Probiotics are often advised to be taken separately from antibiotics due to their sensitivity to antibiotic activity and gastrointestinal (GI) stress. However, Clostridium butyricum spores, as next-generation probiotics, may withstand concurrent use with antibiotics due to their unique structural adaptations. This study systematically evaluated the survival rates and morphological changes of C. butyricum spores exposed to 10 clinically relevant antibiotics in simulated gastric/intestinal fluids, exploring their feasibility for co-administration with antibiotics. Survival rates of C. butyricum spores were tested against 10 antibiotics across four classes (β-lactams, macrolides, aminoglycosides, and tetracyclines) in simulated GI fluids. Time–kill curves analyzed spore survival over 0–4 h, while scanning electron microscopy (SEM) observed spore wall integrity and morphological changes under different conditions. The spore survival rates remained >89% in intestinal fluid and >60% in gastric fluid across all antibiotics. SEM revealed gastric acid and proteolysis increased spore wall fragmentation, reducing resistance, whereas the intestinal environment preserved spore integrity. This study was the first to demonstrate that C. butyricum spores can survive simultaneous exposure to antibiotics in the gastrointestinal tract, challenging traditional probiotic usage guidelines. The findings support their co-administration with antibiotics to simplify dosing regimens and improve medication adherence. Such an approach advances antimicrobial stewardship by optimizing therapeutic strategies for antibiotic–probiotic combinations.

Most probiotics require separate administration from antibiotics due to sensitivity issues. Clostridium butyricum, however, exhibits intrinsic resistance, making it a promising candidate for combined therapy against diarrhea. In this study, a diarrhea model was established in mice induced by Escherichia coli, followed by treatment with azithromycin (AZM), C. butyricum (RH₂), or their combination (COM) to assess therapeutic efficacy. The results demonstrated that mice in RH₂ and COM groups achieved full body weight recovery and significant alleviation of diarrhea, accompanied by normalized fecal E. coli loads, preserved tissue integrity, reduced pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), and elevated anti-inflammatory IL-10. In contrast, AZM treatment led to sex-specific disparities in weight recovery and E. coli loads, and both sexes experienced relapse-prone diarrhea. Furthermore, the AZM group displayed shortened colons, sustained inflammatory infiltration, epithelial damage, and elevated IL-1β and male-specific IL-6. Gut microbiota analysis revealed that the COM group retained beneficial genera (e.g., Parabacteroides, Blautia) from the AZM group while uniquely enriching Lachnospiraceae taxa (e.g., NK4A136_group, FCS020_group). Untargeted metabolomics demonstrated the COM group activated GABA/arginine pathways, enhancing anti-inflammatory and barrier functions, whereas azithromycin disrupted butyrate synthesis and steroid metabolism. These findings highlight the advantage of combining C. butyricum with azithromycin for intestinal protection.