiotics. The activity of skin mucus extracts was increased with the increase in concentration, and high activity was recorded against A. hydrophila, S. aureus and P. aeruginosa at all concentrations. Variations in mean ± SE values of the ZOI of various mucus extracts used against identified bacteria were observed to be significantly (P > 0.05) higher against Fosfomycin. The acidic skin mucus extract from L. rohita, C. idella, and G. catla showed higher ZOI against A. hydrophila (44 ± 1; 44 ± 1; 42.3 ± 2.51 mm respectively), S. aureus (45.33 ± 1.15; 40.33 ± 1; 40.6 ± 1.52 mm respectively) and P. aeruginosa (44 ± 1; 40.6 ± 0.57; 44 ± 1 mm respectively) (Table 1–3). While C. mrigala and H. molitrix acidic extracts exhibited the least ZOI against A. hydrophila (29 ± 2;35 ± 1 mm respectively), S. aureus (31.6 ± 1.52;32.66 ± 0.577 mm respectively) and P. aeruginosa (39.6 ± 1.52; 33.66 ± 0.577 mm respectively) at 4 mg/mL concentration for each shown in (Table 4–5). Although aqueous and organic mucus extracts also showed potent bactericidal activity against identified bacteria, but the ZOI was not remarkably as high as in acidic mucus extract (Table 1–5). In the case of organic (ethanol) extracts of L. rohita, C. idella and G. catla skin mucus exhibited higher bactericidal activity against A. hydrophila (32 ± 1; 34 ± 1; 32 ± 1 mm respectively), S. aureus (38 ± 1; 38.66 ± 1.52; 37 ± 1 mm respectively) and P. aeruginosa (37 ± 1; 25 ± 1; 38 ± 1 mm respectively) which were significantly (P > 0.05) higher among all the identified bacterial strains compared with Fosfomycin (15.33 ± 3.21; 14.66 ± 1.15 mm) as well (Table 1–3). The aqueous extract of L. rohita, C. idella and G. catla also showed maximum inhibitory effect at 4 mg/mL against A. hydrophila (31 ± 1; 32 ± 1; 31 ± 1 mm respectively) S. aureus (26 ± 1; 32.33 ± 2.08; 25 ± 1 mm respectively) and P. aeruginosa (26 ± 1; 32.6 ± 1.52; 26 ± 1 mm respectively) among all identified pathogenic bacteria (Table 1–3). Further, the aqueous extract of C. mrigala showed no remarkably bactericidal activity on initial concentration while with the increase of concentration it showed more activity (Table 5). However, skin mucus of L. rohita and C. idella was observed to be less active against S. marcescens and K. pneumoniae. and same is shown in Table 1–2 and Fig. 3, for G. catla, the acidic and ethanolic mucus extracts for both K. pneumonia (29 ± 1 mm; 21.33 ± 3.21 mm) and S. marcescens (39 ± 1 mm: 26.33 ± 0.57 mm) showed a maximum bactericidal effect. Photographic images of ZOIs of acidic, aqueous, and organic skin mucus extracts and one antibiotic against identified bacterial strains have been shown in Figs. 3–4, respectively.

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Fig. 3

Bactericidal activity of skin mucus of a) L. rohita, b) C. idella and c) G. catla against different identified bacteria. Each MHA Petri plate was punched with three different wells and named A) Acidic, AQ) Aqueous, and O) Organic with the positive control (Fosfomycin). The figures clearly showed that the acidic skin mucus extract of all three species has high bactericidal activity at 4 mg/mL concentration against identified bacteria.

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Fig. 4

Bactericidal activity of skin mucus of a) H. molitrix and b) C. mrigala against different identified bacteria. Each MHA Petri plate was punched with three different wells and named A) Acidic AQ) Aqueous, O) Organic, and with the positive control (Fosfomycin). The figures clearly showed that acidic skin mucus extract of both species has high bactericidal activity at 4 mg/mL concentration against identified bacteria.

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3.4 MIC of potent acidic mucus extracts

The acidic extracts of five selected species were further explored for MIC activities against all identified pathogenic bacteria. The inhibitory concentration of acidic skin mucus extract was observed to differ for diverse tested bacterial species. MIC results were found to be different for acidic mucus extracts on different bacterial pathogens. A. hydrophila, P. aeruginosa, and S. aureus were found to be the most susceptible bacteria against acidic extracts of L. rohita, G. catla, and C. Idella at a concentration of 16 µg/mL. The same bacteria as S. aureus and P. aeruginosa were observed to be the most susceptible against skin mucus of H. molitrix and C. mrigala fish at the 32 µg/mL concentration. Among the skin mucus from five Cyprinid species, L. rohita, C. Idella, and G. catla have the highest inhibitory activity as they inhibited 3 bacterial species at a concentration of 16 µg/mL compared to the activity of H. molitrix and C. mrigala (Table S3). The acidic mucus extract of C. Idella, L. rohita, and G. catla exhibited the ability to kill the bacteria even at a lower concentration (Fig. S2).

3.5 Biochemical characterization of mucus extracts

The change in colour from blue to purple or violet of different mucus extracts (acidic, organic, aqueous) showed the presence of proteins. Due to the presence of peptide bonds, the copper ions in the reagent undergo a charge reduction from + 2 to + 1, changing the colour from purple to blue. Among all the species, the acidic extract of L. rohita, C. idella, and G. catla has the highest protein content (303.6 ± 1.52, 250 ± 1.53, 240 ± 1.53 µg/mL, respectively) compared to C. mrigala and H. molitrix (90 ± 1.52: 100.79 ± 1 µg/mL respectively). Similarly, in the case of carbohydrates content, the colour change in the skin mucus sample from pale yellow to blue dark green showed the presence of carbohydrates, where carbohydrate gets dehydrated when reacting with concentrated H2SO4 and forming a mixture. This mixture reacts with anthrone reagents to give a bluish-green colored complex. L. rohita, C. idella, and G. catla have shown the highest concentration of carbohydrates (100 ± 1.52, 80 ± 1.32, 67 ± 1 µg/mL respectively) compared to C. mrigala and H. molitrix (50 ± 1.52; 40.5 ± 1.52 µg/mL respectively). Furthermore, the presence of lipids was confirmed by changing colour from dark pink to pale pink by adding a standardized alkali solution. The free fatty acids test showed that all the species have the least quantity of lipids compared to proteins and carbohydrates. The lipids content in L. rohita, C. idella, and G. catla (4.07 ± 0.05, 3.1 ± 1.52, 2.52 ± 1 g/mL, respectively) were found more compared to C. mrigala and H. molitrix (1.57 ± 1.53; 0.5 ± 1 g/mL respectively) (Table 6).