Biofilm formation of wild-type D39 and Δrgg (SP68) and Over-EXP (SP44) mutants on eukaryotic cells. Cultures were grown in CDM++ to an OD600 of 0.1 and inoculated on top of a fixed lung cell line (A549). Every 12 h, the medium was changed to avoid bacterial lysis. At 36 h, the supernatant was removed, the biofilm was resuspended in rich medium, the wells were scraped, and the material obtained was immediately refrigerated for plating. The y axis shows the number of biofilm CFU recovered from wells as fold change compared to wild-type D39, and each value is the mean and the standard error of the mean of at least three independent biological experiments. ****, P < 0.0001.