Fig. 6: LCA-induced biofilms display increased antibiotic tolerance and can enhance the competitive fitness of EcN.

A The fraction of surviving cells after 24 h of treatment with 0.5 µg/mL ciprofloxacin, for LCA-induced biofilm vs. planktonic cells collected separately from the same cultures. Error bars represent the standard deviation (n = 3 replicates). ** p < 0.01, Welch’s t test. B The fraction of surviving cells after 24 h of treatment with 0.5 µg/mL ciprofloxacin, for LCA-treated vs. DMSO-treated cultures in which biofilm and planktonic cells were not collected separately. Error bars represent the standard deviation (n = 3 replicates); n.s. not significant (p > 0.05, Welch’s t test). C Growth curves in the presence of 1% DMSO vs. 100 µM LCA for EcN and the other enteric strains used in competition experiments. The drop in OD₆₀₀ for EcN upon entering stationary phase in 1% DMSO likely reflects the onset of self-aggregation (see Fig. 1D). Error bars represent the standard deviation (n = 6 replicates). D Quantification of biofilm formation (crystal violet absorption at OD₅₅₀ normalized to OD₆₀₀) after 24 h of treatment with 1% DMSO or 100 µM LCA, for the strains used in competition experiments. Error bars mark the standard deviation (n = 4 replicate wells from a representative experiment). E The percentage of the surface-attached population achieved by EcN, based on CFU counts, after 24 h of growth in pairwise competition with other strains, in the presence of 1% DMSO or 100 µM LCA. Error bars represent the standard deviation (n = 3 replicates). **p < 0.01, DMSO vs. LCA within each strain pairing (Welch’s t test followed by the Benjamini–Hochberg procedure for controlling the false discovery rate).