The Clinical Study Of Fecal Microbiota Transplantation On Slow-transit Constipation And The Optimization Of Treatment Strategy

With the current changes in the pace of life diet,the prevalence of chronic constipation is on the rise.Epidemiological survey showed that as a common intestinal functional disease,chronic constipation(duration>6 months)prevalence rate is 12-17%in the global adult,the prevalence of adult chronic constipation in China is 4%-6%,and increased with age.Slow transit constipation is one of a functional constipation,the main feature is the colon transit time extended.The high amplitude of the colon contraction activity is reduced after eating.As a representative disease of intestinal motility disorders,more and more studies have begun to pay attention to the relationship between gut microbiome and intestinal motility disorder recently.Animal experiments show that gut microbiome is closely related to gastrointestinal motility,compared with normal mice microbiome,sterile mice showed significant gastric emptying delay and intestinal transit time extended;and when one kind of typical intestinal bacteria is introduced to sterile mice,the improvement of intestinal transit time will be observed.In recent animal studies,many research have proposed the new mechanism of gut microbiome on the role of intestinal motility,studies have confirmed that damage to gut microbiome could affect the formation of short-chain fatty acids,decrease glucagon-like peptide(GLP-1)generation,and thus inhibit intestinal transport function.We also used real-time PCR and DGGE techniques to detect the diversity of fecal and colonic mucosal populations in patients with slow-transit constipation compared with healthy controls.More and more evidence suggest that gut microbiome may play an important role in the development of slow transit constipation.In recent years,many researchers have tried to reconstruct the gut microbiome with fecal microbiota transplantation(FMT),so as to achieve the goal of treating the disease.At the same time,some small sample studies were carried out to use FMT in the treatment of constipation.So it is highly necessary to systematically describe the mechanism of FMT in the role of slow transit constipation,launch high quality clinical trial and establish standardized clinical treatment pathway.In this study,we used the high-throughput gut microbiome sequencing technology(16S rRNA)and gas chromatography-mass spectrometry(GC-MS)to analyze the characteristics of gut microbiome and metabolites in slow-transit constipation patients and healthy control and explore the role of microbiome and its metabolites in the development of slow transit constipation;Then,we carried out a high-quality randomized controlled trials to explore the clinical efficacy and safety of FMT in slow transit constipation patients;Finally,we find there are two questions during clinical application,some of the patients have to accept the repeat transplantation and experienc the invasive approach(nasojejunal tube),so we invent a orallly acid-resistant hypromellose capsules FMT.A variety of bacterial and dead cell observation and counting techniques(plate count method,laser confocal microscopy and flow cytometry)were used to observe the quality of FMT capsules.The aims of this studies is from the clinical requirement to explore in four levels(phenomenon-mechanism-intervention-application),and ultimately return to clinical practice,established a scientific FMT clinical pathway for the treatment of slow transit constipation.Specific research contents are the following three parts.PART I Analysis of gut microbiome and metabolites characteristics in patients with slow transit constipationObjective:To clarify the characteristics of gut microbiome and metabolites(SCFAs)in patients with slow transit constipation.Methods:Eighteen slow transit constipation patients and 17 healthy controls fecal samples were collected.Using the Illumina sequencing platform,High-throughput sequencing(16S rRNA amplification)was used to analyze the diversity and composition of fecal microbial communities.At the same time,the properties and abundance of SCFAs in two groups were detected by gas chromatography-mass spectrometry(GC-MS).Results:The results showed that the diversity of gut microbiome in patients with slow transit constipation was significantly higher than that in healthy controls,and there were significant difference when compared the species and their composition at different classification levels between the two groups.From the phylum level,STC group of patients with Firmicutes significantly reduced,Bacteroidetes increased;From the level of order,Clostridiales significantly reduced,Bifidobacteriales was significantly increased;From the genus level,Ruminococcus,Bifidobacterium was significantly increased;and in the genus Prevotella was significantly reduced.The contents of the two strains Prevotella spp.(Prevotella stercorea,Prevotella_copri)were significantly absent.The study also found that Bifidobacterium as a probiotic was significantly higher in the STC group when compared with the healthy control group,while the Lactobacillus content was not significantly different between the two groups.The results of GC-MS showed that the total SCFAs concentration and acetate concentration in STC group were significantly lower than those in healthy control group,and the other five kinds of SCFAs had no significant change.The correlation analysis showed that there was a positive correlation between the concentration of total and SCFAs and the concentration of acetate.Conclusion:There are significant differences in the main components of gut microbiome between STC patients and healthy controls,and there are also differences in species and their composition at different classification levels.The diversity of gut microbiome in STC patients is significantly higher than that in healthy controls.However,tradiational probiotics(Bifidobacterium,Lactobacillus)content is not lacking,the conclusion let us re-think of the scientific and necessity of using probiotics in the treatment of constipation;The decrease in Prevotella is associated with the reduction of SCFAs,Prevotella-SCFAs-Intestinal motility may explain the relationship between gut microbiome and STC patients.The study was the first time to systematic analysis the characteristic of gut microbiome in STC constipation patients.It is of great importance to recognize the characteristics of gut microbiome in STC patients and follow-up intervention.PART ? Fecal microbiota transplantation in patients with slow-transit constipation:A randomized,clinical trialObjective:Fecal microbiota transplantation has been proposed as a therapeutic approach for chronic constipation.Method:This randomized,controlled trial aimed to compare the effects of conventional treatment alone(control)with additional treatment with FMT(intervention)in patients with slow-transit constipation(STC).Adults with STC were randomized to receive intervention or control treatment.The control group received education,behavioral strategies,and oral laxatives,etc.The intervention group was additionally provided 6 days of FMT.The primary endpoint was the clinical cure rate(proportion of patients achieving a mean of more than three complete spontaneous bowel movements[CSBMs]per week].Secondary outcomes and safety parameters were assessed throughout the study,includeed long-term clinical follow-up,safety assessment and post-transplantation characteristics of flora.Results:Sixty patients were randomized to either conventional treatment alone(n=30)or FMT(n = 30)through a nasointestinal tube.There were significant differences between the intervention group and control group in the clinical improvement rate(intention-to-treat[ITT]:53.3%vs.20.0%,P = 0.009),clinical cure rate(ITT:36.7%vs.13.3%,P = 0.04),mean number of CSBMs per week(ITT:3.2± 1.4 vs.2.1± 1.2,P = 0.001),and the Wexner constipation score(ITT:8.6 ± 1.5 vs.12.7± 2.5,P<0.00001).Compared with the control group,the intervention group showed better results in the stool consistency score(ITT:3.9 vs.2.4,P<0.00001)and colonic transit time(ITT:58.5 vs.73.6 h,P<0.00001).The intervention group had more treatment-related adverse events than did the control group(50 vs.4 cases).In the FMT group,the clinical cure rate and clinical improvement rate were reduced to 30.0%and 43.3%in the FMT group at 24 weeks,26.7%and 40.0%at 48 weeks follow up.There was a significant difference in the diversity of gut microbiome between FMT group and conventional treatment group[Shannon index(pre-FMT 3.5 ± 0.4 vs.2.8 ±0.4,P<0.0001;2 weeks:3.2 ± 0.7 vs.2.9 ± 0.3,P = 0.03;4 weeks:2.9 ± 1.5 vs.3.0 ±1.5,P = 0.67;12 weeks:3.0 ± 1.2 vs.2.7 ± 1.4,P = 0.47;24 weeks:3.3 ± 0.5 vs.2.8 ±2.3,P = 0.54],the diversity index of the FMT group was gradually stable at the 4th week after transplantation,and then gradually returned to the pre-treatment state.There was no significant change in the diversity of the bacteria in the conventional treatment group,the composition of the flora is close to the composition of donor gut microbiome.Conclusion:The efficacy of FMT in the treatment of slow transit constipation is better than that of the conventional control group.The changes of gut microbiome in FMT group were correlated with the clinical symptoms,and the relationship between gut microbiome and constipation was further clarified.However,the long-term efficacy of FMT group was poor,with the trend of efficacy decline.We are looking forword to resolve the repeat transplantation and the invasive approach questions for patients in future study.PART ? Exploration and development of capsulized fecal microbiota transplantation and the optimization of clinical treatment strategiesObjective:To explore the quality of capsulized fecal microbiota transplantation(Capsulized FMT)and to optimize the clinical treatment strategies in slow-transit constipation(STC).Methods:The vacuum freeze-drying technique and acid-resistant hydroxypropyl-methyl cellulose capsule shell were used to prepare the whole bacteria spectrum viable capsules.The quality of the capsules was confirmed by the observation and counting technique of bacterial dead and alive cells.A total of 15 patients with STC in Jinling Hospital,Medical School of Nanjing University,were enrolled in the study,who received capsulized FMT for 6 days,and followed up for 12 weeks after treatment.Rate of clinical remission,Bristol Stool Form Scale,Wexner constipation scale,and bowel movement per week were evaluated at each study visit.To establish a clinical treatment pathway for capsulized FMT.Results:The microbial plate count showed that there were no significant difference in the main four kinds of bacteria[bifidobacteria(6.18 ± 0.34)vs.(7.06 ±0.10)P>0.05,Lactobacillus(6.27 ± 0.30)vs(8.72 ± 0.51)P>0.05,E.cols(5.46 ± 0.28)vs,(5.67 ±0.32)P>0.05,Bacteroides(8.12± 0.14)vs.(8.54± 0.21)P>0.05]when compared capsulized FMT with fresh stool suspension.The LIVE/DEAD BacLight Bacterial Activity Kit was used to stain,and the proportion of dead/live cells was not significantly different under confocal microscopy when compared the capsules FMT and suspension FMT cells.Flow cytometry showed that the proportion of viable bacteria in FMT bacteria was 28.45%±4.62%,FMT capsules showed 28.23%±5.36%viable cells,and there was no significant difference between two groups(P<0.001).Compared with pre-FMT treatment,the rate of clinical improvement and remission based on clinical activity at week 12 was 53.3%(8/15)and 40%(6/15),respectively.The patients'stool frequency increased significantly[(2.2 ± 1.5)times/week vs.(3.1 ±2.1)times/week]after eight weeks of treatment,meanwhile,Bristol scale[(6.7±1.3)vs.(5.1±1.8)points],Wexner constipation scores demonstrated a significant reduction[(13.7 ± 3.5)vs.(10.1 ± 2.3)]and GIQLl score increased remarkable[(87.2± 14.6)vs.(110.9 ± 10.5)],those differences were statistically significant(P<0.05).Capsulized FMT efficacy was stable and no serious adverse events occurred during the whole follow-up.Conclusion:FMT quality control results suggest that FMT capsules have reached the same standard as fresh stool suspension.Clinical application results suggest Capsulized FMT is safe and effective for the treatment of STC,short-term treatment effect is good,which can resolve the problems of long-term maintenance of FMT and the invasive pathways effectively,which is of great significance to optimize the traditional FMT clinical strategy.