The Molecular Characteristics Of Hypervirulent Klebsiella Pneumoniae And Risk Factors For Bloodstream Infection And The Regulation Mechanism Of Delayed Apoptosis In Human Neutrophils

Objectives:1.To investigate the epidemiological characteristics and risk factors of bloodstream infections caused by Hypervirulent Klebsiella pneumoniae,and to provide scientific evidence for rational treatment and prevention of bloodstream infections of Hypervirulent Klebsiella pneumoniae.2.To investigate the virulence and molecular epidemiology of Hypervirulent Klebsiella pneumoniae in bloodstream infection and its effects on neutrophil function.3.To investigate the regulation mechanism of delayed neutrophil apoptosis induced by Hypervirulent Klebsiella pneumoniae.Methods:1.Collected 178 strains of Klebsiella pneumoniae isolated from the blood samples of the First Affiliated Hospital of Nanchang University in January 2014-2016 December.The major genotypes and their distribution were determined by repeat sequence polymerase chain reaction(ERIC-PCR)of enterobacteriaceae and multilocus sequence analysis(MLST).2.Polymerase chain reaction(PCR)and sequencing to determine the capsular serotypes and virulence genes,this study defines who meets any of the following: K1,K2,carrying rmpA/rmpA2 gene or high mucoid phenotype positive strains for Hypervirulent Klebsiella pneumoniaestrains,or non Hypervirulent Klebsiella pneumoniae strains;query and statistical analysis of these 178 cases the clinical data of the patients,divided into experimental group and non Hypervirulent Klebsiella pneumoniae strains of Hypervirulent Klebsiella pneumoniae strains of logistic group,using the single factor analysis and multivariate regression analysis of Hypervirulent Klebsiella pneumoniae isolated bloodstream infection risk factors.3.Collect the first affiliated hospital of nanchang university in June,2016-December 2016 hospitalized patients with klebsiella pneumoniae isolated from the blood specimen 55 strains,PCR amplification and sequencing to determine the WZI type,PCR amplification to determine its ompK36 allele(A,B,C group,D),in vitro toxicity of serum resistance,anti neutrophil cell phagocytosis test of Klebsiella pneumoniae;changes by flow cytometry in patients with peripheral blood neutrophil surface expression of molecules,the release amount of ELISA detection in patients with inflammatory factors.4.To establish an in vitro neutrophil infection model and an animal model of sepsis in vivo,and to detect the expression of neutrophil apoptosis regulating proteins by Western-Blot.Results:1.178 bloodstream infections pneumonia klebsiella bacteria strains,main genotyping is ST11 type,which is the main type ST23 on HvKP bacteria strains.Mainly in digestive department,acute,neurosurgery,severe medical,and cloning of sexually transmitted.2.With basic diseases such as diabetes,drainage,blood type(type A),the use of quinolone classes and penicillium carbon alkene antimicrobial agents are the independent risk factors of HvKP bloodstream infections.3.In the bloodstream infections in 55 strains of KP,20 strains HvKP strains,35 of non-Hv KP strain.HvKP strain of MLST classification is given priority to with ST23 and ST65,and non-HvKP strain is given priority to with ST11 type;HvKP strains ompK36 allele is given priority to with group C,and non-HvKP strain is given priority to with group A and group D;HvKP strain of capsular serotyping wzi2-K2 and wzi1-K1 is given priority to,and non-Hv KP strain is given priority to with wzi64-K64 parting.4.In 20 strains HvKP bloodstream infections,with 16 strains showed high levels of serum resistance ability,in the 35 strains of non-HvKP,only four strains showed high levels of serum resistance ability;HvKP strains carrying virulence genes for more than a non-HvKP strains.5.In terms of anti neutrophil phagocytosis,most non-HvKP strain neutral grain consuming rate in 55-80%,and most HvKP strain of neutrophil phagocytosis rate at about 30%.6.Compared with non-HvKP group,HvKP group of patients with PD-1,CD86 expression significantly lowered,and PD-L1,CD80 no obvious change;HvKP infected groups of patients,IL-2,IL-8,IL-1?release quantity increased obviously,while the TNF-?release a quantity to reduce obviously.7.In the early infection of HvKP,HvKP infected group NF-?BP65 protein content was significantly higher than that of non-HvKP infected group;HvKP infected group had a significantly lower than that of non-HvKP ratio of Bax/Bcl-2 HvKP infected group;HvKP infected group of Caspase 3,8,9 protein content was significantly lower than that of non-HvKP infected group.Conclusion:1.In our hospital,the MLST type of bloodstream infection of KP is mainly ST11,while the main strains of HvKP infection are ST23 and ST65.2 This paper on the HvKP risk of bloodstream infection factors,suggesting that we should pay special attention to patients with chronic medical diseases,especially KP patients with diabetes bloodstream infection;secondly should pay attention to aseptic operation and consciousness of drainage drainage time and rational use of carbapenems,quinolone antibacterial drugs.3.The ompK36 allele of HvKP strain is mainly C group,and the capsular serotypes are mainly wzi2-K2 and wzi1-K1.4.Compared with non-HvKP strain,HvKP carried more virulence related genes,higher resistance to serum and lower phagocytosis rate of neutrophils;5.The release of inflammatory factors and up/down-regulation of PMN surface molecules in HvKP infected patients suggest that HvKP infection may alter the function of PMN.6.We speculate that at the early stage of Hv KP infection,HvKP PMN induced delayed apoptosis may be induced by adjusting Bax/Bcl-2 ratio to Caspase-3,8,9 with down-regulation of the expression,and activation of NF-?B,which delayed neutrophil apoptosis.