Nitrocefin

The β-lactamase activity of P. aeruginosa was assessed using UV-Vis Spectrophotometer (Aligent, Santa Clara, CA, USA) by measuring the hydrolysis of nitrocefin (Merck, Germany). The assay mixture consisted of 83 μg of nitrocefin, 167 μg of BSA (Merck, Germany), 10% glycerol(Merck, Germany) and 0.33 mL (0.6 μg/mL of albumin) (Merck, Germany) of cell lysate containing β-lactamase in a final volume of 1.5 mL of 50 mM phosphate buffer. The activity of β-lactamase was monitored by measuring the reduction in absorbance at 390 nm over a 10-min period at 37 °C. Enzyme activity was quantified as μmol of nitrocefin hydrolyzed per minute per milligram of protein, with the calculation based on the molar extinction coefficient of 15,000 M⁻¹ cm⁻¹ for nitrocefin. As a control, P. aeruginosa ATCC25922 was used in this method. Additionally, sub-minimum inhibitory concentrations (MIC) of natural products were employed as anti-ESBLs in the study [37 (link)].

Samples were grown overnight in LB-L media, then subcultured 1:100 in LB-L media and grown for 8 h at 37 °C and 225 rpm. The cultures were pelleted by one centrifugation step of 2272×g for 10 min and the supernatant was passed through a 0.45 μm filter. Samples were then subjected to a nitrocefin hydrolysis assay, per the substrate vendor (Sigma). 100 μL of reaction buffer (0.1 M phosphate, 1 mM ethylenediaminetetraacetic acid, 50 g mL⁻¹ nitrocefin (EMD Millipore), 0.5% dimethyl sulfoxide, pH 7) was mixed with 10 μL culture supernatant and the absorbance at 486 nm was observed over time. The reaction was performed in a disposable UV-Transparent cuvette (BrandTech, part# 759215) without stirring at 37 °C. The absorbance at 486 nm was measured every 5 s for 60 s with a UV–Vis spectrophotometer (Nanodrop, part# 2000c). The reaction was linear for the first 40 s of all reactions tested and the change in the absorbance as a function of time was determined using a linear least-squares fit. The initial reaction velocity was calculated using an extinction coefficient of 20,500 M⁻¹ cm⁻¹, as specified by the vendor. The experiment was performed on different days in biological triplicate. Error bars represent one standard deviation.

AmpC β-lactamase activity was determined by a nitrocefin hydrolysis assay as previously described (Cavallari et al., 2013 (link); Guerin et al., 2015 (link)). EC isolates were inoculated into LB medium and incubated at 37°C with 250 rpm overnight. It was sub-cultured in LB medium with a concentration of 1:100. When absorbance of OD600 reached 0.8, bacteria were collected and washed once with 1 ml of phosphate buffer (pH 7.0), and resuspended in 1 ml of protein lysate (Sangon Biotech Co., Ltd., Shanghai, China). Samples were placed on ice and lysed by sonication with a microprobe by using a 10-s pulse three times with a 10-s interval during each pulse. The samples were centrifuged at 10,000g for 10 min and supernatant was collected. The concentration of protein in supernatant was determined by a protein quantitative kit (Beyotime, Biotechnology, Shanghai, China). The nitrocefin hydrolysis assay was performed in 250 μl of phosphate buffer (pH 7.0) containing 5 μg of total protein and 50 μg/ml nitrocefin (Sigma-Aldrich; Merck KGaA, St. Louis, MO, United States). The hydrolysis rate of nitrocefin was determined at 486 nm at room temperature every 5 min. AmpC β-lactamase activity was calculated by extinction coefficient of nitrocefin 20, 500 M^–1 cm^–1, each assay was performed independently at least three times.