Isolation And Anti-inflammatory Function Evaluation Of Bacteroides Fragilis From Feces Of Chinese Population

Bacteroides fragilis(B.fragilis)is intestinal symbiotic bacterium widely existing on the surface of human lower gastrointestinal mucosa.It is currently considered as a candidate of next-generation probiotics.Many studies showed that nontoxigenic B.fragilis could improve intestinal barrier,enrich intestinal microecology,prevent intestinal pathogen infection,enhance intestinal immunity and alleviate host inflammation.At present,the research on the functional B.fragilis is mainly limited to the type strain NCTC 9343 from American,and the research on the B.fragilis from Chinese is few,especially the characteristics of the genetic background and physiological function of the strains.In order to analyze the differences and probiotic functions of B.fragilis from the intestinal tract of Chinese,two functional B.fragilis strains with antiinflammatory effects were screened,combining its genome sequencing and in vitro and vivo function evaluation model.First of all,280 fecal samples from 16 different provinces of China were screened for B.fragilis,and 59 B.fragilis strains from different host sources were isolated.The genomic sketches of the strains were determined by the second generation sequencing technology.The comparative genomic analysis combined with 4 known type strains in the NCBI,including NCTC 9343,showed that the guanine(G)and cytosine(C)content of B.fragilis was about 43%,and there were 2274 core genes in total.These genes were mainly involved in the generation of cell wall/membrane/envelope organisms and the transport and metabolism of carbohydrates,inorganic ions and amino acids to meet their basic life needs.Average nucleotide identity(ANI)analysis showed that there were great differences between the strains of B.fragilis from Chinese,which could be divided into two categories.Category 1 contained four type strains,and category 2 were all from Chinese.The ANI index of these two categories was higher than 96%,so there was a high consistency within the categories.The results of carbohydrate enzyme gene analysis showed that six families of carbohydrate enzyme distribution in these two categories was universal,but the number of genes in the synthesis of auxiliary active oxidoreductase,carbohydrate binding module and carbohydrate esterase were different between two categories.Based on the results of species tree and ANI,6 strains of nontoxigenic B.fragilis in category 1 were selected to further evaluate their probiotic function.The systemic inflammatory model of mice was established by intraperitoneal injection of lipopolysaccharide(LPS)to explore the immune effects of B.fragilis intervention on mice.Results showed that different B.fragilis strains had different alleviative effects on the inflammation mice.Among them,B.fragilis FGXGL59E1 and FGXGL107E4 could significantly alleviate LPS-induced inflammation in mice,including significantly reducing the levels of tumor necrosis factor ?(TNF-?)and interleukin-6(IL-6)in serum,alleviating intestinal inflammatory infiltration and mucosal bleeding.In addition,the intervention of B.fragilis FGXGL59E1 and FGXGL107E4 strains could improve the structure of intestinal flora and increase the abundance of Lactobacillus and Akkermansia and the content of short chain fatty acids(SCFAs).The inflammatory model of macrophage RAW264.7 in vitro further showed that B.fragilis FGXGL59E1 and FGXGL107E4 strains could enhance the functional activity of macrophages.The effect of B.fragilis on the activity of macrophages was similar to that of commercial probiotic Lactobacillus rhamnosus GG(LGG).By analyzing the gene distribution of FGXGL59E1 and FGXGL107E4 strains,we found that the low abundance of replication,recombination,signal transduction and defense mechanism genes and high abundance of posttranslational modification,protein turnover and chaperone genes might relate to their probiotic function.To sum up,through the isolation of B.fragilis in different fecal samples,comparative genomic analysis and evaluation of inflammation model in vitro and vivo,two strains of B.fragilis FGXGL59E1 and FGXGL107E4 with anti-inflammatory effects were screened,which were respectively located in cluster 5(including NCTC 9343)and cluster 4 of species tree.These two strains could improve the structure of mouse flora,increase the diversity of intestinal flora,significantly increase the content of beneficial bacteria,promote the formation of short chain fatty acids,enhance the immunity of mouse intestinal barrier and inhibit the LPS-induced inflammatory response.Results of vitro inflammatory cell model also showed that these two strains could significantly enhance the function of macrophages against inflammation.