Characterization of a new integron containing bla(VIM-1) and aac(6')-IIc in an Enterobacter cloacae clinical isolate from Greece.
OBJECTIVE
A clinical isolate of Enterobacter cloacae exhibiting reduced susceptibility to imipenem and a positive EDTA-disc synergy test was studied for carbapenemase production.


METHODS
MICs were determined with standard procedures as well as using a higher inoculum. Isoelectric focusing of cell extracts was used for detection of beta-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-beta-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 beta-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 beta-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.


CONCLUSIONS
We describe a novel class 1 integron containing bla(VIM-1) and aac(6')-IIc genes in an E. cloacae clinical isolate.
Galani
Irene
I
4th Department of Internal Medicine, Molecular Biology Section, University of Athens Medical School, Athens, Greece.
Souli
Maria
M
Chryssouli
Zoi
Z
Orlandou
Konstantina
K
Giamarellou
Helen
H
D000123:Acetyltransferases / Q000235:genetics* / Q000378:metabolism; D000368:Aged; D000595:Amino Acid Sequence; D016470:Bacteremia / Q000382:microbiology; D004269:DNA, Bacterial / Q000032:analysis; D024901:Drug Resistance, Multiple, Bacterial; D016972:Enterobacter cloacae / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification*; D004756:Enterobacteriaceae Infections / Q000382:microbiology; D000077727:Ertapenem; D006115:Greece; D006801:Humans; D015378:Imipenem / Q000494:pharmacology; D032023:Integrons / Q000235:genetics*; D007769:Lactams / Q000494:pharmacology; D008297:Male; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism; D047090:beta-Lactams / Q000494:pharmacology*
D004269:DNA, Bacterial; D007769:Lactams; D047090:beta-Lactams; D015378:Imipenem; D000123:Acetyltransferases; C417264:VIM-1 metallo-beta-lactamase; D001618:beta-Lactamases; D000077727:Ertapenem