gyrA and parC associated with quinolone resistance in Vibrio anguillarum.

Vibrio anguillarum is a Gram-negative commashaped bacterium with polar flagella that is a member of the family Vibrionaceae. It is the cause of vibriosis or haemorrhagic septicaemic disease in wild and cultured marine and freshwater fish (Actis, Tolmasky & Crosa 1999; Austin & Austin 1999). Chemotherapeutic agents, including quinolones, have been widely used for the treatment of vibriosis in cultured fish and multiple drug-resistant strains of V. anguillarum have subsequently arisen (Aoki 1992; Pedersen, Tiainen & Larsen 1995; World Health Organization 1999). Drug-resistance genes on the transferable R-plasmid have been previously characterized, but the R-plasmid of V. anguillarum does not carry any genes for quinolone resistance (Aoki, Egusa & Arai 1974). The mutations in genes and amino acids associated with quinolone resistance in V. anguillarum are still unknown, and it would therefore be useful to characterize these mutations. There are two functional domains related to quinolone resistance in Gram-negative bacteria: DNA gyrase and topoisomerase IV (Hooper 2000). DNA gyrase is composed of two subunits, gyrA and gyrB, whereas topoisomerase IV comprises two subunits, parC and parE. Topoisomerase IV has DNA decatenating and relaxing activities, and plays an essential role in partitioning chromosomes at the terminal stage of chromosome replication (Huang 1996; Hooper 1999, 2000). The molecular mechanisms of quinolone and fluoroquinolone resistance have been described in several organisms (Hooper & Wolfson 1993; Hooper 2000; Chen & Lo 2003) and various point mutations in the quinolone resistance determining regions (QRDRs) of the gyrA and/or parC genes have been reported in some quinolone-resistant fish pathogens (Oppegaard & Sorum 1994; Okuda, Hayakawa, Nishibuchi & Nishino 1999; Ozanne, Benveniste, Tipper & Davies 2005). To understand the mechanism of quinolone resistance in V. anguillarum, the nucleotide sequences of the gyrA, gyrB, parC and parE genes were sequenced and characterized in this study. Twenty-five wild V. anguillarum strains isolated from cultured ayu, Plecoglossus altivelis (Temminck & Schlegel), in Japan, and V. anguillarum strain H775-3 were used (Table 2). The 25 wild strains included 10 oxolinic acid (OA)-sensitive strains [minimal inhibitory concentration (MIC): <0.4 lg mL] and 15 OA-resistant strains (MIC: 6.25–25 lg mL). Vibrio anguillarum were cultured at 25 C in tryptic soy broth (Difco, Baltimore, MD, USA) supplemented with 2% NaCl or in tryptic soy agar supplemented with 2% NaCl. Escherichia coli strain JM109 was grown in 2· yeast tryptone broth or Luria–Bertani broth. The stock solution of OA was prepared with 0.1 n NaOH. MICs were determined by the agar dilution method (Alderman & Smith 2001; Journal of Fish Diseases 2008, 31, 395–399 doi:10.1111/j.1365-2761.2007.00843.x