Internalization assays of S. pyogenes in epithelial cells and macrophages. RDN29 (wild-type strain), EC478 (isogenic luxS-deficient mutant) and EC490 (RDN29 (pEC85)); RDN02 (wild-type strain), RDN306 (isogenic luxS-deficient mutant) and EC482 (RDN29 (pEC85)). Bacterial cultures were used to infect (A) HEp-2 human pharyngeal epithelial cells and (B) RAW 264.7 macrophages in an antibiotic protection assay. For this, bacterial cultures were grown to mid-logarithmic (RAW 264.7) and late-logarithmic (HEp-2) phases, growth time points showing optimal internalization rates in RAW 264.7 and HEp-2, respectively. The percent intracellular invasion represents the average CFU/ml number of viable intracellular bacteria recovered at different times following addition of antibiotics versus the average CFU/ml number of inoculated bacteria × 100. The average values and standard deviations of three independent assays are shown. The asterisks indicate that the differences in survival rates between the luxS-deficient mutants and the wild-type strains were statistically significant (P < 0.01 for Hep-2 epithelial cells, P < 0.05 for RAW 264.7 macrophages). Similar data were obtained for both M1 and M19 serotypes.