The Effect Of Stachyose,Bacteroides Thetaiotao Micron On The Colonization Of Clostridioides Difficile And Gut Microbiota In Mouse Model

Objective:The purpose of this study is to determine the effect of supplementation of stachyose and Bacteroides thetaiotaomicron on the colonization of Clostridioides difficile and gut microbiota using C.difficile-infection mouse model.Methods:1.The effect of stachyose supplementation on the colonization of Clostridioides difficile and gut microbiota in mouse model:C57BL/6J mice were randomly divided into three groups,one group did not receive any treatment(blank group),and the other two groups were daily administrated with stachyose(stachyose group)and PBS(PBS group)respectively for 10 days after the C.difficile infection.On day 10 post infection,the content of C.difficile in feces was measured with Real-time PCR and the variation of intestinal flora was analyzed with 16S rRNA sequencing technique.2.The effect of Bacteroides thetaiotaomicron on the colonization of Clostridioides difficile and gut microbiota nad metablism in mouse model:C57BL/6J mice were randomly divided into four groups,one group did not receive any treatment(blank group),and the other groups were daily administrated with FMT(FMT group),PBS(PBS group)and Bacteroides thetaiotaomicron(Bacteroides thetaiotaomicron group)respectively for 10 days after the C.difficile infection.On day 3 post infection,colon tissue was collected for RNA-sequencing and histopathological analysis.On day 10 post infection,the content of C.difficile in feces was measured with Real-time PCR,the variation of gut microbiota was analyzed with 16S rRNA sequencing technique and the content of bile acids in feces was measured with targeted metabolomics analysis.results:1.The effect of stachyose on the colonization of Clostridioides difficile and gut microbiota in mouse model:On day 10 post infection,the colonization of C.difficile was significantly lower in the stachyose group compared with PBS group.Histopathological analysis showed that Bacteroides thetaiotaomicron has a protective effect on the inflammatory response of intestinal tissues caused by C.difficile.The 16S rRNA sequencing result showed that the ace index(flora richness)of stachyose group and PBS group was different from the blank group,but the ace index of stachyose group was significantly higher than that of PBS group(P<0.05).At the phylum level,the relative abundance of Verrucomicrobia and Proteobacteria in PBS group was significantly higher than that in blank group while the relative abundance of Bacteroidetes and Firmicutes was significantly lower than blank group.The relative abundance of Bacteroidetes and Firmicutes in stachyose group was significantly higher than that in PBS group while the relative abundance of Proteobacteria was significantly lower than that of PBS group.At the species level,compared to blank group,the relative abundance of Parasutterella excrementihominis,Blautia hansenii,Parabacteroides distasonis,Akkermansia muciniphila,Bacteroides thetaiotaomicron was significantly higher and the abundance of TM7 phylum sp.oral clone,Alloprevotella rava,Barnesiella intestinihominis,Lactobacillus animalis was significantly lower in PBS group.Compared to PBS group,the relative abundance of Parabacteroides goldsteinii,Blautia hansenii,Bacteroides thetaiotaomicron was significantly higher while the relative abundance of Parasutterella excrementihomini,Parabacteroides distasonis was significantly lower in stachyose group.2.The effect of Bacteroides thetaiotaomicron on the colonization of Clostridioides difficile and gut microbiota in mouse model:On day 10 post infection,the colonization of C.difficile was significantly lower in the Bacteroides thetaiotaomicron group compared with PBS group.The 16s rRNA sequencing result showed that the shannon index(flora diversity)of Bacteroides thetaiotaomicron group and PBS group was different from the blank group,but the shannon index of FMT group was close to blank group.FMT restored the structure of gut microbiota and diversity of CDI mice.At the phylum level,the relative abundance of Verrucomicrobia and Proteobacteria in PBS group was significantly higher than that in blank group while the relative abundance of Bacteroidetes and Firmicutes was significantly lower than blank group.The relative abundance of Bacteroidetes in Bacteroides thetaiotaomicron group was significantly higher than that in PBS group while the relative abundance of Firmicutes and Verrucomicrobia was significantly lower than that of PBS group.At the species level,compared to blank group,the relative abundance of Clostridioides difficile,Blautia hansenii,Parasutterella excrementihominis,Akkermansia muciniphila was significantly higher and the abundance of Clostridium aldenense,Alloprevotella rava,Barnesiella intestinihominis,Bacteroides thetaiotaomicron was significantly lower in PBS group.Compared to PBS group,the relative abundance of Bacteroides thetaiotaomicron was significantly higher while the relative abundance of Clostridioides difficile,Parasutterella excrementihominis,Akkermansia muciniphila was significantly lower in Bacteroides thetaiotaomicron group.The result of targeted bile acid metabolism analysis showed that the distribution of bile acids in the FMT group was similar to blank group.Compared to PBS group,some content of bile acids in Bacteroides thetaiotaomicron group was tend to blank group.Bacteroides thetaiotaomicron treatment significantly increased the content of DCA、HDCA、TLCA which are secondary bile acids and α-MCA,β-MCA,GCA,NorCA which are primary bile acid in CDI mice and significantly decreased GDCA which is secondary bile acid.α-MCA,NorCA,DCA,TLCA were significantly positively correlated with Barnesiella and significantly negatively correlated with Salmonella,Haemophilus,Parasutterella and Enterococcus.β-MCA and HDCA were significantly negatively correlated with Haemophilus,Parasutterella and Enterococcus and showed no significant correlation or weak positive correlation to Barnesiella.GCA was significantly negatively correlated with Lachnoclostridium and Barnesiella and showed no significant correlation to the others.GDCA was significantly positively correlated with Haemophilus,Parasutterella and Enterococcusand and significantly negatively correlated with Bacteroides.The RNA-sequencing result showed that there were 110 differentially expressed genes between Bacteroides thetaiotaomicron group and PBS group,including 35 up-regulated genes and 75 down-regulated genes.These differentially expressed genes were significantly enriched in six KEGG pathways,including:pancreatic secretion,protein digestion and absorption,fat digestion and absorption,neuroactive ligand-receptor interaction.The KDA genes among differentially expressed genes include Ctrb1、Amy2a5、Cela2a、Spink1、Prss3、Try4、Cpa1、Try5、Try10、2210010C04Rik.They were significantly enriched in pancreatic secretion,protein digestion and absorption and neuroactive ligand-receptor interaction.Conclusions:Both supplementation of stachyose and Bacteroides thetaiotaomicron can effectively reduce the colonization of Clostridioides difficile.Stachyose can improve the richnessof the flora while Bacteroides thetaiotaomicron did not alter the structure and diversity of the gut microbiota.Stachyose and Bacteroides thetaiotaomicron can selectively alter the relative abundance of several key species and Bacteroides thetaiotaomicron may be able to colonize in the gut.Bacteroides thetaiotaomicron treatment restored some content of bile acids to normal level.Differentially expressed genes influenced by Bacteroides thetaiotaomicron mainly focused on the pathway related to metabolism.It is speculated that Bacteroides thetaiotaomicron play a role in regulation of bile acid and metabolism.