Influencing Factors Of Fecal Microbiota Transplantation Colonization Were Analyzed Based On Germ-free Animals And Evaluation Of FMT Intervention Effect On Ulcerative Colitis

FMT is a treatment scheme that rapidly reconstructs the normal microbiota composition,and has been used in the treatment of recurrent Clostridium difficile infection,ulcerative colitis(UC),etc.Although FMT has a good prospect for clinical application,the unclear efficacy bacteria of FMT lead to its low standardization.Due to their clear microbial background,the germ-free(GF)animal can be used to carry out FMT-related researches.The experiments of animal FMT need to be standardized.Based on this,we propose to analyze the influencing factors of FMT based on GF animals,and establish a system for evaluating the effectiveness of FMT in UC and screening potential bacteria.To explore the differences in microbial colonization caused by changes in donors and receptors,analyze the interaction between host and inheritance,evaluate the rationality of clinically advanced transplantation technologies,evaluate the effect of FMT transplantation based on different donor variables on the intervention of UC,and screen common anti-inflammatory bacteria.Systematically establish an evaluation system based on sterile animal experimental models and experimental methods,carry out research on the standardization of FMT animal experiments and strain screening,which can improve the degree of FMT standardization,and promote translational medicine research on FMT.1.Study on FMT colonization of GF mice by different donorIn this study,the bacteria from different donors were transplanted into GF mice,and the colonization rate of recipient and microbial composition,function,diversity,and similarity of the donor and recipient were detected.The experimental results are as follows:(1)At the species level,the bacterial α diversity of PD_FMT group was significantly higher than paired recipient,while the α diversity of DD_FMT group was significantly lower than that paired recipient,and there is no significant difference in donor and paired recipient among the other groups.(2)Annotated metabolic pathways based on the MetaCyc database,confirmed that the donor and recipient shared a high number of metabolic pathways(247,285,361,336,and 333 in mouse,dog,pig,UC,and HD donor transplantation groups,respectively).(3)The colonization rate study found that the highest colonization rate in the MD_FMT group was 94.12%,followed by 74.07%,46.58%,37.70%,and 27.91%in the DD_FMT group,the UC_FMT group,the HD_FMT group,and the PD_FMT group.(4)Adonis analysis showed that UC_FMT had the largest difference in community structure and function with its recipient mice,while MD_FMT had the smallest difference in community structure and function with its recipient mice.The difference between DD_FMT and recipient after cross-species transplantation was minimal.(5)PCoA analysis found that the community structure of the five groups of recipients was significantly different;(6)The species composition comparison found that the number of species and a-diversity among the five groups of recipients mice were significantly different.The predominant phyla in the recipient mice of the MD_FMT group were Verrucomicrobia and Firmicutes,and the predominant phyla in the four groups of cross-species transplant recipient mice was Bacteroidetes.(7)The functional comparison showed that the functional α diversity was significantly different among the recipients.The MD_FMT recipient mice was significantly lower than that of the four groups of cross-species transplant recipient mice.In conclusion,combined with the results of colonization rate,species α diversity and shared metabolic pathways,it is feasible to establish different donor FMT models using GF mice.Comprehensive analysis of the differences between recipients after colonization with different donor bacteria showed that donor factors significantly affected the composition,community structure,and functional α diversity of recipients after FMT.2.To study the effect of deliver way on the colonization of FMT based on GF miceIn this experiment,HD flora were used to transplant into upper and lower gut to GF C57BL/6j mice,the colonization rate of recipient and the microbial species composition,function,diversity,and similarity between the donor and the recipient were detected.The experimental results are as follows:(1)Adonis analysis showed that the community structure and function of LGT_mice group were more similar to those of donors.(2)The number of species and α diversity in the donor group and the LGT_mice group were significantly higher than those in the GT_mice group.(3)At the genus and species level,the colonization rates of the LGT_mice group were 54.55%and 41.84%,which were significantly higher than those of the GT_mice group,47.73%and 35.71%,respectively.(4)PCoA analysis showed that the community structures of the two groups were significantly different at the genus level.(5)LEfSe analysis showed that 18 kinds of bacteria such as Bacteroides_intestinalis were enriched in LGT_mice group,and 13 kinds of bacteria such as Collinsella_aerofaciens were significantly enriched in GT_mice group.33 kinds of donor bacteria were colonized in LGT_mice and GT_mice,2 kinds of donor bacteria were only colonized in GT_mice group,and 8 kinds of donor bacteria were only colonized in LGT_mice group.(7)Annotating metabolic pathways based on the MetaCyc database,LefSe analysis showed that the dominant metabolic pathway in the LGT_mice group was L_histidinedegradationⅢ,and the dominant metabolic pathway in the GT_mice group was inosine-5’-phosphate biosynthesis Ⅰ.In conclusion,the donor community structure,diversity and functional reproducibility were better after FMT colonization in the LGT_mice group.Lower gut transplantation is recommended for clinical FMT.The differences in community structure and colonization characteristics between groups indicated that deliver ways affected the results of FMT colonization,and the deliver ways should be selected according to the colonization characteristics of FMT-effective bacteria.3.Colonization of GF pigs and different strains of mice with healthy human floraIn this experiment,the HD donor flora was transplanted into GF pigs,BALB/c mice,C57BL/6j mice,NPG mice,KM mice,and the colonization rate of recipient and the microbial species composition,function,diversity,and similarity between the donor and the recipient were detected.The experimental results are as follows:(1)Adonis analysis showed that the microbial community structure and function of HAMP pigs were more similar to those of donors,and the community structure of BALB/c mice was more similar to that of donors than C57BL/6j mice.(2)The average colonization rate was 45.90%for HAMP pigs,33.13%for BALB/c mice,30.63%for C57BL/6j mice,34.28%for NPG mice,and 22.28%for KM mice.(3)PCoA analysis showed that species,immunodeficiency,and genetic factors all had significant community structures and functions;(4)Colonization commonality analysis showed that 29 kinds of donor bacteria could colonize all recipients,A.putredinis has a high colonization abundance in all recipients.Functional analysis found that it was involved in 36 metabolic pathways including peptidoglycan biosynthesis.(5)Studies of colonization characteristics show that 11 kinds of bacteria including Lactobacillus_plantarum can only colonize into HAMP,while 6 kinds of bacteria including Bacteroides_eggerthii can only colonize into GF mice,KEGG annotation of Level 2 metabolic pathways found that the abundance of carbohydrate,amino acid,and lipid metabolic pathways differed among recipients.Therefore,recipient factors significantly affect the results of FMT colonization.The comparison of recipient colonization results of different species shows that the donor reproducibility of HAMP pig community structure and function is better.Since mice can carry out high-throughput human FMT experimental research,it is recommended to use a mouse-to-pig research system for preclinical research.The comparison of the colonization results of the genetic strains showed that the immunedeficient mice had significantly decreased resistance to foreign bacteria colonization.Compared with the KM mice,the inbred mice had better colonization effect and the BALB/c mice had better reproducibility of the donor community.It is recommended to use inbred BALB/c mice for FMT experiments.The nutritional preferences of microbiota in different intestinal environments are related to differences in colonization,and FMT studies should comprehensively consider differences in colonization among recipients.4.Based on the DSS-induced colitis model to explore the effectiveness of human FMT in UC and the screening of FMT potential bacteriaBased on the conclusion in the previous section,BALB/c mice with better colonization effect of human flora were selected as the research object.The experimental results are as follows:(1)Pre-administration of HD bacteria significantly relieved the symptoms of colitis compared to the UC bacteria.(2)HD bacteria significantly reduced the levels of inflammatory markers MPO and EPO,and various pro-inflammatory factors,in colitis mice,and increased the secretion of the antiinflammatory factor IL-10.UC bacteria significantly reduced the levels of EPO and pro-inflammatory factor IL-1β.In addition,both groups of donors showed intestinal barrier protection ability,and HD donors had better intestinal barrier protection ability than UC donors.(3)Metagenomic sequencing indicated higher species diversity and higher abundance of anti-inflammatory bacteria in the HD intervention group,including Akkermansia muciniphila.In the UC intervention group,Bacteroides stercoris,IBD-related bacterium Ruminococcus gnavus were more abundant.On the basis that both FMT treatments had curative effects,38 potential anti-inflammatory core bacteria were screened at the species level.(4)Metabolomics analysis showed that the two types of FMT significantly modulated the metabolism of DSS-induced mice.Moreover,compared with the UC intervention group,indoleacetic acid with antiinflammatory effects were significantly enriched in the HD intervention group.In summary,HD and UC donors can alleviate DSS-induced colitis,and provide anti-inflammatory microorganisms,SCFAs,and SB As to regulate cytokine homeostasis and protect intestinal barrier.A total of 38 bacterial species may exert core anti-inflammatory properties.The HD donor group has better curative effect was associated with higher species diversity and enrichment of anti-inflammatory bacteria such as AKK and the anti-inflammatory metabolite indoleacetic acid.In summary,this study based on GF animals to explore the effects of donors,transplantation routes,and recipients on FMT colonization,to evaluate the efficacy of variable FMT donor intervention in UC mice,and to screen potential effective bacteria.The conclusions are as follows:1.It is feasible to analyze the donor flora of different species using GF mice.Donor factors significantly affected recipients community structure and functional α diversity after FMT.2.The recipient community structure was significantly affected by the deliver ways.Lower gut FMT showed better reproducibility in donor community structure,diversity and function,and it was recommended that lower gut transplantation be the first choice for clinical FMT.3.The reproducibility of donor community structure and function in GF pigs was better,and the reproducibility of donor community in BALB/c mice was better than that in C57BL/6j mice,and the nutrient preference of flora was related to differences in colonization.4.HD and UC donors alleviate DSS-induced colitis symptoms by providing antiinflammatory microorganisms,SCFAs,and SB As to regulate cytokine homeostasis and protect intestinal barrier.A total of 38 bacterial species may exert core antiinflammatory properties.The HD donor group has better curative effect was associated with higher species diversity and enrichment of anti-inflammatory bacteria such as AKK and the anti-inflammatory metabolite indoleacetic acid.