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

Background

• 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

Objective

• 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.

Methods

• 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.

Results

• 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.

Conclusions

• 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.

References

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