The Study Of Microbiota Richness And Diversity In Liver Tissues Of Autoimmune Liver Disease Patients

Background and objectsWe explored the difference in yield and purity of DNA extraction using manual lysis and microbial kit methods.In order to provide clues for the study of the pathogenesis of autoimmune liver disease(AILD),we analyzed differences in the microbiota richness and diversity among liver tissues in different subtypes of AILD,as well as investigated the presence of specific bacteria in liver tissues of with AILD patients and control groups.MethodsFrom August 2017 to August 2018,AILD patients who underwent liver biopsy in the general hospital of Tianjin medical university,were chosen for 16 SrRNA gene sequencing.Liver tissues obtained by liver biopsy in AILD patients were collected aseptically.DNA was extracted by methods of microbiological kit and manual pyrolysis,and the effects of the two methods on the sequencing results were compared.A total of 12 patients diagnosed as AILD,including 4 cases of AIH,4 cases of PBC and 4 cases of PBC-AIH OS,as well as 4 cases of hepatic cyst patients as controls,were chosen for specimen of aseptic liver biopsy using Microbiological kit method.Meanwhile,18 patients diagnosed as AILD,including 10 cases of AIH,8 cases of PBC-AIH OS,as well as 2 cases of hepatic cyst patients as controls,were collected by manual lysis.Among them,16 SrRNA gene sequencing was performed in 6 samples using both microbial kit and manual cleavage methods.Further,we explored the differences in the abundance and diversity of the liver microbiota in each group by using the method of manual cleavage DNA extraction.Linear discriminant analysis effect size was used to find out the specific bacteria.Then we explored the correlation between the liver bacteria and clinical indicators using Spearman correlation analysis,as well as predicted the metabolic function of the 16 SrRNA gene sequences.ResultsBacteria were detected in each samples.Using manual pyrolysis and microbiological kit for DNA extraction,there was no statistically significant difference in the effects of on DNA yield(t=2.502,P=0.054).There was no statistically significant difference in the effects of OD260/OD280(t=2.039,P=0.097),an indicator reflecting the extraction purity.At the level of phylum,genus and species level,the DNA extraction by manual pyrolysis on the number of OTU(t=6.096,6.056,6.096,P <0.01)and Alpha diversity(Chao1 richness index,ACE richness index,Simpson diversity index,Shannon diversity index)(t=7.553,7.673,4.714,9.339,P <0.01)were significantly better than DNA extraction by the microbial kit method.The composition of liver tissue colonies measured by the two extraction methods was mainly Proteobacteria.At the genus level,Pseudomonas was the dominant strain using the microbial kit method,while Pelomonas was the dominant strain using the manual pyrolysis method.Compared with the control group,the microbiota richness decreased in the AIH and PBC-AIH group,while the microbiota diversity increased in the two groups.There was no statistical difference among groups.By using manual lysis,we found that Lactobacilliales(LDA=4.30,P=0.002),Lactobacilliaceae(LDA=4.28,P=0.002)and Lactobacilli(LDA=4.30,P=0.002)were enriched in the control group.The abundance of Klebsiella pneumoniae in AIH group was 3.27 times than that in control group(0.661%vs0.202%).The abundance of Klebsiella pneumoniae in OS group was 4.76 times than that in control group(0.961%vs0.202%).In the analysis of the relationship between the specific bacterial abundance of each subtype and the clinical indicators,we found that the abundance of Lactobacillus was negatively correlated with the ALT?IgG?ANA?AST?IgM level(r=-0.9461,-0.8945,-0.8776,-0.8144,-0.5564,P<0.05),and positively correlated with the C4 and C3 levels(r=0.8848,0.8913,P <0.05).The abundance of Klebsiella pneumoniae was negatively correlated with HDL(r=-0.4655,P=0.0386).The prediction and analysis of the metabolic function of the microflora showed that compared with the control group,lipid metabolism and other metabolic pathways in the AILD group were weakened.Serum TC(r=-0.55,P=0.04)and LDL(r=-0.55,P=0.04)level were correlated with the glycerolipid metabolism.Serum TG(r=-0.498,P=0.026)level was negatively correlated with the synthesis and degradation of ketone bodies.ConclusionsBy comparing the results of the two extraction methods,we found that the manual pyrolysis method was more suitable for the extraction of liver DNA than the microbiological kit method.Klebsiella pneumoniae is also specific to AILD group.Compared with the control group,the Lactobacillus abundance decreased in AILD group.What is more,the Lactobacillus abundance was negatively correlated with disease activity in AILD patients.The predictive analysis of the metabolic function showed that lipid metabolism and other metabolic pathways were reduced in the AILD group.Liver microflora,which was closely related to the pathogenesis of AILD,may be a potential target for diagnosis and treatment.Our study indicated that liver microbiota,involved in the pathogenesis of AILD,might be a potential therapeutic target and diagnostic biomarker.