FIG. 3.

E. sakazakii invasion properties. (A) Gentamicin protection assay performed either with or without serum added to the cell culture medium during the 1.5 h of bacterial infection. (B) At various times after infection (10, 30, 60, 90, and 120 min), Caco-2 cell monolayers were either treated with Triton X-100 to obtain the total number of cell-associated E. sakazakii cells or supplemented with gentamicin to determine the number of intracellular E. sakazakii cells. (C) Dependence of the infection rate on the number of cells present initially. Caco-2 cells were challenged with various numbers of bacteria (MOI, 2 to 250), and the number of gentamicin-protected E. sakazakii cells was determined after 1.5 h. The results are expressed as percent invasion (number of gentamicin-protected bacteria/initial number of bacteria × 100). (D) Effect of cell age and differentiation state on invasion and persistence. A gentamicin protection assay with E. sakazakii ATCC 29544 and E. coli XL-1 (control) was performed using Caco-2 cell monolayers, and polarity was generated over time by prolonged incubation. Fresh medium was added every 2 days. The data are the means and standard deviations of three independent experiments.