An endolysin-expressing probiotic attenuates Clostridioides difficile infection in germ-free and dysbiotic microbiota-associated mice

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.