| Probiotics are widely favored by consumers because of their probiotic functions such as regulating intestinal microecology,regulating immunity,anti-inflammatory and anti-cancer.As a result,a variety of probiotic products,such as yoghurt and probiotic OTC preparations,have been derived,and their global consumption is huge.Although some probiotic strains have been widely used,their safety,metabolic capacity and potential complementarity of probiotic strains are still not well understood.Especially,there are few large-scale systematic studies on the relative abundance differences and potential roles of common probiotic strains in different disease cohorts.Researchers and producers have difficulty in precisely developing and producing probiotic products,and consumers also lack sufficient basis for choosing products.The development of high-throughput sequencing has led to the popularization of probiotic genome and intestinal flora sequencing.In particular,metagenome-wide association studies has investigated the relationship between human microbial flora and many non-communicable diseases,such as obesity,diabetes,liver disease,neurodegenerative diseases and even cancer,which shows the potential of intestinal flora as a source Bank of new probiotics.The abundant published data of intestinal metagenome sequencing data of disease cohort,bacterial genome sequences and advanced research methods make it possible to systematically detect the relative abundance and interaction of common probiotics on a large scale and to evaluate their roles in intestinal microbial communities and compare their safety,functional differences and complementarity.Therefore,on the basis of large-scale intestinal metagenome data of disease cohorts and probiotic genome data,this study carries out large-scale data mining,combined with large-scale isolation and culture experiments of intestinal bacteria and high-throughput single bacterial genome sequencing analysis,in order to clarify the relationship between common probiotics,human intestinal microflora and health and disease,and to clarify the genomic characteristics of human intestinal cultured microorganisms.All of these work are of great significance in guiding the precise application of common probiotics and new intestinal probiotics.This study collected the names of probiotics labeled on yogurt and OTC probiotics products in China's domestic market,mainly involving 15(sub)species,defined as major common probiotics[or Major Traditional Probiotics(MTPS)]in this study.A total of 1815 fecal metagenome sequencing data were collected,covering eight common disease cohorts(Crohn's disease,colorectal adenoma-cancer,ankylosing spondylitis,liver cirrhosis,type ? diabetes,obesity,rheumatoid arthritis and atherosclerotic cardiovascular disease).To investigate the major roles and the roles of MTPS in each cohort,the methods of case-control study and co-abundance based bacterial interaction were used.The results showed that Crohn's disease was mainly due to the lack of potential probiotics,while the increase of potential pathogens was the main cause of liver cirrhosis and atherosclerotic cardiovascular disease.Overall,MTPS played a non-dominant role in the association of intestinal flora of diseases,and its role in disease prevention or treatment might be inferior to that of other intestinal bacteria.And the enrichment direction of some species of MTPS was inconsistent.These results indicates that attention should be paid to the species/strains genetic diversity,in order to guide the precise use of common probiotics,and the development of new intestinal viable bacteria resources to construct intestinal bacteria bank,in order to provide more basic resources with key roles for disease prevention or treatment.A total of 444 strains of MTPS with the published genome sequence were analyzed by pangenomic comparison.The self-defined pangenome index was introduced to inspect the influencing factors on the MTPS genetic diversity.The results showed that the pangenome index of different species was markly different,and may be influenced by multiple factors including isolation sources,genome characteristics and genome number in species,indicating that species with open-level pangenome should be more carefully selected.The differences of potential virulence factors and antibiotic resistance genes between potential friendly niche group and potential unfriendly niche group were compared.The results showed that the potential friendly niche group were safer.The metabolic advantage of species/strain specificity was analyzed,and the complementarity of strain compounding was quantified based on the function of KEGG module.The results showed that the greater the genetic distance between strains,the more likely it was to achieve stronger compounding,which needs to be verified by co-culture experiments.As the representative of MTPS,Lactobacillus acidophilus KLDS1.0901 was preliminarily investigated its cholesterol degradation ability and so on,and compared it with the cholesterol degradation ability of eight published intestinal bacteria.The results showed that the cholesterol degradation ability of KLDS 1.0901 was much weaker than that of the eight intestinal bacteria.In this study,6487 microbial colony were obtained from 154 fecal samples of healthy Chinese volunteers by using 11 media and large-scale anaerobic culture methods.Through 16S rDNA sequencing and comparison,1759 strains of important or novel strains were screened out to be performed whole genome sequencing.Finally,an intestinal bacteria bank containing 1520 bacteria and its high-quality culturable genome reference(CGR)were constructed.These bacteria can be classified into 338 species-level genome clusters,covering the main phylum of human intestinal bacteria,including Firmicutes,Bacteroidetes,Actinobacteria,Proteobacteria and Fusobacteria,and covering 38 low abundance(<1%)genera.Among them,134 clusters are new strains and 50 clusters are members of the new genus.Comparing the annotation information of species in CGR with the information of patients-depleted species of eight cohorts,it was found that patient-depleted species which were annotated to accurate species was 444 times,of 169 species-times could be captured by CGR,and the overall capture rate was 38%.Comparing CGR with existing dataset IGCR(Integrated Gene Catalog-associated References),the results show that CGR significantly improves the alignment ratio and resolution of metagenome analysis(p<0.0001).IGCR plus CGR,the number of gene sets and protein clusters increased by 22%and 16%.A total of 936 intestinal metagenome sequencing data from Chinese,American,Danish and Spanish were used to evaluate the alignment ratio improvement.It was found that IGCR plus CGR,the average alignment rates increased by 24.88%,16.95%,18.47%and 20.12%,respectively.The alignment rate improvement of intestinal metagenome data from Chinese was the highest,which may be due to that all the samples were come from Chinese.In this study,multi methods of functional annotation were used to demonstrate the genomic functional characteristics of CGR.The analyses of KEGG metabolic pathway indicates that the pathway involving carbohydrate and amino acid metabolism is the core function of intestinal microorganisms.Virulence factor and antibiotic resistance genes are abundant in Proteobacteria,suggesting that Proteobacteria may be a opportunistic pathogen bank.Amino acid and vitamin B synthesis genes are widely present in various intestinal bacteria,suggesting that intestinal micro flora may be an alternative source of nutrients lacking in vegetarian diet.Pangenomic analysis showed that the genome of intestinal bacteria were rather variable for Bacteroidetes,slightly variable for Firmicutes and Proteobacteria,and rather conservative for Actinobacteria.The housekeeping gene functions were significantly(p<0.05)enriched in the core genome,while the gene functions related to environmental adaptability and strain specificity were significantly(p<0.05)enriched in the dispensable genome. |
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