RESEARCH PAPER Turkish Journal of Fisheries and Aquatic Sciences www.trjfas.org ISSN 1303-2712 DOI: 10.4194/1303-2712-v19_02_06 Antibiotic Resistance and Virulence-Associated Gene Profile of Edwardsiella tarda Isolated from Cultured Fish in Japan Sudu Hakuruge Madusha Pramud Wimalasena 1 , Hansani Nilupama Kumari Senarath Pathirana 1 , Benthotage Chamara Jayasankha De Silva 1 , Sabrina Hossain 1 , Emi Sugaya 2 , Toshihiro Nakai 3 , Gang-Joon Heo 1, * 1 Veterinary Medical Center and College of Veterinary Medicine, Chungbuk National University, Cheongju, 28644, Korea 2 Faculty of Life Science and Biotechnology, Fukuyama University, Fukuyama, Hiroshima 729-0292, Japan 3 Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8528, Japan E-mail: gjheo@cbu.ac.kr Phone: +82 43 2612617 Abstract Edwardsiella tarda is one of the most significant bacterial pathogens to fish aquaculture worldwide. Here, we evaluated antibiotic resistance profiles, virulence genotypes and phylogenetic relationship of 30 Edwardsiella tarda isolates derived from cultured fish in Japan. The E. tarda isolates were phylogenetically (16S rRNA) clustered into two clads. The isolates of clad 1, which were mainly originated from olive flounder Paralichthys olivaceus and Japanese eel Anguilla japonica, had all of the known 11 virulence genes (ank, katB, LuxS, citC, astA, evpP, mukF, gadB, fimA, hlyA, esa) for E. tarda and mostly one or two of three aminoglycoside resistance genes, i.e. aac(6')-Ib, armA and strA-strB, if exists. In contrast, the isolates of clad 2, which were mainly originated from red seabream Pagrus major and black seabream Acanthopagrus schlegelii, lacked four (ank, katB, LuxS, citC) of the 11 virulence genes and, eight of them harbored mainly a β-lactam resistance gene (blaCTX-M). In infection experiments with zebrafish, representative isolates of the clad 1 showed higher virulence than those of the clad 2. These results reveal that the virulence profiles may be applicable for prediction of pathogenicity in fish. Phylogenetic analysis revealed that E. tarda isolates derived from olive flounder and red seabream are ancestor differentiated. Keywords: Edwardsiella tarda, edwardsiellosis, antibiotic resistance genes, virulence gene, pathogenicity, phylogenetics, Paralichthys olivaceus, Pagrus major Introduction In recent years, disease outbreaks caused by bacterial pathogens have affected fish farming all over the world. Edwardsiellosis caused by Edwarsiella tarda has been reported worldwide in economically important fish species, including Japanese eel Anguilla japonica, red sea bream Pagrus major, olive flounder Paralichthys olivaceus, channel catfish Ictalurus punctatus, Asian catfish Clarias batrachus and turbot Scophthalmus maximus (Alcaide, Herraiz, & Esteve, 2006; Castro et al., 2016; Kim et al., 2014; Xu & Zhang, 2014). At present, 17 antibiotics (antibacterial drugs) are approved for aquaculture use and from them 5 drugs are used to treat edwardsiellosis in Japan (Ministry of Agriculture, Forestry and Fisheries http://www.maff.go.jp/j/syouan/suisan/suisan_yobo/index.html). However, the massive use of antibiotics in