Characterization of the fecal and mucosa-associated microbiota in dogs with colorectal epithelial tumors

Abstract

Colorectal epithelial tumors occur spontaneously in dogs, and the pathogenesis seems to parallel that of humans. The development of human colorectal tumorigenesis has been linked to alterations in the composition of the intestinal microbiota. This study characterized the fecal- and mucosa-associated microbiota in dogs with colorectal epithelial tumors (n = 10). The fecal microbiota was characterized by 16S rDNA analysis and compared with that of control dogs (n = 13). We also determined the mucosa-associated microbiota composition in colonic tumor tissue (n = 8) and in adjacent non-tumor tissue (n = 5) by 16S rDNA- and rRNA profiling. The fecal microbial community structure in dogs with tumors was different from that of control samples and was distinguished by oligotypes affiliated with Enterobacteriaceae, Bacteroides, Helicobacter, Porphyromonas, Peptostreptococcus and Streptococcus, and lower abundance of Ruminococcaceae, Slackia, Clostridium XI and Faecalibacterium. The overall community structure and populations of mucosal bacteria were not different based on either the 16S rDNA or the 16S rRNA profile in tumor tissue vs. adjacent non-tumor tissue. However, the proportion of live, potentially active bacteria appeared to be higher in non-tumor tissue compared with tumor tissue and included Slackia, Roseburia, unclass. Ruminococcaeceae, unclass. Lachnospiraceae and Oscillibacter. Colorectal tumors are rarely diagnosed in dogs, but despite this limitation, we were able to show that dogs with colorectal tumors have distinct fecal microbiota profiles. These initial results support the need for future case-control studies that are adequately powered, as well as age-matched and breed-matched, in order to evaluate the influence of bacteria on colorectal cancer etiopathogenesis and to determine whether the bacteria may have potential as biomarkers in clinical settings.

Fig 1. The relative abundance of OTUs at the genus level in fecal samples of control dogs and dogs with colorectal tumors (polyps, adenoma, carcinoma).

The data are based on 16S rDNA and shows the 10 most abundant OTUs in each sample. Numbers at each bar base correspond to the “Dog id” in Table 1.

Fig 2. The bacterial community structure based on weighted UniFrac distance metric in fecal samples from dogs with tumors and control dogs.

The nMDS plot shows the bacterial community structure in control dogs (orange, n = 13) and dogs with colorectal tumors (black, n = 10) based on the 16S rDNA data. Differences among these groups were significant (PERMANOVA, Pseudo-F = 3, p = 0.02 and ANOSIM, R Statistics = 0.27, p = 0.02).

Fig 3. Differentially abundant bacterial taxa in fecal samples from dogs with tumors and control dogs.

A bar plot showing the LDA score (effect size) of the oligotypes that were differentially abundant in fecal samples of control dogs (red, n = 13) and dogs with colorectal tumors (green, n = 10) as determined by Linear Discriminant Effect Size (LEfSe) analysis (α = 0.05, LDA score > 2.0). The number after the taxa name corresponds to the oligotype number (ot. no.).

Fig 4. A non-metric multidimensional scaling (nMDS) plot based on the weighted UniFrac distance metric showing the bacterial community structure for paired mucosal samples at the 16S rDNA (brown) and 16S rRNA (orange) level from eight dogs with colorectal tumors.

Numbers at each bar base correspond to the “Dog id” in Table 1. Differences between these groups were not significant (PERMANOVA p>0.1).

Fig 5. The relative abundance of OTUs at genus level in mucosal samples based on paired 16S rRNA and 16S rDNA data from 8 dogs with colorectal tumors (polyp, adenoma and carcinoma).

Numbers at each bar base correspond to the “Dog id” in Table 1. The 10 most abundant OTUs in each sample are shown.

Fig 6. A non-metric multidimensional scaling (nMDS) plot based on the weighted UniFrac distance metric showing the microbial community structure based on tumor (black) and adjacent non-tumor tissue (orange) from five dogs with colorectal tumors.

The data are based on the 16S rDNA data. Labels adjacent to data points correspond to the “Dog id” in Table 1. Differences between these groups were not significant (PERMANOVA p>0.1).

Fig 7. A scatterplot showing the ratio of live, potentially active bacteria (RNA/DNA) in tumor vs. non-tumor tissue.