Murine thigh infection model.

The neutropenic murine thigh infection model was used for the in vivo study of antofloxacin. Animals were maintained in accordance with the standards of the National Standards for Laboratory Animals of China (GB 14925-2010). All animal studies were approved by the Animal Research Committees of the South China Agriculture University. Six-week-old, specific-pathogen-free, female ICR mice weighing 24 to 27 g (Guangdong Medical Lab Animal Center, Guangzhou, China) were used in this experiment. Neutropenia (neutrophil count, ≤100/mm³) was induced by two doses of cyclophosphamide injected intraperitoneally 4 days (150 mg/kg) and 1 day (100 mg/kg) before experimental infection. An early-logarithmic-phase bacterial suspension (0.1 ml) consisting of approximately 10⁶ to 10⁷ bacteria was inoculated intramuscularly into each posterior thigh muscle of the mice. Antofloxacin was subcutaneously administered 2 h after thigh infection. After 24 h of treatment, the thighs (four per group) were immediately removed upon euthanasia and homogenized in 3 ml of sterile saline. Each homogenate was serially diluted 10-fold, and 100-μl aliquots of each dilution were plated on MHA plates for counting of the number of CFU of bacteria in each thigh.

This infection model was also used to confirm plasmid stability and for bioluminescent strain characterization, performed by measurement of the bacterial counts in homogenized thigh cultures. Plasmid stability was assessed by comparing the bacterial counts after plating on MHA with and without ampicillin. Bioluminescent strain characterization was done by comparing the bacterial counts of E. coli isolates carrying pAKlux2 with those of E. coli isolates lacking the plasmid.