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.

This study identified the optimal multi-enzyme dose rate at three energy levels based on the production performance of broiler chickens. A 42-day grow out trial was conducted using 576 day-old mixed-sex ROSS308 broiler chickens in a 3 × 4 factorial arrangement in a completely randomized design. Diets consisting of three metabolizable energy (ME) levels: standard energy (STD), 150 kcal/kg energy reduction (STD-150), and 200 kcal/kg energy reduction (STD-200), were cross factored with four multi-enzyme inclusion levels (0, 350, 700, and 1000 g/ton). The average daily feed intake and feed conversion ratio increased linearly (p < 0.001) as the dietary ME was reduced, and the multi-enzyme addition improved the feed conversion ratio (p < 0.05) and mitigated the negative effect of the reduced energy diets (RED) on feed intake and feed conversion ratios. Carcass composition, organ weights, and meat quality were not affected by the experimental diets. The RED decreased abdominal fat weight (p < 0.05). Total ash, calcium, and phosphorous contents of the tibia bone were improved (p < 0.04) when the RED were supplemented with the multi-enzyme. Super-dosing multi-enzymes in RED mitigates the negative effect of ME reduction on growth performance while maintaining organ development and meat quality and improving bone mineral content.

Considerable research has been conducted into the efficacy of individual probiotics in broiler production, however information on the most effective combinations of synergistic Bacillus probiotic is lacking. This study investigated the impact of different Bacillus strain combinations in broiler chickens, as well as in vitro enzyme production. In experiment one, a total of 576 Ross 308 broilers at 1 d old were grown for 21 d across 6 treatments of maize-soybean diets (n = 12 pens per treatment) to compare three different strain combinations (formulation 1 [F1]: 3 strains Bacillus amyloliquefaciens; F2: Bacillus coagulans and 2 strains B. amyloliquefaciens; F3: B. coagulans, Bacillus licheniformis and 2 strains B. amyloliquefaciens; F5: Bacillus subtilis, B. licheniformis and 2 strains B. amyloliquefaciens), positive control (PC), and a negative control antibiotic treatment group (NC). In Exp. 2, a total of 360 one-day-old ROSS308 broilers were used to test five treatments (n = 9) including PC, NC, F1 and F5 (selected from Exp. 1), and F4 (Bacillus pumilis and 2 strains B. amyloliquefaciens) in a maize-soybean diet. B. amyloliquefaciens F1 demonstrated a significant improvement in feed conversion ratio (FCR) compared to F2 at d 14 (1.49 vs 2.10; P = 0.038) and the body weight (BW) at d 21 (847.0 g vs 787.4 g) compared to other combinations (P = 0.027). The FCR at d 21 tended to be lower in birds fed F1 (1.46 vs 1.66) compared to the control (P = 0.068). Probiotic treatments had significantly improved nutrient digestibility compared to the PC and NC. Also, probiotic treatments supported the growth of Streptococcus, a common commensal genus and reduced the abundance of genera that correlated with low weight gain such as Akkermansia. Experiment two revealed that F4 improved FCR (P < 0.001) and BW at 28 d (P = 0.014). In vitro testing showed a high production of protease and amylase by Bacillus. Thus, the addition of Bacillus probiotics, particularly containing B. amyloliquefaciens strains and Bacillus pumilus, into the diet of broiler chickens improves production performance, nutrient digestibility, and allows the proliferation of beneficial gut microbiota.

The supplementation of antimicrobial growth promoters (AGPs) has been banned in many countries because of the emergence of antimicrobial-resistant pathogens in poultry products and the environment. Probiotics have been broadly studied and demonstrated as a promising AGP substitute. Our study is centred on the effects of a multi-strain Bacillus-based probiotic product on broiler production performance and gut microbial profile in a dexamethasone-induced leaky gut challenge. Two hundred and fifty-six broiler chicks were hatched and randomly assigned into four groups (wheat-soybean meal basal diet (BD) = non-supplemented control (C), BD supplemented with dexamethasone in week 4 (CD), BD containing a probiotic from day one (P), and BD containing a probiotic from day one and supplemented with dexamethasone during challenge week 4 (PD)). The production performance and caecal, gizzard, jejunal lumen and jejunal mucosa swab microbiota were studied by 16S rRNA gene sequencing. The Bacillus probiotic product significantly improved production performance and altered caecal gut microbiota (p ≤ 0.05), but no significant impact on microbiota was observed in other gut sections.

Probiotics provided from hatch have a major influence on microbiota development, and together with environmental and bedding microbiota, shape the microbial community of the litter. We investigated the influence of probiotic supplementation and a leaky gut challenge induced using dexamethasone (DEX) on the litter microbial community and litter parameters. The probiotic product was a mix of three Bacillus amyloliquefaciens strains. The litter microbiota were compared to the microbial communities from other gut sections. The litter samples had higher microbial diversity compared to the caecum, gizzard, jejunum, and jejunal mucosa. The high similarity between the litter phylum-level microbiota and gizzard microbiota detected in our study could be a consequence of ingested feed and litter passing through the gizzard. Moreover, the litter microbial community is fundamentally distinct from the intestinal microbiota, as evidenced by the number of genera present in the litter but absent from all the intestinal sections and vice versa. Furthermore, LEfSe analysis identified distinct microbial taxa across different groups, with specific genera associated with different treatments. In terms of litter quality, the birds in the DEX groups had a significantly higher moisture content, indicating successful leaky gut challenge, while probiotic supplementation did not significantly affect the moisture levels. These findings provide comprehensive insights into the distinct microbiota characteristics of litter.

The concept of designer microbiota in chicken is focused on early exposure of the hatchlings to pathogen-free microbiota inoculum, limiting the early access to harmful and pathogenic microorganisms, thus promoting colonisation of the gut with beneficial and natural poultry microbiota. In this study, we controlled colonisation of the intestine in broiler chickens in a large-scale industrial setting via at-hatch administration of a commercial product containing a highly diverse microbiota originating from the chicken caecum. The treatment significantly transformed the microbiota membership in the crop, proventriculus, jejunum and caecum and significantly altered the taxa abundance in the jejunum, jejunum mucosa, and caecum estimated using PERMANOVA and unweighted and weighted UniFrac distances, respectively. The treatment also improved the growth rate in chickens with no significant alteration in feed conversion ratio. A comparison of inoculum product microbiota structure revealed that the inoculum had the highest Shannon diversity index compared to all investigated gut sections, and the number of Observed Species second only to the caecal community. PCoA plots using weighted or unweighted UniFrac placed the inoculum samples together with the samples from the caecal origin.