Structure-fucntion relationship of the NDM-1 metallo-beta-lactamase: Importance of Trp-87 in enzyme structure, function and stability.

Background. NDM-1, an acquired subclass B1 metallo-beta-lactamase (MBL), represents a new worrisome global health issue, due to its rapid dissemination among clinical isolates in several countries and further aggravated by the unavailability of clinically-useful MBL inhibitors. NDM-1 thus represents an extremely relevant target for MBL inhibitors, and its structure-function relationship deserves investigation. In a previous study performed on VIM-2, the highly conserved Trp-87 residue was identified as an essential determinant for the enzyme stability and folding. In this work, we probed the impact of Trp-87 substitutions in NDM-1 on the enzyme properties and bacterial resistance. Methods. The role of Trp-87 of NDM-1 was investigated by saturation mutagenesis. A library of blaNDM-1 mutants was obtained by means of a mutagenic PCR, using plasmid pLBII-NDM-1 as the template, and the recombinant plasmids transformed in E. coli. The clones carrying the various blaNDM-1 mutants genes were subjected to sequence analysis and their antimicrobial susceptibility profile determined using the broth microdulition technique as recommended by CLSI. Various NDM-1 variants were also subjected by biochemical characterization using kinetic assays. Results. Antimicrobial susceptibility data obtained with E. coli strains producing the various Trp-87 NDM-1 variants consistently showed a significant reduction of the MIC values with several β-lactam antibiotics (e. g., the ceftazidime MIC values were decreased up to 128-fold, as compared to that shown by the strain producing the wild-type NDM-1). However, the substitution of Trp-87 did not compromise per se the activity of the purified enzyme, as revealed by kinetic assays. These observations are very similar to those previously obtained with the VIM-2 MBL, indicating that residue Trp-87, although not directly involved in catalysis, is a crucial determinant for conferring β-lactam resistance, likely due to its relevance in enzyme stability and proper in vivo folding. Conclusions. These data overall support and validate the hypothesis that the conserved Trp-87 of MBLs is an essential determinant for enzyme stability and in vivo folding and thus would represent an important structural determinant to consider for the development of rationally-designed MBL inhibitors

Probing the role of R2-loop deletions and substituions on the functional properties of the acquired AmpC-type β-lactamase CMY-2.

Background: CMY-2 is a plasmid-encoded AmpC-type β-lactamase (pAmpC) belonging to the CIT cluster which shows the broadest dissemination and represents an important mechanism of β-lactam resistance in Enterobacteriaceae. The crystal structure of CMY-10, a pAmpC belonging to the MOX cluster, revealed subtle differences in the R2-loop of the enzyme (due to a 3-AA deletion) that were associated with a higher hydrolytic activity towards imipenem. In this work, we wanted to probe the impact of modifications in the R2-loop of CMY-2 on the functional features of the enzyme. Methods: Wild-type cmy-2 gene was cloned in vector pLB-II and propagated in E. coli DH5α. Using mutagenic PCR, several CMY-2 laboratory variants were obtained in which (a) a 3-AA deletion was introduced in the R2-loop (CMY-2Δ319-321, CMY-2Δ320-322, etc.) or (b) the whole R2 loop of CMY-2 was substituted by that found in the structurally-equivalent position of CMY-10 (CMY-2loop10). The resulting recombinant vectors were transformed in E. coli. The β-lactam susceptibility of the recombinant E. coli strains carrying the cloned cmy-2 mutants were determined according to CLSI or using E-test. The hydrolytic activity of the various CMY-2 variants were investigated by standard kinetic measurements. Results: The various E. coli strains producing the CMY-2 laboratory variants were resistant to ampicillin and cephalothin, indicating that these modified proteins were functional. However, the 3-AA deletion in CMY-2 (such as in mutant CMY-2Δ319-321) apparently increased the susceptibility to FOX and CAZ, while imipenem MIC was unchanged. Similar results were observed with E. coli strains producing the CMY-2loop10 variant. Moreover, enzymatic assays did not show significant differences in imipenem hydrolysis between the wild-type and the CMY-2 variants. Conclusions: Several CMY-2 variants carrying deletions or substitutions in the R2-loop were obtained and characterized. Despite these variants were functional, they did not decrease imipenem susceptibility when produced in E. coli, indicating that other mutations might be required in CMY-2 to enhance its activity towards imipenem

Microbiological analysis of hospital wastewaters: impact of water treatment on microbial biodiversity and antimicrobial resistance.

Background. Hospital effluents are considered an important source of both microorganisms and chemical substances (e. g. drug residues, disinfectants) that might impact the environment, often requiring pre-treatment before they can be released to public wastewater treatment plants. In order to investigate how wastewater treatment could affect the microbial biodiversity and its associated antimicrobial resistance, we performed a microbiological and molecular analysis of samples collected at the inlet and outlet of our local wastewater treatment plant. Methods. Samples were collected at the University Hospital of Siena wastewater treatment plant during Feb and Oct 2012. Microbial aerobic count was determined on Nutrient Agar and McConkey Agar plates at 30 or 37 °C. Identification of microorganisms was carried out using a Vitek MS. Antimicrobial susceptibility testing was performed as recommended by CLSI. Determinants of antimicrobial resistance were analyzed using either phenotypic tests or molecular methods. Results. A comparative analysis of pre- and post-treatment samples (total bacterial counts in the various samples ranged 5×104 – 2×106 CFU/ml) revealed important differences in both microbial biodiversity and its associated antimicrobial resistance: (a) the percentage of Gram-negative (GN) bacteria in post-treatment samples was significantly lower than that found in pre-treatment samples (5 vs 40% total, respectively) and (b) a higher proportion of antibiotic-resistant GN isolates was found in post-treatment samples, with 59% isolates showing ceftazidime resistance (vs 6% in pre-treatment samples). Approx. 10% of CAZ-resistant GN isolates exhibited resistance to carbapenems, fluoroquinolones and aminoglycosides and belonged to genera Citrobacter, Klebsiella and Shewanella. Molecular analysis revealed the presence of KPC-producing Klebsiella pneumoniae and VIM-2-producing Citrobacter spp. isolates (including C. braaki in which such determinant was not previously reported). Conclusions. The hospital wastewater samples contained both environmental and clinically-relevant bacterial species. Despite post-treatment samples showed a lower prevalence of GN species, a higher of rate antibiotic resistance was found

Crystal structure of the extended-spectrum β-lactamase BEL-1, in native form and in complex with imipenem and moxalactam.

Background. BEL-1 is a class A ESBL detected in P. aeruginosa clinical isolates from Belgium, which is quite divergent from other ESBLs (max. identity, 54% with GES-type enzymes). This enzyme is efficiently inhibited by clavulanate, imipenem and moxalactam. A BEL-1 variant showing the single Leu162Phe substitution (BEL-2) and conferring a higher level of resistance to CAZ, CTX and FEP was also isolated, which showed much lower turnover rates and Km values as compared to those of BEL-1, especially with oxyiminocephalosporins. Methods. BEL-1 was produced in E. coli using a T7-based promoter expression plasmid and purified to near homogeneity by means of two cationic ion exchange chromatography steps. Crystals of BEL-1 were obtained at pH 5.6, and subsequently soaked with imipenem and moxalactam for 30-60 min. Diffraction data were collected and the structure of native BEL-1 determined by molecular replacement, using the coordinates of the Toho-1 beta-lactamase (PDB code, 1IYS) as the search model. Results. The structure of BEL-1, in the native form and in complex with imipenem and moxalactam, was obtained at 1.6-1.9-angstroms resolution. In the acyl-enzyme complexes with imipenem and moxalactam, the C6 α-substituent interacts with conserved residue Asn-132. More surprisingly, the omega-loop, which includes the catalytically-relevant residue Glu-166, was found in different conformations in the various subunits, resulting in a approx. 180° rotation outwards the active site of the Glu-166 side chain or displacements of its Cα atom up to approx. 10 angstroms. Leu-162, located at the beginning of the omega-loop, is surrounded by Phe-72, Leu-139, Leu-148 and Leu-169 (contact distances, 3.8-4 angstroms). This small hydrophobic cavity could not reasonably accommodate the bulkier Phe-162 found in BEL-2 without altering neighbouring residues or the omega-loop itself, thus causing an important alteration of the enzyme kinetic properties. Conclusions. The structure of BEL-1 in complex with imipenem and moxalactam shows an important variation in the conformation adopted by the omega-loop. It also provides a structural rationale supporting the large impact of Leu162Phe substitution on beta-lactam hydrolysis

Purification and biochemical characterization of the VIM-1 metallo-β-lactamase

VIM-1 is a new group 3 metallo-β-lactamase recently detected in carbapenem-resistant nosocomial isolates of Pseudomonas aeruginosa from the Mediterranean area. In this work, VIM-1 was purified from an Escherichia coli strain carrying the cloned bla(VIM-1) gene by means of an anion-exchange chromatography step followed by a gel permeation chromatography step. The purified enzyme exhibited a molecular mass of 26 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an acidic pI of 5.1 in analytical isoelectric focusing. Amino-terminal sequencing showed that mature VIM-1 results from the removal of a 26-amino-acid signal peptide from the precursor. VIM-1 hydrolyzes a broad array of β-lactam compounds, including penicillins, narrow- to expanded-spectrum cephalosporins, carbapenems, and mechanism-based serine-β-lactamase inactivators. Only monobactams escape hydrolysis. The highest catalytic constant/K(m) ratios (> 106 M-1 · s-1) were observed with carbenicillin, azlocillin, some cephalosporins (cephaloridine, cephalothin, cefuroxime, cefepime, and cefpirome), imipenem, and biapenem. Kinetic parameters showed remarkable variability with different β-lactams and also within the various penam, cephem, and carbapenem compounds, resulting in no clear preference of the enzyme for any of these β-lactam subfamilies. Significant differences were observed with some substrates between the kinetic parameters of VIM-1 and those of other metallo-β-lactamases. Inactivation assays carried out with various chelating agents (EDTA, 1,10-o-phenanthroline, and pyridine-2,6-dicarboxylic acid) indicated that formation of a ternary enzyme-metal-chelator complex precedes metal removal from the zinc center of the protein and revealed notable differences in the inactivation parameters of VIM-1 with different agents