[Detection of plasmid-mediated carbapenem-hydrolyzing beta-lactamase KPC-2 in a strain of carbapenem-resistant Enterobacter cloacae].

OBJECTIVE: To investigate the mechanism of carbapenem resistance in Enterobacter cloacae.

METHODS: A carbapenem-resistant strain of E. cloacae (strain ZY1465) was isolated. Antibiotic susceptibilities were determined by agar dilution method. Conjugation experiments were carried out in mixed broth cultures. Plasmid DNA preparations were obtained by using an alkaline lysis technique and were digested by various endonucleases; The crude beta-lactamase extracts of E. cloacae and E. coli transconjugant were subjected to analytical isoelectric focusing (IEF). Specific PCR amplification and DNA sequence analysis were preformed to confirm the beta-lactamase type. Outer membrane proteins (OMPs) were isolated and examined by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

RESULTS: The E. cloacae isolate showed resistance against carbapenems. The MICs of imipenem and meropenem were both 32 microg/ml. The isolate was also resistant strongly against penicillins, cephalosporins, cefoxitin, aztreonam, quinolones, and aminoglycosides. Conjugation studies with E. coli resulted in the transfer of reduced carbapenem susceptibility from E. cloacae isolate. Plasmid restriction analysis showed identical restriction profiles between the transconjugants of E. cloacae ZY1465 and Serratia marcescens ZN008. Isoelectric focusing demonstrated six beta-lactamases, with the isoelectric points (pIs) of 5.4, 6.7, 7.3, 7.8, 7.9, and 8.6, in E. cloacae ZY1465, and only one beta-lactamase with the pI of 6.7 in transconjugant. Specific PCR amplification and DNA sequence analysis confirmed that E. cloacae ZY1465 harbored TEM-1, KPC-2, DHA-1, CTX-M-14, CTX-M-3 and chromosomal AmpC (not detected in IEF) genes. Urea-SDS-PAGE analysis of OMPs showed that E. cloacae ZY1465 lacked an OMP of approximately 38 000 Da which was present in E. cloacae ATCC13047.

CONCLUSION: It is the first detection of plasmid-mediated carbapenem-hydrolyzing beta-lactamase KPC-2 in a clinical isolate of E. cloacae from China. Production of multiple beta-lactamases, especially KPC-2 and loss of an OMP in E. cloacae ZY1465 result in resistance to carbapenems.