Role of beta-lactam hydrolysis in the mechanism of resistance of a beta-lactamase-constitutive Enterobacter cloacae strain to expanded-spectrum beta-lactams

Abstract

Enterobacter cloacae strains producing chromosomally mediated beta-lactamase constitutively show high degrees of resistance to most of the third-generation beta-lactams. It has been proposed that this resistance is due to the nonhydrolytic binding or trapping of beta-lactams by the enzyme. We found that the outer membrane of E. cloacae strain 55M indeed had permeability to cefazolin about 14-fold lower than that of Escherichia coli, and that the number of beta-lactamase molecules produced by this constitutive mutant was exceptionally large (2 X 10(5) per cell). These conditions are expected to produce a low degree of resistance, but could not explain the high resistance level of the mutant. We showed that the beta-lactamase of this strain hydrolyzed third-generation beta-lactams at measurable rates. Although the V max for these compounds was less than 0.01% of that for cefazolin, the enzyme could hydrolyze them at rates comparable to the rate for cefazolin when the substrate concentration was near 0.1 microM, a concentration thought to be physiologically relevant for the inhibition of cell growth, because of the exceptionally high affinity of the enzyme to many third-generation compounds. Calculations based on kinetic parameters of the enzyme, outer membrane permeability, and affinity toward penicillin-binding proteins succeeded in predicting the MICs for several third-generation beta-lactams. The data suggest that hydrolysis may be more important than nonhydrolytic binding for the expression of the resistant phenotype, and that studies on the susceptibility of beta-lactams to beta-lactamases should be carried out at physiologically relevant, very low concentrations of the drug, rather than the customary very high concentrations, such as 100 microM.