PROPERTIES OF IRT-14 (TEM-45), A NEWLY CHARACTERIZED MUTANT OF TEM-TYPE BETA-LACTAMASES

IRT-14 (TEM-45) is a new mutant TEM-type beta-lactamase that was isola ted from clinical Escherichia coli P37 and that confers resistance to broad-spectrum penicillins with reduced sensitivity to beta-lactamase inhibitors, The MICs of amoxicillin alone and of amoxicillin combined with 2 mu g of clavulanic acid or 2 mu g of tazobactam per ml were 4,0 96, 2,048, and 1,024 mu g/ml, respectively. The strain was susceptible to cephalosporins, aztreonam, moxalactam, and imipenem, The enzyme wa s purified to homogeneity and values of the kinetic parameters k(cat), K-m, and k(cat)/K-m were determined for different substrates. This en zyme, with a pI of 5.2, was found to have reduced affinity for broad-s pectrum penicillins and cephalosporins, The values of 50% inhibitory c oncentrations of clavulanic acid, sulbactam, tazobactam, and brobactam are correlated with the higher K(m)s for substrates, The resistance o f E. coli P37 to mechanism-based inactivators results from a higher le vel of production of the TEM-derived enzyme due to the G-to-T substitu tion at position 162 (G-162-->T) in the promoter region of bla(TEM) an d from the structural modifications resulting from the Met-69-->Leu an d Arg-275-->Gln substitutions that characterize IRT-14 beta-lactamase.