Detection and Characterization of VIM-31, a New Variant of VIM-2 with Tyr224His and His252Arg Mutations, in a Clinical Isolate of Enterobacter cloacae

Abstract

We report the first description of the metallo-␤-lactamase VIM-31, a new variant of VIM-2 with Tyr224His and His252Arg mutations, in Enterobacter cloacae 11236, which was isolated from blood specimens of a patient with colonic adenocarcinoma in Belgium. bla VIM-31 was found on a class 1 integron located on a self-transferable but not typeable 42-kb plasmid. Compared to values published elsewhere for VIM-2, the purified VIM-31 enzyme showed weaker catalytic efficiency against all the tested betalactam agents (except for ertapenem), resulting from lower k cat (except for ertapenem) and higher K m values for VIM-31. The worldwide spread of metallo-␤-lactamases (i.e., VIM, IMP, and NDM enzymes) in carbapenem-resistant Gram-negative bacteria presently constitutes a major public health issue.So far, 33 distinct bla VIM alleles have been described (www .lahey.org/Studies) for a variety of Gram-negative opportunistic pathogens, but only VIM-1, VIM-2, VIM-4, VIM-7, and VIM-27 enzymes (7,12,15,22,31) have been characterized biochemically and structurally. While VIM-1-derived enzymes have been reported largely for Enterobacteriaceae from around the world, especially from Greece but also recently from Belgium (4), VIM-2 has been associated mostly with Pseudomonas aeruginosa (32). The occurrence of VIM-2 has nevertheless also been reported for clinical isolates of Enterobacteriaceae from Asia (17, 24, 39), Mexico (28), Argentina (16), and Tunisia (10). Moreover, a recent report showed the presence of VIM-2 in Enterobacter ludwigii isolated from sewage water of a hospital, highlighting the involvement of environmental bacteria as potential reservoirs for the dissemination of clinically relevant metallo-␤-lactamase resistance genes (37). In many subclass B1 (e.g., IMP and NDM) and B2 (e.g., CphA) metallo-␤-lactamases, the amino acid at position 224 (a conserved Lys residue), close to the active site, plays an important role in substrate binding (2, 27). In the case of VIM enzymes, Lys224 is not present and is replaced by His224 in VIM-1 and by Tyr224 in VIM-2. The side chains of histidine and tyrosine residues are much shorter than that of a lysine residue, thereby preventing the interaction of these two amino acids with the carboxylate moiety (C 4 or C 3 ) of the substrate. For VIM enzymes, it is hypothesized that Arg228, with its long side chain, may replace Lys224 in interacting with the substrate (15,22).We report here the detection and characterization of VIM-31, a new variant of VIM-2 with Tyr224His and His252Arg mutations, in an Enterobacter cloacae clinical isolate recovered from an elderly patient in Belgium. MATERIALS AND METHODSBacterial strains and antimicrobial susceptibility. Enterobacter cloacae 11236 was recovered in June 2011 from blood culture specimens from a patient hospitalized in Brussels, Belgium. The isolate was identified to the species level by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) with a Microflex LT mass spectrometer (Bruker Daltonik GmbH, Leipzi...

The three‐dimensional structure of VIM‐31 – a metallo‐β‐lactamase from Enterobacter cloacae in its native and oxidized form

“…It should be noted that the comparison of the kinetic properties of VIM‐31 to the VIM‐2 variant was performed using previously determined data from Bogaerts et al . and from Docquier et al . .…”

“…). However, with the exception of cefoxitin ( k cat / K m ratio for VIM‐2/VIM‐31 is 54), VIM‐31 and VIM‐2 exhibit similar kinetic behaviour . Nevertheless, VIM‐31 presents decreased K m values compared to VIM‐2 for benzylpenicillin, cefoxitin and ceftazidime (the K m ratio for VIM‐31/VIM‐2 is > 3) (Table ).…”

“…VIM-31 presents decreased k cat values for piperacillin, cefepime, cefoxitin, nitrocefin and meropenem (the k cat ratio for VIM-2/VIM-31 is > 4) ( Table 2). It should be noted that the comparison of the kinetic properties of VIM-31 to the VIM-2 variant was performed using previously determined data from Bogaerts et al [20] and from Docquier et al [8]. Because these experiments were performed by different groups in different laboratories, differences by factors of 2 or 3 should not be considered.…”

“…This Tyr224His substitution is also present in VIM‐1 , VIM‐4 , VIM‐7 , VIM‐12 , VIM‐14 , VIM‐19 and VIM‐27 . VIM‐31 exhibits globally lower catalytic efficiencies than VIM‐2 (because of lower k cat and higher K m values) .…”

B1-Metallo-β-Lactamases: Where Do We Stand?

“…Similarly, compared to the parental VIM-2, VIM-31 contains two mutations (Y224H and H252R) in the C -terminal part of the protein and exhibits globally lower catalytic efficiencies. The catalytic features of this variant are thought to derive from the Y224H substitution, since the H252R substitution, situated on the α4 helix and distant from the active site of the VIM enzyme, renders unlikely any effect on substrate binding and/or catalysis [113]. …”