Enterotype-based Analysis of Gut Microbiota along the Conventional Adenoma-Carcinoma Colorectal Cancer Pathway

The dysbiosis of human gut microbiota is strongly associated with the development of colorectal cancer (CRC). The dysbiotic features of the transition from advanced polyp to early-stage CRC are largely unknown. We performed a 16S rRNA gene sequencing and enterotype-based gut microbiota analysis study. In addition to Bacteroides- and Prevotella-dominated enterotypes, we identified an Escherichia-dominated enterotype. We found that the dysbiotic features of CRC were dissimilar in overall samples and especially Escherichia-dominated enterotype. Besides a higher abundance of Fusobacterium, Enterococcus, and Aeromonas in all CRC faecal microbiota, we found that the most notable characteristic of CRC faecal microbiota was a decreased abundance of potential beneficial butyrate-producing bacteria. Notably, Oscillospira was depleted in the transition from advanced adenoma to stage 0 CRC, whereas Haemophilus was depleted in the transition from stage 0 to early-stage CRC. We further identified 7 different CAGs by analysing bacterial clusters. The abundance of microbiota in cluster 3 significantly increased in the CRC group, whereas that of cluster 5 decreased. The abundance of both cluster 5 and cluster 7 decreased in the Escherichia-dominated enterotype of the CRC group. We present the first enterotype-based faecal microbiota analysis. The gut microbiota of colorectal neoplasms can be influenced by its enterotype.


Supplementary
: Phylum-level microbial composition Bcateroidetes, Proteobacteria, and Firmicutes are the most dominant phyla in stool samples.
These 3 phyla covered more than 95% of our sequenced reads. Total 10 phyla exist in all groups, except the phylum, Gemmatimonadetes, only found in cancer group. The 10 phyla share the same relative abundance order in three groups with minor variations of percentages.  Table S2. Major common bacteria Top 20 genera in all and each groups based on relative abundance. All groups share 16 common genera with different order. All these 25 genera occupied more than 90% of the relative abundance in each group.     Table S5. NC correlated CAGs The co-abundance groups (CAG) between the normals and CRCs clustered by Pearson's correlation analysis.
Cluster 2 contained ten genera (red marked) which were clustered in every group with high correlation coefficient.   Figure S1A. Shannon diversity index by group across enterotypes Shannon diversity index of each sample in three enterotypes. Enterotype I & II have similar Shannon diversity index in each group. Enterotypes III has slightly higher Shannon diversity index, and the cancer group also varies slightly more without significance.

NAC correlated
E3 Figure S5C. Boxplots of relative abundance of significant CAG Summation of relative abundance of NAC correlated cluster 6 showed significant lower abundance in CRC group than normal and adenoma groups. Figure S6A. Spearman's correlation analysis of normals and adenomatous polyps Genera appearance greater than 50% in cancer group were included to calculate the correlation coefficient by relative abundance. Only microflora of normals and adenomatous polyps were employed.
All E1 E2 E3 Figure S6B. Boxplots of relative abundance of CAGs clustered by normals and adenomatous polyps Summation of relative abundance of 6 NA correlated CAGs were illustrated based on all samples and samples of each enterotype.

NA correlated
E3 Figure S6C. Boxplots of relative abundance of significant CAG Summation of relative abundance of NA correlated cluster 5 showed significant lower abundance in CRC group than normal and adenoma groups. Figure S7A. Spearman's correlation analysis of adenomatous polyps and CRCs Genera appearance greater than 50% in cancer group were included to calculate the correlation coefficient by relative abundance. Only microflora of adenomatous polyps and CRCs were employed.
All E1 E2 E3 Figure S7B. Boxplots of relative abundance of CAGs clustered by adenomatous polyps and CRCs Summation of relative abundance of 7 NC correlated CAGs were illustrated based on all samples and samples of each enterotype.

AC correlated
E3 Figure S7C. Boxplots of relative abundance of significant CAG Summation of relative abundance of AC correlated cluster 5 showed significant lower abundance in CRC group than adenoma groups.

Enterotype I -Bacteroides dominated
Normal n = 42 Cancer n = 24 Adenoma n = 57 Figure S8A. Using the steady CAG (cluster 2) as a standard to identify specific genus that differed between groups -Enterotype I The correlation coefficient of Clostridium and Coprococcus with cluster 2 varied in normal, adenoma, and cancer groups in enterotypes 1.

Enterotype II -Prevotella dominated
Normal n = 13 Cancer n = 7 Adenoma n = 16 Figure S8B. Using the steady CAG (cluster 2) as a standard to identify specific genus that differed between groups -Enterotype II The correlation coefficient of 9 genera with cluster 2 varied in normal, adenoma, and cancer groups in enterotypes 2.

Enterotype III -Escherichia dominated
Normal n = 49 Cancer n = 31 Adenoma n = 44 Figure S8C. Using the steady CAG (cluster 2) as a standard to identify specific genus that differed between groups -Enterotype III The correlation coefficient of 7 genera with cluster 2 varied in normal, adenoma, and cancer groups in enterotypes 3.

Normal n = 13
Adenoma n = 16 Cancer n = 7 Figure S9. Network analysis of stool microbiota using Pearson's correlation coefficients (Enterotype II) Correlation coefficients network between 10 genera of CAG 2 and 9 other genera.