Sources of Diversity of Carbapenem Resistance Levels in Klebsiella Pneumoniae Carrying blaVIM-1

J Antimicrob Chemother. 2006 Sep;58(3):669-72. doi: 10.1093/jac/dkl302. Epub 2006 Jul 26.

Abstract

Objectives: To elucidate the mechanisms responsible for the diversity of beta-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 beta-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. beta-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 bla(VIM)- 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 beta-lactam resistance to Escherichia coli through conjugative, IncN plasmids that exhibited differences in the RFLP and hybridization patterns with bla(VIM)- and IS26-specific probes. The Vipm-16 plasmid, mediating the higher imipenem MICs among transconjugants, carried two copies of bla(VIM-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 bla(VIM-1)-carrying plasmid probably mediated by IS26, generating isolates with imipenem MICs ranging from susceptibility to resistance.

MeSH terms

  • Anti-Bacterial Agents / pharmacology*
  • Bacterial Outer Membrane Proteins / genetics*
  • Carbapenems / pharmacology*
  • Cloning, Molecular
  • Genes, Bacterial*
  • Klebsiella pneumoniae / drug effects
  • Klebsiella pneumoniae / enzymology
  • Klebsiella pneumoniae / genetics*
  • Klebsiella pneumoniae / isolation & purification
  • Microbial Sensitivity Tests
  • Plasmids
  • Polymerase Chain Reaction
  • beta-Lactam Resistance / genetics*
  • beta-Lactamases / genetics*

Substances

  • Anti-Bacterial Agents
  • Bacterial Outer Membrane Proteins
  • Carbapenems
  • beta-Lactamases