Researchers Examine Changes to the Microbiota Composition and Metabolic Profiles of Patients with Recurrent Clostridium difficile Infection (rCDI) Following Treatment with Faecal Microbiota Transplant (FMT)


This study aimed to examine changes to the microbiota composition and metabolic profiles of seven patients with recurrent Clostridium difficile infection (rCDI), following treatment with faecal microbiota transplant (FMT).



This study aimed to examine changes to the microbiota composition and metabolic profiles of seven patients with recurrent Clostridium difficile infection (rCDI), following treatment with faecal microbiota transplant (FMT).


16S rDNA sequencing and 1H NMR were performed on faecal samples from the patients (pre-, post-FMT, and follow-up) and the associated donor samples. Sparse partial-least-square analysis was used to identify correlations between the two datasets.


The patients’ microbiota post-FMT tended to shift towards the donor microbiota, specifically through proportional increases of Bacteroides, Blautia, and Ruminococcus, and proportional decreases of Enterococcus, Escherichia, and Klebsiella. However, although cured of infection, one patient, who suffers from chronic alcohol abuse, retained the compositional characteristics of the pre-FMT microbiota. Following FMT, increased levels of short-chain fatty acids, particularly butyrate and acetate, were observed in all patients. Sparse partial-least-square analysis confirmed a positive correlation between butyrate and Bacteroides, Blautia, and Ruminococcus, with a negative correlation between butyrate and Klebsiella and Enterococcus.


Clear differences were observed in the microbiota composition and metabolic profiles between donors and rCDI patients, which were largely resolved in patients following FMT. Increased levels of butyrate appear to be a factor associated with resolution of rCDI.


Although Clostridium difficile is present in the intestines of ∼3–5% of healthy adults,1 the occurrence of C. difficile infection (CDI) in healthy individuals is relatively uncommon due to the protective effect of the gut microbiota. The incidents and severity of CDI has risen significantly over the last decade, and it is now recognised as the main causative agent of healthcare-associated infectious diarrhoea in hospitals worldwide.2 The standard treatment for CDI is the administration of metronidazole for mild to moderate infections, and oral vancomycin or fidaxomicin for severe infections and relapses. The ability of C. difficile to form spores, coupled with the increase in antibiotic-resistant strains, can lead to persistence of infection, relapses, and the administration of more antibiotics, which further depletes the commensal bacteria. This creates an environment that is more favourable to C. difficile, thus setting up a cycle of relapse and re-infection. It is estimated that 20-30% of patients who develop a first episode of CDI go on to have at least one relapse, and of these, a further 60% develop further episodes of relapses.3 This increases the need for further antibiotics, the risk of antibiotic-resistance in the gut commensal flora, and costs to the health service, with each episode of CDI estimated to cost approximately £7000 in 2010.4

Faecal microbiota transplants (FMT) represents an effective alternative to antibiotics to treat recurrent CDI (rCDI), with a primary cure rate as high as 91%.5 The central tenet behind FMT is that the introduction of a healthy bacterial community into the intestines produces an environment that is less favourable to C. difficile by increasing colonisation resistance and reinstating a protective effect. The advantages of this treatment are that it is quick, cost-effective, and could help to eradicate antibiotic resistant strains of C. difficile.

It is known that a dysbiotic gut microbiota increases the risk of developing CDI, however whether there is a common element within this community composition that could help to determine if a patient is at greater risk of rCDI is as yet unknown. The reduction in diversity within the dysbiotic gut microbiota would also suggest a reduction in metabolic potential through the loss of gene diversity. The functional redundancy6 within the gut microbiota suggests, that metabolic function is more relevant than which species are present or absent. Whilst a number of studies have looked at the changes in microbiota composition due to FMT,7, 8, 9, 10 we know little about the changes to the metabolic capacities of the altered microbiota. The aim of this study was to assess FMT-induced changes in both the microbial community structure and metabolite profiles of the gut microbiomes of seven patients with rCDI, as well as those of their associated FMT donors.

Patients and methods


Patients were selected as candidates for the FMT procedure if they had at least two confirmed recurrences of CDI. C. difficile testing was based on a two stage algorithm in line with Public Health England recommendations.11 This involves screening faecal samples by glutamate dehydrogenase enzyme immunoassay (Techlab, USA), followed by C. difficile toxin testing by enzyme immunoassay (Techlab, USA). Glutamate dehydrogenase positive, toxin negative samples were further tested for the presence of toxigenic genes by PCR. FMT exclusion criteria included immunocompromised patients, those aged less than 16, and those with severe comorbidities which would make the patient unfit for endoscopy. FMT was introduced into clinical care at Norfolk and Norwich University Hospital following approval by the New Therapies committee, and was performed in accordance with the Helsinki Declaration of 1975. Patients were consented for the study by a clinician following a detailed discussion of the procedure with the patient or their next of kin. All patient data is fully anonymised.

Donor screening

The faecal donors used for the cohort of patients who underwent FMT in this study were both healthy Caucasian males with a BMI between 24 and 27 kg/m2, aged 36 (D05) and 30 (D03) years of age, respectively. Potential donors were asked to complete a questionnaire adapted from van Nood et al.12 regarding their medical history and lifestyle habits to identify risk factors for potentially transmittable diseases. Eligible candidates provided blood and stool samples for laboratory screening tests. Blood samples were screened for hepatitis A, B, C, and E antibodies, HIV 1 & 2, human T-lymphotropic virus 1 & 2, Epstein-Barr virus, Cytomegalovirus, syphilis, Entamoeba histolytica, Strongyloides stercoralis, and Treponema pallidum. Stool samples were tested for the presence of C. difficile or its toxins, Helicobacter pylori antigen, Norovirus, methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, extended-spectrum β-lactamase-producing organisms, carbapenemase-producing Enterobacteriaceae, Escherichia coli O157, Salmonella spp., Shigella spp., and Campylobacter species. In addition, microscopy was used to investigate for ova, cysts, and parasites. Prior to the donation of stool samples for each FMT procedure, donors were asked to refrain from eating peanuts and shellfish, and to complete a short screening questionnaire to confirm no changes to health or lifestyle since the last donor screening that may put the patient at risk.

Faecal suspension preparation

Donor faeces were collected in a sterile container on the day of the procedure, and transferred to a sterilised class II safety cabinet (Walker Ltd, UK). A maximum of 80 g of donor stool was homogenised with sterile saline (0.9%), to a ratio of 5 ml saline per gram of stool, in a strainer bag (BA6141/STR; Seward Limited, UK) using a Stomacher® 400 Circulator (Seward Limited, UK) set to 230 RPM for a duration of 1 min. The filtered faecal preparation was drawn up into labelled sterile 60 ml syringes using nasojejunal tubing connected to the Luer lock. The syringes were secured with sterile Luer lock caps and transported immediately to the hospital. Aliquots of the donor faecal sample were immediately stored at −20 °C until analysis.

Faecal suspension infusion

Patients were prescribed oral vancomycin 500 mg four times daily for 4 days, with the last dose received the night before the procedure. Also, on the day before the FMT procedure, a bowel lavage is performed using 4 l of macrogol solution (Klean-Prep, Norgine). Patients were taken to the endoscopy unit for insertion of nasojejunal tube the night before the procedure. Our FMT protocol was adapted from that of van Nood et al.12 On the day of FMT infusion, the patient’s headrest was elevated to 45°, patency of the nasojejunal tube was checked by flushing with water, and 420 ml of faecal suspension was delivered slowly by the patient’s bedside in the isolation room via a nasojejunal tube using the prefilled syringes. This was performed at a rate of ∼20 ml per minute with a break of 5-10 minutes applied halfway through the procedure. Post-infusion instructions were to monitor observations, and record bowel motions. Patients could take on fluids one hour after the procedure, and were observed overnight before discharge the next day at the earliest. Although there are no agreed durations of follow-up post-FMT,13 van Nood et al.12 used two endpoints to measure cure, namely no relapse after 5 weeks, and no relapse after 10 weeks. Resolution was defined as type 4 or less on the Bristol stool chart or stool normal for the patient e.g. in case of percutaneous endoscopic gastrostomy feeding. We followed patients up by telephone or in person if they were re-admitted into the hospital for an unrelated illness. Post-FMT samples were collected after a minimum of 10 days post-FMT, and postal kits were provided to patients who were willing to donate a ‘follow-up’ sample up to 2 weeks later.

Sample analysis

Faecal microbiota analysis

Faecal samples were collected from recipients within 9 days prior to FMT, however the pre-FMT sample for patient R13 was not collected within this timeframe, and a previously frozen sample obtained whilst the patient was suffering from the same episode of CDI was used. Further samples were collected for all recipients following the procedure (‘post-FMT’ range: 11–141 days; ‘follow-up’ range: 4–14 days after post-FMT sample), and stored at −20 °C until analysis. The DNA was extracted using the FastDNA SPIN Kit for Soil (MP Biomedicals, UK) with a bead-beating step.14 DNA yield was quantified using the Qubit fluorometer prior to the samples being sent to the Earlham Institute (UK), where the V4 hypervariable region of the 16S rRNA genes were amplified using the 515F and 806R primers with built-in degeneracy.15 The amplicons were sequenced using paired-end Illumina sequencing (2 × 250 bp) on the MiSeq platform (Illumina, USA). Sequencing data, for the 21 samples that had an appropriate level of sequencing depth, were analysed using the Quantitative Insights Into Microbial Ecology (QIIME) 1.9 software and RDP classifier 16S rRNA gene sequence database.16,17 The trimmed reads were filtered for chimeric sequences using ChimeraSlayer, bacterial taxonomy assignment with a confidence value threshold of 50% was performed with the RDP classifier (version 2.10), and trimmed reads clustered into operational taxonomic units at 97% identity level. Alpha diversity and rarefaction plots were computed using the Chao1 index. Weighted and unweighted UniFrac distances were used to generate beta diversity principal coordinates analysis plots, which were visualised using the Emperor tool.

Faecal metabolite analysis

A known mass (∼ 100 mg) of thawed faecal samples were added to sterile tubes. The faecal waters were generated by adding the phosphate buffer (prepared in D2O) to 8.3% w/v. Homogenised faecal waters were centrifuged at 16,200 x g at room temperature for 5 min. The supernatants were filter sterilised (0.2 µm) and placed in a 5 mm NMR tube. The 1H NMR spectra were recorded at 600 MHz on a Bruker Avance spectrometer (Bruker BioSpin GmbH, Germany) running Topspin 2.0 software and fitted with a cryoprobe and a 60-slot autosampler. Each 1H NMR spectrum was acquired with 1280 scans, a spectral width of 12,300 Hz, and an acquisition time of 2.67 s. The “noesypr1d” pre-saturation sequence was used to suppress the residual water signal with a low-power selective irradiation at the water frequency during the recycle delay and a mixing time of 10 ms. Spectra were transformed with a 0.3 Hz line broadening, and were manually phased, baseline corrected, and referenced by setting the TSP methyl signal to 0 ppm. The metabolites were quantified using the software Chenomx® NMR Suite 7.0TM.

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