Characterization of ertapenem resistance in Klebsiella pneumoniae from Croatia

Abstract

ABSTRACT Klebsiella pneumoniae isolates with reduced susceptibility to carbapenems were recently reported in USA, UK, Turkey and some other countries of the world. Recently eight Klebsiella pneumoniae strains with reduced susceptibility to carbapenems were isolates in four different hospitals in Croatia. The aim of the study was to determine the mechanisms of ertapenem resistance in these strains. Antibiotic susceptibilities were determined by broth microdilution method. Transferability of ertapenem resistance was determined by conjugation (broth mating method) using E. coli A15 R- as recipient. Production of metallo β-lactamases was detected by double-disk synergy test and E test. Β-lactamases were characterized by PCR with primers specific for extended-spectrum β-lactamases, plasmid-mediated ampC β-lactamases, metallo β-lactamases of VIM and IMP series, KPC and OXA-48 β-lactamases. Genotyping of the strains was performed by pulsed-field gel electrophoresis. All strains were resistant to ceftazidime, cefotaxime, ceftriaxone, piperacillin alone and combined with tazobactam, amoxycillin/clavulanate, gentamicin and ciprofloxacin. All except one strains showed resistance to ertapenem, intermediate susceptibility or resistance to meropenem and intermediate susceptibility or full susceptiblity to imipenem. One strain was resistant to all three carbapenems. Ertapenem resistance was not transferable by conjugation to E. coli recipient in neither of our strains. PCR revealed the presence of blaSHV and blaCTX-M genes. Multiplex PCR was positive for group 1 CTX-M β-lactamases. The strain resistant to all three carbapenem was positive by E test for MBLs. However, PCR was negative for VIM and IMP β-lactamases. No KPC or plasmid-mediated ampC β-lactamases were found. The strains were not clonally related as shown by PFGE and displayed distinct PFGE fingerprints. This is the first report of carbapenem resistant Klebsiellae in Croatia. Ertapenem resistance in Klebsiella was previously reported in UK, Turkey and Israel mainly due to the production of CTX-M β-lactamases of group 1 combined with porin loss (OmpK36 or OmpK35). The characterization of outer membrane porins needs to be done to clarify the mechanisms of ertapenem resistance in our strains. Further testing is necessary to determine the mechanism of carbapenem resistance in the strain with positive E test–MBL.