The Dynamic Changes Of Bacillus Subtilis In The Gut Based On High-throughput Sequencing

BackgroundIntestinal flora played an important role in gut microbiota maintaining and the pathogenesis of many diseases such as intestinal irritable bowel syndrome(IBS).The study found that diarrhea IBS patients can be cured by short-term use of probiotics for abnormal intestinal mucosal barrier function,the symptoms of abdominal pain and abdominal distention,and reducing the total IBS symptom scores.However,how probiotics colonize the gut and influence the intestinal microecology is unclear.This experiment designed and constructed the "gene tracer",using 16SrDNA high-throughput sequencing technologies to analyze the dynamic change of probiotics and the intestinal flora.At present,researches on gut microbiota mainly applied traditional molecular biological techniques,such as fluorescent quantitative PCR and denaturing gradient gel electrophoresis(DGGE).However,these methods have some limitations.lllumina high-throughput sequencing technologies are able to sequence all DNA sequences of gut microbes,then analyze of the diversity and abundance of gut microbes comprehensively,enhancing the comprehensive understanding of the intestinal microflora.This experiment adopted high-throughput sequencing method to explore the dynamic change of the probiotic B.subtilis in the gut and its effects on gut microbiota abundance and diversity,compared technology of high-throughput sequencing with traditional technology-real-time fluorescent quantitative PCR,further confirmed that the certain technology has an superiority in tracing probiotic quantitatively.ObjectiveTo investigate the situation of Bacillus subtilis colonizing the gut,and how the time duration infecting gut microbiota.Methods1.Building gene tracer:Quantitatively explore the dynamic changes of probiotic B.subtilis in the gut after intaking B.subtilis.To differentiate the B.subtilis and the intestinal flora,the gene tracer was transducted in the animal experimental part.The forward and reverse primers are 338F-5' ACTCCTACGGGAGGCAGCA,806R-5'GGACTACHVGGGTWTCTAAT respectively.After this tracing the marked B.subtilis had an unique sequence,and the relative abundance represented the quantity of the B.subtilis and traced the dynamic alteration of this probiotic in gut.2.Animal experiment:Eighteen C57BL/6J female mice were randomly divided into three groups including the control group(C group),two days' intervention group(TW group)and ten days' intervention group(TE group).The C group was treated with normal saline each day from day 0 to day 9;the TW group was treated with live B.subtilis each day from day 0 to day 1,then with normal saline at the same amount from day 2 to day 9;the TE group was treated with live B.subtilis at the same amount each day from day 0 to day 9.All mice were given an unrestricted diet from day 0 to day 20.Feces were collected on day 0,4,8,12,16,20 in TE group and C group,and day 0,2,4,7,12,16,20 in TW group to extract DNA,then Illumina Miseq platform was used for high-throughput sequencing,and fluorogenic quantitative PCR was used to compare with that.Results1.After treated with live B.subtilis,the relative abundance of Bacillus genus microbes in TW group and TE group decreased.2.The variation tendencies of Firmicutes and Bacteriodetes are not apparent,and fluctuate fluently during all the period.On the day 20 the relative abundance of the Firmicutes was higher in TW and TE group.As the time passing,the abundance of Proteobacteria became lower.The variation of Verrucomicrobia accord to such a law:on the day 4,the relative abundance of Verrucomictobia was higher in TW group than C group,but lower than TE group,and kept this tendency as time passed.Although it became higher on the 16th day,it decreased on the day 20.However,the relative abundance of Verrucomicrobia in TE group was lower than TW and C group all the time.3.It has been found that the intestinal flora structure of TE group was similar on each day except day O.Yet the intestinal flora structure of TW group was a little different on each day.On the day 20,the microbiota structure of TW and TE group are similar.fluorogenic quantitative PCR confirmed that there was no Bacillus subtilis planted in the gut.ConclusionB.subtilis could regulate intestinal flora structure possibly via interacting with intestinal microbia but not via colonizing.Otherwise,long-term use of the probiotic changes intestinal flora structure more quickly than short-term does.