FIG 5.

Analysis of biofilm formation, dispersion, and subsequent adherence to HT-29 cells. (A) Analysis of glucose requirement in biofilm formation. Single colonies of S. flexneri 2457T were inoculated into medium (LB) with or without 0.4% (wt/vol) bile salts at the indicated percentage (wt/vol) of glucose. Crystal violet (left) and FITC-ConA (right) staining was performed, and the average OD₅₄₀ and fluorescence at 488 nm, respectively, ± SE are presented. Bile salts increased bacterial aggregation, and the addition of glucose further increased this phenotype. However, EPS production was dependent on the presence of glucose. For both the crystal violet and FITC-ConA staining, all differences between medium alone and the corresponding condition with bile salts are indicated with a double dagger (‡, P < 0.001). Differences in biofilm formation between each of the glucose plus bile salts concentrations relative to 0.0% glucose plus bile salts concentration are indicated with an asterisk (*, P < 0.01). (B) Analysis of bile salt removal in biofilm dispersion. S. flexneri 2457T biofilms were gently washed with PBS and resuspended in prewarmed PBS or PBS with the indicated treatments for 30 min at 37°C. The supernatants were subsequently removed and dilution plated, and CFU were determined. Dispersion was minimal in the presence of bile salts (*, P < 0.01), regardless of the presence of glucose. The average dispersion ± SE relative to the PBS control is plotted for three independent experiments in which technical triplicates were performed for each assay. For reference, the average recovery for the PBS treatment was 7.6 × 10⁷ CFU while the average recovery for the PBS with bile salts treatment was 7.8 × 10⁵ CFU. (C) Analysis of S. flexneri 2457T adherence following dispersion from the biofilm. Bacteria were grown overnight in media (LB) without or with a combination of glucose and bile salts to initiate biofilm formation. On the next day, bacteria were collected, washed with 1× PBS, and analyzed for adherence to HT-29 cells. Strain 2457T consistently had significantly increased adherence following biofilm dispersion (*, P < 0.0001) compared to bacteria recovered from medium without glucose and bile salts. The average percent recovery of adherent bacteria ± SE is plotted for three independent experiments, each of which had technical triplicates.