Antibiotic efflux and permeability resistance mechanisms

We demonstrated the role of active efflux as a mechanism responsible for the intrinsic resistance of P. aeruginosa to specific antibiotics, like temocillin, or macrolides.
For macrolides, we demonstrated also that it regains activity when bacteria are cultivated in clinically-relevant media (serum, broncho-alveolar lavage), because of an increased permeability of the bacterial outer membrane in these specific environments. We are now studying the impact of efflux on resistance to temocillin in an international collection of strains isolated from cystic fibrosis patients. We also took advantage of the existence of this collection with impressive proportion of multiresistant strains for evaluating the impact of a novel inhibitor of beta-lactamase currently in phase III of clinical development on resistance to ceftazidime, one of the first-line drugs in these patients (Figure 6).

In parallel, we study the efflux of antibiotics from phagocytic cells and try to identify, at the phenotypic and genotypic levels, the transporter(s) involved. We also study the consequences of this active efflux in terms of cellular toxicity of antibiotics and of modulation of their activity against intracellular bacteria.

Figure 5. Penetration of the fluoroquinolone delafloxacin in S. aureus biofilms as a predictor of activity. Top: Confocal images of biofilms from three clinical isolates of S. aureus incubated with delafloxacin [blue], and labelled with live/dead staining [red: dead; green: live]. Middle: The graph compares the relative penetration of the antibiotic within the depth of the corresponding biofilm, expressed in percentage of the added concentration. Bottom: Correlation between relative potency of delafloxacin, its penetration within biofilms and the proportion of polysaccharides in biofilms, based on data obtained with 8 bacterial strains. Relative potency is estimated as C25 (concentration needed to reduce of 25 % viability within biofilms, penetration within biofilms) is determined in confocal microscopy, and the ratio of calcofluor white [evaluating polysaccharide content] fluorescence to crystal violet absorbance [evaluating biomass] is calculated based on quantitative determinations . The shaded areas show the normal contour density contour.		Figure 6: Effect of avibactam (4mg/L) on the activity of ceftazidime against 334 isolates of P. aeruginosa collected from cystic fibrosis patients. A: Cumulative MIC distribution with indication of MIC50, MIC90 and percentage of susceptibility according to the interpretive criteria of EUCAST (S ≤ 8 mg/L; R > 8 mg/L) and CLSI (S ≤ 8 mg/L; R > 16 mg/L). The dotted line points to the limit between susceptible and resistant strains according to EUCAST. B: Reduction in the MIC (± SD) of ceftazidime (expressed in number of dilutions) when combined to avibactam as a function of the ceftazidime MIC. The data were used to fit a log Gaussian equation (R2 = 0.979) allowing to calculate that the maximal amplitude of change (no. of dilutions; 4.3 ± 0.14) occurred for an MIC of 229 ± 29 mg/L. C: Correlation between MICs of ceftazidime alone and ceftazidime/avibactam for each individual strain in the collection using quantile density contour analysis. Colours (from warm [red] to cold [blue]) are indicative of the number of strains for each MIC combination. The dotted lines point to the MIC value above which the isolates are considered resistant strains according to EUCAST interpretive criteria and the figures indicate the percentage of strains in each quadrant. Chalhoub et al, 2015