Detection Of Carbapenemase Genes And Epidemiology Study In Enterobacteriaceae Isolates

Objectives To investigate the drug resistance and prevalence of carbapenemases in Enterobacteriaceae clinical isolates collected from our hospital and to study molecular epidemiology and resistance mechanism of carbapenemase-producing isolates.Methods Nonrepetitive Enterobacteriaceae isolates were collected from May to October 2010 in the clinical microbiology laboratory of our hospital. Enterobacteriaceae isolates resistant to carbapenems were screened by disk diffusion method. The modified Hodge test and EDTA-synergy test were used to screen carbapenemase-producing isolates.The blaKPC, blaIMP, blaVIM and blaNDM-1 genes were detected by specific PCR and the amplified products were sequenced. Minimal inhibitory concentrations (MICs) were measured using two-fold agar dilution method. Conjugation test and ERIC-PCR were performed to analyze the resistant molecular mechanism and molecular epidemiology of carbapenemase-producing isolates.Results In all the 738 strains, the resistant rates to imipenem,meropenem and ertapenem were 1.36%(10/738),2.17%(16/738) and 5.96%(44/738), respectively.27 strains were positive in the modified Hodge test and 20 strains were positive in EDTA-disk synergy test.24 isolates were found to carry carbapenemase genes.4 K. pneumoniae isolates possessed blaKPC-2 gene.9 strains harboured IMP-4 type carbapenemase. A novel IMP-type metallo-enzyme, named as IMP-30, was discovered in 9 K.pneumoniae isolates. The GenBank accession number was HQ875573. Plasmid DNA extracted from carbapenemase-producing strains was found to carry carbapenemase gene by specific PCR methods. The blaKPC gene-harboring plasmid was successfully transferred to the recipient strain. But conjugation test in IMP producing isolates failed. ERIC-PCR typing results showed that the KPC-2 producing K. pneumoniae isolates belonged to the same clone type, however, IMP-producing isolates had many epidemic cloning strains.Conclusions IMP-type carbapenemase is the main type of carbapenemase in clinical carbapenem-resistant Enterobacteiaceae isolates in our hospital, followed by KPC type. A novel IMP-subtype gene, named as IMP-30, is found in K. pneumoniae isolates. Many cloning types of carbapenemase-producing strains are spreading in our hospital. Enough importance should be attached to the carbapenemase-producing strains by clinicians. Effective measures should be taken to control the spread of resistant isolates.