Piperacillin sodium salt

C109 was synthesized as previously described (12 (link)). Antibiotic working stock solutions were prepared as follows: C109, 10 mg/ml in dimethyl sulfoxide (DMSO); trimethoprim, 50 mg/ml in DMSO (Sigma); doxycycline hyclate, 25 mg/ml in H₂O (Sigma); chloramphenicol, 20 mg/ml in ethanol (EtOH; Sigma), meropenem, 10 mg/ml in DMSO (Sigma); tobramycin sulfate, 10 mg/ml in H₂O (Alfa Aesar); ceftazidime, 10 mg/ml in 0.1 M NaOH (Sigma); ciprofloxacin, 10 mg/ml in 0.1 M HCl (Sigma); novobiocin sodium, 10 mg/ml in H₂O (Sigma); and piperacillin sodium salt, 10 mg/ml in H₂O (Sigma). IPTG and L-rhamnose were obtained from Sigma.

E. coli K12 MG1655 (ATCC 700926) cells (5 × 10⁵ CFU mL⁻¹ mid-log phase in CAMHB) were exposed to various MIC-folds (MIC and 1/4×, 1/2×, 2×, 4×, 8×, 16×, 32× MIC) of arenicin-3 (MIC 0.25 μg mL⁻¹), AA139 (MIC 0.125 μg mL⁻¹), piperacillin sodium salt (Sigma-Aldrich, Cat # P8396; MIC 2 μg mL⁻¹), and colistin sulfate (Sigma-Aldrich, Cat # C4461; MIC 0.03 μg mL⁻¹). ATP was measured using the BacTiter-Glo^TM Microbial Cell Viability Assay (Promega) following the manufacturer’s instructions. The assay was performed for three biological replicates at 37 °C, with luminescence recorded using a Tecan Infinite M1000 Pro plate reader. The fold reduction of ATP was calculated as: Fold reduction ATP = 1 − (L_treat − L_media/L_control − L_media); where L_treat is the luminescence of treated cells, L_media is the luminescence of CAMHB without cells, and L_control is the luminescence of untreated cells. Resultant graphs show mean (n = 7) and std error for each data point, prepared in Prism 8. DMSO was included as a control as piperacillin was solubilized in DMSO, with final assay concentration of 2.5%.