| Objectives Through genome-wide sequencing analysis of Klebsiella pneumoniae(KP),a comprehensive understanding of its genomics and genetic background can clarify its homology and transmission pathways as well as the virulence factors and resistance genes it carries.Methods The KP infected by the patients in the neurointensive care unit of a top three hospital in Tangshan City from January 2015 to December 2018 and the KP isolated from the ward environment were collected and tested for drug sensitivity.Pulse field gel electrophoresis(PFGE)technique was used to analyze the homology between strains.Randomly select one strain of KP pathogens with no homology in 2015,2016,and 2017,select KP2 strains with homology in 2018 and 2017,analyze their clinical situation,and use the drug sensitive paper method(K-B method)to detect the drug sensitivity of 5 strains of KP,polymerase chain reaction amplification(PCR)was used to detect the drug resistance genes carried on their chromosomes and plasmids.Perform genome-wide sequencing on 5 representative strains to understand the genetic characteristics of KP;compare genomics analysis to understand the genomic differences between 5 representative KP strains.At the same time,phylogenetic trees were constructed to understand the genetic evolution of Klebsiella pneumoniae.Genome functional annotation to understand the metabolic characteristics of representative KP and predict the virulence factors and drug resistance genes it carries.Results 1 From 2015 to 2018,the NICU detected 107 KP strains.In the pulsed field gel electrophoresis results,there were 15 sets of KP with 100% homology and 20 sets of KP with 80% homology.2 The clinical infection status of 5 strains of KP showed that 5 strains of KP showed resistance or intermediary to ampicillin and piperacillin,and all 5 strains were negative for the detection of extended-spectrum ?-lactamase(ESBL),but for ampicillin All are completely resistant.3 The whole genome sequencing results of 5 strains showed that the size of 5 strains of KP genome was between 5,469,543 b and 5,722,999b;the GC content was between 56.94% and 57.26%.4 Comparative genomics analysis,86% of KP SNP occurred in the gene coding region(CDS),of which about 46% were non-synonymous mutations;single nucleotide polymorphism(SNP)differences between the same clones Very small,the same popular clones are significantly increased,the number of SNPs between non-homologous KPs vary greatly;the same clones have the same unique gene family;a small sequence of genes occurs at a certain position in the genome Insertions or deletions,of which some insertion deletion mutations(I / D)are located in the CDS region.5 The functional classification of clusters of adjacent proteins(COG)shows that more than 88% of predicted gene protein products meet the four orthologous protein function types,and the number of metabolic related gene proteins is up to 46%;Kyoto Gene and Genome Encyclopedia(KEGG)metabolic pathway database annotation results show that 60% to 66% of predicted genes are involved in metabolism,cellular processes,environmental information processing,genetic information processing,human diseases and other five types of metabolic pathway functions,of which 90% is sugar,Amino acids,fats,nucleotides,cofactors,vitamins and other metabolic pathway genes.6 52 kinds of virulence genes were found in the KP genome,mainly adhesion,iron absorption,secretory system type virulence genes,etc.Among them,there are up to 17 virulence factors expressed by adhesion virulence genes;There are as many as 72 drug genes,and each KP carries between 20 and 49 drug resistance genes,with the most efflux pump drug resistance genes.Clinically the same popular clone line and the same clone KP strain have the same antimicrobial sensitivity,while the KP strain without homology has a large difference in drug resistance.Conclusions KP can be propagated and colonized in the ward environment or patients in a clonal manner.In the same clone KP strain,not only the number of SNPs differs very little but also their unique gene families are the same,in the same popular clone line KP strains,the number of SNPs increases,and their unique gene families also appear different;In non-homologous KP strains,the number of SNPs and their unique gene families are very different.At the same time,KP has powerful energy and genetic material metabolism function genes and information storage and replication,recombination,repair and other complex biological function genes,and the type and number of these genes carry no obvious relationship with KP homology.However,the virulence genes carried by KP can be transmitted in the form of clones,but a large number of drug resistance genes carried are not transmitted in the form of clones,and most of them are in an inactive state,activating the function of efflux pump resistance gene is the main mechanism for its resistance.Figure20;Table19;Reference 100... |
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