Molecular Characterization Of Enterobacteriaceae With Decreased Susceptibility To Carbapenems

【Objective】To study the mechanism of carbapenme-resistant Enterobacteriaceae.【Method】49 Enterobacteriaceae strains with decreased susceptibility to carbapenem were isolated from 16 teaching hospitals in China from 2004 to 2008. MICs of antimicrobial agents were determined by agar dilution method. Modified Hodge test (MHT) and EDTA synergistic test was used to detect carbapenemase. Conjugation test and plasmid profile analysis was carried out to analyze the transferable genes. The encoding genes of beta-lactamases including carbapenemases, Qnr, aac(6')-Ib and integron were detected by PCR. Electrophoretic analysis of OMPs by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. RT-PCR was performed to investigate the expression level of ompK35 and ompK36 porins. The homogeneity of strains was analyzed by pulse-field gel electrophoresis (PFGE).【Results】Thirty-six of 49 isolates had intermediate resistance or were resistant to imipenem (MIC,>4μg/ml),31 had intermediate resistance or were resistant to meropenem (MIC,>4μg/ml) and 47 had intermediate resistance or were resistant to ertapenem (MIC,>2μg/ml). Fourty-five isolates were susceptible to tigecycline while 47 were susceptible to polymyxin B. PFGE showed that most isolates had poor homogenicity except a few clonal-disseminated strains. Sixteen isolates were detected to carry carbapenemase genes.4 Klebsiella pneumoniae strains and 2 Escherichia coli strains, which were all isolated from Hangzhou city, carried blaKPC-2.3 Klebsiella pneumoniae strains,3 Enterobacter cloacae strains and 2 Citrobacter freundii strains carried blaIMP-4.2 Klebsiella pneumoniae strains carried blaIMP-8.Resistance genes-harboring plasmids were successfully transferred from 23 of 49 strains to E.coli C600 while 13 carbapenemase-producing transconjugants were acquired. Nine different structures of class 1 integrons were dectected, and most integrons carried genes mediating resistance to aminoglycoside and trimethoprim. Isolates with combination of carbapenemase and porin loss showed relatively high MICs of carbapenems. Among the 33 non-carbapenemase-producing strains,29 lost or decreased-expressed both two major porins (OmpK35 and OmpK36 for Kpneumoniae, OmpF and OmpC for E.coli, E. cloaceae, E. aerogenes and C.freundii) and 28 of them produced ESBLs or AmpC or both enzymes simultaneously. RT-PCR revealed that the resistant level of Klebsiella pneumoniae to carbapenems was related to the combined expression level of OmpK35 and OmpK36.【Conclusion】Moderate-to-high level carbapenem resistance in most isolates was mediated by Loss or decreased expression of both major porins (OmpK35/36 or OmpF/C) combined with production of AmpC or ESBLs enzymes, while KPC-2, IMP-4 and IMP-8 carbapenemase production was associated with low-to-moderate level carbapenem resistance in Enterobacteriaceae in China.