The synthetic peptide GATR-3 shows significant antibacterial and biofilm-inhibition activity against shellfish- and oyster-associated bacteria Vibrio vulnificus and Vibrio parahaemolyticus

• GATR-3 has strong MIC activity against V. vulnificus and V. parahaemolyticus . • Nanomolar EC 50 (10 mM phosphate + 1 % w/v NaCl) indicates GATR-3 salt tolerance. • In bacterial media, GATR-3 outperforms LL-37 and rivals Mastoparan-AF. • LL-37 is strongly cationsensitive; activity is only seen in low cation buffers. • Supports GATR-3 development as a topical therapy for Vibrio wound infections. The bacteria Vibrio vulnificus and Vibrio parahaemolyticus are of concern because of the severe wound infections that they can cause, as well as their potential antimicrobial resistance and biofilm formation. Antimicrobial peptides (AMPs) are promising alternatives owing to rapid bactericidal, membrane-disruptive activity and a low propensity for resistance. GATR-3 is a rationally designed α-helical AMP, active against gram-negative bacteria, that eradicates Acinetobacter baumannii biofilms and shows a favorable cytotoxicity profile. We evaluated GATR-3 against multiple Vibrio strains. GATR-3 has strong antibacterial (MIC) activity for V. vulnificus MO6 and V. parahaemolyticus SAK11, V. vulnificus JY1701, V. parahaemolyticus NY477, and Vibrio alginolyticus . Some Vibrio strains ( V. mimicus, Vibrio cholerae ) were resistant to GATR-3, likely due to differences in their LPS. We report the antibacterial activity of Mastoparan-AF against V. vulnificus strains. GATR-3 mostly outperformed Mastoparan-AF peptide’s antibacterial activity and far exceeded LL-37 peptide activity, which was markedly inhibited by salt and cations. In 3-hr bactericidal “ Vibrio -adapted” EC 50 assays, GATR-3 was highly potent against V. vulnificus and V. parahaemolyticus strains; LL-37 and MastoparanAF were more active against V. parahaemolyticus than GATR-3; but was less active against V. vulnificus than GATR-3. Biofilm quantification demonstrated that V. vulnificus MO6 and V. parahaemolyticus NY477 were strong biofilm-formers. GATR-3 at 14 and 38 µg/mL inhibited MO6 and NY477 biofilm formation by 50 % (MBIC₅₀), whereas Mastoparan-AF required ∼64 and 13 µg/mL, respectively. GATR-3′s potency, tolerance to physiological salt, and its biofilm-inhibiting activity, combined with prior low cytotoxicity data, support further development of GATR-3 as a candidate peptide for future use in treatment as a potential topical adjuvant for Vibrio vulnificus and V. parahaemolyticus wound infections in humans.