Study On The Diversity Of Intestinal Flora In Patients With Acute Pancreatitis And Its Correlation With The Severity Of The Diseas

Part I:Diversity of Gut microbiome in Acute Pancreatitis uncovered through MetagenomicsObjective:Acute pancreatitis(AP)is one of the most common digestive system diseases requiring acute hospitalization,characterized by severe abdominal pain and elevated serum concentrations of pancreatic enzymes.Considering the limited treatment options currently available for AP and the poor prognosis for patients with severe AP,new therapeutic options are required.In recent years,the interactions between the gut microbiome and AP have gained substantial attention,but most studies are only restricted to 16S rRNA-based analysis of the gut microflora.This study aims to detect the characteristics of microbial community structure and diversity in patients with AP through metagenomic approaches,and to provide a theoretical basis for seeking potential therapeutic targets in treatment of AP.Methods:The fecal samples of healthy volunteers and AP patients were collected by rectal swabs.High-throughput metagenomic sequencing was used to analyzed the differences in the composition and function of intestinal microflora in AP patients,and to evaluate the effects of disease severity on the intestinal microflora.Results:1.A total of 12 AP patients and 12 healthy controls were enrolled in this study,and there was no statistically significant difference between the two groups with respect to age,sex or BMI(body mass index).Compared with the control group,the alpha diversity of case group was significantly declined(P<0.001),and the relative abundances of unclassified Enterococcus sp.,Enterococcus faecium,Candida albicans,and Enterococcus faecalis were significantly increased,while the relative abundances of Prevotella copri,unclassified Streptococcus sp.,unclassified Lachnospiraceae sp.,and Eubacterium rectale were significantly decreased.2.In the function analysis of intestinal microflora,the up-regulated clusters of orthologous groups of proteins(COG)functions in case group included Carbohydrate transport and metabolism,Transcription,and Cytoskeleton,while downregulated COG functions contained Coenzyme transport and metabolism,and Cell motility in contrast to the control group.The up-regulated genes included putative transposase,iron complex transport system permease protein,cellobiose phosphotransferase system(PTS)EIIC component,while downregulated genes contained starch-binding outer membrane protein,transketolase,U32 family peptidase.The up-regulated functional modules included Pentose phosphate pathway,Semi-phosphorylative EntnerDoudoroff pathway,Ascorbate degradation,and Cationic antimicrobial peptide resistance,while downregulated functional modules contained Citrate cycle,C5 isoprenoid biosynthesis,and Dicarboxylate-hydroxybutyrate cycle.3.In the subgroup analysis according to varying AP severity,the relative abundance of Escherichia coli increased,while the relative abundance of unclassified Bacteroides sp.declined as the disease severity increased.In contrast to the moderately severe group,the up-regulated genes in severe group included Putative surface-exposed virulence protein and Molybdenum cofactor biosynthesis,while downregulated genes contained ribosomal protection tetracycline resistance protein,and glucosamine-6-phosphate deaminase.Conclusions:There were significant differences in the intestinal microflora between AP patients and healthy humans,characterized by decreased diversity,increased opportunistic pathogens,and decreased probiotics.With regard to gene functions and metabolic pathways,the genes associated with virulence,carbohydrate transport and metabolism in AP patients were significantly up-regulated compared with healthy humans,which indicated that the virulence enhancement of opportunistic pathogens was implicated in the occurrence and development of AP.As the disease progressed,the relative abundance of opportunistic pathogens increased,the relative abundance of probiotics declined,and the genes associated with virulence and metabolism were significantly up-regulated.Part II:Diversity of Gut microbiome in Acute Pancreatitis uncovered through CulturomicsObjective:There remain certain limitations of Metagenomics for analyzing the intestinal microflora,which cannot evaluate the activity or physiological status of microbiota nor isolate target strains for mechanistic studies.Therefore,this study aims to further investigate the diversity of intestinal microflora in patients with acute pancreatitis(AP)by combining the Culturomics.By screening,culturing,and identifying the specific strains closely associated with disease progression or remission,our study provides a rationale for further validation in germ-free animal models and detailed exploration of molecular mechanism.Methods:The rectal swabs and fecal specimens were collected from AP patients and healthy volunteers.We accomplished the large-scale cultivation,isolation,and identification of samples using the yeast extract,casitone and fatty acid(YCFA)medium and the Modified Gifu Anaerobic Medium(MGAM),to evaluate the diversity and composition of intestinal microflora in AP patients through the strains isolated from samples.Results:A total of 438 isolates from 25 genera and 44 species including 3 new species were isolated and identified,after cultivation of one fecal specimen and two rectal swabs from health humans,and two rectal swabs from AP patients.The results of isolates from rectal swabs of AP patients and healthy humans were merged,respectively.At the genus level,the dominant genera in case group included the Enterococcus,Klebsiella,and Paenibacillus,while the dominant genera in control group contained the Enterococcus,Bacteroides,Paenibacillus,Clostridium,and Shigella.At the level of species,the dominant species in case group included the Enterococcus faecium,Klebsiella grimontii,and Paenibacillus lautus,while the dominant species in control group contained the Enterococcus faecium,Paenibacillus lautus,Shigella flexneri,and Bacteroides faecis.Conclusions:The overall results from the Culturomics were consistent with the Metagenomics,and there were significant differences in the intestinal microflora between AP patients and healthy humans,characterized by decreased diversity,decreased probiotics,and increased opportunistic pathogens.New species can be discovered by the Culturomics,which is beneficial to the database construction of human gut microbiome and has important implications for further study on the relationship between intestinal microecology and human health.