Amikacin

Nine antibiotics were used in the present study. Tetracycline (active ingredient 95.0%, dissolvent: ddH₂O, TET), ampicillin (USP grade, d: 1 mol/L HCl, AMP), ciprofloxacin (a.i. 90.0%, d: 1 mol/L NaOH, CPFX), sulfadiazine (a.i. 98.0%, d: DMSO, SUD), amikacin (a.i. 67.4%, d: ddH₂O, AMI), oxyTetracycline (a.i. 95%, d: 0.1 mol/L HCl, OTC), kanamycin (a.i. 75.0%, d: ddH₂O, KAN) and erythromycin (a.i. 94.1%, d: ddH₂O, EM) were kindly provided by Beijing Solarbio Science & Technology Co., Ltd., Beijing, China; rifampicin (a.i. 98.0%, d: DMSO, RFD) was generously provided by Wuhan Pytbio Bioengineering Co., Ltd., Wuhan, China. The above antibiotics were dissolved and diluted with the corresponding solvents to 50 mg/mL, a stock solution was prepared, and the mixture was stored at 4 °C for subsequent dilution in this study. Additionally, tryptone soybean agar medium (TSA) was used for the isolation and purification of bacteria; nutrient broth agar medium (NA) was used for the propagation of bacteria; 15% glycerol–nutrient broth medium (15% glycerol–NB) was used for the cryopreservation of bacteria; 0.3% agar–NB was used for the determination of bacterial motility; and Luria–Bertani medium (LB) and Luria–Bertani agar medium (LA) were used for the determination of bacterial resistance to antibiotics.

The phagocytosis assay was performed using the mouse macrophage cell line RAW 264.7 (ATCC catalogue number TIB-71) and peritoneal macrophages as previously described⁵³ . Peritoneal macrophages were collected from 7-week-old female C57BL/6J mice on the 5th day after intraperitoneal injection of 1 ml 5% thioglycollate broth (BD Difco, catalogue number 211716)⁵⁴ . Briefly, 5 × 10⁵ cells were infected with bacteria at a multiplicity of infection of 50 for 1 h. The cells were then washed three times with 1× PBS and treated with 500 μl Dulbecco’s modified eagle medium (Servicebio, catalogue number G4515) containing 300 μg ml⁻¹ gentamycin (Sangon Biotech, catalogue number A506614) or amikacin (Solarbio, catalogue number A9660) for 1 h to eliminate the extracellular bacteria. After washing three times with 1× PBS, the cells were treated with 1 ml of 1% TritonX-100 (Sangon Biotech, catalogue number A110694) for 10 min to release the intracellular bacteria. Then intracellular bacteria were counted by serial dilution agar plating method.