Isolation and Characterization of Bacteria from the Intestine of Clarias batrachus for Probiotic Organism

Abstract

Clarias batrachus (Linn.) in India is at a deteriorating state. The use of probiotic organism is one of the alternatives to promote fish nutrition in worldwide aquaculture. An extensive study was performed to isolate and identify probiotic bacteria from the gut of the C. batrachus. Quantitative and qualitative analysis of bacterial flora associated with the intestine of C. batrachus were carried out. Total viable bacterial count in the intestine of catfish was 1.61 × 1010cfu/g. Thirty-two different bacterial isolates were selected from the intestinal microflora of C. batrachus. Gram-positive rod-shaped bacteria dominated (81%) the populations in catfish. The five intestinal isolates (PKA1, PKA2, PKA17, PKA18 and PKA19) showed antagonistic properties against common fish pathogens - Vibrio harveyi, Vibrio vulnificus and Vibrio parahaemolyticus. The strain PKA17, PKA18 and PKA19 were identified as Lysinibacillus sphaericus, Bacillus cereus and Bacillus thuringiensis respectively by the 16S rDNA sequencing.

Introduction

The Asian catfish, Clarias batrachus (L.) is widely recognized for its nutritional and economic significance (Ganguly et al. 2017). The species is found in a variety of habitats including fresh and brackish water, low-land, streams, swampy or paddy fields and shows exceptionally well tolerance level at varied environment (Talwar and Jhingran 1991). It has good growth rate, efficient feed conversion ratio and sustainability in oxygen-depleted, hyper-ammonium, stressful water condition (Argungu et al. 2013).

However, the species is presently at a deteriorating state in the Asian countries due to the exploitation of its natural habitats, intermittent periods of drought, reclamation of wetlands and uncontrolled introduction of exotic fishes. Another major cause of species deterioration is attributed by pathogenic infections (Ahmed et al. 2012). In recent decade, prevention and control of aquatic diseases have focused on the use of probiotics instead of chemotherapeutic agents and antibiotics as these agents may result in unfavorable alteration of the microbiota and involve in the emergence of antibiotic resistant bacteria (Aarestrup 1999; Nayak and Mukherjee 2011). Probiotics confer protection against pathogens by production of antimicrobial substances and endorse fish nutrition by breakdown of indigestible components and production of vitamins (Gatesoupe 1999; Bandyopadhyay and Das Mohapatra 2009).

The selection of probiotics should be done in species-specific manner to overcome ineffectiveness (Ghosh et al. 2007; Hai et al. 2007). There is growing awareness on the influence of intestinal bacterial composition of fish for probiotic potentiality. The intestinal flora of endothermic animals serves both digestive function and as a defence barrier against infections (Sissons 1989). Therefore, studies on the composition and characteristics of the dominant intestinal microflora are crucial to obtain effective probiotic strain. However, in order to establish as a potential probiotic the organism must possess properties like antibiotic sensitivity, cell surface hydrophobicity or bile salt tolerance activity etc. (Mehmet et al. 2015).

Screening of probiotics to date has concentrated on the search for microbes active against pathogens (Chythanya et al. 2002). In the present investigation, an effort has been taken to isolate suitable probiotic strain from the intestine of C. batrachus through characterization and evaluation of antagonistic properties on the common fish pathogens Vibrio harveyi, Vibrio vulnificus, Vibrio parahaemolyticus, Aeromonas hydrophila. They were also tested against two non-pathogenic gram (+ve) bacteria Bacillus subtilis and Bacillus cereus. In addition, the biochemical properties and cell-surface hydrophobicity of the candidate probiotics were also determined.

Materials and Methods

Collection of Samples from the Intestine of C. batrachus

Healthy C. batrachus were collected from a cultivation pond (Ramsagar; Bankura District, West Bengal, India) assuming that they might have a well-established pattern of intestinal microflora. The pond was constructed in the year of 1993 has no history of use of probiotics or any other bacterial adjuvant. The weight of those fish was about 270.5 ± 11.76 (Mean ± SD) g. Catfishes were immediately transferred to water collected from the culture pond and carefully brought into the laboratory in live condition. They were then sacrificed and the intestinal tracts were aseptically taken out by horizontal dissection starting from rectum towards the head. The length and weight of the catfish intestines was about 14.5 ± 2.72 cm and 2.9 ± 0.62 g respectively. Gut contents were then removed by scrapping and the intestines were washed thoroughly with 0.9 (w/v) % of sterile Normal Saline Solution (NSS) by the help of sterile syringe to remove non-adherent microflora. The fish intestines were crushed separately to make homogenized solution and vortexed to obtain microbial solution (Irianto and Austin 2002).

Isolation and Cultivation of Bacteria

The stock solution was further serially diluted (10−1 to 10−10). 100 µL of 10−6, 10−8 and 10−10 dilutions were spread plated on Nutrient Agar (NA), Man Rogossa Sharp (MRS) Agar, Tryptone Soya Agar (TSA), Pseudomonas agar and Bacillus agar media (Barman et al. 2011). The plates were then incubated at 30 °C temperature for 24–48 h to obtain isolated colonies.

Morphological Characterization of the Isolates

Colonies were enumerated to determine the microbial load of intestine. Colony morphology namely configuration, elevation, margins, surface, pigments, size were observed (Uddin and Al-Harbi 2012). Gram character of each bacterial isolate was studied and their shape and size were recorded. Pure cultures of the isolates were maintained at 4 °C as stocks and were sub-cultured on slants in every 6 weeks.

Pathogens

Catfish pathogens such as Vibrio harveyi (MTCC 7954), Vibrio vulnificus (MTCC 1145), Vibrio parahaemolyticus (MTCC451) were obtained from Microbial Type Culture Collection and Gene Bank, IMTECH, Chandigarh. Aeromonas hydrophila SBK1 (Accession No. HM802878.1) was obtained from the department of Microbiology of Vidyasagar University. They were used as target pathogens along with two gram (+ve) bacterial strain Bacillus subtilis and Bacillus cereus to detect antagonistic activity of the bacterial isolates.

Selection of Probiotic Bacteria

The in vitro antagonistic tests of intestinal bacterial isolates were carried out by using agar well diffusion method (Gram et al. 2004). First, 100 µL of known concentrations (12 × 103 cfu/mL) of target pathogens were spread on separate TSA plates and allowed for 3–5 min drying. Wells of 5 mm diameter were then bored on these plates by the sterile well borer. 5–10 µL of melted agar (15 g Agar + 1 L distilled water) was added at the bottom of the each well to prevent the seepage of the test bacterial suspension. 50 µL of known concentration (24 × 103 cfu/mL) of concerned bacterial suspension were then inoculated in the specified bored wells and the plates were incubated at 30 °C for 24 h. The test was performed in triplicates. After incubation, the intestinal microbial strains that produced clear inhibitory zone against the test pathogenic bacteria with a diameter greater than 1 mm were selected to be an antibacterial substance producer or probiotic strain (Vignolo et al. 1993).

The isolates containing antibacterial activity against target fish pathogens were further cultivated in tryptone soya broth (TSB) media (Irianto and Austin 2002) for maximum growth. They were grown at different pH (5.0–11.0), temperature (10–65 °C) and salt concentrations (0.1–10.0) to obtain their growth range along with optimum growth requirements (Barman et al. 2011). The test was done in triplicates.

Biochemical Analysis of the Bacterial Isolates

The biochemical characteristics of the major intestinal isolates (PKA17, PKA18 and PKA19) were studied according to Bergey’s manual of systematic bacteriology (O’hara et al. 2000)

Cell Surface Hydrophobicity

The hydrophobicity phenotype of bacterial cell surface is correlated to their adhesive capacity and colonization of the gut (Ouwehand et al. 1999). The isolates were grown separately in tryptone soya broth (TSB) at 37 °C for 24 h. The culture was then centrifuged at 4000 × g for 10 min, washed twice with phosphate-buffered saline (PBS) and finally resuspended in PBS. The cell suspension was diluted with PBS up to an optical density of 1.00 at 620 nm. Of this sample, 3 mL was added to 0.6 mL of xylene, vortexed for 1 min and incubated at 37 °C until phase separation was observed. The absorbance (A) of the aqueous phase was subsequently measured. The decrease of the absorbance was taken as hydrophobicity dimension of the cell surface and calculated according to the formula (Savage 1992):

$$ {\text{Hydrophobicity }}\left( \% \right) = \frac{{A_{0} - A_{f} }}{{A_{0} }} \times 100 $$

where, A0 = initial absorbance, Af = final absorbance

Chromosomal DNA Isolation for 16 s rDNA Amplification and Sequencing

Three morphologically different isolates were subjected to molecular identification through 16S rDNA sequencing. Chromosomal DNA was isolated using XcelGen Bacterial gDNA Mini kit (XG2411-01) and confirmed using agarose gel electrophoresis. Partial sequence of 16S rDNA of PKA17 and PKA18 were amplified using universal forward primer (8F) and reverse primer (1492R) where as PKA19 rDNA were amplified using forward primer (27F) and reverse primer (1492R). The quality of PCR products were evaluated on 1.2% agarose gel. The PCR amplicon was purified with ExoSap enzymatic purification. After purification, the products were subjected to Sanger sequencing using ABI, 3730XL DNA analyzer using BdT v3.1 chemistry. Each forward and reverse reaction of PCR amplified products were sequenced separately. Forward and Reverse DNA sequencing reaction of PCR amplicons of respective samples were carried out with specific primer using BDT v3.1 cycle sequencing kit on ABI 3730xl genetic analyzer.

Phylogram Construction

Nucleotide BLAST was performed using NCBI online server (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Twelve similar sequences for PKA17, thirteen similar sequences for PKA18 and ten similar sequences for PKA19 were retrieved and used for multiple sequence alignment using ClustalX2 (Larkin et al. 2007), phylograms were constructed using Phylip 3.69 (Tuimala 1989) and trees were represented using Dendroscope (Huson et al. 2007) software.

Result and Discussion

Isolation and Enumeration of Bacteria

Bacteria comprise a significant portion of planktonic biomass and are responsible for contributing productivity in aquatic systems (Muylaert et al. 2002). They play a fundamental role in nutrient recycling by decomposition and mineralization of organic matter (Azam et al. 1983). Thus, the quality of aquatic species can be regulated by monitoring the bacteria of growing ponds. The growth rate of bacteria is mostly controlled by their rapid response to physical, chemical or environmental condition. Ganguly et al. (2017) evaluated the physico-chemical parameters of a C. batrachus cultivation pond throughout the year and reported its correlation with the bacterial load of pond water and sediment.

During the present study, the bacterial count (grown in TSA media) of the pond water ranged from 8.3 ± 2.75 × 103 to 2.8 ± 1.45 × 104 cfu/mL whereas the total viable bacterial load in the intestine of catfish was 1.61 ± 2.9 × 1010 cfu/g. Similar kind of observation of bacterial count using TSA media was reported by Uddin and Al-Harbi (2012). In their findings, the bacterial count was 9.2 ± 5.5 × 103 to 6.6 ± 5.1 × 104 cfu/mL in pond water and 2.7 ± 3.4 × 1010 to 1.0 ± 4.5 × 1011 cfu/g in the intestine of the Clarias gariepinus. The intensive feeding rate may promote the bacterial load in the gut of the catfish (Al-Harbi and Uddin 2010).

Selection of Probiotic Bacteria

The study of bacterial composition reveals that gram-positive rod-shaped bacteria dominated the populations 70% in pond water and 81% in catfish intestine. Thirty-two different bacterial isolates were screened from the intestinal microflora of C. batrachus and morphologically characterized. During the in vitro antagonistic tests against V. harveyi, V. vulnificus, V. parahaemolyticus, A. hydrophila it was found that the inhibitory effects of those bacterial isolates varied with different parameters (8 ± 1 to 24.93 ± 0.12 mm diameter). The five intestinal isolates PKA1, PKA2, PKA17, PKA18 and PKA19 showed maximum antagonism against target pathogens (Table 1). PKA1 and PKA2 were grown for 48 h where as PKA17, PKA18 and PKA19 for 24 h to get proper colony morphology. Their colony and gram characters were examined (Table 2). The inhibitory effects of bacterial isolates may be due to the competition for nutrients or presence of bacteriocins, siderophores or enzymes (Verschuere et al. 2000). The experimental results suggested that, these isolates with antibacterial properties may be used as a probiotic strain to inhibit the growth of invading pathogen and to increase the survival rate of the catfish (Ayoola et al. 2013). Dahiya et al. (2012) eliminated pathogenic Micrococcus sp. and increase the immunity of the C. batrachus by the in vivo use of probiotics. Kumar et al. (2013) isolated Lactobacillus plantarum and Lactobacillus casei from the intestine of C. batrachus and evaluated their role as probiotic against Aeromonas hydrophila and Edwardsiella tarda in vitro and found to be successful. The lactic acid bacteria isolated from Clarias gariepinus showed apparent bactericidal activity against fish pathogens Salmonella typhimurium and Escherichia coli (Hamid et al. 2012).

Table 1 Inhibitory effects of bacteria isolated from the intestine of C. batrachus against target pathogens
Table 2 Morphological character and gram nature of selected bacteria isolates

The growth of the isolates was observed in a wider range of pH, temperature and NaCl concentration. PKA1, PKA2 and PKA19 strains require pH 7 and PKA17, PKA18 strains require pH 6 for their optimum growth (Fig. 1). PKA1, PKA17, PKA18 and PKA19 grew best at 37 °C whereas PKA2 at 30 °C temperature (Fig. 2). 0.1% salt concentrations results maximum growth for all of these isolates (Fig. 3). Finally, three bacterial isolates were selected for further study on the basis of higher zone of inhibition against fish pathogens.

Fig. 1
figure1

Growth of bacterial isolates of C. batrachus at various pH

Fig. 2
figure2

Growth of bacterial isolates of C. batrachus at various temperatures

Fig. 3
figure3

Growth of bacterial isolates of C. batrachus at various salt concentration

The biochemical properties of PKA17, PKA18 and PKA19 were found according to their characteristics (Table 3). The in vitro hydrophobicity test of these isolates (PKA17, PKA18 and PKA19) demonstrated high adherence towards non-polar solvent xylene (Table 4). The cell-surface hydrophobicity varied with the strains. PKA19 (76.4%) showed significantly (P ≤ 0.05) higher hydrophobicity followed by PKA17 and PKA18. High cell-surface hydrophobicity is significative to adhesion and plays a pivotal role in the formation of biofilm and removal of contaminants (Krasowska and Sigler 2014). In the present study, all the test isolates exhibited high cell-surface hydrophobicity and ensured their capacity of adherence to the intestinal wall. The use of those probiotics (PKA17, PKA18 and PKA19) may consequence the cultivation of C. batrachus for the generation of high-quality livestock product in terms of size, production time and health.

Table 3 Biochemical characteristics of the isolate PKA17, PKA18 and PKA19
Table 4 Test of hydrophobicity

Molecular Identification of Three Antibacterial Isolates

According to the sequencing result, the amplified products for PKA17 were 747 base pair, PKA18 were 677 and PKA19 were 1460 base pair long (Fig. 4) which showed corresponding single bands. The 16S rDNA partial sequences were subjected to nucleotide BLAST against non-redundant database. With respect to 100% query coverage and 94% identity from PKA17 BLAST result, twelve sequences were selected for phylogram construction. According to phylogeny constructed against 100 bootstrap value, PKA17 showed similarity with Lysinibacillus sphaericus OUG29GKBB (Accession No. KM972671.1) as shown in Fig. 5. On the other hand for the construction of PKA18 tree, thirteen sequences were selected having 98% query coverage and 96% identity values. PKA18 showed similarity with Bacillus cereus strain Gut16 (Accession No. KU156696.1) as shown in Fig. 6. With respect to 100% query coverage and 99% identity from PKA19 BLAST result, ten sequences were selected for phylogram construction. According to phylogeny constructed against 100 bootstrap values, PKA19 showed similarity with Bacillus thuringiensis Bt 53 (Accession No. KY784654.1) as shown in Fig. 7. So, the above study indicated that the three isolated organism PKA17, PKA18 and PKA19 belongs to Lysinibacillus sphaericus, Bacillus cereus and Bacillus thuringiensis species and the accession numbers provided by GenBank were KX580190.1, KX826079.1 and MF139049.1 respectively.

Fig. 4
figure4

PCR product of 16S rDNA genes of PKA17, PKA18 and PKA19 bacterial isolates of C. batrachus

Fig. 5
figure5

Phylogenetic analysis of isolate PKA17 and related bacteria based on 16 s rDNA sequencing

Fig. 6
figure6

Phylogenetic analysis of isolate PKA18 and related bacteria based on 16 s rDNA sequencing

Fig. 7
figure7

Phylogenetic analysis of isolate PKA19 and related bacteria based on 16 s rDNA sequencing

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Ganguly, A., Banerjee, A., Mandal, A. et al. Isolation and Characterization of Bacteria from the Intestine of Clarias batrachus for Probiotic Organism. Proc Zool Soc 72, 411–419 (2019). https://doi.org/10.1007/s12595-018-0283-x

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Keywords

  • Aquaculture
  • Clarias batrachus
  • Probiotic
  • Antagonistic effect