Isolation and characterization of bacteriocin-producing E. coli isolates from a poultry slaughterhouse, and cell-free production and evaluation of native and engineered bacteriocins

Antimicrobial resistance (AMR) is a growing public health threat, particularly in food animal production. Poultry acts as a major reservoir for AMR dissemination, underscoring the need for sustainable alternatives to antibiotics. Bacteriocins are promising candidates, especially when produced by strains adapted to gut environments. In this study, over 1,900 isolates from broiler chicken gastrointestinal tracts and slaughterhouse water were screened for antimicrobial activity against Escherichia coli and Salmonella spp. Of the 384 active isolates, 17 genetically distinct E. coli strains were selected for whole-genome sequencing (WGS) and bioinformatic analysis. All harbored at least one bacteriocin gene cluster (BGC), with colicin M and microcin V being most prevalent. One strain, E. coli GTI-2H6, lacked virulence and AMR genes, identifying it as a safe probiotic candidate. An in vitro cell-free protein synthesis (IV-CFPS) protocol enabled rapid production and functional validation of six native colicins and microcins. To enhance antimicrobial spectra, hybrid colicins were rationally engineered by recombining domains from colicins E1, E7, and M. Several hybrids displayed expanded activity against both E. coli and Salmonella spp. These findings demonstrate the value of poultry-associated microbiota as a source of bacteriocins and support modular engineering and IV-CFPS as tools for developing next-generation antimicrobials.