Penicillin g

STEC were distributed into 24-well plates and incubated at 37 °C in 5% CO₂ until the cells were confluent. Cells were then inoculated with HPS4-YC at different MOI of 10, 100 or 1000. Plates were centrifuged at 800 × g for 10 min. After 2 h of incubation at 37 °C with 5% CO₂, cells were washed three times with PBS to remove nonadherent bacteria. For the adhesion assay, the cells were lysed with double-distilled water and diluted appropriately with PBS. The number of adherent bacteria was determined by spreading on supplemented TSA. For the invasion assay, extracellular bacteria were killed by the addition of DMEM containing 100 µg/mL gentamicin (Solarbio, China) and 5 μg/mL penicillin G (Solarbio, China) for an additional 1 h. Antibiotic-treated cells were washed three times in PBS, lysed with double-distilled water, and diluted appropriately with PBS before spreading on supplemented TSA to determine bacterial counts. Assays were performed in triplicate and repeated three times.

HeLa229 cells were seeded in 6-well plates at a density of 6×10⁵ cells per well and were cultured overnight prior to chlamydial inoculation. After the medium was removed, C. trachomatis, diluted in 250 μL SPG, was directly inoculated onto the cell monolayers at an appropriate multiplicity of infection (MOI) as indicated for individual experiments for 2 h at 37°C with 5% CO₂. An equal amount of SPG without C. trachomatis was used to mock infect HeLa229 cells as a control. During chlamydial inoculation, 6-well plates were rocked slowly every 30 min to ensure the inoculum fully contacted with the cell monolayers. Following infection, the inoculum was removed and replaced with DMEM supplemented with 10% FBS. In the penicillin inhibition experiment, 100 U/mL penicillin G (Solarbio, Beijing, China) was added to the medium at the same time. Cells were cultured at 37°C in a 5% CO₂ incubator. At 0, 12, 24, 36 and 44 h post-infection, cells were washed twice with 1× phosphate-buffed saline (PBS), scraped off the plate, and then suspended in 250 μL PBS per well. The cell suspensions were transferred into 1.5-mL Eppendorf tubes, vortexed with 3-mm glass beads for 1 min, and then centrifuged at 500 g for 10 min at 4°C. The supernatants were collected and used in C5 cleavage assays and hemolysis assays.

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 THP‐1 cell line used in this study was purchased from the Cell Bank of the Chinese Academy of Sciences. THP‐1 cells were cultured in Roswell Park Memorial Institute medium (RPMI 1640; Gibco; Thermo Fisher Scientific) for subculture containing 10% of heat‐inactivated fetal bovine serum (Gibco; Thermo Fisher Scientific) and 1% penicillin‐streptomycin solution (100 U/ml penicillin G, 0.1 mg/ml streptomycin sulfate; Solarbio). Cell cultures were maintained in an incubator with 5% CO₂ at 37°C.