Lactococcus garvieae, the causative agent of lactococcosis, has evolved strains that are highly resistant to antibiotics. Here, the 20,034-bp sequence of L. garvieae conjugative plasmid pKL0018 was determined. It contained two ermB genes and one tetS gene and a backbone more than 96% identical to that of pRE25, an Enterococcus faecalis plasmid from dry sausage.Lactococcus garvieae, a gram-positive bacterium that causes lactococcosis, has caused serious economic damage to fish aquaculture worldwide (11). The appearance of L. garvieae strains resistant to macrolides and tetracycline following antibiotic treatment practices has compounded the problem. The newly acquired resistance was attributed partly to a transferable R plasmid(s) carried by the resistant strains, based on studies done as early as 1990 (1).Of 170 strains of L. garvieae isolated from cultured Seriola species (yellowtail, amberjack, and kingfish) from nine prefectures in Japan in 2002, most have been reported to have high frequencies of erythromycin (EM), lincomycin (LCM), and oxytetracycline resistance, and all isolates possessed ermB and tetS genes (6). Moreover, the yellowtail-derived L. garvieae isolates appeared to be homogenous and very different from isolates obtained from other fish, terrestrial animals, and food plants (4,5,6). Recently, of 146 L. garvieae strains isolated from 1999 to 2006 from yellowtail farms in three prefectures in Japan, 46 strains had high levels of resistance to EM, LCM, and tetracycline and were found to be carrying transferable R plasmids that carry ermB and tetS genes, as evidenced by conjugation, Southern blotting, and PCR methods (7).In this study, the complete nucleotide sequence of R plasmid pKL0018 from a multiple-drug-resistant, gram-positive L. garvieae strain isolated from yellowtail was determined and analyzed.L. garvieae strains were cultured in Todd-Hewitt broth (Difco) and 2% NaCl at 25°C. Enterococcus faecalis strain OG1SS was used as recipient cells of R plasmid. The cells were cultured in Todd-Hewitt broth (Difco) at 37°C. R plasmid pKL0018, isolated from EM-, LCM-, and tetracycline-resistant L. garvieae strains from Kagoshima, Japan, in 2000, was sequenced using cloning vector pBluescript (Stratagene).R plasmid was transferred in mixed culture with E. faecalis OG1SS for 18 h at 37°C and was selected by culture in medium containing 150 g/ml of streptomycin and 100 g/ml of EM. Purified plasmid DNA was randomly fragmented by sonication, cloned into the sequencing vector pBluescript (Stratagene), and transformed into Escherichia coli JM109 by electroporation. Clones were then sequenced using a Thermosequenase sequencing kit (Amersham-Biotech) with an LC4200 (Li-Cor) automated DNA sequencer. Data were assembled and analyzed using the ATSQ program in GENETYX v. 7.0 (SDC Software Development Co.), BLASTX of NCBI (http://www.ncbi.nlm.nih.gov), ClustalW (http://www.ebi.ac.uk/Tools/clustalw2/), and BLAST 2 sequences (http://ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi).Sequence of R plasmid pKL0018. The R plas...