Assessing Streptococcal Cellulitis in Mice

The ability of S. pyogenes to cause cellulitis in mice after subcutaneous inoculation was assessed using a procedure described elsewhere [17 (link)]. In brief, S. pyogenes 1529, which was clinical isolates from severe invasive disease in Japan [17 (link)], was harvested after 16-hour growth on brain heart infusion agar (Eiken Chemical, Tokyo, Japan) containing 0.3% yeast extract (BHY agar) mixed in 1 mL of phosphate buffered saline (0.15 M, pH 7.2, PBS) and then centrifuged at 2,000 × g for 2 min. The pellets were diluted in 1 ml PBS to 1×10⁸ CFU and then injected 1 × 10⁶ CFU under the skin surface of inbred 3-week-old female Slc:ICR mice (Japan SLC, Shizuoka, Japan) using a 27-gauge needle. The number of CFU injected was verified for each experiment by plating the bacteria on BHY agar and counting CFU. The general status of mice was observed daily. In the LCEEs-treated groups, mice were gavaged with each LCEE of the fruits, leaves, or stems (1 g/kg/day) on days −1, 0, 1, and 2 after S. pyogenes inoculation, respectively. Mice in the control group were given an equal volume of PBS and were infected using the same method (Figure 1). The experimental procedures were conducted according to Nagoya City University Guidelines for the Care and Use of Laboratory Animals, and the study protocol was approved by the local Animal Ethics Committee of Nagoya City University (H24-M11).