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

Abstract

Objectives: A clinical isolate of Enterobacter cloacae exhibiting reduced susceptibility to imipenem and a positive EDTA-disc synergy test was studied for carbapenemase production. 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 β-lactamases. PCR assays with primers specific for the bla_VIM gene and the conserved segments of class 1 integrons and sequence analyses were carried out to identify the gene and to map the metallo-β-lactamase encoding integron. Transferability of the gene was assessed with conjugation experiments using the filter mating technique. To identify the location of the bla_VIM-1 gene, Southern hybridization was carried out in genomic DNA using an internal fragment of the bla_VIM-1 gene as a probe, amplified by PCR. Results: The isolate was resistant to extended-spectrum β-lactams. The MICs of carbapenems were below the resistance breakpoints but rose above resistance breakpoints when an inoculum of 10⁸ cfu/mL was used. Isoelectric focusing detected a β-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 bla_VIM-1 gene in the variable region of a class 1 integron which also carried the aac(6′)-IIc gene. The bla_VIM-1 probe hybridized with an approximately 130 kb fragment of genomic DNA, suggesting a chromosomal location of the gene. Conclusion: We describe a novel class 1 integron containing bla_VIM-1 and aac(6′)-IIc genes in an E. cloacae clinical isolate.