Efficacy and safety evaluation of artificial intelligence-identified antimicrobial peptides for use against avian pathogenic Escherichia coli in the poultry industry

ABSTRACT The overuse of antibiotics in both veterinary and human medicine has resulted in the emergence of antibiotic-resistant bacteria, prompting a search for effective alternatives. Antimicrobial peptides (AMP) are short, often cationic, peptide-based molecules with antimicrobial and immunomodulatory activity, which makes them promising alternatives to conventional antibiotics in poultry production. From a prior machine-learning-guided screen of 875 candidate AMPs, 62 exhibited activity against avian pathogenic Escherichia coli (APEC) and low in vitro hemolytic and cytotoxic activity. We selected three lead AMPs from this list (named TeRu4, TeBi1, and PeNi4), and evaluated their in vitro and in vivo efficacy, safety, and immunomodulatory potential for use in poultry farming. In animal experiments, AMPs were administered via in ovo injection on day 18 of embryonic development. In APEC challenge trials, yolk sacs were inoculated with APEC post-hatch to assess early chick mortality, while in pen trials, birds were raised in a commercial production setting for 35 days. For challenged birds, TeBi1 (10 μg/egg) significantly reduced bacterial detection in the air sac and pericardium, increased body weight by 50% and reduced cytokine transcript levels by 10-30% on day 7 post hatch. In HD11 chicken macrophage-like cultured cells, TeRu4 (16 μg/mL) suppressed lipopolysaccharide (LPS)-induced pro-inflammatory cytokine transcript levels. In pen trials, TeRu4 (20 μg/egg) increased the survival probability of female birds by 4.9%, while TeBi1 (20 μg/egg) increased the survival probability of all birds by 4.4%, by day 35. Gene expression analysis revealed AMP- and sex-specific cytokine responses. In pen trials, no significant differences were observed in mean weights, feed conversion ratio (FCR), and flock uniformity on day 35. These findings demonstrate that the three selected AMPs are safe antibiotic alternatives that improve survival, modulate immune responses, and maintain normal growth performance in broiler chickens. Competing Interest Statement Birol I. is a founder, Chief Scientific Officer, and equity holder of Amphoraxe Life Sciences Inc., which has licensed intellectual property related to antimicrobial peptides described in this manuscript. The following authors are named inventors on related intellectual property: Helbing C.C., Fraser E., Hof F., Corrie L., Warren R.L., Thompson V.C., Yanai A., Birol I., and Hoang L.M.N. Kotkoff M. is also a stakeholder on the IP. Demirsoy E., Parkin T.I., and Dema A.H. received internship funding from Amphoraxe through Mitacs. Amphoraxe contributed to aspects of the work, including study design for selected experiments, resource allocation, collaborative experimental activities, internship support, and participation in manuscript preparation. The authors retained control over data collection, analysis, interpretation, and the decision to submit the manuscript for publication. All other authors declare no competing interests. Footnotes The primary revisions in this version consist of additional statistical analyses of the results and minor textual edits to enhance clarity and flow.