Impacts Of Long-term Prophylactic Use Of Rifaximin-α On Gut Microbiota And Resistome In Patients With Liver Cirrhosis

Backgrouds:Liver diseases are very common in China,it is estimated that the number of liver cirrhosis patients is expected to be 1.6 million in 2019.The incidence of hepatic encephalopathy in these patients was at least 30%-45%.Ornithine Aspartate,Lactulose,Lactitol,Rifaximin-α are the main drugs for the prevention and treatment of hepatic encephalopathy at present.Rifaximin is a derivative of rifamycin and its distinguishing characteristic compared with other antibiotics is,after oral administration,its very low systemic absorption.Rifaximin was also recommended for the treatment of hepatic encephalopathy patients and for the recurrence of hepatic encephalopathy prevention in Guidelines for the treatment of hepatic encephalopathy of the China in 2013The human gut microbiota including bacteria,archaea,fungi,and viruses,is diverse.The microbiota not only help maintain a variety of important physiological functions,such as energy homeostasis,but also affect human psychology and behavior Imbalance of the intestinal microbiota can cause a variety of serious diseases.Intestinal microbiota as a community,contains a variety of bacterias,is the human microbial gene pool,may also be the antibiotic resistance gene pool,Gene exchanges between different bacterias were frequent,which promoted the spread of antibiotic resistance genesThere is little research on the changes of intestinal microbiota and resistome before and after long-term prophylactic use of rifaximin-α in patients with liver cirrhosis,the research on the mechanism is also rare currentlyObjective:The purpose of this project is to explore the impacts of long-term prophylactic use of rifaximin-α on gut microbiota and resistome in patients with liver cirrhosis by metagenomic methods and to provide basis for the rational long-term prophylactic use of rifaximin-α in clinicalMethods:The eligible clinical patients were selected through the inclusion and exclusion criteria.After the get patient’s informed consent,he took rifaximin-α for 12 weeks,and the patients were regularly checked according to the research flow chart.The medical history the combined medication was recorded.Complete blood count,Clinical urine tests,liver and kidney function test,CTP score,assessment of patient compliance,and recording of adverse reactions were done as planed.patients’ fecal specimens were collected one day before the patient took rifaximin-α,and one,two,four,six,eight,ten,and twelve weeks after taking the drug.The metagenomic DNA was extracted,and Next-generation high-throughput sequencing was performed.Bioinformatics was used to analyze the changes in the gut microbiota structure,diversity,and microbiota function of patients during drug administration.The changes and types of intestinal drug-resistant genes were also cheked.Finally,we the analysis the effects of rifaximin-αon the genome of the strains in the gutResults:Part 1 Clinical study of long-term rifaximin-α prophylactic use in patients with liver cirrhosis1.A total of 37 patients were included in this study.Among them,21 patients completed the medication cycle,with an average age of 56 years and a male to female ratio of 13:8.Among the patients who completed the course of treatment,5 patients suffered hepatic encephalopathy and 4 patient had diarrhea.2.In the course of taking rifaximin,the blood ammonia concentration of patients with liver cirrhosis decreased,and the results of NCT and DST improved,Ather biochemical indicators did not change significantly.3.A total of 164 stool samples from 21 patients who completed the study were collected.Part 2 Impacts of long-term prophylactic rifaximin-a use on gut microbiota in patients with liver cirrhosis1.A total of 151 stool samples of 20 patients were selected for sequencing.Per sample data was greater than 10G.2.Based on the principal component analysis,there is no significant difference in the composition of the intestinal microbiota among different patients.According to the time of medication to analysis,patients also had no significant difference in intestinal microbiota at different times.3.During the administration of rifaximin-α,neither the Shannon index nor the Simpson index changed significantly,and were stable at 2.3 and 0.8,respectively.No significant change in the diversity of the intestinal microbiota.4.The occurrence of hepatic encephalopathy in patients is caused by the imbalance of the Escherichia,Klebsiella,Weillonella and other strains,and the diversity is reduced during the episode.Rifaximin-α has rarely inhibitory effect on these strains.5.Compared with the healthy control group,we identified 43 metagenomic species(MGS)with statistical differences between healthy patients and patients with liver cirrhosis,6.19 MGS was enriched in the healthy group and 24 was enriched in the patient group.During the administration of rifaximin-α,9 of the 19 MGS strains were enriched,while 17 of 24 MGS strains enriched in the liver cirrhosis group were decreased.7.During the taking of rifaximin-α,most of the intestinal flora metabolic pathways remained stable.Same pathways involved in the metabolism of branched amino acid,citrulline biosynthesis,chitin degradation et al.was changed.Among them,the lipopolysaccharide and branched amino acid metabolism pathway were reduced.Part 3 Impacts of long-term prophylactic rifaximin-α use on gut resistome in patients with liver cirrhosis1.A total of 1,845 antibiotic resistant genes were noted in the gut microbiota of patients with liver cirrhosis.According to the type of antibacterial drug,these drug-resistant genes were divided into 37 types.The five most abundant types are penicillins,macrolides,quinolones,tetracyclines,and chloramphenicol.According to the abundance analysis,the most abundance resistant drug gene was the tetQ gene,followed by the efflux pump gene.2.The number and abundance of resistance genes in patients with liver cirrhosis did not change significantly during taking rifaximin-α,the number and abundance of drug resistance genes related to rifamycin-α did not change significantly during administration.3.The most type of plasmid in the intestinal flora of patients with liver cirrhosis is the IncFIB plasmid.The type and number of the plasmid did not change significantly during the patients taking rifaximin-α.4.The strains of patients with liver cirrhosis carry about 1200 types of insert sequences.The insertion sequences did not change significantly during taking rifaximin-α.Part 4 Impact of long-term prophylactic rifaximin-α use on genome of strains in patients with liver cirrhosis1.Long-term prophylactic use of rifaximin-α in patients with liver cirrhosis will cause a total of 35 strains in the intestinal to be replaced by different strains of the same species.Among them,10 strains were shared by all patients.2.Long-term prophylactic use of rifaximin-α in patients with liver cirrhosis can cause different numbers of single nucleotide variations(SNV)in 43 strains in the intestinal microbiota.There are 20 strains contained more than 100 SNVs,9 of 20 strains were MGS identied in Part 2,which were abnormal in the gut of patients with liver cirrhosis.3.The strain that undergoes strain replacement or SNV has a different growth rate compared to the original strain,which results in changes in the strain abundance during taking rifaximin-α.Conclusions:1.Patients with liver cirrhosis are well tolerated by long-term prophylactic rifaximin-αuse.Long-term use could improve neurophysiological functions and decreased the ammonia in blood2.Long-term prophylactic rifaximin-α use in patients with liver cirrhosis has little effect on the structure,function and diversity of the intestinal microbiota3.Compared with healthy controls,patients with liver cirrhosis had abnormally abundant strains.Rifaximin-α decreased the strains enriched in the intestinal tract of liver cirrhosis and increased the abundance of strains enriched in the intestines of healthy controls.4.Long-term prophylactic rifaximin-α use in patients with liver cirrhosis does not change the type and abundance of antibiotic resistance genes in the intestinal,and the resistance genes related to rifamycin have not changed significantly.The number of mobile elements remains stable5.Long-term prophylactic rifaximin-α use could lead to intestinal strain replacement or SNV.These mutations cause changes of the growth rate,which is the mechanism of Rifaximin-α regulating the abnormal intestinal microbiota abundance in patients with liver cirrhosis.