Sulbactam

The Minimum Inhibitory Concentrations (MIC) of recombinant BL21(DE3) cells harboring bla_KPC-2 gene were determined by micro dilution method for different carbapenems alone and in combination with inhibitors (clavulanic acid, sulbactam, tazobactam, ZINC01807204 and ZINC02318494) against BL21 control cells (cells with null vector). The recombinant E. coli cells were induced with IPTG prior to performing experiments. The antimicrobial agents, imipenem, meropenem, ertapenem, clavulanic acid, sulbactam and tazobactam were obtained from Sigma Aldrich (USA) and compounds ZINC01807204 and ZINC02318494 were procured from Vitas M (Netherlands). The inhibitors were taken at a fixed concentration of 4 µg/ml. The results were interpreted according to the guidelines laid by Clinical Laboratory Standards Institute (CLSI) [12] . The MIC was determined as the lowest concentration that totally inhibits visible bacterial growth.

2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), FeCl3.6H2O, and analytical grade phenolic standards (catechin, protocatechuic acid, gallic acid, ellagic acid, gentisic acid, chlorogenic acid,
p-hydroxybenzoic acid, chicoric acid, 4-hydroxybenzaldehyde,
p-coumaric acid, rosmarinic acid, rutin, oleuropein, naringenin, quercetin, kaempferol, and hesperidin), dimethyl sulfoxide (DMSO), ofloxacin (OFX), netilmicin (NET), sulbactam (SCF), and maxipime were obtained from Sigma-Aldrich (St. Louis, MO, USA). Mueller Hinton Broth (MHB) and Mueller Hinton Agar (MHA) were obtained from Merck (Kenilworth, NJ, USA).

The minimum inhibitory concentrations (MICs) of ampicillin (AMP) and ceftriaxone (CEF) against ST^AS and ST^MDR were determined using a broth microdilution method. Antibiotic stock solutions were prepared to obtain a final concentration of 1024 μg/mL dissolved in water. Each antibiotic stock solution (100 μL) was serially (1:2) diluted with TSB in 96-well microtiter plates and 10⁵ CFU/mL of test strains were inoculated with and without sulbactam (SUL; Sigma Chemicals; St Louis, MO, USA) at 8 μg/mL. The plates were incubated at 37 °C for 18 h to determine MICs that are the lowest concentrations, where no bacterial growth was observed.

colistin (sulfate; CAS# 1264-72-8) and sulbactam (CAS# 68373-14-8) were obtained from Sigma-Aldrich (Saint Louis, USA). Stock solutions (5.12 mg/ml of each) were prepared using Milli-Q water and filtered through 0.22-μm syringe filters (Sartorius, Melbourne, VIC, Australia). All organic solvents used in metabolite sample extraction and the mobile phase of liquid chromatography (LC) were LC–mass spectrometry (LC-MS) grade and purchased from Merck Millipore (Bayswater, VIC, Australia).
A. baumannii AB090342 was collected from a patient (60–65 years old) with ventilator-associated pneumonia who received intravenous colistimethate sodium (150-mg colistin base activity every 12 h for 10 days) (Han et al., 2018a (link)). The patient was enrolled in a clinical study that was approved by the institutional review board of Sir Run Run Shaw Hospital (Zhejiang, China) and Huashan Hospital (Shanghai, China), and an informed consent form was obtained from the patient before the study. Minimum inhibitory concentrations of colistin (0.5 mg/L) and sulbactam (128 mg/L) were determined by broth microdilution.

Embelin (purity > 98%), meropenem, biapenem, cefepime ampicillin, cefradine, clavulanic acid, sulbactam, and tazobactam were purchased from Sigma Co. (United States). ATCC BAA-2146 was purchased from the American Type Culture Collection.