A Multi-Factorial Observational Study on Sequential Fecal Microbiota Transplant in Patients with Medically Refractory Clostridioides difficile Infection

Abstract

Fecal microbiota transplantation (FMT) is highly effective in recurrent Clostridioides difficile infection (CDI); increasing evidence supports FMT in severe or fulminant Clostridioides difficile infection (SFCDI). However, the multifactorial mechanisms that underpin the efficacy of FMT are not fully understood. Systems biology approaches using high-throughput technologies may help with mechanistic dissection of host-microbial interactions. Here, we have undertaken a deep phenomics study on four adults receiving sequential FMT for SFCDI, in which we performed a longitudinal, integrative analysis of multiple host factors and intestinal microbiome changes. Stool samples were profiled for changes in gut microbiota and metabolites and blood samples for alterations in targeted epigenomic, metabonomic, glycomic, immune proteomic, immunophenotyping, immune functional assays, and T-cell receptor (TCR) repertoires, respectively. We characterised temporal trajectories in gut microbial and host immunometabolic data sets in three responders and one non-responder to sequential FMT. A total of 562 features were used for analysis, of which 78 features were identified, which differed between the responders and the non-responder. The observed dynamic phenotypic changes may potentially suggest immunosenescent signals in the non-responder and may help to underpin the mechanisms accompanying successful FMT, although our study is limited by a small sample size and significant heterogeneity in patient baseline characteristics. Our multi-omics integrative longitudinal analytical approach extends the knowledge regarding mechanisms of efficacy of FMT and highlights preliminary novel signatures, which should be validated in larger studies.

Keywords: Clostridioides difficile; fecal microbiota transplantation; host-microbial interactions; immunosenescence; systems biology.

Conflict of interest statement

T.M.M. has received consultancy fees from Takeda. B.H.M. has received consultancy fees from Finch Therapeutics Group. J.R.M. has received consultancy fees from Cultech Ltd. Port Talbot, UK, and Enterobiotix Ltd. Glasgow, Scotland. G.L. is founder and CEO of Genos, a private research organization that specializes in high-throughput glycomic analysis and has several patents in this field. M.P.-B. and F.V. are employees of Genos. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. The remaining authors declare that they have no competing interests.

Figures

Figure 1

Schematic of pipeline. (A) Multidimensional, longitudinal assays performed in patients receiving sequential fecal microbiota transplantation (FMT) either by enema or colonoscopy with severe or fulminant Clostridioides difficile infection; (B) Delivery route for each patient; (C) Methodologies utilized; (D) Treatment and sampling strategy with timelines.

Figure 2

Reversal of immunosenescence features in patients with severe or fulminant Clostridioides difficile infection post-sequential FMT. (A) Representative flow cytometry plots show the kinetics of peripheral CD28−ve CD57+ve senescent CD8 T cells in FMT responders (n = 2) (mean ± S.D data for patient’s 2 and 3) and a non-responder patient (n = 1) (patient 1). Percentage of peripheral (B) naïve CD8 T cells; (C) B cells; and (D) Unswitched memory B cells in responders at the start of the trial, post-FMT cycle 1, post-FMT C\cycle 2, post-final FMT cycle, and 1 week and 2 weeks after FMT. (E) Representative flow cytometry plots show the kinetics of peripheral CD24hi CD38hi regulatory B cells in FMT responders and non-responder patient at the start of the trial, post final FMT cycle and 1 week after FMT.

Figure 3

Heat map of normalized frequency values of selected immune subset parameters in the responders (patient 2 and 3 combined) and non-responder patient (patient 1) at different time points. Patients 1, 2 and 3 were clustered using hierarchical clustering (Euclidian distance based). High and low normalized frequency values are indicated in red and blue, respectively. Different immune subset percentages for FMT responders (n = 2; patients 2 and 3)) and non-responder (n = 1; patient 1) are indicated at the end of the column.

Figure 4

In vitro evaluation of antibody-mediated neutralization of Toxin B (patient 2, FMT responder). Sera were serially diluted and incubated with whole purified toxin B (toxinotype 0, strain VPI 10463, ribotype 087) before addition to VERO cells. Cytotoxicity was assessed by counting the number of rounded and non-rounded healthy cells, expressed as percentage protection. For patient 2, detection of neutralization against Toxin B became apparent post-FMT cycle 2 with 100% protection from Vero cell rounding observed with the most concentrated serum tested (1:4 dilution) compared to 0% at the 2 earlier time points. The degree of protection increased from this point over the course of treatment, with protective efficacy clearly detected even in the lowest dilution tested, 1:16. For 1:8 diluted sera, the mean percentage of healthy, non-rounded, protected cells increased from 36.57% at post-FMT cycle 2 to 62.43% pre-final FMT to 66.42% 1 week post-final FMT to 100% 2 weeks post-final FMT. Unlike this patient, patients 1 and 3 displayed no neutralization against Toxin B, and none of the patients showed neutralization against Toxin A throughout the treatment. Controls for this assay showed 100% rounding for cells incubated with the appropriate toxin alone and 100% healthy non-rounding for cells incubated with the respective serum dilution alone. Sera from patients 1 and 3 showed no neutralization against Toxin B. No neutralization was shown against Toxin A for patients 1, 2 or 3. Data from triplicate values +/− SD.

Figure 5

Fecal metataxonomic changes at the phylum level in relation to sequential FMT in severe or fulminant CDI patients. 16S rRNA gene sequencing of DNA extracted from stool samples, presented as relative abundance plots. Participant samples presented as: three stool donors; patient 1(001), earliest to latest timepoint; patient 2 (002), earliest to latest timepoint; patient 3 (003), earliest to latest timepoint; and the fourth patient, earliest to latest timepoint.

Figure 6

K-means clustering of trendlines of 562 valid measurements. Results shown for FMT responders (n = 2) (success group; (A)) and FMT non-responder (n = 1) (failure case; (B)) with each cluster indexed. Clusters are regrouped into four categories as highlighted in each row of the subplots: increased after FMT (up, red), increased after FMT but recovered (up-down, yellow), decreased after FMT but recovered or unchanged (down-up or unchanged, green), and decreased after FMT (down, blue).

Figure 7

Longitudinal TCR repertoire analysis. (A) TCR repertoire clonality calculated as the inverse of the Shannon entropy on sampled peripheral blood mononuclear samples to 1000 TCR sequences. (B) Temporal clustering performed with Mfuzz R package for the 50 most abundant TCRs, alpha and beta, for each time point for patient 1 (FMT failure/non-responder), patient 2 (FMT success/responder) and patient 3 (FMT success/responder). The figure shows 3 exemplary clusters out of 6 for each patient. Thin grey lines in the background represent single clonotypes. The median value of the temporal trajectories of TCR alpha (violet) and beta (green) chains for each cluster is displayed. (C) Exemplary TCR CDR3 amino acid sequences of the most interesting temporal clusters containing TCRs increasing or decreasing in abundance during and after FMT for patients 2 and 3 and TCRs with high abundance at baseline and during disease recurrence for patient 1.