Metallo-β-lactamases (MBLs) cause resistance of Gram-negative bacteria to β-lactam antibiotics and are of serious concern, because they can inactivate the last-resort carbapenems and because MBL inhibitors of clinical value are still lacking. We previously identified the original binding mode of 4-amino-2,4-dihydro-5-(2-methylphenyl)-3H-1,2,4-triazole-3-thione (compound IIIA) within the dizinc active site of the L1 MBL. Herein we present the crystallographic structure of a complex of L1 with the corresponding non-amino compound IIIB (1,2-dihydro-5-(2-methylphenyl)-3H-1,2,4-triazole-3-thione). Unexpectedly, the binding mode of IIIB was similar but reverse to that of IIIA. The 3 D structures suggested that the triazole-thione scaffold was suitable to bind to the catalytic site of dizinc metalloenzymes. On the basis of these results, we synthesized 54 analogues of IIIA or IIIB. Nineteen showed IC values in the micromolar range toward at least one of five representative MBLs (i.e., L1, VIM-4, VIM-2, NDM-1, and IMP-1). Five of these exhibited a significant inhibition of at least four enzymes, including NDM-1, VIM-2, and IMP-1. Active compounds mainly featured either halogen or bulky bicyclic aryl substituents. Finally, some compounds were also tested on several microbial dinuclear zinc-dependent hydrolases belonging to the MBL-fold superfamily (i.e., endonucleases and glyoxalase II) to explore their activity toward structurally similar but functionally distinct enzymes. Whereas the bacterial tRNases were not inhibited, the best IC values toward plasmodial glyoxalase II were in the 10 μm range.

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Objectives: To investigate the first Italian outbreak of bloodstream infections caused by multidrugresistant (MDR) Klebsiella pneumoniae producing metallo-b-lactamase (MBL), which occurred in three wards of one large tertiary-care hospital in Genoa, Italy, from September 2004 to March 2005.Methods: MBL production was screened by an imipenem-EDTA disc synergy test and confirmed by a conventional hydrolysis test. Antibiotic susceptibility was determined by broth microdilution or disc diffusion. PFGE was used to study the genetic relatedness of isolates. PCR and sequencing were carried out to identify the b-lactamase genes and to analyse the genetic context of the MBL gene. Outer membrane protein (OMP) profiles were analysed by SDS-PAGE.Results: Nine cases of bloodstream infections caused by an MDR strain of K. pneumoniae producing the VIM-1 MBL and the SHV-5 extended-spectrum b-lactamase (ESBL) were identified. The isolates exhibited various carbapenem resistance levels (imipenem MICs ranged from 4 to 64 mg/L) and were resistant to other b-lactams, fluoroquinolones, trimethoprim/sulfamethoxazole and chloramphenicol. The isolate with the highest imipenem MIC also lacked the k36 OMP. The bla VIM-1 gene cassette was part of the variable region of a class 1 integron that also included an aac(6 0 )-IIc cassette. The ESBL and MBL genes were transferable by conjugation.Conclusions: This is the first report on the emergence of an MDR strain of K. pneumoniae producing the VIM-1 MBL, causing an outbreak of bloodstream infections in an Italian hospital. The strain evolved through OMP alterations generating a mutant with increased carbapenem resistance.

Metallo-␤-lactamase (MBL)-producing bacteria are emerging worldwide and represent a formidable threat to the efficacy of relevant ␤-lactams, including carbapenems, expanded-spectrum cephalosporins, and ␤-lactamase inactivator/␤-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.

The Wellcome Trust and Innovative Medicines Initiative 2 Joint Undertaking.

Susceptibility to several ␤-lactams and ␤-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-␤-lactamase (MBL) activity and to carry a new subclass B3 MBL gene, named bla POM , 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. Pseudomonas otitidis is a newPseudomonas species that has recently been recognized in association with otic infections in humans, including acute otitis externa, acute otitis media, and chronic suppurative otitis media (2). Genotypically and phenotypically, P. otitidis is closely related to Pseudomonas aeruginosa (2), and this similarity likely accounts for the belated identification of this new pathogenic species within the Pseudomonas genus.The susceptibility of P. otitidis was previously investigated with several antimicrobial agents, including aminoglycosides, fluoroquinolones, macrolides, ␤-lactams, tetracycline, chloramphenicol, and polymyxin B, and overall, the behavior of this species appeared to be similar to that of P. aeruginosa (2). However, of ␤-lactams, only piperacillin was tested, while no information is available on the susceptibility of this species to antipseudomonal cephalosporins, aztreonam, and carbapenems.In this work, we investigated the susceptibility of P. otitidis to several ␤-lactams and the production of ␤-lactamase activity by this species. Results revealed that P. otitidis strains constitutively produce a novel subclass B3 metallo-␤-lactamase (MBL), that was named POM (after P. otitidis metallo-␤-lactamase), which is active on carbapenems and other ␤-lactams. MATERIALS AND METHODSBacterial strains. The P. otitidis strains investigated in this work included 19 strains from the collection previously described by Clark et al. (2) and a clinical isolate from South Korea (isolate YMC-Po/06) cultured from the puru...

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.

BackgroundsStreptococcus pneumoniae expresses three distinct sialidases, NanA, NanB, and NanC, that are believed to be key virulence factors and thus, potential important drug targets. We previously reported that the three enzymes release different products from sialosides, but could share a common catalytic mechanism before the final step of product formation. However, the kinetic investigations of the three sialidases have not been systematically done thus far, due to the lack of an easy and steady measurement of sialidase reaction rate.ResultsIn this work, we present further kinetic characterization of pneumococcal sialidases by using a direct spectrophotometric method with the chromogenic substrate p-nitrophenyl-N-acetylneuraminic acid (p-NP-Neu5Ac). Using our assay, the measured kinetic parameters of the three purified pneumococcal sialidase, NanA, NanB and NanC, were obtained and were in perfect agreement with the previously published data. The major advantage of this alternative method resides in the direct measurement of the released product, allowing to readily determine of initial reaction rates and record complete hydrolysis time courses.ConclusionWe developed an accurate, fast and sensitive spectrophotometric method to investigate the kinetics of sialidase-catalyzed reactions. This fast, sensitive, inexpensive and accurate method could benefit the study of the kinetics and inhibition of sialidases in general.