The agricultural industry utilizes antibiotic growth promoters to promote livestock growth and health. However, the World Health Organization has raised concerns over the ongoing spread of antibiotic resistance transmission in the populace, leading to its subsequent ban in several countries, especially in the European Union. These restrictions have translated into an increase in pathogenic outbreaks in the agricultural industry, highlighting the need for an economically viable, non-toxic, and renewable alternative to antibiotics in livestock. Probiotics inhibit pathogen growth, promote a beneficial microbiota, regulate the immune response of its host, enhance feed conversion to nutrients, and form biofilms that block further infection. Commonly used lactic acid bacteria probiotics are vulnerable to the harsh conditions of the upper gastrointestinal system, leading to novel research using spore-forming bacteria from the genus Bacillus. However, the exact mechanisms behind Bacillus probiotics remain unexplored. This review tackles this issue, by reporting antimicrobial compounds produced from Bacillus strains, their proposed mechanisms of action, and any gaps in the mechanism studies of these compounds. Lastly, this paper explores omics approaches to clarify the mechanisms behind Bacillus probiotics.

Antibiotic growth promoters (AGPs) suppress the growth of infectious pathogens. These pathogens negatively impact agricultural production worldwide and often cause health problems if left untreated. Here, we evaluate six Bacillus strains (BPR-11, BPR-12, BPR-13, BPR-14, BPR-16 and BPR-17), which are known for their ability to survive harsh environmental conditions, as AGP replacements in animal feed. Four of these Bacillus strains (BPR-11, BPR-14, BPR-16 and BPR-17) showed antimicrobial activity against the pathogenic strains Clostridium perfringens, Escherichia coli and Staphylococcus aureus at 25 μg/mL, with BPR-16 and BPR-17 also able to inhibit Pseudomonas aeruginosa and Salmonella enterica at 100 μg/mL. Further chemical investigation of BPR-17 led to the identification of eight metabolites, namely C16, C15, C14 and C13 surfactin C (1–4), maculosin (5), maculosine 2 (6), genistein (7) and daidzein (8). Purified compounds (1–4) were able to inhibit all the tested pathogens with MIC values ranging from 6.25 to 50 μg/mL. Maculosin (5) and maculosine 2 (6) inhibited C. perfringens, E. coli and S. aureus with an MIC of 25 μg/mL while genistein (7) and daidzein (8) showed no activity. An animal trial involving feeding BPR-11, BPR-16 and BPR-17 to a laboratory poultry model led to an increase in animal growth, and a decrease in feed conversion ratio and mortality. The presence of surfactin C analogues (3–4) in the gut following feeding with probiotics was confirmed using an LC–MS analysis. The investigation of these Bacillus probiotics, their metabolites, their impacts on animal performance indicators and their presence in the gastrointestinal system illustrates that these probiotics are effective alternatives to AGPs.

Background/Objectives: Bacillus probiotic mixtures have been used as a novel source of antimicrobial production. However, there is a need to emphasize the potential of co-cultured Bacillus strains and to understand the underlying mechanisms involved in their combination formulation. Methods: Bacillus strains BPR-11, BPR-16, and BPR-17, were cultured either as mono or multi-cultures. The bacterial growth and density were evaluated by measuring their optical density. The chemical profiles of their bioactive extracts were analysed by LC-MS, and their antimicrobial activity were tested against selected pathogens. Results: Our results reveal that co-cultured strains significantly increased bacterial growth, with an optical density level of, 2.67 which was significantly higher than the monocultures of BPR-11 (2.24), BPR-16 (2.32), and BPR-17 (2.22). Furthermore, the ethyl acetate extracts from these bacterial cultures showed that the combined co-culture (F1) exhibited the highest antimicrobial activity, with MICs of 25 µg/mL against C. perfringens, E. coli, and S. aureus, and 50 µg/mL against P. aeruginosa and S. enterica. In contrast, the monocultures BPR-16 and BPR-17 showed moderate activity with MICs of 50 µg/mL against C. perfringens, E. coli, and S. aureus, and 100 µg/mL against P. aeruginosa and S. enterica. BPR-11 had the lowest antimicrobial activity, with MICs of 100 µg/mL against C. perfringens, E. coli, and S. aureus, and no activity against P. aeruginosa and S. enterica. Metabolite profiling showed that the extract from the co-culture had a marked increase in the production of antimicrobial metabolites, including C₁₃-C₁₆ surfactin C. Lastly, the metabolism study of surfactin C analogues suggested that they were highly stable (99%) when incubated with cytochromes P450 over 120 min. Conclusions: Together, these findings highlight the potential for multi-strain co-culturing to develop new antimicrobials and provide valuable insights into the synergistic effects for antimicrobial production.