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

John W Ribis·César Nieto·Nicholas V DiBenedetto·Anchal Mehra·Pola Kuhn·Qiwen Dong·Irene Nagawa·Imane El Meouche·Bree B Aldridge·Mary J Dunlop·Rita Tamayo·Abhyudai Singh·Aimee Shen
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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.

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Clostridioides difficileAnimalsTime-Lapse ImagingMiceClostridium InfectionsSingle-Cell AnalysisHumansPhenotypeJournal ArticleResearch Support, N.I.H., Extramural

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John W Ribis, César Nieto, Nicholas V DiBenedetto, Anchal Mehra, Pola Kuhn, Qiwen Dong, Irene Nagawa, Imane El Meouche, Bree B Aldridge, Mary J Dunlop, Rita Tamayo, Abhyudai Singh, Aimee Shen. 2025-05-21. Unique growth and morphology properties of Clade 5 Clostridioides difficile strains revealed by single-cell time-lapse microscopy.. https://doi.org/10.1371/journal.ppat.1013155

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