Fusobacterium is associated with colorectal adenomas

Abstract

The human gut microbiota is increasingly recognized as a player in colorectal cancer (CRC). While particular imbalances in the gut microbiota have been linked to colorectal adenomas and cancer, no specific bacterium has been identified as a risk factor. Recent studies have reported a high abundance of Fusobacterium in CRC subjects compared to normal subjects, but this observation has not been reported for adenomas, CRC precursors. We assessed the abundance of Fusobacterium species in the normal rectal mucosa of subjects with (n = 48) and without adenomas (n = 67). We also confirmed previous reports on Fusobacterium and CRC in 10 CRC tumor tissues and 9 matching normal tissues by pyrosequencing. We extracted DNA from rectal mucosal biopsies and measured bacterial levels by quantitative PCR of the 16S ribosomal RNA gene. Local cytokine gene expression was also determined in mucosal biopsies from adenoma cases and controls by quantitative PCR. The mean log abundance of Fusobacterium or cytokine gene expression between cases and controls was compared by t-test. Logistic regression was used to compare tertiles of Fusobacterium abundance. Adenoma subjects had a significantly higher abundance of Fusobacterium species compared to controls (p = 0.01). Compared to the lowest tertile, subjects with high abundance of Fusobacterium were significantly more likely to have adenomas (OR 3.66, 95% CI 1.37-9.74, p-trend 0.005). Cases but not controls had a significant positive correlation between local cytokine gene expression and Fusobacterium abundance. Among cases, the correlation for local TNF-α and Fusobacterium was r = 0.33, p = 0.06 while it was 0.44, p = 0.01 for Fusobacterium and IL-10. These results support a link between the abundance of Fusobacterium in colonic mucosa and adenomas and suggest a possible role for mucosal inflammation in this process.

Figure 1. Abundance of Fusobacterium in rectal mucosal biopsies from adenoma cases and non-adenoma controls.

qPCR results show that Fusobacterium is more abundant in cases than controls.

Figure 2. Representative fluorescence in situ hybridization targeting Fusobacterium sp. in colorectal mucosal biopsy sections using bacterial 16S rRNA probes.

Fig. 2A–B are composite images of Cy3 and DAPI views of sections hybridized with a Fusobacterium-specific probe. Fusobacterium species is localized within the mucus layer of colorectal sections (A) 20X and 40X. Fusobacterium species is localized within the crypts of colorectal section (B) 20X and 40X. Fig. 2C (20X and 40X) is a positive control and shows sections stained with general bacteria probe (Eub 388). General bacteria, including most Eubacteria species, are localized to the mucus layer above the epithelium. White arrows point to bacteria either in mucus layer above the colonic epithelium or within the crypt.

Figure 3. Correlations between Fusobacterium abundance and local cytokine gene expression in adenoma cases and non-adenoma controls.

Results suggest a significant positive correlation between Fusobacterium abundance and local inflammation in cases but not controls. The Correlations were significant for IL-10 (r = 0.44, p = 0.01) and TNF-α (r  = 0.33, p = 0.06). *p<0.05.

Figure 4. Log Abundance of Fusobacterium in matched normal colon and colorectal cancer tissue.

Fusobacterium abundance was evaluated in DNA samples from normal colon and tumor tissue by qPCR using Fusobacterium-specific primers. Results suggest that Fusobacterium is increased in colon cancer tissue compared to normal tissue (t-test p = 0.0005).