Establishment Of “Visible” Patient-derived Bacteria Xenograft As An Infection Model Using Zebrafish

According to the bacterial drug resistance monitoring report of inpatients and outpatients in Bethune Third Hospital of Jilin University in 2021,two strains of Escherichia coli,Klebsiella pneumoniae and Staphylococcus aureus,which are common in blood samples of patients with bacteremia,were selected as the experimental strains of subsequent in vivo models after comprehensive evaluation.The fluorescent bacteria platform was constructed by electroporation method and dye method,and the fluorescent bacteria were injected into zebrafish by microinjection technology to establish a visual human bacterial xenograft(PBX)infection model.By observing the changes of the average fluorescence intensity of bacteria in zebrafish,the survival rate of zebrafish before and after antibiotic treatment,and the migration and quantity changes of neutrophils,we initially constructed an antimicrobial susceptibility test platform in vivo.At the same time,through the zebrafish secondary infection experiment,we tracked the recruitment and sterilization of neutrophils in vivo,established the zebrafish inflammatory preconditioning(In P)model,further explored the role of In P and neutrophils in infectious diseases,and provided a basis for revealing the molecular mechanism of In P.The visualized patient-derived bacteria xenograft infection model of zebrafish and the inflammatory preconditioning model have certain significance in the treatment of clinical bacterial infection.The experimental results are as follows:1.Construction of a visualized patient-derived bacteria xenograft infection model:This study identified the strains isolated and cultured from six clinical samples by MALDI-TOF-MS,and established a fluorescent bacterial staining platform by electroporation and dye methods respectively,and obtain stable fluorescent strains.The fluorescent strain was injected into zebrafish by microinjection technology,and the zebrafish PBX infection model was successfully constructed.2.Construction of an antimicrobial susceptibility test platform in vivo: Treating zebrafish infected by bacteria with different antibiotics,and the average fluorescence intensity of three non-drug resistant bacteria in zebrafish significantly decreases.The survival rate of zebrafish significantly increased;For zebrafish infected with drugresistant bacteria,the results showed that levofloxacin for E.coli 2,ceftazidime for K.pneumoniae 2,penicillin and oxacillin for S.aureus 2 had poor efficacy in the treatment of bacterial infection.The average fluorescence intensity of bacteria basically did not change significantly,and the survival rate of zebrafish declined.At the same time,neutrophils gradually migrated from the hematopoietic tissue at the tail to the infected site within 2-4 hours after infection.The average fluorescence intensity of neutrophils at the infected site gradually increased,indicating that neutrophils play a bactericidal role.3.Construction of zebrafish secondary infection model: Compared with the Model group,more neutrophils were gathered in the In P group,and the survival rate of zebrafish in the In P group was significantly higher than that in the Model group.Conclusions:1.Through the fluorescent bacteria staining platform,a visualized patient-derived bacteria xenograft infection model can be constructed in zebrafish.2.The zebrafish infection model can be used for preliminary screening of antibacterial drugs,and an experimental detection platform for antimicrobial susceptibility in vivo can be established.3.A secondary infection model was established in zebrafish,which proved that zebrafish in In P group had higher resistance to bacterial infection.