Optimization Of Human Gut Microbiota Culturomics For Construction And Application Of Human Gut Microbiota Bank

The human microbiome is closely related to health,which brings new opportunities for the development of medical diagnosis and treatment.Current research methods mostly use 16S r RNA gene amplicons and metagenomic sequencing or combined with metabolomics analysis to explore the relationship between the microbiome and disease.In recent years,the development of culturomics can obtain intestinal microbial organisms,which has laid the material foundation for the study of the relationship between microbiome and disease.Culturomics is a culture method that uses a variety of culture conditions and uses MALDI-TOF mass spectrometry and 16S r RNA sequencing to identify bacteria species.Since most of the bacteria in the gut are anaerobic bacteria,and the nutritional requirements are different,many are difficult to culture.Therefore,the first consideration of culturomics is to use a variety of media and culture conditions?aerobic and anaerobic culture,different temperature,etc.?to cultivate as many species as possible.It can be seen that this technology is a time-consuming and labor-intensive work.How to isolate more strains under a variety of culture conditions while reducing the workload as much as possible is the research direction of this technology.Relying on this technology can establish a human gut microbiota bank,and provide more material basis for the subsequent experimental research of diseases and flora.This study first optimized the culture strategy of human gut microbiota culturomics.Most bacteria in the human gut are difficult to culture,and culturomics has been designed to overcome this issue.Culturomics makes it possible to obtain living bacteria for further experiments,unlike metagenomics.In this study,we performed a 30-day continuous enrichment in blood culture bottles and cultured bacterial isolates from pre-cultures removed at different time points.We compared the bacteria isolated from the enriched culture with or without adding fresh medium after each pre-culture was removed.We also compared“experienced”colony picking?i.e.,picking 2 to 3 colonies for each recognized colony type?and picking all the colonies from each plate.Finally,the workload of culturomics is optimized to reduce the workload on the basis of ensuring the number of isolated bacteria species.In total,from five fecal samples,106 species were isolated,including three novel species and six that have not previously been isolated from the human body.Adding fresh medium to the culture increased the rate of bacterial species isolation by 22%compared with the non-supplemented culture.Picking all colonies increased the rate of bacterial isolation by only 8.5%compared with experienced colony picking.After optimization through statistical analysis and simulation,sampling aerobic and anaerobic enrichment cultures at six and seven time-points,respectively,is likely to isolate>90%of bacterial species,reducing the workload by 40%.Then analyzed and identified a novel bacteria in the human gut microbiota bank of our laboratory?temporarily named:"DONG20-135^T"?.Identify the novel bacteria from the perspective of phenotype,chemotaxonomic,and genetics.The results showed that the bacteria isolated from the human gut were obligately anaerobic.Cells were Gram-stain-negative,rod-shaped,non-motile,non-spore-forming.The major fatty acids were C_16:0and C_18:1?9c,and the 16S r RNA gene sequence shared a low identity?<92.7%similarity?and distantly relationship to any described species.The phylogenetic tree based on 16S r RNA gene sequences and the protein-concatamer tree showed that strain DONG20-135^Tformed a distinct lineage within the family Erysipelotrichaceae.Average nucleic acid consistency?ANI?of this strain compared to neighboring strains was lower than 69.3%and 92.1%,and this value was lower than the threshold of the defined new species 95%-96%.average amino acid consistency?AAI?values of48.9%-62.5%,which is also below the new species threshold of 60%-80%.Based on the results of phenotypic,chemotaxonomic and genome analyses,strains DONG20-135^Trepresents a novel genus of the family Erysipelotrichaceae.Finally,the preliminary application of the human gut microbiota bank in this laboratory,that is,the comparison of Escherichia coli strain differences.Metagenomics has been widely used to link the gut microbiota and disease,but all analyses have been based on bacterial genus-or species-level comparisons,providing no strain-level information to identify the specific bacteria involved in the development of this disease.Therefore,we selected 158 E.coli strains from human gut microbiota bank for genome sequencing and bioinformatics analysis,and classified them at strain level.Then we tested the interaction between bacteria and the host at the cell-level and animal-level to verify the different effect of bacteria on host at strain-level.The experimental results show that 158 strains of E.coli were divided into five phylogroups:A,D,B1,B2,and F.Some strains were from the same individual but were divided into different phylogroups,and some strains were from different individuals but were divided into the same phylogroups,which suggests that the same individual may have different strains of the same bacteria.A total of 36 strains were selected from each group for cell experiments to monitor the levels of three cytokines?IL-1?,TNF-?,and IL-6?in the cell culture medium,of which the three cytokines in groups A and D had the most differences obvious.Two strains were selected from each of the A and D phylogroups for animal experiments,and the differences in the three cytokines in the mouse serum were also detected.The results also showed that there were significant differences between the A and D phylogroups,which suggests that the relationship between bacteria and disease must be established at the bacterial strain level in the future.In this experiment,first of all,the culture conditions of the culturomics were optimized,and innovatively added new culture conditions and achieved a good effect of bacteria isolation.At the same time,we optimized the strategy to reduce the workload and improve efficiency.Then we analyzed the novel bacteria isolated in the experiment.At last,we analyzed the influence of different bacteria strains from patients on the host.This indicated the importance of the strain level when studying the relationship between bacteria and disease health,which will also be the development trend of the study of the relationship between bacteria and disease in the future.Meanwhile,it will be an important reference value for such research.