CHARACTERIZATION AND AMINO-ACID-SEQUENCE OF IRT-4, A NOVEL TEM-TYPE ENZYME WITH A DECREASED SUSCEPTIBILITY TO BETA-LACTAMASE INHIBITORS

The clinical isolate Escherichia coli PEY was highly resistant to amox ycillin, ticarcillin and piperacillin associated to beta-lactamase inh ibitors such as clavulanic acid, sulbactam, tazobactam and brobactam b ut susceptible to cephalosporins, aztreonam and imipenem. The suscepti bility to mecillinam indicated that this phenotype was not related to hyperproduction of the TEM-1 beta-lactamase. E. coli PEY produced a ne w plasmid-mediated inhibitor-resistant beta-lactamase of pI 5.2, which was named IRT-4. The determination of the amino acid sequence (Swiss- Prot accession number, P00810) of the purified protein indicated that IRT-4 differed from TEM-1 by two substitutions: Leu for Met-69 (ABL nu mbering) and Asp for Asn-276. A Met-69-Leu variant of TEM-1, obtained by site-directed mutagenesis, has been described as resistant to clavu lanate. The Asp for Asn-276 substitution has not been reported previou sly. The side chains of Asp-276 and Arg-244 were expected to interact. Determinations of 50% inhibitory concentrations of beta-lactamase inh ibitors and substrate profile of IRT-4 suggested that such an ionic bo nd was implicated in the alteration of the mechanistic process of TEM- 1 beta-lactamase.