Study On The Plasmid-mediated Carbapenemase In A Clinical Isolate Of Enterobacter Cloacae And The Mechanism Of Transmission

Carbapenemases refer to a category of β- lactamases with highly activity in hydrolyzing carbarpenams e.g. imipenem or meropenem. Currently, the reported carbapenemases belonged to classes A, B, or D in Ambler molecular classification scheme .In Enterobacteriaceae bacteria, the reported carbapenemase are mainly of classes A and B. The coding genes of carbapenemases can be located in transferable plasmid, therefore can be transmitted among different species. It results in much difficulty for clinicians to effectively treat the infections caused by such highly resistant strains.We investigated the resistance mechanism of a strain of carbapenem-resistant E. cloacae isolated from the blood sample of a patient with uremia in our hospital in August 2001. The minimal inhibitive concentration (MIC) were detected by E test. Isoelectric focusing electrophoresis(IEF), three dimensional test, conjugation experiment, Southern blot, polymerase chain reaction(PCR), restriction endonuclease to digest , cloning and screening , shot gun sequencing and walking sequencing methods were carried out for analyzing the enzyme activity, the resistance gene, mechanism of transmission.The carbapenemase produced by Enterobacter cloacae had the band with pIs 8.1. The carbapenemase had some characteristics of Class 2f enzyme which can hydrolyze penicillins and carbapenem antibiotics. However, it has low potency to hydrolyze cefotaxime and ceftazidime. The activity of hydrolysising imipenem could be inhibited by clavulanic acid, but not inhibited by cloxacillion and EDTA. The imipemen-resistant gene was located on a about 80kb plasmid and the amino acid sequence have 99% of homology with chromosome-mediated IMI-1. In addition, at each flanking side of the encoding gene there is an identical enzyme which had 71% homology with Tn903.In brief, The Enterobacter cloacae resistant to imipenem was caused by a novel plasmid-mediated IMI-3 carbapenemase (GenBank accession number : AY780889) . This study also confirmed that IMI-3 encoding gene was transferred from 2f carbapenemase gene located in chromosome to plasmid via Tn903.In China, It is the first time to report plasmid-mediated carbapenemase in Enterobacteriaceae bacteria.