Hepes

Penicillin G, Piperacillin, Ceftriaxone, Cefepime, Cefazolin, Meropenem were purchased from Solarbio Co., Ltd. (Beijing, China). Imipenem was bought from Fresenius Kabi (Bad Homburg, Germany). HEPES was obtained from Sangon Biotech Co., Ltd. (Shanghai, China). All other reagents were purchased from Sigma-Aldrich Co (St. Louis, USA). The controlled pore glass (CPG) beads (125–140 μm particle diameter and 50 nm pore size) were originally purchased from VEB Trisola, Steinach, Germany. The construction of plasmid encoding NDM-1, expression and purification of recombinant NDM-1(rNDM-1) were performed as previously described (Meng et al., 2021a (link); Meng et al., 2021b (link)). The purity of the purified rNDM-1 is > 95%, The unit activity of rNDM-1 to hydrolyze Meropenem is 108 IU/mg.

The isolation method of mouse skin primary KCs was described previously^{68 (link)}. In brief, shaved back skin was incubated in 1% Typsin without EDTA at 37 °C for 1 h. After separating the epidermis from the dermis, the epidermis was cut into small pieces enough to enter the tip of a 10-ml pipette. Single-cell suspensions were prepared by repeated pipetting with a 10-ml pipette and filtering through a 100-μm cell strainer. Primary KCs were used for protein and RNA extractions.
For isolation of immune cells, mouse back skin was separated at the indicated time point, then cut into small pieces and incubated in RPMI 1640 medium containing 1 mg/ml collagenase IV (Sigma, #C5138), 50 μg/ml DNase I (Sangon Biotech, #A610099, Shanghai, China), 10 mM HEPES (Sangon Biotech, #E607018), and 10% FBS (Gbico, #12657-029) at 37 °C for 90 min. Digested skin pieces were passed through a 74-μm nylon mesh, and the suspensions were added additional RPMI 1640 to inactivate enzyme activity. Skin leukocytes were isolated by 30% and 70% percoll (GE Healthcare, #17-0891-01) at 1260 g for 20 min. The pellets were resuspended in PBS, and the cell number was counted.

Meropenem (MEM) and ceftazidime (CAZ) were obtained from USP (Rockville, MD, USA). Penicillin G (PEN), cefoxitin (FOX), cefaclor (CEC), ceftriaxone (CTRX), cefepime (FEP) and tazobactam (TAZ) were from Solarbio (Beijing, China). Avibactam (AVI), vaborbactam, fosfomycin (FOS), amikacin (AMK) and imipenem (IMI) were purchased from MedChemExpress (MCE, Monmouth Junction, NJ, USA). The purity of all antibiotics used in this study was higher than 95%. The propylamino-derivatized controlled pore glass (CPG) beads, with a diameter of 125–140 µm and pore size of 50 nm, were obtained from Steinachglas (Steinach, Germany). HEPES was purchased from Sangon Biotech (Shanghai, China). All other chemical reagents were purchased from Sigma-Aldrich (Shanghai, China) unless stated otherwise. Molecular biology enzymes were bought from Takara Bio (Dalian, China), and the primers were purchased from Sangon Biotech (Shanghai, China).

Three human colorectal cancer cell lines: HT29 cells (colorectal adenocarcinoma with p53 and APC mutation), LS174T cells (colorectal adenocarcinoma with KRAS mutation) and HCT116 cells (colorectal carcinoma with KRAS mutation) as well as normal colon, CCD841 CoN cells were obtained from American Type Cell Culture (Manassas, VA, USA). All cell lines were cultured in an incubator under the condition of 37°C temperature, humidified 5% CO₂ air atmosphere. Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Corning, NY, USA), supplemented with 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich, St Louis, MO, USA), 25mM HEPES (Sangon Biotech, Shanghai, China) and 1% Penicillin/Streptomycin (Merck, Germany).

Human bladder cancer BIU-87 cells (a kind gift from Professor Xiaofeng Yang of the Department of Urology, our hospital, China) were cultured in RPMI-l640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 0.05 g/L streptomycin (North China Pharmaceutical Group Corp., Shijiazhuang, China), 0.05 g/L penicillin (North China Pharmaceutical Group Corp., Shijiazhuang, China), 0.8 g/L NaHCO₃ (Tianjin DaMao Chemical Reagent Company), 3.6 g/L HEPES (Sangon Biotech Co. Ltd, Shanghai, China) and 10% fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials Co. Ltd, Hangzhou, China). The cells were incubated at 37 °C in a humidified environment containing 5% CO₂ (NU-5500E incubator, Nuaire, Plymouth, MN, USA). For the experiments, cells in the logarithmic growth phase were digested into suspension with 0.25% trypsin (Sangon Biotech Co. Ltd, Shanghai, China).