Fig. 4: OprF is required for sub-MIC antibiotic-induced biofilm stimulation.
a Sub-MIC antibiotics fail to stimulate biofilm formation in an oprF::FRT mutant (N = 3). b Expression of OprF from pHERD30T in trans restores (either fully or partially) the biofilm response to sub-MIC cefixime, thiostrepton, and tobramycin (N = 3). c Multiple antibiotics fail to stimulate biofilm formation of an oprF mutant (N = 3). Notably, the oprF mutant was ~16x more sensitive than the wild type to trimethoprim. d Expression of a truncated OprF that lacks the C-terminal PG-binding region (oprFΔC-term) from pHERD30T restores (either fully or partially) antibiotic-induced biofilm formation in an oprF mutant (N = 2). e OmpA, the OprF homolog in E. coli, is required for biofilm stimulation by sub-MIC cefixime, novobiocin, or tetracycline in E. coli K12 (N = 2). Planktonic growth (OD600, yellow) and biofilm (A600, purple) are reported as percentage of the vehicle control. A two-way ANOVA followed by Tukey’s multiple comparisons test was performed for the biofilm formation values for each panel. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Each biological replicate has 3 technical replicates, and a representative biological replicate is shown with the circle or triangle symbols representing individual data points. The graphs show the maximum amount of biofilm observed from a dose-response assay (such as those shown in Fig. 1) for each antibiotic/strain. Maximum biofilm formation occurred at 5 µM for cefixime, 10 µM for thiostrepton, 0.2 µM for tobramycin, 200 µM carbenicillin, 4 µg/mL for chloramphenicol, 0.25 µg/mL ciprofloxacin, 150 µg/mL novobiocin, and 16 µg/mL trimethoprim. For oprF mutant strains that are not complemented, maximum biofilm formation tended to occur at the lowest concentration of antibiotic as biofilm formation decreased at higher antibiotic concentrations. Error bars represent the standard deviation. Source data are provided in Supplementary Data Set 1.
