For susceptibility to oxacillin, an inoculum of 107 CFU/ml was prepared and the plate was incubated at 37°C for 24 hours on Mueller-Hinton agar + 2% NaCl. Antibiotic

disks were obtained from Biorad, Marne la Coquette, France. The 17 tested antibiotics were: benzyl penicillin (10 UI), oxacillin (5 μg), cefoxitin screen (30 μg), gentamicin (10 UI), tobramycin (10 μg), kanamycin (30 μg), vancomycin (30 μg), teicoplanin https://www.selleckchem.com/products/mek162.html (30 μg), fusidic acid (10 μg), buy GF120918 fosfomycin (50 μg), rifampicin (30 μg), trimethoprim/sulfamethoxazole (1.25/23.75 μg), erythromycin (15 μg), lincomycin (30 μg), pristinamycin (15 μg), linezolid (30 μg) and tetracyclin (30 UI). Toxin detection Phenotypic detection of toxins For the phenotypic detection of toxins radial gel immunodiffusion selleck compound was performed. The production of Panton-Valentine Leukocidin (PVL) and epidermolysins A (ETA) and B (ETB) were

evidenced from culture supernatants after 18 h of growth in Yeast Casamino-acid Pyruvate (YCP) medium [67] by radial gel immunodiffusion in 0.6% (wt/vol) agarose with component-specific rabbit polyclonal Arachidonate 15-lipoxygenase and affinity-purified antibodies [68, 69]. Genotype detection of toxins Presence of genes encoding for the 12 toxins, for which we don’t have antibody, was detected by Multiplex PCR using specific primers (Table 1) previously used for [70]. Then, the genes encoding for enterotoxins A (sea), B (seb), C (sec), D (sed), E (see), G (seg), H (seh), I (sei) and tsst were analyzed. Additionally, genes encoding PVL, ETA and ETB were also detected. Briefly, total DNA was purified

using QIAamp® DNA Mini Kit (Qiagen, GmbH, Germany) with a Gene Amp® PCR System 9700 (Perkin-Elmer, Norwalk, USA) and amplified in a total volume of 50 μl containing 25 pmoles of each primer, 50 ng of total DNA, 1.5 mM MgCl2, 200 μM of dNTP mixture, 1× PCR reaction Buffer and 5 units of Taq™ DNA polymerase (Invitogen™). The thermal cycling conditions included an initial denaturation step (2 min at 92°C) followed by 35 cycles of amplification comprising three steps: 2 min denaturation for 92°C, 1 min annealing at 50°C, 2 min extension at 72°C. The reaction was terminated with 3 min extension at 72°C. PCR products were analysed by electrophoresis through 1.4% (wt/vol) agarose gel (Euromedex, Mundolsheim, France).