(2004, 2008). Lancefield serotyping (Lancefield, 1933) was performed using Pastorex Strep (Bio-Rad, Marnes-la-Coquette, France) according to the manufacturer’s protocol. Biochemical and enzymatic characterizations were performed using the API 20 STREP® and the API ZYM® systems (bioMerieux, Marcy-l’Etoile, France), respectively. All the isolates were cultured on blood agar (Columbia

agar base; Becton Dickinson) containing 5% sheep blood (Nippon Bio-Test Laboratories) at 37 °C for 24 h, and fresh colonies were evaluated according to the manufacturer’s instructions. learn more The antimicrobial susceptibility of the strains was determined using the disk diffusion method on Muller–Hinton agar (Difco Laboratories, Detroit, MI). The following R428 molecular weight chemotherapeutic agents (microgram per disk) were used in the disk diffusion method: oxytetracycline (30) (Eiken Chemical

Co. Ltd, Tokyo, Japan), erythromycin (15) (Oxoid, UK), florfenicol (30) (Oxoid), lincomycin (10) (Oxoid), and ampicillin (10) (Oxoid). The strains were considered resistant to oxytetracycline if the diameter of the inhibition zone around the disk was less than 19 mm (Constable & Morin, 2002). The presence of tet(L), tet(O), tet(S), and tet(M) genes that encode tetracycline resistance was investigated for all the resistant isolates by PCR according to the method reported previously (Agersøet al., 2002). Internal fragments representing 85% of the sodA gene of 23 fish isolates were amplified using the universal primer set and sequenced according to the method reported by Nomoto et al. (2008). The nucleotide sequences were analyzed using bioedit version 7.0 (Hall, 1999). The phylogenetic analysis was carried out using the neighbor-joining method using mega version 3 (Kumar

et al., 2004). The restriction enzyme-digested chromosomal Y-27632 2HCl DNA was analyzed by BSFGE, a modified pulsed-field gel electrophoresis (PFGE) technique (Madinabeitia et al., 2009). Streptococcus dysgalactiae strains were cultured on THA at 37 °C for 24 h, and the preparation of genomic DNA and DNA digestion with the restriction enzyme ApaI were carried out according to the previously described method (Nomoto et al., 2006). Macrorestriction fragments digested by ApaI were separated using a 1% agarose horizontal gel using the BSFGE system (Genofield; Atto, Tokyo, Japan). The biased sinusoidal electric field was applied for 20 h at DC 48 V and AC 288 V at a frequency of 0.005 Hz (initial) and 0.330 Hz (final). After gel electrophoresis, the gel was stained and visualized under UV light. The macrorestriction patterns were then calibrated and analyzed using the gene profiler software package along with treecon software (version 4.05; Scanalytics Inc., Fairfax, VA).