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.