Properties of extended-spectrum beta-lactamases constructed by site-directed mutagenesis.

Plasmids carrying three types of TEM-type extended-spectrum beta-lactamase (ESBL) genes, encoding TEM-3, TEM-5, and TEM-9, respectively, were constructed by site-directed mutagenesis. ESBL producers were prepared by transformation of Escherichia coli JM109 with a plasmid carrying one gene of either the three TEM types, an SHV-type, or a Toho-1 group gene. This strategy with the same vector and host strain can exclude the contribution of other factors to susceptibility, and is useful in Japan, where few TEM-type ESBL producers have been isolated. In vitro antibacterial activities of 23 beta-lactam antibiotics were tested against the ESBL producers by the agar dilution method, and the results were compared. The minimum inhibitory concentrations (MICs) of penicillins tested were more than 32 micro g/ml against both the parental RTEM and ESBL producers, but they were substantially decreased by a combination with beta-lactamase inhibitors. Compared with the MICs against the ESBL-nonproducing host strain, the MICs of the cephalosporins tested for the ESBL producers were increased more than eight times in most cases and in several cases soared to more than 2048 times against a Toho-1 ESBL producer. On the other hand, the MICs of carbapenem, cephamycin, and penem antibiotics were generally comparable to those against the host strain, and were increased by 32 times at most. Kinetic analysis revealed that extended-spectrum cephalosporins were hydrolyzed only slightly to moderately by the TEM-type ESBLs, while carbapenems and a cephamycin were scarcely hydrolyzed, and rather inhibited or inactivated the mutant enzymes.