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The production of metallo-␤-lactamase (MBL) is an important mechanism of resistance to ␤-lactam antibiotics, including carbapenems. Despite the discovery and emergence of many acquired metallo-␤-lactamases, IMP-type determinants (now counting at least 27 variants) remain the most prevalent in some geographical areas. In Asian countries, and notably Japan, IMP-1 and its closely related variants are most widespread. Some other variants have been detected in other countries and show either an endemic (e.g., IMP-13 in Italy) or sporadic (e.g., IMP-12 in Italy or IMP-18 in the United States) occurrence. The IMP-18-producing Pseudomonas aeruginosa strain PS 297 from the southwestern United States carried at least two class 1 integrons. One was identical to In51, while the other, named In133 and carrying the bla IMP-18 gene cassette in the third position, showed an original array of five gene cassettes, including aacA7, qacF, aadA1, and an unknown open reading frame (ORF). Interestingly. In133 differed significantly from In96, the bla IMP-18 -carrying integron identified in a P. aeruginosa isolate from Mexico. The meropenem and ertapenem MIC values were much lower for Escherichia coli strains producing IMP-18 (0.06 and 0.12 g/ml, respectively) than for strains producing IMP-1 (2 g/ml for each). Kinetic data obtained with the purified enzyme revealed lower turnover rates of IMP-18 than of other IMP-type enzymes with most substrates.

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The IMP-13 metallo-␤-lactamase was overproduced in Escherichia coli BL21(DE3) and purified by chromatography. Analysis of kinetic parameters revealed some notable differences with other IMP-type enzymes, noteworthily a higher catalytic efficiency toward ticarcillin and piperacillin and a marked preference for imipenem over meropenem.Metallo-␤-lactamases (MBLs) are characterized by their ability to efficiently hydrolyze carbapenems and by their insensitivity to the commercially available ␤-lactamase inhibitors (10). Several types of acquired MBLs have been described worldwide for Gram-negative nonfermenters (GNNFs), among which the IMP-and VIM-type enzymes are the most widespread (9, 10). The IMP-13 MBL has 92.3% amino acid sequence identity with IMP-2 and 82.5% with IMP-1, being quite divergent from other variants. It has been identified in Pseudomonas aeruginosa clinical isolates from different countries in Europe and South America, sometimes associated with nosocomial outbreaks (2,5,8,12,13,15,16). The aim of this study was to purify and analyze the kinetic parameters of IMP-13 MBL.The bla open reading frame was amplified by PCR using primers IMP13-F (5Ј-CCGAATTCATATGAAGAAAT TATTTGTTTTATGT-3Ј, containing the EcoRI and NdeI restriction sites) and IMP13-R (5Ј-AGGGATCCTTAGTTACT TGGTGATGATGTTTT-3Ј, containing the BamHI restriction site). The source of the bla IMP-13 gene was a P. aeruginosa isolate from Argentina (13). The NdeI-BamHI fragment was cloned into the pLBII vector (1) and into the pET-9a expression vector (Novagen, Inc., Madison, WI) to obtain recombinant plasmids pLBII-IMP13 and pET-IMP13, respectively. The authenticity of the cloned fragments was confirmed by sequencing. Escherichia coli DH5␣ (Gibco Life Technologies, Gaithersburg, MD) was transformed with the pLBII-IMP13 plasmid, and the MICs of a representative panel of ␤-lactam antibiotics were determined at 37°C in Mueller-Hinton broth using the broth microdilution test according to the CLSI (3). The pET-IMP13 plasmid was introduced into E. coli BL21(DE3) (Novagen) to overproduce the enzyme. E. coli BL21(DE3)(pET-IMP13) was grown overnight in P0.5G noninducing broth, and 1.5 ml of the culture was then inoculated in 1 liter of autoinducing ZYP-5052 medium containing 100 g/ml of kanamycin (14). The highest specific activity, measured spectrophotometrically with total enzyme extracts, using 200 M imipenem as the substrate, were achieved in the supernatant fraction of the autoinducing broth (being 2-to 4-fold higher than that obtained in the whole-cell extract at 24 and 48 h, respectively), while the total activities in the supernatant and the cellular fraction were roughly similar.Enzyme purification was carried out as follows. The supernatant fraction was clarified with solid ammonium sulfate at a final concentration of 40% saturation. The enzyme was then precipitated with addition of solid ammonium sulfate to 75% saturation. After centrifugation, the isolated precipitate was resuspended in 40 ml of 50 mM HEPES buffer containing 50 M ZnSO 4 (pH 7.0...

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