Amber N. McCoy

Fusobacterium Is Associated with Colorectal Adenomas

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.

Increased rectal microbial richness is associated with the presence of colorectal adenomas in humans.

Differences in the composition of the gut microbial community have been associated with diseases such as obesity, Crohn’s disease, ulcerative colitis and colorectal cancer (CRC). We used 454 titanium pyrosequencing of the V1–V2 region of the 16S rRNA gene to characterize adherent bacterial communities in mucosal biopsy samples from 33 subjects with adenomas and 38 subjects without adenomas (controls). Biopsy samples from subjects with adenomas had greater numbers of bacteria from 87 taxa than controls; only 5 taxa were more abundant in control samples. The magnitude of the differences in the distal gut microbiota between patients with adenomas and controls was more pronounced than that of any other clinical parameters including obesity, diet or family history of CRC. This suggests that sequence analysis of the microbiota could be used to identify patients at risk for developing adenomas.

Differential Gene Expression between African American and European American Colorectal Cancer Patients

The incidence and mortality of colorectal cancer (CRC) is higher in African Americans (AAs) than other ethnic groups in the U. S., but reasons for the disparities are unknown. We performed gene expression profiling of sporadic CRCs from AAs vs. European Americans (EAs) to assess the contribution to CRC disparities. We evaluated the gene expression of 43 AA and 43 EA CRC tumors matched by stage and 40 matching normal colorectal tissues using the Agilent human whole genome 4x44K cDNA arrays. Gene and pathway analyses were performed using Significance Analysis of Microarrays (SAM), Ten-fold cross validation, and Ingenuity Pathway Analysis (IPA). SAM revealed that 95 genes were differentially expressed between AA and EA patients at a false discovery rate of ≤5%. Using IPA we determined that most prominent disease and pathway associations of differentially expressed genes were related to inflammation and immune response. Ten-fold cross validation demonstrated that following 10 genes can predict ethnicity with an accuracy of 94%: CRYBB2, PSPH, ADAL, VSIG10L, C17orf81, ANKRD36B, ZNF835, ARHGAP6, TRNT1 and WDR8. Expression of these 10 genes was validated by qRT-PCR in an independent test set of 28 patients (10 AA, 18 EA). Our results are the first to implicate differential gene expression in CRC racial disparities and indicate prominent difference in CRC inflammation between AA and EA patients. Differences in susceptibility to inflammation support the existence of distinct tumor microenvironments in these two patient populations.

Differences in microbial signatures between rectal mucosal biopsies and rectal swabs

There is growing evidence the microbiota of the large bowel may influence the risk of developing colorectal cancer as well as other diseases including type-1 diabetes, inflammatory bowel diseases and irritable bowel syndrome. Current sampling methods to obtain microbial specimens, such as feces and mucosal biopsies, are inconvenient and unappealing to patients. Obtaining samples through rectal swabs could prove to be a quicker and relatively easier method, but it is unclear if swabs are an adequate substitute. We compared bacterial diversity and composition from rectal swabs and rectal mucosal biopsies in order to examine the viability of rectal swabs as an alternative to biopsies. Paired rectal swabs and mucosal biopsy samples were collected in un-prepped participants (n = 11) and microbial diversity was characterized by Terminal Restriction Fragment Length polymorphism (T-RFLP) analysis and quantitative polymerase chain reaction (qPCR) of the 16S rRNA gene. Microbial community composition from swab samples was different from rectal mucosal biopsies (p = 0.001). Overall the bacterial diversity was higher in swab samples than in biopsies as assessed by diversity indexes such as: richness (p = 0.01), evenness (p = 0.06) and Shannon’s diversity (p = 0.04). Analysis of specific bacterial groups by qPCR showed higher copy number of Lactobacillus (p < 0.0001) and Eubacteria (p = 0.0003) in swab samples compared with biopsies. Our findings suggest that rectal swabs and rectal mucosal samples provide different views of the microbiota in the large intestine.

Altered Tissue Metabolites Correlate with Microbial Dysbiosis in Colorectal Adenomas

Several studies have linked bacterial dysbiosis with elevated risk of colorectal adenomas and cancer. However, the functional implications of gut dysbiosis remain unclear. Gut bacteria contribute to nutrient metabolism and produce small molecules termed the “metabolome”, which may contribute to the development of neoplasia in the large bowel. We assessed the metabolome in normal rectal mucosal biopsies of 15 subjects with colorectal adenomas and 15 nonadenoma controls by liquid chromatography and gas chromatography time-of-flight mass spectrometry. Quantitative real-time PCR was used to measure abundances of specific bacterial taxa. We identified a total of 274 metabolites. Discriminant analysis suggested a separation of metabolomic profiles between adenoma cases and nonadenoma controls. Twenty-three metabolites contributed to the separation, notably an increase in adenoma cases of the inflammatory metabolite prostaglandin E2 and a decrease in antioxidant-related metabolites 5-oxoproline and diketogulonic acid. Pathway analysis suggested that differential metabolites were significantly related to cancer, inflammatory response, carbohydrate metabolism, and GI disease pathways. Abundances of six bacterial taxa assayed were increased in cases. The 23 differential metabolites demonstrated correlations with bacteria that were different between cases and controls. These findings suggest that metabolic products of bacteria may be responsible for the development of colorectal adenomas and CRC.

Fusobacterium spp. and colorectal cancer: cause or consequence?

Although increasing evidence suggests a relationship between bacterial dysbiosis and colorectal cancer (CRC), few studies have identified specific microbial etiologic factors. Recent studies have implicated overabundance of Fusobacterium in association with colorectal adenomas and cancer. Two articles published in Cell Host & Microbe provide insights into the Fusobacterium-CRC relationship.

Colonic Diverticula Are Not Associated With Mucosal Inflammation or Chronic Gastrointestinal Symptoms

BACKGROUND & AIMS: Colonic diverticulosis has been reported to be associated with low‐grade mucosal inflammation, possibly leading to chronic gastrointestinal symptoms. However, there is poor evidence for this association. We aimed to determine mucosal inflammation and whether diverticula are associated with chronic gastrointestinal symptoms. We explored whether inflammation was present among symptomatic participants with and without diverticula. METHODS: We analyzed data from a prospective study of 619 patients undergoing a screening colonoscopy from 2013 through 2015 at the University of North Carolina Hospital in Chapel Hill, North Carolina. Among our participants, 255 (41%) had colonic diverticula. Colonic mucosal biopsy specimens were analyzed for levels of interleukin 6 (IL6), IL10, and tumor necrosis factor messenger RNAs by quantitative reverse‐transcriptase polymerase chain reaction, and numbers of immune cells (CD4+, CD8+, CD57+, and mast cell tryptase) by immunohistochemistry. Gastrointestinal symptoms were assessed using Rome III criteria. Proportional odds models were used to estimate odds ratios (ORs) and 95% confidence interval (CIs). RESULTS: After adjustment for potential confounders, there was no association between diverticulosis and tumor necrosis factor (OR, 0.85; 95% CI, 0.63–1.16), and no association with CD4+ cells (OR, 1.18; 95% CI, 0.87–1.60), CD8+ cells (OR, 0.97; 95% CI, 0.71–1.32), or CD57+ cells (OR, 0.80; 95% CI, 0.59–1.09). Compared with controls without diverticulosis, biopsy specimens from individuals with diverticulosis were less likely to express the inflammatory cytokine IL6 (OR, 0.59; 95% CI, 0.36–0.96). There was no association between diverticulosis and irritable bowel syndrome (OR, 0.53; 95% CI, 0.26–1.05) or chronic abdominal pain (OR, 0.68; 95% CI, 0.38–1.23). There was no evidence for inflammation in patients with symptoms when patients with vs without diverticulosis were compared. CONCLUSIONS: We found no evidence that colonic diverticulosis is associated with mucosal inflammation or gastrointestinal symptoms. Among patients with symptoms and diverticula, we found no mucosal inflammation.

Maternal Gut Microbiome Biodiversity in Pregnancy

Objective To measure maternal gut microbiome biodiversity in pregnancy. Materials and Methods In phase 1, maternal fecal samples were collected by rectal swab in 20 healthy pregnant women (14‐28 weeks gestation) to measure bacterial abundance. In phase 2, fecal samples were collected from 31 women at enrollment (<20 weeks gestation, baseline) and at 36 to 39 weeks of gestation (follow‐up). We assessed cluster analysis to assess bacterial community profiles at the phylum level longitudinally through pregnancy. DNA was extracted from swabs, followed by PCR of the bacterial 16s rRNA gene and multiplex high‐throughput sequencing (Ion Torrent). Results In phase 1, 16 of 20 samples yielded usable data. White women (n = 10) had greater abundance of Firmicutes (23 ± 0.15 vs. 16% ± 0.75, p = 0.007) and Bacteroidetes (24 ± 0.14 vs. 19% ± 0.68, p = 0.015) compared with non‐White women (n = 6). In the 11 paired specimens, Bacteroidetes increased in abundance from baseline to follow‐up. Compared with women who gained weight below the median gestational weight gain (GWG, <15.4 kg), those who gained above the median GWG had increased abundance of Bacteroidetes (p = 0.02) and other phyla (p = 0.04). Conclusion Maternal microbiome biodiversity changes as pregnancy progresses and correlates with GWG.

An Aberrant Microbiota is not Strongly Associated with Incidental Colonic Diverticulosis

Colonic diverticula are protrusions of the mucosa through weak areas of the colonic musculature. The etiology of diverticulosis is poorly understood, but could be related to gut bacteria. Using mucosal biopsies from the sigmoid colon of 226 subjects with and 309 subjects without diverticula during first-time screening colonoscopy, we assessed whether individuals with incidental colonic diverticulosis have alternations in the adherent bacterial communities in the sigmoid colon. We found little evidence of substantial associations between the microbial community and diverticulosis among cases and controls. Comparisons of bacterial abundances across all taxonomic levels showed differences for phylum Proteobacteria (p = 0.038) and family Comamonadaceae (p = 0.035). The r-squared values measuring the strength of these associations were very weak, however, with values ~2%. There was a similarly small association between the abundance of each taxa and total diverticula counts. Cases with proximal only diverticula and distal only diverticula likewise showed little difference in overall microbiota profiles. This large study suggests little association between diverticula and the mucosal microbiota overall, or by diverticula number and location. We conclude that the mucosal adherent microbiota community composition is unlikely to play a substantial role in development of diverticulosis.

Mo1869 Gut Bacteria and Inflammation Are Associated With Diverticulosis

Diverticulosis is a common GI condition among older adults in the United States. However, limited information exists on the etiologic risk factors for diverticulosis. We hypothesized that the gut microbiome and chronic inflammation contribute to the development of diverticulosis.We evaluated the association between gut bacteria, mucosal inflammation and diverticulosis in a case-control study. Methods:Participants (n=196) were consented patients who underwent screening colonoscopy at UNC Hospitals. Subjects were classified as case if diverticula were present or control if no diverticula were present. The abundance of specific bacteria in the sigmoid mucosa was determined by qPCR based on the bacterial 16s rRNA gene for Bacteroides, Bifidobacterium, Clostridium, E. coli, Lactobacillus, and Eubacteria.Mucosal mRNA gene expression of cytokines of IL-6, IL-10, IL-17, and TNF-α and housekeeping gene HMBS were assessed by qRT-PCR. Immune cells in the lamina propria were stained for CD4, CD8, CD57 and Mast cell tryptase (MCT) by immunohistochemistry (IHC). The IHC stained sections were digitally imaged using the Aperio ScanScope XT. Analysis of biomarkers was performed using Definiens Tissue Studio software with the Composer_ Nuclei_and_Simulated_Cells algorithm. The percent cells with strong (+3), medium (+2) and weak (+1) positive signal, was used to compare biomarker levels among tissue samples. Statistical analysis included t-test and logistic regression to compare cases and controls as well as Spearman correlation to assess association between bacteria and inflammation. Results:We observed a higher relative abundance of Clostridium, Bacteroides, Bifidobacterium, E. coli, and Eubacteria in cases compared to controls. Cases had lower density of CD8 compared to controls. Although the cytokine gene expression levels of IL-6, IL-10, IL-17, and TNF-α did not differ between cases and controls, there was a significant positive correlation between CD57 expression and IL-6 (r=0.23, p=0.03), CD57 and TNFα (r0.33, p=0.001) and CD57 and IL-10 (r=0.22, p= 0.03) among cases. The association between bacterial abundance and immune cell density also varied depending on case-control status. Among cases, MCT expression showed positive correlations with Bacteroides (r=0.21, p= 0.05), Clostridium (r=0.18, p=0.08) and inverse correlation with Bifidobacteria (r=-0.26, p= 0.01). Among controls, CD57 expression was inversely correlated with Lactobacillus (r=0.19, p=0.06). Conclusions: Our findings suggest that gut bacteria and inflammation are associated with diverticulosis. This data combined with further research could have clinical implications for the prevention and treatment of diverticulosis.