| Objective1.To study the epidemiological characteristics of carbapenem-resistant Klebsiella pneumoniae(CRKP)and provide evidence for further preventions and control of CRKP infection.2.To investigate the distribution of integrons in clinical isolates of carbapenem-resistant and their relationships to bacterial resistance to antimicrobial agents.3.To analysis the diversity of the genetic environment of the blaKPC-2 harboring Klebsiella pneumoniaes.Methods1.A total of 84 non-duplicated CRKP isolates were collected from various specimens of patients at Fengxian Hospital affiliated of Anhui University of Science and Technology from Januaiy 2016 to December 2017.The universal carbapenemase genes,including blaKPC,blaNDM,blaVIM,blaVES,blaIMP,blaGES,blaoxA-23,were screened by PCR.PCR analysis and gene sequencing were used to analyze the serum capsule typing and virulence gene.MLST were performed to analyze the molecular epidemiology of isolates.Different ST type strains were selected according to the situation to determine whether the carbapenem resistance gene was located on the conjugative plasmid through a conjugation test.2.For the 84 non-duplicated CRKP isolates,Classes 1 and 2 integrase genes were screened by PCR.The variable regions of integrons were amplified by long fragment PCR.The types of promoters and gene cassette arrays of variable regions were determined by sequencing,and the relationship between integrator and bacterial resistance to antimicrobial agents was analyzed by statistical methods.3.A total of 79 blaKPC-2 harboring CRKP isolates were collected from various specimens of patients at Fengxian Hospital affiliated of Anhui University of Science and Technology from January 2016 to December 2017.The genetic environment of the blaKPC-2 gene was investigated by Junction PCR,Mapping PCR,genome sequencing assembling,reverse PCR to analyze the diversity of genetic environment of blaKPC-2.Results:1.79 isolates were positive for blaKPC and were all confirmed to be blaKPC-2.11 isolates were positive for blaNDM and were all confirmed to be blaNDM-5,of them,6 isolates were positive both for blaKPc-2 and blaNDM-5.blaVIM,blIMP,blaGES,blaoxA-23 and blaVEB were all negative.The hypervirulent members of K.pneumoniae,did not detected in this study.84 CRKP had eight different MLST patterns and had various clonal groups,the main of which was ST290.Of 79 blaKPC-2 positive isolates,the main serotype is wzi21-K21,accounting for 55.7%(44/79),and the dominant clone was ST290.7 of 11 blaNDM-s positive isolates were ST340.The blaKPC-2 gene of ST290,ST15,ST1066 stains were successfully transferred by conjugation.2.Of 84 CRKP,Class 1 integrase gene intll was detected in 76 isolates(90.4%),while class 2 integrase gene intI2 was not detected.Two gene cassette arrays of variable regions,aadA2 and dfrA12-orfF-addA2 were detected in 75 intl1-positive isolates.Among them,26 were aadA2 and which promoter was PcHl,49 were dfrA12-orfF-addA2 and which promoter was PcWTGN-10.3.The genetic environment of blaKPC-2 was diverse,the Tn1721－blaKPc-2-ΔTn3-IS26 was the dominant chimeric structure,it also shows five distinct platforms(i-v)in 79 blaKPC-2 harboring CRKP,showing the polymorphism of the blaKPC-2 surrounding genetic environment,this suggested that there may be different mechanisms for the horizontal transmission of blaKPC-2 gene.Five plasmid replicon types(IncFIA,IncFIIS,IncFrepB,IncN,IncT)were detected in 79 blaKPC-2 positive isolates.Sixty-five isolates carried two or more plasmid replicons.As to the blaKPC-2 positive ST290 isolates,35 of 40 were IncFIA+IncFIIS,indicated strong plasmid compatibility and prone to acquire antimicrobial resistance genes through plasmids transmission.Conclusion:Antimicrobial resistance in carbapenem-resistant Klebsiella pneumoniae was serious,which closely related with blaKPc-2 and blaNDM-5 gene;CRKP strains had 8 clonal types,indicating that the transmission of CRKP was polyclonal.This study reported for the first time the prevalence of blaKPC-2 carrying K.pneumoniae ST290.In addition,blaNDM-5 in K.pneumoniae ST340 and co-carrying of blaKPC-2 and blaNDM-5 in K.pneumonia were also reported for the first time;The detection rate of class 1 integrase gene intl1 in carbapenem-resistant Klebsiella pneumoniae was high,which were associated with high antimicrobial resistance rates of CRKP;The genetic environment of blaKPC-2 genes was diverse,there may be exist new mechanisms for the horizontal transmission of blaKPC-2 gene.Figure[4]table[11]reference[40]... |
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