Rabbit Infective Endocarditis and Sepsis.

The infective endocarditis experiments were performed as previously described (34). Male and female New Zealand White rabbits (∼2 kg; Bakkom Rabbitry) were used for the experiments, and males and females responded similarly. Animal care and surgical procedures used were as described in the University of Iowa IACUC protocol number 6121907. Rabbits were anesthetized with ketamine (25 mg/kg) and xylazine (25 mg/kg; Akorn) and administered pain-relieving medications (buprenorphine 0.05 mg/kg twice daily subcutaneous; Reckitt Benckiser Healthcare) throughout the experiment. Each animal’s neck was shaved, and the left carotid artery was exposed by a 5-cm incision. A plastic catheter was inserted through the left carotid artery into each animal until it abutted the aortic valve. The catheter was then tied in place and left for 2 h. After 2 h, the catheter was removed and the animal sutured closed. The animals were injected intravenous through the marginal ear veins with 5 × 10⁸ S. aureus cells (MN8-WT + pRMC2, MN8ΔsrrAB + pRMC2, MN8ΔsrrAB + pRMC2::srrAB, and MN8ΔsrrAB + pRMC2::srrAB_C501A) grown in 10 μg/mL chloramphenicol. Chloramphenicol (2.5 mg) was injected subcutaneous into the rabbits daily to maintain plasmids. Plasmid stability of MN8 strains in rabbits was verified by plating vegetation homogenates in the presence of chloramphenicol, which showed colony growth indicating retention of plasmids (16, 45, 70). The rabbits were monitored for health status for up to 4 d. During this time, animals that simultaneously failed to exhibit escape behavior and maintain upright positions (a test 100% predictive of lethal infection) were killed with 1 mL/kg of Euthasol (Virbac). The animals were killed after 4 d or prematurely as indicated earlier. Their hearts were removed and vegetation dissected, weighed, and homogenized for colony-forming unit (CFU) determination. Four animals were used for the MN8 wild-type control group, and six animals each were used for remaining strain groups. Statistical analyses of the vegetation size and bacterial counts were performed (P values were calculated using unpaired t test) in SigmaPlot (Systat Software).