Clinical isolates of Escherichia coli producing multiple TEM mutants resistant to beta-lactamase inhibitors.

Twenty clinical isolates of Escherichia coli resistant to amoxycillin and ticarcillin, both in combination with clavulanic acid, were studied. The ranges of MICs for these strains as determined by the agar dilution method were as follows: amoxycillin, 2048- > 4096 mg/L; ticarcillin, 512- > 4096 mg/L; piperacillin, 32-256 mg/L; mecillinam, 0.5-8 mg/L; and cephalothin 4-16 mg/L. Combining amoxycillin with beta-lactamase inhibitors, each at a fixed concentration of 4 mg/L, had only modest potentiating effects on the activities of this agent, the ranges of MICs falling to 256- > 2048 mg/L in the presence of clavulanic acid or sulbactam and to 64-1024 mg/L and 128-2048 mg/L in the presence of tazobactam and brobactam respectively. The pI values for the beta-lactamases produced by the 20 isolates were 5.2 for 15 strains, 5.4 for four strains and 7.4 for a single strain. Colony hybridization with oligonucleotides was performed in order to detect substitutions of arginine at position 241 (Arg-241) and methionine at position 67 (Met-67). Based on this technique, the four beta-lactamases with pI values of 5.4 were grouped into two oligotypes (+ = hybridization, - = non-hybridization)-Arg-241+, Met-67- (n = 3) and Arg-241+, Met-67+ (n = 1); in one of the three mutants which did not hybridize with the Met-67 probe, leucine had been substituted for methionine at position 67. The beta-lactamases with pI values of 5.2 which were identified in 15 strains were grouped into the following three oligotypes: Arg-241-, Met-67+ (n = 7); Arg-241-, Met-67- (n = 6); and Arg-241+, Met-67- (n = 2). In three of the 13 mutants which failed to hybridize with the Arg-241 probe, serine residues had replaced arginine residues at position 241. Substitutions of Arg-241 or Met-67 led to reduced affinities of the mutant enzymes for the beta-lactams tested. The results of the hybridization studies demonstrate that, amongst E. coli clinical isolates, there is a diversity of mutant TEM enzymes mediating resistance to beta-lactamase inhibitors.