Neutropenic murine thigh infection model.

Mice were immunosuppressed using cyclophosphamide at 150 mg/kg on day −4 and at 100 mg/kg on day −1. Prior to infection on day 1, mice were anaesthetized with 2.5% isofluorane–97.5% oxygen. Once anesthesia was confirmed by the absence of pedal reflex, both thighs were infected by intramuscular injection with 50 μl of bacterial suspension containing ∼5 × 10⁵ CFU. While still under anesthesia, mice were administered 0.03 mg/kg buprenorphine for pain relief (this was readministered after 8 and 16 h postinfection). Mice were returned to cages in a warming cabinet with frequent observations until full recovery from anesthesia. Treatment was initiated 2 h postinfection by i.v. injection of a 5-ml/kg test article via the lateral caudal vein for doses of 150 to 600 cefepime mg/kg/day combined with 6 to 600 enmetazobactam mg/kg/day and via an indwelling JVC using programmed syringe pumps for doses of 1,200 mg/kg/day cefepime combined with 6 to 600 mg/kg/day enmetazobactam. Animals in pretreatment groups were euthanized 2 h postinfection, and animals of treatment groups were euthanized 26 h postinfection, both by a pentobarbitone overdose followed by cervical dislocation. Immediately following confirmation of death, thighs were removed and weighed. Thighs were placed individually in bead-beating tubes containing 2 ml of PBS plus 10% glycerol and subjected to mechanical disruption. Organ homogenates were diluted in PBS and cultured quantitatively on drug-free agar, resulting in two data points per animal (right and left thighs). Numbers of CFU per gram of tissue from each treatment group were converted to the log₁₀ of the group geometric mean [log₁₀(CFU/g)]. The terminal bioburden resulting from a specific treatment regimen was expressed as the log₁₀(CFU/g) difference between pretreatment and treatment groups [Δlog₁₀(CFU/g)]. Results from the dose fractionation (q4/q4 fractionation) and dose-response study employing isolate 1077711 confirmed the reproducibility of the in vivo data.