High-dose multi-strain Bacillus probiotics enhance treatment and reduce antibiotic usage in children with persistent diarrhea through immune and microbiota modulation

Persistent diarrhea remains medical challenge to date regarding both its etiology and therapeutic interventions. Here, we conducted a randomized, double-blind, controlled clinical trial to evaluate the effectiveness of high-dose multi-strain Bacillus spore probiotics (LiveSpo DIA30) containing B. subtilis, B. clausii, and B. coagulans at 5 billion CFU/5 mL ampoule, in the supportive treatment of persistent diarrhea in children. Our findings revealed a significant 3-day shorter recovery period, 1.60-fold enhanced efficacy, and a 9.47-fold increase in odds (all p-values < 0.0001) for effectively resolving diarrhea by day 5 with the Bacillus spores. Notably, the group receiving LiveSpo DIA30 (Dia30 group) experienced a reduction in antibiotic treatment duration by 2 days compared to the Control group, equivalent to a 25% decrease in antibiotic usage. After 5 days of treatment, LiveSpo DIA30 demonstrated significant reductions in elevated blood pro-inflammatory cytokines, including IL-17 (26.62%; p = 0.0178), IL-23 (25.13%; p = 0.0256), TNF-α (19.09%; p = 0.038), and in fecal sIgA (24.24%; p = 0.0433). Analysis of 16S rRNA metagenome revealed that Dia30 group exhibited a notable increase in density of Actinomycetota and Bacillota phylum, along with Actinomycetaceae, Lactobacillaceae, and Streptococaceae families. Lacticaseibacillus rhamnosus, a beneficial gut species, was not detectable at day 0 but reached a density of 0.91% (p = 0.015) in Dia30 group by day 5. Additionally, Dia30 group showed a significant reduction in density of the Proteobacteria phylum, Enterobacteriaceae family, and harmful species Escherichia fergusoni (682.8-fold; p = 0.011). In conclusion, this clinical trial presents robust clinical evidence, supported by laboratory testing data, demonstrating the efficacy of multi-strain and high-concentration Bacillus spore probiotics in rapidly alleviating symptoms and reducing antibiotic usage in children with persistent diarrhea. This is archived by improving the native gut microbiota and modulating immunological responses. Trial registration: ClinicalTrials.gov, Identifier No: NCT05812820, 14/4/2023. © The Author(s) 2025.