Genomic and Phenotypic Characterization of the Nontoxigenic Clostridioides difficile Strain CCUG37785 and Demonstration of Its Therapeutic Potential for the Prevention of C. difficile Infection.

Symptoms of Clostridioides difficile infection (CDI) are attributed largely to two toxins, TcdA and TcdB. About 17-23% of C. difficile isolates produce binary toxin, which enhances C. difficile pathogenesis. Previously, we engineered the nontoxigenic C. difficile strain CCUG37785 (designated as CCUG37785) to express immunogenic fragments of TcdA and TcdB as an oral mucosal CDI vaccine candidate. In this study, we performed genomic and phenotypic analyses of CCUG37785 and evaluated its potential use for preventing and treating CDI. Whole genome sequencing showed that CCUG37785 is ribotype ST3 and lacks toxin genes. Comparative analyses of PaLoc and CdtLoc loci of CCUG37785 revealed 115-bp and 68-bp conserved fragments in these regions, respectively. Phenotypic comparisons between CCUG37785 and C. difficile R20291 (an epidemic hypervirulent BI/NAPI/027 strain, designated as R20291) found that CCUG37785 exhibited significantly higher adhesion and sporulation, significantly lower spore germination and biofilm formation, and comparable motility to R20291. We also showed that oral inoculation of CCUG37785 spores prior to infection with R20291 spores provided mice almost full protection against developing CDI. However, oral inoculation of CCUG37785 spores after infection with R20291 spores only provided minor protection against CDI. Further analysis showed that mice pretreated with CCUG37785 spores secreted significantly less R20291 spores, while mice treated with CCUG37785 spores after infection with R20291 secreted a comparable amount of R20291 spores to mice infected with R20291 spores only. Our data both highlight the potential use of CCUG37785 for the prevention of primary and recurrent CDI in humans and support its use as an oral mucosal vaccine carrier against CDI.