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bioRxiv · 10.1101/2024.02.13.580212

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

Ribis, J. W.·Nieto-Acuna, C. A.·Dong, Q.·DiBenedetto, N.·Mehra, A.·Nagawa, I. L.·El Meouche, I.·Aldridge, B. B.·Dunlop, M. J.·Tamayo, R.·Singh, A.·Shen, A.
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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. Clade 5 strains are highly prevalent in agricultural animals and can cause zoonotic infections, suggesting that these strains 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. Author SummaryThe Clade 5 strains of the Clostridioides difficile species 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 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 other C. difficile clades and that they frequently form long chains. These chaining properties are driven by the environmentally responsive expression of a non-canonical signal transduction system, which our analyses suggest is selected against during murine infection. Collectively, our analyses reveal that Clade 5 strains have distinct growth properties that may promote their persistence in the environment.

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Ribis, J. W., Nieto-Acuna, C. A., Dong, Q., DiBenedetto, N., Mehra, A., Nagawa, I. L., El Meouche, I., Aldridge, B. B., Dunlop, M. J., Tamayo, R., Singh, A., Shen, A.. 2024-02-14. Unique growth and morphology properties of Clade 5 Clostridioides difficile strains revealed by single-cell time-lapse microscopy. https://doi.org/10.1101/2024.02.13.580212

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