Large antibiotic-resistance plasmid of Edwardsiella tarda contributes to virulence in fish.

Edwardsiella tarda, an enteric gram negative bacterium, infects a wide range of fish and causes a systemic fish disease called edwardsiellosis. E. tarda CK41, isolated from Japanese flounder diagnosed with edwardsiellosis, has exhibited a high degree of resistance to multiple antibiotics, including kanamycin, tetracycline, streptomycin, among others. As the bacterial antibiotic-resistance genes are usually contained in plasmids, we hypothesized that E. tarda CK41 may harbor one or more plasmids for antibiotic resistance. We showed the existence of plasmids in E. tarda CK41, and the size of the plasmid, designated as pCK41, was estimated to be approximately 70 kb. Escherichia coli DH5α transformed by the pCK41 plasmid exhibited an antibiotic-resistance phenotype against kanamycin (30 μg/mL), tetracycline (30 μg/mL), and streptomycin (10 μg/mL), indicating the existence of at least 3 antibiotic-resistance genes in pCK41. Through a procedure for pCK41 plasmid curing, a plasmid-cured strain, designated as E. tarda CK108, was identified, which was unable to grow in the presence of either kanamycin or tetracycline. As virulence-associated genes are occasionally encoded in bacterial plasmids, we examined the virulence of E. tarda CK108 in Japanese flounder. The virulence of plasmid-cured E. tarda CK108 was lower (survival rate 80%) than that of CK41 (20%), indicating the existence of virulence-associated genes in pCK41. The strain also appeared to be attenuated in both goldfish and zebrafish pathogenesis models. To analyze genes for antibiotic resistance and virulence in pCK41, the entire nucleotide sequences of pCK41 were determined (GenBank accession number: HQ332785). A total of 84 open reading frames (ORFs) were annotated. The pCK41 plasmid consists of potential virulence genes, transposases, plasmid maintenance genes, antibiotic-resistance genes (including kanamycin, tetracycline, and streptomycin), conjugal transfer genes, and unknown ORFs. These results suggest that pCK41 is a virulence plasmid of substantial importance in the E. tarda pathogenesis to fish.