ISOLATION AND CHARACTERIZATION OF Aeromonas AND ASSOCIATED BACTERIAL STRAINS FROM STINGING CATFISH FARMS

Abstract

Bangladesh has emerged as one of the leading nations in aquaculture production and the main aquaculture species are major and minor carps, exotic carps, catfish, snakehead, shrimp, prawn etc. Disease outbreak is a major challenge to the expansion of aquaculture production. In this present study, 56 samples comprising infected and apparently healthy Heteropneustes fossilis, water and sediment were collected from 10 stinging catfish farms under 6 districts in Bangladesh such as Mymensingh, Dinajpur, Kishoreganj, Noakhali, Narayanganj and Munshiganj. Clinical signs were recorded and a total of 977 bacterial colonies were marked after spreading samples from enrichment in buffered peptone water at preliminary stage of bacterial isolation from which 135 well-formed colonies were subjected to streak plate culture and preserved for further study. Highest viable bacteria were found as 4.30×108 CFU/g in intestine and lowest as 0.34×108 CFU/g in liver of diseased H. fossilis. Total bacterial load was in a range of 1.56×108 CFU/g to 3.74×108 CFU/g in lesion, 0.84×108 CFU/g to 4.30×108 CFU/g in intestine, 0.34×108 CFU/g to 3.53×108 CFU/g in liver , 0.38×108 CFU/g to 4.17×108 CFU/g in gill , 0.67×108 CFU/g to 3.54×108 CFU/g in slime, 1.49 ×108 CFU/g to 4.21×108 CFU/g in sediment and 1.52×108 CFU/ml to 3.60×108 CFU/ml in water. Observing cultural characteristics, doing gram staining and bacterial microscopy, 27 isolates were separated for performing biochemical tests. Aeromonas spp., Bacillus spp., Escherichia coli, Pseudomonas spp., Klebsiella spp., Enterobacter spp., Flavobacterium spp., Staphylococcus spp., Edwardsiella spp., Salmonella spp., and Clostridium spp. were recorded as the presumptive species. Polymerase chain reaction was done with universal 27F and 1492R bacterial primers for 16S rRNA gene amplification of 13 gram-negative isolates and band of 1464bp was observed by electropherogram. Obtained sequences from the amplicons were edited via BioEdit software, confirmed by BLAST tool of GenBank and validated by the construction of phylogenetic tree in Maximum Likelihood (ML) method using MEGA software version 6.0. From the confirmed species, the presence of Aeromonas hydrophila (7.40%), Enterobacter ludwigii (6.67%), Klebsiella pneumoniae (5.93%) and Pseudomonas mendocina (3.70%) were recorded, where Aeromonas hydrophila was the predominant species and found in collected samples from all farms. This study implicates that not only Aeromonas spp. are causing disease in stinging catfish but also the involvement of other strains such as Pseudomonas, Enterobacter, Klebsiella and so on can be opportunistic pathogens for ultimately leading increased mortality of catfishes.