Fig. 7: Kaempferol inhibits ROS detoxification during colistin treatment.

a Sub-MIC amounts of colistin (1.22 μg/ml) induce ROS production, while kaempferol alone (at 0.375 mM) does not. The kaempferol and colistin combination treatment (colistin at 1.22 μg/ml and kaempferol at 0.375 mM) induces levels of ROS that significantly exceed those induced by colistin alone. The positive control consisted of hydrogen peroxide at a concentration lethal to A. baumannii (10% v/v) (Supporting Information Fig. S3). b Growth of sodB and sodC transposon mutants in the presence of DMSO, kaempferol, colistin and the combination of kaempferol and colistin (colistin was used at 1.22 μg/ml and kaempferol at 0.375 mM). The sodB mutant is more susceptible to sub-MIC amounts of colistin compared to wild-type A. baumannii or the sodC mutant. c Growth of AB5075 derivative strains overexpressing either sodB or sodC from a miniTn7-based IPTG-inducible system compared to an empty-vector control. The overexpression of sodB could not restore the growth after treatment with kaempferol and colistin (colistin was used at 1.22 μg/ml and kaempferol at 0.375 mM). However, the overexpression of sodC could significantly alleviate growth inhibition. Statistical comparisons were performed between the overexpression strains and the empty-vector control. d Fluorescence measurements from a transcriptional gfp fusion to the sodB and sodC promoter regions (PsodB and PsodC, respectively) after growth for 2 h in the presence of DMSO or the combination of kaempferol and colistin (colistin was used at 1.22 μg/ml and kaempferol at 0.375 mM). For both conditions (carrier control or combination treatment) sodB is expressed at higher levels than sodC. e Growth inhibition of A. baumannii in the presence of 0.375 mM kaempferol concentration in combination with sub-MIC amounts of colistin (1.22 μg/mL) is not rescued by Cu^2+, Mg^2+, Zn²⁺ or Ca²⁺ supplementation. All assays were carried out in biological triplicate, with three technical repeats. Analysis consisted of one-way ANOVA for (a) and two-way ANOVA for (b), comparing the treated samples with the DMSO carrier control. Two-way ANOVA analysis was carried out for (c) and (e) comparing the treated the supplemented samples and the H₂0 control. Two-way ANOVA analysis was carried out for (d) between PsodB::gfpmut3 and PsodC::gfpmut3. Average values ± S.D. are represented. Significance is indicated as ns = non-significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.