Isolation and screening of lactic acid bacteria producing anti-Edwardsiella from the gastrointestinal tract of wild catfish (Clarias gariepinus) for probiotic candidates

2.1 Gut sampling dan bacterial isolation

Fish gut sampling was conducted according to a modified protocol of Amin et al. [10]. In brief, a total of nine wild catfish, Clarias gariepinus, with an average weight of 139.2 + 53.5 g and an average length of 24.5 + 4.1 cm) were caught at the Brantas River, East-Java Indonesia (−7.445550, 112.467981) using a cast net. Then, the fish were killed by spiking their brain using a dissecting needle. Dead catfish were disinfected using 70% ethanol by spraying on the entire surface of the fish to kill bacteria on the fish’s body surface. Afterward, the fish were rinsed with sterile distilled water. The fish belly was sliced ventrally from the anus to the interbrancial membrane, and the digestive tract was taken out and placed on a sterile plate. The digestive tract was then weighed and mixed with a sterile phosphate-buffered saline (PBS, pH 7.2) solution to make a 10-fold dilution rate in a stomacher bag, followed by homogenization using a stomacher (Laboratory Paddle Blender/Stomacher 3–400 mL/500 W, Model SC11L). 100 µL of homogenate was then spread on de Man Rogosa Sharpe Agar (MRSA, Merck 10661) medium, pH of 5.9 previously added 10 g/L calcium carbonate as an indicator and incubated anaerobically for 7 days at 30°C. Pure isolates were then tested for Gram staining, catalase and oxidase activities, and glucose fermentation. Those isolates Gram-positive, catalase and oxidase negatives, and able to ferment glucose were considered potential LAB members and chosen for further assays.

1. Ethical approval: The research related to animal use has been complied with all the relevant national regulations and institutional policies for the care and use of animals.

2.2 Preparation of cell-free supernatant (CFSn)

Screening for anti-edwardsiella activity was carried out according to a protocol previously described by Amin et al. [11] with slight modification. Briefly, each LAB was subcultured in 10 mL of MRS broth (MRSB, Merck 10661) and then incubated at room temperature anaerobically for 24 h. Bacterial cells were then harvested by centrifugation at 15,000 rpm for 10 min at 4°C. The supernatant was taken out using a 1,000 µL micropipette pH was adjusted to 6.6–6.8 with the addition of 1 M sodium hydroxide (NaOH, Merck 106498) to remove the antimicrobial effect of the organic acids. Both supernatants were then sterilized by filtering through a syringe filter size of 0.22 µm (Merck MillexTM – GC Sterile Syringe Filter) and stored at 4°C until further analysis.

2.3 Preparation of indicator pathogen

An indicator bacterial pathogen used for antimicrobial activity was Edwardsiella ictaluri NCIMB 13272 which was obtained from a collection of Fish Quarantine Standard Laboratory (Fish Quarantine and Inspection Agency, Surabaya Indonesia). The bacterial isolate was first inoculated into brain heart infusion broth and incubated aerobically at 30°C for 48 h. Thereafter, the bacterium solvent was subcultured by streaking on Tryptic Soy Agar (TSA, Merck 105458) at 30°C for 48 h. A single colony was picked up and diluted in PBS to reach ∼1 × 10⁸ CFU/mL or 0.5 McFarland. Thereafter, 100 µL E. ictaluri dilution was spread onto a Mueler Hinton (MH) agar (Merck 1.05437.0500) and air-dried for 5 min.

2.4 Screening for anti-edwardsiella activity

The antimicrobial assay was performed according to a protocol previously described by Amin et al. [11] with slight modifications; 6 mm wells were punctured in each MH agar plate previously impregnated with E. ictaluri with a Pasteur pipette. Subsequently, 5 µL of CFSn was added to the wells and incubated aerobically at 30°C. In addition, sterile MRS broth was used as a substitute for CFSn as negative control, and each treatment had two replicates. After 48 h incubation, a clear zone formed around the wells was observed and measured for antimicrobial activity [12].

2.5 Phenotypic and molecular identification

The selected LAB was then identified based on phenotypical and molecular methods. The phenotype assay was carried out using a conventional method as described by Cowan [13]. The assay consisted of Gram stain, catalase, oxidase, carbohydrate fermentation, motility, Voges Proskauer, indole, Hydrogen sulfate (H₂S) production, OF test, the ability to grow at 5, 15°C, and 6.5% NaCl medium.

Furthermore, a molecular assay was performed based on bacterial partial 16 s rRNA gene sequences. First, each bacterial DNA was extracted using a Bacterial DNA isolation kit (Geneaid Biotech Ltd) according to the manufacturer’s instructions. Subsequently, the bacterial DNA was used as a template for polymerase chain reaction (PCR) by targeting the bacterial 16 S rRNA gene sequence. The primers used for the target DNA amplification were 27F (5′-AGA GTT TTG ATC CTG GCT CAG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′). The PCR was performed using SENSOQUEST Labcycler according to the protocol developed by Loh et al. [14]. The amplified PCR product was then sent for DNA sequencing. Partial 16 s rRNA sequences were analyzed by alignment using Bioedit software and identified by comparing them to existing data at the National Center for Biotechnology Information (NCBI) using the BLAST (http://blast.ncbi.nlm.nih.gov) to find out the closest species from these bacteria [15]. The phylogeny tree was created using MEGA X software using the statistical method of Neighbor-joining with 1,000 replication bootstraps [16].

2.6 Viability in gastrointestinal conditions

Simulation of the survival rate of LAB to tolerate and pass through the fish stomach was assessed with the method performed by Geraylou et al. [17]. Each LAB was subcultured on MRS broth and incubated aerobically at 30 + 2°C for 24 h. LAB was harvested by centrifugation at 6,000 rpm for 15 min at 4°C. The supernatant was removed, and then, the pellet was rinsed with sterile PBS twice. The last centrifuged pellet was resuspended in sterile PBS, and the concentration of LAB was adjusted to 10⁸ CFU/mL; 50 µL of LAB supernatants was added to 5 mL of a simulated gastric juice. A simulated gastric juice was prepared by mixing 3 mg/mL pepsin (Merck 7197) in 0.85% NaCl (Merck 6404) (w/v), and the pH was adjusted to 4 (according to the pH in the catfish stomach) with the addition of 1 M HCl. After 3-h incubation at 30 + 2°C, the viability of bacteria was assessed by total plate count. Each LAB had two replicates.

The probiotic candidates were then exposed to a simulation of the intestinal fish according to a protocol of Geraylou et al. [17] with slight modifications. LAB in the gastric juice simulation was then adjusted to pH 7.4 with the addition of 1 M NaOH. Thereafter, 1 mg/mL of pancreatin (Merck 7130) and 0.3% (w/v) bile salt from catfish were added according to the last volume. Bile salt was taken from catfish by piercing the gallbladder of catfish and taking its liquid with a syringe then stored at −20°C [18] until further used. The samples were then incubated at 30 ± 2°C for 4 h. LAB viability was determined using total plate count after 4 h of incubation

2.7 Adhesion capacity (AC) of LAB to intestinal mucus

The adhesive of LAB isolates to the intestinal mucus of fish was examined using the method used by Geraylou et al. [17]. The preparation of fish intestinal mucus was carried out according to Nikoskelainen et al. [18]. Briefly, catfish were fasted for 2 days and then killed as previously protocol. Subsequently, the catfish was cut in the ventral part using scissors from the anus to the head, and the intestine (pylorus to rectal of the intestine) was removed using a scalpel and placed on a sterile plate. The inner surface of the intestine was rinsed with sterile PBS (pH 7.2) and scraped the mucus using a rubber spatula. The intestinal mucus was transferred to a sterile tube and then centrifuged at 13,000 rpm for 30 min at 4°C to remove particulates and cellular material. The supernatant was filtered out with a 0.22-µm syringe filter. The protein of mucus was adjusted to 0.5 mg/mL in PBS using the Bradford method [19] and stored at −70°C until further tests were carried out. Thereafter, a 96-well microplate was prepared, then coated with 150 µL of catfish gut mucus, and left for one night at 4°C with plastic covered. The mucus was removed and washed twice with 100 µL PBS sterile and then discarded; this was carried out to remove mucus that was not attached to the microplate.

About 100 µL LAB inoculum (–1.0 × 10⁸ CFU/mL) was added to 96 culture cell wells, and a wavelength of 600 nm was recorded (A ₀). The wells were then incubated for 1 h, and unattached LAB cells were discarded. Meanwhile, the attached bacterial cells were fixed by incubation at 60°C for 20 min, followed by staining with 0.1% crystal violet (100 µL/mL) for 45 min. Crystal violet was then removed and washed with sterile PBS three times to remove excess dye. The dye bound to the bacterial cells was dissolved by adding 100 µL of 20 mM acetate buffer, pH 4.3 to the well. Thereafter, bacterial AC on the catfish intestinal mucus was determined by measuring absorbance of dissolved dye using a microplate reader at a wavelength of 600 nm (A ₁). A microplate filled only with catfish intestinal mucus was used as a negative control (A _control). Bacterial adhesion was determined with two replications for each LAB, and each test was carried out on eight repetitions to correct the variations. The percentage of bacterial adhesion was calculated using a formula previously described by Amin et al. [20].

where AC is the adhesion capacity (%); A _Ctrl is the absorbance values of control (stained intestinal mucus); A ₀ is the absorbance values of the tested LAB used in the assay (10⁸ CFU/mL); A ₁ is the absorbance values of LAB attached to intestinal mucus.

2.8 Antibiotic susceptibility assay

This assay was conducted to determine the susceptibility of LAB to certain antibiotics. The standard Kirby-Bauer method was carried out to examine each LAB. Each LAB was subcultured on MRS agar and incubated at 30°C for 24 h and subsequently diluted in PBS until it had turbidity to a 0.5 McFarland standard or equivalent to 10⁸ CFU/mL. Within 15 min after adjustment, a sterile cotton swab was dipped into the adjusted suspension, rotated several times, and pressed firmly on the inside wall of the tube above the fluid level to remove excess fluid from the swab. The bacteria were inoculated to MHA by streaking the swab over the entire sterile agar surface. This procedure was repeated two more times, and the plate was rotated to ensure an even distribution of inoculum. The lid jar was left for 3–5 min at room temperature to dry. Three antibiotic disks containing enrofloxacin (CT0639B), oxytetracycline, and novobiocin (CT0038B) were pressed on the surface of MH agar and incubated at 35°C for 16–18 h. The clearance zone formation was interpreted based on the Clinical and Laboratory Standards Institute (CLSI) in which >20 mm was considered sensitive (S), 15–19 mm was considered intermediate (I), and dan <14 mm was considered resistant (R). LAB which was sensitive to these antibiotics will produce a clear zone around the disc.

2.9 Safety evaluation of probiotic candidate

To confirm that the candidate probiotics are not harmful to the host, each LAB was given to catfish fingerling by in vivo experiment according to Nayak and Mukherjee [21]. One hundred and forty catfish fingerlings (7−9 cm) were divided into seven groups: six groups for each LAB and one for negative control with two replicates. Before treatment, fish were kept in the tank (25 L with an aerator) and fed with a commercial pellet for a week. All LAB was inoculated on MRSA and then incubated for 24 h at 37°C. LAB colonies were suspended in PBS at a concentration of 10⁸ CFU/mL. The experiment was carried out by injecting 0.1 mL of each LAB intraperitoneally (IP) into the previously prepared catfish. The negative control group of fish was injected with 0.1 mL of sterile PBS. All fish were observed for survival rate for 7 days, and the mortality of each treatment was recorded.

2.10 Data analysis

The results of the experiment including survival in gastrointestinal conditions, AC in catfish intestinal mucus, and safety evaluation of LAB were analyzed either using paired T test or one-way analysis of variants (ANOVA). Differences within the experiment were assessed with the Tukey posthoc test. All statistic was performed using SPSS 22. Antimicrobial activity and antibiotic resistance were evaluated with descriptive analysis.

3.1 Isolation and LAB screening

A total of 29 LAB isolates showing a clearance zone on the MRS agar were isolated from the intestinal tract of nine wild-caught catfish. Preliminary phenotypical assay indicated that all these 29 isolates showed to be: Gram-positive, catalase-negative, and oxidase-negative and were able to ferment glucose. Six of these 29 isolates (6.5%) had antagonistic activity against E. ictaluri, indicated by the formation of a clearance zone >10 mm (Table 1). The largest diameter of inhibitory activity was found for isolates ZMW55 and ZMW102 as these isolates inhibited more than 19-mm diameter clearance zone, while the lowest diameter of the inhibition zone was observed from isolate ZMW150

3.2 Phenotypic and genotypic identification of LAB

The results from phenotypic tests for the six LAB are presented in Table 2. The cell morphology of 4 LABs was spherical-shaped while the remaining two have rod-shaped. All LAB were Gram-positive and grew in aerobic or anaerobic conditions. Negative results were obtained for the motility test, catalase, oxidase, and indole tests, as well as were unable to produce H₂S. The isolates were also able to utilize glucose and maltose. Meanwhile, only three isolates were able to utilize arabinose (ZMW50, ZMW96, and ZMW102). Two isolates were able to utilize salicin. None of these LAB isolates grew at 5°C, producing indole activity as well as gas production. But only a single isolate (ZMW102) was able to grow at 15°C. From these phenotype assays, ZMW50, ZMW94, and ZMW96 were identified as Enterococcus sp. ZMW55 which did not grow in NaCl 6.5% was identified as Lactococcus sp., and ZMW101 and ZMW102 were identified as Weissella sp. (Table 2).

Based on partial 16 s rRNA gene sequences, isolates ZMW50, ZMW55, ZMW94, ZMW96, ZMW101, and ZMW102 were identified as Enterococcus faecalis (99.89%), Lactococcus lactis ssp. lactis (99.93%), Enterococcus hirae (100%), Enterococcus faecalis (99.93%), Weissella confusa (100%), and Weissella cibaria (99.79%), respectively. This result is consistent with the phenotypic test. All six LAB had been submitted to Genbank NCBI. The phylogenetic tree of the six LAB constructed based on partial sequences of 16 s rRNA is presented in Figure 1.

Figure 1

Phylogenetic tree of LAB isolates based on 16 s rRNA gene sequence. Dots (•) is LAB isolates from gastrointestinal tracts of wild-caught catfish in the present study. The numbers on the lines indicate the distance between the branches. The bootstrap value for each node is displayed at the base of the branch. The Neighbor joining method was used as a model with Maximum Composite Likelihood Substitution; E. coli is used as the outgroup.

3.3 Viability in the stomach and intestinal conditions

Each LAB was firstly exposed to the simulation stomach Juice for 3 h, followed by intestinal juice for 4 h. The results all tested LAB had good viability after passing through the gastrointestinal tract juice simulation, ranging from 5.07 log CFU to 6.38 log CFU. The viability of all LABs was first decreased after being exposed to stomach juice for 3 h. E. faecalis (isolate ZMW50, ZMW96), E. hirae, W. confusa and W. cibaria decreased significantly, except for Lactococcus lactis which still showed high viability after 3 h incubation (Figure 2). Although all tested bacterial strains survived the simulation of the stomach, all LAB isolates showed a decrease after being exposed to the simulated stomach juice which consisted of pepsin 3 mg/mL, 0.85% NaCl and pH 4.

Figure 2

Viability of six LAB in simulation gastrointestinal condition. Before simulation, simulation stomach juice, simulation intestinal juice. All values are means with standard deviations represented by vertical bars of two replicates. Blue bars represent initial viability; orange bars represent viable cell number after simulation of stomach juice; green bars represent viable cell number after exposure simulation of the intestinal juice. Different letters indicate significant differences (p < 0.05).

Further simulation was performed by incubating each LAB in the simulated intestinal juice for 4 h. The result showed that there was a different result in survival for each LAB. E. hirae showed a significant difference in increasing the viability after 3-h incubation. However, viable cells of two LAB (E. faecalis ZMW50, and ZMW96) increased after being exposed to simulated intestinal juice for 4 h, and the number of viable cells was not a significant difference compared to the initial concentration of both LAB, p > 0.05. While viable cells of three LAB (L. lactis, W. confusa, and W. cibaria) counted at the end of stomach exposure were not significantly different before and after 4 h exposure to the simulated intestinal juice (Figure 2).

3.4 AC to intestinal mucus

All six LAB were able to adhere to the mucus of catfish gastrointestinal tracts, ranging from 3.42 to 16.72%. However, the adherence capacity was significantly different among the LAB isolates, p < 0.05. The highest AC was obtained from Weisella ciberia (16.72%), followed by Weisella confusa (9.00%), Enterococcus faecalis (5.59%), and Lactococcus lactis (4.56%). Meanwhile, the lowest AC was observed from Enterococcus faecalis (3.57%), though there were no significant differences between L. lactis, E. hirae, and E. faecalis (ZMW96; Figure 3).

Figure 3

AC of six LAB isolates expressed in percentage. Bars represented average values and standard deviation of eight replicates. Different letters indicate significant differences in adhesive capacity at p < 0.05.

3.5 Antibiotic susceptibility

The six LAB species showed inhibition zones (11–32 mm) after being challenged with enrofloxacin, oxytetracycline, and novobiocin. Diameters of the inhibition zones were interpreted based on CLSI. The six LABs were highly susceptible to oxytetracycline indicated by an inhibition zone >20 mm. Five LAB were susceptible to enrofloxacin (Lactococcus Lactis ZMW55, Enterococcus hirae ZMW94, and Weissella confusa ZMW101 were categorized as highly susceptible with a diameter of inhibition zone >20 mm; two intermediate susceptibles in diameter of inhibition zone ranging from 15–19 mm). Meanwhile, one LAB (Weissella cibaria ZMW102) was found to be R to enrofloxacin indicated by a diameter of inhibition zone <14 mm. The result showed also that 5 LAB were intermediately susceptible to Novobiocin, but one LAB (Enterococcus faecalis ZMW96) was R to Novobiocin. Based on these results, two LAB (E. faecalis (ZMW96) and W. cibaria) were excluded from probiotics candidates (Table 3).

3.6 Safety evaluation of LAB

Pathogenicity of the six LAB was assessed by in vivo trials in which each LAB was IP injected into 20 catfish juveniles and reared for 7 days. The results showed that no mortality was observed, except in E. faecalis (ZMW50 and ZMW96) which caused 5.2% mortality (one of 19 fish). Similarly, no mortality was observed in 20 fish injected with PBS (negative control), which were similar to those in catfish injected with L. lactis, E. hirae, W. confusa and W. cibaria (Figure 4).

Figure 4

Survival rate of catfish on pathogenicity test by IP injection method. The error bar shows the standard deviation (n = 2). There was no significant difference in all treatments with control (p > 0.05).