In situ visualization of Clostridioides difficile phenotypic heterogeneity and single-cell morphology during gut infection

Phenotypic heterogeneity refers to the ability of clonal populations of the same species to display distinct phenotypes despite experiencing the same environment. Bacteria exhibit phenotypic heterogeneity across diverse cellular processes, which can provide competitive fitness advantages both within the host and against surrounding microbiota. However, visualizing phenotypic heterogeneity at single-cell resolution within dense microbial communities is technically challenging. Here, we present a method for visualizing this heterogeneity by combining spectrally compatible reporters to track the spatial distribution of gene expression in individual bacterial cells in the mammalian gut. Using toxin gene expression in Clostridioides difficile as a model for visualizing phenotypic heterogeneity, we demonstrate that, while C. difficile primarily occupies the lumen, a subpopulation of C. difficile associates with the colonic epithelium independent of toxin production. We further show that heterogeneity in C. difficile toxin gene expression is independent of location in the gut and that a toxin gene overexpressing mutant unexpectedly forms filamentous cells during the acute phase of infection. Thus, our reporter system provides quantitative, single-cell resolution of bacterial behavior within the intact gut environment and establishes a broadly applicable platform for investigating phenotypic heterogeneity in dense microbial communities.