Mouse thigh infection model.

The antimicrobial synergy of the polymyxin B-zidovudine combination against NDM-producing K. pneumoniae 02 was evaluated in a neutropenic murine thigh infection model. All animal experiments were approved by the Monash University Animal Ethics Committee and conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Female Swiss mice (8 to 10 weeks old) were employed in the neutropenic murine thigh infection model. Mice were injected intraperitoneally (i.p.) with cyclophosphamide on days −4 (150 mg/kg) and −1 (100 mg/kg) to induce neutropenia (6, 7, 35, 51, 52). On day 0, mice were injected 50 μl of an early logarithmic phase bacterial suspension (∼10⁷ CFU/ml) into each thigh to achieve an inoculum of ∼10⁶ CFU/ml. Neutropenic mice infected with NDM-producing K. pneumoniae 02 were treated with saline or antibiotics 2 h post-bacterial inoculation. There were a total of four treatment groups, namely, (i) 0.9% saline-treated group as the control, (ii) 10 mg/kg polymyxin B thrice daily (8 hourly; maximum daily dose, 30 mg/kg/day), (iii) 200 mg/kg zidovudine thrice daily (8 hourly; maximum daily dose, 600 mg/kg/day), and (iv) combination of 10 mg/kg polymyxin B thrice daily and 200 mg/kg zidovudine thrice daily. Polymyxin B was administered via subcutaneous injection, while zidovudine was administered via i.p. injection. The polymyxin B dose was selected to mimic the PK of polymyxin B in humans (5, 28), whereas zidovudine doses were based on the LD₅₀ in rodents (38). At 0 and 24 h, the bacterial burden was determined. Mice were humanely killed, and thighs were aseptically removed and homogenized. The homogenate was filtered and subsequently serially diluted with 0.9% saline and spiral plated onto nutrient agar with subsequent incubation at 35°C for 24 h. Colonies were counted using a ProtoCOL colony counter, and CFU values were expressed as log₁₀ CFU/thigh. The limit of detection was 164 CFU/thigh (equivalent to one colony per plate). Tukey’s multiple comparison test was used to compare the groups at 24 h and a P value of <0.05 was considered statistically significant.