Citation: Tran, C.; Horyanto, D.; Stanley, D.; Cock, I.E.; Chen, X.; Feng, Y. Antimicrobial Properties of Bacillus Probiotics as Animal Growth Promoters. Antibiotics 2023, 12, 407. https://doi.org/10.3390/ antibiotics12020407 Academic Editors: Tamara P. Russo and Antonio Santaniello Received: 30 January 2023 Revised: 14 February 2023 Accepted: 17 February 2023 Published: 17 February 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). antibiotics Article Antimicrobial Properties of Bacillus Probiotics as Animal Growth Promoters Charlie Tran 1 , Darwin Horyanto 2,3 , Dragana Stanley 2 , Ian E. Cock 4 , Xiaojing Chen 3 and Yunjiang Feng 1, * 1 Griffith Institute for Drug Discovery (GRIDD), Griffith University, Brisbane, QLD 4111, Australia 2 Institute for Future Farming Systems, Central Queensland University, Rockhampton, QLD 4702, Australia 3 Bioproton Pty Ltd., Brisbane, QLD 4110, Australia 4 School of Environment and Science, Griffith University, Brisbane, QLD 4111, Australia * Correspondence: y.feng@griffith.edu.au Abstract: 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. Keywords: animal feed; antimicrobials; Bacillus; omics; probiotics; spore-forming 1. Introduction Antimicrobial growth promoters (AGPs) are currently utilised in the agricultural industry to improve livestock production, feed-energy conversion and to prevent the spread of infectious diseases [1–3]. These AGPs eliminate the microbiota in the gut and allow the host to access more nutrients [4,5]. They also halt the production of toxins produced by pathogens and improve livestock production [4,5]. However, there has been a steady rise of antimicrobial resistance due to the misuse of these AGPs. This has driven restrictions and bans of AGPs in countries throughout the EU, U.S. and Indonesia [6]. These regulations have been linked to decreases in livestock production and higher rates of food-borne infections, highlighting a need for safer and more effective alternatives to AGPs, such as probiotics [6–9]. Probiotics are live microorganisms that provide a range of benefits to their hosts once consumed [10,11]. Their benefits include the production of enzymes that assist in breaking down indigestible material, providing competition for nutrients in the gut to inhibit pathogenic bacterial growth and the production of antimicrobial metabolites [12–16]. These mechanisms are linked to an observed increase in animal growth and a reduction in Antibiotics 2023, 12, 407. https://doi.org/10.3390/antibiotics12020407 https://www.mdpi.com/journal/antibiotics