The Study Of Virulence Factors And In Vivo Evolution In Klebsiella Pneumoniae

Part ?: Hypervirulent Klebsiella pneumoniae(hv KP)is emerging around the AsianPacific region and it is the major cause of the community-acquired pyogenic liver abscesses.Multidrug-resistant hypervirulent Klebsiella pneumoniae(MDR-hv KP)isolates were reported in France,China,etc.However,the international ally agreed definition for hv KP and the virulence level of hv KP are not clear.In this study,56 hv KP isolates were collected from March 2008 to June 2012 and investigated by string test,capsule serotyping,multilocus sequence typing(MLST),virulence gene detection and serum resistance assay.Among the 56 K.pneumoniae isolates,64.3% had the hypermucoviscosity phenotype,meanwhile,64.3% were the K1 serotype and 19.6% were the K2 serotype.Within the K1 serotype,94.4% were ST23,and within the K2 serotype,ST65,ST86 and ST375 accounted for the same percentage of 27.3%.According to the 50% lethal dose of Galleria.mellonella infection model,hv KP isolates were divided into high virulence level group and moderate virulence level group.MLST was a relatively reliable evaluation method to discriminate the virulence level of hv KP.Part ?: Hypermucoviscosity phenotype of K.pneumoniae is often used as the synonym of hv KP.The mucoid evolution mechanism of ST11 CR-hv KP was not clear.In this study,two isolates(FS and GZ-1)were successively collected with different mucoid phenotype from a single patient.The antimicrobial susceptibility test,PFGE(pulsed-field gel electrophoresis),string test,India ink stain,G.mellonella infection model and sequencing were performed between the two isolates and isogeneic knockout isolates.FS and GZ-1 were ST11,carbapenem resistant and KPC producing.The results of PFGE and sequencing showed that both isolates had close phylogenic relationship,which contained same plasmids and antimicrobial resistance genes.The mutation on gene ptk caused the shift of hypermucoviscous phenotype,and GZ-1?ptk lost the hypermucoviscous phenotype.GZ-1?ptk was less virulent,cit and rfb was down-regulated in the knockout isolate.Part ?: Bloodstream infections(BSIs)are associated with high morbidity and mortality,where the pks gene cluster plays a major role in the occurrence and prevalence of BSIs;however,the prevalence and characteristics of the pks gene cluster in K.pneumoniae is currently limited in mainland China.In this study,571 bloodstreamsourced K.pneumoniae isolates were collected and 117 isolates were pks gene cluster positive.pks-positive isolates were significantly more susceptible to antimicrobial agents.The pks-positive isolates(5.1%)were multidrug-resistant with infrequent extendedspectrum beta-lactamase(ESBL)production.The dominant sequence type of pks-positive K.pneumoniae was ST23(78/117)and ST65(20/117),and virulence genes were common in pks-positive isolates.The pks-positive K.pneumoniae was divided into two major clades,which consisted of ST23 and ST65,respectively.The clinical information of the community-onset K.pneumoniae bloodstream infection patients showed that the patients infected with pks-positive isolates had a higher proportion of liver abscesses and a lower proportion of biliary obstructions(p <0.05).The infection of the pks-positive isolates caused a much higher expression of ?H2AX than the pks-negative isolates in HeLa cells(p <0.05).