Determination of β-lactamase activity.

Crude lysates from overnight cultures of E. coli DH5α harboring pK_CTX-M plasmids were obtained as described before (47). β-Lactamase activity was determined spectrophotometrically by measuring the hydrolysis of 100 μM CEF as the substrate. One unit of β-lactamase activity was defined as the amount of enzyme hydrolyzing 1 nmol substrate per min (in 20 mM phosphate buffer, pH 7.0) at 25°C. The specific activity was defined as the units of β-lactamase per milligram of protein, determined by the Bio-Rad protein assay kit (Bio-Rad, USA).