REEVALUATION OF THE FACTORS INVOLVED IN THE EFFICACY OF NEW BETA-LACTAMS AGAINST ENTEROBACTER-CLOACAE

The roles of outer membrane permeability, beta-lactamase stability, and inhibition of penicillin-binding proteins in the activity of new beta-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 techniques that avoided certain possible artifacts of the traditional methods V(max) values were calculated from the numbers of enzymes molecules produced per cell and from catalytic constant (K(cat)) values, which were obtained with purified beta-lactamase. Minimal periplasmic antibiotic concentrations needed to inhibit bacterial cell wall synthesis were estimated from the Zimmerman-Rosselet equation. All the beta-lactams tested formed relatively stable complexes with purified beta-lactamase. The antibiotics that exhibit low affinity for beta-lactamase apparently needed higher periplasmic concentrations to inhibit cell wall synthesis, suggesting a possible correlation between the affinity of beta-lactamase and the affinity of penicillin-binding proteins for the new beta-lactams. By using these estimates of outer membrane permeability, beta-lactamase hydrolysis, and cell wall-inhibiting concentrations, MIC could be theoretically predict ed to within 1 dilution for five beta-lactams in three isogenic E. cloacae strains with differences in antibiotic susceptibility due to different porin or beta-lactamase contents. for five-beta-latams in three isogenic C. claoacae strains with differences in antibiotic susceptibility due to different porin or beta-lactamase contents.