Unique growth and morphology properties of Clade 5Clostridioides difficilestrains revealed by single-cell time-lapse microscopy

Abstract

AbstractClostridioides difficileis a major One Health threat as a gastrointestinal pathogen of both humans and agricultural animals. TheC. difficilespecies consists of five main clades, with Clade 5 currently undergoing speciation from Clades 1-4. Notably, Clade 5 strains are highly prevalent in agricultural animals and can cause zoonotic infections, suggesting that Clade 5 strains have evolved phenotypes that distinguish them from Clade 1-4 strains. Here, we compare the growth properties of Clade 5 strains to Clade 1, 2, and 4 strains using anaerobic time-lapse microscopy coupled with automated image analysis. These analyses reveal that Clade 5 strains grow faster than Clade 1, 2, and 4 strains and are more likely to form long chains of cells. Comparative genomic analyses revealed that the orientation of the invertiblecmrswitch sequence likely drives the cell chaining phenotype of Clade 5 strains during growth in rich media. Interestingly, during murine infection, Clade 5 strains with a high frequency of thecmrswitch in the ON orientation shifted to a largelycmrOFF state, suggesting that thecmrOFF state may be positively selected during infection. Taken together, our data suggest that Clade 5 strains have distinct growth properties that allow them to inhabit diverse ecological niches.ImportanceThe Clade 5 strains of theClostridioides difficilespecies are so phylogenetically divergent that they almost meet the threshold of being a distinct species. Although these strains are ubiquitously isolated from agricultural and environmental settings and an important source of zoonotic and community-acquired infections, it is unclear whether they have distinct phenotypic properties that allow them to efficiently colonize diverse hosts or persist in the environment. By combining a novel anaerobic time-lapse microscopy method with automated image analysis, we discovered that Clade 5 strains grow faster than strains from otherC. difficileclades and that they frequently form long chains. These chaining properties appear to be driven by the environmentally responsive expression of a non-canonical signal transduction system, which we also found is likely selected against during growth in mice. Collectively, our analyses reveal that Clade 5 strains have distinct growth properties that may promote their persistence in the environment.