Isolation of Plesiomonas shigelloides from Infected Cichlid Fishes using 16S rRNA Characterization and its Control with Probiotic Pseudomonas sp.

Background: Bacterial diseases are reported to cause heavy mortalities in both cultured and wild fi shes throughout the world. In any fi eld, diagnosis of disease plays a main role in curing the disease. In aquaculture, antibiotics are used to control the infectious diseases caused by bacteria. The use of antibiotics has been questioned as it experiences problems of bacterial resistance to antimicrobials and unacceptable residues in aquaculture products and environment. Recently, many studied have been performed in the application of probiotic as a control measure of infectious diseases. Therefore, this study has designed for the isolation of possible probiotic bacteria which inhibit the growth of specifi c pathogenic bacteria isolated from a fresh water ornamental fi sh. Materials, Methods & Results: Diseased cichlid fi shes were brought to the laboratory for the isolation of causative bacteria. A total of three strains were isolated (APG01, APG02 and FA3) from the serially diluted samples of gut, gill and spleen. The isolated pathogenic bacteria were injected at the concentration of 10 5 cfu/mL to healthy cichlid fi shes to fiout its virulency in vivo at varying time duration (12, 24, 36 and 48 h) of exposure for 15 days. Among the three isolates, only APG01 caused 100% mortality of cichlid fi shes at 48 h of exposure. APG01 was subjected to 16S rRNA characterization and confi rmed as Plesiomonas shigelloides. The nucleotide sequence was submitted to NCBI Banklt and provided with the accession number. To study the in vitro inhibition of Plesiomonas shigelloides, a possible probiotic bacteria (Pseudomonas strain BPH2) was isolated from fermented rice. In vitro antibacterial activity was studied by agar well diffusion assay with 10, 50 and 100 µL of probiotic strain at the concentration 10 7 cfu/mL. Maximum zone of inhibition of 23 ± 0.78 mm was produced by 50 µL and 100 µL Pseudomonas strain BPH2. Also, the antibacterial effect of cell free supernatant was studied and observed the zone of inhibition of 21 ± 1.00 mm against P. shigelliodes. The susceptibility and reproducibility of probiotic bacteria at varying pH was tested by observing the growth in the media with the pH ranging from 5 to 9. The growth was measured by absorbency at 600 nm for every 8 h. Pseudomonas strain BPH2 was found susceptible at the pH ranging from 6 to 9. Discussion: In agar well diffusion assay, Pseudomonas strain BPH2 isolated from fermented rice showed maximum inhibitory activity against P. shigelloides APG01. In the report of Lactococcus lactis isolated from fresh milk showed a maximum inhibitory effect of 14.77 ± 1.17 mm against the pathogenic strain Aeromonas hydrophila in the agar well diffusion assay. While comparing this result, the inhibitory activity of Pseudomonas strain BPH2 is maximum. The pH tolerance study revealed that pH could signifi cantly infl uence the viability and growth of probiotic Pseudomonas strain BPH2. Lactobacillus mesenteroides showed survival and growth at the pH 3 - 7 with highest viability and growth rate at neutral conditions. The probiotic strain used in this study showed viability between the pH 6 to 8. Hence Pseudomonas strain BPH2 failed to show susceptibility at the acidic pH, it might be questionable to use as a gut associated probiotic. Other than that this strain can survive in fresh aquatic environment and this strain might be effectively used in the aquaculture system like water and soil to control the bacterial disease.