Clinical observations

Clinical observations of the volunteers were performed during the first month post-FMT. The AEs were registered for all the subjects 8 to 10 hours after taking the capsules (Additional file 6: Table S2). There was no emerging AE past the first 24 hours. V1 and V3 exhibited only grade 1 gastrointestinal AEs post-FMT. The second volunteer (V2) developed a SIRS. Dynamic monitoring of clinical blood cell counts, biochemical blood analysis, and evaluation of lymphocyte subpopulations are presented in Fig. 2. On day 2 of treatment, all laboratory tests were in the normal range, except for increased blood neutrophil counts from 59.1% (5.1 ×109/l) to 70.6% (8.9 ×109/l), and from 61.4% (6.3 ×109/l) to 70.7% (6.9 ×109/l), for V1 and V2, respectively. Blood lymphocyte counts showed a decrease from 31.7% to 23.6%, at similar absolute lymphocyte numbers (2.8 ×109/l and 3.0 ×109/l). V3 exhibited a relative decrease of lymphocyte counts, both in percentage (30.1% to 17.8%) and in absolute values (2.8 ×109/l to 1.7 ×109/l). A limited number of immune parameters included counts of total leukocytes and their main subpopulations. Gross changes in total leukocyte and neutrophil counts were observed in V1 and V2. Meanwhile, the CD3+/CD4+ and CD4+/CD8+ ratios seemed to increase by day +8 after FMT, with a rapid reversal to normal values. These changes do not mean any prolonged immune depression, as compared, e.g., to leukopenia observed following cytostatic therapy. Rather, they resemble a systemic acute response to antigenic stimulation.

Fig. 2 Dynamics of neutrophil counts, lymphocyte counts (a) and lymphocyte sub-populations after FMT (b). The second volunteer (V2) developed SIRS Full size image

For V2, we observed a number of pronounced symptoms, which required additional therapy. Ciprofloxacin was administered at a daily dose of 500 mg for 3 days, and the 2nd round of FMT in this subject was canceled. V2 developed a clinical pattern of the systemic inflammatory response (fever, with the one-time rise of body temperature to 39.1 ∘C, with shivers and tachycardia of 102 per minute on the day after administration). The blood changes corresponded to acute bacterial infection: leukocytosis to 16.7 ×109/l, neutrophils 90.6% (15.1 ×109/l), absolute lymphopenia (0.9%, 0.2 ×109/l). Blood smear counts showed an increase in band forms, 10% (1.67 ×109/l), segmented forms, 80% (13.36 ×109/l); toxic granulation in neutrophils and decrease of lymphocytes, 4% (0.66 ×109/l). C-reactive protein levels were within normal ranges, a marginal increase of γ-glutamyl transpeptidase to 56.7 U/l (normal range: 0-55 U/l) and ALT to 62 U/l (normal range: 0-50 U/l) was noted on day +2. Clinical chemistry parameters of V1 and V3 were within normal ranges during the treatment course.

The lymphocyte subpopulations were examined before FMT, as well as on day +9 and day +30. By day +9, an increased percentage and absolute numbers were observed for T-helpers CD3+CD4+, CD19+CD23+ cells; CD4/CD8 ratio; as well as a decrease in lymphocyte subpopulations, i.e., T-cytotoxic CD3+CD8+ lymphocytes, and NK cells (CD3-CD16+56+). By day 30, a reverse dynamics to normal values was revealed. The number of recipients was insufficient to evaluate the statistical significance of the observed changes. However, we could be assumed an association between adverse effects and immune system perturbation.

Gut microbiome changes after FMT

16S rRNA gene sequencing (16S seq) data analysis was performed in two independent laboratories and the results were consistent in both assays. Summary sequencing statistics are presented in Additional file 6: Table S3. The NMDS bi-dimensional plot obtained with using Aitchison distance and 16S seq taxonomic data is presented in Fig. 3. It shows the convergence of the recipients’ gut taxonomic profiles to the donor profile within 300 days after the FMT. Interestingly, the gut metagenomic profile of V1 showed a dramatic change after the second FMT round procedure from the same donor (2 days after the second FMT round). However, further samples showed a return to the donor pattern. Additionally, analysis using NMDS and unweighted UniFrac distance confirmed previous results (see Additional file 1: Figure S1A).

Fig. 3 Movement of recipient samples to the donor during the observation time based on 16S rRNA gene sequencing taxonomic composition. Bi-dimensional plot obtained by Aitchison distance with the aid of DEICODE. Donor samples: X. Volunteer’s samples: red / blue / green colors (see figure legend). The lines denote the evolution of the volunteer’s samples in time (different time points). The days after FMT procedure (or baseline for donor samples) denoted by color numbers Full size image

Shotgun metagenomic sequencing was another method for studying changes in the intestinal microbiota profile of the recipients, which yielded 23.1 ± 3.7 M of 250 bp reads per sample (98.3 Gbp in total) after quality control. Seventeen metagenomic samples were sequenced with the shotgun method (6 for the V1, 4 for the V2 and V3 and 3 samples for the donor). The sequencing summary statistic is presented in Additional file 6: Table S3. A total of 74 genera were detected in all samples. The dataset of relative abundances of bacterial genera is shown in Additional file 6: Table S4. The shotgun sequencing confirmed the 16S seq data with a similar pattern of changes towards the donor profile (Fig. 4a). Similar results were obtained by NMDS bi-dimensional visualization using Bray-Curtis dissimilarity (see Additional file 1: Figure S1B).

Fig. 4 Shifts of the taxonomic profile of microbiota in volunteers towards donor values over the observation time. The figure is based on shotgun sequencing data. a on-metric multidimensional scaling bi-dimensional plot of MetaPhlAn2 taxonomic profile (genera level relative abundances), based on the Aitchison distance. The lines denote the evolution of the volunteer’s samples in time (different time points). The days after FMT procedure (or baseline for donor samples) denoted by numbers. b CoDa dendrogram which characterizes association of bacterial families, balances presented as edges. Decomposition of total variance by balances between groups of families is shown by vertical bars. Mean values of balances is shown by anchoring points of vertical bars. Color of vertical bars corresponds to time points. Color rectangles highlighted families belonging to important balances. The arrows direction indicates the predominance of this balance part in the donor. MOTUs with no family information are collapsed into the no-name family Full size image

For constructing the model of microbiota succession caused by FMT, the balance dendrogram (CoDa dendrogram) was used. This approach allows identifying specific balances (ratio between taxonomic abundances) that are involved in the reshaping of the microbiome of recipients [31, 38]. This model describes the intensity of taxonomic reshapes when moving the recipients’ profiles to the donor-specific parameters (see Fig. 4b). Immediately, on the fifth day after FMT, the recipients relatively increased the content of Prevotellaceae, unknown Burkholderiales, Erysipelotrichaceae, Vellonellaceae, and Desulfovibrionaceae; however, the shift towards Prevotellaceae, unknown Burkholderiales, was more pronounced at day 5. At the same time, the relative increase of Lachnospiraceae, Oscillospiraceae, Rumminococaceae, Sphingomonadaceae, Bradyrhizobiaceae, Bifidobacteriaceae and Coriobacteriaceae occurred less quickly and more smoothly. Also, the relative abundance of Enterobacteriaceae, Bacteroidaceae, Porphyromonadaceae, Rikenellaceae, unknown Bacteroidales, Eubacteriaceae, and Streptococcaceae decreased gradually towards the donor-like profile.

Altogether, the obtained results show a directed change in the gut microbiota composition of volunteers, namely pre-FMT profiles of the recipient microbiota have changed after FMT and become similar to the donor microbiota.

Identification of donor bacteria in the recipient metagenomes

Taxonomic profiling methods may reveal general changes of the taxonomic profile for the gut microbiota. However, it is important to examine the engraftment of the donor bacteria in recipients. To assess the engraftment of the donor bacteria using the obtained shotgun sequencing data, we used genome-resolved metagenomics – an approach allowing to restore bacterial genome from the metagenomic data (metagenome-assembled genomes – MAGs). This method is based on the metagenomic assembly and clustering of contigs through a metagenomic binning procedure and others specific manipulations (see “Methods” section). As a result, 243 MAGs were assembled for all metagenomic samples both from donor and recipients. For the donor 46 MAGs were obtained, for each of the volunteers 87, 56, and 54 MAGs, respectively (note that these MAGs represent microbes from both pre- and post-FMT time points). Further, based on 43 marker single-copy proteins, the place at the dendrogram for each MAG was determined (see Additional file 2: Figure S2), and appropriate taxonomic annotation was ascribed with the CheckM tool. We detected 14 donor-like MAGs in which 100% amino acid similarity of marker proteins was observed (see Fig. 5a). The changes in the relative abundance of these 14 MAGs are shown in Additional file 3: Figure S3. The similarity of the nucleotide sequence (average nucleotide identity – ANI) between donor and recipient MAGs was also high (see Fig. 5b). Anvi’o visualization for the mapping results of the reads from recipient samples in the donor MAGs is shown in Fig. 5c.

Fig. 5 Comparison of similarity between donor and recipients metagenome-assembled genomes (MAGs). a The AA distance based on 43 marker proteins between all donor MAGs and all MAGs of all recipients. Arrow shows that some MAGs in donor and recipient is present with absolute similarity of marker genes sequence. b The average nucleotide identity (ANI) between similar donor and recipients MAGs. The MAGs with 100% AA similarity of 43 marker proteins were selected. c Anvi’o plot denoted prevalence of donor MAGs across all metagenomic samples. Detection value (proportion of nucleotides in a contig that are covered at least 1x (according to http://merenlab.org/2017/05/08/anvio-views) was used as an abundance metric, which is shown as color brightness. Black color denotes detection value of donor MAGs in the donor samples, red – in the V1 samples, blue – in the V2 samples, green – in the V3 samples. DONOR BIN – clusters of metagenome-assembled genomes similar to the donor bacteria. The days after FMT denoted by numbers. The mapping of recipient metagenomic reads to donor MAGs was performed with 100% similarity Full size image

DONOR_BIN_26 didn’t show the 100% amino acid homology with MAG of V1 and V3. However, they were similar in their nucleotide composition. These discordances could be explained by some metagenomic assembly artefacts and binning, thus resulting in chimeric contigs. The given approach shows a rather big number of false-negative results; however, it allows to detect successful cases from nonspecific findings. Of 14 donor MAGs with complete amino acid sequence similarity in marker proteins, 10 may be considered as successfully engrafted in at least one recipient. The DONOR_BIN_28, DONOR_BIN_47, DONOR_BIN_22, DONOR_BIN_22, DONOR_BIN_22 did not enter this list due to the following reasons: (1) nucleotide identity from recipient MAGs (threshold <99.90% ANI); (2) they were covered by reads after FMT (not 100% certainty of their donor origin). By taxonomic annotation, these "strong" colonizers belong to the following orders: Bacteroidales (n=5), Clostridiales (n=3), Selenomonadales (n=1). Many donor MAGs didn’t show 100% similarity with recipients MAGs in the two parameters described above. However, this MAGs appeared in the recipients after FMT. This can be explained by the chimeric contigs and/or sequencing errors when assembling recipients MAGs. In addition, similar recipient bacteria can increase after FMT.

Ten MAG clusters, similar in amino acid sequences of marker proteins, were exclusively present in the recipient metagenomes (see Additional file 3: Figure S3). Based on the criteria of an nucleotide similarity (ANI >99.90%), there is evidence that the observed changes of several genera of bacteria abundances were donor-independent. Either these expanding bacteria were in donor samples, but were not found due to insufficient read coverage, or the FMT procedure induces the relative expansion of certain types of recipient bacteria (for example, see Additional file 3: Figure S3A). We have also revealed 4 cases of similar MAG sets in V2 and V3 that decreased relatively after FMT in both recipients. V2 and V3 have similar patterns of decrease and increase of some similar MAGs (see Additional file 3: Figures S3 D, F, H, I, J and Additional file 4: Figure S4). More detailed information about MAGs assembly is presented in Additional file 6: Table S5.

Additionally, SNV-profiling using mOTUs2 pipeline and metaSNV was performed. We detected that after FMT the number of mOTUs identical to donor were increased. The results are present in Additional file 5: Figure S5.