A Study On The Molecular And Epidemical Mechanism Of Carbapenem-Resistance Enterobacteriaceae

BackgroundsEnterobacteriaceae are important causes of community-acquired and hospital-associated infections,the severe abuse of antibiotics has promoted the rapid dissemination of Enterobacteriaceae resistant to drugs.At present,drug-resistance of Enterobacteriaceae has gradually become a serious and universal problem,resulting in the inefective clinical treatment of antimicrobial agents,increasing the cost of clinical treatment,shortering period of new antimicrobials application and development,threatening the health of human directly. Carbapenems,such as imipenem and meropenem,diffuse easily into bacteria,and have a very broad spectrum of activity as the drugs, especially against many Gram-negative bacteria possessing extended-spectrumβ-lactamases(ESBLs) and AmpC enzymes.However,carbapenem-resistant Gram-negative bacteria, particularly Acinetobacter baumannii and Pseudomonas aeruginosa,has been observed in some areas,but in Enterobacteriaceae is rare.In our study,we aimed to investigate the molecular epidemiology and mechanism of carbapenem resistance in Klebsiella pneumoniae, Escherichia coli and Serratia marcescen.ObjectiveTo investigate the molecular epidemiology and mechanism of carbapenem resistance of Enterobacteriaceae collected from ICUs.Methods1.Enterobacteriaceae resistance to carbapenem were isolated from patients in Nanjing general hospital,and antimicrobial susceptibility were determined by the disk diffusion and agar dilution method.2.Enterobacterial repetitive intergenic consensus-PCR(ERIC-PCR) and pulsed-field gel electrophoresis(PFGE) were performed to analyze the molecular epidemiology of carbapenem-resistance isolates.3.Isoelectric focusing,three-dimensional-test and EDTA-Na₂-disk synergy test were performed to analyzeβ-lactamases.4.Specific PCR,DNA sequencing and southern blotting were carried to confirm carbapenemase genotype.5.plasmid extraction by using an alkalinelysis technique were subjected to determine transformation assay,conjunction experiment were determined to revealed the transferability of plasmid,and elimination of plasmid were carried out to analyze its transmission mechanism.6.SDS-PAGE was subjected to analyze the alterations of outer membrane proteins(OMPs) of Enterobacteriaceae resistance to carbapenem.Results1.All the clinical isolates were resistant toβ-lactam including penicillins,cephalosporins,aztreonam,carbapenem antimicrobial agents andβ-lactamase inhibitors,but uncertain to fluoroquinolones and aminoglycosides2.PFGE and ERIC-PCR showed the Klebsiella pneumoniae, Escherichia coli and Serratia marcescen belonged to different genotypes.3.IEF,three-dimensional-test of enzyme extraction, EDTA-Na₂-disk synergy test revealed that all isolates produced KPC-2 carbapemase with pI of 6.7,andβ-lactamase inhibitor didn't significantly inhibited the hydrolyzing carbapenems.4.PCR,DNA sequencing,southern blotting revealed that all isolates encoded carbapenem-hydrolyzing enzyme gene,KPC-2.5.Southern blotting with plasmid of Klebsiella pneumoniae, plasmid extraction and transformation assays of Escherichia coli revealed that KPC-2 were encoded on an approximately 56-kb plasmid. Conjunction tests of Serratia marcescen indicated that carbapenem-resistance could be transferred from parent isolates to recipient.6.SDS-PAGE analysis revealed that there are alterations in outer membrane proteins(OMPs) of Escherichia coli and Serratia marcescens resistant to carbapenem compared to isolates susceptibility to carbapenem.ConclusionsProduction of KPC-2 carbapenemase mainly contributed to reduced susceptibility of carbapenem in Klebsiella pneumoniae, Escherichia coli and Serratia marcescens.The alterations in outer membrane proteins(OMPs) may as a cofactor in high-level drug-resistance in Escherichia coli and Serratia marcescens.Vertical transmission and horizontal transmission plasmid-mediated are probably the principal epidemical mechanism.