Figure 7. Chemical inhibition of the DSB system sensitizes multidrug-resistant clinical isolates to colistin.

(A) Addition of a small-molecule inhibitor of DsbB to a colistin-resistant clinical E. coli isolate expressing MCR-1 results in sensitization to colistin. (B) Chemical inhibition of the DSB system in the presence of colistin (final concentration of 2 μg/mL) results in drastic changes in cell morphology for the E. coli clinical isolate used in panel (A), while bacteria remain unaffected by single treatments (DSB inhibitor or colistin). Images show representative scanning electron micrographs of untreated cells (top row, left), cells treated with the DSB inhibitor (top row, middle), cells treated with colistin (top row, right), and cells treated with both the DSB inhibitor and colistin (bottom row). Scale bars are at 400 nm. (C) Chemical inhibition of the DSB system results in sensitization of four additional colistin-resistant E. coli strains expressing MCR enzymes. For panels (A) and (C), graphs show MIC values (µg/mL) from four biological experiments, each conducted in technical quadruplicate, to demonstrate the robustness of the observed effects. (D) Use of the DSB system inhibitor on the same clinical E. coli isolate tested in panel (A), results in intermediate resistance for ceftazidime as defined by EUCAST. The graph shows MIC values (μg/ml) from two biological experiments, each conducted as a single technical repeat. For panels (A), (C), (D), MIC values determined using Mueller-Hinton agar (MHA) in accordance with the EUCAST guidelines (dark blue bars) are comparable to the values obtained using defined media (M63 agar, white bars); use of growth media lacking small-molecule oxidants is required for the DSB system inhibitor to be effective. For all panels, red dotted lines indicate the EUCAST clinical breakpoint for each antibiotic, and purple dotted lines indicate the EUCAST threshold for intermediate resistance.