Figure 4.

The combination of pEt_20 and rifampicin reverses rifampicin resistance phenotype developed in A. baumannii (BAA‐1709) mutants. a) The rifampicin‐resistant mutants were selected by culturing the bacteria (≈10⁸ CFUs) on agar containing rifampicin at 1×, 2× and 5× MBC, i.e., 3.9, 7.8, and 19.5 µg mL⁻¹, respectively) for three days. The table shows tremendous MIC and MBC elevation for all the three mutants at each concentration, while the polymer at 0.5× MIC (i.e., 7.8 µg mL⁻¹) reversed rifampicin resistance phenotype in the mutants, lowering MIC and MBC by up to ≈2.5 × 10⁵‐fold. At 7.8 µg mL⁻¹, the polymer did not exert any bactericidal effect (≈0% killing efficiency as compared to CFU at 0 h). b) Fold reduction in rifampicin MIC. c) Fold reduction in rifampicin MBC against a mutant developed with rifampicin at 2× MBC in the presence of pEt_20 and colistin sulfate at their 0.5× MIC (i.e., 7.8 and 0.5 µg mL⁻¹). Although colistin sulfate reduced rifampicin MIC and MBC in the mutants, it is much less effective than pEt_20. d) Killing kinetics of rifampicin (0.5× MIC against A. baumannii 1709, 2.0 µg mL⁻¹), pEt_20 (0.5× MIC against both A. baumannii 1709 and rifampicin‐resistant A. baumannii 1709 mutants, 7.8 µg mL⁻¹), pEt_20/rifampicin combination (pEt_20: 7.8 µg mL⁻¹, rifampicin: 2.0 µg mL⁻¹), colistin sulfate (0.5× MIC against both A. baumannii 1709 and rifampicin‐resistant A. baumannii 1709 mutants, 0.50 µg mL⁻¹) and colistin sulfate/rifampicin combination (colistin sulfate: 0.50 µg mL⁻¹, rifampicin: 2.0 µg mL⁻¹) against rifampicin‐resistant mutants (developed from 2× MBC). Combination of pEt_20 and rifampicin killed rifampicin‐resistant A. baumannii 1709 (3 log reduction in bacteria count in 4 h), while the combination with colistin sulfate did not show any bactericidal activity (≈0% killing efficiency as compared to CFU at 0 h). Limit of detection: 100 CFU mL⁻¹. The data are representative of three biological replicates.