β-Lactamase activity assay

The β-lactamase activity was evaluated by using a nitrocefin hydrolyzing assay with minor modifications [11]. WT-ST, CI-ST, and CA-ST cells cultured with and without 1/2 MIC ceftriaxone were centrifugated at 3000×g for 20 min at 4 °C. The cell-free supernatants were mixed with 10 μL of 1.5 mM nitrocefin and incubated at 37 °C for 30 min. The absorbance was recorded every 5 min at 515 nm using a microplate reader (BioTek Instruments, Inc., Norwood, MA, USA). According to the protocol of β-lactamase activity assay kit (ancam, Seoul, Korea), the standard curve was generated and then the β-lactamase activity was expressed as μmol/min/mL.