Isolation,Identification And Comparison Of Intestinal Desulfovibrio In Patients With Liver Cirrhosis

Desulfovibrio,is a gram-negative bacteria,which is a sulfate-reducing bacteria widely found in human intestines.At present,many studies have shown that Desulfovibrio is closely related to ulcerative colitis,chronic kidney disease and other diseases,and found that the abundance of Desulfovibrio in gut microflora of patients with various liver diseases increased.Besides endotoxin（LPS）,the metabolites of Desulfovibrio,such as hydrogen sulfide（H₂S）and trimethylamine（TMA）,may be important factors affecting the occurrence and development of liver disease.The gut microflora is highly complex,which is not only diverse at the species level of bacteria,but also different in subtypes of the same species,so the isolation of a single strain of bacteria is necessary to study its function.In this study,Desulfovibrio desulfuricans（D.desulfuricans）and Cupidesulfovibrio oxamicus（C.oxamicus,it was a member of Desulfovibrio before a study reclassified as Cupidesulfovibrio of Desulfovibrionaceae in 2021）were isolated from the feces of 9patients with liver cirrhosis.D.desulfuricans JN-54～JN-63 isolated from the intestines of healthy volunteers and D.desulfuricans JN-B13～JN-B21 and C.oxamicus JN-B22～JN-B35were from patients.The strain morphology,desulfurization performance,tolerance of simulated digestion,H₂S metabolism,choline metabolism and antibiotic sensitivity were compared with the D.desulfuricans isolated from healthy volunteers.The effects of culture supernatants of different D.desulfuricans strains on different hepatocytes were further explored.Finally,the whole genome of D.desulfuricans was resequenced,and the genetic evolution and gene mutation of the population were analyzed based on the sequencing information.The main work are as follows:（1）Desulfovibrio was isolated and identified from feces of patients with liver cirrhosis,and the strain morphology,desulfurization performance,tolerance of simulated digestion,H₂S metabolism,choline metabolism and antibiotic sensitivity were compared.In this study,a total of 24 Desulfovibrio strains were isolated.Compared with the D.desulfuricans isolated in the previous study,it was found that there were little morphological differences among different strains,but there were some differences among different groups of strains in the phylogenetic tree based on 16S r RNA gene.According to the results of source classification,the strains from patients with liver cirrhosis had stronger desulfurization performance,tolerance to simulated digestion and the ability to produce H₂S and choline metabolism than those from healthy people.In the antibiotic sensitivity test,it was also found that the strains from patients showed stronger antibiotic resistance,and some strains evolved to be resistant,even multiple drug-resistant strains.At the same time,the results of comparative studies are also different among different strains,highlighting the individual differences between strains and the necessity of exploring the differences between the same species but different individual strains.（2）The effects of different D.desulfuricans culture supernatants on the cell activity and cytokine relative expression of human normal hepatocyte L-02 cell,human hepatic stellate LX-2 cell and human hepatoma Hep G2 cell were investigated.The results were analyzed from two aspects of different sources and different strains.After being stimulated by the culture supernatant of the strain,the activity of the three kinds of cells detected by CCK-8 decreased in varying degrees,and the proliferation inhibition of L-02 and Hep G2was dose-dependent.Compared with healthy people and standard strains,the culture supernatant of D.desulfuricans from patients increased the VEGF expression and ROS level of L-02 cells,decreased the expression ofα-SMA,type I collagen and fibronectin in LX-2 cells,promoted the level of IL-6,IL-8 and TNF-αin Hep G2 cells,and showed damage to all kinds of hepatocytes.（3）The genetic and evolutionary relationship between the two sources of strains was analyzed by whole genome resequencing,and the phylogenetic analysis dendrogram construction of mutation sites,PCA analysis,LD attenuation analysis and differential gene analysis were carried out.The results showed that D.desulfuricans JN-B（Isolated from the intestines of patients with liver cirrhosis）had more mutation sites than D.desulfuricans JN（Isolated from the intestines of healthy volunteers）,and there were great differences between mutation sites,which showed two groups of independent evolutionary clusters in the e phylogenetic analysis dendrogram.PCA analysis further confirmed the difference between the two groups,and LD attenuation analysis showed that the strains from patients with liver cirrhosis had lower genetic diversity.The SNP distribution map of the genome showed differences in the density and location of SNP distribution between the two groups,and SNP analysis of key enzyme genes in the H₂S metabolic pathway and choline metabolism pathway was preliminary analyzed.To sum up,the results of this study compared the differences of growth and metabolism of different Desulfovibrio from patients with liver cirrhosis and healthy people,and compared the damage of their metabolites on hepatocytes.Finally,the genetic and evolutionary relationship was analyzed at the gene level,which laid a theoretical foundation for further exploring the role of D.desulfuricans in the development of liver cirrhosis.