The effect of microbiota-based investigational drug RBX2660 on the gut microbiome and resistome revealed by a placebo-controlled clinical trial CURRENT STATUS: REVIEW

Background Intestinal microbiota restoration can be achieved by replacing a subject’s perturbed microbiota with that of a healthy donor. Recurrent Clostridioides difficile infection (rCDI) is one key application of such treatment. Another application of interest is depletion of 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 decrease of ARG abundance during the first 7 days post-treatment. However, the microbiome and resistome shifts towards healthier configurations were more significant and longer lasting in RBX2660 recipients compared to placebo. We identified 18 taxa and 21 metabolic functions distinguishing the baseline microbiome of non-transplanted patients, and the majority of features were correlated to intrinsic vancomycin resistance. We also identified 7 patient-specific and 3 RBX2660-specific ARGs and tracked their dynamics post treatment. 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 By including a placebo group, we distinguished the effects of RBX2660 from baseline post-antibiotic microbiome dynamics. Antibiotic discontinuation alone resulted in significant recovery of gut microbial diversity and reduced ARG 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.

In the current study, we explored the effect of a microbiota-based investigational drug RBX2660, a suspension of healthy donor microbiota [26][27][28][29], on the intestinal microbiome and resistome of recipients treated for rCDI. In an international, multicenter, randomized, and blinded phase 2b study, rCDI patients received either placebo (control group), one dose, or two doses of RBX2660 (Fig. 1), with more patients being recurrence-free after either RBX2660 regimen than placebo [26]. Through shotgun metagenomic sequencing, we demonstrate considerable shifts of taxonomic and resistome structures common to both placebo-and RBX2660-treated patients likely from discontinuation of antibiotics, particularly during the first week after treatment. By controlling for placebo effects, we could also distinguish taxonomic and resistome changes specific to RBX2660 treatment. Furthermore, we identified discriminative features strongly correlated with microbiota transplant and demonstrated an overall decrease in AROs as well as introduction of a few AROs by RBX2660.

Study cohorts and sample collection
Fecal specimens from a total of 66 patients and their corresponding RBX2660 products were collected during a multicenter, randomized, blinded, and placebo-controlled phase 2b study for the treatment of rCDI (Fig. 1) [26]. 21 patients received 2 doses of placebo (14 females, 9 CDI recurrence, median age 63 years), 22 patients received 1 dose of RBX2660 and 1 dose of placebo sequentially (15 females, 5 CDI recurrence, median age 63 years), and 23 patients received 2 doses of RBX2660 (15 females, 8 CDI recurrence, median age 68 years) [26]. Each RBX2660 dose derives from a single donor, and RBX2660 dose selection was not constrained to ensure a single donor was represented in patients that received two RBX2660 doses (Supplementary Table 1). The first dose of study drug (RBX2660 or placebo) was administered 24-48 hours following completion of antibiotic treatment for CDI, and the second treatment was administered 7 ± 3 days later (Fig. 1). Patients who experienced a new rCDI episode within 60 days after the first dose were moved to open-label treatment and received two additional doses of randomized RBX2660. Patient fecal specimens were collected at selected time points from baseline (day 0) through 365 days after the first dose. AROs from each fecal sample were isolated on selective media plates (Methods, Supplementary Table 2).

RBX2660 shifted taxonomic structures of patients' intestinal microbiome towards those of healthy donors
A comparison of alpha diversity (Shannon diversity) of the gut microbiota between RBX2660 product and rCDI patients revealed that rCDI patients had significantly lower alpha diversity than donors before the treatment (Fig. 2a) as previously described [29]. Following study drug administration, the alpha diversity of all rCDI patients' microbiota increased to near-RBX2660 levels regardless of the treatment group, with the steepest increase during the first week (Fig. 2b). The taxonomic composition of the gut microbiota of RBX2660 recipients and placebo recipients both changed over time (Fig. S1). Analysis of intra-subject Bray-Curtis dissimilarities indicated that the largest taxonomic shift occurred during the first week in all treatment groups (Fig. S2a).
Prior to treatment, the majority of patients had distinct gut microbiome taxonomic compositions from those of RBX2660 (Fig. 2c). Bray-Curtis dissimilarities between recipient and corresponding RBX2660 product were calculated to assess the level of taxonomic transformation towards that of RBX2660. For placebo recipients, the dissimilarity was measured from a pseudo-donor (DS00) profile calculated from the average species-level taxonomic profile of all RBX2660 products in this study (Fig. 2c). The mean Bray-Curtis dissimilarity of DS00 from RBX2660 products was 0.4926, which was lower than the inter-RBX2660 Bray-Curtis distance of 0.6274. Bray-Curtis dissimilarities between patients and RBX2660 demonstrate that RBX2660 administration effectively changed recipients' microbiome structure towards RBX2660 at a larger magnitude and for a longer duration as compared to placebo (Kruskal-Wallis test, P = 0.043 at day 30, P = 0.028 at day 60, Fig. 2d).
Placebo recipients exhibited similar direction of taxonomic structural changes toward RBX2660, but the change was not as dramatic as that of RBX2660 recipients (Fig. 2e). When comparing groups based on rCDI treatment success, treatment-failure patients who experienced a new rCDI episode within 60 days after treatment and treatment-success patients did not exhibit significant difference regarding taxonomic structural change (Fig. S2b −  between baseline (Distance BL ) and selected time point (Distance T ), divided by the distance from donor at baseline: (Distance BL -Distance T )/Distance BL . DS00 was used for placebo recipients, who were then used to determine taxonomic transplantation success. RBX2660 recipients were categorized as transplanted or non-transplanted based on whether their transplant index was higher (transplanted) or lower (non-transplanted) than the maximum value of the placebo group (Fig. 3a) (Fig. 3b).
difficile [41]) are predicted to be vancomycin susceptible. As compared to the transplanted patients, the 4 non-transplanted patients did not exhibit any other distinctive taxonomic characteristics in terms of alpha diversity and composition of Bacteroidetes, Firmicutes, and Proteobacteria phyla (

Cessation of antibiotics is a major factor for resistome regression
Prior to treatment, rCDI patients showed a similar resistome alpha diversity (Wilcoxon signed-rank test, P = 0.18, Fig. 4b) but significantly higher ARG abundance (Wilcoxon signed-rank test, P < 0.0001, Fig. 4a) as well as distinct resistome composition from RBX2660 products (Fig. S4). Specifically, major facilitator superfamily (MFS) and resistance-nodulation-cell division (RND) efflux pumps were the major ARG families present in rCDI patients before the treatment, whereas CfxA beta-lactamase, tetracycline-resistant ribosomal protection proteins, and Erm 23S rRNA methyltransferases were representative of the RBX2660 resistome (Fig. S4).
Over time, microbiome ARG abundance significantly decreased in all treatment arms including the placebo group (Fig. 4c). We tracked individual changes in resistome composition of each patient for 60 days using t-Distributed Stochastic Neighbor Embedding (t-SNE) analysis [42] and resistome transplantation indices defined analogously to the microbiome transplantation index. rCDI patients showed distinctive resistome compositions as compared to those of RBX2660 prior to the treatment, but over time their resistome compositions converged to become similar to RBX2660 (Fig. 4d). The To identify features distinguishing patient and RBX2660 resistomes, we used a Random Forest Classifier ( Fig. S5a − b). Of the top 10 features of importance, 7 ARGs, namely MFS efflux pump, RND efflux pump, OXY β-lactamase, Pmr phosphoethanolamine transferase, undecaprenyl pyrophosphate related proteins, ATP-binding cassette (ABC) efflux pump, small multidrug resistance (SMR) efflux pump, and tetracycline resistant ribosomal protein, were specific to patients' baseline resistomes.
Class A β-lactamases (CfxA and CblA) and a tetracycline resistance protein, which are frequently identified in otherwise healthy populations [43][44][45][46], were classified as RBX2660-specific ARGs ( Fig. 5a). Abundances of all selected ARGs were significantly altered in recipients one week after study drug administration. (Fig. 5b − k). The regression of patient-origin ARGs occurred in all patients without statistically significant differences among placebo and RBX2660 recipients (

RBX2660 effectively cleared AROs compared to placebo but introduced new AROs
We identified both persisting and newly introduced AROs based on whole genome sequence analyses of isolates from both blind and open-label treatment patients. ARO isolates were Escherichia coli (n = 104), vancomycin-resistant Enterococcus (VRE) (n = 25) and other species (n = 135). The majority of donor derived AROs were E. coli (Fig. 6). We selected E. coli and VRE, the plurality of screened AROs, for further analyses based on availability of donor-patient matched pairs and longitudinal samples.
Pairwise average nucleotide identity (ANI) was above 97% for all E. coli isolates (Fig. S7), with more than 99.43% identity for all VRE (Fig. S6). Core genome phylogeny indicated the E. coli were mostly of the B2 and D phylogroups. Isolates not only clustered together based on the patient of origin, but also with their corresponding donor (Fig. S7).
In general, RBX2660 recipients demonstrated faster clearance of AROs as compared to placebo recipients (Fig. 6). Simultaneously, new AROs from RBX2660, mostly E. coli, were introduced to corresponding patients. Calculation of single nucleotide polymorphism (SNP) distances (Methods 5.7) revealed many of these AROs were likely clonal, with a median of 6 SNPs for all pairwise distances indicating near-identical genomes (Supplementary Table 3). We sorted post-treatment ARO E. coli into RBX2660-origin or patient-origin strains and determined clonal persistence following RBX2660 intervention. In some cases, we observed clonal persistence of patient AROs (e.g., patients R1-05 and R2-18), while in some we observed strain replacement by donor-derived AROs (e.g., patient R2-16).
Interestingly, patients receiving the same donor stool did not display identical trends. Patient R2-21 received the same donor stool as R2-18 yet only R2-21 engrafted the donor ARO (Fig. 6).
Isolate ARGs did not indicate a changing resistance profile for these ARO lineages over time. For instance, E. coli isolates exhibited an average of 60 predicted ARGs, and these numbers remained stable throughout the time frame of this investigation (Supplementary Table 4). The 15 RBX2660origin AROs which were engrafted to corresponding recipients harbored beta-lactamase genes such as AmpC (12 AROs Table 4). Antibiotic susceptibility testing (AST) corroborated these findings on the phenotypic level with all introduced AROs being resistant to ciprofloxacin and levofloxacin, and 60% (9/15) resistant to ampicillin (Fig. S8). Approximately half were resistant or intermediate to trimethoprim-sulfamethoxazole (7) and doxycycline (7), and a few were resistant to ampicillinsulbactam (3) and cefazolin (4), while all were susceptible to cefotetan, ceftazidime, meropenem, imipenem, piperacillin-tazobactam, ceftazidime-avibactam, amikacin, aztreonam, tigecycline, and nitrofurantoin (Supplementary Table 2). The introduced AROs were Enterobacteriaceae and resistant to a median of 4 antibiotics, which was less than that of the patient-derived Enterobacteriaceae AROs (median resistance to 7 antibiotics, Supplementary Table 2). The most resistant isolate introduced from RBX2660 was an E. coli strain which was engrafted into patient R1-09. It was retrieved at 5 subsequent time points (final stool collected at 12 months, all < 20 SNPs, Fig. 6, Supplementary

Discussion
In this study, we investigated factors underlying changes in the microbiome derived from RBX2660 in a randomized, placebo-controlled clinical trial [26]. Consistent with a previous evaluation using 16S rRNA methods [29] but in higher resolution with shotgun metagenomic sequencing, we demonstrated that not only RBX2660 recipients but also placebo recipients exhibited noticeable changes toward a healthier microbiome. All patients increased alpha diversity (Fig. 2b), shifted taxonomic structure ( Fig. 2e and S2a), and decreased ARG burden (Fig. 4c) regardless of treatment. Based on the shifts observed among placebo-recipients, some of these changes could be accredited to the natural trajectory of recovery after antibiotic discontinuation, as has been observed [10, 47]. Nonetheless, we distinguished RBX2660-derived effects from placebo effects through a simple yet novel metric, the transplantation index. The transplantation index accounts for long-term changes in the microbiome while controlling for individual variation in baseline composition. With this index, we found that RBX2660 recipients exhibited stronger and longer-lasting changes than placebo recipients.
During the first week, both single and double RBX2660 treatments caused notable taxonomic transformation toward RBX2660, while double RBX2660 resulted in an increase in the ratio of transplanted patients compared to a single dose (Fig. 3a). Despite their apparent difference in transplantation indices, one and two RBX2660 doses showed equivalent clinical efficacy [26], so further exploration is needed to clarify the impact of transplantation efficiency on clinical efficacy.
Likewise, although early-stage transplantation by day 7 appeared to be an important factor determining durable transplant by day 60, it did not always secure successful prevention of additional CDI recurrences and vice versa (Fig. 3a). Taxonomic changes of the non-transplanted patients toward corresponding donors were not distinguishable from those of placebo patients (Fig. 3a). 1 single-dose recipient (R1-04) and 3 double-dose recipients (R2-01, R2-03, and R2-14) failed to maintain their transplanted state at day 7 until day 60 and eventually reverted to below the taxonomic transplantation threshold. The cause for this instability is unknown, but may reflect multifactorial microbial interactions [48].
In an effort to predict transplantation success, we identified baseline taxonomic features that had strong correlations with taxonomic non-transplantation. Species with intrinsic vancomycin resistance were discriminative baseline features of the 4 patients who failed to acquire or maintain irrespective of the dose of RBX2660. The decrease in the 7 ARGs was steepest during the first week of the clinical trial ( Fig. 5 and S8c − i). These outcomes suggest that antibiotic discontinuation and recovery of the microbiota post-antibiotics [47] could be the drivers of the changes in resistome during this clinical trial. The patient-specific 7 ARGs were discriminative features of the resistome disturbed by antibiotic treatment for CDI. As efflux pumps and ribosomal protection proteins are ubiquitous in commensal microbiota [55], further studies comparing ARGs and their functional consequences are required for better defining microbiome health and disturbance.
RBX2660-derived effects were also observed. Patients gained RBX2660-origin beta-lactamases in a dose-dependent manner while simultaneously losing patient-specific ARGs (Fig. 5i and 5j). At the isolate level, RBX2660 simultaneously introduced and eradicated AROs in patients during the process of transplantation (Fig. 6). Previous studies have also demonstrated some efficacy of FMT for eradicating AROs [56], but to our knowledge this is the first to comprehensively track clonality for patient-derived ARO isolates. Most introduced AROs were antibiotic resistant E. coli that are commonly present in a healthy population [57,58] (Fig. 6). The introduced AROs were found in patients longitudinally for up to one year post-treatment ( Fig. 6 and Supplementary Table 2). In some patients, the RBX2660 ARO replaced that of the patient, while the patient strain persisted in others.
Persisting AROs derived from patients R1-05 and R2-18 showed higher phenotypic resistance than their corresponding RBX2660-derived isolates, which failed to engraft. On the other hand, patient R2-21 received the same RBX2660 product as R2-18 but the RBX2660 ARO was engrafted. Patient R2-21 lacked baseline AROs and perhaps provided a "clean slate" for the ARO engraftment.
We identified one ESBL-producing E. coli strain from a donor carrying AmpC and CTX-M-14, whose RBX2660 product was administered to one patient, R1-09. The patient was a single-dose recipient,

Conclusions
We thoroughly examined the impact of RBX2660 on the taxonomic structure, resistome, and ARO colonization of recipients during a randomized and placebo-controlled clinical trial. This study is based on samples from a completed placebo-controlled clinical trial of intestinal microbiota restoration, which enabled us to determine microbiome effects of the microbiota-based drug. Significant changes in both taxonomic and resistome structures following placebo administration, implicating microbiota recovery following cessation of antibiotic treatment as a major contributor to microbiota recovery.
Still, RBX2660 administration transplanted healthy microbiota in the recipients in a dose-dependent manner. V. atypica and intrinsic vancomycin resistant species were discriminative features of patients showing long-lasting microbiota transplantation and resisting microbiota transplantation, respectively. RBX2660 led to more dynamic transformation of the resistome than placebo, but antibiotic discontinuation was the major driving force behind the reduction of patient-origin ARGs, while RBX2660 simultaneously introduced RBX2660-origin ARGs in a dose-dependent manner. RBX2660 more efficiently decolonized AROs than placebo but simultaneously introduced new AROs. Genomic outcomes of intestinal microbiota restoration with RBX2660 in the current study show both latent limitations of microbiota transplantation as well as its potential benefits and highlight the importance of the design and quality control of microbiota-based drugs.

Study cohort, drug, and specimen
Subjects were recruited from among 17 centers in the United States and Canada from 10 December least two recurrences after a primary episode (total three CDI episodes) and had completed at least two rounds of oral antibiotic therapy or ii) had at least two episodes of severe CDI resulting in hospitalization. They were randomly assigned to one of three treatment groups: placebo, single, or double doses of RBX2660. All treatments were blinded and delivered by enema [26]. The second dose was administered approximately 7 days after the first dose. For patients that received two RBX2660 doses, donor selection was random and not constrained to provide a single representative donor per patient.
The selection and screening of donors for RBX2660 were performed as previously described [27,28].  [60,61]. Each isolate was frozen in tryptic soy broth with glycerol at -80˚C.

Antibiotic susceptibility testing
Antibioticl susceptibility testing was performed through Kirby Bauer disk diffusion, and the resulting zone sizes were interpreted according to the M100 document from the Clinical and Laboratory Standards Institute [62].

DNA extraction and sequencing
Metagenomic DNA was extracted from approximately 100 mg of stool samples using DNeasy PowerSoil Kit (Qiagen) following the manufacturer's protocol excepting the stool lysis step: stool samples were lysed by 2 rounds of bead beating for 2 min (total 4 min) at 2,500 oscillations/min using a Mini-Beadbeater-24 (Biospec Products). Samples were chilled on ice for 2 min between the two bead beating rounds. Extracted DNA was quantified using a Qubit fluorometer dsDNA HS Assay (Invitrogen) and stored at −20°C until the library preparation. Metagenomic DNA was diluted to 0.5 ng/μL before preparing the sequencing library. Libraries were prepared using the Nextera DNA Library Prep Kit (Illumina) as previously described [63]. The libraries then were purified through the Agencourt AMPure

Microbiome composition and comparison
Microbiome taxonomic composition was predicted by MetaPhlAn v2.0 [73] and controlled for relative abundance. Genus-level composition plots were obtained by grouping together genus present in less than 50% of samples as "Other." DS00 pseudo-donor microbiome was obtained by averaging the species-level taxonomic profiles of all donor microbiomes. Bray-Curtis distances were calculated using the vegan package [74] and visualized as PCoA plots via the ape package [75] in R 3.5.3. LEfSe [30] identified baseline taxonomic and metabolic features distinguishing transplanted and nontransplanted patients (alpha value for the factorial Kruskal-Wallis test = 0.05, threshold on the logarithmic LDA score = 2). HUMAnN2 [76] was employed for metabolic pathway prediction.

Resistome identification and random forest classifier
ARGs in the microbiome were identified using ShortBRED [77] with CARD [54]. Isolate ARGs were identified with RGI and CARD [54,78]. The resulting genes were manually curated into more general ARG families (n = 64). A subset of 70% of available resistomes were then used to train a random forest classifier distinguishing patient baseline and donor stool resistomes (training set n=103), which was then tested on the remaining samples (test set n=45). The random forest classifier was built with the package scikit-learn (https://scikit-learn.org/stable/index.html) on Python 3.7.3, with trees averaging 12 nodes and a maximum depth of 4.

ARO tracking and SNP calling
SNPs were called using Bowtie2 [79], SAMtools and BCFtools [80], with the first isolate from the patient or donor used as the reference genome. Reads from subsequent isolates of the same species were aligned against the reference with Bowtie2 (-X 2000 --no-mixed --very-sensitive --n-ceil 0,0.01).
BAM files were obtained and sorted with SAMtools (view and sort), which were then converted to pileup files (mpileup). BCFtools view generated VCF files, and variants were called, with the following criteria: minimum coverage of 10 reads per SNP, major allele frequency above 95%, and FQ-score of -85 or less. Indels were excluded. VCF files for each patient were compiled with BCFtools merge, after which SNPs were parsed and counted using custom python and R scripts.   administration in a dose-dependent manner (i−j, red, Kruskal-Wallis test). Tetracycline resistant ribosomal protection protein was a RBX2660-specific ARG, but its 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 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