Characterization of a Functional Metallo-β-Lactamase from the Marine Bacterium Haliangium ochraceum

Background: Acquired metallo-β-lactamases (MBLs) are worrisome resistance determinants conferring broad-spectrum β-lactam resistance in clinically-relevant pathogens such as Enterobacteriaceae, Pseudomonas and Acinetobacter. However, the origin of these acquired determinants is still poorly understood. Exploiting the increasing number of microbial genome sequences available in public databases, we specifically investigated the presence of genes encoding for proteins showing homology with acquired subclass B1 enzymes in the genomes of environmental bacteria belonging to the class of δ-Proteobacteria. Methods: Using genome data mining (with the protein sequence of VIM-2 as the search template and the tblastn algorithm available at the NCBI web site), a gene encoding a subclass B1 MBL homologue (locus tag, Hoch_6144) was identified in the genome of Haliangium ochraceum DSMZ 14365. The Hoch_6144 ORF was cloned in plasmid vector pET-9a. The resulting plasmid (pET-HAL-1) were electroporated in E. coli BL21(DE3). The MBL was purified by chromatography and its functional properties investigated by means of kinetic analysis. Results: The MBL homologue encoded by gene Hoch_6144, named HAL-1, shared the closest ancestry with FIM-1, NDM-1 and VIM-2 acquired MBLs (identity range, 41-46 %). Significant β-lactam-hydrolyzing activity was found in crude extracts of E. coli BL21(DE3)/pET-HAL-1, that was inhibited >95% in the presence of 5 mM EDTA. HAL-1 was successfully produced and purified (>95%) using three chromatography steps. Purified HAL-1 exhibited a broad-spectrum of activity, with catalytic efficiencies for the hydrolysis of β-lactams comparable to that of VIM-2. Interestingly, HAL-1 showed low Km values for most tested substrates, at the exception of ceftazidime. Strikingly, doripenem (the latest carbapenem antibiotic to receive FDA approval) was hydrolyzed 45-fold more efficiently by HAL-1 than by the acquired VIM-2 MBL. Conclusions: In this work, we described a new functional MBL identified in a marine bacterium of the class δ-Proteobacteria, Haliangium ochraceum. HAL-1 exhibits functional properties very close to that of clinically-relevant subclass B1 enzymes and highlights the potential of δ-Proteobacteria as a reservoir of functional subclass B1 MBL determinants

Genetic context and biochemical characterization of the IMP-18 metallo-beta-lactamase identified in a Pseudomonas aeruginosa isolate from the United States.

The production of metallo-β-lactamase (MBL) is an important mechanism of resistance to β-lactam antibiotics, including carbapenems. Despite the discovery and emergence of many acquired metallo-β-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 μg/ml, respectively) than for strains producing IMP-1 (2 μg/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

Metallo-β-Lactamase Production by Pseudomonas otitidis: a Species-Related Trait ▿

Susceptibility to several β-lactams and β-lactamase production was investigated in a collection of 20 strains of Pseudomonas otitidis, a new Pseudomonas species that has been recently recognized in association with otic infections in humans. All strains appeared to be susceptible to piperacillin, cefotaxime, ceftazidime, and aztreonam, while resistance or decreased susceptibility to carbapenems was occasionally observed. All strains were found to express metallo-β-lactamase (MBL) activity and to carry a new subclass B3 MBL gene, named blaPOM, that appeared to be highly conserved in this species. P. otitidis, therefore, is the first example of a pathogenic Pseudomonas species endowed with a resident MBL. The POM-1 protein from P. otitidis type strain MCC10330 exhibits the closest similarity (60 to 64%) to the L1 MBL of Stenotrophomonas maltophilia. Expression in Escherichia coli and Pseudomonas aeruginosa revealed that, similar to L1 and other subclass B3 MBLs, POM-1 confers decreased susceptibility or resistance to carbapenems, penicillins, and cephalosporins but not to aztreonam. Expression of the POM MBL in P. otitidis is apparently constitutive and, in most strains, does not confer a carbapenem-resistant phenotype. However, a strong inoculum size effect was observed for carbapenem MICs, and carbapenem-resistant mutants could be readily selected upon exposure to imipenem, suggesting that carbapenem-based regimens should be considered with caution for P. otitidis infections

Mutational Analysis of VIM-2 Reveals an Essential Determinant for Metallo-β-Lactamase Stability and Folding▿

Metallo-β-lactamase (MBL)-producing bacteria are emerging worldwide and represent a formidable threat to the efficacy of relevant β-lactams, including carbapenems, expanded-spectrum cephalosporins, and β-lactamase inactivator/β-lactam combinations. VIM-2 is currently the most widespread MBL and represents a primary target for MBL inhibitor research, the clinical need for which is expected to further increase in the future. Using a saturation mutagenesis approach, we probed the importance of four residues (Phe-61, Ala-64, Tyr-67, and Trp-87) located close to the VIM-2 active site and putatively relevant to the enzyme activity based on structural knowledge of the enzyme and on structure-activity relationships of the subclass B1 MBLs. The ampicillin MIC values shown by the various mutants were affected very differently depending on the randomized amino acid position. Position 64 appeared to be rather tolerant to substitution, and kinetic studies showed that the A64W mutation did not significantly affect substrate hydrolysis or binding, representing an important difference from IMP-type enzymes. Phe-61 and Tyr-67 could be replaced with several amino acids without the ampicillin MIC being significantly affected, but in contrast, Trp-87 was found to be critical for ampicillin resistance. Further kinetic and biochemical analyses of W87A and W87F variants showed that this residue is apparently important for the structure and proper folding of the enzyme but, surprisingly, not for its catalytic activity. These data support the critical role of residue 87 in the stability and folding of VIM-2 and might have strong implications for MBL inhibitor design, as this residue would represent an ideal target for interaction with small molecules