FMT gives sustained C. difficile eradication in children with and without IBD. FMT‐restored diversity is sustained in children without IBD. In those with IBD, bacterial diversity returns to pre‐FMT baseline by 6 months, suggesting IBD host‐related mechanisms modify faecal microbiome diversity.

Eight children underwent FMT for CDI; five had IBD. All had resolution of CDI symptoms. All tested had eradication of C. difficile at 10–20 weeks and 6 months post‐FMT. Pre‐FMT patient samples had significantly decreased bacterial richness compared with donors ( P = 0.01), in those with IBD ( P = 0.02) and without IBD ( P = 0.01). Post‐FMT, bacterial diversity in patients increased. Six months post‐FMT, there was no significant difference between bacterial diversity of donors and patients without IBD; however, bacterial diversity in those with IBD returned to pre‐FMT baseline. Microbiome composition at 6 months in IBD‐negative patients more closely approximated donor composition compared to IBD‐positive patients.

Children with a history of recurrent CDI (≥3 recurrences) underwent FMT via colonoscopy. Stool samples were collected pre‐FMT and post‐FMT at 2–10 weeks, 10–20 weeks and 6 months. The v4 hypervariable region of the 16S rRNA gene was sequenced. C. difficile toxin B gene polymerase chain reaction was performed.

To investigate C. difficile eradication and microbiome changes with FMT in children with and without IBD.

Little data are available regarding the effectiveness and associated microbiome changes of faecal microbiota transplantation (FMT) for Clostridium difficile infection (CDI) in children, especially in those with inflammatory bowel disease (IBD) with presumed underlying dysbiosis.

Introduction Clostridium difficile infection (CDI) is the leading cause of nosocomial diarrhoea in the USA and is increasing in prevalence both in adult1 and paediatric populations.2 Those with inflammatory bowel disease (IBD) are at increased risk of CDI.3-5 Recurrence of CDI can occur in 20–30% after initial infection, with frequency of recurrence increasing further after subsequent infections.6, 7 Faecal microbiota transplantation (FMT) is an effective treatment for recurrent CDI as shown in randomised controlled trials.8, 9 In adults, it has been shown that CDI is associated with a decrease in microbiota diversity that is restored after FMT.10-12 In small studies in children, FMT appears to be effective for recurrent CDI.13-15 However, little data exist in children regarding FMT‐associated microbiome changes with this intervention. Such studies are warranted, as there are known differences in the developing microbiome of children compared with adults.16 Moreover, children with IBD have an underlying dysbiosis 17, 18 and are disproportionately susceptible to CDI19; yet, there are little data regarding whether FMT gives sustained clearance of C. difficile in these patients and the microbiome changes that occur. Thus, the goal of our study was to investigate whether there is sustained C. difficile eradication after FMT for recurrent CDI in children with and without IBD, and to evaluate associated microbiome changes. We hypothesised that FMT would be less effective for sustained eradication of C. difficile in children with IBD given their underlying IBD‐associated dysbiosis.

Materials and methods Children, under 18 years of age, with and without IBD, underwent FMT as clinically indicated for the treatment of recurrent CDI. All of the patients required the following: (i) at least three recurrences of CDI, each with at least three episodes of diarrhoea per day; (ii) C. difficile positive for each recurrence on laboratory testing either by C. difficile toxin polymerase chain reaction (PCR) or enzyme immunoassay (ELISA) of C. difficile toxins and (iii) had symptoms that resolved or improved with CDI antibiotic treatment. Related donors were used for the FMT; all donors completed a health screening questionnaire, had a history and physical examination by their primary medical doctor and had blood and stool screened for potential pathogens following previously published protocols.20 Faecal microbiota transplantation recipients stopped antibiotics to treat CDI 48 h prior to the FMT procedure. Donors were given a mild laxative on the day prior to the procedure (one capful of Miralax or 200 mg of Colace) and donor stool was collected within 12 h of FMT. Up to 100 g of donor stool (range 38–100 g, median 100 g, mean 92 g) was vortexed with 400 mL of nonbacteriostatic saline and filtered. Prepared stool was then delivered to recipient via colonoscopy, primarily in the caecum, with a small amount of stool delivered through the rest of the colon as colonoscope was withdrawn. Recipients remained lying flat for 2 hours after the FMT procedure and received loperamide to aid retention of stool; children <43 kg received 2 mg loperamide after FMT and 1 mg before discharge and those >43 kg received 4 mg after FMT and 2 mg prior to discharge. Baseline information for FMT recipients was collected including age, gender, race, medications, antibiotic use, gastric acid suppression use, probiotic use, whether the patient had IBD, and if so IBD phenotype, location and IBD medications. Donor information recorded included age, gender, race, relationship with recipient, medications, recent antibiotic use (with exclusion of those who had received antibiotics within the last 3 months) and medical history. Follow‐up clinic visits and/or phone calls were conducted at 1–4 days, 2–10 weeks, 10–20 weeks and 6 months after FMT. Adverse effects were screened for at each time point including fever, chills, malaise, fatigue, anorexia, abdominal pain, diarrhoea, constipation, nausea and vomiting. Stool was collected from both donor and recipient prior to FMT, and from recipients at 2–10 weeks, 10–20 weeks and 6 months after FMT; the samples were immediately frozen at −80 °C until analysis. DNA extraction was performed on faecal samples using the MoBio PowerSoil DNA Isolation Kit (Carlsbad, CA, USA). Golay barcoded primers were used to amplify the v4 hypervariable region of the 16S rRNA gene.21 Sequencing was performed on the Illumina MiSeq platform using 2 × 150 bp paired‐end reads. Paired‐end reads were joined with FastqJoin from EA‐utils,22 and only reads that matched in the overlapping region at ≤6% error were kept. De‐multiplexed sequences were filtered for quality and clustered into operational taxonomical units using QIIME 1.8 software.23 Taxonomy was assigned using the open reference method and the Greengenes 16S database (version gg_13_8).24 QIIME was used to assess richness (via the observed species metric), to generate taxa plots, and to calculate UniFrac distances and generate unweighted principal coordinate analysis (PCoA) plots, visualised using EMPeror software.25 All alpha and beta diversity analyses were performed with samples rarefied to a sequencing depth of 8000. Two‐sample nonparametric t‐tests are used to calculate P‐values for alpha diversity analyses. Post‐FMT statistics were calculated using the 6 month timepoint. Differentially abundant taxa were identified using Linear Discriminant Analysis Effect Size (LefSe) software.26 Default settings were used to determine significance thresholds (i.e. alpha % 0.05 for the Kruskal–Wallis test among classes and R 2.0 for the logarithmic linear discriminant analysis score). Polymerase chain reaction of the toxin B gene was performed on recipient stool samples at 10–20 weeks and 6 months after FMT using the commercial BD GenOhm C. difficile assay (BD Diagnostics, Inc, Sparks, MD, USA) directly on stool as per the manufacturer's instructions,27 which has been shown to have high sensitivity and specificity for detection of C. difficile from frozen paediatric stool samples.28 For a subset of stool samples where the genus Clostridium was identified on 16SrRNA sequencing, selective anaerobic culture of stool samples for C. difficile was performed following previously described methods.28 Institutional Review Board approval was obtained for this study at both the Johns Hopkins Hospital and INOVA Fairfax hospital.

Results Demographics and stool collection Eight children between the ages of 6 and 17 years of age received FMT for recurrent CDI. Five also had IBD; four with Crohn's disease and one with ulcerative colitis. All IBD patients had colonic involvement. Baseline demographics for recipients and donors, underlying disease and medications are shown in Table 1. Stool samples were collected prior to FMT from the donor and recipient, and at 2–10 weeks and 10–20 weeks after FMT in all cases; six of eight patients provided stool samples 6 months after FMT, three with IBD (all Crohn's disease) and three without IBD. Table 1. Patient and donor demographics, clinical response to FMT and C. difficile PCR post‐FMT Patient Donor Results Patient C. difficile toxin B gene PCR Age (years) Race Underlying illness IBD location Medical therapy at FMT Age (years) Relation Resolved C. difficile symptoms Adverse effects 12–20 weeks 6 months 1 10 Caucasian Crohn's Disease Pancolitis Adalimumab

Methorexate

Oral corticosteroids

Mesalazine (mesalamine) enemas 45 Father Yes No Negative Negative 2 15 Asian Crohn's Disease Ileocolonic Oral mesalazine 42 Mother Yes No Negative Unknown 3 16 Caucasian Crohn's Disease Ileocecal Oral corticosteroids 56 Father Yes No Negative Negative 4 17 Caucasian Ulcerative Colitis Pancolitis Oral mesalazine 50 Father Yes No Negative Unknown 5 13 African American Crohn's Disease Ileocolonic Infliximab 44 Mother Yes No Negative Negative 6 16 Caucasian POTSa N/A Fludrocortisone 24 Brother in law Yes No Negative Negative 7 6 Caucasian Mitochondrial disease, cecostomy N/A None 36 Father Yes No Negative Negative 8 12 Asian None N/A None 26 1st Cousin Yes C. difficile‐negative diarrhoea Negative Negative Clinical results and adverse effects Seven of eight patients had resolution of symptoms associated with CDI within 1–3 days after only one FMT and no patients required a repeat FMT. Transient mild abdominal pain was reported in two patients immediately after the procedure, with no other immediate or delayed side effects of FMT reported. Three of the five IBD patients were being treated with systemic corticosteroids and/or tumour necrosis factor (TNF) α antagonists at the time of FMT without post‐FMT adverse effects reported. No exacerbation of IBD symptoms was reported and no escalation in IBD therapies was required in the 10 weeks post‐FMT in both patients with Crohn's disease and the one patient with ulcerative colitis. Although CDI and IBD symptoms can be very difficult to distinguish from each other, after the initial improvement of diarrhoea attributed to CDI clearance, baseline symptoms in patients with IBD, both with Crohn's disease and ulcerative colitis remained relatively stable but without any apparent improvement over time and without the ability to wean off medications for IBD. However, as this study was not designed to assess the effectiveness of FMT for IBD, more robust assessments with clinical disease activity indices and follow‐up endoscopies were not conducted. Six months post‐FMT, one patient with Crohn's Disease (patient 3) on oral corticosteroids prior to FMT was transitioned to a TNF α antagonist and another patient with Crohn's Disease (patient 5) transitioned to a different TNF α antagonist due to decreasing response; all other patients with IBD remained on stable medications for IBD at 6 months post‐FMT. One patient (patient 8) without IBD had prolonged diarrhoea, faecal urgency and intermittent faecal incontinence after FMT which was different in nature and less severe compared to their CDI. These symptoms included 4–5 loose, nonbloody stools per day without nocturnal bowel movements; with CDI the patient had up to 20 liquid stools in 24 h, including bloody and nocturnal stools. Post‐FMT symptoms fit a pattern consistent with irritable bowel syndrome, however were debilitating enough to cause school absence due to faecal incontinence. Extensive work‐up for infectious aetiologies, including multiple repeat C. difficile toxin B PCR tests were negative. Loperamide was not tolerated, and symptoms gradually improved at 2 months after FMT with the use of behavioural psychology and cholestyramine. At 6 months, this patient's diarrhoea had fully resolved and no medications were being used. Clearance of C. difficile At 12–20 weeks after FMT, all patients had a negative C. difficile toxin B PCR. In the six patients who provided samples 6 months after FMT, all still had a negative C. difficile toxin B PCR. Microbiome diversity Pre‐FMT patient stool samples had significantly decreased bacterial diversity compared with donors (P = 0.01; Figure 1a), in both those with IBD (P = 0.02) and without IBD (P = 0.01). There was no difference in pre‐FMT diversity between those with and without IBD (P = 1.0). Six months after FMT, diversity was restored to the same level of donors in patients without IBD (P = 0.54), but remained low in IBD patients (P = 0.07) (Figure 1b). Time course analyses revealed that bacterial diversity increased at 2–10 weeks after FMT in patients with IBD, then decreased to pre‐FMT levels by 6 months (Figure 1c). In contrast, in patients without IBD, bacterial diversity increased and was sustained at 6 months, with no difference between recipient and donor diversity at 2–10 weeks (P = 1.0), 10–20 weeks (P = 1.0) and 6 months (P = 0.54) after FMT. Figure 1 Open in figure viewer PowerPoint (a) Alpha diversity before and after faecal microbiota transplant (FMT) for paediatric patients with C. difficile‐associated disease in comparison to their adult stool donors. While there is decreased diversity as measured by the observed species metric (richness) between recipients and donors before FMT (P = 0.01), diversity increases by the first timepoint (2–10 weeks) post‐FMT and matches donor diversity by 6 months (P = 1.0). (b) When analysed by inflammatory bowel disease (IBD) status, both those with IBD (P = 0.02) and without IBD (P = 0.01) had significantly decreased bacterial diversity compared with donors pre‐FMT. However, 6 months after FMT, diversity is restored to the same level of donors in patients without IBD (P = 0.54), but remains low in IBD patients (P = 0.07). (c) Alpha diversity pre‐FMT and over time post‐FMT in patients with IBD. After FMT there is an initial increase in diversity which returns to baseline levels by 6 months. To confirm this difference in diversity after FMT in IBD‐positive and ‐negative patients was not a result of donor stool diversity, the alpha diversity was compared between donors who gave stool to IBD‐positive patients and those who gave stool to IBD‐negative patients; no difference was found in alpha diversity between the two donor groups. Microbiome composition In general, the microbiome composition of FMT recipients shifted towards that of the donor over the study timepoints as demonstrated by PCoA of unweighted UniFrac distances (Figure 2a). Taxonomic plots demonstrating the relative abundance of microbiome components also showed that the microbiome composition of the recipients became more similar to the donors following FMT (Figure 3). Overall, there was increasing Bacteroidetes post‐FMT (6.4% pre‐FMT, 20.9% 2–10 weeks post‐FMT, 32.6% 10–20 weeks post‐FMT, 29.4% 6 months post‐FMT vs. 45.6% in donor). Figure 2 Open in figure viewer PowerPoint (a) Principal Coordinate Analysis (PCoA) of faecal microbiome composition at all timepoints from eight paediatric patients with CDI treated with FMT and their eight corresponding stool donors. Pre‐FMT samples and donor samples group separately on opposite ends of the PC1 axis. Following FMT, samples from FMT recipients shift towards the donors. Samples from recipient 8 are highlighted and also follow this pattern of change in microbiome composition despite the patient's prolonged C. difficile‐negative diarrhoea after FMT. (b) PCoA before FMT and 6 months after FMT comparing recipients with and without IBD to donor. Recipient microbiome composition shifts towards donor mostly in those without IBD. Figure 3 Open in figure viewer PowerPoint Taxa plots demonstrating relative abundance at the phylum and order level in paediatric patients with CDI treated with FMT, as well as in their adult stool donors. Pre‐FMT samples are most notable for a relative abundance of Proteobacteria, predominantly from the order Enterobacteriales. Following FMT, the composition of the stool microbiome of CDI patients became more similar to donor stools, with a relative abundance of Bacteroidales from the phylum Bacteroides noted and a decrease in the previously seen Enterobacteriales. It was noted, however, that analysis of microbiome composition by PCoA based on IBD status demonstrated that samples from non‐IBD patients cluster with the donors post‐FMT, while samples from IBD patients continued to group separately (Figure 2b). While linear discriminant analysis of differentially abundant taxa using LefSe software found no differences in microbiome composition of IBD‐positive and IBD‐negative patients prior to FMT, there were several differentially abundant taxa present in both groups prior to FMT compared to donors (Table 2, Figure S1a,b). At a phylum level, in patients pre‐FMT, relative abundance of the phylum Proteobacteria was present in both those with and without IBD (41% IBD, 50.3% non‐IBD vs. 5.5% in donor), predominantly from the order Enterobacteriales. Table 2. Differentially abundant taxa as determined by LEfSe in recipients vs. donors prior to FMT, by IBD status Phylum Class Order Family Genus (A) IBD negative vs. donors, before FMT Increased in IBD‐negative samples Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Enterobacter Klebsiella Pleasiomonas Proteus Morganella Lactobacillaceae Pediococcus Veillonellaceae Veillonella Leptotrichiaceae Leptotrichia Aerococcaceae Increased in donor samples Coriobacteriia Coriobacteriaceae Coriobacteriaceae Ruminococcaceae Faecalibacterium Rikenellaceae Odoribacteraceae Mogibacteriaceae Parabacteroides Lachnospira Dorea Coprococcus Blautia Anaerostipes Dorea Coprococcus Blautia Anaerostipes Holdemania (B) IBD positive vs. donors, before FMT Increased in IBD‐positive samples Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Pseudomonadales Moraxellaceae Acinetobacter Pseudomonadaceae Actinobacillus Sphingomonadace Sphingomonadaceae Comamonadaceae Acidovorax Bacilli Bacillales Leuconostocaceae Bacillus Corynebacteriaceae Corynebacterium Mycobacteriaceae Mycobacterium Veillonellaceae Megasphaera, Veillonella Peptostreptococcace Increased in donor samples Bacteroidetes Bacteroidia Bacteroidales Odoribacteraceae Odoribacter Porphyromonadaceae Parabacteroides Bacteroidaceae Bacteroides Rikenellaceae Anaerostipes Lachnospira Roseburia Oscillospira Six months following FMT, differentially abundant taxa differed between IBD and non‐IBD patients as well as when compared to donors (Table 3). Notably, when compared to non‐IBD patients, IBD‐positive patients had a relative increase in Fusobacterium, including increases at all its higher taxonomic classifications (Table 3a, Figure S2a). In addition, IBD‐positive patients displayed several differentially abundant taxa compared to donor samples (Table 3b, Figure S2b), whereas non‐IBD patients displayed only two differentially abundant taxa compared to donors, a 3‐fold increase in Bacteroides fragilis and an approximately 2.5‐fold decrease in the genera Acidaminococcus (Table 3c, Figure S3). Lastly, comparisons of pre‐FMT and 6 months post‐FMT samples revealed multiple significant changes in differentially abundant taxa in the IBD‐negative group, whereas the only sustained change in those with IBD was a greater than 5 log increase in Bacteroides (not shown). After FMT, Proteobacteria abundance decreases at 2–10 weeks in both groups (3.7% IBD and 2.4% non‐IBD); however, at 6 months post‐FMT again increases in those with IBD (21.2% IBD and 3.6% non‐IBD). Table 3. Differentially abundant taxa as determined by LEfSe, 6 months after FMT Phylum Class Order Family Genus (A) IBD positive vs. IBD negative, 6 months after FMT Increased in IBD‐negative samples Christensenellaceae Christensenella Odoribacteraceae Odoribacter Porphyromonadaceae Parabacteroides Clostridium Phascolarctobacterium Increased in IBD‐positive samples Fusobacteria Fusobacteria Fusobacteriales Fusobacteriaceae Fusobacterium Veillonella Dialister (B) IBD positive vs. donor, 6 months after FMT Increased in IBD‐positive samples Xanthomonadales Xanthomonadaceae Stenotrophomonas Rhizobiales Bradyrhizobiaceae Comamonadaceae Limnohabitans Veillonella Increased in donor samples Coriobacteriia Coriobacteriales Coriobacteriaceae Porphyromonadaceae Parabacteroides S24_7 Deltaproteobacteria Desulfovibrionales Desulfovibrionales Bilophila Lachnospira Phylum Class Order Family Genus Species (C) IBD negative vs. donor, 6 months after FMT Increased in IBD‐negative samples Bacteroides fragilis Increased in donor samples Acidaminococcus As a control, microbiome composition of FMT donors for IBD patients was compared to donors for non‐IBD patients. Of the five differentially abundant taxa found, only Phascolarctobacterium (increased in IBD‐negative patients' donors) was noted to also be differentially abundant in the post‐FMT analyses (increased in IBD‐negative patient samples). At a genus level, the relative abundance of Clostridium for all recipients was similar pre‐ and post‐FMT (0.2% pre‐FMT, 0.1% 2–10 weeks post‐FMT, 0.1% 10–20 weeks post‐FMT, 0.1% 6 months post‐FMT vs. 1.1% in donor). To determine whether the Clostridium detected represented C. difficile, a subset of stool samples where the genus Clostridium was identified in sequencing [5 donor samples, 13 recipient samples (8 IBD samples and 5 non‐IBD samples)], representing each time point post‐FMT, underwent selective anaerobic culture for C. difficile. All cultures were negative for C. difficile. Microbiome changes in patient with prolonged diarrhoea The microbiome of patient 8 without IBD was examined individually given the prolonged C. difficile‐negative diarrhoea following FMT. Bacterial diversity increased and was sustained at 6 months after FMT. In addition, there were similar changes compared with other patients in composition at a phylum level with a decrease in relative abundance of Proteobacteria and increasing Bacteroidetes after FMT. Immediately post‐FMT, microbiome composition of this recipient also shifted towards donor (Figure 2a). Thus, despite this seeming return to normal gut microbial composition, the child experienced an adverse clinical course; however, it is possible that nonbacterial or occult microbiome components may have played a role in the prolonged diarrhoea.

Discussion To our knowledge, this is the first, and largest, study to address longitudinal microbiome changes with FMT for recurrent CDI in children both with and without underlying IBD. The key finding was that FMT gave clinical improvement and sustained eradication of C. difficile in both children with and without IBD. Initially, there was increased bacterial diversity in patients after FMT; however, by 6 months post‐FMT, the increased bacterial diversity in patients without IBD was sustained and in striking contrast, in those with IBD, decreased bacterial diversity returned. While there was clear distinction between the low diversity of IBD patients compared with donors at 6 months (Figure 1b), the P‐value did not reach statistical significance, likely due to the smaller number of samples available at this particular timepoint. These results are consistent with IBD host‐driven loss of microbiome diversity over time and support the hypothesis that host genetic polymorphisms impact microbiota composition.29 Additionally, most of the patients with IBD were receiving potent immunosuppressive medications and another hypothesis is that immunosuppression may also contribute to structuring the microbial community. However, one study in immunocompromised patients showed FMT was successful for treating recurrent CDI30; although sequencing was not performed in this study, these results suggest that immunosupression alone may not prevent successful microbiome engraftment. Furthermore, it is possible that there was ongoing intestinal inflammation in patients with IBD after FMT, even if this was subclinical; intestinal inflammation has been associated with decreased microbiome diversity and abundance of Proteobacteria,31 as seen in the patients with IBD in our study 6 months after FMT. In this study, FMT clinically improved symptoms of CDI in all children within a few days as shown in studies both in adults 8, 32 and children.13-15, 33 No child required a second FMT procedure for CDI and there were no recurrences of CDI within the follow‐up period. Apart from transient abdominal pain and cramping after the procedure, as previously described with FMT,9 seven of eight children tolerated the procedure well without any overt short‐ or long‐term side effects during follow‐up. There have been reports of patients with IBD and CDI receiving FMT having worsening colitis 34 and bacteraemia,35 however no such side effects were found in children receiving FMT in this study despite many being on potent immunosuppressive agents including corticosteroids and TNF α antagonists. There has also been a recent report of substantial unexplained weight gain after FMT36; in our study no child had unexpected weight gain after FMT in the follow‐up period and potential donors were excluded if they were overweight or obese. Not only was there clinical improvement of CDI but patients also had sustained clearance of C. difficile, tested by PCR at 10–20 weeks and 6 month post‐FMT. This is important to elucidate, because it is known that children with IBD have an increased rate of asymptomatic carriage compared to those without IBD,28 however following FMT no patient in the study had asymptomatic carriage of toxigenic C. difficile based on PCR assay. In addition, even though the Clostridium genus was detected in many faecal samples, C. difficile was not detected by selective anaerobic culture, considered the gold standard for detection of faecal C. difficile.37 This study confirmed that, prior to FMT, there was decreased bacterial diversity compared to healthy donors in those with recurrent CDI, as previously seen in mice38 and adult human studies.10, 39-42 After FMT, microbiota diversity increased in faecal samples, as also demonstrated by Song et al.9 Overall, there is a shift towards donor composition following FMT with the relative abundance of Proteobacteria decreasing and Bacteroidetes increasing after FMT, as previously described.10, 12, 43 However, subgroup analysis by IBD status revealed that 6 months post‐FMT, those with IBD displayed several differentially abundant taxa compared with donors in contrast to those without IBD, with only two differentially abundant taxa. Of note, a relative abundance of Proteobacteria recurred at 6 months in those with IBD, whereas those without IBD maintained low levels of Proteobacteria. Intestinal microbiome dysbiosis is well described in IBD in addition to an increased relative abundance of Proteobacteria.17, 18, 44 Of particular interest, an increase in Fusobacterium was identified in those with IBD compared to those without IBD at 6 months post‐FMT. Fusobacterium spp., especially Fusobacterium nucleatum, has previously been found with increased frequency in those with IBD 45 and has been linked to carcinogenesis.46-48 Patients with IBD are at increased risk of recurrent CDI49 compared to those without IBD. Decreased diversity of the microbiome is reported as associated with recurrent CDI.50 Hence, we speculate that the loss of microbiota diversity with time in patients with IBD after FMT places this patient group at increased risk for recurrent CDI; this differs from prior studies in non‐IBD adults that suggest that FMT breaks the cycle of recurrent CDI.51 Our finding of nonsustained improvement in bacterial diversity after FMT in IBD patients may also provide insight into why a single FMT infusion may be of limited benefit fo r the treatment of IBD52, 53; further investigation into whether serial FMT in IBD gives a more sustained engraftment of donor microbiome is warranted. Although FMT was well tolerated in this series, patient 8 developed mild prolonged diarrhoea that resolved 6 months post‐FMT which was consistent with a diagnosis of irritable bowel syndrome per Rome III criteria.54 In addition, in our personal experience treating adults with CDI using FMT, it is not uncommon for patients to note diarrhoea (or constipation) post‐procedure that is of a different nature than their CDI symptoms. Post‐infectious irritable bowel syndrome (IBS) is well described,55 including after C. difficile infection although this is less common.56 It is difficult to elucidate in this patient whether prolonged diarrhoea was related to the original C. difficile infection, the FMT procedure itself and/or sustained colonic inflammation yielding IBS. Microbiome sequence analysis provided no insight into the reason for this child's clinical course. The main limitation of this study is the relatively small number of patients, although this is the largest study to date in children examining microbiome changes with FMT for recurrent CDI. Larger studies are needed to confirm the difference in bacterial diversity seen here between patients with and without IBD after FMT and to better elucidate the changes in the microbiome between the two groups. A further limitation of this study was that not all patients had a follow‐up stool sample at 6 months, although all patients had samples 10–20 weeks after FMT and were clinically followed up for at least 6 months.

Conclusions Faecal microbiota transplantation gives sustained eradication of C. difficile in children with and without IBD. There is increased bacterial diversity in patients after FMT. However, by 6 months post‐FMT, those with IBD have decreased bacterial diversity compared with donors, whereas those without IBD have diversity similar to donors. This suggests that FMT‐restored faecal diversity is sustained in children without IBD, but not in IBD patients. This may provide insight into why IBD patients are susceptible to recurrent CDI.

Authorship Guarantor of the article: Suchitra Hourigan. Author contributions: Suchitra K Hourigan: conceptualised and designed the study, drafted the initial manuscript, and approved the final manuscript as submitted. Lea Ann Chen: conducted microbiome sequencing and analysis, critically revised the manuscript and approved the final manuscript as submitted. Zoya Grigoryan: conducted microbiome sequencing and analysis and approved the final manuscript as submitted. Greggy Laroche: enrolled many patients included, prepared samples and approved the final manuscript as submitted. Melissa Weidner: enrolled many patients included, prepared samples and approved the final manuscript as submitted. Cynthia Sears: conceptualised and designed the study, critically revised the manuscript and approved the final manuscript as submitted. Maria Oliva‐Hemker: conceptualised and designed the study, critically revised the manuscript and approved the final manuscript as submitted.

Acknowledgements We thank the Inova Health System Seed Grant for supporting this project. Declaration of personal interests: None. Declaration of funding interests: This study was funded in part by the Inova Health System Seed Grant, grant number CC150816.

Supporting Information Filename Description apt13326-sup-0001-FigS1a.pdfapplication/PDF, 254.3 KB Figure S1. Cladograms of differentially abundant taxa as determined by LEfSe analysis in recipients vs. donors prior to FMT, by IBD status. (a) IBD negative vs. donors, before FMT. (b) IBD positive vs. donors, before FMT. apt13326-sup-0002-FigS1b.pdfapplication/PDF, 297.7 KB apt13326-sup-0003-FigS2a.pdfapplication/PDF, 460.4 KB Figure S2. Cladograms of differentially abundant taxa as determined by LEfSe analysis following FMT, by IBD status. (a) IBD positive vs. IBD negative, after FMT. (b) IBD positive vs. donor, after FMT. apt13326-sup-0004-FigS2b.pdfapplication/PDF, 248.7 KB apt13326-sup-0005-FigS3.pdfapplication/PDF, 70.1 KB Figure S3. Histogram of linear discriminant analysis as determined by LEfSe in IBD negative vs. donors, after FMT. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.