Colorectal Cancer Patients With Clinical Stage Of Intestinal Flora Diversity Analysis And Strain Culture

ObjectiveThe intestinal flora plays an important role in the occurrence and development of colorectal tumors and has been increasingly confirmed in recent years.With the rapid development of sequencing technology,new culture schemes and ideas have been provided for the culture of the intestinal flora.The TNM staging of colorectal tumors is divided into stages ?,?,?,and ? based on tumor size,lymph node metastasis,and distant metastasis,providing a basis for clinical treatment and prognosis.However,the changes of the flora in each stage of colorectal tumors,and which kinds of flora occupy the core position are still an unknown result.In addition,the intestine is the main gathering place for human flora,but the cultured flora is only 1%of the number of intestinal flora,and the strains are isolated from the patient's feces at each stage through the culture medium.Harmful strains.These studies provide a theoretical basis for the prevention and treatment of colorectal tumors,and explore whether different strains of medium can be used to develop target strains in the stool of colorectal tumor patients,creating conditions for subsequent experiments.Methods1.Fecal specimens were collected from 8 normal subjects,6 stage ? patients,8 stage? patients,and 7 stage ? patients;2.Take part of the samples and send them to Meiji Company for 16srRNA gene sequencing;and use its biological cloud platform to analyze flora diversity.3.Take a part of the specimens to isolate and culture the strains,determine the single bacteria by PCR and then send the sequencing to the strains to determine the strains and store them.4.Combined with the diversity analysis and the results of strain isolation and culture,check the biology of the cultured strains and their correlation with disease in Pubmed.5.Select possible bacteria after consulting the literature,and take the supernatant after low-speed centrifugation after incubation in the liquid culture medium,and filter,and co-culture the filtrate with colorectal tumor cells for CCK8 test to determine whether it affects tumor cell proliferation.Results1.The analysis of the community Bar chart can find that the composition of the dominant species is basically similar between the 4 groups,but the abundance of the dominant species is different.The figure shows that the analysis at the subordinate level found that the main genus between the 4 groups is:Bacteroides?Bacteroides?,prevotella 9?Pseudomonas?,Faecallbacterium?Sphaerococcus?,Shigella-Escherichia?Shigella?,Lachnoclostridium?Trichospira?,Agathobacter,Blautia,Akkermansia Genus).However,Bacteroides?Bacteroides?group ?,?,and ? groups were reduced compared to group N;prevotella 9?Pseudomonas?and Faecallbacterium?faecal bacteria?group ?,?,and ? groups were compared to N Group Shigella-Escherichia?Shigella?group ? and N group decreased,while group ?,group ? increased,especially in group ? significantly increased;Lachnoclostridium?Trichorium?increased in group ?,group ?,and group ?compared to group N;Agathobacter decreased in group ?,group ?,and group ?compared to group N;Blautia compared to group N in group ? and ? The group increased.Compared with group N,Akkermansia increased significantly in groups ?and ?.2.Through the community pie chart of the four groups at the door level,it can be found that Firmicutes,Bacteroidetes,and Proteobacteria are the dominant bacteria in the four groups.These three dominant bacteria are in each group.Hehe occupies more than 90%of each group,and the abundance of Firmicutes in the three groups except the N group has the highest proportion in each group.In group N:Firmicutes?46.05%?,Bacteroidetes?48.19%?,Proteobacteria?5.33%?,Actinobacteria?0.36%?,Fusobacteria?0.03%?,Verrucomicrobia?0.01%?,Synergistetes?0.01%?.In group ?:Firmicutes?47.74%?,Bacteroidetes?45.36%?,Proteobacteria?2.43%?,Actinobacteria?1.30%?,Fusobacteria?2.83%?,Verrucomicrobia?0.21%?,Synergistetes?0.02%?,group ? Compared with group N,Firmmicutes,Actinobacteria,Fusobacteria,Verrucomicrobia,Synergistetes,etc.accounted for a higher proportion of bacteria,while Bacteroidetes,Proteobacteria decreased.In Group ?:Firmicutes?43.47%?,Bacteroidetes?34.97%?,Proteobacteria?13.60%?,Actinobacteria?0.56%?,Fusobacteria?3.68%?,Verrucomicrobia?3.68%?,Synergistetes?0.00%?,Group ?Compared with group N,Proteobacteria,Actinobacteria,Fusobacteria,Verrucomicrobia and other mycobacteria increased in proportion,while Firmicutes,Bacteroidetes,Synergistetes decreased.In group ?:Firmicutes?44.81%?,Bacteroidetes?32.85%?,Proteobacteria?9.73%?,Actinobacteria?0.70%?,Fusobacteria?1.22%?,Verrucomicrobia?7.12%?,Synergistetes?3.42%?,group ?Compared with group N,Proteobacteria,Actinobacteria,Fusobacteria,Verrucomicrobia,Synergistetes,etc.accounted for a higher proportion of bacteria,while Firmicutes,Bacteroidetes decreased.Conclusions:1.The abundance of the normal group is lower than that of the colorectal cancer patient group;but the normal group is more diverse than the tumor patient group;secondly,in the comparison of the alpha diversity of the three groups of tumor patients,although there is no statistics However,the community richness and community diversity in the advanced tumor group?Group ? and ??are lower than those in the early group?Group ??.2.The composition of the dominant species in the four groups is basically similar at the genus level,but the abundance of dominant species is somewhat different.3.In the difference test between groups,it was found that 12 genera at the genus level had statistical differences between each period.Among them,the reduction of Agathobacter,Lachnospira?Lactospirillum?,Lachnospiraceae_NC2004_group,Lachnospiraceae_ND3007_group,Tyzzerella₃ may be related to the occurrence of colorectal tumors.The number of Bifidobacterium?Bifidobacterium?,Peptostreptococcus?Peptostreptococcus?,and Prevotella?Prestobacterium?in Phase? patients is significantly higher than that of other groups,which may be the driving bacteria for colorectal tumors.These bacteria may be able to As an auxiliary indicator in the diagnosis of early colorectal tumors.Eggerthella,Gemella?Twinococcus?and Erysipelatoclostridium increase significantly in the later stages?stage ? and ??of colorectal tumors,and may be the driving bacteria for tumor development.4.In the prediction of 16S rRNA gene function,it was found that the biological functions were in the tumor groups such as Biosynthesis of amino acids,ABC transporters,Carbon metabolism,Ribosome,Purine metabolism,Pyrimidine metabolism,Quorum sensing,Two-component system?Group ?,Group ?,Group??was significantly higher than the normal group.5.Diversity analysis shows that the microbial composition of the N group and the samples of the disease is clearly distinguished.With the development of colorectal cancer,the microbial structural composition of each group differs greatly,and gradually deviates from the N group from stage ? to ?.Microbial structure.It shows that the core flora is obviously different in each stage of colorectal tumor.6.The vast majority of purely cultured bacteria derived from samples of colorectal cancer patients are pathogenic bacteria,suggesting that the gradual enrichment of pathogenic bacteria in the development of colorectal tumors affects the progress of the tumor.7.In the pure culture experiment,4 potential new species were found?similarity<97%?,which needs further verification;during the pure culture of the strain,a large number of Shigella were isolated,which may be associated with colorectal tumors.Related,but the carcinogenic effect of Shigella has not been reported.