The murine appendiceal microbiome is altered in spontaneous colitis and its pathological progression

Abstract

Background: Inflammatory bowel disease (comprising ulcerative colitis and Crohn's disease) is a multifactorial disease that is extensively associated with stool microbiome changes (dysbiosis). Appendicitis and appendectomy limits subsequent colitis, clinically, and in animal models. We wanted to examine how the appendiceal and stool microbiome fared in our spontaneous colitic Winnie (Muc2(-/-)) mice model.

Methods: Two C57BL/6 and 10 Winnie mice at ages 12 and 15 weeks were euthanized for stool and caecal patch samples. DNA was extracted using the QIAamp DNA Stool Mini Kit then the V1-V3 hypervariable region of the 16S rRNA gene was sequenced using the Roche/454 GS FLX + pyrosequencing instrument. A Galaxy metagenomic pipeline was used to define phyla and families at sequence similarity threshold of ≥ 80%.

Results: Bacteriodetes was decreased in 15-week Winnie mice appendices compared to corresponding stool samples (P < 0.01). Proteobacteria was increased in appendices of Winnie mice compared to corresponding stool samples (P < 0.05). The Bacteroidetes family Rikenellaceae could be identified only in 15-week-old Winnie mice appendices. A higher quantity of Acetobacteraceae (Proteobacteria phylum) was present in 15-week Winnie mice when compared to 12-week Winnie mice (P < 0.01). Helicobacteraceae (Proteobacteria phylum), which is prominent in all Winnie mice, is absent in control mice.

Conclusions: The appendiceal dysbiosis observed in our Winnie mice is commensurate with, and adds to extant literature data. The presence of Helicobacteraceae (Proteobacteria) only in colitic Winnie mice (but not control mice) is consistent with reports of increased Helicobacter in IBD patients. Bacteroides (Bacteroidetes) decreases may be a reflection of reduced anti-inflammatory commensal species such as B. fragilis. Further research is warranted to expand and delineate the relationship between IBD and the appendix microbiome.

Keywords: Appendix; Bacteroidetes; Colitis; Inflammatory bowel disease; Microbiome; Proteobacteria; Stool; Winnie.

Figure 1

The mouse caecal lymphoid patch (appendix). Normal murine caecal lymphoid patch (≈human appendix) – This is the pale milky-white area encircled with a ring. [License number to reproduce image from John Wiley and Sons – 3415650102997].

Figure 2

Overview of most common bacterial colonies in mouse appendices and stool. The Bacteriodetes phylum is the most abundant phylum in appendix and stool samples, from both C57BL/6 and Winnie mice. The Proteobacteria phylum is second most abundant in stool samples from both C57BL/6 and Winnie mice. The number of samples varied from 2–5 for each group.

Figure 3

Bacterial phyla signature of mouse appendix and stool specimens. Bacterial phyla estimates in appendix and stool samples from Winnie and C57BL/6 mice aged 12 or 15 weeks were assessed: (A) Relative abundance estimate of phylum Actinobacteria. (B) Relative abundance estimate of phylum Bacteroidetes. (C) Relative abundance estimate of phylum Firmicutes. (D) Relative abundance estimate of phylum Proteobacteria. Bacteriodetes was significantly lesser in 15-week Winnie appendices compared to 15-week Winnie stool samples (P < 0.01). Proteobacteria was significantly increased in Winnie mice appendices (12 weeks) in contrast to stool samples (P < 0.05). Proteobacteria was increased in Winnie group appendices (15 weeks) compared to stool samples (P < 0.05). No microbiome signature differences were observed between Winnie stool samples, and between Winnie appendix samples. Error bars represent standard error of the mean. The number of samples varied from 2–5 for each group.

Figure 4

Constituents and proportion of Bacteroidetes families in appendices. (A) Relative proportions of different families in Bacteroidetes appendix samples from C57BL/6; and 12-week-, and 15-week Winnie mice. There are no statistically significant differences. (B) Individual Bacteroidetes family preponderances appendix samples from C57BL/6; and 12-week-, and 15-week Winnie mice. Rikenellaceae are found only in 15-week-old Winnie mice. C57BL6: 2 samples; 12-week Winnie: 4 samples; 15-week Winnie: 5 samples.

Figure 5

Constituents and proportion of Proteobacteria families in appendices. (A) Relative proportions of different families in Proteobacteria appendix samples from C57BL/6; and 12-week-, and 15-week Winnie mice. Acetobacteraceae are increased in 15-week Winnie mice when compared to 12-week Winnie mice (P < 0.05). (B) Individual Proteobacteria family preponderances appendix samples from C57BL/6; and 12-week-, and 15-week Winnie mice. Helicobacteraceae was not present in C57BL/6 controls. Rhizobiaceae, Pasteurellaceae, Xanthomonadaceae, Vibrionaceae, Sphingomonadaceae, Bartonellaceae, Pseudomonadaceae, and Campylobacteraceae which had trace presence in 12-week Winnie mice appendices, flatten out at the 15-week time-point. C57BL6: 2 samples; 12-week Winnie: 4 samples; 15-week Winnie: 5 samples.

Figure 6

The role of the appendiceal microbiome in experimental spontaneous colitis. In this study, we note significant differences in appendiceal Proteobacterial compostion between controls and colitic (Winnie) mice. Acetobacteraceae is decreased in colitic mice. Helicobacteraceae, an unusual inhabitant of control mice appendices, is found abundantly in colitic mice. Rikenellaceae, a family belonging to the phylum Bacterioidetes, is found only in 15-week-old Winnie mice, but neither earlier, nor in controls.