A COMPLEX MUTANT OF TEM-1 BETA-LACTAMASE WITH MUTATIONS ENCOUNTERED IN BOTH IRT-4 AND EXTENDED-SPECTRUM TEM-15, PRODUCED BY AN ESCHERICHIA-COLI CLINICAL ISOLATE

Escherichia coli GR102 was isolated from feces of a leukemic patient, It expressed different levels of resistance to amoxicillin or ticarcil lin plus clavulanate and to the various cephalosporins tested, The dou ble-disk synergy test was weakly positive. Production of a beta-lactam ase with a pI of 5.6 was transferred to E. coli HB101 by conjugation. The nucleotide sequence was determined by direct sequencing of the amp lification products obtained by PCR performed with TEM gene primers, T his enzyme differed from TEM-1 (blaT-1B gent) by four amino acid subst itutions: Met-->Leu-69, Glu-->Lys-104, Gly-->Ser-238 and Asn-->Asp-276 . The amino acid susbstitutions Leu-69 and Asp-276 are known to be res ponsible for inhibitor resistance of the IRT-4 mutant, as are Lys-1O4 and Ser-238 substitutions for hydrolytic activity of the extended-spec trum beta-lactamases TEM-15, TEM-4, and TEM-3. These combined mutation s led to a mutant enzyme which conferred a level of resistance to coam oxiclav (MIC, 64 mu g/ml) much lower than that conferred by IRT-4(MIC, 2,048 mu g/ml) but higher than that conferred by TEM-15 or TEM-1 (MIC , 16 mu g/ml). In addition, the MIC of ceftazidime for E. coli transco njugant GR202 (1 mu g/ml) was lower than that for E. coli TEM-15 (16 m u g/ml) and higher than that for E. coli IRT-4 or TEM-1 (0.06 mu g/ml) . The MICs observed for this TEM-type enzyme were related to the kinet ic constants K-m and k(cat) and the 50% inhibitory concentration, whic h were intermediate between those observed for IRT-4 and TEM-15. In co nclusion, this new type of complex mutant derived from TEM-1 (CMT-1) i s able to confer resistance at a very low level to inhibitors and at a low level to extended-spectrum cephalosporins, CMT-1 received the des ignation TEM-50.