Study On Antimicrobial Resistant Mechanisms Of Carbapenem-resistant Enterobacteriaceae

Objectives: To investigate the antimicrobial resistant mechanisms of CRE, construct monitoring system of acquired carbapenemase.Methods: Clinical isolates of Enterobacteriaceae in our hospital which zone diameters of meropenem were not larger than 21mm were collected from January 2007 to June 2010. Antibiotic susceptibility was performed to select CRE. Then, phenotype and gene screenings were performed. Integron insertion sequence and genetic structure of carbapenemase genes were also detected by PCR method. Pulsed-field gel electrophoresis (PFGE) and Southern blot were used to analyze the plasmids of CRE. Multilocus sequence typing (MLST) was used to determine the genotypes and homology of these isolates. In addition, out membrane proteins (OMPs) were examination by PCR and SDS-PAGE.Results: 11 isolates of CRE were collected and confirmed, mostly were Klebsiella pneumoniae (7/11). Susceptibility of antimicrobial agents indicated that all these strains resistant to most antimicrobials, including carbapenems (meropenem 8~64μg/ml, imipenem 4~64μg/ml, ertapenem 4~64μg/ml). However, susceptibilities of aminoglycosides and fluoroquinolones were significantly different. Using PCR method to detect resistant genes, the result showed that 6 isolates were IMP-4 positive and 3 isolates were KPC-2 positive, including one isolate carrying both blaIMP-4 and blaKPC-2 genes. Integron insertion sequences were amplified and sequenced. All of them did not include carbapenemase encoding genes. Genetic structure of carbapenemase genes was analyzed, suggesting that blaKPC-2 located in an integration structure of a Tn3-based transposon and partial Tn4401 segment. PFGE showed that most CRE contained three or more plasmids. Out membrane proteins were detected by SDS-PAGE, indicating that only one isolate, Kox656, lacked OMPs (OmpK35 and OmpK36). Kpn6617 and Kpn6099 were assigned to a novel sequence type, ST476, by MLST。Besides, blaKPC-2 encoding plasmid in Kpn6617 was transmissible. Further evidence showed the homology of blaKPC-2-harbouring plasmids among KPC-2 positive isolates. However, the probing data for blaIMP-4 suggested a diversity of blaIMP-4-harbouring plasmids.Conclusions: The most common carbapenemase-resistant Enterobacteriaceae was K.pneumoniae in our hospital. Producing carbapenemases, which type mostly IMP-4, was the most reason that bacteria resistant to carbapenem. A novel carbapenemase-resistant K.pneumoniae encoding both blaKPC-2 and blaIMP-4 was detected, probalbly due to transmission of the blaIMP-4-harbouring plasmid into KPC-2-producing K.pneumoniae (ST476). The concomitant presence of these genes is alarming and poses therapeutic as well as infection control problems.