An endolysin-expressing probiotic attenuates Clostridioides difficile infection in germ-free and dysbiotic microbiota-associated mice
- 1Oklahoma State University
Abstract
Disruption of the intestinal microbiota increases susceptibility to Clostridioides difficile infection (CDI), creating an opportunity for prophylactic interventions before symptomatic disease develops. We investigated whether Escherichia coli Nissle 1917 (EcN) engineered to express the bacteriophage-derived endolysin CD27L could enhance pathogen inhibition and attenuate CDI. Using a plasmid-cured EcN chassis and pMUT2-derived vectors, we generated strains expressing CD27L under constitutive or anaerobically regulated promoter control. The constitutive strain, BioEcN1, reduced viable C. difficile recovery relative to parental EcN and was selected for animal studies. BioEcN1 remained recoverable from monoassociated mice without in vivo antibiotic selection, with similar recovery on selective and nonselective media supporting construct retention. Prophylactic administration reduced clinical disease, body-weight loss, and intestinal histopathological injury in germ-free mice. Observed survival was 100% in BioEcN1-treated challenged mice versus 50% in untreated challenged mice during the monitored period. Protection was accompanied by lower fecal and cecal pathogen burdens. In mice harboring a human-derived dysbiotic microbiota, prophylactic BioEcN1 also reduced clinical and stool-consistency scores, with lower early fecal pathogen recovery and less colon shortening. These findings establish preclinical proof of concept for prophylactic administration of an endolysin-expressing probiotic to attenuate CDI.
Related
- Phage endolysin enables targeted manipulation of the small intestinal microbiota and uncovers niche overlap between oral and butyrate-producing taxa
- Fecal microbiota transplantation restores gut microbiota diversity and butyrogenic taxa more effectively than fidaxomicin in recurrent Clostridioides difficile infection
- The Microbiota Dictates Vendor-Derived Differences in a Murine Clostridioides difficile Infection Model
- Mechanistic prediction of fecal microbiota transplant engraftment and Clostridioides difficile colonization resistance with μODE
- C3a receptor signalling in ObRb-expressing neurons promotes leptin resistance and obesity-associated cardiac remodelling
BibTeX
@misc{cheemadan2026endolysinexpressing,
title = {{An endolysin-expressing probiotic attenuates Clostridioides difficile infection in germ-free and dysbiotic microbiota-associated mice}},
author = {A. Cheemadan and P. Harnvoravongchai and S. More and P. Kashyap and J. Scaria},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.07.757339},
url = {https://www.biorxiv.org/content/10.64898/2026.10.07.757339v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - An endolysin-expressing probiotic attenuates Clostridioides difficile infection in germ-free and dysbiotic microbiota-associated mice
AU - A. Cheemadan
AU - P. Harnvoravongchai
AU - S. More
AU - P. Kashyap
AU - J. Scaria
PY - 2026
DA - 2026/10/07
PB - bioRxiv
DO - 10.64898/2026.10.07.757339
UR - https://www.biorxiv.org/content/10.64898/2026.10.07.757339v1
AB - Disruption of the intestinal microbiota increases susceptibility to Clostridioides difficile infection (CDI), creating an opportunity for prophylactic interventions before symptomatic disease develops. We investigated whether Escherichia coli Nissle 1917 (EcN) engineered to express the bacteriophage-derived endolysin CD27L could enhance pathogen inhibition and attenuate CDI. Using a plasmid-cured EcN chassis and pMUT2-derived vectors, we generated strains expressing CD27L under constitutive or anaerobically regulated promoter control. The constitutive strain, BioEcN1, reduced viable C. difficile recovery relative to parental EcN and was selected for animal studies. BioEcN1 remained recoverable from monoassociated mice without in vivo antibiotic selection, with similar recovery on selective and nonselective media supporting construct retention. Prophylactic administration reduced clinical disease, body-weight loss, and intestinal histopathological injury in germ-free mice. Observed survival was 100% in BioEcN1-treated challenged mice versus 50% in untreated challenged mice during the monitored period. Protection was accompanied by lower fecal and cecal pathogen burdens. In mice harboring a human-derived dysbiotic microbiota, prophylactic BioEcN1 also reduced clinical and stool-consistency scores, with lower early fecal pathogen recovery and less colon shortening. These findings establish preclinical proof of concept for prophylactic administration of an endolysin-expressing probiotic to attenuate CDI.
ER -