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We describe 12 VIM-1-producing strains (7 Enterobacter cloacae, 2 Klebsiella pneumoniae and 3 clonal Klebsiella oxytoca strains) detected among clinically relevant Enterobacteriaceae isolates from routine cultures at the Hospital del Mar (Barcelona, Spain) from December 2006 to May 2007.Susceptibility to carbapenems was evaluated with the MicroScan system. β-Lactamases were identified by PCR and sequencing. Clonal relationships between the isolates were analysed by PFGE. Transferability of the enzymes was tested by conjugation. Plasmid characterization was performed by PCR-based replicon typing and PFGE with S1 nuclease digestion of whole genomic DNA. The PFGE gels were then transferred and hybridized.The disc diffusion method correctly identified five of the seven E. cloacae isolates as intermediate or resistant strains. All isolates produced the VIM-1 enzyme. Three E. cloacae and three K. oxytoca strains were also CTX-M-9-producing strains, and one E. cloacae was also a CTX-M-3-producing strain. The plasmids carrying the blaVIM gene, of unknown incompatibility group, had a size of ∼75 kb (eight strains) or 40 kb (three strains) and also contained the qnrS and the aac(6′)-Ib-cr genes. In the remaining strain the blaVIM-1 gene was found in an HI2 plasmid of 290 kb together with blaCTX-M-9, qnrA, qnrS and the aac(6′)-Ib-cr genes.The results showed a linkage between the blaVIM-1 and the qnrS and the aac(6′)-Ib-cr genes, and between the blaCTX-M-9 and the qnrA genes.