Relationship Between Intestinal Microbiota And Infection Of Liver Abscess Caused By Klebsiella Pneumoniae

Objection:We found that Klebsiella pneumoniae serotype K1 isolate caused pyogenic liver abscess(KLA)in mice by oral inoculation.For better understand the relationship between intestinal microbiota and KLA infection,intestinal microbiota composition were analyzed by using high-throughput 454 pyrosequencing.The fecal microbiota transplantation(FMT),which has been identified to restore the intestinal microbiota,was also used in this study to explore the relationship between intestinal microbiota and KLA infection.Methods:1.KLA infection mice model:6-week-old C57BL/6 male mice were given 10~6 CFU K.pneumoniae serotype K1 strain by oral inoculation.2.After 48 h of oral inoculation,all the mice were sacrificed and the tissue of liver,contents of cecum and serum were retrieved for the following analysis.Based on the pathology and liver function serum tests,mice with Kp1002 inoculation were divided into two groups:KLA group and NonKLA group.3.DNA were extracted from cecal contents and used for PCR amplification.The PCR products were sequenced by using 454 pyrosequencing.The intestinal microbiota composition were compared among three groups:Healthy group,NonKLA group and KLA group.4.Constructed the KLA infection model and the fecal microbiota transplantation intervention treatment was conducted at the same time.The feces came from the fresh morning stool of the allogeneic healthy mice,and the fecal bacteria solution of 300 mg/mL was prepared.After 24hours,0.3mL/piece of fecal bacteria solution was gavaged once.After 24 hours of fecal microbiota transplantation(48 hours of bacterial gavage),mice in the bacterial gavage group were divided into Kp1002+NonKLA group and Kp1002+FMT+NonKLA group according to the results of serum liver function test.5.The influence of fecal microbiota transplantation on the infection rate of KLA:the infection rate of KLA after 48 hours of gavage and 24 hours after the intervention of fecal microbiota transplantation was compared by liver function test after blood collection.6.ELISA was used to detect the changes in defensins,mucin and immunoglobulin in cecal mucus of mice,and the changes of defensins and mucin in gastric mucus,the Changes in serum levels of inflammatory cytokines among the Blank group,Kp1002+NonKLA group and Kp1002+FMT+NonKLA group.7.RT-qPCR and WB were used to detect mRAN expression levels and related protein expression levels of interleukin-1?(IL-1?),tumor necrosis factor-?(TNF-?)and mucin2(MUC2)in colon tissue of each group.Results:1.High-throughput 454 pyrosequencing was performed on the V1-V3 hypervariable region of the bacterial 16S rRNA gene to examine the effect of K.pneumoniae infection on the composition of the cecal microbiota.In particular,the Lactobacillus was significantly decreased in KLA-infected mice,which may be closely related to KLA infection.2.Fecal microbiota transplantation reduced the KLA infection rate caused by K1 serotype K.pneumoniae by oral gavage.3.ELISA results showed that after the fecal microbiota transplantation intervention,Kp1002+FMT+NonKLA group compared with Kp1002+NonKLA group,the Kp1002+FMT+NonKLA group showed decrease in the concentration of?-defensin 1,?-defensin 6,and the concentration of MUC2 and SIgA was increased in the cecum mucus;In the gastric mucus,the concentration of?-defensin 1 was decreased;the concentrations of IL-1?and TNF-?in serum were decreased;all of them had statistical significance(P<0.05).There was no difference between Kp1002+FMT+NonKLA group and Blank group(P>0.05).4.The results of RT-qPCR and WB showed that Kp1002+FMT+NonKLA group compared with Kp1002+NonKLA group,the mRNA and the protein expressions of IL-1?and TNF-?in colon tissue were lower than the Kp1002 group.The mRNA expression of MUC2 and protein was higher than that the Kp1002+NonKLA group;all of them had statistical significance(P<0.05).And there was no significant difference in IL-1?,TNF-?,MUC2mRNA and related protein expression levels between the Kp1002+FMT+NonKLA group and Blank group.Conclusions:1.K.pneumoniae serotype K1 strain by oral inoculation,leading to significant changes in the composition of microbiota of the cecum contents in mice.Especially at the level of genus,the abundance of Lactobacillus in KLA-infected mice decreased significantly,which may be closely related to KLA infection.2.After the fecal microbiota transplantation,the infection rate of KLA was significantly reduced.Meanwhile,the concentration of defensins in gastrointestinal tissue,the expression of MUC2 in intestinal mucosal protein,and the concentration of serum immune factors were also changed.