Study subjects and treatment outcome

The current study was a substudy from a randomized controlled trial (RCT) of FMT versus vancomycin (STD) for patients with CDI. Consecutive CDI subjects enrolled into this RCT were invited to participate in a substudy of assessment of fecal microbiota. Patients were included if they had three or more loose or watery stools per day for at least two consecutive days or eight or more soft or loose stools in 48 h and a positive-stool test for C. difficile based on a two-step testing algorithm in our hospital, a positive-glutamate dehydrogenase screening test followed by a positive-polymerase chain reaction (PCR) test of C. difficile. A total of 31 subjects with CDI and 24 healthy household controls were recruited and stool samples at baseline were obtained for analyses of fungal and bacterial microbiomes. Among them, 24 CDI subjects consented to have stool samples collected serially after treatment for microbiome analysis. Totally, 16 CDI subjects were treated with FMT and 8 were treated with vancomycin, and they were followed up at baseline and at weeks 2, 4, 10, and 16 after treatment (Supplementary Figure 2). Subjects in FMT group received 5 days of vancomycin (125 mg, four times daily) followed by donor infused stool via nasojejunal route and those who had STD received oral vancomycin 500 mg orally four times per day for 10 days. A computer-generated randomization schedule was used to assign patients to the treatment sequences. All patients kept a stool diary and were questioned about stool frequency and consistency and medication use. The study was approved by The Joint Chinese University of Hong Kong, New Territories East Cluster Clinical Research Ethics Committee (The Joint CUHK-NTEC CREC, CREC Ref. No.: 2014.183-T; Clinical Trial registry, NCT02570477). We collected stool samples from new consecutive CDI patients. From this cohort, we included 12 CDI patients with antibiotics exposure, 12 CDI patients with no antibiotics exposure at inclusion, and an additional 17 healthy controls to determine effect of antibiotics on C. albicans. In addition, five new CDI subjects were treated with FMT (using two new donors with undetectable fecal C. albicans as determined by quantitative PCR) and consented to have stool samples collected serially after treatment for C. albicans quantitative analysis to confirm the findings.

Treatment response was defined as an absence of diarrhea or persistent diarrhea that could be explained by other causes with a negative stool test for C. difficile toxin, while relapse was defined as diarrhea with a positive-stool test for C. difficile toxin. Treatment cure is defined as symptom resolution and a negative C. difficile toxin in stool until the last follow-up (last follow-up is referred to as the last stool collection time-point, as shown in Supplementary Figure 2). Out of 16, 9 subjects who had FMT (FMT1–FMT9), and 5 of the 8 patients (STD1–STD5), who had vancomycin were cured of CDI (termed responders, Supplementary Data 3) at a median follow-up of 16 weeks. CDI recipients FMT11 and FMT12 shared the same donor, and this donor was termed “Donor11”. Clinical data of the subjects and collected stool samples are shown in Supplementary Datas 3 and 4. None of the patients had received antibiotics or proton pump inhibitors after FMT.

Study design

Patient inclusion criteria

1. C. difficile infection was defined as diarrhea (≥3 soft, loose or watery stools per day for at least 2 consecutive days or ≥8 soft or loose stools in 48 h). 2. A positive-stool test for C. difficile toxin (on a two-step testing algorithm, a positive-GDH screening test followed by a positive-PCR test of C. difficile). 3. Age ≥ 18. 4. Written informed consent obtained.

Patient exclusion criteria

1. The presence of human immunodeficiency virus (HIV) infection with a CD4 count of less than 240. 2. Pregnancy. 3. Gastrointestinal bleeding. 4. Acute coronary syndrome.

Donor screening

Donors included individuals who are spouses or partners, first-degree relatives, other relatives, friends, and individuals unknown to the patient. They were screened with a questionnaire and excluded if they had taken antibiotics within the preceding 3 months; were on major immunosuppressive agents, including chemotherapeutic agents; had known or recent exposure to HIV, hepatitis B or C; had a current communicable disease; participated in high-risk sexual behaviors; used illicit drugs; traveled within 6 months to areas with endemic diarrheal illnesses; or had history of IBD, irritable bowel syndrome or chronic diarrhea, gastrointestinal malignancy or polyposis. In addition, donor was screened for HBsurface Ag, Anti-HBc, Anti-HCV, Anti-HIV, Syphilis EIA, stool microscopy, culture and sensitivity, stool cyst, ova, parasite, norovirus, and C. difficile (cytotoxin and PCR assay). All subjects and collected stool samples are listed in Supplementary Data 4.

The donors for the FMT group were healthy household controls and the donor stool samples analyzed were the same samples used for FMT. All subjects provided written informed consent.

Family members provided donor stool for subjects randomized to FMT arm. Cure after FMT or vancomycin therapy was defined as symptom resolution and negative C. difficile toxin in stool at last follow-up by PCR assay. Relapse was defined as diarrhea with a positive-stool test for C. difficile toxin.

This was a randomized, but not blinded study. However, for mycobiome and bacterial microbiome analyses on stool samples, assessments were initially performed by analysts who were blinded to the clinical outcome of the studied subjects. When the profiled mycobiome and bacterial microbiome data were available for each individual subject, correlation was then made to associate microbiome profiles with treatment outcomes of subjects.

Infusion of donor stool

In subjects who received FMT, a nasoduodenal tube was inserted with radiology guidance. Donor feces was diluted with 500 ml of sterile saline (0.9%), blended and the supernatant was strained with filter paper and poured in a sterile bottle. Within 6 h after collection of feces by the donor, the solution was infused through a nasoduodenal tube (2–3 min per 50 ml). The tube was removed 30 min after the infusion, and patients were monitored for 2 h. In subjects who received FMT, a minimum of 50 g of donor stool was collected on the same day of infusion and used within 6 h of collection.

Fecal DNA extraction

Fecal DNA was isolated as described below. A 100 mg fecal sample was prewashed with 1 ml ddH 2 O and pelleted by centrifugation at 10,000×g for 1 min. The fecal pellet was resuspended in 800 μl TE buffer (pH 7.5), supplemented with 1.6 μl 2-mercaptoethanol and 500 U lyticase (Sigma), and incubated at 37 °C for 60 min. The sample was then centrifuged at 10,000×g for 2 min and fecal DNA was subsequently extracted from the pellet using ZR Fecal DNA miniPrep kit (Zymo Research, Orange, CA) according to the protocal. Briefly, fecal pellet was added to the BashingBeadLysis Tube with 750 μl Lysis solution, and then processed at maximum speed for 10 min. The lysates were centrifuged at ≥10,000×g for 1 min. The supernatant was transferred to a Zymo-SpinTM IV Spin Filter in a collection tube and centrifuged at 7000×g for 1 min. About 1200 μl of fecal DNA binding buffer was added to the filtrate in the collection tube, followed by concentration and purification in a new filter tube. Finally, a total of 50 μl eluted DNA with a concentration at 20–100 ng/μl was prepared for each sample.

Fungal ITS2 sequencing and quality control

The final fecal DNA for fungal sequencing was amplified based upon internal transcribed spacer 2 (ITS2) region using primers as below and PrimeSTAR HS DNA Polymerase kit (TaKaRa, Japan). The primer pairs are ITS2-F: 5′-GCATCGATGAAGAACGCAGC-3′, ITS2-R: 5′-TCCTCCGCTTATTGATATGC-3′. ITS2 amplicons were generated with 38 cycles of 3-step PCR: 98 °C 10 s, 59 °C 10 s, and 72 °C 30 s. PCR samples were then sequenced on the Illumina MiSeq PE300 platform (2 × 300 bp, BGI, China), 151,524 ± 97,694 (mean ± SD) clean sequences obtained on average (sequence statistics in Supplementary Data 5).

Raw reads were filtered by SOAPnuke (v 1.5.3) (http://soap.genomics.org.cn/) developed by BGI as follows: (i) adapters removed, (ii) read removed if N base is more than 3% of the read, (iii) read removed if bases with quality low than 20 were more than 40% of read, and (iv) all duplicates removed. Quality control and data analysis were further implemented in PIPITS (v 1.4.5)35. Briefly, PIPITS_PREP prepares raw reads from Illumina MiSeq sequencers for ITS extraction; PIPITS_FUNITS extracts ITS2 from the reads; and PIPITS_PROCESS analyses the reads to produce OTU abundance tables and the Ribosomal Database Project (RDP) taxonomic assignment table for downstream analysis. The quality trimmed and ITS2 extracted reads were aligned to fungi UNITE database exploiting RDP classifier 2.10 for taxonomic assignment to produce OTU abundance table (based on sequence identity ≥ 97% identity) and phylotype abundance tables at different taxonomic levels, for downstream analysis.

The fungal OTU and phylotype abundance data were imported into R 3.2.3. Richness, diversity, and evenness calculation were performed using the estimated richness function of the phyloseq package. Spearman correlation and their significance were calculated using the cor and cor.test functions in R, respectively. For the fungal–bacterial taxa comparisons, Spearman correlations were calculated for the relative abundance of the differentially presented fungal taxa and the bacterial taxa determined to be significantly associated with disease by Lefse analysis. Correlation plots were generated using the corrplot package in R. Heatmaps were generated using the pheatmap package in R.

Quantitative PCR for detection of total fungal load in human fecal DNA samples

Total fungal loads in human stools were quantified by TaqMan qPCR analysis (Premix Ex TaqTM, TaKaRa) of extracted human fecal DNA using primers36: Fungi-quant-F 5′-GGRAAACTCACCAGGTCCAG-3′; Fungi-quant-R 5′-GSWCTATCCCCAKCACGA-3′, and probe: 5′-TGGTGCATGGCCGTT-3′.

Quantitative PCR for detection of C. albicans in human fecal DNA samples

C. albicans loads in human stools were quantified by qPCR analysis (SsoAdvanced SYBR Green Supermix, Bio-Rad) of extracted human fecal DNA using C. albicans specific primers: C.albicans-F 5′-CCTGTTTGAGCGTCGTTTCTC-3′; C. albicans-R 5′-TTTGGTTAGACCTAAGCCATTGTCA-3′. C. albicans absolute abundance was determined using standard curve constructed with reference genomic DNA (gDNA) of C. albicans.

LefSe linear discriminant analysis and multivariate analysis

To compare differences in the configurations of fungal and bacterial microbiomes between CDI patients and healthy controls, between FMT responders and nonresponders, between FMT treatment and vancomycin (STD) treatment, LefSe analyses were performed on the Huttenhower lab Galaxy server (http://huttenhower.sph.harvard.edu/galaxy/) by importing the fungal and bacterial relative abundance values and associated sample metadata, with FDR-adjusted q value < 0.05 considered significant and effect size calculated. MaAsLin (Multivariate Analysis by Linear Models) was implemented to identify associations between clinical metadata (age, gender, household, CDI severity, and underlying IBD) and fungal community abundance matrix on the Huttenhower lab Galaxy server (http://huttenhower.sph.harvard.edu/galaxy/).

Nonmetric multidimensional scaling analysis

The difference in fecal fungal community structures between controls and CDI was performed via NMDS plot based upon Bray–Curtis dissimilarities among all subjects. The two ellipses correspond to the fecal mycobiota community dispersion range within each group, controls and CDI, respectively, drawn using the function veganCovEllipse in the Package Vegan in R basing upon the Least-Squares criterion for estimation of the best fit to an ellipse from a given set of mycobiota communities.

Bacterial 16S rRNA sequencing and data analysis

The final fecal DNA samples were subject to bacterial 16S rRNA V4 region amplification and sequenced on the Illumina MiSeq PE250 platform (2 × 250 bp, BGI, China), 132,081 ± 65,429 (number ± SD) sequences obtained on average (sequence statistics in Supplementary Data 6). Quality control and data analysis were implemented in mothur (v 1.38.0) as previously described37. Any sequences with ambiguous bases and anything longer than 275 bp were removed, and aligned against the nonredundant Greengenes database (v 13.8)38 using the NAST algorithm. Any sequences that failed to align with the V3–4 region were discarded. The remaining sequences were trimmed to the same alignment coordinates over which they fully overlapped, followed by removal of homopolymers and detection for the presence of chimeras by UChime.

The resulting sequences were classified against the Greengenes database and annotated with deepest level taxa represented by pseudo-bootstrap confidence scores of at least 80% averaged over 1000 iterations of the naive Bayesian classifier. Any sequences that were classified as either being originated from archaea, eukarya, chloroplasts, mitochondria, or unknown kingdoms, were removed. The annotated sequences were assigned to phylotypes according to their consensus taxonomy with which at least 80% of the sequences agreed. Closed reference OTUs sharing 97% identity were clustered as well and assigned taxonomy according to the Greengenes database. LefSe analysis was performed to define bacterial taxa associated with CDI and healthy controls. The relative abundance of these abundance-differential taxa identified by LefSe in pre-FMT baseline samples and post-FMT last follow-up samples were plotted using pheatmap R package.

Calculation of donor transferred OTUs in recipients

In samples after FMT, if a fungal or bacterial OTU was not present in the recipient baseline sample but present both in the corresponding donor baseline sample and in the recipient post-FMT sample, the OTU was defined as “donor derived”; if an OTU was not present in the corresponding donor baseline sample, but detected both in the recipient baseline sample and in the recipient post-FMT sample, the OTU was defined as “recipient exclusive”; if an OTU was present across the recipient baseline sample, the recipient post-FMT sample and the corresponding donor baseline sample, the OTU was defined as “donor–recipient co-existed”.

Mouse husbandry and model of C. difficile infection

Studies were conducted on 4- to 6-week-old female C57BL/6 that were reared in groups of nine. Individual mice were randomized after arrival. Mice were subjected to a previously described model of CDI39. Briefly, mice were given an antibiotic cocktail of kanamycin (0.4 mg/mL), gentamicin (0.035 mg/mL), colistin (850 U/mL), metronidazole (0.215 mg/mL), and vancomycin (0.045 mg/mL) (all antibiotics were purchased from Sigma–Aldrich, St. Louis, MO) in their drinking water for 3 days. Mice were then given 2 days of recovery before administration of 107 spores of C. difficile (strain HK31423, Ribotype 002, a clinical isolate during an outbreak at Prince of Wales Hospital in Hong Kong) in phosphate-buffered saline (PBS) via oral gavage. Animal grouping and research scheme were designed as shown in Fig. 4a. On day 1 poststool infusion, diarrhea was evaluated by stool water content, calculated as stool weight loss after air drying at 70 °C for 4 h. Cecums were harvested, fixed in 4% formalin solution and embedded in paraffin on day 1 after FMT. Sections were stained with hemotoxylin and eosin for histological assessment40. In experiment for investigating the effect of vancomycin administration on the presence of Candida in mice, seven 4- to 6-week old female C57BL/6 mice were given vancomycin (0.1 mg/mL) in their drinking water for 3 days. Stool samples before and after vancomycin regime were collected and subsequently determined for Candida levels by qPCR analysis (SsoAdvanced SYBR Green Supermix, Bio-Rad) using primers41: pan-Candida-F 5′-GCAAGTCATCAGCTTGCGTT-3′; pan-Candida-R 5′-TGCGTTCTTCATCGATGCGA-3′. All animal experiments were approved and performed in compliance with the Animal Experimentation Ethics Committee (AEEC) of The Chinese University of Hong Kong.

C. albicans administration and donor stool infusion in mice

C. albicans (10231, from ATCC, USA) was administered to mice (2 × 108 cfu per mouse) via gavage after 3-day antibiotic treatment or supplemented in donor stool slurry at the time of donor stool infusion. Human stool from a healthy volunteer (Chinese, male, age 28 years), without presence of C. albicans as measured by qPCR, was obtained with informed consent. For stool microbiota infusions, approximately 2 g of stool samples were cut in an anaerobic chamber and suspended in 10 ml of PBS. Mice were colonized by oral gavage of 150 μl of fecal slurry with or without supplementation of C. albicans on day 2 after C. difficile challenge. For antifungal experiment, animal grouping and research scheme were designed as shown in Supplementary Figure 12a. Mice was initially colonized with C. albicans (2 × 108 cfu per mouse) after 3 days of antibiotic cocktail treatment in the drinking water, followed by 4 days of fluconazole treatment supplemented in the drinking water (0.5 mg/mL, Sigma). Then the mice were subjected to C. difficile administration (107 spores per mice) through gavage after a consecutive 1.5-day antibiotic cocktail- and 1.5-day free water-drinking. Human stool infusion was performed 2 days later after C. difficile gavage. Both C. difficile load and C. albicans load were enumerated by cultivation on day 0 before FMT and day 1 after FMT.

Quantification of C. difficile and C. albicans burdens in mouse feces

Mouse stool were collected both before and after stool infusion. Fecal C. difficile and C. albicans burdens on day 0 before and day 1 after stool infusion were measured by cultivation. Samples were diluted in PBS and, respectively, plated on taurocholate cycloserine cefoxitin fructose agar for quantification of C. difficile burden, on Sabouraud dextrose agar (SDA) for quantification of C. albicans load. Stool samples prior to C. albicans colonization from antibiotic-treated mice were plated on SDA to ensure that mice were C. albicans culture negative.

Administration of P. brocae, and A. penicillioides, and donor stool infusion in mice

A 4- to 6-week-old female C57BL/6 that were reared in groups of eight. Animal grouping and research scheme were designed as shown in Supplementary Figure 13a. P. brocae (CBS116042, from Westerdijk Fungal Biodiversity Institute, Netherlands) and A. penicillioides (CBS112373, from Westerdijk Fungal Biodiversity Institute, Netherlands) were administered to mice, respectively (approximately 0.5 g/kg), via gavage after 3-day antibiotic treatment or supplemented in donor stool slurry at the time of donor stool infusion. Colonization of P. brocae or A. penicillioides in mice was confirmed by PCR with detectable genomic DNA of respective species in fecal DNA after oral administration of each fungus. For stool microbiota infusions, approximately 2 g of stool samples were cut in an anaerobic chamber and suspended in 10 ml of PBS. Mice were colonized by oral gavage of 150 μl of fecal slurry with or without supplementation of these fungi on day 2 after C. difficile challenge.