Murine infection model.

Murine models were used to test the virulence of both the WT and agr mutant strains as we previously reported (36,–38, 40). Cultures were grown overnight at 37°C in a shaking incubator set at 200 rpm from frozen glycerol stocks for two selected wild-type and agr mutant strain pairs (strains 116 and 399) in 5 ml of TSB. Subcultures were prepared to a total volume of 350 ml, consisting of 34.65 ml of TSB and 350 μl of an overnight culture, and then grown to an OD₆₀₀ of 0.5 at 37°C in a shaking incubator at 200 rpm. Ten milliliters of this culture was then added to 40 ml of Dulbecco’s phosphate-buffered saline (DPBS) and centrifuged at 3,500 rpm for 10 min. The pelleted cells were then resuspended in sterile saline to reach a concentration of 2 × 10⁷ CFU/50 μl and placed on ice. Five BALB/c mice were used to test each strain, resulting in the use of 20 mice in total. Mice were anesthetized with 1 to 5% isoflurane. The abdominal skin of each mouse was shaven with a microtome blade; the mice were then labeled, weighed, and prepped for sterile injection. Insulin syringes were used to intradermally inoculate each mouse with 50 μl of a specified S. aureus suspension. The mice were monitored daily, with the lesion size (in square centimeters) as well as weight loss being recorded as markers of disease for up to 2 weeks. All mouse experiments were approved by the University of Iowa Institutional Animal Care and Use Committee.