Isolation and Molecular Characterization of Bacteria from Diseased Farm Fish

Abstract

Present study was carried out for three years duration from March 2012 to February 2015 to isolate and characterize the bacteria collected from diseased farmed carp fishes and pond water. From the collected diseased carps (Puntius sarana, Labeo rohita, L. bata, Catla catla, Cirrhinus mrigala, Hypophthalmichthys molitrix) and water samples total nine isolates were differentiated and sub-cultured at 30-37C in the laboratory. The bacteria were identified on the basis of their morphological, physiological and biochemical characteristics and confirmed by sequencing of 16S rRNA gene sequence analysis. Among the nine isolates six were Gram-negative rods and three were Grampositive. Gram-negative six isolates can utilize citrate, and two isolates can ferment lactose. Four isolates were found as catalase positive, while two were weak positive; three were oxidase positive. All the nine isolates were found as fermentative type of bacteria. None of the isolates reduce sulphur, five produced indole; three were motile on motility indole urea media and showed flagellar movement on hanging-drop method. Six isolates showed positive results for methyl red test, while four of them were Voges- Proskauer positive. Sucrose was utilized by all the isolates where one of the isolate could only ferment sucrose. Among the nine isolates six showed resistance against bacitracin. All the isolates are more or less sensitive to gentamicin, neomycin, cephradine, doxycycline, tetracycline, ceftriaxone, ciprofloxacin, pefloxacin, mecillinam and nitrofurantoin. The highest colony forming units/ml was calculated as 65-71×1010 and lowest was 11-16×108. Two isolates were found ampicillin resistant, and MIC for this antibiotic ranged from 320-640 μg/ml and MBC was in the range of 320-1280 μg/ml for the rest of the isolates. The MIC and MBC were ranged from 320-640 μg/ml and 320-1280 μg/ml respectively for the antibiotic tetracycline as all the isolates were sensitive to this antibiotic. Isolates showed their optimum growth in the range of temperature 28–30C, pH 6.8–7.5 and salinity 1%. Five different primers were used and the combination of 8F–806R and 8F–1492R gave the more contrast single band in between 700–800 bp in comparison to 1kb plus DNA ladder. NCBI BLAST nucleotide sequence first matching revealed the nine isolates as Klebsiella oxytoca strain KB (98%), Klebsiella oxytoca strain SHD-1 (99%), Klebsiella oxytoca strain AIMST 8.Cp.16 (99%), Pantoea sp. strain F6-PCAi-T3P21 (84%), Aeromonas sp. strain OS6 (97%), Aeromonas veronii biovar sobria strain ER.1.24 (99%), Brevibacillus borstelensis strain 1CK49 (99%), Brevibacillus borstelensis strain 1CK49 (91%) and Bacillus sp. BVC99 (99%). Among the identified isolates six belongs to the family Enterobacteriaceae, two from the family Paenibacillaceae and one from the family Bacillaceae.