V. Sumathi1, V. Sridevi1, D. C. Reddy2 and V. Kalarani*1

1Department of Biotechnology, Sri Padmavati Mahila Visvavidyalayam, (Women’s University), TIRUPATI- 517 502, A.P., INDIA.

2Department of Fishery Science and Aquaculture, Sri Venkateswara University,

TIRUPATI-517 502, A.P., INDIA.

e-mail : kala.dandala@gmail.com*

Introduction

World aquaculture has grown tremendously over the last fifty years and became the fastest growing food producing sector in the world.  Global intensification and commercialization of aquaculture invariably resulted in disease problems.  For decades, outbreaks of infectious diseases caused by a variety of bacteria such as Flavobacterium, Vibrio and Mycobacterium are focused as primary constraints to the culture of many aquatic species, impeding both economic and social development. Hence, making aquaculture products disease-free and more acceptable to consumers has become the primary challenge.

The limitations associated with the use of antibiotics and vaccinations point to the need for the development of a more user-friendly methodology, which became the focus of attention of international aqua culturists. Probiotics, “live micro-organisms that confer a health benefit on the host” were suggested to be the safe alternative to control proliferation of pathogenic bacteria (Decamp and Moriaty, 2005). The diverse range of bacteria belonging to Bacillus, Lactobacillus, Enterococcus, Pediococcus, Bifidobacteria and the yeast, Saccharomyces (Watson et al., 2008) have shown potential as probiotic organisms for aquaculture application. Several studies emphasized the role of gut colonizing probiotics in reducing mortality of the host, alleviating harmful organisms and producing polyamines and digestive enzymes.  However, more attention has to be paid on the selection of a suitable probiotic in order to achieve the desired benefit in the host species. Isolation and identification of putative probiotic from the host, in which the probiotic is intended for effective use, has already been claimed as an elegant logic.  Thus, identification of gut inhabitant bacteria with probiotic nature to design species-specific feed formulations with the inclusion of indigenous probiotic strain attains priority for successful disease management in aquaculture.

Bacteria are generally identified by sequencing specific sections of the genomic DNA and searching for a close match in the database to find out their taxonomy/genus/species.   Among molecular tools,16s rRNA gene is generally accepted as the potential target for identification and phylogenetic analysis of bacteria (Amann et al., 1995) and this technique is a valuable tool for rapid and fairly reliable identification of bacteria. For long, Bacillus spp. are known to be responsible for the exclusion of pathogenic strains  due to their ability to produce antagonistic antibiotics, amino acids and enzymes. Therefore the present study documents Bacillus species inhabiting the gut of Zebrafish as probiotics against common pathogenic bacteria of freshwater fishes.

Materials and methods

Test species

              Zebrafish, Danio rerio Hamilton obtained from Southern Aquafarms, Chennai, India were maintained in concrete tanks of 50 L capacity at a stocking density of 100 / tank. They were acclimatized with optimal pH (8.0 ± 0.2) and temperature (26 ± 0.50C).

Isolation and propagation of Bacillus

Fish starved for over 12 h were used for experimentation. Gut was dissected out and homogenized aseptically using sterile phosphate buffer (pH 7.0) and centrifuged at 6000 rpm for 10 minutes.  Supernatant obtained was serially diluted using sterile phosphate buffer and the suspect colonies were isolated and purified using nutrient agar. The purified discrete colonies were further inoculated on Bacillus selective medium, incubated at 37ºC for 18 hours. The colonies from the selective medium were subjected to standard microbial screening techniques such as differential staining, motility test, methyl red test, spore formation and catalase test (Pacarynuk et al., 2004). Pure culture was stored for further antagonistic and DNA studies.

Assessment of antagonistic activity

         Antimicrobial activity of probiotic strain was assayed separately using agar diffusion method (Benkerroum et al., 1993).  Protein was extracted from probiotic bacteria (10⁵CFU/ml) and partially purified by sonication followed by dialysis at 4°C. Sterile luria agar medium was prepared and 100µl of about 10⁵ CFU/ml of each pathogenic bacteria were evenly spread on the surface of the medium. Discs (0.5 mm) prepared with 100% (49.6µg/ml), 75% (36.8µg/ml), 50% (24.7µg/ml), and 25% (12.1µg/ml) of protein extracts were placed in each of the plates containing pathogenic strains (Escherichia coli / Pseudomonas fluorescens / Klebsiella aerogenes / Xanthomonas maltophilia). Another set of four discs prepared by dipping in 5ug/50uL of Gentamicin / Chloramphenicol / Ofloxacin / Erythromycin were placed separately in each of the plates containing the same pathogens and the set is considered as the positive control. All the plates were incubated for 24 hours at 37ºC and the zone of inhibition was observed against respective controls.

DNA extraction and 16s rRNA gene amplification  

Broth culture (16h) was used for DNA isolation using phenol:chloroform extraction followed by ethanol precipitation. From the isolated DNA, 16S rRNA gene fragment was amplified using universal primers 27F (5’AGAGTTTGACCTGGCTCAG 3’) and 1492R (5’ GGTTACCTTGTTACGACTT 3’) (Weisburg et al., 1991).

Amplification was carried out using gradient thermal cycler (Eppendorf, Germany) up to 35 cycles with initial denaturation at 94°C for 3 minutes, denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute, extension at 72°C for 1 minute and final extension at 72°C for 5 minutes. 10 µl of PCR amplified 16S rRNA gene product was separated through 2% agarose gel electrophoresis using 1% TAE buffer (for 1h at 80V). The band was visualized and documented using a gel documentation system.

 Analysis of PCR product

Sequencing of PCR product of 16S rRNA gene was carried out in a commercial automated DNA Sequencer (Applied Biosystems  Inc, Hyderabad). The sequence information of the bacterial isolate was compared with the similar sequences that are available in the GenBank of National Centre for Biotechnological Information (NCBI) (www.ncbi.nlm.nih.gov) using BLAST.

Results and discussion

Fig.1 Bacillus strain isolated from the gut of Zebra fish

Antagonistic activity test carried out in the present study using 100%, 75%, 50%, and 25% of proteins extracted from Bacillus spp. showed inhibitory effect of the genus against Escherichia coli, Pseudomonas fluorescens, Klebsiella aerogenes and Xanthomonas maltophilia (Fig. 2). The  impact of protein extracts on zone of inhibition against all the pathogenic strains was examined. The zone of inhibition of 0.2 ± 0.03 cm at 25 % showed gradual increase to 0.6 ± 0.01 at 100 % against E. coli. Likewise the inhibition zone of 0.1 ± 0.03 cm at 25 % increased to 0.3 ± 0.03 cm at 100% against P.  fluorescens. Similar concentration dependent inhibition was observed against K. aerogenes and X. maltophilia with increase in the zone of inhibition from 0.2 ± 0.03 and 0.1 ± 0.03 at 25 % to 0.4 ± 0.03 and 0.2 ± 0.03 cm at 100% respectively (Fig. 3). Thus the inhibitory effect of Bacillus spp. was found to be significantly higher at all the concentrations tested against E. coli compared to the rest of pathogenic bacteria. Antagonistic studies carried out earlier by Ghosh et al. (2007) considering B. subtilis demonstrated effective inhibition of P. fluorescens and Aeromonas hydrophila species in L. rohita. Chantharasophon et al. (2011) also demonstrated inhibitory role of Bacillus against growth of A. hydrophila in Nile tilapia.

Fig. 2 Antagonistic activity of different concentrations (100, 75, 50, 25 %; C= LB medium) of protein extracts from zebrafish gut inhabiting  Bacillus spp. against a) E. coli , b)  P. fluorescens,  c) K. aerogenes  and  d) X. maltophilia.

e) EPC, f) PPC, g) KPC & h) XPC represent respective positive controls.

Fig. 3 Inhibition zone of different concentrations of proteins extracted from Bacillus species and antibiotics (PC = Positive Control) against E.coli, P. fluorescens, K. aerogenes and X. maltophilia.

(Values are Mean ± SD (n=5) of 5 individual observations)

Isolated DNA of Bacillus run through agarose gel against 500 bp marker exhibited a discrete band with molecular weight of > 5000 bp. 16s rRNA gene amplification carried out through PCR and run through 2% agarose gel showed a clear band of 1600 bp (Fig.4 lane2). Partial sequence of 16s rRNA gene product obtained using forward primer amplification provided the sequence of 689 bases (Fig. 5). Further, the homology search made using BLAST revealed 98% similarity of the product with that of Bacillus thuringiensis  (www.ncbi.nlm.nih.gov/blast/) (Gene Bank accession number JQ894337). Furthermore, Bacillus spp. showing 99 % homology with B. substilis (Geethanjali and Anitha Subash, 2011) and 99.9% homology with B. brevis (Chantharasophon et al., 2011) were more prevalent in the gut of laboratory reared L. rohita and Niletilapia respectively.

Fig. 4   PCR product of DNA isolated from Zebrafish gut inhabitant Bacillus spp.  amplified using 16S rRNA primers. (Lane 1 - 5000 bp Marker; Lane 2 - amplified gene product)

Fig. 5  Partial sequence of PCR product of 16S rRNA gene of Bacillus spp. isolated from the gut of laboratory reared Zebrafish (www.ncbi.nlm.nih.gov)

Conclusion

Non-pathogenic nature and probiotic role of Bacillus species demands its use as an antagonistic agent against fish pathogens. The present study for the first time document, the occurrence of Bacillus in the gastrointestinal tract of laboratory reared Zebrafish. The strain showed high homology to Bacillus thuringiensis and significantly inhibited the growth of Escherichia coli / Pseudomonas fluorescens / Klebsiella aerogenes / Xanthomonas maltophilia, and thus provide strong evidence as a feed probiont for disease management in fresh water fish. Further studies on complete sequencing of 16S rRNA gene and phylogenetic status of the newly isolated Bacillus are in progress.

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