Identification of plasmid-mediated carbapenem-hydrolyzing β-lactamase KPC-2 in Enterobacteriaceae / 中华检验医学杂志

ABSTRACT

Objective To investigate the molecular epidemiology and mechanism of earbapenem resistance of Serratia marcescens,Klebsiella pneumoniae and Escherichia coli isolates from intensive care units(ICUs).Methods Twenty-one S.marcescens,ten K.pneumoniae and one E.coli isolates with carbapenem resistance or reduced carbapenem susceptibility were recovered from two ICUs in our hospital from April 2006 to Febmary 2007.Pulsed-field gel electrophoresis(PFGE)and enterobacterial repetitive intergenic consensus-PCR(ERIC-PCR)were performed to analyze the molecular epidemiology of isolates.Antibiotic susceptibilities were determined bv agar dilution method.Conjugation experiments were carried out in mixed broth cultures.Plasmid DNA was obtained bv using an alkalinelysis technique and was digested by various endonucleases.Elimination of plasmid from S.marcesceus isolates were performed by repeated SDS treatment.The crude β-lactamase extracts of original isolates and E.coli transconjugants were subjected to isoelectric focusing(IEF);Specific PCRs and DNA sequencing were preformed to confirm the genotype of β-lactamases.Results ERIC-PCR indicated that all S.marcescens isolates belonged to a clonal strain.PFGE indicated that ten K. pneumoniae isolates were indistinguishable or closely related to each other.The MICs of imipenem and meropenero for all isolates were 2 to 8 μg/ml except K.pneumoniae K10(128 and 256 μg/ml).Conjugation studies with E.coli(EC600)resulted in the transfer of reduced carbapenem susceptibility from original isolates(MICsfrom≤0.125 μg/ml to 1-2μg/ml).IEF,PCR and DNA sequence analysis confirmed that S.marcescens isolates produced KPC-2(pI of 6.7)and a β-lactamase(pI 6.5).k pneumoniae isolates produced TEM-1(pI 5.4),KPC-2,CTX-M-14(pI 7.9),and a β-lactamase(pI 7.3).E.coli El produced KPC-2,CTX-M-15(pI 9.0),and a β-laetamase(pI 7.3).Only a KPC-2 was detected in E.coli transeonjugants.Plasmid restricfion analysis using EcoR Ⅰ,Hind Ⅲ,and Bcu Ⅰ showed identical restrietion patterns among all E.coli transconjugants.SDS-PAGE and ompK 35/36 gene sequence analysis of OMPs revealed that K.pneumoniae K10 failed to express OmpK36 because of insertional inactivation by an insertion of ISEcp1.Conclusions Carbapenem-non-susceptible S.marcescens.K.pneumoniae and E.coli were epidemic in two ICUs in our hospital.Resistance or reduced susceptibility to carbapenems in these strains is mainly due to production of KPC-2.Presence of KPC-2 combined with porin deftciency result in high-level carbaoenem resistance in K.pneumoniae.The game blaKPC2-encoding plasmid spreads among the three different genera.