The targeted depletion of potential gut pathogens is often challenging because of their intrinsic ability to thrive in harsh gut environments. Earlier, we showed that Campylobacter jejuni (C. jejuni) exclusively uses the Type-VI Secretion System (T6SS) to target its prey such as Escherichia coli (E. coli), and phenotypic differences between T6SS-negative and T6SS-positive C. jejuni isolates toward bile salt sensitivity. However, it remains unclear how the target-driven T6SS functionality prevails in a polymicrobial gut environment. Here, we investigated the fate of microbial competition in an altered gut environment via bacterial T6SS using a T6SS-negative and -positive C. jejuni or its isogenic mutant of the hemolysin-coregulated protein (hcp). We showed that in the presence of bile salt and prey bacteria (E. coli), T6SS-positive C. jejuni experiences enhanced intracellular stress leading to cell death. Intracellular tracking of fluorophore-conjugated bile salts confirmed that T6SS-mediated bile salt influx into C. jejuni can enhance intracellular oxidative stress, affecting C. jejuni viability. We further investigated whether the T6SS activity in the presence of prey (E. coli) perturbs the in vivo colonization of C. jejuni. Using chickens as primary hosts of C. jejuni and non-pathogenic E. coli as prey, we showed a marked reduction of C. jejuni load in chickens cecum when bile salt solution was administered orally. Analysis of local antibody responses and pro-inflammatory gene expression showed a reduced risk of tissue damage, indicating that T6SS activity in the complex gut environment can be exploited as a possible measure to clear the persistent colonization of C. jejuni in chickens.

The main goal of this study was to evaluate the effect of nutraceuticals vs. in-feed antibiotics on performance, blood lipids, antioxidant capacity, cecal microbiota, and organ histomorphology of broiler chickens. A total of 320 one-day-old male broiler chickens were distributed into 5 treatment groups with 8 replicates each. The control group was fed on a basal diet without any additives (NC); the antibiotic group was fed on a basal diet supplemented with 100 mg kg-1 avilamycin (PC); the algal group was fed on a basal diet supplemented with a mixture of Spirulina platensis and Chlorella vulgaris (1.5 g + 1.5 g/kg feed) (SP+CV); the essential oil group was fed with a basal diet containing 300 mg/kg feed rosemary oil (REO); and the probiotics group (a mixture of 1 × 1011 CFU/g Bacillus licheniformis, 1 × 1011 CFU/g Enterococcus facieum, 1 × 1010 CFU/g Lactobacillus acidophilus, and 2 × 108 CFU /g Saccharomyces cerevisiae) was fed with a basal diet supplemented with 0.05% probiotics (PRO). The experiment lasted for 35 d. A beneficial effect of SP+CV and PRO (P < 0.01) was noticed on final body weight, body weight gain, feed conversion ratio, and breast yield. The dietary supplementation with SP+CV, REO, and PRO increased (P < 0.001) broilers\' cecal lactic acid bacteria count compared to the control. Lower cecal Clostridium perfringens and Coliform counts (P < 0.001) were noticed in chickens fed the PC and supplemental diets. Malondialdehyde (MDA) concentration was decreased, while glutathione peroxidase (GPx), superoxide dismutase, and catalase enzymes were increased in the breast and thigh meat (P < 0.001) of broiler chickens fed SP+CV, REO, and PRO diets. Dietary SP+CV, REO, and PRO supplementation decreased (P < 0.001) serum total lipids, cholesterol, triglycerides, low-density lipoprotein, and MDA, but increased serum high-density lipoprotein and GPx compared to PC and NC. No pathological lesions were noticed in the liver, kidney, or breast muscle among broilers. The SP+CV, REO, and PRO groups had greater (P < 0.001) intestinal villi height and crypt depth while lower goblet cell densities (P < 0.01) than the control. The present findings suggest that PRO and SP+CV, followed by REO could be suitable alternatives to in-feed antibiotics for enhancing the performance, health, and meat quality of broiler chickens.

Edwardsiella piscicida causes significant economic losses to the aquaculture industry worldwide. Phage-based biocontrol methods are experiencing a renaissance because of the spread of drug-resistant genes and bacteria resulting from the heavy use of antibiotics. Here, we showed that the novel Edwardsiella phage EPP-1 could achieve comparable efficacy to florfenicol using a zebrafish model of Edwardsiella piscicida infection and could reduce the content of the floR resistance gene in zebrafish excreta. Specifically, phage EPP-1 inhibited bacterial growth in vitro and significantly improved the zebrafish survival rate in vivo (P = 0.0035), achieving an efficacy comparable to that of florfenicol (P = 0.2304). Notably, integrating the results of 16S rRNA sequencing, metagenomic sequencing, and qPCR, although the effects of phage EPP-1 converged with those of florfenicol in terms of the community composition and potential function of the zebrafish gut microbiota, it reduced the floR gene content in zebrafish excreta and aquaculture water. Overall, our study highlights the feasibility and safety of phage therapy for edwardsiellosis control, which has profound implications for the development of antibiotic alternatives to address the antibiotic crisis.

Finding effective antibiotic alternatives is crucial to managing the re-emerging health risk of Clostridium perfringens (CP) type A/G-induced avian necrotic enteritis (NE), a disease that has regained prominence in the wake of governmental restrictions on antibiotic use in poultry. Known for its antimicrobial and immunomodulatory effects, the use of bovine lactoferrin (bLF) in chickens is yet to be fully explored. In this study, we hypothesized that bLF can accumulate in the small intestines of healthy chickens through gavage and intramuscular supplementation and serves as a potential antibiotic alternative. Immunohistochemistry located bLF in various layers of the small intestines and ELISA testing confirmed its accumulation. Surprisingly, sham-treated chickens also showed the presence of bLF, prompting a western blotting analysis that dismissed the notion of cross-reactivity between bLF and the avian protein ovotransferrin. Although the significance of the route of administration remains inconclusive, this study supports the hypothesis that bLF is a promising and safe antibiotic alternative with demonstrated resistance to the degradative environment of the chicken intestines. Further studies are needed to determine its beneficial pharmacological effects in CP-infected chickens.

Amino acid-fermenting Clostridia have undesirable effects in agricultural systems, which can be mitigated by antibiotics, but resistance necessitates alternatives. Here, we demonstrate the efficacy of cannabidiol on growth and ammonia inhibition of five agriculturally relevant Clostridia: Clostridium sporogenes, Peptostreptococcus spp., Clostridioides difficile, Acetoanaerobium sticklandii, and Clostridium aminophilum.

Phytogenic feed additives (PFAs) often referred to as phytobiotics or botanical feed additives, are natural compounds derived from various plants, herbs, spices and other botanical sources. These feed additives are intended to serve a variety of purposes, including an immune system regulator, an antimicrobial, an antimutagenic, an antioxidant and a growth promoter. They are composed of bioactive compounds extracted from plants, including essential oils, polyphenols, terpenoids and flavonoids. They are mostly utilized as substitute antibiotic growth promoters in nonruminant (swine and poultry) livestock production, owing to the prohibition of antibiotic usage in the feed industry. It has been thoroughly examined to ascertain their impact on intestinal health and activity, correlation with animals\' effective health and well-being, productivity, food security and environmental impact. The potential uses of these feed additives depend on the properties of herbs, the comprehension of their principal and secondary components, knowledge of their mechanisms of action, the safety of animals and the products they produce. They are gaining recognition as effective and sustainable tools for promoting animal health and performance while reducing the reliance on antibiotics in nonruminant nutrition. Their natural origins, multifaceted benefits and alignment with consumer preferences make them a valuable addition to modern animal farming process. However, because of their inconsistent effects and inadequate knowledge of the mechanisms of action, their usage as a feed additive has been limited. This review offers a comprehensive assessment of the applications of PFAs as an effective feed supplement in swine and poultry nutrition. In summary, this comprehensive review provides current knowledge, identifies gaps in research and emphasizes the potential of phytogenic additives to foster sustainable and healthier livestock production systems while addressing the global concerns associated with antibiotic use in livestock farming.

The development of antibiotic-resistant microorganisms prompted the investigation of possible antibiotic substitutes. As a result, the purpose of the current study is to assess the effect of dietary Spirulina platensis extract as an antibiotic alternative on Japanese quail (Coturnix japonica) growth, antioxidant status, blood parameters, and cecal microorganisms. There was a total of 150 Japanese quails used in this study, divided equally among 5 experimental groups (10 birds per group with 3 replicates): group 1 (G1) received a basal diet without any S. platensis extract, group 2 (G2) received a basal diet supplemented with 1 mL S. platensis extract/kg, group 3 (G3) received a basal diet supplemented with 2 mL S. platensis extract/kg, group 4 (G4) received a basal diet supplemented with 3 mL S. platensis extract/kg, and group 5 (G5) received a basal diet supplemented with 4 mL S. platensis extract/kg from d 7 until d 35. The results showed that compared to the control birds in G1, Japanese quail supplemented with 4 mL of S. platensis extract/kg of diet (G5) had significantly better live body weight, body weight gain, feed intake, feed conversion ratio, digestive enzymes, blood parameters, liver and kidney functions, lipid profile, antioxidant profile, immunological parameters, and cecal microorganism\'s count. There were no significant changes in the percentage of carcasses, liver, and total giblets among all the 5 groups. Only gizzard percentage showed a significant increase in G2 compared to birds in G1. In addition, intestinal pH showed a significant drop in G2 and G5 compared to birds in G1. After cooking the quail meat, the juiciness and tenderness increased as S. platensis extract levels increased, whereas aroma and taste declined slightly as S. platensis extract levels increased. Furthermore, when a high concentration of S. platensis extract was used, the lightness of the meat reduced while its redness and yellowness increased. The disk diffusion assay showed that S. platensis extract had significant antibacterial activity against Staphylococcus aureus, Listeria monocytogenes, Campylobacter jejuni, and Salmonella typhi, with inhibition zones ranging from 16 to 42 mm. This activity may be attributable to the volatile chemicals in S. platensis extract, of which Geosmin and 2-methylisoborneol are the primary components. In the diet of Japanese quails, it is possible to draw the conclusion that the extract of S. platensis can be utilized as a feed additive and as an alternative to antibiotics.

Peptide nucleic acids (PNAs) are synthetic molecules that are like DNA/RNA, but with different building blocks. PNAs target and bind to mRNAs and disrupt the function of a targeted gene, hence they have been studied as potential antibacterials. The aim of this systematic review was to provide an in-depth analysis of the current status of PNAs as antibacterial agents, define the characteristics of the effective PNA constructs, and address the gap in advancing PNAs to become clinically competent agents. Following the PRISMA model, four electronic databases were searched: Web of Science, PubMed, SciFinder and Scopus. A total of 627 articles published between 1994 and 2023 were found. After screening and a rigorous selection process using explicit inclusion and exclusion criteria, 65 scientific articles were selected, containing 656 minimum inhibitory concentration (MIC) data. The antibacterial activity of PNAs was assessed against 20 bacterial species. The most studied Gram-negative and Gram-positive bacteria were Escherichia coli (n=266) and Staphylococcus aureus (n=53), respectively. In addition, the effect of PNA design, including construct length, binding location, and carrier agents, on antibacterial activity was shown. Finally, antibacterial test models to assess the inhibitory effects of PNAs were examined, emphasising gaps and prospects. This systematic review provides a comprehensive assessment of the potential of PNAs as antibacterial agents and offers valuable insights for researchers and clinicians seeking novel therapeutic strategies in the context of increasing rates of antibiotic-resistant bacteria.

Streptococcus iniae, a zoonotic Gram-positive pathogen, poses a threat to finfish aquaculture, causing streptococcosis with an annual economic impact exceeding $150 million globally. As aquaculture trends shift towards recirculating systems, the potential for horizontal transmission of S. iniae among fish intensifies. Current vaccine development provides only short-term protection, driving the widespread use of antibiotics like florfenicol. However, this practice raises environmental concerns and potentially contributes to antibiotic resistance. Thus, alternative strategies are urgently needed. Endolysin therapy, derived from bacteriophages, employs hydrolytic endolysin enzymes that target bacterial peptidoglycan cell walls. This study assesses three synthetic endolysins (PlyGBS 90-1, PlyGBS 90-8, and ClyX-2) alongside the antibiotic carbenicillin in treating S. iniae-infected hybrid striped bass (HSB). Results demonstrate that ClyX-2 exhibits remarkable bacteriolytic potency, with lytic activity detected at concentrations as low as ∼15 μg/mL, approximately 8-fold more potent than the PlyGBS derivatives. In therapeutic effectiveness assessments, both carbenicillin and ClyX-2 treatments achieved significantly higher survival rates (85 % and 95 %, respectively) compared to placebo and PlyGBS-based endolysin treatments. Importantly, no statistical differences were observed between ClyX-2 and carbenicillin treatments. This highlights ClyX-2 as a promising alternative for combating S. iniae infections in aquaculture, offering potent bacteriolytic activity and high survival rates.

Maintaining animal gut health through modulating the gut microbiota is a constant need when antibiotics are not used in animal feed during the food animal production process. Prebiotics is regarded as one of the most promising antibiotic alternatives for such purpose. As an attractive prebiotic, the role and mechanisms of neoagarooligosaccharides (NAOS) in promoting animal growth and gut health have not been elucidated. In this study, we first cloned and expressed marine bacterial β-agarase in yeast to optimize the NAOS preparation and then investigated the role and the underlying mechanisms of the prepared NAOS in improving chicken gut health and function. The marine bacterial β-agarase PDE13B was expressed in Pichia pastoris GS115 and generated even-numbered NAOS. Dietary the prepared NAOS promoted chicken growth and improved intestinal morphology, its barrier, and digestion capabilities, and absorption function. Metagenomic analysis indicated that NAOS modulated the chicken gut microbiota structure and function, and microbial interactions, and promoted the growth of spermidine-producing bacteria especially Faecalibacterium. Through integration of gut metagenome, gut content metabolome, and gut tissue transcriptome, we established connections among NAOS, gut microbes, spermidine, and chicken gut gene expression. The spermidine regulation of genes related to autophagy, immunity, and inflammation was further confirmed in chicken embryo intestinal epithelium cells. We also verified that NAOS can be utilized by Faecalibacterium prausnitzii to grow and produce spermidine in in vitro experiments. Collectively, we provide a systematic investigation of the role of NAOS in regulating gut health and demonstrate the microbial spermidine-mediated mechanism involved in prebiotic effects of NAOS, which lays foundation for future use of NAOS as a new antibiotic alternative in animal production.