title: Sources of diversity of carbapenem resistance levels in Klebsiella pneumoniae carrying blaVIM-1.
abstract: OBJECTIVE
  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.
authors.affiliation: Laboratory of Bacteriology, Institute Pasteur Hellenique, Athens, Greece.
authors.forename: A
authors.initials: A
authors.lastname: Loli
authors.forename: L S
authors.initials: LS
authors.lastname: Tzouvelekis
authors.forename: E
authors.initials: E
authors.lastname: Tzelepi
authors.forename: A
authors.initials: A
authors.lastname: Carattoli
authors.forename: A C
authors.initials: AC
authors.lastname: Vatopoulos
authors.forename: P T
authors.initials: PT
authors.lastname: Tassios
authors.forename: V
authors.initials: V
authors.lastname: Miriagou
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D015780:Carbapenems / Q000494:pharmacology*; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D007711:Klebsiella pneumoniae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D008826:Microbial Sensitivity Tests; D010957:Plasmids; D016133:Polymerase Chain Reaction; D018440:beta-Lactam Resistance / Q000235:genetics*; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D015780:Carbapenems; D001618:beta-Lactamases