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

Abstract

Clostridioides difficile is a gastrointestinal pathogen of both humans and agricultural animals and thus a major One Health threat. The C. difficile species consists of five main clades, with Clade 5 currently undergoing speciation from Clades 1–4. Since Clade 5 strains are highly prevalent in agricultural animals and a frequent cause of zoonotic infections, these strains may have evolved phenotypes that distinguish them from Clade 1–4 strains. Here, we compare the growth properties of Clade 5 strains to those of Clade 1–4 strains using anaerobic time-lapse microscopy coupled with automated image analysis. Our analyses indicate that Clade 5 strains grow faster and are more likely to form long chains of cells than Clade 1–4 strains. Using comparative genomic and CRISPRi analyses, we show that the chaining phenotype of Clade 5 strains is driven by the orientation of the invertible cmr switch sequence, with chaining strains exhibiting a bias to the cmr-ON state. Interestingly, Clade 5 strains with a bias towards the cmr-ON state shifted to a largely cmr-OFF state during murine infection, suggesting that the cmr-OFF state is under positive selection during infection. Collectively, our data reveal that Clade 5 strains have distinct growth properties, which may allow them to inhabit diverse ecological niches.