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This study was conducted in Middle Upper Egypt to determine the prevalence of <jats:italic>A. hydrophila<\/jats:italic> among the diseased <jats:italic>Oreochromis niloticus<\/jats:italic> (<jats:italic>n<\/jats:italic>\u2009=\u2009100) and <jats:italic>Clarias gariepinus<\/jats:italic> (<jats:italic>n<\/jats:italic>\u2009=\u2009100) at Assiut and Sohag Governorates. <jats:italic>A. hydrophila<\/jats:italic> isolates (<jats:italic>n<\/jats:italic>\u2009=\u200944) were assessed for antimicrobial susceptibility and biofilm production. Moreover, PCR was performed to analyze the incidence of some genes in 20 isolates of <jats:italic>A. hydrophila<\/jats:italic>. The results showed that 24% and 20% of the examined <jats:italic>O<\/jats:italic>. <jats:italic>niloticus<\/jats:italic> and <jats:italic>C<\/jats:italic>. <jats:italic>gariepinus<\/jats:italic> were infected with <jats:italic>A. hydrophila<\/jats:italic> respectively, with all (100%) showing a variety of clinical signs of septicemia. <jats:italic>A. hydrophila<\/jats:italic> isolates were all biofilm producers, with varied degrees of biofilm production. 79.5% of the isolates were multidrug-resistant and had a high multiple antimicrobial resistance index &gt; 0.2. PCR analysis revealed that all isolates carried <jats:italic>act<\/jats:italic> and <jats:italic>blaTEM<\/jats:italic> genes but not carried <jats:italic>int2<\/jats:italic> gene. Additionally, <jats:italic>sul1<\/jats:italic>,<jats:italic> aer<\/jats:italic>,<jats:italic> tetA<\/jats:italic>,<jats:italic> int1<\/jats:italic>, and <jats:italic>qnrA<\/jats:italic> genes were present in 75%, 60%, 55%, 55% and 45% of them, respectively. This study highlights the high incidence of multidrug-resistant pathogenic <jats:italic>A. hydrophila<\/jats:italic> in the infected fishes, posing a serious risk to humans and fish.<\/jats:p>","DOI":"10.1038\/s41598-025-89465-4","type":"journal-article","created":{"date-parts":[[2025,2,18]],"date-time":"2025-02-18T14:02:44Z","timestamp":1739887364000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Phenotypic and genotypic characterization of Aeromonas hydrophila isolated from freshwater fishes at Middle Upper Egypt"],"prefix":"10.1038","volume":"15","author":[{"given":"Usama H.","family":"Abo-Shama","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amany A. 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