**3. Results**

#### *3.1. Baseline Characteristics*

We included 139 RTRs (age 58.3 ± 12.8 years; 50% males) at a median post-transplantation time of 6.0 (1.5–12.5) years and 105 healthy controls (age 59.2 ± 10.6 years; 57% males). Mean BMI was 27.7 ± 5.4 kg/m<sup>2</sup> for RTRs and 27.2 ± 6.0 kg/m<sup>2</sup> for controls. In total 3 (3%) healthy controls and 38 (27%) RTRs had diabetes mellitus (*p* < 0.001). RTRs had a significantly higher HbA1c, 40.0 (37.0–46.0) compared to healthy controls, 37.5 (36.0–40.0) (*p* < 0.001). RTRs had a significantly lower eGFR of 48.3 ± 16.7 mL/min/1.73 m<sup>2</sup> compared with 69.0 ± 19.2 mL/min/1.73 m<sup>2</sup> for controls (*p* < 0.001). In total 7 (5%) RTRs used antibiotics, 115 (83%) RTRs used antihypertensive medication, 96 (69%) RTRs used PPIs, and 66 (47%) RTRs used statins. Cyclosporine was used by 25 (18%) RTRs, tacrolimus by 79 (57%) RTRs, azathioprine was used by 13 (9%) RTRs, mycophenolate mofetil by 100 (72%) RTRs, and prednisolone by 133 (96%) RTRs (Table 1).


All characteristics are presented as means ± standard deviation unless otherwise stated. IQR—interquartile range.

#### *3.2. Diversity of the Gut Microbiome*

The median Shannon diversity index, a measure for the diversity of the gu<sup>t</sup> microbiome, was significantly lower in RTR samples with 3.4 (3.1–3.8) *vs*. 3.7 (3.5–4.0) for healthy controls (*p* < 0.001). The median operational taxonomic units (OTUs) per sample was 256 (214–304) for RTRs and 314 (260–351) for healthy controls (*p* < 0.001) (Figure 1). The diversity between samples was further assessed using beta diversity analysis. The gu<sup>t</sup> microbiome was significantly different between RTRs and healthy controls (*p* < 0.01). A separation in gu<sup>t</sup> microbiota composition can be observed between RTRs

and healthy controls in the principal coordinate plot (Figure 2). A permutational multivariate analysis of variance using distance matrices (ADONIS) was performed to estimate the variation explained in the gu<sup>t</sup> microbiome by different variables. In total, 5.8% of the variation of the gu<sup>t</sup> microbiome of RTRs and healthy controls was significantly explained by sample type (RTR or healthy control, *p* < 0.001). Furthermore, using ADONIS, baseline characteristics including medication use were tested in the gu<sup>t</sup> microbiome of RTRs. Within the gu<sup>t</sup> microbiome of RTRs age (1.2%), BMI (1.1%), and eGFR (1.0%) significantly explained variation within the gu<sup>t</sup> microbiome. Furthermore, the use of PPIs (1.2%) and the use of mycophenolate mofetil (1.0%) significantly explained variation within the gu<sup>t</sup> microbiome of RTRs. Age was positively correlated to the Shannon diversity index (*p* < 0.01). Use of mycophenolate mofetil and use of antibiotics was negatively correlated to the Shannon diversity index (*p* < 0.01) (Figure 3).

**Figure 1.** This is a figure showing the diversity of the gu<sup>t</sup> microbiome of renal transplant recipients (RTRs) compared to healthy controls: (**A**) a boxplot depicting the Shannon diversity index, which is a measure for the diversity of the gu<sup>t</sup> microbiome, was significantly lower in RTRs compared to healthy controls (*p* < 0.001); (**B**) a boxplot showing the number of observed operation taxonomic units (OTUs) between RTRs and healthy controls (*p* < 0.001).

**Figure 2.** Principal coordinate analysis of 139 RTRs and 105 healthy controls. The principal coordinates plot shows principal coordinates for the Bray–Curtis distance, a measure for the composition of the gu<sup>t</sup> microbiome, for RTRs and healthy controls. Separation in the composition of the gu<sup>t</sup> microbiome between RTRs and healthy controls can be observed. PCo1 is principal coordinate 1 and PCo2 is principal coordinate 2. The gu<sup>t</sup> microbiome of RTRs is significantly different from that of healthy controls in the first coordinate (PCo1 *vs*. PCo2: *p* < 0.01). RTR or healthy control status significantly explained 5.8% of variation in the gu<sup>t</sup> microbiome (*p* < 0.001).

**Figure 3.** Depiction of variables that are associated with variation in the gu<sup>t</sup> microbiome within RTRs. In the bar plots, the x-axis represents the percentage of explained variance in the gu<sup>t</sup> microbiome of RTRs expressed as the Bray–Curtis distance. The heatmap depicts significant negative correlations (red) and positive correlations (blue) with the Shannon diversity index (*p* < 0.01). These variables were tested only in the gu<sup>t</sup> microbiome of RTRs. Bars in orange represent variables which significantly explain variance in gu<sup>t</sup> microbiota composition (*p* < 0.05).

#### *3.3. Composition of the Gut Microbiome*

We analyzed the gu<sup>t</sup> microbiome at different taxonomic levels: phylum, class, order, family, genus, and species. Using MaAsLin, we were able to identify significant differences in taxa abundances between RTRs and healthy controls while correcting for age, sex, BMI, smoking, use of antihypertensive medication, use of antibiotics, use of statins, use of PPIs, and read depth. In total, we found significant alterations in 127 of the 447 bacterial taxa abundances in the gu<sup>t</sup> microbiome of RTRs (*p*FDR < 0.10) (Table 2). On the phylum level we found that RTRs have significantly higher levels of Proteobacteria and lower levels of Actinobacteria (*p*FDR < 0.10) (Figure 4). Within the phylum Proteobacteria, the species *E. coli* was significantly more abundant in the gu<sup>t</sup> microbiome of RTRs (*p*FDR < 0.10). Within the phylum Actinobacteria multiple species had a lower abundance within the gu<sup>t</sup> microbiome of RTRs, especially multiple *Bifidobacterium* species (*p*FDR < 0.10). The predominant phylum Firmicutes was not significantly different in RTRs compared to healthy controls. However, within the phylum Firmicutes there were many significantly different species in the gu<sup>t</sup> microbiome of RTRs compared to healthy controls (Figure 4 and Table S1). An extensive overview of MaAsLin results for complete taxonomy is provided in Table S1.


**Table 2.** Overview of significantly altered taxa between renal transplant recipients and healthy controls.

1 Total number of taxa with an abundance >0.1%; 2 *p*FDR < 0.10.

**Figure 4.** This figure depicts the abundance of phyla and species for RTRs and healthy controls. Bar plots represent the mean proportion and differences in mean proportions with 95% confidence intervals are depicted on the right. Taxa that are depicted were filtered for a difference in mean proportion >0.2%. *p*FDR < 0.10 was considered as statistically significant and indicated in the plot with a star (\*).
