Increased Antimicrobial Resistance in a Novel CMY-54 AmpC-Type Enzyme with a GluLeu217-218 Insertion in the Ω-Loop.

During a Spanish surveillance study, a natural variant of a CMY-type ß-lactamase related to CMY-2 with a GluLeu217-218 insertion in the Ω-loop (designated CMY-54) was found to increase the minimum inhibitory concentractions to ß-lactams in a clinical strain of Escherichia coli. The aim of this study was to characterize CMY-54 by genetic, microbiological, and biochemical analysis. The blaCMY-54 gene is encoded by a plasmid of around 100 kb that hybridizes with K and FIB probes. The genetic context of blaCMY-54 and blaCMY-2 genes was found to be very similar. E. coli expressing CMY-54 under isogenic conditions showed a clear fourfold to eightfold increase in MICs to penicillins, cefotaxime, ceftazidime, and aztreonam compared with CMY-2. The catalytic efficiencies of pure CMY-2 and CMY-54 proteins correlated with their microbiological parameters. CMY-2 protein was more resistant to thermal denaturation than CMY-54, indicating that the Ω-loop of CMY-54 may be wider and more relaxed and probably enables better accommodation of these antimicrobials. Otherwise, the higher stabilization of CMY-2 may induce a slight reduction of the dynamics of this enzyme and primarily affect the hydrolysis of some of the bulkiest antibiotics. In summary, the GluLeu217-218 insertion observed in CMY-54 compared to CMY-2 produces a ß-lactamase with a distinctive catalytic efficacy for ß-lactam antimicrobials likely caused by an increased flexibility slightly affecting the active site shape, highlighting the relevance of single mutations on the hydrolytic spectrum in class C ß-lactamases.

Genetic and kinetic characterization of the novel AmpC ß-lactamases DHA-6 and DHA-7.

During a Spanish surveillance study, two natural variants of DHA ß-lactamases, DHA-6 and DHA-7, were found, with the replacements Ala226Thr and Phe322Ser, respectively, with respect to DHA-1. The DHA-6 and DHA-7 enzymes were isolated from Escherichia coli and Enterobacter cloacae clinical isolates, respectively. The aim of this study was to genetically, microbiologically, and biochemically characterize the DHA-6 and DHA-7 ß-lactamases. The blaDHA-6 and blaDHA-7 genes were located in the I1 and HI2 incompatibility group plasmids of 87.3 and 310.4 kb, respectively. The genetic contexts of blaDHA-6 and blaDHA-7 were similar to that already described for the blaDHA-1 gene and included the qnrB4 and aadA genes. The MICs for cephalothin, aztreonam, cefotaxime, and ceftazidime were 8- to 32-fold lower for DHA-6 than for DHA-1 or DHA-7 expressed in the same isogenic E. coli TG1 strain. Interestingly, the MIC for cefoxitin was higher in the DHA-6-expressing transformant than in DHA-1 or DHA-7. Biochemical studies with pure ß-lactamases revealed slightly lower catalytic efficiencies of DHA-6 against cephalothin, ceftazidime, and cefotaxime than those of DHA-1 and DHA-7. To understand this behavior, stability experiments were carried out and showed that the DHA-6 protein displayed significantly higher stability than the DHA-1 and DHA-7 enzymes. The proximity of Thr226 to the N terminus in the tertiary protein structure in DHA-6 may promote this stabilization and, consequently, may induce a slight reduction in the dynamic of this enzyme that primarily affects the hydrolysis of some of the bulkiest antibiotics.

New mutations in ADC-type ß-lactamases from Acinetobacter spp. affect cefoxitin and ceftazidime hydrolysis.

OBJECTIVES: Two natural variants of ADC-type ß-lactamases of Acinetobacter spp., ADC-1 and ADC-5, differ by nine mutations in their protein sequence. ADC-5 hydrolyses cefoxitin better than ADC-1 and the opposite is true for ceftazidime. We produced single and combined mutations in ADC-5 and characterized the variants microbiologically and biochemically to determine which amino acid residues are involved in the hydrolysis of ß-lactam antibiotics in this family of ß-lactamases. METHODS: Site-directed mutagenesis, with blaADC-5 as a source of DNA, was used to generate nine single mutated and three combined mutated enzymes. The proteins (wild-type and derivatives) were then expressed in isogenic conditions in Escherichia coli. MICs of ß-lactams were determined using Etest strips. ADC-1, ADC-5, ADC-5-P167S and ADC-5-P167S/D242G/Q163K/G342R were also purified and the kinetic parameters determined for ceftazidime, cefoxitin, cefalotin and ampicillin. RESULTS: Single mutations did not significantly convert the hydrolysis spectrum of the ADC-5 enzyme into that of the ADC-1 enzyme, although among all studied mutants only the quadruple mutant (ADC-5-P167S/D242G/Q163K/G342R) displayed microbiological and biochemical properties consistent with those of ADC-1. CONCLUSIONS: Although some single mutations are known to affect cefepime hydrolysis in ADC-type ß-lactamases, little is known about ceftazidime and cefoxitin hydrolysis in this family of ß-lactamases. Hydrolysis of these antibiotics appears to be positively and negatively affected, respectively, by the Q163K, P167S, D242G and G342R amino acid replacements.

Characterization of the new AmpC ß-lactamase FOX-8 reveals a single mutation, Phe313Leu, located in the R2 loop that affects ceftazidime hydrolysis.

A novel class C ß-lactamase (FOX-8) was isolated from a clinical strain of Escherichia coli. The FOX-8 enzyme possessed a unique substitution (Phe313Leu) compared to FOX-3. Isogenic E. coli strains carrying FOX-8 showed an 8-fold reduction in resistance to ceftazidime relative to FOX-3. In a kinetic analysis, FOX-8 displayed a 33-fold reduction in kcat/Km for ceftazidime compared to FOX-3. In the FOX family of ß-lactamases, the Phe313 residue located in the R2 loop affects ceftazidime hydrolysis and alters the phenotype of E. coli strains carrying this variant.

Characterization of a novel IMP-28 metallo-ß-lactamase from a Spanish Klebsiella oxytoca clinical isolate.

An isolate of Klebsiella oxytoca carrying a novel IMP metallo-ß-lactamase was discovered in Madrid, Spain. The bla(IMP-28) gene is part of a chromosomally located class I integron. The IMP-28 k(cat)/K(m) values for ampicillin, ceftazidime, and cefepime and, to a lesser extent, imipenem and meropenem, are clearly lower than those of IMP-1. The His306Gln mutation may induce important modifications of the L3 loop and thus of substrate accessibility and hydrolysis and be the main reason for this behavior.

Distant and new mutations in CTX-M-1 beta-lactamase affect cefotaxime hydrolysis.

The CTX-M ß-lactamases are an increasingly prevalent group of extended-spectrum ß-lactamases (ESBL). Point mutations in CTX-M ß-lactamases are considered critical for enhanced hydrolysis of cefotaxime. In order to clarify the structural determinants of the activity against cefotaxime in CTX-M ß-lactamases, screening for random mutations was carried out to search for decreased activity against cefotaxime, with the CTX-M-1 gene as a model. Thirteen single mutants with a considerable reduction in cefotaxime MICs were selected for biochemical and stability studies. The 13 mutated genes of the CTX-M-1 ß-lactamase were expressed, and the proteins were purified for kinetic studies against cephalothin and cefotaxime (as the main antibiotics). Some of the positions, such as Val103Asp, Asn104Asp, Asn106Lys, and Pro107Ser, are located in the (103)VNYN(106) loop, which had been described as important in cefotaxime hydrolysis, although this has not been experimentally confirmed. There are four mutations located close to catalytic residues-Thr71Ile, Met135Ile, Arg164His, and Asn244Asp-that may affect the positioning of these residues. We show here that some distant mutations, such as Ala219Val, are critical for cefotaxime hydrolysis and highlight the role of this loop at the top of the active site. Other distant substitutions, such as Val80Ala, Arg191, Ala247Ser, and Val260Leu, are in hydrophobic cores and may affect the dynamics and flexibility of the enzyme. We describe here, in conclusion, new residues involved in cefotaxime hydrolysis in CTX-M ß-lactamases, five of which are in positions distant from the catalytic center.

Structure-function studies of arginine at position 276 in CTX-M beta-lactamases.

OBJECTIVES: In order to assess whether or not the Arg-276 of CTX-M-type enzymes is equivalent to the Arg-244 of IRT-TEM-derivative enzymes, we replaced the former with six different amino acids, some of them previously described as involved in resistance to beta-lactamase inhibitors in TEM-IRT derivatives. We also investigated the role of Arg276 in cefotaxime hydrolysis. METHODS: By site-directed mutagenesis and by use of the bla(CTX-M-1) gene as template, Arg-276 was replaced with six different amino acids (Trp, His, Cys, Asn, Gly and Ser). MICs of beta-lactams alone and in combination with beta-lactamase inhibitors were established. The seven enzymes (CTX-M-1 wild-type and six derived mutants) were purified by affinity chromatography, and kinetic parameters (k(cat), K(m), k(cat)/K(m)) towards cefalotin and cefotaxime were determined. Clavulanic acid IC(50) values were also assessed with all enzymes. RESULTS: No increase in MICs of beta-lactam/beta-lactamase inhibitor combination was detected with any of the six CTX-M-1-derived mutants, in agreement with the clavulanic acid IC(50) values. The MICs of cefotaxime were clearly lower for the Escherichia coli harbouring the Trp, Cys, Ser and Gly CTX-M-1 mutant enzymes than for CTX-M-1, and these enzymes showed a clearly reduced catalytic efficiency towards cefotaxime. As regards cefalotin, there was a moderate reduction in catalytic efficiency for Cys and His. CONCLUSIONS: Arg-276 in CTX-M-type beta-lactamases is not equivalent to Arg-244 in IRT-type enzymes. Position Arg-276 appears to be important for cefotaxime hydrolysis in CTX-M-type enzymes, although different effects were obtained regarding the replaced amino acid.