3D imaging for the quantification of spatial patterns in microbiota of the intestinal mucosa

Improving our understanding of host-microbe relationships in the gut requires the ability to both visualize and quantify the spatial organization of microbial communities in their native orientation with the host tissue. We developed a systematic procedure to quantify the 3D spatial structure of the native mucosal microbiota in any part of the intestines with taxonomic and high spatial resolution. We performed a 3D biogeographical analysis of the microbiota of mouse cecal crypts at different stages of antibiotic exposure. By tracking eubacteria and four dominant bacterial taxa, we found that the colonization of crypts by native bacteria is a dynamic and spatially organized process. Ciprofloxacin treatment drastically reduced bacterial loads and eliminated Muribaculaceae (or all Bacteroidetes entirely) even 10 days after recovery when overall bacterial loads returned to pre-antibiotic levels. Our 3D quantitative imaging approach revealed that the bacterial colonization of crypts is organized in a spatial pattern that consists of clusters of adjacent colonized crypts that are surrounded by unoccupied crypts, and that this spatial pattern was resistant to the elimination of Muribaculaceae or of all Bacteroidetes by ciprofloxacin. Our approach also revealed that the composition of cecal crypt communities is diverse and that bacterial taxa are distributed differently within crypts, with Lactobacilli laying closer to the lumen than Bacteroidetes, Ruminococcaceae, and Lachnospiraceae. Finally, we found that crypts communities with similar taxonomic composition were physically closer to each other than communities that were taxonomically different. Significance StatementMany human diseases are causally linked to the gut microbiota, yet the field still lacks mechanistic understanding of the underlying complex interactions because existing tools cannot simultaneously quantify microbial communities and their intact native context. In this work, we provide a new approach to tissue clearing and preservation that enables visualization, in 3D and at scales ranging from centimeters to micrometers, of the complete biogeography of the host-microbiota interface. We combine this new tool with sequencing and multiplexed labelling of the microbiota to provide the field with a platform on which to discover patterns in the spatial distribution of microbes. We validated this platform by quantifying the distribution of bacteria in the cecal mucosa at different stages of antibiotic exposure. This approach will enable researchers to formulate and test new hypotheses about host-microbe and microbe-microbe interactions.