Fig. 3: Determination of the protection from antibiotic treatments due to the bacterial behaviour during infection caused by a mutation in mexZ. | Nature Communications

Fig. 3: Determination of the protection from antibiotic treatments due to the bacterial behaviour during infection caused by a mutation in mexZ.

From: Mutations in the efflux pump regulator MexZ shift tissue colonization by Pseudomonas aeruginosa to a state of antibiotic tolerance

Fig. 3

a Colony Forming Units (CFUs) of PAO1, mexZ*, and mexZ*ΔlecA in the apical (red) and basolateral (blue) ALI compartments, and attached to the cell layer (green), after 7 h of infection in fully differentiated BCi-NS1.1 cells, a timepoint after which antibiotics were added. Data are presented as mean values ± SEM of the results from three biological replicates with three technical replicates each. Statistical significance was determined by two-sided t-test assuming equal variances for CFU measurements and indicated as ** (p < 0.005) and *** (p < 0.0005): attached CFUs PAO1 vs mexZ* p = 0.00007; Attached CFUs mexZ* ΔlecA vs mexZ* p = 0.0025. b Fold change of Transepithelial Electrical Resistance (TEER) (Ω·cm2) of mock ALI cell layers uninfected (grey) and after 7 h of infection or 7 h of infection followed by 12 h of treatment with tobramycin (TOB), ceftazidime (CAZ) of ciprofloxacin (CIP) of PAO1 (orange), mexZ* (blue) or mexZ*ΔlecA (purple) strains respect to the TEER before starting the infection experiment. Data are presented as mean values ± SEM of the results from three biological replicates. Statistical significance with respect to the mock uninfected control cells was determined by two-sided t-test assuming equal variances for TEER measurements and indicated as *** (p < 0.0005) and **** (p < 0.00005): PAO1 7hpi (p = 0.19), mexZ* 7hpi (p = 0.15), mexZ*ΔlecA 7hpi (p = 0.06), PAO1 7hpi + 12 h TOB (p = 0.33), mexZ* 7hpi + 12 h TOB (p = 0.000027), mexZ*ΔlecA 7hpi + 12 h TOB (p = 0.00046), PAO1 7hpi + 12 h CAZ (p = 0.08), mexZ* 7hpi + 12 h CAZ (p = 0.00017), mexZ*ΔlecA 7hpi + 12 h CAZ (p = 0.51), PAO1 7hpi + 12 h CIP (p = 0.38), mexZ* 7hpi + 12 h CIP (p = 0.000034), mexZ*ΔlecA 7hpi + 12 h CIP (p = 0.000051). c CFUs of PAO1, mexZ*, and mexZ*ΔlecA in the apical (red) and basolateral (blue) ALI compartments, and attached to the cell layer (green), after 7 h of infection followed by 12 h of treatment with tobramycin (TOB), ceftazidime (CAZ) or ciprofloxacin (CIP) in fully differentiated BCi-NS1.1 cells. Data are presented as mean values ± SEM of the results from three biological replicates with three technical replicates each. Statistical significance for CFU measurements after antibiotic treatments in the infection system regarding PAO1 was determined by two-sided t-test assuming equal variances and indicated as ** (p < 0.005), *** (p < 0.0005) and **** (p < 0.00005): mexZ* 7hpi + 12 h TOB (p = 0.0000025), mexZ*ΔlecA 7hpi + 12 h TOB (p = 0.0036), mexZ* 7hpi + 12 h CAZ (p = 0.0000057), mexZ*ΔlecA 7hpi + 12 h CAZ (p = 0.65), mexZ* 7hpi + 12 h CIP (p = 0.000064), mexZ*ΔlecA 7hpi + 12 h CIP (p = 0.00038). d CFUs of PAO1 (orange), mexZ* (blue), and mexZ*ΔlecA (purple) present in the concentration corresponding to half of the PAO1 MIC of tobramycin (TOB), ceftazidime (CAZ), or ciprofloxacin (CIP) in 100 μL of Müller Hinton (MH) medium. Data are presented as mean values ± SEM of the results from three biological replicates with three technical replicates each. Statistical significance for CFU measurements in the MIC determination set up in MH regarding PAO1 was determined by two-sided t-test assuming equal variances and indicated as * (p < 0.05) and ** (p < 0.005): mexZ* TOB (p = 0.0036), mexZ*ΔlecA TOB (p = 0.031), mexZ* CAZ (p = 0.4), mexZ*ΔlecA CAZ (p = 0.66), mexZ* CIP (p = 0.019), mexZ*ΔlecA CIP (p = 0.00066). Source data are provided as a Source Data file.

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