Fig. 1: Loss of cytochrome bd increases biofilm antibiotic sensitivity.

a PVC-associated air-liquid interface biofilms were grown for 48 h, treated with antibiotics for 72 h, and biomass was quantified using the crystal violet assay. Biofilm biomass was quantified for wild-type UTI89 and isogenic cytochrome bd-deficient strain ∆cydAB. Data were normalized to the untreated control of each strain. The mean absorbance value in each treatment group was compared to the untreated control of the same strain (asterisks), and to the mean value of the other strain in the same treatment group (horizontal line with asterisks) using a two-tailed Welch’s t-test. Data represent at least three biological replicates with at least eight technical replicates each. Each dot represents an independent well. b–d Dose response curves depicting the total biomass of biofilms after treatment with decreasing concentrations of ampicillin (b), gentamicin (c), or ciprofloxacin (d). Data were analyzed using a two-way ANOVA to evaluate overall differences between strain across the range of concentrations tested (horizontal line with asterisks), with multiple comparisons used to evaluate differences in mean at each concentration (asterisks). Data represent three biological replicates with three technical replicates each. Each dot represents the mean value of a biological replicate. Solid lines connect mean values at each concentration. e Representative images of antibiotic treated biofilms stained with STYO9 and imaged by confocal laser scanning microscopy. At least five images were acquired along the air-liquid interface of three biological replicates. Scale bar is 20 µm. f Survival of bacteria in colony biofilms after antibiotic treatment. Colony biofilms were grown on YESCA agar for 72 h, and biofilms were transferred to a new plate with or without antibiotics. After 24 h of antibiotic treatment, biofilms were homogenized and plated to enumerate CFUs. Mean values in each treatment group were statistically compared using a two-tailed unpaired t-test. Data are representative of at least five biological replicates. Each dot represents a biological replicate. For all graphs, data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.