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Sir, We read with great interest the article by Merino et al., which assessed the role of loop L3 in the evolution of VIM-1-like enzymes, including an evaluation of substitution at amino acid position 228. Several VIM enzymes, including VIM-2, possess an arginine at position 228, which is postulated to play a critical role in hydrolytic activity against carbapenems. We recently detected a new VIM-2 variant that differs from its ancestor only by the presence of a serine residue at position 228 instead of arginine. In this study, we describe the genetic characterization of VIM-23 and the comparative antimicrobial susceptibility profile of this enzyme and VIM-2. During 2008, 110 Enterobacteriaceae isolates were consecutively collected from medical centres located in Mexico as part of the SENTRY Antimicrobial Surveillance Program. Isolates were tested for susceptibility by the reference broth microdilution method. Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were concurrently tested for quality assurance; all the results were within published ranges. The modified Hodge test (MHT) and PCRs targeting the carbapenemaseencoding genes blaIMP, blaVIM, blaSPM-1, blaKPC, blaSME, blaIMI, blaNMC-A, blaGES and blaOXA-48 were carried out for isolates showing elevated carbapenem MIC values (MIC of imipenem or meropenem, ≥2 mg/L). Among four carbapenem-resistant Enterobacter cloacae isolates, one (6196A) was MHT-positive and yielded a positive PCR result for blaVIM. This E. cloacae was recovered from the blood of an 18-year-old male burn patient in Guadalajara, Mexico. Strain 6196A was resistant to most b-lactams (Table 1), including carbapenems, but was susceptible to aztreonam (MIC, 0.5 mg/L). This isolate was susceptible to gentamicin (MIC, ≤2 mg/L), tetracycline (MIC, ≤2 mg/L), tigecycline (MIC, 0.5 mg/L), fluoroquinolones (ciprofloxacin MIC, 1 mg/L) and polymyxin B (MIC, ≤0.5 mg/L). Sequencing revealed that E. cloacae 6196A carried a metallo-b-lactamase (MBL)-encoding gene distinct from blaVIM-2 by a single point mutation. The deduced amino acid sequence revealed an Arg228Ser modification, also present in VIM-1. This novel variant was named VIM-23. blaVIM-23 was embedded in a class 1 integron that carried an open reading frame (orf) and aacA7. This cassette arrangement was identical to the blaVIM-2-containing integrons detected in one Klebsiella oxytoca and two E. cloacae recovered in the 2005–07 period from the same medical centre. Plasmid alkaline lysis and S1 nuclease preparations were resolved in agarose gels and hybridized with a blaVIM-2 probe in order to identify the location of the MBL gene. All four VIMproducing isolates recovered from this site (three VIM-2carrying strains previously identified and the VIM-23 producer) had several plasmid bands; however, hybridization signals were noted from one high molecular weight plasmid band present in each of the four isolates of approximately 450 kb. PFGE was used to evaluate clonality among E. cloacae strains producing VIM-2 (2005 and 2007) and VIM-23 (2008). The E. cloacae 6196A PFGE pattern was distinct from those of the two genetically unique VIM-2-producing E. cloacae isolates (data not shown). The genes encoding VIM-23 and VIM-2 were cloned into the pPCRScriptCam SK+ plasmid vector (Stratagene Cloning Systems, La Jolla, CA, USA). Colonies obtained after transformation in XL10-Gold Kan ultracompetent E. coli were selected on plates containing 50 mg/L chloramphenicol and the presence and orientation of the insert was confirmed by sequencing. Recombinant strains were tested for their susceptibility to several b-lactam agents by Etest (bioMérieux, Marcy l’Étoile, France) according to the manufacturer’s instructions. In the same genetic background, VIM-23 showed similar MICs of imipenem, meropenem, doripenem, ampicillin and piperacillin alone and combined with tazobactam (≤2-fold variations; Table 1) when compared with blaVIM-2-carrying E. coli. However, compared with VIM-2-producing E. coli, the recombinant strain producing VIM-23 had lower MICs of ertapenem, ceftazidime, cefotaxime and cefepime (4-fold) and cefoxitin (.64-fold) (Table 1). Merino et al. concluded that an arginine residue at position 228 in VIM-1-type enzymes led to a more open active site, increasing hydrolysis against imipenem and also against ceftazidime and cefepime. The MIC results obtained for VIM-23producing recombinant E. coli confirmed that a serine residue at position 228 resulted in lower MIC values of (and thus hydrolytic activity against) cephalosporins and ertapenem. However,

Comment on: Role of changes in the L3 loop of the active site in the evolution of enzymatic activity of VIM-type metallo- -lactamases