Bacterial pathogens pose a significant challenge for fish production. Escherichia coli is a Gram-negative bacterium being among the leading contributors of economic loss to fish farmers worldwide. E. coli infects a variety of fish species making a concern for public health. In order to gain a better understanding of this issue, a study was conducted to isolate and identify E. coli present in Labeo rohita , Catla catla , Cirrhinus mrigala , Hypophthalmichthys molitrix , and Ctenopharyngodon idella sampled from fish farms of Kasur and Muzaffargarh. E. coli was isolated from liver, intestine, stomach and kidney of fish samples using MacConkey and Eosin Methylene blue (EMB) agar media. DNA was extracted using Genomic DNA Purification Kit and E. coli was detected by amplification of virulence genes viz. stx2 and eaeA and antibiotic resistance genes viz. tetA and sul1 in E. coli by PCR using species-specific primers. E. coli was recovered from 103 (41.2% prevalence) fish samples of five species. Phenotypic and morphological characterization revealed pink, smooth, and circular colonies of E. coli on MacConkey agar while dark purple and circular colonies on EMB agar media. Biochemical tests proved E. coli positive in catalase, indole, urease, methyl red and motility tests while negative results in Gram-staining, oxidase, citrate, Voges-Proskauer, H2 S and indole production tests. E. coli isolates proved to be resistant against pencillin , sulfamethoxazole and tetracycline while sensitive against ampicillin, and erythromycin, and intermediate resistance against amoxicillin and cefotaxime . Maximum prevalence of stx2 gene of E. coli was recorded in intestine and liver of L. rohita and C. mrigala . Phylogenetic tree analysis of our isolated E. coli strains revealed 97% similarity with E. coli strains isolated in previously studies. The results concluded that shiga toxic gene was the most significant pathogenic gene of E. coli . High stocking density, low water quality parameters and unchecked application of antimicrobial agents causes emergence of pathogenic bacteria in fish farms.
Keywords : MacConkey agar, Mueller Hinton agar (MHA), Labeo rohita , Phylogenetic tree analysis, Biochemical tests