Tissue-Associated Bacterial Alterations in Rectal Carcinoma Patients Revealed by 16S rRNA Community Profiling

Abstract

Sporadic and inflammatory forms of colorectal cancer (CRC) account for more than 80% of cases. Recent publications have shown mechanistic evidence for the involvement of gut bacteria in the development of both CRC-forms. Whereas, colon and rectal cancer have been routinely studied together as CRC, increasing evidence show these to be distinct diseases. Also, the common use of fecal samples to study microbial communities may reflect disease state but possibly not the tumor microenvironment. We performed this study to evaluate differences in bacterial communities found in tissue samples of 18 rectal-cancer subjects when compared to 18 non-cancer controls. Samples were collected during exploratory colonoscopy (non-cancer group) or during surgery for tumor excision (rectal-cancer group). High throughput 16S rRNA amplicon sequencing of the V4-V5 region was conducted on the Ion PGM platform, reads were filtered using Qiime and clustered using UPARSE. We observed significant increases in species richness and diversity in rectal cancer samples, evidenced by the total number of OTUs and the Shannon and Simpson indexes. Enterotyping analysis divided our cohort into two groups, with the majority of rectal cancer samples clustering into one enterotype, characterized by a greater abundance of Bacteroides and Dorea. At the phylum level, rectal-cancer samples had increased abundance of candidate phylum OD1 (also known as Parcubacteria) whilst non-cancer samples had increased abundance of Planctomycetes. At the genera level, rectal-cancer samples had higher abundances of Bacteroides, Phascolarctobacterium, Parabacteroides, Desulfovibrio, and Odoribacter whereas non-cancer samples had higher abundances of Pseudomonas, Escherichia, Acinetobacter, Lactobacillus, and Bacillus. Two Bacteroides fragilis OTUs were more abundant among rectal-cancer patients seen through 16S rRNA amplicon sequencing, whose presence was confirmed by immunohistochemistry and enrichment verified by digital droplet PCR. Our findings point to increased bacterial richness and diversity in rectal cancer, along with several differences in microbial community composition. Our work is the first to present evidence for a possible role of bacteria such as B. fragilis and the phylum Parcubacteria in rectal cancer, emphasizing the need to study tissue-associated bacteria and specific regions of the gastrointestinal tract in order to better understand the possible links between the microbiota and rectal cancer.

Keywords: 16S rRNA gene sequencing; Bacterial diversity and community composition; Bacteroides fragilis; mucosa-associated microbiota; rectal cancer.

Figure 1

Alpha and beta diversity for non-cancer and rectal-cancer samples. (A) Rarefaction curves showing the average number of observed OTUs for both groups. Error bars represent ± standard error of the mean. Blue: Non-cancer samples; red: Rectal-cancer samples. (B) Rarefaction curves showing the average number of observed OTUs for NC samples and for smaller (pT2) and larger rectal tumors (pT3). Error bars represent ± standard error of the mean. (C) Boxplots showing alpha diversity in rectal-cancer samples and non-cancer samples using different metrics (Observed OTUs, Shannon index and Simpson index). (D) Principal Coordinate Analysis (PCoA) ordination plots for four distance metrics (Bray-Curtis, Jensen-Shannon Divergence, Weighted and Unweighted UniFrac). Ellipses represent the 95% confidence level assuming a multivariate t-distribution.

Figure 2

Enterotyping analysis reveals the presence of two community types. (A) Fitting to the Dirichlet Multinomial Mixture model indicates optimal classification into two community types. (B) Distribution of rectal-cancer samples and non-cancer samples in both enterotypes (P = 0.0001, Fisher's exact test). (C) Non-metric dimensional scaling (NMDS) ordination plot of Jensen–Shannon divergence values between samples. Red, community type-1; green, community type-2. (D) Relative abundances of the top 8 most abundant genera in the two community types.

Figure 3

Genera and OTU level differential abundance signatures. (A) Volcano plot for all 260 genera found in our samples. Red points indicate genera with an adjusted p-value <0.05; green points indicate genera with an adjusted p-value <0.05 and log₂FC > 1. Points circled in black are genera shown in the adjacent boxplot. (B) Boxplots showing log abundances for 5 genera with significant increases (top) and 5 genera with significant decreases in rectal-cancer samples (bottom). (C) Volcano plot for 1492 OTUs found in our samples. Points color scheme is the same as in (A). (D) Boxplots showing log abundances for 5 OTUs with significant increases (top) and 5 OTUs with significant decreases in rectal-cancer samples (bottom).

Figure 4

Alternative approaches demonstrating the presence of B. fragilis. (A) B. fragilis ddPCR quantification correlates with HTS-derived data. Using linear regression we obtained a correlation of R² = 0.78, P < 0.001. Blue: Non-cancer samples; red: Rectal-cancer samples. (B) Boxplot showing log10 of the ddPCR ratio found for B. fragilis after normalizing for RNAseP values for both groups. Blue: Non-cancer samples; red: Rectal-cancer samples. (C,D) Immunohistochemisty analysis of two B. fragilis-positive rectal-cancer samples, demonstrating the presence of this microbe (antibodies are labeled in red and shown with arrows) using magnification of 1000X.