ENAapplication/xmlftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR327/025/SRR32797225/SRR32797225_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR327/026/SRR32797226/SRR32797226_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR327/029/SRR32797229/SRR32797229_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR327/028/SRR32797228/SRR32797228_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR327/028/SRR32797228/SRR32797228_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR327/027/SRR32797227/SRR32797227_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR327/025/SRR32797225/SRR32797225_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR327/029/SRR32797229/SRR32797229_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR327/027/SRR32797227/SRR32797227_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR327/026/SRR32797226/SRR32797226_1.fastq.gzprimaryOK200GenomicsTufts University/Tufts Medical Centerhttps://www.ebi.ac.uk/ena/browser/view/PRJNA1238863Clostridioides difficileClostridioides 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 genetic 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.pathogen:prioritypathogen:bacteriumpathogenbacteriaENAfalseClostridioides difficileUnique growth and morphology properties of Clade 5 Clostridioides difficile strains revealed by single-cell time-lapse microscopy2025-09-242025-03-22PRJNA12388631496