We investigated factors underlying changes in the microbiome derived from RBX2660 in a randomized, double-blind, placebo-controlled clinical trial [26]. Consistent with a previous evaluation [29] but in higher resolution using shotgun metagenomic sequencing, we demonstrated RBX2660 dose-dependent changes in the microbiome. All patients initially increased alpha diversity (Fig. 2b) and shifted taxonomic structure (Fig. 2e and S2a) 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 [10, 50].
We hypothesized that it would be possible to distinguish RBX2660-derived effects from the microbiome recovery after antibiotic discontinuation by assessing both extent and direction of microbiome shifts of placebo recipients as thresholds. To test the hypothesis, we developed a simple yet novel metric, the transplantation index. The transplantation index accounts for long-term changes in the microbiome toward corresponding RBX2660 while controlling for individual variation in baseline composition. We also computed pseudo transplantation indices based on dissimilarities between patients and other random RBX2660s and compared them with the original transplantation indices in order to validate the transplantation index. The dose-dependent increase in pseudo indices (Fig. S5) is additional evidence that RBX2660 shifted patients’ intestinal microbiome toward those of healthy donors. Some of the pseudo indices were lower than zero, indicating that the transplantation index well reflects individual directionality of recipient’s microbiome shift toward respective RBX2660 (Fig. S5). With the highest transplantation index among placebo recipients as threshold, we demonstrated that RBX2660 recipients exhibited stronger and longer-lasting microbiome changes toward corresponding RBX2660 than placebo recipients (Fig. 3a). Double dose administration resulted in an increase in the ratio of transplanted patients compared to single dose (Fig. 3a). Statistically significant differences between the original and pseudo transplantation indices of double dose recipients, but not single dose (Fig. S5), connoted that double dose administration allows more RBX2660-specific microbiome shift than single dose.
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 transplantation by double RBX2660 administration by day 60 (R2-01, R2-02, R2-03, and R2-14, Fig. 3c). 94% of all patients (64/68) including the 4 patients had received vancomycin, with the remainder receiving metronidazole or fidaxomicin prior to study drug (Fig. 1). Previously reported microbiome signatures of vancomycin administration including lower diversity, lower Firmicutes, and higher Proteobacteria abundances [10, 51, 52] could not distinguish the 4 non-transplanted patients from transplanted patients (Fig. S6c − g). The specific abundance of intrinsically vancomycin-resistant species therefore could be an indicator of more severe microbiome disturbance by vancomycin. Interestingly, the baseline abundance of V. atypica was significantly and positively correlated with durable taxonomic transplantation of RBX2660 microbiome in both the single and double dose arms (Fig. 3b). V. atypica has long been known as an oral bacteria that communicates and develops oral plaque biofilm with lactic acid bacteria [53, 54], but a recent study has highlighted its capacity to build metabolomic networks via a peculiar metabolic function—converting lactate to propionate—in the host gut [55]. Further studies combining both metagenomic and metabolomic analyses are required to uncover the mechanism underlying the positive role of V. atypica in durable microbiota transplantation.
We further characterized the taxonomic transplantation by identifying transplantation-specific taxa enrichments. We discovered that abundances of 2 genera, Barnesiella and Coprobacillus, are significantly correlated with taxonomic transplantation status (Fig. 3d). Barnesiella, which exhibited positive correlation with taxonomic transplantation, also has been linked to clearance of VRE colonization in mice [56]. Although not identified by ZIBR, the genus Clostridium was abundant in non-transplanted patients compared to engrafted, with a significant difference between the groups by day 60. Among identified species features, both Bacteroides species were overrepresented in transplanted patients, reflecting the previous report on their correlation with the healthy gut microbiome [57, 58].
We also hypothesized that microbiome features of patients are also associated with the prevention of CDI recurrence during the RBX2660 clinical trial. Although the treatment-failure patients exhibited higher Bray-Curtis dissimilarities to corresponding RBX2660 products than those of other successful patients, the differences were not statistically significant (Fig. S4a − c). This is likely due to limited number of treatment-failure samples after baseline, as patients were omitted from the current blinded study for the standard-of-care treatment at failure determination. Thus, to identify baseline features correlated to rCDI prevention, we performed general linear model-based multivariate statistical analyses. The model identified K. pneumoniae as a species associated with treatment failure from all patients (Fig. S4d) or only placebo recipients but did not from RBX2660 recipients. Baseline abundance of K. pneumoniae might indeed be a rCDI-associated feature, such as a biomarker of the imbalanced microbiome [59] that underlies CDI, but not correlate with the outcomes of RBX2660 recipients whose microbiomes were affected by RBX2660. Together with the higher efficacy for RBX2660 on the rCDI prevention than placebo [26], the model outputs suggest that RBX2660 transplantation restored the disturbed intestinal microbiota to outcompete C. difficile. Nevertheless, we reckoned that taxonomic transplantation by RBX2660 might not be the only cause of clinical efficacy on rCDI prevention. Despite their apparent difference between transplantation indices of single and double dose recipients (Fig. 3a), the two treatment arms showed equivalent clinical efficacy [26]. 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 rCDI and vice versa (Fig. 3a).
Beyond taxonomic shifts, we determined the distinct ARG compositions of patients and RBX2660 through a Random Forest classifier (Fig S5b). Of the top 10 ARGs of importance, antibiotic efflux pumps dominated the patient baseline resistome, while tetracycline resistant ribosomal protection protein was the most enriched ARG in RBX2660 (Fig. 5k and S4). These differences between rCDI patients and RBX2660 became narrowed in all the three treatment arms over time (Fig. 4c, 4d, and S4). For instance, the abundance of the 7 patient-specific ARGs decreased irrespective of the dose of RBX2660 (Fig. 5), and the decrease was steepest during the first week of the clinical trial (Fig. 5 and S8c − i). These outcomes suggest that antibiotic discontinuation has a significant effect on natural recovery of the microbiome post-antibiotics [50] could be the drivers of the changes in resistome during this clinical trial.
Despite the natural recovery after antibiotic discontinuation, we hypothesized that transplantation of RBX2660 microbiota shaped patient resistome. We demonstrated significant correlation between the transplantation indices of taxonomic and resistome structures (Fig. 4e). RBX2660 recipients exhibited more dynamic increase in both resistome and taxonomic transplantation indices than placebo recipients, indicating simultaneous transplantation of resistome and microbiome (Fig. 4e). At the ARG level, 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 the efficacy of FMT for eradicating AROs [60], but to our knowledge this is the first to comprehensively track clonality for both RBX2660- and patient-derived ARO isolates. Most introduced AROs were antibiotic resistant E. coli that are commonly present in a healthy population [61, 62] (Fig. 6). The introduced AROs were found in patients longitudinally for up to 1 year post-treatment (Fig. 6 and Supplementary Table 2).
We identified one ESBL-producing E. coli strain from a RBX2660 product carrying AmpC and CTX-M-14, whose RBX2660 product was administered to one patient, R1-09. The patient was a single-dose recipient, with recorded treatment success (i.e. no recurrence of CDI and absence of diarrhea for 8 weeks post-treatment) and no known clinical disease resulted from the trial. ESBL-producing E. coli are not inherently more virulent than other strains but can pose a therapeutic challenge if infection occurs [63]. Of note, this trial enrolled patients from December 2014 to November 2015, prior to recognition of ESBL as an important aspect of donor screening. At that time, donor stools were screened for carbapenem-resistant Enterobacteriaceae (CRE) but not ESBL, whereas Rebiotix now screens all donor stools for both CRE and ESBL. Moreover, to date there have been no adverse infection events due to bacterial transmission from RBX2660 in any clinical trials. In light of a recent death caused by ESBL-producing E. coli bacteremia in an immunocompromised patient after FMT [21], our findings highlight the importance of a controlled and regulated donor screening program as well as mandatory, monitored safety reporting. Likewise, our findings prompt a general consideration of risk factors for infections from intestinal microorganisms in any life biotherapeutic investigational product.