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Inhibitor-resistant TEM (IRT) β-lactamases derive from TEM-1 or TEM-2 enzymes by point mutations in the corresponding coding gene (http://www.lahey.org/studies). Inhibitor-resistant SHV (IRS) β-lactamases are less commonly identified than IRT enzymes, and mainly in Klebsiella pneumoniae strains (3, 4, 5, 8, 9). In this study, we performed a phenotypic, molecular, and biochemical characterization of a new IRS enzyme, SHV-84, produced by a clinical Escherichia coli strain.
E. coli INSRA4590 was isolated from a patient admitted in 1999 to Centro Hospitalar de Coimbra, Portugal. Antimicrobial susceptibility, isoelectric focusing for isoelectric point determination, identification of β-lactamase-encoding genes, and extraction and purification of a new β-lactamase were performed as previously described (1, 7). Antimicrobial susceptibility results were interpreted by using French Society of Microbiology criteria (2). E. coli INSRA4590 exhibited high-level resistance to the penicillins tested (except piperacillin) and to the combination of amoxicillin and clavulanic acid but was susceptible to the combination of piperacillin and tazobactam (Table (Table1);1); it was susceptible to all cephalosporins tested and to aztreonam.
The clinical strain expressed SHV-1 with a pI of 7.6 and the new SHV-84 β-lactamase with a pI of 7.4, which differed from SHV-1 by the amino acid substitution Lys234Arg. This mutation has been described in other IRS enzymes, such as SHV-56 (5) and SHV-72 (also encountered in Portugal) (8), in which it was associated with the substitution Leu35Gln and the substitutions Ile8Phe and Ala146Val, respectively. The recombinant SHV-encoding plasmid (pBK-SHV-84) and the corresponding transformant E. coli DH5α were obtained as previously described (8). The SHV-84-producing transformant DH5α-SHV-84 exhibited a β-lactam resistance phenotype similar to that of the clinical strain (Table (Table1),1), and the corresponding β-lactamase had a pI of 7.4.
The kinetic constants of the purified enzyme (≥99% pure; data not shown) and the concentrations of inhibitors required to inhibit enzyme activity by 50% (IC50s) were determined as previously reported (6, 8) and compared with those of SHV-1. Unlike SHV-72, SHV-84 showed a lower affinity for penicillins (Km, 64 to 101 μM) and decreased catalytic activity against these antibiotics (kcat, 216 to 1,042 s−1) than SHV-1 (Km, 11 to 31 μM; kcat, 220 to 1,937 s−1) (Table (Table1).1). In SHV-72, mutations other than Lys234Arg might confer increased affinity of the enzyme for penicillins, as well as better catalytic activity against those antibiotics. SHV-84, like SHV-72, exhibited lower affinity for cephalothin (Km, 169 to 141 μM) than SHV-1. No hydrolysis of extended-spectrum cephalosporins was detected. However, SHV-84 was less susceptible to clavulanic acid (IC50, 2.21 μM) than SHV-1 (IC50, 0.17 μM), as observed for SHV-72 (IC50, 1.72 μM) (Table (Table1)1) (8). The IC50 of tazobactam for SHV-84 was 3.5-fold lower than that for SHV-1 (IC50s, 0.03 and 0.15 μM, respectively).
In conclusion, this study underlines the importance of the Lys234Arg substitution in resistance to clavulanic acid in nature, since we demonstrate that this mutation alone is responsible for decreased susceptibility to β-lactamase inhibitors. These results corroborate those previously obtained by molecular dynamic simulation in a study using the model of the mutant SHV-72 enzyme, in which the authors suggested a change in the positioning of the Ser130 side chain induced by Arg234 (8).
The new blaSHV nucleotide sequence was submitted to the EMBL Nucleotide Sequence Database as blaSHV-84 with accession number AM087453.
This work was supported financially by the project POCTI/2001/ESP/43037 from Fundação para a Ciência e a Tecnologia, Lisbon, Portugal, awarded to M. Caniça. V. Manageiro was supported by grant SFRH/BD/32578/2006 from Fundação para a Ciência e a Tecnologia, Lisbon, Portugal.
Published ahead of print on 8 March 2010.