**4. Discussion**

We have shown that the gu<sup>t</sup> microbiome of RTRs is different compared to the gu<sup>t</sup> microbiome of healthy controls. Interestingly, we demonstrated that RTRs have dysbiosis characterized by general loss of microbial diversity. We found that RTRs have an increased abundance of Proteobacteria, a decrease in Actinobacteria, and a loss of butyrate-producing bacteria. Finally, we found that age, BMI, eGFR, the use of PPIs, and the use of mycophenolate mofetil are determinants of the gu<sup>t</sup> microbiome of RTRs and that age, BMI, and the use of mycophenolate mofetil correlate to the diversity of the gu<sup>t</sup> microbiome.

In a pilot study of Lee et al., significant changes were seen in the gu<sup>t</sup> microbiome of RTRs when pre-transplantation samples were compared to post-transplantation samples. The diversity, although not significant, was lower after transplantation. Proteobacteria and Enterobacteriales were increased in the gu<sup>t</sup> microbiome post-transplantation [8]. We observed a significant loss of diversity in the composition of the gu<sup>t</sup> microbiome in RTRs with a similar increase in Proteobacteria. It is known that a lower diversity is associated with various diseases such as inflammatory bowel disease (IBD), metabolic disease and cardiovascular disease, as stated by the Human Microbiome Consortium [21]. Previous research in a cohort of allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients demonstrated that a lower diversity of the gu<sup>t</sup> microbiome was associated with a higher mortality risk [9]. Additionally, allo-HSCT recipients who were deceased had a higher level of Gammaproteobacteria, Enterobacteriales, and Enterobacteriaceae [7]. These findings are strikingly similar to the results of our study. We also observed a lower diversity and higher levels of Proteobacteria, Gammaproteobacteria, Enterobacteriaceae, *Escherichia*, *Streptococcus*, and *Lactobacillus* in the gu<sup>t</sup> microbiome of RTRs. However, it is unknown whether these changes in the composition of the gu<sup>t</sup> microbiome are associated with mortality in RTRs.

The lower diversity observed in the gu<sup>t</sup> microbiome of RTRs sugges<sup>t</sup> that RTRs su ffer from dysbiosis. We found increased levels of Proteobacteria which has previously been proposed as a marker for dysbiosis in the gu<sup>t</sup> microbiome [22]. Furthermore, we observed a loss of butyrate-producing bacteria in RTRs. Butyrate is a short-chain fatty acid (SCFA) that plays a key role in maintaining gu<sup>t</sup> health. Butyrate is associated with trans-epithelial fluid transport, reduction of inflammation and oxidative stress, reinforcement of the epithelial barrier, and has potential protective properties against colorectal cancer [23]. In this study, lower levels of *Faecalibacterium prausnitzii, Gemmiger formicilis*, *Eubacterium rectale*, *Coprococcus catus*, *Coprococcus comes* and *Roseburia* were observed in the gu<sup>t</sup> microbiome of RTRs. These are all well-known butyrate-producing bacteria [24]. The decrease of butyrate production in RTRs could be detrimental to their gu<sup>t</sup> health. Furthermore, animal studies show that butyrate has immunomodulatory properties through the e ffect on regulatory T-cells (Treg), which in turn plays a key role in suppressing inflammatory responses. Increasing butyrate in the gu<sup>t</sup> improved renal dysfunction and reduced local and systemic inflammation in mice [25,26]. These results have also been observed in allogeneic bone marrow transplant recipients. Reduced butyrate altered gene regulation and resulted in fewer Treg cells. Restoring butyrate levels led to improved junction integrity, decreased apoptosis, and improved graft versus host disease [27]. Dysbiosis and a loss of butyrate-producing bacteria in the gu<sup>t</sup> microbiome of RTRs could therefore have detrimental effects on gu<sup>t</sup> health. Further research is needed to study the clinical consequences of the loss of butyrate-producing bacteria in RTRs.

In another study of Lee et al., a loss of diversity and a loss of butyrate-producing bacteria was also observed in RTRs with post-transplantation diarrhea. In this study, post-transplantation diarrhea was not associated with common infectious diarrheal pathogens but rather with dysbiosis [4]. RTRs had lower levels of *Ruminococcus*, *Coprococcus*, and *Dorea* in this study. These findings are in accordance with results from our study, which also demonstrated lower levels of *Ruminococcus* in the microbiome of RTRs. However, we did not observe higher levels of *Coprococcus* and *Dorea* in RTRs. One reason for this might be that we included patients more than one year after transplantation while Lee et al. included patients within the first year after transplantation. In addition, we corrected for various factors that influence the composition of the gu<sup>t</sup> microbiome, which was not done in the study by Lee et al. [8].

We found many significant di fferences in taxa abundance in RTRs compared to healthy controls. Some of these di fferences in the composition of the gu<sup>t</sup> microbiome might indicate that RTRs su ffer from increased inflammation in the gut. For example, the abundance of Proteobacteria was much higher in RTRs compared to healthy controls. This was also observed in patients with severe intestinal inflammation and inflammatory bowel disease (IBD), colorectal cancer, necrotizing enterocolitis, and irritable bowel syndrome (IBS) [28]. Increased oxygen availability in the gu<sup>t</sup> colonocytes could

explain these findings, since it is associated with inflammation in the gu<sup>t</sup> and drives the expansion of aerobic Proteobacteria, Enterobacteriaceae, and *E. coli* while lowering the levels of anaerobic bacteria such as *Bifidobacterium* and butyrate-producing bacteria [22,28]. In this study, RTRs indeed had an increased abundance of Proteobacteria, Enterobacteriaceae, and *E. coli*, and lower levels of Clostridia and Bifidobacteria. These similarities in the gu<sup>t</sup> microbiome composition of RTRs and IBD patients sugges<sup>t</sup> that RTRs may be su ffering from inflammation in the gut, which could lead to a loss of epithelial barrier function and diarrhea [29].

In this study, we showed that use of PPIs and mycophenolate mofetil was associated with variation within the gu<sup>t</sup> microbiome of RTR. Imhann et al. demonstrated that the use of PPIs changes the gu<sup>t</sup> microbiome, especially an increase of *Streptococcus* species was found in PPI users [30]. We found multiple *Streptococcus* species that had a higher abundance in the gu<sup>t</sup> microbiome of RTRs. This could be due to the use of PPIs. The influence of immunosuppressive medication on the gu<sup>t</sup> microbiome is not ye<sup>t</sup> well studied in RTRs. In a previous murine study, prednisolone, tacrolimus, and mycophenolate mofetil changed the gu<sup>t</sup> microbiome [6]. Mycophenolate mofetil has multiple side e ffects including diarrhea [31]. In our study, the use of mycophenolate mofetil was significantly correlated to a lower diversity of the gu<sup>t</sup> microbiome. A lower diversity of the gu<sup>t</sup> microbiome is more prevalent in less healthy individuals. More research is needed to investigate the interplay between the use of mycophenolate mofetil, the gu<sup>t</sup> microbiome, and clinical outcomes.

Improving the observed dysbiotic state of RTRs might have clinical implications concerning long-term outcome after renal transplantation. Important issues are that many RTRs su ffer from cognitive decline and development of skin cancer [32,33]. Dysbiosis might contribute to cognitive decline due to an e ffect of the gu<sup>t</sup> microbiome on the gut–brain axis [34]. The same mechanism might apply to the occurrence of skin cancer, due to an e ffect of the gu<sup>t</sup> microbiome on the gut–skin axis [33,35]. Improving the dysbiotic state of the gu<sup>t</sup> microbiome in RTRs may therefore be a modifiable factor that allows for inhibition of cognitive decline and skin cancer after renal transplantation. Changing the diet of RTRs might be an intervention that allows for improvement of the gu<sup>t</sup> dysbiosis of RTRs. Diet has been identified as an important potentially modifiable factor influencing the gu<sup>t</sup> microbiome [20]. After transplantation, many RTRs adhere to the diet that was prescribed prior to transplantation [36]. This diet includes a protein restriction, which is meant to limit and prevent uremic symptoms and progression of decline of renal function [37]. It also includes a phosphorus restriction to prevent hypophosphatemia, and a potassium restriction to prevent occurrence of hyperkaliemia, cardiac arrhythmias, and acute cardiac death [37]. Improving diet and eating habits may have a positive e ffect on the composition of the gu<sup>t</sup> microbiome, which could ultimately translate into a beneficial e ffect on cognitive function [38]. Future, larger cohort studies could focus on the influence of diet on the gu<sup>t</sup> microbiome in RTRs, and on potential sex di fferences therein. Potential sex di fferences in the gu<sup>t</sup> microbiome might be of interest, but cohorts to allow for determining those likely need to be large, because previously reported sex di fferences in the gu<sup>t</sup> microbiome are small [20,39].

We showed that there are many di fferences in the gu<sup>t</sup> microbiome of RTRs compared to healthy controls. However, the current study should be interpreted within its limitations. The main indication for renal transplantation is chronic kidney disease (CKD). Alterations in the gu<sup>t</sup> microbiome of patients with CKD are already present before transplantation. Patients with CKD also su ffer from a lower diversity of the gu<sup>t</sup> microbiome and dysbiosis. A lower colonization by Bifidobacteria and an increase in Enterobacteriaceae was observed in patients with CKD [26]. These findings are similar to our findings which sugges<sup>t</sup> that the dysbiosis observed in RTRs may already be present pre-transplantation and does not recover post-transplantation [8]. Moreover, we measured the composition of the gu<sup>t</sup> microbiome using 16S rRNA sequencing instead of metagenomic sequencing. Therefore, we were unable to analyze metabolic pathways of bacteria. Furthermore, SCFA were not measured in the current study. Therefore, it remains unknown whether the observed loss of butyrate-producing bacteria also leads to a decreased production of butyrate. Future studies should include pre-transplantation patients and study how the gu<sup>t</sup> microbiome develops after transplantation. Focus should be on the metagenome of RTRs to study the effects of dysbiosis, the metabolism genes, and metabolites. Furthermore, current studies of the gu<sup>t</sup> microbiome mainly focus on bacteria. However, at the kingdom level of the gu<sup>t</sup> microbiome, there are many more micro-organisms such as archaea, fungi, eukaryotes, as well as viruses which could also play an important role in the gu<sup>t</sup> microbiome of immunosuppressed patients [40].

In conclusion, the gu<sup>t</sup> microbiome of RTRs more than one year post-transplantation is significantly different from that of healthy controls. The gu<sup>t</sup> microbiome of RTRs contains more Proteobacteria and less Actinobacteria and there is a loss of butyrate-producing bacteria which could be detrimental to gu<sup>t</sup> health. The use of mycophenolate mofetil and antibiotics is associated with variation in the gu<sup>t</sup> microbiome of RTRs and correlated to a lower diversity. The results of this study are preliminary and require replication in a larger cohort. Nevertheless, we demonstrate that RTRs suffer from dysbiosis more than one year post-transplantation and that the use of mycophenolate mofetil correlates to a lower diversity.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2077-0383/9/2/386/s1, Table S1: MaAsLin results for RTRs compared to healthy controls.

**Author Contributions:** Conceptualization, J.C.S., H.J.M.H., and S.J.L.B.; data curation, J.C.S., M.F.E., M.v.L., and A.W.G.-N.; formal analysis, J.C.S., S.H., and S.J.L.B.; investigation, J.C.S.; methodology, J.C.S., S.H., A.V.V., H.J.M.H., and S.J.L.B.; resources, H.J.M.H. and S.J.L.B.; software, J.C.S. and S.H.; supervision, R.K.W., H.J.M.H., and S.J.L.B.; writing—original draft, J.C.S.; writing—review and editing, J.C.S., R.M.D., S.H., M.F.E., A.V.V., M.v.L., A.W.G.-N., R.K.W., H.J.M.H., and S.J.L.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** R.M. Douwes is supported by NWO/TTW in a partnership program with DSM, Animal Nutrition and Health, The Netherlands; gran<sup>t</sup> number: 14939.

**Acknowledgments:** The authors thank Rebekka van den Bosch, Rudi Tonk, Carien Bus-Spoor, and Ranko Gacesa for technical assistance.

**Conflicts of Interest:** The authors declare no conflict of interest.
