Antimicrobial resistance (AMR) poses a significant challenge to animal production due to the widespread use of antibiotics. Therefore, there is an urgent need for alternative antimicrobial strategies to effectively manage bacterial infections, protect animal health, and reduce reliance on antibiotics. This study evaluated the use of emerging approaches and procedures for the isolation, identification, and characterization of bacteriocin-producing bacteria and their bacteriocins, sourced from the gastrointestinal tract (GIT) of meat-producing pigs. Out of 2056 isolates screened against Gram-positive and Gram-negative indicator strains, 20 of the most active antimicrobial isolates were subjected to whole genome sequencing (WGS) for the prediction of coding DNA sequences (CDS) and the identification of bacteriocin gene clusters (BGC) and their functions. The use of an in vitro cell-free protein synthesis (IV-CFPS) protocol and the design of an IV-CFPS coupled to a split-intein mediated ligation (IV-CFPS/SIML) procedure made possible the evaluation of the production and antimicrobial activity of described and putatively novel bacteriocins. A colony MALDI-TOF MS procedure assisted in the identification of class I, II, and III lanthipeptides. MALDI-TOF MS and a targeted proteomics, combined with a massive peptide analysis (LC-MS/MS) approach, has proven valuable for the identification and biochemical characterization of previously described and novel bacteriocins encoded by the isolated bacteriocin-producing strains.
Keywords: AMR; IV-CFPS/SIML; LC-MS-MS; MALDI-TOF MS; bacteriocin gene clusters (BGC); bacteriocin-producing bacteria; bacteriocins; in vitro cell-free protein synthesis (IV-CFPS); whole genome sequencing (WGS).