Development Of A Risk Prediction Model Of Invasive Klebsiella Pneumoniae Liver Abscess Syndrome Based On Clinical-imaging Features And Mechanism Study

Objective: In recent years,Klebsiella pneumoniae(KP)has become the main pathogen of pyogenic liver abscess.Liver abscess-associated KP is defined as hypervirulent KP(hv KP)due to its high viscosity and high virulence phenotype.The incidence of liver abscess caused by hv KP(KPLA)is increasing year by year.KPLA is often complicated by extrahepatic migratory infections(EMI).KPLA combined with EMI is clinically defined as invasive KPLA syndrome(IKPLAS).Occult symptoms and delayed diagnosis and treatment of IKPLAS often lead to catastrophic sequelae.Therefore,timely and accurate prediction of the risk of IKPLAS will help to improve the prognosis of KPLA patients.In addition,accurately revealing the pathogenesis of IKPLAS will provide new insights for its prevention and treatment.The purposes of the current study are to: 1.develop and validate a prediction model of IKPLAS;2.screen and verify the specific virulence factors of hv KP that cause invasive Klebsiella pneumoniae liver abscess(IKPLA);3.preliminarily explore the molecular mechanism of T6 SS promoting IKPLAS.Methods:1.Development and validation of a risk prediction model of IKPLAS based on clinical and imaging characteristics of KPLA patientsKPLA patients from 4 tertiary hospitals from January 2011 to December 2020 were included in this study.Patients from hospital A were classified into the development cohort,and patients from other three hospitals were classified into the validation cohort.Clinical and imaging data of these KPLA patients were collected.KPLA combined with EMI was defined as IKPLAS group,while KPLA without EMI was defined as non-IKPLAS group.In the development cohort,logistic regression analysis was used to screen independent risk factors related to IKPLAS,and these risk factors were incorporated into the risk scoring formula.Then,we calculated the risk score of each risk factor and developed the risk prediction model.In the development and validation cohorts the predictive efficacy of the risk prediction model was evaluated and the incidence of IKPLAS was estimated using the model.2.To screen and verify the specific virulence factors of hv KP that cause IKPLA The pus samples of 16 KPLA patients from January 2021 to December 2021 were collected for 16 S r RNA gene sequencing.A total of 243 hv KP isolates from January 2015 to December 2021 were collected.PCR was used to detect capsule type(K1/K2)of hv KP,core genes(Hcp,Vgr G and Icm F)of T6 SS and other common virulence genes(rmp A,aerobactin,etc.).Twenty-five hv KP strains were selected for multi-locus sequence typing(MLST)and the carrying status of T6 SS gene in hv KP with different sequence types(ST)was compared 14 hv KP strains were screened for RT-PCR analysis based on the analysis results of MLST and the transcription and expression levels of Hcp gene were compared.Representative hv KP strains from different sources were selected for western blotting to compare the expression levels of Hcp at the protein level.The precipitation amounts of T6SS(+)and(-)hv KP suspensions were compared by centrifugal precipitation method to verify the correlation between T6 SS and high viscosity phenotype of hv KP.3.Preliminarily explore the molecular mechanism of T6 SS promoting IKPLAS Peripheral blood of healthy adults was collected and neutrophils were isolated.Typical KP strains including T6SS(+),T6SS(-)hv KP and c KP were selected to construct neutrophilic infection model and mouse hv KP infection model through oral administration.The effect of hv KP on pyroptosis of neutrophil was quantitatively analyzed by flow cytometry in vitro.Hoechst 33342 staining was used to observe the changes of nuclear apoptosis after neutrophil infection.The effects of hv KP on neutrophil activity were detected using Cell Counting Kit-8(CCK-8)kits.The survival of KP in serum and neutrophils was observed by serum and neutrophils killing test.The expression of pyroptosis related proteins was detected by western blotting.Mice infected with hv KP were used to construct liver abscess model,and the survival rate and the inflammation level of liver and lung tissues were compared between the two groups.Results: 1.The development of the risk prediction model based on the clinical and imaging characteristics of KPLA patients can effectively predict the risk of IKPLAS A total of 382 KPLA patients were included in this study.Patients from hospital A(n=296)were classified into the development cohort,and patients from other three hospitals(hospitals B-D,n=86)were classified into the validation cohort.The overall incidence of EMI was 19.1%(development cohort,n = 55;validation cohort,n = 18,P > 0.05).In the development cohort,four independent risk factors(age ≤ 40 years,fasting blood glucose >7mmol/L,no rim enhancement,and thrombophlebitis on CT),significantly associated with EMI,were incorporated into the scoring system.The area under curve(AUC)of the receiver operating characteristic curve(ROC)in the development and validation cohorts was 0.931(95% confidence interval [CI]: 0.93–0.95)and 0.831(95% CI: 0.86–0.91),respectively.The calibration curves fitted well.The incidence of EMI was 3.3% and 56.5%for the low-(total scores ≤ 4)and high-risk(total scores > 4)groups in the development cohort,and 3.2% and 66.7% in the validation cohort(all P < 0.001),respectively.2.Screening and verification of T6 SS as IKPLAS-related hv KP specific virulence factorPICRUSt2 predicted that the T6SS-related genes were notably enriched in the IKPLA group.PCR detection of T6 SS hallmark genes showed that 197(81.1%)were T6 SSpositive strains.The T6SS-positive strains detection rate of IKPLA group was higher than KPLA group(97.1% versus 78.4%;P<0.05).RT-PCR showed that the hcp expression level was markedly increased in IKPLA isolates(P<0.05).The centrifugation precipitation test found that the T6SS-positive bacterial suspension was more turbid and contained less sediment than the T6SS-negative and c KP strain(all P<0.05).3.T6 SS inhibits pyroptosis of neutrophils and increases immune escape of KP and promotes IKPLASThe results of flow cytometry indicated that the pyroptosis level of neutrophils in T6SS(+)KP group was lower than that in T6SS(-)group,and Hoechst 33342 staining indicated that the number of apoptosis nuclei in neutrophils induced by T6SS(+)KP was lower than that in T6SS(-)KP and c KP.CCK-8 assay suggested that the cellular activity of neutrophils infected by T6SS(+)KP was higher than that of T6SS(-)group.In vitro,serum and neutrophil killing tests showed that the survival rate of T6SS(+)KP was higher than that of T6SS(-)and c KP strains.The expression level of Caspase-1 in neutrophils lysates of T6SS(+)KP infection was lower than that of T6SS(-)group.In animal experiments lower expression level of Caspase-1 in liver parenchyma(P<0.05),shorter survival time,higher mortality,and higher expression level of IL-6 in liver and lung tissues(P<0.05)were observed in T6SS(+)hv KP infected mice.Conclusion: The risk prediction model based on the clinical and imaging characteristics of KPLA patients can effectively predict the risk of IKPLAS.T6 SS is a virulence factor closely related to IKPLAS and is associated with high virulence phenotype of hv KP.HVKP resists the immune killing of neutrophils by inhibiting the pyroptosis of neutrophils through T6 SS,thus promoting the hematogenous dissemination of hv KP.