Impact of investigational microbiota therapeutic RBX2660 on the gut microbiome and resistome revealed by a placebo-controlled clinical trial

Abstract

Background: Intestinal microbiota restoration can be achieved by complementing a subject's perturbed microbiota with that of a healthy donor. Recurrent Clostridioides difficile infection (rCDI) is one key application of such treatment. Another emerging application of interest is reducing antibiotic-resistant genes (ARGs) and organisms (AROs). In this study, we investigated fecal specimens from a multicenter, randomized, double-blind, placebo-controlled phase 2b study of microbiota-based investigational drug RBX2660. Patients were administered either placebo, 1 dose of RBX2660 and 1 placebo, or 2 doses of RBX2660 via enema and longitudinally tracked for changes in their microbiome and antibiotic resistome.

Results: All patients exhibited significant recovery of gut microbiome diversity and a decrease of ARG relative abundance during the first 7 days post-treatment. However, the microbiome and resistome shifts toward average configurations from unperturbed individuals were more significant and longer-lasting in RBX2660 recipients compared to placebo. We quantified microbiome and resistome modification by RBX2660 using a novel "transplantation index" metric. We identified taxonomic and metabolic features distinguishing the baseline microbiome of non-transplanted patients and taxa specifically enriched during the process of transplantation. We elucidated the correlation between resistome and taxonomic transplantations and post-treatment dynamics of patient-specific and RBX2660-specific ARGs. Whole genome sequencing of AROs cultured from RBX2660 product and patient samples indicate ARO eradication in patients via RBX2660 administration, but also, to a lesser extent, introduction of RBX2660-derived AROs.

Conclusions: Through shotgun metagenomic sequencing, we elucidated the effects of RBX2660 in the microbiome and resistome. Antibiotic discontinuation alone resulted in significant recovery of gut microbial diversity and reduced ARG relative abundance, but RBX2660 administration more rapidly and completely changed the composition of patients' microbiome, resistome, and ARO colonization by transplanting RBX2660 microbiota into the recipients. Although ARGs and AROs were transmitted through RBX2660, the resistome post-RBX2660 more closely resembled that of the administered product-a proxy for the donor-than an antibiotic perturbed state.

Trial registration: ClinicalTrials.gov, NCT02299570 . Registered 19 November 2014 Video Abstract.

Keywords: Antibiotic-resistant organisms; Clostridioides difficile infection; Microbiome; Microbiota-based therapy; Placebo; Resistome.

Conflict of interest statement

Rebiotix provided access to study specimens and data, and reviewed the manuscript prior to submission, but was not involved in this study’s design, specimen processing, data analysis, or interpretation. Erik R. Dubberke is a consultant for Sanofi, Pfizer, Synthetic Biologics, BioK+, and Rebiotix, and has a grant from Pfizer.

Figures

Fig. 1

Study design for the use of RBX2660 to prevent recurrent Clostridioides difficile infection (rCDI). Total of 66 patients with a history of rCDI were treated with RBX2660 in a randomized and blinded manner. Placebo (white triangle) and RBX2660 (brown triangle) were administered and fecal samples (black circle) were collected at the indicated time points. Patients who were declared a new episode of rCDI within 60 days (white square) were moved to open-label treatment

Fig. 2

RBX2660 shifted taxonomic structures of the gut microbiome of recipients toward a healthy state. a RBX2660 products exhibited significantly higher alpha diversity than patient samples before treatment (Wilcoxon signed-rank test) based on the metagenomic taxonomic profiling data. b Alpha diversity of all patients including placebo recipients increased similarly after treatment. Changes in alpha diversity were significant for the first week after treatment, but there was no statistically significant difference among treatment groups (Kruskal-Wallis test). c Principal coordinates analysis (PCoA) showed a species-level clustering of RBX2660 (white) and pseudo-donor sample DS00 (yellow) distinct from patient baseline samples (violet). d Bray-Curtis distance between taxonomic structures of patients and corresponding RBX2660. D1 and D2 indicate the first dose and the second dose, respectively. DS00 was used for calculating the Bray-Curtis distance of placebo recipients. The decrease in Bray-Curtis distances was steepest during the first week after treatment (black, Wilcoxon signed-rank test). RBX2660 recipients showed a more dynamic decrease in Bray-Curtis distances than placebo recipients by day 60 (red, Kruskal-Wallis test). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. e Upper panels: PCoA describing the direction of changes in taxonomic structures of RBX2660 recipients. Corresponding RBX2660 products and all placebo recipients were included. Lower panels: adjusted P values of PERMANOVA and relevant pairwise comparisons (Pillai-Bartlett non-parametric trace and Benjamini-Hochberg FDR correction). P values of comparisons between placebo and RBX2660 recipients (red asterisks, left y-axis), placebo recipients and RBX2660 (circle, right y-axis), single-dose recipients and RBX2660 (triangle, right y-axis), and double-dose recipients and RBX2660 (square, right y-axis) of PCoA plots were presented in corresponding lower panels

Fig. 3

Discriminative taxonomic features of RBX2660 transplantation. a Transplantation index of patients on day 7 and 60. We defined the taxonomic transplantation as a state showing a higher transplantation index than that of all placebo recipients (green). The patients who were declared rCDI within 60 days were marked (x). The white square represents the patient who exhibited a lower transplantation index for the first dose but a higher transplantation index for the second dose than placebo patients (R2-21, Fig. S7a). b Higher baseline relative abundances of Veillonella atypica in patients who showed durable taxonomic transplantation by day 60 in both single and double RBX2660 treatment groups (Wilcoxon signed-rank test, P = 0.027). c Linear discriminant analysis effect size (LEfSe) determined baseline taxonomic features of the obstinate non-transplanted patients who exhibited lower transplantation indices than placebo recipients at day 60 after double RBX2660 treatment. Thirteen species among 18 taxonomic features were intrinsically vancomycin resistant (violet square, including E. casseliflavus of low resistance). There was no taxonomic feature specific to transplanted patients determined by LEfSe. Genus (d) and species enrichment (e) associated with the taxonomic transplantation (transplanted, green; non-transplanted, purple) were identified through a two-part zero-inflated Beta regression model with random effects (ZIBR) test. *P ≤ 0.05, **P ≤ 0.01

Fig. 4

RBX2660 fluctuated resistome structures of patients via the taxonomic transplantation. a Alpha diversity of baseline patient resistomes was comparable to that of RBX2660 (P = 0.18). b However, baseline patient resistomes had a greater antibiotic-resistant gene (ARG) reads per kilobase per million sample reads (RPKM, Wilcoxon signed-rank test). c Significant decrease in ARG RPKM was observed over time in all treatment groups (Wilcoxon signed-rank test with Benjamini-Hochberg FDR correction, FDR < 0.05). Bars indicate mean of individual ARG relative abundances. D1, the first dose; D2, the second dose. d Patients and RBX2660 products were clustered separately in t-distributed stochastic neighbor embedding (t-SNE) analysis of resistome structures at day 0. Patient resistome became similar to RBX2660 over time, but the speed of change varied for each patient regardless of RBX2660 dose and taxonomic transplantation index. e RBX2660 simultaneously fluctuated both taxonomic and resistome structures more dynamically as compared to placebo. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001

Fig. 5

Recipients adopted a resistome profile similar to that of donors. a Ten most important patient-specific (violet) and RBX2660-specific (white) antibiotic-resistant gene (ARG) families were identified through random forest classifier. b–k Relative abundance of the selected 10 ARGs in RBX2660 (D) and patients who received placebo (gray), single RBX2660 (red), and double RBX2660 (blue). Relative abundance of patient-specific ARGs decreased over time in all patients without statistically significant difference among treatment arms (b–h). Relative abundance of the two RBX2660-specific beta-lactamases in patients increased by RBX2660 administration in a dose-dependent manner (i–j, red, Kruskal-Wallis test). Tetracycline-resistant ribosomal protection protein was a RBX2660-specific ARG, but its relative abundance in placebo recipients also increased after the treatment (k). These changes were significant during the first week after the treatment (black, Wilcoxon signed-rank test). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001

Fig. 6

RBX2660 effectively cleared antibiotic-resistant organisms (AROs) compared to placebo and simultaneously introduced new AROs. We specifically tracked patient-derived (blue dot) and RBX2660-derived AROs (red dot). Patients with no ARO detected from both the baseline sample and corresponding RBX2660 were excluded. Persistency (solid line), disappearance (dash line), and introduction (curved line) of the AROs were determined by genomic comparison of AROs (the “ARO tracking and SNP calling” section). Squares indicate the sample availability (blue, patient baseline samples; red, RBX2660; gray, patient samples after RBX2660 administration). Patients with no samples after day 7 were marked with red. 1R0-03 showed 2–3 separate lineages of E. coli prior to day 30, which were reduced to 1 lineage by day 60. 2Patient R2-16 received the same RBX2660 product twice. 3Although the two RBX2660 products for patient R2-05 were prepared from different donor samples, ARO E. coli strains screened from those appeared to be clonal (distance = 8 SNPs)