Probiotics have been widely used to prevent primary Clostridioides difficile infection (pCDI); however, there are fewer studies on their therapeutic aspects for pCDI. In this study, high doses of Bifidobacterium breve YH68 were used alone or in combination with vancomycin (VAN) and metronidazole (MTR) to treat pCDI mice. Mouse feces were collected from preinfection, postinfection, and posttreatment stages. Subsequently, the C. difficile number and toxin level in feces were detected by plate count method and C. difficile toxin enzyme-linked immunosorbent assay (ELISA). Simultaneously, 16S rRNA amplicon sequencing and untargeted metabolomics were employed to explore the changing patterns and characteristic markers of fecal microbiota and metabolome. The results indicated that high doses of YH68 used alone or in combination with VAN and MTR were more effective than the combination of VAN and MTR for pCDI mice and improved their final survival rate. This probiotic strain and its combination with antibiotics reduced C. difficile numbers and toxin levels in the feces, downregulated proinflammatory cytokine levels in colon tissue, and alleviated cecum tissue hyperplasia. Meanwhile, the level of fecal microbiota diversity increased significantly in pCDI mice after treatment, with an increase in the relative abundance of , , , unidentified, and , and this process was accompanied by elevated levels of secondary bile acid, butyric acid, and gentamicin C1a and reduced levels of primary bile acid and indoles. Most notably, the combination of YH68 with VAN and MTR diminished the damaging effect of antibiotic treatment alone on the microbiota. Our findings suggested that high doses of YH68 used in combination with VAN and MTR have a better therapeutic effect on pCDI mice than the combination of VAN and MTR alone. Many studies have focused on the preventive effects of probiotics against pCDI, but few studies have investigated in depth the therapeutic effects of probiotics, especially at the postinfection stage. We demonstrated that high doses of Bifidobacterium breve YH68 used alone or in combination with vancomycin (VAN) and metronidazole (MTR) exerted outstanding efficacy in the treatment of pCDI mice. This probiotic-antibiotic combination regimen has the potential to be a new option for the clinical treatment of pCDI.
