Alexa R. Weingarden

Standardized frozen preparation for transplantation of fecal microbiota for recurrent Clostridium difficile infection.

OBJECTIVES:While fecal microbiota transplantation (FMT) is historically known to be an effective means to treat recurrent Clostridium difficile infection (CDI) refractory to standard antibiotic therapies, the procedure is rarely performed. At least some of the reasons for limited availability are those of practicality, including aesthetic concerns and costs of donor screening. The objective of this study was to overcome these barriers in our clinical FMT program.METHODS:We report clinical experience with 43 consecutive patients who were treated with FMT for recurrent CDI since inception of this program at the University of Minnesota. During this time, we simplified donor identification and screening by moving from patient-identified individual donors to standard volunteer donors. Material preparation shifted from the endoscopy suite to a standardized process in the laboratory, and ultimately to banking frozen processed fecal material that is ready to use when needed.RESULTS:Standardization of material preparation significantly simplified the practical aspects of FMT without loss of apparent efficacy in clearing recurrent CDI. Approximately 30% of the patients had underlying inflammatory bowel disease, and FMT was equally effective in this group.CONCLUSIONS:Several key steps in the standardization of donor material preparation significantly simplified the clinical practice of FMT for recurrent CDI in patients failing antibiotic therapy.

High-throughput DNA sequence analysis reveals stable engraftment of gut microbiota following transplantation of previously frozen fecal bacteria.

Fecal microbiota transplantation (FMT) is becoming a more widely used technology for treatment of recurrent Clostridum difficile infection (CDI). While previous treatments used fresh fecal slurries as a source of microbiota for FMT, we recently reported the successful use of standardized, partially purified and frozen fecal microbiota to treat CDI. Here we report that high-throughput 16S rRNA gene sequencing showed stable engraftment of gut microbiota following FMT using frozen fecal bacteria from a healthy donor. Similar bacterial taxa were found in post-transplantation samples obtained from the recipients and donor samples, but the relative abundance varied considerably between patients and time points. Post FMT samples from patients showed an increase in the abundance of Firmicutes and Bacteroidetes, representing 75–80% of the total sequence reads. Proteobacteria and Actinobacteria were less abundant (< 5%) than that found in patients prior to FMT. Post FMT samples from two patients were very similar to donor samples, with the Bacteroidetes phylum represented by a great abundance of members of the families Bacteroidaceae, Rikenellaceae and Porphyromonadaceae, and were largely comprised of Bacteroides, Alistipes and Parabacteroides genera. Members of the phylum Firmicutes were represented by Ruminococcaceae, Lachnospiraceae, Verrucomicrobiaceae and unclassified Clostridiales and members of the Firmicutes. One patient subsequently received antibiotics for an unrelated infection, resulting in an increase in the number of intestinal Proteobacteria, primarily Enterobacteriaceae. Our results demonstrate that frozen fecal microbiota from a healthy donor can be used to effectively treat recurrent CDI resulting in restoration of the structure of gut microbiota and clearing of Clostridum difficile.

Microbiota transplantation restores normal fecal bile acid composition in recurrent Clostridium difficile infection

Fecal microbiota transplantation (FMT) has emerged as a highly effective therapy for refractory, recurrent Clostridium difficile infection (CDI), which develops following antibiotic treatments. Intestinal microbiota play a critical role in the metabolism of bile acids in the colon, which in turn have major effects on the lifecycle of C. difficile bacteria. We hypothesized that fecal bile acid composition is altered in patients with recurrent CDI and that FMT results in its normalization. General metabolomics and targeted bile acid analyses were performed on fecal extracts from patients with recurrent CDI treated with FMT and their donors. In addition, 16S rRNA gene sequencing was used to determine the bacterial composition of pre- and post-FMT fecal samples. Taxonomic bacterial composition of fecal samples from FMT recipients showed rapid change and became similar to the donor after the procedure. Pre-FMT fecal samples contained high concentrations of primary bile acids and bile salts, while secondary bile acids were nearly undetectable. In contrast, post-FMT fecal samples contained mostly secondary bile acids, as did non-CDI donor samples. Therefore, our analysis showed that FMT resulted in normalization of fecal bacterial community structure and metabolic composition. Importantly, metabolism of bile salts and primary bile acids to secondary bile acids is disrupted in patients with recurrent CDI, and FMT corrects this abnormality. Since individual bile salts and bile acids have pro-germinant and inhibitory activities, the changes suggest that correction of bile acid metabolism is likely a major mechanism by which FMT results in a cure and prevents recurrence of CDI.

Dynamic changes in short- and long-term bacterial composition following fecal microbiota transplantation for recurrent Clostridium difficile infection

BackgroundFecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridium difficile infection (CDI) that often fails standard antibiotic therapy. Despite its widespread recent use, however, little is known about the stability of the fecal microbiota following FMT.ResultsHere we report on short- and long-term changes and provide kinetic visualization of fecal microbiota composition in patients with multiply recurrent CDI that were refractory to antibiotic therapy and treated using FMT. Fecal samples were collected from four patients before and up to 151 days after FMT, with daily collections until 28 days and weekly collections until 84 days post-FMT. The composition of fecal bacteria was characterized using high throughput 16S rRNA gene sequence analysis, compared to microbiota across body sites in the Human Microbiome Project (HMP) database, and visualized in a movie-like, kinetic format. FMT resulted in rapid normalization of bacterial fecal sample composition from a markedly dysbiotic state to one representative of normal fecal microbiota. While the microbiome appeared most similar to the donor implant material 1 day post-FMT, the composition diverged variably at later time points. The donor microbiota composition also varied over time. However, both post-FMT and donor samples remained within the larger cloud of fecal microbiota characterized as healthy by the HMP.ConclusionsDynamic behavior is an intrinsic property of normal fecal microbiota and should be accounted for in comparing microbial communities among normal individuals and those with disease states. This also suggests that more frequent sample analyses are needed in order to properly assess success of FMT procedures.

Resolution of Severe Clostridium difficile Infection Following Sequential Fecal Microbiota Transplantation

Over the past two decades Clostridium difficile infection (CDI) has steadily risen in both incidence and severity, correlating with emergence of new, hypervirulent strains of the bacterium.1 Most of the immediate mortality associated with CDI is associated with severe infection that can lead to fulminant colitis and multisystem organ failure.2 Early surgical intervention, typically a subtotal colectomy, can be lifesaving, but is nevertheless associated with postoperative mortality of approximately 50%.3, 4 In 1958, Eiseman and colleagues originally reported fecal microbiota transplantation (FMT) as an alternative to surgical therapy for treatment of refractory pseudomembranous colitis.5 However, in recent years the focus of FMT practice has been primarily on multiply recurrent forms of CDI, while only a handful of case reports exist on the use of FMT in acute, severe, or fulminant CDI.6–8 Between March 2011 and February 2012 we treated four patients with FMT for severe CDI that was refractory to antibiotic therapy. Patients satisfied clinical parameters consistent with severe disease including fever, abdominal pain and distension, WBC count ≥ 20 × 10−9/L, albumin ≤ 2.5 g/dL, thickened colon on abdominal CT and presence of ascites.9 Subtotal colectomy was considered in all patients, but was felt to be prohibitively risky or was refused by the patient (Patient 4). Systemic antibiotics, including metronidazole, were discontinued for at least 48 hours prior to the procedure. Vancomycin, which is poorly absorbed, was discontinued 12–24 hours prior to the procedure, and the patients were administered 2–3 liters of polyethylene glycol electrolyte solution via NG tube or orally. FMT was performed via colonoscopy by an experienced endoscopist using a standardized preparation of concentrated fecal microbiota as we previously described.10 Patient 1 received the preparation the day it was processed. For Patients 2–4, the microbiota preparation was frozen in 10% (v/v) glycerol and stored frozen at −80°C until used. The colonoscope was advanced gently to the farthest point possible maintaining minimal loop formation and using carbon dioxide for insufflation. Pseudomembranous colitis was present on colonoscopy for the initial FMT in all cases. Each patient in this series had a unique clinical narrative and individual histories are provided in Appendix I (Supplementary Materials). All patients had a prompt positive clinical response to the procedure. This was especially dramatic in Patient 1 who was monitored in the medical intensive care unit, where hemodynamic improvement was evident within a few hours after FMT, with lessening vasopressor and ventilatory support and defervescence. A precipitous fall in white blood cell (WBC) count from 25 × 109/L to near normal levels was seen over 24 h, and the CRP began decreasing within hours of the procedure. The improvement, however, was not sustained. Symptoms and signs of CDI returned in Patients 1 and 2 within 3 to 5 days. Patient 1 underwent subtotal colectomy on day 5 post-FMT. Patient 2 was restarted on vancomycin on day 5 after FMT and continued on vancomycin for 2 weeks as an outpatient, which was followed by a second FMT. During this second procedure his colon was noted to be normal without any residual pseudomembranes. After the experience with these two patients, we recommended re-initiation of antibiotics after a holding period of several days following the first FMT in Patients 3 and 4, with the plan to repeat the FMT on an outpatient basis after completing the antibiotic course. This plan was implemented in Patient 3, who was started on fidaxomicin on day 2 following her first FMT and had the second FMT done on day 14. Patient 4 was also started on fidaxomicin on day 3 after her first FMT, but refused to undergo the second FMT. She ultimately succumbed to fulminant CDI, and elected comfort care in a hospice program. Patients 2 and 3 have not had recurrence of CDI in over a year of follow-up despite receiving subsequent antibiotics for different indications, including pneumonia and urinary tract infection.

Changes in Colonic Bile Acid Composition following Fecal Microbiota Transplantation Are Sufficient to Control Clostridium difficile Germination and Growth

Fecal microbiota transplantation (FMT) is a highly effective therapy for recurrent Clostridium difficile infection (R-CDI), but its mechanisms remain poorly understood. Emerging evidence suggests that gut bile acids have significant influence on the physiology of C. difficile, and therefore on patient susceptibility to recurrent infection. We analyzed spore germination of 10 clinical C. difficile isolates exposed to combinations of bile acids present in patient feces before and after FMT. Bile acids at concentrations found in patients’ feces prior to FMT induced germination of C. difficile, although with variable potency across different strains. However, bile acids at concentrations found in patients after FMT did not induce germination and inhibited vegetative growth of all C. difficile strains. Sequencing of the newly identified germinant receptor in C. difficile, CspC, revealed a possible correspondence of variation in germination responses across isolates with mutations in this receptor. This may be related to interstrain variability in spore germination and vegetative growth in response to bile acids seen in this and other studies. These results support the idea that intra-colonic bile acids play a key mechanistic role in the success of FMT, and suggests that novel therapeutic alternatives for treatment of R-CDI may be developed by targeted manipulation of bile acid composition in the colon.

Interaction of gut microbiota with bile acid metabolism and its influence on disease states

Primary bile acids serve important roles in cholesterol metabolism, lipid digestion, host-microbe interactions, and regulatory pathways in the human host. While most bile acids are reabsorbed and recycled via enterohepatic cycling, ∼5% serve as substrates for bacterial biotransformation in the colon. Enzymes involved in various transformations have been characterized from cultured gut bacteria and reveal taxa-specific distribution. More recently, bioinformatic approaches have revealed greater diversity in isoforms of these enzymes, and the microbial species in which they are found. Thus, the functional roles played by the bile acid-transforming gut microbiota and the distribution of resulting secondary bile acids, in the bile acid pool, may be profoundly affected by microbial community structure and function. Bile acids and the composition of the bile acid pool have historically been hypothesized to be associated with several disease states, including recurrent Clostridium difficile infection, inflammatory bowel diseases, metabolic syndrome, and several cancers. Recently, however, emphasis has been placed on how microbial communities in the dysbiotic gut may alter the bile acid pool to potentially cause or mitigate disease onset. This review highlights the current understanding of the interactions between the gut microbial community, bile acid biotransformation, and disease states, and addresses future directions to better understand these complex associations.

Ursodeoxycholic Acid Inhibits Clostridium difficile Spore Germination and Vegetative Growth, and Prevents the Recurrence of Ileal Pouchitis Associated With the Infection

Goals: To test whether ursodeoxycholic acid (UDCA) is inhibitory to Clostridium difficile and can be used in the treatment of C. difficile-associated ileal pouchitis. Background: The restoration of secondary bile metabolism may be the key mechanism for fecal microbiota transplantation (FMT) in treating recurrent C. difficile infections (RCDI). Therefore, it is possible that exogenous administration of inhibitory bile acids may be used directly as nonantibiotic therapeutics for this indication. The need for such a treatment alternative is especially significant in patients with refractory C. difficile-associated pouchitis, where the efficacy of FMT may be limited. Study: We measured the ability of UDCA to suppress the germination and the vegetative growth of 11 clinical isolate strains of C. difficile from patients treated with FMT for RCDI. In addition, we used oral UDCA to treat a patient with RCDI pouchitis that proved refractory to multiple antibiotic treatments and FMT. Results: UDCA was found to be inhibitory to the germination and the vegetative growth of all C. difficile strains tested. Fecal concentrations of UDCA from the patient with RCDI pouchitis exceeded levels necessary to inhibit the germination and the growth of C. difficile in vitro. The patient has remained infection free for over 10 months after the initiation of UDCA. Conclusions: UDCA can be considered as a therapeutic option in patients with C. difficile-associated pouchitis. Further studies need to be conducted to define the optimal dose and duration of such a treatment. In addition, bile acid derivatives inhibitory to C. difficile that are able to achieve high intracolonic concentrations may be developed as therapeutics for RCDI colitis.

Emergence of fecal microbiota transplantation as an approach to repair disrupted microbial gut ecology

In the recent years fecal microbiota transplantation (FMT) has emerged as an effective therapeutic option for patients with refractory Clostridium difficile infection that is not responding to antibiotic therapy. It results in implantation of donor microbiota into recipients and restoration of normal distal gut microbial community structure. We anticipate that this form of therapy represents merely the first entry into a new class of therapeutics. There is great interest in application of FMT or defined microbial consortia to treatment of many diseases associated with dysbiosis. However, many challenges remain in development as our understanding of microbial ecology within the human body and microbiota-host interactions remain limited. Future advances in this field will be critically depending on detailed mechanistic understanding.

Intestinal microbiota, fecal microbiota transplantation, and inflammatory bowel disease

ABSTRACT Inflammatory bowel disease (IBD) is a complex set of diseases that lead to chronic inflammation in the gastrointestinal tract. Although the etiology of IBD is not fully understood, it is well-known that the intestinal microbiota is associated with the development and maintenance of IBD. Manipulation of the gut microbiota, therefore, may represent a target for IBD therapy. Fecal microbiota transplantation (FMT), where fecal microbiota from a healthy donor is transplanted into a patients GI tract, is already a successful therapy for Clostridium difficile infection. FMT is currently being explored as a potential therapy for IBD as well. In this review, the associations between the gut microbiota and IBD and the emerging data on FMT for IBD will be discussed.