Quantitative whole-tissue 3D imaging reveals bacteria in close association with mouse jejunum mucosa

Because the small intestine (SI) epithelium lacks a thick protective mucus layer, microbes that colonize the thin SI mucosa may exert a substantial effect on the host. For example, bacterial colonization of the human SI may contribute to environmental enteropathy dysfunction (EED) in malnourished children. Thus far, potential bacterial colonization of the mucosal surface of the SI has only been documented in disease states, suggesting mucosal colonization is rare, likely requiring multiple perturbations. Furthermore, conclusive proof of bacterial colonization of the SI mucosal surface is challenging, and the three-dimensional (3D) spatial structure of mucosal colonies remains unknown. Here, we tested whether we could induce dense bacterial association with jejunum mucosa by subjecting mice to a combination of malnutrition and oral co-gavage with a bacterial cocktail (E. coli and Bacteroides spp.) known to induce EED. To visualize these events, we optimized our previously developed whole-tissue 3D imaging tools with third-generation hybridization chain reaction (HCR v3.0) probes. Only in mice that were malnourished and gavaged with the bacterial cocktail did we detect dense bacterial clusters surrounding intestinal villi suggestive of colonization. Furthermore, in these mice we detected villus loss, which may represent one possible consequence that bacterial colonization of the SI mucosa has on the host. Our results suggest that dense bacterial colonization of jejunum mucosa is possible in the presence of multiple perturbations and that whole-tissue 3D imaging tools can enable the study of these rare events.