Ashley S. Weiss

Clostridioides difficile causes severe disease in adults but commonly colonizes infants asymptomatically. The consequences of early-life colonization on host development remain unknown. In a neonatal mouse model, C. difficile colonization drove proinflammatory and tissue repair responses in the intestinal epithelium, enriching injury-associated intestinal stem cell populations and skewing differentiation toward secretory lineages. Despite transient colonization, exposure to C. difficile early in life led to persistent changes into adulthood. Epithelial responses were toxin dependent, as colonization with nontoxigenic strains or maternal vaccination with a C. difficile –targeted messenger RNA–lipid nanoparticle vaccine protected neonates. Human infant intestinal epithelial cells were sensitive to C. difficile toxins, and biopsies from colonized infants exhibited altered intestinal stem cell behavior. This study redefines C. difficile as an underappreciated early-life pathogen with lasting effects on host development.

To adapt and persist in the gastrointestinal tract, many enteric pathogens, including Clostridioides difficile , employ strategies such as phase variation to generate phenotypically heterogeneous populations. Notably, the role of the gut microbiota and polymicrobial interactions in shaping population heterogeneity of invading pathogens has not been explored. Here, we show that Enterococcus faecalis , an opportunistic pathogen that thrives in the inflamed gut during C. difficile infection, can impact the phase-variable CmrRST signal transduction system in C. difficile . The CmrRST system controls multiple phenotypes, including colony morphology, cell elongation, and cell chaining in C. difficile . Here, we describe how interactions between E. faecalis and C. difficile on solid media lead to a marked shift in C. difficile phenotypes associated with phase variation of CmrRST. Specifically, E. faecalis drives a switch of the C. difficile population to the cmr- ON state, leading to chaining and a rough colony morphology. This phenomenon is most pronounced with E. faecalis , as other enterococcal species and select Gram-negative enteric bacteria do not show a similar effect. These results suggest that the composition of the polymicrobial environment in the gut is critical to influencing C. difficile population heterogeneity. Our findings shed light on the complex role that microbial ecology and polymicrobial interactions can have in the phenotypic heterogeneity of invading pathogens. IMPORTANCE: Clostridioides difficile is an enteric pathogen with critical implications for public health. The microbial ecosystem in which C. difficile resides shapes the behavior and fitness of C. difficile ; however, the mechanisms underlying these interactions are not well defined. Here, we demonstrate that Enterococcus faecalis , an opportunistic pathogen known to co-colonize the gut with C. difficile , influences phase variation and downstream growth phenotypes in C. difficile . This phenomenon represents a new paradigm by which co-residing bacteria can modulate phase variation dynamics in C. difficile or other enteric pathogens. Understanding factors that influence C. difficile behavior may elucidate new therapeutic strategies, especially in complex polymicrobial infections.