Isolation,Identification And Functional Evaluation Of Butyrate-Producing Bacteria From Human Gut

The incidence of inflammatory bowel diseases(IBD)has been increasing during the last three decades,and become one of the most prevalent diseases that threatening the health and living quality of human beings.Though genetic predisposition is one of the the etiology of IBD,and more than 160 human gene loci have been identified to be associated with the susceptibility of IBD,the rapid increasing of IBD incidence can not be ascribed to the genetic variation.The environmental factors including gut microbiota may play key role during the development of IBD.The enrichment of certain opportunitic pathogens and the depletion of butyrate-producing bacteria(BPB)was found to be the key features of the gut microbiota structure in IBD patients.Introducing the BPB back into the gut of IBD patients may be a novel breakthrough of the microbiome targeted therapy for IBD,so BPB was proposed to be the next generation probiotics for IBD treatment.However,as a functional group in the gut microbiota,BPB showed high phylogenetic diversity with at least four different metabolic pathways for butyrate production.There are still many novel BPB strains in the human gut.The anti-inflammatory effects of several BPB strains were investigated by cell line or mice models,but the impact of the inoculating BPB strains on the host microbiota have not been fully evaluated.In this study,we isolated the typical BPB strains from human gut,and evaluated the physilogical function of the strains by dextran sodium sulphate(DSS)-induced colitis mice model,the interation between BPB strain and host microbiota and health was carefully analyzed.First of all,we isolated 29 BPB candidates from the fresh fecal sample of a healthy adult male volunteer.Physiological,biochemical and morphological characteristics of three representative strains were tested,comparative genomic analysis was performed after the whole genome sequencing.According to the nearest neighbor of 16 S rRNA gene sequences,3 BPB strains were named with Anaerostipes hadrus BPB5(similarity=99.73%),Eubacterium sp.BPB21(similarity=96.00%)and Eubacterium rectale BPB22(similarity=99.80%)respectively.All the three strains were obligated anaerobic,produced butyrate and hydrogen in YCFAGSC medium with little consumption of acetate.10.81 mM butyrate was produced by BPB5,and BPB22 could produce 22.44 mM butyrate using the same medium.BPB21 had the peculiar morphology: cells stayed in line after division to form the filamentous apperance,several lines of cells twisted with each other in a clockwise direction but with closed ends.BPB5 could ferment more kinds of carbon sources than the other two strains.Sucrose,sorbose,glycerol and inulin were exclusively metabolized by BPB5,and there were more genes associated with the utilization of sucrose and inulin in the genome of BPB5 compared to the other two strains.Each of the strains had at least one completed metabolic pathway for butyrate production within the genome.Both BPB5 and BPB22 used butyryl coenzyme A: acetate-CoA transferase to carry the last step of butyrate production,wihle BPB21 used butyrate kinase.The results of safety assessment based on the whole genome sequences of the three strains showed no known gene of virulence factor or strong antigen in any genome.Number of the other risk factors(eg.antibiotic resistence gene,inserting sequence)within three BPB were similar with typical probiotic strains and significantly lower than typical pathogens.Taken together,we got three BPB strains without virulence factor in the genomes,but the physiological functions need further investigation.Then,we evaluated the physiological function of two human-derived butyrate-producing strains using the DSS-induced colitis mice model.Here we found that one of the strain,Eubacterium rectale BPB22,could promote the recovery after the withdrawal of DSS.But augmentation of the other strain,Anaerostipes hadrus BPB5,significantly aggravated colitis in the DSS-treated mice while exerted no detrimental effect in healthy mice.We explored how the interactions among the BPB strains and the host gut microbiota may contribute to this differential impact on the hosts.Butyrate production and severity of colitis were assessed in both healthy and DSS-treated mice,and gut microbiota structural changes were analysed using high-throughput sequencing.BPB5-inoculated healthy mice showed no signs of colitis,but increased butyrate content in the gut.In DSS-treated mice,neither BPB5 nor BPB22 increased butyrate content,but BPB5 induced significantly more severe disease activity index and much higher mortality,while BPB22 alleviated the colitis after the end of DSS treatment.BPB5 didn't induce significant changes of gut microbiota in healthy hosts,but expedited the structural shifts 3 days earlier toward the disease phase in BPB5-augmented than DSS-treated mice.Co-abundance group(CAG)network analysis of 83 OTUs responding to the treatments showed dramatic increase in a CAG containing Akkermansia as a dominant member 3 days earlier in BPB5-augmented than DSS-treated animals.The abundance of Akkermansia was positively correlated with the disease activity index.The analysis of potential metabolic cooperation based on the whole genome sequences showed that BPB5 may support the earlier and faster increasing of Akkermansia by providing 13 essential metabolic compounds.Taken together,two BPB stains isolated from the same sample exerted the opposite physiological function in the DSS-induced colitis mice model.The differential response of gut microbiota in healthy and DSS-treated mice to the same BPB strain with drastically different health consequences emphasized the importance of gut microbiota during the development of colitis.The results indicated the strain-specific function of gut bacterial members,and this function also depended on the health status and gut microbiota structure of the host.These results suggest that rigorous and comprehensive safety assessments should be done before the clinical inoculation of any potentially beneficial bacteria.Animal model with dysbiotic gut microbiota should be employed for the safety assessment of probiotic candidates,the impact of the inoculated strain to the indigenous microbiota should be adequatly evaluated.