Shotgun metagenomics was used to analyse the viromes of these donors and recipients. This corresponded to 134 faecal samples collected for 14 recipient patients pre-FMT and at intervals up to 1-year post-FMT, as well as samples from each of the 3 donors collected at the corresponding time points. From 2 recipients (P11 and P14), no pre-FMT sample was available, while some post-FMT samples were unavailable for 3 recipients. Metadata corresponding to these donors and recipients can be found in Additional file 1: Table S1 and in the original study examining the bacteriome of these individuals [4]. Viral DNA was sequenced on an Illumina HiSeq to a mean depth of 1,277,374 paired-end reads per sample and assembled with metaSPAdes. In order to avoid contamination with bacterial DNA, confounding estimates of viral transfer, only those contigs that contained known viral genes or were predicted as viral with VirSorter [12] were used for further analysis resulting in a total of 7064 metagenomic viral contigs with a mean length of 4.9 kb containing an average of 5.7 known viral genes per contig. The bacteriophage community was dominated by dsDNA and ssDNA phages. The majority of RNA viruses found in the gut are plant and human viruses [13], and thus, these were not investigated here, as the current study focused on DNA containing viral particles of the virome. Nonetheless, the impact and involvement of eukaryote and other RNA viruses are of interest, especially as it has been previously reported that the eukaryotic virome in the gut is altered post-FMT in ulcerative colitis (UC) patients [14]. Whether this is UC-specific phenomenon or a general effect of FMT is yet to be established.

The impact of FMT on the DNA virome beta diversity was measured using Bray-Curtis distance. Principal coordinate analyses of the virome demonstrated that FMT resulted in gut virome remodelling in a donor-specific fashion with each set of donor-recipient groups forming distinct groups centred on the donor (Fig. 1a), strikingly more distinct than that observed based on bacterial profiles as measured with 16S (Fig. 1b). In order to examine the level of donor-recipient similarity over time, we plotted the Spearman correlation between each recipient sample and its donor sample at each time point, the correlation between recipients and their own pre-FMT (rCDI) sample, and their correlation with other donors and finally donor similarity over time to their own T-0 sample (Fig. 1c). We found that for 2 months post-FMT, the recipient’s viromes were significantly more similar to that of their donors than it was to their own pre-FMT virome (Wilcoxon rank sum test, P = 0.01288). Moreover, for up to 12 months post-FMT, the recipients were found to be equally similar to their own pre-FMT sample and their donor’s sample. This demonstrates a lasting shift in the recipient gut virome towards a donor-specific one, and given that bacteriophages are usually strain-specific, this presumably reflects a long-term alteration to the fine structure of the microbiome.

Fig. 1 Viral profiles of donors and recipients. a Bray-Curtis PCoA plot based on virome abundance demonstrating that post-FMT recipients cluster strongly with their donor. b Bray-Curtis PCoA plot based on 16S abundance data demonstrating the bacterial genus level group clustering of donors and recipients. P value and R2 values were generated with PERMANOVA as implemented in the Adonis function in the Vegan package in R. c Plot shows similarity of individual recipient viromes at each time to themselves at the first time point, i.e., to their CDI sample (pink line), to their donor (green line), or to other donors (blue line) over time. Donor virome similarity to their original donated sample was also investigated over the 12-month period (red line). The y-axis values represent Spearman’s rho, with the mean and 95% confidence interval plotted per time point. Statistical significance, as assessed by the nonparametric Wilcoxon rank sum test, between the recipient’s similarity to their donor and recipient similarity to themselves pre-FMT is indicated by asterisks. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 Full size image

Recurrent CDI is marked by changes in the gut virome relative to healthy controls [8]. We investigated the abundance of viral taxonomic groups by utilising a read classification approach based on amino acid similarity to the Non-Redundant (nr) database at NCBI [15]. The vast majority of classifiable reads were identified as being from the Caudovirales and Microviridae taxa, indicating the samples were predominantly composed of bacteriophages as has been previously found with the human gut virome [16]. In agreement with Zuo et al., we found that rCDI is marked by a disturbance in the enteric gut virome characterised by increased Caudovirales (Wilcoxon test, P = 0.0009806), decreased Microviridae (Wilcoxon test, P = 0.01093), and increased Anelloviridae abundance (Wilcoxon test, P = 0.04509) when compared to our healthy donors. (Fig. 2). Furthermore, we have expanded upon previous observations by demonstrating that these differences are not only immediately resolved by FMT but persist for at least 12 months (Fig. 2). We also observed an increase in unclassified viruses (Wilcoxon test, P = 0.00039) in the rCDI virome and observed that these individuals have decreased levels of CrAssphage (Wilcoxon test, P = 0.0005647) (Fig. 2), which represents the most prominent group of bacteriophages in the human gut [17, 18]. This group of CrAssphage-like bacteriophage is thought to primarily predate on members of the phylum Bacteroidetes, which has previously been described as decreased in the CDI gut microbiota [19]. Jalanka et al. observed a 2.7-fold increase in the members of the Bacteroidetes phylum in this cohort related to Bacteroides vulgatus and Prevotella oralis using 16S rRNA profiling [2]. No single bacteriophage, virus, viral family, or viral contig could be detected as universally present post-FMT suggesting that no single bacteriophage or DNA virus was responsible for disease resolution. The only statistically significant difference in viral richness or diversity was observed with a reduction in Chao1 richness in samples collected at 2 weeks post-FMT when compared with the original CDI patient samples (paired Wilcoxon rank sum test, P = 0.0425, Additional file 2: Figure S1). This initial fluctuation in richness post-FMT was also observed by Zuo et al. [8], and we believe it may constitute an initial loss of unique donor/recipient phages as a consequence of loss of their bacterial host due to engraftment and shifting of other members of the bacterial population following FMT.

Fig. 2 Abundances of viral taxonomic groups. a Boxplots showing relative abundances of viral taxonomic groups identified in this data by the Kaiju metagenomic classifier. Unclassified viruses refer to sequences classified as viral but not to any specific taxonomic group. Wilcoxon rank sum test was used to determine statistical differences in relative abundance between the donors and recipients pre-FMT and is indicated by asterisks; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Whiskers indicate the highest or lowest occurring value within 1.5*IQR (interquartile range) of the upper or lower quartile Full size image

Chehound et al. [9] observed that certain temperate bacteriophages were transferred with greater efficiency than other members of the virome during FMT. We aimed to assess the prevalence of integrase genes within our viral contigs to determine the extent to which temperate phages were transferred in this study. Of the set of 7064 contigs assembled here, 916 could be classified into a viral order or family, and 182 of these were found to contain integrase genes. A further 486 contigs that could not be classified to a known viral group also contained a known integrase. Only 51 of these 1402 (916 + 486) were observed to be transferred from any donor to any recipient at any time point; however, it is possible many others were below the detection threshold. Thus, our analysis indicates that the collective virome contains at least several hundred temperate phages, and some of these are indeed transplanted. In addition to temperate phage transfer, we also observed the transplantation of lytic phages such as CrAssphage (deemed lytic as it contains no known integrase gene and has not been observed in bacterial genomes) and members of the predominantly lytic Microviridae family [20]. We believe this strongly indicates that transfer of lytic bacteriophages occurs during FMT and that these either transplanted with their host or acquired a new host within the recipient’s microbiome.

Previous work has highlighted the differential colonisation of donor bacterial strains depending on the donor-recipient pairing post-FMT [4, 21,22,23,24]. Thus, we sought to explore viral colonisation across the 3 donors and 12 recipients (those with pre-FMT samples) for up to 1 year after treatment. Contig relative abundances were summed by origin (transferred and non-transferred contigs specific to the donor, unique recipient contigs, those common to both, or those newly detected post-FMT) (Fig. 3). We observed that the relative abundance of transferred contigs (those absent in CDI samples, present in the donor sample, and present post-FMT) is highly variable, ranging from almost the entire sample to a fraction of a percent depending on the donor-recipient pair. Individual’s viromes are highly unique [25]; thus, engraftment would be expected and is observed to be donor-specific (Fig. 1a). We therefore sought to investigate the colonisation patterns of specific bacteriophages. In all but one case, these phages were absent from the recipient and are examples of donor transfer but with differing recipient colonisation. A number of bacteriophages, including a member of the Microviridae family, a contig of 86 kb classified as a Caudovirales, CrAssphage, and an unclassified viral contig of 44 kb, none of which possess a known integrase, were transferred to all recipients but with varying degrees of engraftment and persistence over time (Fig. 4a, b). It is possible that the abundance of these lytic phages expands and contracts overtime due to fluctuations in host populations. Moreover, in some FMT recipients, high proportions of contigs were detected that were not detected in either CDI samples or in the donor. This suggests that these bacteriophages were either below the threshold of detection at the earlier time points, were the result of newly induced prophage, or were acquired exogenously by recipients.

Fig. 3 Relative viral abundances in donors and recipients. Relative abundance plots of donors per recipient and per time point with contig abundances summed by their origin, i.e., if they were transferred contigs unique to the donor (green), non-transplanted donor contigs (blue), unique recipient contigs (pink), contigs found in both donors and recipients (red), or contigs found in neither donors or pre-FMT at donation (yellow). In the case of two recipients (P11 and P14), pre-FMT samples were not available and so these individuals were omitted from this analysis Full size image

Fig. 4 Relative abundance of individual bacteriophages in different donor-recipient pairs. a Line graphs representing relative abundance of different individual bacteriophages in the recipients of donor 2—a novel Caudovirales and Microviridae—and b recipients of donor 3—CrAssphage and an unclassified viral contig. A donor timeline depicting the relative abundance the bacteriophage in question has also been included in each case, with the time of donation to each recipient marked therein. As donor 1 only had a single recipient, it was not possible to determine if differential colonisation patterns were associated with this donor Full size image

Given that all recipients here achieved clinical resolution, it is impossible to ascertain what impact the colonisation of specific microbes played in the success of each FMT treatment. One plausible explanation is that a healthy, diverse, and stable ecosystem per se, even without specific or prescribed microbial components, can resolve rCDI. In such a scenario, bacteriophages could play a crucial role in maintaining ecosystem stability. Research to date has not established any difference in rCDI resolution following the use of related or unrelated healthy donor samples [26]. However, for large-scale implementation of FMT and especially for other indications, banks of screened faeces from healthy donors provide a more logistically sound approach. As highlighted here, and in previous studies [22], donor-recipient compatibility may be a key factor in the extent and stability of microbial colonisation. However, determination of donor-recipient microbiome similarity and/or compatibility in relation to disease resolution is still lacking. Virome analysis represents another view of the microbiome that could improve the selection of optimal donor-recipient pairs.