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JAC Advance Access originally published online on July 26, 2006
Journal of Antimicrobial Chemotherapy 2006 58(3):669-672; doi:10.1093/jac/dkl302
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Sources of diversity of carbapenem resistance levels in Klebsiella pneumoniae carrying blaVIM-1

A. Loli1, L. S. Tzouvelekis1,2, E. Tzelepi1, A. Carattoli3, A. C. Vatopoulos4, P. T. Tassios2 and V. Miriagou1,*

1 Laboratory of Bacteriology, Institute Pasteur Hellenique Athens, Greece 2 Department of Microbiology, Medical School, University of Athens Athens, Greece 3 Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanita Rome, Italy 4 Department of Microbiology, National School of Public Health Athens, Greece

Received 26 May 2006; returned 28 June 2006; revised 3 July 2006; accepted 4 July 2006

*Corresponding author. Tel: +30-210-6478810; Fax: +30-210-6423498; E-mail: miriagou{at}mail.pasteur.gr

Objectives: To elucidate the mechanisms responsible for the diversity of ß-lactam resistance phenotypes among isolates of a VIM-1-producing Klebsiella pneumoniae (VPKP) strain that is endemic in Greek hospitals.

Methods: Five VPKP clinical isolates were studied. MICs of ß-lactams were determined by agar dilution. PFGE of XbaI-digested genomic DNA was used for typing. Profiles of outer membrane proteins (OMPs) were determined by SDS–PAGE. Selected isolates were transformed with a plasmid encoding the Omp36K porin. ß-Lactamase activities were analysed by IEF and imipenem hydrolysis was assessed by spectrophotometry. VIM-1-encoding, self-transmissible plasmids were characterized by replicon typing, RFLP and hybridization with blaVIM- and IS26-specific probes. Characterization of integrons was performed by PCR, cloning and sequencing.

Results: Isolates exhibited highly similar PFGE patterns. Imipenem MICs were 2, 4, 16, 32 and 64 mg/L. The isolate with the highest imipenem MIC (Vipm-64) lacked a 36 kDa OMP. Expression of a cloned OmpK36 in this isolate reduced the imipenem MIC to susceptibility levels. Imipenem-hydrolysing activity was significantly higher in Vipm-16 as compared with the other isolates that expressed similar amounts of VIM-1. All isolates transferred ß-lactam resistance to Escherichia coli through conjugative, IncN plasmids that exhibited differences in the RFLP and hybridization patterns with blaVIM- and IS26-specific probes. The Vipm-16 plasmid, mediating the higher imipenem MICs among transconjugants, carried two copies of blaVIM-1. Cloning and sequencing showed In-e541-like integrons truncated at the 5'CS by insertion of IS26 elements at two different positions.

Conclusions: A VIM-1-producing strain of K. pneumoniae has evolved through OMP alterations and rearrangements in the blaVIM-1-carrying plasmid probably mediated by IS26, generating isolates with imipenem MICs ranging from susceptibility to resistance.

Keywords: K. pneumoniae , metallo-ß-lactamases , ß-lactam resistance


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