(**c**)

**Figure 9.** *Cont*.

**Figure 9.** The ileal microbiome profile, taxonomic analysis: (**a**) Pie chart of the microbiome abundance profile of the terminal ileum. The inner circle represents the family level, and the outer circle represents the genus level. Microbiome profiles classified according to tumor location and MSS status: RSCC (**b**), LSCC (**c**), MSS (**d**) and MSI (**e**).

From five LSCC patients, we also had specimens of the terminal ileum. In these patients, the top taxa at the genus level were *Streptococcus* (37% versus 10% in patients with RSC) and *Enterobacter* (19% versus 2% in patients with RSC). Due to the small sample size, no significant differences were observed in regard to sidedness. The core microbiome analysis further revealed that the ileal microbiome of the RSCC and LSCC patients as well as of the MSS and MSI patients harbored a diverse core microbiome (Figure 10c,d). The differential abundance analysis with the highest power to compare groups, especially for less than 20 samples per group, revealed five significantly different features for MSS status: *Enterobacter*, *Actinomyces* and *Streptococcus* for the MSS patients and *Eisenbergiella* and *Parasutterella* for the MSI patients. The original LEfSe analysis revealed three significantly different features, *Actinomyces, Abiotrophia* and *Atopobium*, in the MSS patients (*p* value < 0.05, LDA > 3.0), but the FDR-adjusted *p* values revealed no differences.

Between the ileal samples and preoperative stool samples, the alpha (Observed index *p* value < 0.01, [*t* test] statistics: −2.61) and beta diversity (PCoA Jensen–Shannon (PER-MANOVA) F value: 18.525, R-squared: 0.23592, *p* value < 0.001) clustered significantly differently (Supplementary Figure S3). The LEfSe analysis revealed 23 genera with a significantly different abundance (*p* value < 0.05, LDA > 3.0, FDR-adjusted *p* value < 0.05, Supplementary Figure S4).

Next, we compared ileal samples and tumor tissue and interestingly did not reveal a significant difference in the alpha and beta diversity (Figure 11b). The original LEfSe analysis revealed only one significantly different abundant genus, *Atopobium* (*p* value < 0.05, LDA > 3.0), in specimens of the terminal ileum, and the FRD-adjusted analysis (<0.05) revealed no significant differences.

**Figure 10.** Heatmap of the core microbiome of the terminal ileum (defined as genera present in >50% of samples), based on tumor location: RSCC (**a**) and LSCC (**b**), and on MSS status: MSI (**c**) and MSS (**d**).

**Figure 11.** The tumor microbiome–ileal microbiome association: PCoA using Jensen–Shannon divergence of beta diversity between ileal and healthy colon tissue was significantly different, *p* value < 0.05 (**a**), while no significant differences were observed between ileal samples and tumor samples (**b**).

Additionally, between the ileal samples and healthy colon tissue samples, no significant differences in alpha diversity were observed, while the beta diversity was significantly different (PCoA Jensen–Shannon (PERMANOVA) F value: 3.8652, R-squared: 0.063505, *p* value < 0.03; [PERMDISP] F value 4.7804, *p* value < 0.03; Figure 11a). The LEfSe analysis revealed three genera with significantly different abundances in specimens of the terminal ileum: *Streptococcus*, *Gemella* and *Granulicatella* (*p* value < 0.05, LDA > 3.0).

2.2.5. The Stool Microbiome Structure: Sequential Analysis before and after Surgery Revealed Major Changes

Due to bowel preparation, perioperative antibiotic prophylaxis and surgery, the stool microbiome underwent major changes before and after surgery. The ratio between *Firmicutes* and *Bacteroidetes* (regarded as dysbiosis) was decreased: the preoperative stool samples harbored 41% *Firmicutes* and 51% *Bacteroides*, while the postoperative samples consisted of 29% *Firmicutes* and 60% *Bacteroides* (Figure 12a). The microbiome composition differed strikingly at the genus level between the timepoints (beta diversity analysis (PERMANOVA) F value: 14.506; R-squared: 0.18019; *p* value < 0.001) (Figure 12c). Bacterial richness and evenness were significantly lower in the postoperative stool samples, and the postoperative stool samples were characterized by a significant increase in the abundance of *Enterococcus* (*<sup>p</sup>* value < 2.20 × 109), LDA –5.84), a lactic-acid-producing bacterial genus that includes potentially pathogenic strains.

**Figure 12.** The stool microbiome preoperative and postoperative showed major differences: (**a**) Phylum level abundance profile of preoperative and postoperative samples. Comparison of pre- and postoperative stool revealed significant differences at the genus level: (**b**) alpha diversity and (**c**) beta diversity, (*p* < 0.001).
