biostudies-literatureUnknownCid-Rojas FNIAID NIH HHS<i>Clostridioides difficile</i> is a Gram-positive and obligate anaerobic spore-former pathogen. <i>C. difficile</i> spores are essential for the transmission and recurrence of <i>C. difficile</i> infections (CDI). A major challenge in sporulation studies in <i>C. difficile</i> is the low yield and asynchronous nature of this process. In this work, a hypersporulating strain, derivative of R20291, with an early sporulation onset and enhanced sporulation efficiency was isolated by serendipity. This strain had 1000-fold higher sporulation efficiency than the parental R20291 strain in sessile culture conditions. Electron micrographs revealed that spores of both strains have similar hair-like projections, electron-dense outer exosporium layer features. Whole genome sequencing and genomic analysbioRxiv : the preprint server for biology2025.11.25.690273https://www.ebi.ac.uk/biostudies/studies/S-EPMC12687779biostudies-literatureCharacterization of a hypersporulating strain derivative &lt;i&gt;of Clostridioides difficile&lt;/i&gt; R20291.PMC12687779R01 AI177842Cid-Rojas FParedes-Sabja DfalseCharacterization of a hypersporulating strain derivative &lt;i&gt;of Clostridioides difficile&lt;/i&gt; R20291.<i>Clostridioides difficile</i> is a Gram-positive and obligate anaerobic spore-former pathogen. <i>C. difficile</i> spores are essential for the transmission and recurrence of <i>C. difficile</i> infections (CDI). A major challenge in sporulation studies in <i>C. difficile</i> is the low yield and asynchronous nature of this process. In this work, a hypersporulating strain, derivative of R20291, with an early sporulation onset and enhanced sporulation efficiency was isolated by serendipity. This strain had 1000-fold higher sporulation efficiency than the parental R20291 strain in sessile culture conditions. Electron micrographs revealed that spores of both strains have similar hair-like projections, electron-dense outer exosporium layer features. Whole genome sequencing and genomic analys2025-01-01T00:00:00Z2025 Dec2026-06-04T03:15:55.251Z2026-06-04T03:11:13.125ZS-EPMC126877794137751510.1101/2025.11.25.690273