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

Abstract

Clostridioides difficileis a major One Health threat as an important gastrointestinal pathogen of both humans and agricultural animals. TheC. difficilespecies can be subdivided into 5 main clades, with Clade 5 currently undergoing speciation from Clades 1-4. Since Clade 5 strains are found more frequently in agricultural animals and can cause zoonotic infections, Clade 5 strains likely 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 an anaerobic time-lapse microscopy system coupled with automated image analysis. These analyses revealed that Clade 5 strains grow faster than Clade 1, 2, and 4 strains and are more likely to form long chains of cells. Notably, the chaining phenotype was not shared among all Clade 5 strains examined, since 1 of the 9 strains did not form chains. Genomic analyses of the Clade 5 strains revealed that the orientation of thecmrswitch, an invertible DNA element controlling the expression of a signal transduction system that regulates chaining, correlates with the propensity of a given Clade 5 strain to form chains. Taken together, Clade 5 strains appear to have distinct growth properties that allow them to inhabit more diverse ecological niches.