A crypt-specific core microbiota resides in the mouse colon

Abstract

In an attempt to explore the microbial content of functionally critical niches of the mouse gastrointestinal tract, we targeted molecular microbial diagnostics of the crypts that contain the intestinal stem cells, which account for epithelial regeneration. As current evidence indicates, the gut microbiota affects epithelial regeneration; bacteria that are likely to primarily participate in this essential step of the gut, microbiota cross talk, have been identified. We show in this article that only the cecal and colonic crypts harbor resident microbiota in the mouse and that regardless of the line and breeding origin of these mice, this bacterial population is unexpectedly dominated by aerobic genera. Interestingly, this microbiota resembles the restricted microbiota found in the midgut of invertebrates; thus, the presence of our so-called "crypt-specific core microbiota" (CSCM) in the mouse colon potentially reflects a coevolutionary process under selective conditions that can now be addressed. We suggest that CSCM could play both a protective and a homeostatic role within the colon. This article is setting the bases for such studies, particularly by providing a bona fide--and essentially cultivable--crypt microbiota of reference.

Importance: Metagenomic typing of the whole-gut luminal microbiome was recently provided, revealing great opportunities for physiological and physiopathological analysis of the host-microbiota interface. On this basis, it appears increasingly important to analyze which niches of the gut exposed to a particular microbiota are of major functional importance, specifically focusing on the crypt, which accounts for permanent epithelial renewal, and to analyze how this microbiota compares to its luminal counterpart in composition and quantity. Crypt-specific core microbiotas may show themselves as important elements regarding crypt protection and homeostasis of its functions.

FIG 1

Bacteria reside in the murine proximal colonic crypts. Warthin-Starry staining (A to D) or FISH (E to H) with the pan-bacterial probe Eub338 at different levels of the intestine of C57BL/6 mice is shown: small intestine (A and E), cecum (B and F), proximal colon (C and G), or distal colon (D and H). Black and white arrows indicate the presence of bacteria. Scale bars, 10 µM.

FIG 2

Unweighted Unifrac Bray-Curtis principal coordinate analysis shows dissimilarity in samples. Axis 1, percent variation explained (24.2%); axis 2, percent variation explained (17.17%). Squares and triangles represent crypt and luminal samples, respectively. Each murine strain is represented by a color code (C57BL/6 from provider 1, purple; C57BL/6 from provider 2, blue; BALB/c, green; C3H/HeN, orange).

FIG 3

Proportional abundances of the 20 most-represented OTUs in mice belonging to different strains at the crypt and luminal levels. These family-level phylogenetic relative contributions are based on 16S rRNA gene frequencies.

FIG 4

Acinetobacter and the Firmicutes are localized mainly in the crypt and lumen of the colon, respectively. Hybridization of an Alexa555-labeled Acinetobacter-specific probe on colonic sections from C57BL/6 (A), BALB/c (B), or C3H/HeN (C) or of an Alexa555-labeled Firmicutes-specific probe on colonic sections from C57BL/6 (D) is shown.

FIG 5

Acinetobacter colonizes the colonic crypt. (A) qRT-PCR amplification using phylum- and family-specific primers with DNA extracted from the feces of SPF C57BL/6 mice or from germfree BALB/c mice before and after 7, 15, and 26 days of colonization with conventional microbiota. (B and C) Warthin-Starry (B) or FISH using an Alexa555-labeled Acinetobacter-specific probe (C) on colonic crypt sections after 26 days of colonization of germfree BALB/c mice with a conventional microbiota. Scale bars, 10 µM.