Phenotype and molecular characterization of the first inhibitor-resistant TEM-derived beta-lactamase identified in Portugal.

Abstract

Since the description of the first TEM-derived β-lactamases conferring resistance to clavulanate, a number of these enzymes have emerged in various parts of the world, as profiled by Jacoby and Bush (http://lahey.org/studies/webt.htm). There is little information available on resistance to β-lactam-β-lactamase inhibitor combinations in members of the family Enterobacteriaceae in Portugal (2, 4). Here, we report the first phenotypic and molecular characterization of a Portuguese clinical isolate harboring an inhibitor-resistant TEM-derived β-lactamase (IRT). Escherichia coli strain INSRA99 was isolated in January 1999 from the urine of a 20-year-old woman hospitalized at SAMS Hospital in Lisbon. In disk diffusion tests at the National Institute of Health in Lisbon, the strain was resistant to amoxicillin and amoxicillin-clavulanate. Its phenotype and genotype were studied to determine if the clavulanate resistance was due to an inhibitor-resistant TEM-derived β-lactamase (IRT)-, an AmpC-, or OXA-type-producing mechanism or was due to the hyperproduction of TEM β-lactamases. Resistance was cotransferred to E. coli strain HB101. MICs of various β-lactams, alone or in combination with β-lactamase inhibitors (Table ​(Table1),1), were determined by the agar dilution method, as described in the 2000 report of the Antibiogram Committee of the French Society for Microbiology 2000-2001 (G. Carret, J. D. Cavallo, H. Chardon, C. Chidiac, P. Choutet, P. Courvalin, H. Dabernat, H. Drugeon, L. Dubreuil, F. Goldstein, V. Jarlier, R. Leclercq, M. H. Nicolas-Chanoine, A. Philippon, C. Quentin, B. Rouveix, J. Sirot, and C.-J. Soussy). Strains producing TEM-1, IRT-2, OXA-1, and AmpC enzymes were used as references. TABLE 1. MICs of β-lactam antibiotics for E. coli INSRA99, the transconjugant E. coli HB101-RA99, and other E. coli strains E. coli INSRA99 showed resistance to amoxicillin and ticarcillin, alone and in combination with β-lactamase inhibitors, and susceptibility to the other β-lactams (Table ​(Table1).1). The transconjugant E. coli HB101-RA99 showed less resistance than E. coli INSRA99, being susceptible to ticarcillin-clavulanic acid and to the other β-lactams. Isoelectric focusing showed a single enzyme with a pI of 5.2. A 1,092-bp blaTEM DNA fragment obtained by PCR and purified with Qiaquick spin columns (Qiagen, Hilden, Germany) was sequenced as previously described (3) with an ABI377 sequencer (Applied Biosystems, Foster City, Calif.). The sequence revealed a blaTEM-1-derived gene with the nucleotide substitution C929→A (Arg244→Ser). This mutation explains why the pI of INSRA99 β-lactamase is more acidic than that of TEM-1. We conclude that the enzyme is TEM-30 (IRT-2) (1). The coding regions of blaINSRA99 and blaHB101-RA99 are similar to that of the parental blaTEM-1B, but 1 nucleotide position in the promoter region, G162→T, differs, corresponding to the P4 promoter (5). The presence of T162 in the Pribnow box of blaINSRA99, blaHB101-RA99 in this study, and blaP20 (3) promoters may contribute to overproduction of the inhibitor-resistant enzymes produced by the corresponding E. coli strains. This promoter mutation was first identified in a blaESBL (6) and has been described in blaIRT genes (3): it was mostly associated with a TEM-2-like framework. However, the blaTEM-1B frameworks of the blaINSRA99 gene and of other blaIRT-2 genes (7) associated with the promoter variation enlarge the molecular diversity and indicate convergent evolution of blaTEM and blaIRT genes. In this study, the susceptibility profile of E. coli, the pI of the β-lactamase produced, and the molecular analysis of the gene coding for that enzyme are in agreement and suggest that there is a selective pressure from β-lactamase inhibitors in Portugal. The worldwide spread of resistance to β-lactamase inhibitors may impede the use of β-lactams for effective treatment of bacterial infections.