FIG. 6.
Intracellular survival of S. pyogenes Manfredo wild-type (□) and isogenic mutant (■) bacteria in Hep-2C cells. Epithelial cells (5 × 105 cells/well) were incubated for 24 h in 24-well plates in antibiotic-free DMEM containing 10% fetal calf serum and 3 mM glutamine. The resulting monolayers were washed three times with fresh medium before streptococci were added. Infected monolayers were incubated for 90 min at 37°C in a humidified atmosphere of 5% CO2 and then washed three times with PBS to remove extracellular bacteria. Medium supplemented with gentamicin (200 μg ml−1) was added, and after a further 90 min of incubation (T = 0), the cells were washed three times with PBS and then either trypsinized and lysed with ice-cold sterile distilled H2O or incubated for a further 3 or 5 h in fresh DMEM plus gentamicin (5 μg ml−1) before being lysed. Numbers of released viable bacteria were determined by plating out serial dilutions. Control wells with no monolayers were set up and treated in the same way. Results show the mean number of viable streptococci recovered from the monolayers at each time point and the SEM and are representative of results obtained in at least two separate experiments.