Columbia agar base

We used plates containing Columbia agar base (Becton Dickinson Norway AS) supplemented with 15 mg/L NAD (BioNor Laboratories AS, Norway) and 15 mg/L hemin (Sigma Aldrich Norway AS), as well as chocolate agar plates (Oxoid).
Selective agar plates were Columbia agar plates containing rifampicin (10 mg/L) (rif plates) and rifampicin (10 mg/L) + azithromycin (30 mg/L) (rif+azm plates). The plates were incubated at 35°C, 5% CO₂ for 24 or 48 h before colony counting.

Skim-milk agar was prepared by Columbia agar base (Becton, Dickinson and Company, Sparks, MD, USA) supplemented with 3% skim milk. GAS strains were sub-cultured on the Skim-milk agar and incubated at 5% CO₂, 37°C incubator for 12-16 h. Casein hydrolysis by GAS strains results in the appearance of a clear zone around bacterial colonies. For Western blot analysis, 30 μl of GAS culture supernatant was subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer to the polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% skim milk in PBST buffer (PBS containing 0.2% of tween-20) at 37°C for 1 h, and SpeB protein was detected by the anti-SpeB antibody (Toxin Technology, Inc., Sarasota, FL, USA) according to the manual. After hybridization, the membrane was washed with PBST and hybridized with the secondary antibody, peroxidase conjugated goat anti-rabbit IgG (1:10,000 dilution; Cell Signaling Technology, Inc., Danvers, MA, USA) at room temperature for 1 h. The blot was developed using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific Inc., Rockford, IL, USA) and the signal was detected by the Gel Doc XR+ system (BioRad).