In vitro efficacy of synthetic antimicrobial peptide SET-M33 against poultry isolates with diverse antimicrobial resistance phenotypes

Antimicrobial resistance is an impactful One Health issue. One of its drivers is the extensive use of antibiotics in both human and animal production systems, and despite regulatory restrictions on antibiotic use in poultry farming, antimicrobial resistance remains a major challenge. Consequently, animals are at higher risk of harder-to-treat diseases and play a role as resistance reservoirs, highlighting the need for alternative antimicrobial strategies. Toward this end, antimicrobial peptides (AMPs) have emerged as promising candidates due to their broad-spectrum activity and lower propensity to induce resistance. However, the effectiveness of AMPs against poultry pathogens, and in particular multidrug-resistant strains, is largely unclear. Here, we evaluated SET-M33, a synthetic tetrabranched AMP, against four species of clinically relevant pathogens in poultry, namely, Escherichia coli , Salmonella enterica , Enterococcus faecalis , and Enterococcus cecorum . Using a panel of 141 field isolates, we found that SET-M33 broadly inhibited bacterial growth at low micromolar concentrations (median minimum inhibitory concentrations of 2.5 and 5 μM for gram-negative and gram-positive strains, respectively), including in multidrug-resistant isolates. To examine the potential impact of SET-M33 on host cells, we established an in vitro co-cultivation system using chicken intestinal organoids. We found that SET-M33 retains its antimicrobial activity in organoid-microbe co-cultures at concentrations that preserve host cell viability. These findings demonstrate the potential of SET-M33 as a new antimicrobial agent against pathogens in poultry. IMPORTANCE Antimicrobial resistance is an important One Health issue that affects human and animal health. In particular, antimicrobial resistance remains a challenge in poultry farming, where animals are at a higher risk of harder-to-treat diseases and play a role as resistance reservoirs. To solve this issue, we need alternative antimicrobial strategies, and antimicrobial peptides have emerged as particularly promising candidates. Here, we examined the efficacy of one such new antimicrobial, the synthetic antimicrobial peptide SET-M33, against a large panel of poultry pathogen isolates. We found that SET-M33 was highly effective against the vast majority of isolates, including strains that are multidrug resistant or resistant to other antimicrobial peptides. To examine the potential impact of SET-M33 on the host, we established an in vitro co-cultivation system using chicken intestinal organoids. We found that SET-M33 retains its antimicrobial activity in organoid-microbe co-cultures at concentrations that preserve host cell viability.