The Influence Of Intestinal Bacteroides Structure And Composition On The Progress Of Ulcerative Colitis

Bacteroides,one common member of the intestinal flora with high abundance,are accounting for about 25%of the total number of intestinal microbiota.Accumulating studies have demonstrated that Bacteroides are associate with the occurrence and progress of Inflammatory Bowel Disease.It is noteworthy that the relationship is associate with the abundance and ecological distribution of some Bacteroides species in the intestinal tract.Thus,in order to elucidate the complex relationship between different Bacteroides species and host health and disease,ongoing studies should be focused on the identification of Bacteroides species.However,current methods lack efficacy and can be used to identify a few Bacteroides species.Thus,it is need to develop a new method to clarify the relative abundances of different Bacteroides spp.present in complex intestinal microbial communities.In this paper,we aimed to establish a Illumina MiSeq high-throughput sequencing-based method to evaluate the composition of the Bacteroides community in the intestine,analyze the abundance and structure of Bacteroides in the intestinal tract of healthy people from different regions of China and isolate the Bacteroides strains,explore the change of Bacteroides species level during the progress of colitis to find the potential Bacteroides species that could alleviate colitis;verify the roles of Bacteroides in alleviating colitis by constructing colitis model in mice as foundation for further application.The results are listed as follows:First,we established an Illumina MiSeq high-throughput sequencing-based method based on the rps D(30S ribosomal protein S4)gene of Bacteroides for analyzing the composition of the Bacteroides community in complex samples.We first obtained the core gene sequences of different Bacteroides species by using the molecular biological methods.Based on these sequences,a core gene fragment rps D gene with high resolution was selected as the screening marker.We used this primer set to develop a novel primer,Bac-rps D-F:5'-AWC DAG AAT HGC MCG TAA-3'and Bac-rps D-R:5'-YRT CCCAYT CCA ACC A-3',based on the MiSeq Illumina sequencing platform that enabled an accurate assessment of the Bacteroides compositions in complex samples.There is a good linear relationship,Y=1.0076x-0.1012(R~2=0.9974),between the different Bacteroides content mixed in the simulated samples and the corresponding Bacteroides quantity obtained by sequencing,and the detection limit can reach 10~3CFU/m L.These results showed that the method has high accuracy and specificity in the detection of Bacteroides species.Besides,the novel method could be used to analyze the structure and composition of Bacteroides in health human and mice fecal samples.The results indicated that this study provides a high-throughput analysis method for analyzing the structural composition of Bacteroides species in the intestinal tract.Second,the fecal samples of healthy people in different regions of China were collected.The abundance and structure of Bacteroides in intestinal tract of these fecal samples were analyzed by using the novel method.The results showed that the abundance of Bacteroides vulgatus,Bacteroides ovatus and Bacteroides caccae are high in the intestinal tract of healthy Chinese people.Besides,we have also isolated the Bacteroides strains from fecal samples of healthy people in different regions of China by using species-specific screening medium.Finally,we have isolated more than 600 Bacteroides strains,which belonging to 23 different species,including 19 species of Bacteroides,3 species of Parabacteroides,1 species of Prevotella and 1 potential novel species of Bacteroides.Among them,the number of Bacteroides fragilis,Bacteroides thetaiotaomicron et al.are higher than other Bacteroides species,which consistent with those of the abundance of Bacteroides species in the fecal samples of health human.Besides,the genomic information of more than 100 Bacteroides strains were sequenced.The results showed that 100 Bacteroides strains mainly contained five families of carbohydrate active enzymes:CBMs family,CEs family,GHs family,GTs family and PLs family,and there are great differences in glucoside hydrolases in different Bacteroides species.Third,we have analyzed the composition of Bacteroides in the intestinal tract of different batches of mice based on MiSeq sequencing technology.The result showed that there are significantly difference of Bacteroides composition in different mice.Among these,Bacteroides acidifaciens,Bacteroides uniformis and B.vulgatus were common species of all mice.Besides,the progress of colitis in mice were induced by giving mice sterile water supplemented with DSS.We have evaluated the relative abundance of different Bacteroides species in the development of colitis in mice.The result showed that the abundance of all Bacteroides species has no significant change,whereas the abundance of B.uniformis shows a downward trend in the model group,suggested that B.uniformis might have a potential role in alleviating colitis.Thus,the B.uniformis was selected as the main research object in the following study.Finally,three B.uniformis strains(FNMHLBE1K1,FNMHLBE10K2 and FTJS3E4),selected based on their differing genomic characteristics,were evaluated for their ability to alleviate DSS-induced ulcerative colitis(UC)by using C57BL/6J mice.We analyzed the anti-inflammatory effects of the strains and the changes they caused in intestinal epithelial permeability.The results showed that different B.uniformis strains exerted different effects on the assessed parameters of the mice.Oral intake of B.uniformiss FNMHLBE1K1 could help alleviate the severity of UC.The results of real-time quantitative polymerase chain reaction(q RT-PCR)showed that B.uniformis FNMHLBE1K1 intake uploaded the relative expression of the tight-junction proteins Occludin.Besides,oral B.uniformiss FNMHLBE1K1 could increase the abundance of Alloprevotella?Parabacteroides significantly.This study indicates the therapeutic potential of B.uniformis FNMHLBE1K1 for the prevention of UC and provides scientific support for the screening of next-generation probiotics.