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Transfer and carriage of naturally occurring Pseudomonas aeruginosa plasmids harbouring metallo-beta-lactamase to Enterobacteriaceae strains

Abstract number: 1134_01_9

Castanheira M., Mendes R.E, Toleman M.A, Jones R.N, Walsh T.R.

Background:  

The presence of metallo-beta-lactamase (MBL) genes in clinical strains of some Enterobacteriaceae is worrisome since carbapenems have been used clinically to counteract extended-spectrum beta-lactamase producing pathogens. Herein, we assessed the frequency and the viability of plasmids carrying MBL genes transferred into Enterobacteriaceae.

Methods:  

Twenty-one carbapenem-susceptible and intermediate resistant Enterobacteriaceae (Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae and Citrobacter freundii) clinical isolates from different genotypes were screened for absence of plasmids and activity against carbapenems. The isolates were transformed with wild-type plasmids (24 kb–400 kb) harbouring the MBL genes blaIMP-1, blaVIM-7, blaSPM-1 and blaGIM-1 by electroporation and conjugation. Plasmids were isolated using Qiagen commercial kits. Selection was performed with a range of ceftazidime (20–100 mg/ml) or imipenem (10–50 mg/ml) incorporated into the medium. The plasmids RSF1010 and pK18 were used as controls during the transformation experiments. PCR reactions for the internal regions of the MBL genes and activity assays against imipenem were performed to confirm the transfer of the gene.

Results:  

Twelve isolates containing no plasmids and showing high-level of sensibility to carbapenems were transformed with the four MBL harbouring plasmids. Transformants could not be recovered in 11 from the 12 isolates, although the controls demonstrated positive transfer at high frequencies. Evidence of transfer was obtained in plates containing 20, 30 and 40 mg/ml of imipenem with an imipenem intermediate-resistant C. freundii isolate (MIC 8 mg/ml) transformed with blaVIM-7 plasmid. The presence of the MBL gene in the transformants was confirmed by PCR and sequencing; however, no activity against imipenem could be observed.

Conclusions:  

The spread of the MBL genes in Enterobacteriaceae is a great concern. However, data from this pilot study indicates that in-vitro transfer does not happen at a high frequency and may be strain dependent.

Session Details

Date: 01/08/2007
Time: 00:00-00:00
Session name: XXIst ISTH Congress
Subject:
Location: Oxford, UK
Presentation type:
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