Fig. 2: Robust decline of ciprofloxacin resistance in different nfxB defective clinical strains of Pseudomonas aeruginosa in antibiotic-free environments.

Susceptibility to antibiotics from different structural families was analysed in populations (1 to 4) from 8 clinical isolates (64 populations) submitted to ALE assays in the absence of antibiotic (rich medium -LB- or rich medium supplemented with 10 µg/mL of dequalinium chloride -DC-) for 100 bacterial generations. Intensity of the colour is proportional to the log-transformed fold change of MIC of each replicate population (Rep) with respect to the MIC of the respective parental strain. Statistical significance was computed by non-parametric Kruskal–Wallis (Rank Sums) test followed by each-pair comparison test using the Wilcoxon method corrected by FDR. Only differences with FDR adjusted p-values of <0.05 were considered statistically significant. Changes of MICs above or below an increase or a decrease of 2-fold, respectively, were considered biologically relevant to classify a population as “resistant” (purple) or “susceptible” (yellow) with respect to its respective parental strain, as previously described²⁶. Detailed information on the biological and statistical significance of the comparisons is presented in the Source Data. An important decline of ciprofloxacin MIC was observed in 43 out of 64 populations evolved in antibiotic-free environments (LB or DC), presenting 42 of these populations MIC values below the EUCAST clinical breakpoint (Fig. 3 and Supplementary Table 2). CIP ciprofloxacin, TOB tobramycin, IPM imipenem, CAZ ceftazidime, ATM aztreonam, FOF fosfomycin.