Metagenomic insights into the effects of Clostridium butyricum and Bacillus subtilis probiotics on the gut microbiome and metabolic pathways of industrial broilers in Thailand

Probiotic supplementation has become increasingly important in broiler production due to its safety and well-documented health benefits. The gut microbiome of broilers plays a vital role in feed digestion and maintaining intestinal homeostasis, which directly influences the efficacy of probiotics under specific farm conditions. This study aims to investigate the effects of single Bacillus subtilis probiotics and double-strain probiotics of Clostridium butyricum and B. subtilis supplementation on the gut microbiome of broilers in industrial farms. We evaluated sequencing data obtained from broilers supplemented with these probiotics through amplicon sequencing and metagenomic analysis. Our study revealed that probiotics significantly influence the cecal microbiome and its functionality in broilers. The use of double-strain probiotics increased butanoate metabolism, as well as the metabolism of glycine, serine, and threonine. This suggests their contribution from microbial gut species, including Alistipes onderdonkii, Alistipes finegoldii, Bacteroides uniformis, and Phocaeicola dorei. Supporting this finding, network analysis shows more connections between probiotics and commensal cecal microbiota, highlighting a cascade-linked association with butanoate-producing microbiota. Furthermore, single-strain B. subtilis probiotic supplementation uniquely enhanced arginine and proline metabolism, likely due to the presence of species such as Bacteroides sp. zj-18, Bacteroides cellulosilyticus, and Parabacteroides distasonis. Overall, our findings indicate that double-strain probiotics increased richness in the cecal microbial community, reshaped the microbial network, and enriched short-chain fatty acid and amino acid metabolism, contributing to improved gut health and performance in broiler production.