Reevaluation of the factors involved in the efficacy of new β-lactams against Enterobacter cloacae

The roles of outer membrane permeability, β-lactamase stability, and inhibition of penicillin-binding proteins in the activity of new β-lactams against Enterobacter cloacae were reappraised by using several methodological improvements. Outer membrane permeability in intact cells was determined by using a high-pressure liquid chromatography (HPLC)-based technique that avoided certain possible artifacts of the traditional methods. V(max) values were calculated from the numbers of enzyme molecules produced per cell and from catalytic constant (K(cat)) values, which were obtained with purified β-lactamase. Minimal periplasmic antibiotic concentrations needed to inhibit bacterial cell wall synthesis were estimated from the Zimmermann-Rosselet equation. All the β-lactams tested formed relatively stable complexes with purified β-lactamase. The antibiotics that exhibited low affinity for β-lactamase apparently needed higher periplasmic concentrations to inhibit cell wall synthesis, suggesting a possible correlation hetween the affinity of β-lactamase and the affinity of penicillin-binding proteins for the new β-lactams. By using these estimates of outer membrane permeability, β-lactamase hydrolysis, and cell wall-inhibiting concentrations, MIC could be theoretically predicted to within 1 dilution for five β-lactams in three isogenic E. cloacae strains with differences in antibiotic susceptibility due to different porin or β-lactamase contents.