Increased Microbial Diversity Found in Successful Recipients of Next-Generation FMT for Recurrent Clostridium difficile Infection

Sahil Khanna, MBBS; Courtney Jones, BS; Lee Jones, MBA
Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN; Rebiotix Inc., Roseville, MN

Increased Microbial Diversity in Patentis Receiving Successful FMT for Recurrent C Diff Image

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Gut Microbiota for World Health
March 5-6, 2016, Miami, FL


  • Patients with recurrent Clostridium difficile infection (CDI) have been found to have markedly decreased fecal microbiota diversity.1-3
  • Diversity increases after successful fecal microbiota transplantation (FMT).4,5
  • While the fecal microbiota of patients with recurrent CDI becomes more diverse, it remains dynamic following FMT.5


  • We evaluated the gut microbiota of patients with recurrent CDI after successful and unsuccessful treatment with RBX2660, a Microbiota Restoration Therapy (MRT) sourced from live human-derived microbes.


  • The fecal microbiota of a subset of 17 patients who participated in the previously reported Phase 2 PUNCH CD study assessing RBX2660 for recurrent CDI were characterized at 7 and 60 days post treatment using 16S rRNA gene sequencing in a post-hoc analysis.
  • RBX2660 was delivered via enema.
  • The patients (median 69.2 years; 65% female; 94% white) included 8 successes with 1 dose of RBX2660; 6 with 2 doses and 3 failures with 2 doses.
  • Success was defined as the absence of CDI-associated diarrhea (passage of three or more unformed stools in 24 or fewer consecutive hours for at least two consecutive days through 8 weeks after the last dose of RBX2660).
  • Failure was defined as a recurrence of CDI symptoms < 8 weeks after the last dose of RBX2660; going back on antibiotic treatment for CDI or a CDI-related hospitalization.

Patient Selection

  • The patient cohort sequenced and analyzed was representative of the outcomes seen in the PUNCH CD study:
    • Single-dose success
    • Single-dose failure (opted out of 2nd dose of RBX2660)
    • Two-dose success
    • Two-dose failure

Sample Collection

  • Patients supplied stool samples at baseline (pre-treatment) and at 7 days, 30 days, 60 days and 180 days (approximately) after treatment with RBX2660.
  • Sample collection was restarted using the same schedule if a second dose of RBX2660 was received.
  • Samples were collected at the patient’s home (or care facility) and shipped overnight to Rebiotix in a cold pack.
  • Aliquots of stool were stored at -80 °C until completion of the sample cycle.

Sequencing and Analysis

  • All sequencing and analysis was performed by the CFAR Viral/Molecular High Density sequencing core at the University of Pennsylvania (Bushman Lab).
  • DNA sequencing was performed on an Illumina MiSeq platform.
  • The 16S sequences were clustered into operational taxonomic units (OTUs) and used to determine within-sample diversity and taxonomic composition at each time point. Weighted Unifrac analysis was used to determine differences in between-sample diversity.
  • Box and whisker plots were used to represent microbial diversity. Samples were rarefied to an even depth of 20,000 OTUs per sample to account for variation in sequencing effort.
  • Abundance-weighted Unifrac distances were calculated and used to examine the shared community structures between samples.


  • There was no significant difference in diversity between successes and failures with RBX2660 at 7 days. A trend toward higher diversity was seen in patients who were successfully treated with RBX2660.
  • At day 60, there was a significantly increased microbial richness in patients with successful treatments compared with failure, P =0.008. Successfully treated patients had increased microbial diversity, regardless of whether they had 1 or 2 doses of RBX2660, Figure 1.
  • Unifrac analysis suggested that the microbial diversity of successfully treated patients was similar; the microbial diversity of failures was dissimilar by PERMANOVA test, P=0.05, Figure 2.


  • Patients with recurrent CDI who responded to treatment with RBX2660 had a more diverse gut microflora at day 60 compared with patients who failed treatment but not at day 7.
  • The results suggest a time dependence for re-establishing a diverse microbiome.
  • The normal microbial diversity associated with successful treatment of recurrent CDI with RBX2660 is consistent with previously reported results obtained with FMT.
  • The observed correlation between higher microbial diversity and treatment success could potentially serve as an early indicator of treatment outcomes as well as provide a biomarker for use in future MRT products.


  1. Chang JY, Antonopoulos DA, Kalra A. et al. Decreased diversity of fecal microbiome in recurrent
    Clostridium difficile-associated diarrhea. JID; 2008;197:435-8.
  2. Seekatz AM, Young VB. Clostridium difficile and the microbiota. J Clin Invest. 2014;124:4182-9.
  3. Shankar V, Hamilton MJ, Khoruts A. Species and genus level resolution analysis of gut microbiota in
    Clostridium difficile patients following fecal microbiota transplantation. Microbiome. 2014;2:13.
  4. Fuentes S, van Nood E, Tims S, et al. Reset of a critically disturbed microbial ecosystem: Faecal
    transplant in recurrent Clostridium difficile infection. ISME J. 2014;8:1621-33.
  5. Weingarden A, Gonzalez A, Vazquez-Baeza Y, et al. Dynamic changes in short- and long-term bacterial
    composition following fecal microbiota transplantation for recurrent Clostridium difficile infection. Microbiome. 2015;3:10