A clinical Escherichia coli strain highly resistant to the combinations of amoxicillin-clavulanate, ampicillin-sulbactam, and piperacillin-tazobactam was isolated from a patient with a community-acquired urinary tract infection who was previously treated with amoxicillin-clavulanate. These resistances were carried by a 45-kb conjugative plasmid encoding for a single beta-lactamase with a pI of 5.4. Cloning and sequencing of the new beta-lactamase, IRT-3, revealed identity with the blaT1 gene encoding the TEM-1 beta-lactamase except for a replacement of the methionine residue at position 67 by isoleucine and of the methionine residue at position 180 by threonine. Both mutations were segregated by the construction of hybrid genes, and only the mutation at methionine at position 67 was related to resistance to the suicide inhibitors. The inhibitory effects of clavulanate, sulbactam, and tazobactam on the TEM-1 enzyme were substantially decreased in comparison with those on IRT-3, as indicated by the 50% inhibitory concentrations.