Objectives: To elucidate the mechanisms responsible for the diversity of β-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 β-lactams were determined by agar dilution. PFGE of Xba I-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. β-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 β-lactam resistance to Escherichia coli through conjugative, IncN plasmids that exhibited differences in the RFLP and hybridization patterns with bla VIM - and IS 26 -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 IS 26 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 IS 26, generating isolates with imipenem MICs ranging from susceptibility to resistance. © 2006 Oxford University Press.
Sources of diversity of carbapenem resistance levels in Klebsiella pneumoniae carrying blaVIM-1 / Loli, A; Tzouvelekis, Ls; Tzelepi, E; Carattoli, A; Vatopoulos, Ac; Tassios, Pt; Miriagou, V.. - In: JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY. - ISSN 0305-7453. - 58:3(2006), pp. 669-672. [10.1093/jac/dkl302]
Sources of diversity of carbapenem resistance levels in Klebsiella pneumoniae carrying blaVIM-1
Carattoli A;
2006
Abstract
Objectives: To elucidate the mechanisms responsible for the diversity of β-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 β-lactams were determined by agar dilution. PFGE of Xba I-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. β-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 β-lactam resistance to Escherichia coli through conjugative, IncN plasmids that exhibited differences in the RFLP and hybridization patterns with bla VIM - and IS 26 -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 IS 26 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 IS 26, generating isolates with imipenem MICs ranging from susceptibility to resistance. © 2006 Oxford University Press.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
