Pellets were washed once with a 4-ml aliquot of 50 mM
sodium phosphate buffer, pH 7.2, containing 145 mM sodium chloride, and were suspended in 400 μl of the same buffer. Over 99% of the ß-lactamase was associated with the centrifuged cell pellets, and therefore the assay was carried out using the washed cell suspension. A pair of 1.0-ml reaction mixtures was prepared containing 10 μl cell suspension, 10 μl 100 mM EDTA and 880 μl 50 mM sodium phosphate buffer, pH 7.0. The ML323 reaction was initiated by adding 100 μl 500 μM nitrocefin, and one tube was incubated for 3 min and the other for 13 min. The tubes were centrifuged at 12 000 × g for 2 min, and clear supernatant was separated. A486 was determined at 5 and 15 min. Reaction velocity per minute was calculated by subtracting A486 at 5 min from that at 15 min ATM/ATR activation divided by 10. Colour development from 5 to 15 min appeared linear under the conditions. For the cells with low ß-lactamase
activity, 100 μl cell suspension was used and incubated at 24°C for 30 min. One unit of the enzymatic activity was defined as μmol nitrocefin hydrolysis/min/mg protein. Quantification of cellular protein Cell suspensions were mixed with 2.0% of sodium dodecyl sulphate, and the mixture was heated at 100°C for 5 min and then centrifuged at 12 000 × g for 5 min. Protein concentration in the clear supernatant was determined using the BioRad Protein Assay kit (BioRad, Hercules, CA, USA) according to the 17DMAG concentration manufacturer’s instructions. Determination of MIC of antibiotics The MIC of antibiotics was determined by the agar dilution method according to the Clinical and Laboratory Carnitine palmitoyltransferase II Standards Institute manual . Extraction of plasmid DNA Bacterial cells were grown overnight in 5.0 ml brain–heart infusion broth (Becton–Dickinson) containing 10 μg/ml ceftizoxime, and harvested by centrifugation
at 6000 × g for 10 min. Cells were treated with 50 μg/ml lysostaphin at 37°C for 40 min. Plasmid DNA was extracted using the Qiagen Plasmid Mini kit, according to the manufacturer’s instructions. DNA was analysed by agarose gel electrophoresis (1.0%), stained with GelRed and visualised under UV light. Transformation experiments Transformation-competent cells were prepared according to the manufacturer’s instructions of the MicroPulser (BioRad). Transformation experiments were carried out using 250 ng DNA and the MicroPulser according to the manufacturer’s instructions. Transformants were selected on agar plates impregnated with a 1.5-fold MIC equivalent of ampicillin. Statistical analysis The χ2 and Fisher’s tests were carried out using a computer programme embedded in Microsoft Excel. Acknowledgement This study was supported in part by a grant-in-aid from the Food Safety Commission, Japan. References 1. Sakai F, Hanaki H, Barada K, Hirao Y, Inamatsu T, Nakae T, Sunakawa K: A 25-year trace of methicillin-resistant Staphylococcus aureus dissemination in a geriatric hospital in Japan. Internl J Gen Med 2010, 3:399–405. 2.