Study design

The study protocol, in accordance with Good Clinical Practice Guidelines, was approved by the Human Research Ethics Boards at Queen’s University and the University of Guelph, and meets the provisions of the Helsinki Declaration (1964, amended in 2008) of the World Medical Association.

Inclusion criteria for the study included a history of previous CDI, confirmed by C. difficile fecal toxin immunoassay, new onset of symptoms after completing a full course of medication for CDI, positive C. difficile toxin assay confirming recurrent CDI, and age 18 years or older. Patients were assessed by specialists in Infectious Disease and Gastroenterology, and other possible causes of diarrhea were ruled out. Two patients who fulfilled the inclusion criteria were enrolled in the study and written informed consent was obtained.

Microbiology

Development and preparation of RePOOPulate

The RePOOPulate human probiotic or synthetic stool mixture was developed by extensively culturing the microbial diversity from the stool of a healthy, 41-year-old female donor. Sixty-two different bacterial isolates were recovered on various media types (including Brain Heart Infusion agar, Wilkins–Chalgren agar, Reinforced Clostridial Agar, and deMan, Rogosa & Sharpe agar) using strict anaerobic conditions (to recover both strict and facultative anaerobes). Purified isolates were identified by 16S rRNA gene sequencing and were subjected to antibiotic susceptibility profiling. Susceptibility to antimicrobials was determined either by directly measuring susceptibility or through inference based on other cultivated representatives. For instance, in cases where minimum inhibitory concentration breakpoints are not documented, susceptibility was determined using Kirby–Bauer discs for select antibiotics known to have anaerobic activity; if the bacterial lawn grew up to the edge of the disc, then it was considered resistant and that isolate was not used. For isolates where there was a zone of inhibition of questionable significance, an acceptable level of inhibition was inferred based on other cultivated representatives. If there was any doubt, and the organism was at all suspected to be resistant, then it was not used in the mixture.

Thirty-three isolates, representing commensal species that were generally sensitive to a range of antimicrobials and were relatively straightforward to culture, were selected for the final stool substitute formulation. Full-length 16 S rRNA sequences were aligned with the NAST server[6], and were then classified using the GreenGenes classification server[7]. The most specific name in the GreenGenes classification was used and we report the DNA maximum likelihood score for each classification (Table 1).

Table 1 Composition of stool substitute (RePOOPulate) Full size table

To infer a relative ratio of the selected isolates for inclusion in the formulated RePOOPulate product, a comparison of our list of cultured bacterial species was made with the MetaREP metagenomic database collection of stool sample datasets from healthy donors[8]. Using the taxonomy browser, the dataset that most closely matched our profile of cultured isolates (SRS058723) was selected and used as a guide for inference of relative abundance of each species – with the exception that Bifidobacterium spp. were added to higher abundances, reflecting the widely observed underestimated abundances of Actinobacteria, and specifically this genus, in metagenomic analyses of human stool[9, 10]. An approximate ratio based on cultured cell biomass, measured using standard 10 μl microbiological loops, was generated (Table 1).

Each of the 33 isolates was individually cultured on Fastidious Anaerobe Agar (Lab M Ltd. Heywood, Lancashire, UK) under anaerobic conditions, and then cultures were formulated into the predetermined ratio, as described above, in 100 ml pre-reduced sterile 0.9% normal saline to an estimated concentration of 3.5 × 109 colony-forming units/ml. The bacterial suspension was placed in a reduced atmosphere in a double-sealed container at 4°C, and used within 24 hours of preparation.

Isolation and ribotyping of C. difficile from patient stool samples

C. difficile was isolated from stool samples according to methods described previously[11], using selective media of moxalactam norfloxacin broth (CDMN; Oxoid, Nepean, Ontario, Canada) enriched with 0.1% sodium taurocholate. Isolates were typed using the PCR ribotyping method described by Bidet and colleagues[12].

Administration of stool substitute

Antibiotic therapy was withheld for 2 days and the patients underwent standard colon cleansing the evening prior to colonoscopy. The following morning during colonoscopy, one-half (50 ml) of the solution was deposited in the region of the cecum/proximal ascending colon and the other half was drizzled throughout the transverse colon as the colonoscope was withdrawn. Both patients were noted to have significant diverticular disease. Immediately post procedure, patients were maintained in the Trendelenburg position for 60 minutes before being discharged home. Patients were instructed to eat a fiber-rich diet and not to consume products containing probiotics. Patients were followed by a study nurse to obtain stool samples and closely monitor their clinical response.

Sequence analysis

gDNA extraction from stool samples

gDNA was extracted using a protocol involving a combination of bead beating, the E.Z.N.A.® Stool DNA Kit (Omega Bio-Tek, Norcross, Georgia, USA) and the Maxwell® 16 DNA Purification Kit (Promega, Madison, Wisconsin, USA). Briefly, 200 μl stool sample, 300 μl E.Z.N.A. Kit SLX buffer, 10 μl of 20 mg/ml proteinase K (in 0.1 mM CaCl 2 ) and 200 mg glass beads were added to a screw-capped Eppendorf tube and disrupted in a bead beater for 3 minutes. Following subsequent incubation at 70°C for 10 minutes and at 95°C for 2 minutes, 100 μl E.Z.N.A. Kit Buffer P2 was added to each sample and incubated on ice for 5 minutes. Samples were then centrifuged at 14,000 × g for 5 minutes, and the supernatant transferred into new tubes, each containing 200 μl E.Z.N.A. Kit HTR reagent. Following thorough mixing, samples were incubated at room temperature for 2 minutes and centrifuged at 14,000 × g, and the supernatant was transferred into Maxwell® 16 DNA Purification Kit cartridges. The remainder of the DNA extraction protocol was carried out in the Maxwell® 16 Instrument according to the manufacturer’s instructions (Promega).

V6 rRNA amplification

PCR amplification of the bacterial V6 rRNA region was carried out with the left-side primer CWACGCGARGAACCTTACC and the right-side primer ACRACACGAGCTGACGAC. These primer sequences were chosen because they are exact matches to >95% of the rRNA sequences from organisms identified in the human microbiome project (GBG, unpublished observations). In addition the left-side primers contained the standard Ion Torrent (Ion Torrent Systems Inc., Guilford, Connecticut, USA) adapter and key sequence at their 5′ end (CCATCTCATCCCTGCGTGTCTCCGACTCAG). One of the following 5-mer barcodes was located between the 3′ end of the key sequence and the 5′ end of the primer: TATCG, TAGAC, TGCAT, ATGAG, ACAGT, AGATG, CTCAC, CTGTA, CGTGA, CGACT, AACTC, or CCTAT. Duplicate samples did not use the same barcodes. The right-side primer had the other standard Ion Torrent adapter sequence (CCTCTCTATGGGCAGTCGGTGAT) attached to its 5′ end. Amplification was performed for 25 cycles in 40 μl using the colorless GO-Taq hot start master mix (Promega) according to the manufacturer's instructions with the following three-step temperature profile: 95°C, 55°C and 72°C for 1 minute each step, then 5 μl of the resulting amplification were quantified using the QuBit broad-range double-stranded DNA fluorometric quantitation reagent (InVitroGen, Life technologies Inc., Burlington, Ontario, Canada). Samples were pooled at approximately equal concentrations and purified using a Wizard PCR Clean-Up Kit (Promega).

Sequencing

Sequence reactions were carried out on the Ion Torrent 314 and 316 chip platform. Up to 12 samples were multiplexed on each chip through use of individual sequence tags. Data from all runs were pooled when samples were run on more than one chip.

Sequence data processing

Five sequencing reactions were carried out on the Ion Torrent platform: three reactions on a 314 chip and two reactions on the 316 chip. The chips differ only in the density of the spots, and hence in the amount of sequence that can be obtained (the 316 chip is about five to six times as dense as the 314 chip). The sequence was provided in fastq format. All sequences were then filtered according to the following criteria: exact matches to the barcodes used, exact match to the left-side primer including redundant positions in the primer, an exact match to the first six nucleotides of the right-side primer, and a length between the left-side and right-side primer of between 71 and 83 nucleotides. This length was chosen because it encompasses the predicted amplicon product size from all human-associated bacterial organisms that have been cultured and sequenced as part of the human microbiome project.

Approximately 40 to 50% of the reads passed these filters in the most recent Ion Torrent runs; reads not passing the filters were not examined further. Reads were processed as described by Gloor and colleagues[13] except that clustering with USEARCH was performed at 97% identity. Chimera detection was performed with UCHIME (version v5.2.32) using the de novo method[14]. Only four chimeric sequences were observed out of 30,419 unique sequences in the merged dataset, and all were rare. This frequency is similar to that reported previously for amplification and sequencing of the V6 rRNA region using the Illumina platform[13]. Chimeric sequences were not considered an issue in this dataset.

A table of counts for sequences grouped at the 97% operational taxonomic unit (OTU) and 100% identical sequence unit identity level were generated for each sample as before[13], keeping all identical sequence unit or OTU sequences that were represented in any sample at a frequency >0.5%. Reads that were never abundant in any sample (<0.5%) were grouped into the remainder and discarded. Between 12.6 and 51.9% (median 31%) of the identical sequence unit reads and between 1.4 and 17.2% (median 5.8%) of the OTU reads were in the remainder group. These values are approximately five times greater than those observed for identical sequence units sequenced on the Illumina platform but are about equivalent to the Illumina platform observations when reads were clustered.

Taxonomic classification

Classification of the sequences by either the GreenGenes or RDP classifiers proved to be unreliable because of the short length of the V6 region. Classification of the sequences present in the count table was therefore performed using the RDP closest match option on the full-length, high-quality, isolated subset. The maximum number of best hits was identified, and the taxonomic classification of the best match and ties was collected. The classification of those hits was adopted for all levels where the classification was identical across all best matches, otherwise the classification was marked as undefined. The V6 region is not able to resolve the genus or species level of a number of clades, so all analyses were carried out at the family level. This strategy worked for all abundant families – with the exception of the Bifidobacterium, which were annotated as such from BLAST searches of the NCBI microbial 16 S rRNA database. The taxonomic classification was added to the sequence count table and the data were presented in formats that could be accepted by QIIME 1.5.0[15] as follows. Sequence alignments were built using Muscle[16] and a neighbor-joining tree was generated by ClustalW2[17]. Beta-diversity was calculated by the UniFrac algorithm[18]. Tables were imported into MacQIIME, which is an OS X bundled version of QIIME 1.5.0, and were analyzed using the default parameters.