Studies On Molecular Epidemiology Of Carbapenem Resistant Enterobacteriaceae

Objective:Recently,Carbapenem-Resistant Enterobacteriaceae(CRE)have emerged and spread rapidly.Carbapenem-resistant Klebsiella pneumoniae(CRKP)is the most prevalent,especially strains with both high virulence and high resistance(CR-hvKP),is seriously threating the human health.Klebsiella oxytoca is low in clinical isolation,but always leads to serious situation in severe patients.Therefore,an in-depth understanding of its molecular resistance mechanism and epidemiological characteristics is of great significance for the treatment,monitoring and prevention of clinical infections.Firstly,this paper intends to carry out an overall molecular epidemiological analysis of CRE isolates from six representative regions of China,with the molecular resistance and virulence of CRKP as a priority.Secondly,this paper will carry out a research on Carbapenem-resistant Klebsiella oxytoca(CRKO).It is hoped that through these studies,we will further understand the molecular epidemiology of CRE in China,especially Klebsiella,so as to provide a theoretical basis for the treatment and prevention of clinical CRE infections.Methods:1.Epidemiological analysis of molecular resistance of multi-center CRE isolates in China.(1)Strain identification and drug sensitivity experiments:clinical CRE strains from six regions including Beijing,Chengdu,Guangzhou,Kunming,Suzhou and Yinchuan in 2018 were collected.Strain identification and drug sensitivity experiments were conducted by matrix-assisted laser desorption ionization-time-of-flight mass spectrometer(MALDI-TOF MS)and phoenix-100(BD)automatic microbiological analyzer.(2)Detection of carbapenenase genes:carbapenenase genes(blaKPC?blaNDM?blaVIM?blaIMP?blaoXA-48)were identified by PCR amplification,and positive results were further identified by sequencing.(3)Molecular detection of Klebsiella pneumoniae:multiple PCR was used to detect the Sequence typing(ST11,ST258,ST23,ST64/375,ST86),serum capsule typing(wzyK1,wzyK2,WzyKL64,wzykL47)and virulence genes(rmpA,rmpA2,iroN,iutA)of K.pneumoniae.2.Multi-center molecular epidemiological study of carbapenems resistant Klebsiella oxytoca.(1)Strain identification and drug sensitivity experiments:during July 2015 to June 2018,CRKO isolates from Chengdu,Kunming,Suzhou and Yinchuan were collected.Strain identification and drug sensitivity experiments were carried out by the same method as 1.1.(2)Detection of carbapenenase genes:The method is the same as 1.2.(3)Plasmid conjugation experiments:The donor and the recipient strains were co-cultured for plasmid conjugation and positive zygotes were further identified by PCR.(4)Pulsed-field gel electrophoresis(PFGE)analysis:the homology between strains was analyzed by PFGE.(5)Plasmid S1-PFGE and Southern blot experiments:Plasmids integrated with the carbapenemase genes were further identified by these experiments.(6)Plasmid sequencing analysis:representative plasmids were extracted for the sequence determination and further analysis.3.Research on NDM-5 producing Klebsiella pneumoniae sequence type 258 in China.(1)Strain collection and drug sensitivity experiments:The NDM-5 producing K.pneumoniae ST258(Kp2573),carbapenem-sensitive K.pneumoniae(Kp2588)and carbapenem-resistant Escherichia coli(Ec2551)were isolated from the same patient.The minimum inhibitory concentration(MIC)against various antibiotics of these strains was detected by VITEK 2 system.(2)Carbapenemase gene and sequence typing(ST)detection:The method is the same as 1.2 and 1.3.(3)Plasmid conjugation experiment:The zygotes of E.coli Ec2551 and K.pneumoniae Kp2573 were obtained by plasmid conjugation,the same method as 2.3.(4)PFGE and plasmid S1-PFGE:the plasmids and homology of the isolates were analyzed by the same method as 2.4.(5)Next-generation sequencing(NGS)of the genome and plasmid:the entire genome of Ec2551,Kp2573 and Kp2588,as well as the plasmids in Ec2551 and Kp2573 were extracted for further analysis.Results:1.Epidemiological analysis of molecular resistance of multi-center CRE isolates in China.(1)A total of 404 strains were collected in this study,of which Klebsiella pneumoniae was the most common strain,followed by Escherichia coli.blaKPC and blaNDM was the most prevalent.(2)ST11 was the main clone of CRKP,of which KL47 and KL64 was the main serum capsule type with a significant regional difference.Some of the strains carried one or more virulence genes,with the highest virulence gene carrying rate in Suzhou.2.Multi-center molecular epidemiological study of carbapenems resistant Klebsiella oxytoca.(1)A total of 13 CRKO strains were collected in this study.There was a high degree of homologous difference among these isolates.The carbapenemase genes include blaNDM,blaKPC and blaIMP.Among them,blaIMP was mainly detected in Suzhou.(2)blaNDM was the most common carbapenemase gene in CRKO isolates.The integrated plasmids varied in size with IncX3 plasmids of?78kb and IncC plasmids of?170kb were common.3.Research on NDM-5 producing Klebsiella pneumoniae sequence type 258 in China.(1)There was 100%homologous between Klebsiella pneumoniae Kp2573 and Kp2588.And Carbapenem-resistant Kp2573 and E.coli Ec2551 shared the same size plasmid,which was not found in the carbapenem sensitive strain Kp2588.(2)Results of NGS further showed that the core single nucleotide polymorphism(SNP)analysis of Klebsiella pneumoniae Kp2573 and Kp2588 was only single nucleotide difference.In addition,Ec2551 and Kp2573 carried the same b/aNDM-5 gene and the IncX3 plasmid.The results above suggested that carbapenem-resistant Kp2573 might be formed by the obtaining of blaNDM-s gene from Ec2551 to carbapenem-sensitive strain Kp2588.(3)Chinese ST258 and global ST258 had evolved from a a common ancestral strain,but Chinese ST258 strains have separate evolutionary histories.Conclusion:1.Carbapenemase genes varied in different CRE species.Except for Yinchuan,ST11 was the main clone,of which KL47 and KL64 was the main serum capsule type with significant regional difference.Most of the strains in Suzhou carried both virulence and drug resistance genes.2.Resistance genes of carbapenems resistant K.oxytoca were varied but blaNDM was the most common,and blaIMP was mainly popular in Suzhou.Most of the strains were sporadic epidemics with high homologous difference.3.NDM-5-producing K.pneumoniae ST258 in China might be formed by that carbapenem-sensitive ST258 strain obtained NDM-5-harboring IncX3 plasmid from E.coli,and China ST258 and global ST258 had evolved from a common ancestral strain,but with separate evolutionary histories.