Ceftriaxone

MP1102 with the purity of 96.4% was prepared based on our previous protocols (Zhang et al., 2015 (link)) and stored at −20°C before subsequent antibacterial assessments. Ceftriaxone and propidium iodide (PI) were purchased from Dalian Meilun Biological Technology Co., Ltd. and Sigma-Aldrich Shanghai Trading Co. LLC (Shanghai, China), respectively. Other reagents were of analytical grade. S. suis strains (CVCC 3928, 3309, and 606) isolated from piglets and Streptococcus pneumoniae CVCC 2350 were obtained from the China Veterinary Culture Collection (CVCC). S. pneumoniae CGMCC 1.8722 was obtained from China General Microbiological Culture Collection Center (CGMCC). Mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

Susceptibility to antimicrobials was determined in triplicate using the standard agar dilution method recommended by the Clinical and Laboratory Standards Institute (CLSI, 2020) [66 ]. The following eighteen antimicrobials were tested: kanamycin (KAN), gentamicin (GEN), erythromycin (ERY), azithromycin (AZM), norfloxacin (NOR), ofloxacin (OFX), ciprofloxacin (CIP), lomefloxacin (LOM), levofloxacin (LEV), sulfadiazine (SD), trimethoprim (TMP), ceftriaxone (CRO), cefixime (CFM), ampicillin (AMP), amoxicillin (AML), aztreonam (ATM), imipenem (IPM) and tetracycline (TET) (Meilun Biotechnology Co., LTD, Dalian, China). The isolates were grown on TSA plates, suspended in stroke-physiological saline solution to a turbidity equivalent to 0.5 McFarland Standard, and inoculated onto Mueller-Hinton agar plates using a multipoint inoculator (MIT-60P; Sakuma Seisakusyo, Tokyo, Japan). The final inoculum was approximately 10⁴ CFU per spot. Plates were incubated at 37 °C for 16–18 h. The range of 2-fold concentrations used to determine the susceptibility was determined by CLSI criteria. In addition, the results were interpreted in accordance with CLSI criteria (Supplementary Table S3). E. coli ATCC25922 and E. faecalis ATCC29212 were used as quality control strains.