Characterization of a new integron containing blaVIM-1 and aac(6′)-IIc in an Enterobacter cloacae clinical isolate from Greece

Materials and methods: MICs were determined with standard procedures as well as using a higher inoculum. Isoelectric focusing of cell extracts was used for detection of b-lactamases. PCR assays with primers specific for the blaVIM gene and the conserved segments of class 1 integrons and sequence analyses were carried out to identify the gene and to map the metallo-b-lactamase encoding integron. Transferability of the gene was assessed with conjugation experiments using the filter mating technique. To identify the location of the blaVIM-1 gene, Southern hybridization was carried out in genomic DNA using an internal fragment of the blaVIM-1 gene as a probe, amplified by PCR. Results: The isolate was resistant to extended-spectrum b-lactams. The MICs of carbapenems were below the resistance breakpoints but rose above resistance breakpoints when an inoculum of 10 8 cfu/mL was used. Isoelectric focusing detected a b-lactamase with a pI of 6.1, which exhibited imipenem-hydrolysing activity in a microbiological assay. Ceftazidime and imipenem resistance were not transferable by conjugation. PCR assays identified the blaVIM-1 gene in the variable region of a class 1 integron which also carried the aac(6 0 )-IIc gene. The blaVIM-1 probe hybridized with an approximately 130kb fragment of genomic DNA, suggesting a chromosomal location of the gene. Conclusion: We describe a novel class 1 integron containing blaVIM-1 and aac(6 0 )-IIc genes in an