Study on the drug-resistance mechanism of plasmid-mediated KPC-2 carbapenemase possessing Klebsiella pneumoniae isolated from children / 中华微生物学和免疫学杂志

ABSTRACT

Objective To investigate molecular epidemiology and antimicrobial susceptibility of carbapenem-resistant strains of Klebsiella pneumoniae isolated from children.Methods From July 2010 to June 2011,twelve non-replicate clinical isolates of carbapenem-resistant Klebsiella pneumoniae were consecutively collected from children inpatients in the Second Hospital of Wenzhou Medical Colloge.All of the isolates were identified by the automated microbiology systems.Modified Hodge test was used to screen strains producing carbapenemases.Pulsed field gel electrophoresis(PFGE) was performed to analyze the homogeneity of genomic DNA of Klebsiella pneumoniae.KPC,IMP,GIM,SPM,SME,OXA-10,bla(s),VIM gene and integrase gene were amplified by PCR and then sequenced to cofirm the genotypes;Plasmid conjugation experiment was used to study the transfer method of bacterial resistance.Plasmid-curing test were used to initally locate the resistant genes.Results One(8.3％),5(41.7％),7(58.3％),1(8.3％),1(8.3％) and4(33.3％) of12isolates were susceptible to gentamicin,tobramycin,amikacin,ciprofloxacin,levofloxacin and trimoxazole,respectively.All isolates carried KPC-2,TEM-1 and SHV genes(six for SHV-11-like,six for SHV-12-like).Eleven of twelve isolates with KPC-2 gene carried CTX-M genes(4 for CTX-M-14-like,6 for CTX-M-15-like).Two isolates carried OXA-10 genes,and one isolates carried PER-1 gene.None of NDM-1,GIM,SPM,SIM and VIM carbapenemase genes was detected in 12 isolates.All of 12 isolates carried Int 1 genes.The plasmids of 2 isolates were transgerred into the recipients E.coli EC600.PCR and sequence analysis revealed that blaTEM-1 and blaCTX-M-15-like were co-transferred with the KPC-2 gene to the recipients.Elimination of KPC-2-encoding plasmid from Kp7 and Kp12 resulted in imipenem susceptibility in the two isolates.Amplification revealed that KPC-2 gene was lost by the plasmid-curing test.Of the 12 isolates,5 patterns were obtained by PFGE.Pattern B and C were the main drug resistant clones.Conclusion KPC-2 gene are the major carbapenemase genes in Klebsiella pneumoniae isolated from children,including ESBLs and integrase.Some resistance genes can be disseminated by plasmids.