Carbapenemase Activity Determination by CarbaNP Test

Activity of class A/B/D carbapenemases was determined by CarbaNP test (Dortet et al., 2012 (link)) with modifications. Overnight bacterial cell culture in MH broth was diluted 1:100 into 3 ml of fresh MH broth, and bacteria were allowed to grow at 37°C with shaking at 200 rpm to reach an OD₆₀₀ of 1.0–1.4. If required, ampicillin was used at 200 μg/ml. Bacterial cells were harvested from 2 ml of the above culture, and washed twice with 20 mM Tris-HCl (pH 7.8). Cell pellets were resuspended in 500 μl of 20 mM Tris-HCl (pH 7.8), and lysed by soniation, followed by centrifugation at 10,000 ×g at 4 °C for 5 min. 50 μl of the supernatant (the enzymatic bacterial suspension) were mixed with 50 μl of substrate I to V, respectively, followed by incubation at 37°C for a maximum of 2 h. Substrate I: 0.054% phenol red plus 0.1 mM ZnSO₄ (pH 7.8). Substrate II: 0.054% phenol red plus 0.1 mM ZnSO₄ (pH 7.8), and 0.6 mg/μl imipenem. Substrate III: 0.054% phenol red plus 0.1 mM ZnSO₄ (pH 7.8), 0.6 mg/μl mg imipenem, and 0.8 mg/μl tazobactam. Substrate IV: 0.054% phenol red plus 0.1 mM ZnSO₄ (pH 7.8), 0.6 mg/μl mg imipenem, and 3 mM EDTA (pH 7.8). Substrate V: 0.054% phenol red plus 0.1 mM ZnSO₄ (pH 7.8), 0.6 mg/μl mg imipenem, 0.8 mg/μl tazobactam, and 3 mM EDTA (pH 7.8).