Is Autologous Fecal Microbiota Transfer after Exclusive Enteral Nutrition in Pediatric Crohn's Disease Patients Rational and Feasible? Data from a Feasibility Test

Abstract

Background: Exclusive enteral nutrition (EEN) is a highly effective therapy for remission induction in pediatric Crohn's disease (CD), but relapse rates after return to a regular diet are high. Autologous fecal microbiota transfer (FMT) using stool collected during EEN-induced clinical remission might represent a novel approach to maintaining the benefits of EEN.

Methods: Pediatric CD patients provided fecal material at home, which was shipped at 4 °C to an FMT laboratory for FMT capsule generation and extensive pathogen safety screening. The microbial community composition of samples taken before and after shipment and after encapsulation was characterized using 16S rRNA amplicon sequencing.

Results: Seven pediatric patients provided fecal material for nine test runs after at least three weeks of nutritional therapy. FMT capsules were successfully generated in 6/8 deliveries, but stool weight and consistency varied widely. Transport and processing of fecal material into FMT capsules did not fundamentally change microbial composition, but microbial richness was <30 genera in 3/9 samples. Stool safety screening was positive for potential pathogens or drug resistance genes in 8/9 test runs.

Conclusions: A high pathogen burden, low-diversity microbiota, and practical deficiencies of EEN-conditioned fecal material might render autologous capsule-FMT an unsuitable approach as maintenance therapy for pediatric CD patients.

Keywords: Crohn’s disease; autologous FMT; exclusive enteral nutrition; fecal microbiota transfer; pediatric IBD.

Figure 1

Clinical course of study participants and time point of stool donation. (a) Study design. Study participants were asked to donate stool at home during EEN treatment. After immediate transport to the clinic at 4 °C, Fecotainers were packed and shipped overnight at 4 °C to the GMP-certified FMT laboratory. Stool donations were then analyzed for fecal mass and stool consistency; a comprehensive FMT donor safety screening for pathogens was performed; and FMT capsules were produced. (b) Illustration of treatment periods and time points of stool donation for FMT capsule production. Green: induction therapy with EEN. Ochre: maintenance therapy with IFX and MTX. Red triangle: time point of stool collection. (c) Weighted PCDAI (wPCDAI) scores of study participants at baseline, at weeks 2–4 of EEN (EEN week 2–4), at weeks 5–8 of EEN (EEN week 5–8), and 1–2 weeks after completion of EEN (post-EEN). (d) Fecal calprotectin (FCal) concentrations of study participants at baseline, at weeks 2–4 of EEN (EEN week 2–4), at weeks 5–8 of EEN (EEN week 5–8), and 1–2 weeks after completion of EEN (post-EEN). Test for significance was performed using a one-way ANOVA with Tukey´s multiple comparisons test (confidence level of 95%; definition of statistical significance: p < 0.05). Abb.: CD = Crohn’s disease; EEN = exclusive enteral nutrition; FCal = fecal calprotectin; FMT = fecal microbiota transfer; IFX = infliximab; MTX = methotrexate; wk = week; wPCDAI = weighted pediatric Crohn’s disease activity index.

Figure 2

Microbial composition analysis of fecal samples pre- and post-shipment and after encapsulation as determined by 16S rRNA sequencing. (a) Shannon’s effective number of species plotted for fecal samples pre- (■), post-shipment (▲), and after stool encapsulation (●). The test for significance was calculated using a paired t-test (confidence level: 95%, definition of statistical significance: p < 0.05); n.s. = not significant. (b) Beta diversity measured by weighted UniFrac dissimilarity and principal coordinates analysis (PCoA) plotted for fecal samples pre- (■) post-shipment (▲), and after encapsulation (●). A PERMANOVA analysis was used to evaluate the significance between the groups. A pairwise comparison of stool before and after shipment (p = 0.41) and stool before shipment and after encapsulation (p = 0.07) showed no statistically significant difference, respectively. The comparison of stool after shipment and after encapsulation (p = 0.04) showed a statistically significant difference. (c) Stacked bar charts of the relative abundance of the top 20 bacterial genera pre- (stool.bf.ship), post-shipment (stool.af.ship), and after encapsulation (caps.af.enc). (d) A heatmap showing the relative abundances of the top 40 bacterial genera in stool samples from study participants pre- (stool.bf.ship, khaki green), post-shipment (stool.af.ship, light blue), and after encapsulation (caps.af.enc, light red), as determined by 16S rRNA sequencing. A distance tree based on hierarchical clustering illustrates the relationship between the respective samples and genera. Note: Patients FMT-2 and FMT-3 provided fecal material for two test runs (FMT-2a/b and FMT-3a/b). Two samples taken post-transport were analyzed for patient FMT-5, and no capsules could be produced from the fecal material donated by this patient due to stool leakage from the Fecotainer during transport.

Figure 3

Results from infectious disease safety screening. In-depth FMT donor screening was performed on stool samples from the seven study participants. Red boxes indicate detection of the respective pathogen. Patients FMT-2 and FMT-3 provided fecal material for two test runs (FMT2a/b and FMT-3a/b).

Figure 4

Suitability of stool donations from pediatric CD patients for autologous FMT. Numbers from 1 to 7 represent patients FMT-1 to FMT-7. Illustration of quality and quantity of stool donations (Fecotainer: green: standard requirements of FMT laboratory for FMT capsule production met, including fecal biomass >65 g, Bristol stool scale 3–4, and high alpha diversity, represented by Shannon index >2 and a richness above 30 genera, yellow: deviations of fecal weight and/or consistency and/or microbial richness, red: fecal weight and/or consistency insufficient resulting in failure to produce full batch of FMT capsules), stability of microbial composition during transport (Tube: green: comparable microbial composition and absolute abundance pre- and post-transport, yellow: increased absolute abundance of bacteria and/or significant changes in relative microbial composition at the genus level post-transport) and results from FMT donor pathogen screening (Bacterium: green: no pathogens detected, yellow: tolerable pathogens detected, and red: pathogens detected) for the nine FMT capsule production test runs.