Engineered Lactococcus lactis for Oral Delivery of an Antimicrobial Peptide against Enterotoxigenic Escherichia coli in Weaned Piglets.

Food-grade strategies against foodborne pathogens while preserving intestinal health are gaining attention. Here, we developed a regulated delivery platform using Lactococcus lactis engineered to secrete a tandem dimeric antimicrobial peptide (SD). The engineered strain (SDLactis) exhibited stable growth, genetic stability, and tolerance to simulated gastrointestinal conditions. Chloride-responsive SD secretion enabled effective antibacterial activity against enterotoxigenic Escherichia coli (ETEC) K88 in vitro. In ETEC-challenged piglets, oral SDLactis alleviated diarrhea, improved growth, and reduced intestinal injury and inflammation. It strengthened intestinal barrier integrity by upregulating tight junction proteins (ZO-1, Claudin-1,Occludin) and downregulating CFTR. Microbiome analysis revealed that SDLactis partially restored gut microbial diversity, reducing Escherichia-Shigella while enriching Lactobacillus and short-chain fatty acid-producing genera. Overall, food-grade engineered lactic acid bacteria serve as controllable delivery vehicles for antimicrobial peptides, offering a nonantibiotic strategy for pathogen control and gut health management in food and feed applications.