Aeromonas hydrophila is a major waterborne pathogen, which induces various diseases in freshwater fish with the capability for zoonotic potential. This study was applied to investigate the prevalence of A. hydrophila in diseased Nile tilapia fish, genetic characterization of the virulence encoding genes (act, aerA, alt, and ast genes) , and antibiotic susceptibility. Out of the 500 diseased Nile tilapia fish samples, 70% (350/500) Aeromonas species were isolated. From which 53.4% (187/350) of Aeromonas hydrophila strains were identified. A. hydrophila was detected in kidneys, followed by liver, spleen, intestine, and gills. The results of virulotyping displayed the presence of act, and aerA genes in a high percentage of 40%, followed by alt gene (30%) , but ast gene was not detected (0%) in A. hydrophila strains. Based on DNA sequence analysis of three virulence associated-genes (act, aerA, and alt genes) , the phylogenetic tree showed the genetic relationship with related species. Finally, the antibiotic susceptibility tests revealed high resistance toward chloramphenicol (67.4%) , followed by amikacin (51.9%) and gentamicin (47.1%) , whereas a high sensitivity was exhibited toward meropenem (90.9%) , followed by ciprofloxacin (84.2%) , amoxicillin-clavulanic acid (73.3%) and trimethoprimsulfamethoxazole (64.2%) . The multidrug-resistant A. hydrophila strains were observed in 69.0% of strains with six resistance patterns.

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