Fig. 3: Mechanistic insights into synergy between bismuth drugs and CEF.

a Difference UV-vis spectra of CEF upon addition of 0.1–1.0 molar equivalents of CBS. A titration curve at 314 nm is shown as the insert. HEPES buffer at pH 7.4 was used. b Iron uptake by PAO1 detected by calcein-AM fluorescence intensity in the bacterial cells. During 300 s of monitoring, CEF at 50 μg ml⁻¹ was added at 60 s after the initiation of the monitoring. Calcein-AM coordinates with iron in bacterial cells, resulting in fluorescence to be quenched. The concentration of bismuth and iron compounds are 50 and 3 µM, respectively. c Final intensity of fluorescence in PAO1 after 1 h treatment. Mean value of three replicates are shown and error bars indicates ±SEM. p values were determined by one-way ANOVA test. ***p = 0.0002, ns p > 0.99. d Bismuth concentration per protein in cell lysates in the presence of different concentration of CEF determined by ICP-MS. CBS at 16 μM was supplemented to the bacterial cells for 30 min. n = 3 biologically independent samples. Error bars indicates mean ± SEM. p values were determined by two-tailed one sample t-test, **p = 0.0019, ***p < 0.001. e The uptake of bismuth and iron in bacterial cells treated with different concentration of CBS and 0.5 µM of CEF. Bismuth concentration was determined by ICP-MS and Fe concentration was determined by calcein-AM and normalized as percentage. n = 3 biologically independent samples. Mean value of three replicates are shown and error bars indicates ±SEM. f Representative heat plots of microdilution checkboard assay for the combination of CEF and CBS against PAO1ΔPiuA. Abs absorbance, Fe iron citrate, Bi colloidal bismuth citrate, CEF cefiderocol. Source data are provided as a Source Data file.