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

Highlights

• •GATR-3 has strong MIC activity against V. vulnificus and V. parahaemolyticus.
• •Nanomolar EC₅₀ (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.

Abstract

The bacteriaVibrio 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” EC₅₀ 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.

Graphical abstract

Introduction

The rise of antimicrobial resistance and the ability of pathogens to form biofilms pose a major global health threat, leading to persistent infections, treatment failures, and increased morbidity and mortality¹. Drug-resistant infections associated with biofilms complicate treatment in clinical and foodborne settings, where conventional antibiotics often fail to eradicate the bacteria, resulting in chronic disease recurrence and high healthcare costs. It is estimated that biofilms contribute to up to 80 % of microbial infections, highlighting their critical role in the antimicrobial resistance crisis².

Biofilms are complex microbial communities embedded in a self-produced matrix of polysaccharides, DNA and proteins, which facilitate bacterial adhesion to cell surfaces and intercellular interactions. This structural adaptation provides protection against environmental stresses, impairs the diffusion of antimicrobial compounds, and enhances bacterial resistance to antibiotics^1,3.

Vibrio spp. are water-borne bacteria commonly found in marine, estuarine, and freshwater environments, some of which have emerged as significant pathogens affecting both human and animal health. Approximately twelve Vibrio species are recognized as clinically relevant pathogens in humans, such as Vibrio (V.) vulnificus, V. parahaemolyticus, V. cholerae, V. harveyi, V. mimicus, and V. alginolyticus[4], [5], [6], [7], [8], [9]. These bacteria are Gram-negative and mostly halophiles. V. vulnificus and V. parahaemolyticus are naturally present in estuarine and coastal waters^6,10, form biofilms¹¹ and often found associated with shellfish such as oysters and shrimp^7,8,12. These opportunistic pathogens pose a substantial public health risk through their involvement in seafood-borne gastroenteritis, wound infections, and life-threatening septicemia^6,8,13. We are exploring whether AMPs could be developed as part of a topical treatment approach for Vibrio-mediated wound infections.

V. vulnificus and V. parahaemolyticus are of particular clinical concern due to their natural reservoirs in aquatic environments, pathogenic versatility, and capacity to form biofilms^6,11. These bacteria are opportunistic human pathogens and are found associated with shellfish and other marine organisms^7,8,12. Both Vibrio species exhibit increasing antimicrobial resistance and can form protective biofilms or clusters¹¹. These infections can be lethal, emphasizing the need for effective new therapeutic strategies⁶.

Among them, V. vulnificus is regarded as the most virulent, responsible for the majority of seafood-related deaths in the United States despite its low incidence. Primary septicemia following consumption of contaminated shellfish can progress rapidly, with case-fatality rates exceeding 50 % and death often occurs within 72 h post-admission[14], [15], [16]. V. vulnificus can infect open wounds exposed to seawater and these can escalate rapidly into necrotizing fasciitis, with mortality rates of 25 %, increasing to over 50 % in immunocompromised hosts, particularly those with chronic liver disease or other comorbidities^14,17,18.

V. parahaemolyticus is recognized as the leading cause of bacterial seafood-borne gastroenteritis worldwide, representing a major public health concern due to its high incidence and widespread distribution¹⁹. Infection usually results from consuming raw or undercooked seafood and manifests as acute gastrointestinal illness marked by diarrhea, abdominal cramps, nausea, and vomiting^8,13. Most clinically identified strains contain the thermostable direct hemolysin toxin and/or related toxins, which are key virulence determinants²⁰. The incidence and mortality of V parahaemolyticus infections in the United States are approximately 4500 cases annually, resulting in hundreds of hospitalizations and a few deaths. Notably, vibriosis causes an estimated 80,000 illnesses each year in the United States, and V. parahaemolyticus is a major contributor to these infections. Most V. parahaemolyticus infections (which are primarily gastrointestinal) are usually self-limiting and rarely fatal. Less commonly, V. parahaemolyticus can cause wound infections.

Antimicrobial resistance among Vibrio species is a growing concern. In a previous study, V. harveyi strains in India were found to be resistant to azithromycin, bacitracin, cephalexin, cephalothin, erythromycin, aminoglycosides, and β-lactam antibiotics²¹. Similarly, other studies have reported that V. alginolyticus strains isolated in China are also resistant to β-lactam antibiotics ^22,23 V. mimicus can be resistant to nitrofurantoin, ampicillin, and polymyxins, and can cause cholera-like infection and other vibriosis ^24,64,25. V. cholera strains (biotype El Tor and O139) are resistant to polymyxin and colistin^21,22. The emergence of multidrug-resistant Vibrio strains, which are linked to widespread antibiotic use in aquaculture, further exacerbates the clinical challenge, with resistance observed to agents such as ampicillin, penicillin, and tetracycline²⁶. Moreover, the aquaculture environments where Vibrio species thrive are often contaminated with residual antibiotics and heavy metals, both of which exert selective pressure that enhances antimicrobial resistance among marine bacteria and host microbiota²⁷. In addition, Vibrio species form robust biofilms that further increase their tolerance to antibiotics and host defenses, complicating the eradication of infections. Given the severity and clinical challenges, there is an urgent need to develop alternative antibacterial strategies, such as topical antimicrobial peptides, to effectively treat Vibrio infections in conjunction with antibiotics²⁸, particularly for Vibrio wound-infections and necrotizing fasciitis patients.

Antimicrobial peptides are evolutionarily conserved components of innate immunity found across all domains of life. These small, cationic peptides (typically 10–50 amino acids) exhibit amphipathic α-helical structures that enable interaction with negatively charged bacterial membranes, such as the outer membrane of a gram-negative bacterium. These antibacterial peptides generally comprise 10 to 50 amino acids with a molecular weight less than 10 kDa, with a positive net charge ranging from +2 to +9, and a hydrophobicity that is greater than 30 %[29], [30], [31]. Antimicrobial peptides have several potential advantages over traditional antibiotics, such as a broad spectrum of activity, rapid killing, and lower propensity for resistance development due to a rapid killing mechanism and could be highly useful when applied in a combination treatment with existing antibiotics²⁷.

GATR-3 is a synthetic α-helical antimicrobial peptide³² rationally designed from a cryptic peptide, Apo6, which we originally identified in the serum of the American alligator³³. Engineered to enhance its amphipathicity and cationic charge over the parent peptide Apo6, GATR-3 exhibits potent killing activity against multidrug-resistant Gram-negative wound-infecting pathogens, particularly Acinetobacter (A.) baumannii, with a minimum inhibitory concentration (MIC) of 4 µg/mL (1.4 µM) and demonstrates a membrane-targeting, rapid bactericidal mechanism³². Beyond its planktonic activity, GATR-3 effectively inhibits A. baumannii biofilm formation and eradicates preformed biofilms. Its antibacterial and antibiofilm activity, coupled with its robust therapeutic index and minimal toxicity, underscore its promise as a next-generation antimicrobial candidate that could potentially be used as a topical therapeutic against wound infections³².

Given its favorable activity against other gram-negative bacteria³², the present study aimed to evaluate the antibacterial and antibiofilm activity of GATR-3 against clinically relevant strains V. vulnificus and V. parahaemolyticus. Specifically, we determined its antibacterial activity under both standard MIC conditions and Vibrio-adapted bactericidal (EC₅₀) assays and compared its potency with other benchmark antimicrobial peptides (LL-37 and Mastoparan-AF). We also determined the biofilm-inhibitory ability of GATR-3 peptide. Our objective was to establish whether GATR-3 has antibacterial and biofilm-inhibitory activity against the oyster and shellfish-associated Vibrio strains that can cause wound infections, V. vulnificus and V. parahaemolyticus.

Materials and methods

Reagents, media, and equipment

All chemicals and reagents were of analytical grade and used without further purification. Mueller-Hinton Broth (MHB; Thermo Scientific Cat# CM0405B) and Heart Infusion Broth (BD Difco Cat# 256120) were used for bacterial culture and susceptibility testing, while Tryptic Soy Broth (TSB; BD Difco Cat# 211825) served for routine cultivation of Vibrio and Escherichia coli strains. Sodium dihydrogen phosphate (NaH₂PO₄·H₂O; Sigma-Aldrich Cat# 7558–80–7) and Sodium phosphate dibasic (Na₂HPO₄; Sigma-Aldrich Cat# 7558–79–4) were used to prepare a 10 mM phosphate buffer. Sodium chloride (NaCl; Sigma-Aldrich Cat# 7647–14–5) was added to achieve a final concentration of 1 % (w/v) for Vibrio-adapted buffer formulations. Tetracycline hydrochloride (Thermo Fisher Scientific Cat# A39246) was used as an antibiotic reference standard. Ultrapure water (18.2 MΩ·cm, Milli-Q Advantage System, Millipore) was used for all reagent preparations. All antimicrobial peptide solutions were aliquoted and stored in low-binding microcentrifuge tubes (Eppendorf Cat# 022431021) at −80 °C. MIC and EC₅₀ assays were performed in 96-well polypropylene microplates (Corning Cat# 3879) to minimize peptide adsorption. Optical density measurements were performed using an absorbance microplate reader (BioTek 800S, Agilent Technologies, USA). Colony counts were obtained using Mueller-Hinton agar (BD Difco Cat# 225250) plates incubated at 37 °C. Incubation was conducted in Thermo Scientific Microbiological Incubators (Thermo Fisher Scientific Cat#15–015–2634), and bacterial concentrations were standardized using a McFarland Densitometer (DEN-1; Biosan; Cat# BS-050101-AA). All statistical analyses and dose–response curve fittings were performed using GraphPad Prism version 10 (GraphPad Software, San Diego, CA, USA).

Peptides

GATR-3 peptide (KFRNWFSQHFKKFKQKLKNTFA) was synthesized by China Peptides to >95 % purity³². Mastoparan-AF (sequence: INLKAIAALAKKLF-NH2) was synthesized by GenScript Biotech with >95 % purity. LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES-NH2) was obtained from NovoPro with a purity of 97.751 %. All peptides were confirmed by HPLC and ESI-MS. All peptides were synthesized using standard solid-phase Fmoc (9-fluorenylmethoxycarbonyl) peptide synthesis (SPPS). Peptides were solubilized in ultrapure water, aliquoted (100 µg/100 µl), and stored at −80 °C. The physicochemical properties of the peptides are summarized in Supplemental Table 1.

Bacterial strains and culture

Clinical and environmental strains of Vibrio were used (Supplemental Table 2). Vibrio strains were obtained from the laboratory of Dr. J. D. Oliver (University of North Carolina at Charlotte, N.C., USA) and were grown in Heart Infusion Broth (BD Difco Cat# 256,120), E. coli in TSB (BD Difco Cat# 211,825), at 37 °C. Glycerol stocks (50 % v/v) were prepared and stored at −80 °C until use. The Vibrio species and strains used in this study include V. vulnificus MO6; V. vulnificus JY1701; V. parahaemolyticus NY477; V. parahaemolyticus SAK11; V. harveyi ATCC 35084; V. cholerae 9064; V. mimicus ATCC 33653; V. alginolyticus (0–04-D1); V. alginolyticus 8653; V. alginolyticus (22–08–18)¹⁰. E. coli (ATCC 25922) is a reference strain for antimicrobial susceptibility testing by the Clinical and Laboratory Standards Institute (CLSI)³⁴ and is commonly used as a control strain in microbiology research.

Minimum inhibitory concentration (MIC)

MIC assays were performed following CLSI guidelines, with adaptations to testing antimicrobial peptides³⁵. Briefly, the bacteria were cultured in the appropriate media for 16–20 hr at 37 °C. The bacterial inoculum was adjusted to 1 × 10⁸ CFU/mL by McFarland Densitometer, and the adjusted inoculum was diluted to 1 × 10⁶ CFU/mL with Mueller Hinton Broth (Thermo Scientific Cat# CM0405B) (MHB) + 1 % w/v NaCl., and 50 µL was added to each well in a polypropylene 96-well plate (Corning, Cat# 3879) with a final bacterial concentration of 5 × 10⁵ CFU/mL in MHB. The antimicrobial activity of the peptide was tested by making 2-fold dilutions, usually starting from 64 µg/mL to the lowest concentration at 0.125 µg/mL in 50 μL of various dilutions of peptides prepared in ultrapure water. In some cases, the range was expanded to test for higher concentrations. The 96-well plate was then incubated for 16–20 h at 37 °C, and the first clear well was read by eye. The antibiotic tetracycline and the antimicrobial peptide Mastoparan-AF were used as positive controls following previous research showing that tetracycline is effective against many Vibrio species, and Mastoparan-AF is effective against V. parahaemolyticus^36,37. The tetracycline MICs were performed in MHB, following the peptide protocol, and so may slightly differ from reported MICs following the CLSI method using CA-MHB. This choice was made intentionally to maintain consistent assay conditions for direct comparison between peptide and tetracycline results. LL-37 was used as a comparator peptide.

Bactericidal activity assay (EC₅₀)

To assess cation-sensitive activity while preserving Vibrio viability, EC₅₀ was determined in a 10 mM phosphate buffer with added 1 % NaCl, to support the growth of these halophilic species¹⁴. The Vibrio species were grown in TSB overnight at 37 °C, and the E. coli was grown in LB overnight at 37 °C. For EC₅₀ determination, peptides were diluted from a stock solution for variable concentration (10⁻⁷–10² μg/mL). In a 96-well plate, the overnight bacteria were adjusted to 1 × 10⁶ CFU/mL, and 50 μL was added into each well in 10 mM phosphate buffer + 1 % w/v NaCl (Supplemental Table 3) with 50 μL various dilutions of peptides prepared in ultrapure water. The 96-well plate was incubated for 3hr at 37 °C. CFU counts were obtained via spot-plating. After 3 hr incubation, each well was serially diluted, and 8 μL of each dilution was spotted onto cation-adjusted Mueller-Hinton Agar and allowed to dry. The agar plates were then incubated overnight at 37 °C, and the CFU was determined. In these experiments, the antimicrobial peptides LL-37 and Mastoparan-AF were used as controls. After incubation, colonies were counted, and the EC₅₀ was calculated based on untreated bacteria by using GraphPad Prism 10. In GraphPad Prism 10, the number of colonies from peptide-treated samples was normalized by dividing the number of colonies from untreated controls. The data were then analyzed using the nonlinear regression (curve fit) option, selecting “Dose–response”; “Inhibition with log(inhibitor) vs. response (three parameters)”. Separate graphs were generated for each bacterial strain and each peptide.

Biofilm formation assay

We performed the biofilm formation assay for V. vulnificus and V. parahaemolyticus following Nguyen et al., 2024¹¹ and Hammer and Bassler, 200,3³⁸. Briefly, overnight cultures of V. vulnificus or V. parahaemolyticus were grown in TSB with 1.5 % (w/v) NaCl at 30 °C. The following day, the cultures were then diluted to an OD₆₀₀ = 0.1. Then, the diluted cell suspension was aliquoted in triplicate (150 μL per well) into 96-well polystyrene plates (Falcon 353,072, Corning, NY, USA). Each bacterial strain test included eight replicates (n=8). After overnight incubation at 30 °C, non-adherent cells and culture medium were carefully removed from the wells, and the attached cells in the wells were washed 3 times with a 2 mM CaCl₂/MgCl₂ buffer. Plates were then heat-fixed at 70 °C for one hour. The heat-fixed biofilms were then stained with 0.01 % crystal violet solution for 15 min. The plates were washed 3 more times with tap water in a tub following the O’Toole protocol³⁹. To solubilize the stain for reading, 150 µL of 70 % ethanol was added to each well. The solutions were transferred to a new 96-well plate, and the absorbance was measured at a wavelength of 600 nm.

We followed the criteria of Stepanovic et al., 2007 ⁴⁰ for classifying the capacity of V. vulnificus and V. parahaemolyticus for biofilm formation. The criteria are as follows: OD>4×ODc means strong biofilm formation; 2×ODc<OD≤4×ODc, moderate biofilm formation; ODc<OD≤2×ODc, weak biofilm formation; and non-biofilm formation when OD<ODc. The cut-off value (ODc) refers to the control measurement, which was performed in a microtiter plate without cells.

Biofilm inhibition assay

We performed the biofilm inhibition assay to determine the minimum biofilm inhibitory concentration (MBIC) of GATR-3 peptide against V. vulnificus MO6 and V. parahaemolyticus NY477, following our MBIC protocol^32,41 with the following modifications. A range of concentrations of GATR-3 peptide (2 μg/mL-64 μg/mL) in 100 μL of sterilized H₂O was incubated with 100 μL of 0.2 OD₆₀₀ nm V. vulnificus MO6 and V. parahaemolyticus NY477, then biofilm was allowed to form overnight, and was stained and measured as described above. Estimated MBIC₅₀ values were determined by nonlinear regression curve fitting using GraphPad Prism. Data are presented as the mean of biological replicates with corresponding 95 % confidence intervals (CI).

Statistical analysis

Statistical significance for MIC assays was determined via Student’s T-test, p < 0.05 was used as the cutoff for significance. For EC₅₀ assays, GraphPad Prism was used to calculate the 95 % confidence interval of the calculated EC₅₀. Values within the 95 % confidence interval were not considered significantly different. Values outside the 95 % confidence interval were considered significantly different. To compare the biofilm formation, a one-way ANOVA was performed, as well as post hoc Tukey’s HSD tests.

Result

A collection of clinical and environmental Vibrio strains was tested for their sensitivity or resistance to GATR-3 antimicrobial peptide, along with two comparator helical antibacterial peptides, LL-37 and Mastoparan-AF, while tetracycline was included as an antibiotic reference control to validate assay performance.

Minimum inhibitory concentration (MIC) determination for peptides against V. vulnificus and V. parahaemolyticus

GATR-3 and comparator peptides were tested for MIC (under CLSI conditions optimized for peptides, using polypropylene plates and Mueller–Hinton broth (MHB) to minimize cation interference³⁵) (Table 1). In these experiments, GATR-3 showed strong antibacterial activity against several Vibrio strains (Fig. 1). Against V. vulnificus MO6, the MIC value for GATR-3 peptide was very low at 8 μg/mL (2.8 μM). Against V. vulnificus JY1701, GATR-3 had an MIC value of 16 μg/mL (5.6 μM), also showing considerable antibacterial efficacy. Among the Vibrio parahaemolyticus isolates, strain SAK11 had an MIC of 8 µg/mL (2.8 µM), whereas V. parahaemolyticus NY477 required 32 µg/mL for inhibition, which is still considered “Active” when the micromolar concentration is considered (11.19 µM). GATR-3 also displayed robust activity toward V. alginolyticus, with a MIC value of 32 µg/mL (11.19 µM) for strains 0–04-D1 and 22–08–18 and an intermediate activity against V. alginolyticus strain 8653, with MIC=64 µg/mL (22.4 µM).

Table 1.

Minimum Inhibitory Concentration (MIC) values for antimicrobial peptides against Vibrio strains (reported in both μg/mL and μM). Note: the lower the MIC number, the more potent the antibacterial activity. Tetracycline MIC was performed in MBH, following the peptide MIC protocol³⁵, not in CA-MHB. GATR-3 MW 2859.354 g/mol, LL-37 MW 4493.37 g/mol, Mastoparan-AF MW 1513.922 g/mol. Numbers in BOLD indicate peptide concentrations that we classify as “active”. Definitions are: “Active”, <32 μg/mL or <12 μM; “Moderate”, 32–100 μg/mL or equivalent μM; and “Inactive”, >100 μg/mL or equivalent μM.

Bacteria	Strain Type	GATR-3 (μg/mL)	GATR-3 (μM)	LL-37-NH2 (μg/mL)	LL-37-NH2 (μM)	Mastoparan -AF-NH2 (μg/mL)	Mastoparan-AF-NH2 (μM)	Tetra-cycline (μg/mL)	Tetra-cycline (μM)
V. vulnificus MO6	Clinical	8	2.8	128	28.49	16	10.57	0.25	0.56
V. vulnificus JY1701	Environmental	16	5.6	64	14.24	16	10.57	0.25	0.56
V. parahaemolyticus NY477	Clinical	32	11.19	128	28.49	32	21.14	1	2.25
V. parahaemolyticus SAK11	Environmental	8	2.8	256	56.97	16	10.57	0.5	1.13
V. harveyi (ATCC 35084) strain 116B	Isolated from the brown shark	8	2.8	>512	-	8	5.28	<0.125	-
V. alginolyticus 0–04-D1		32	11.19	512	113.95	16	10.57	0.25	0.56
V. alginolyticus 22–08–18		32	11.19	512	113.95	16	10.57	<0.125	-
V. alginolyticus 8653		64	22.4	>512	-	64	42.27	0.25	0.56
V. mimicus ATCC 33653	Clinical	128	44.77	512	113.95	16	10.57	0.25	0.56
V. cholerae 9064		256	89.53	512	113.95	64	42.27	0.25	0.56

Fig. 1.

Minimum Inhibitory Concentration of GATR-3 peptide against Vibrio strains. The log of the MIC values (μg/mL) is plotted to enable rapid comparisons between the MIC activities of the 3 peptides and tetracycline. Definitions are: “Active”, <32 μg/mL or 15–20 μM; “Moderate”, 32–100 μg/mL or equivalent μM; and “Inactive”, >100 μg/mL or equivalent μM.

Interestingly, some strains of Vibrio showed resistance to GATR-3 killing (Fig. 1). Two Vibrio strains were highly resistant to GATR-3 peptide, including V. mimicus ATCC 33653, which has a MIC of 128 μg/mL (44.77 µM), and V. cholerae 9064 at 256 μg/mL (89.53 µM).

Mastoparan-AF peptide demonstrated strong antibacterial activity against these strains (Table 1). Against V. harveyi ATCC 35084, Mastoparan-AF was very effective at 8 μg/mL (5.28 μM), illustrating the cation-independent nature of this antimicrobial peptide. For V. vulnificus MO6 and JY1701, V. mimicus ATCC 33653, V. alginolyticus strains 0–04-D1 and 22–08–18, Mastoparan-AF displayed consistent activity with MIC values of 16 μg/mL (10.57 μM). For V. parahaemolyticus, Mastoparan-AF achieved MICs of 16 μg/mL (10.57 μM) and 32 μg/mL (21.14 μM) for SAK11 and NY477, respectively, approximately equivalent to prior published data. In a published report on the effect of Mastoparan-AF on V. parahaemolyticus CCRC 10806[³⁷], the MIC was reported to be 16 μg/mL, and the minimum bactericidal concentration (MBC) was 32 μg/mL. Strain NY477 required a 2x higher concentration of Mastoparan-AF than CCRC 10806. For V. cholerae 9064 and V. alginolyticus 8653 strains, Mastoparan-AF had an MIC of 64 μg/mL (42.27 μM), and thus they would be considered resistant to this peptide.

All the MIC values determined for LL-37 were high, indicating poor antibacterial activity of this peptide for Vibrio strains (Table 1). For V. vulnificus, LL-37 showed MIC values of 64 μg/mL (14.24 μM) against strain JY1701, considered to be an intermediate MIC value. For V. vulnificus MO6 and V. parahaemolyticus NY477, LL-37 showed MIC values of 128 μg/mL (28.49 μM) and thus was considered ineffective. V. parahaemolyticus SAK11 was resistant to LL-37 at MICs of 256 μg/mL (56.97 μM). Against V. cholerae 9064, V. mimicus ATCC 33,653, and V. alginolyticus strains 0–04-D1 and 22–08–18, LL-37 showed 512 μg/mL (104 μM), thus also resistant. V. alginolyticus strain 8653 and V. harveyi ATCC 35,084 revealed that LL-37 had an MIC value >512 μg/mL and is considered resistant.

Tetracycline was the control (Table 1); V. harveyi ATCC 35084 and V. alginolyticus 22–08–18 had a MIC of < 0.125 μg/mL, V. cholerae, V. mimicus ATCC 33653, V. alginolyticus 0–04-D1 and 8653, V. vulnificus MO6 and JY1701 MICs were 0.25 μg/mL, V. parahaemolyticus SAK11 MIC was 0.5 μg/mL, and V. parahaemolyticus NY477 MIC was 1 μg/mL. Although these MICs are performed under peptide MIC conditions (MHB), our results are in general agreement with the reported CLSI-sensitivity of these strains to tetracycline[42], [43], [44].

We showed that the GATR-3 peptide is highly effective against four Vibrio strains, which can be observed by comparing the micromolar (μM) equivalent values of the MICs. When comparing based on μM values, GATR-3 MIC is comparable at 11.6 μM (32 μg/mL) to Mastoparan-AF at 10.6 μM (16 μg/mL), indicating the effectiveness of GATR-3 peptide in the presence of salt and moderate cations of MHB (Table 1). Of interest, the lowest GATR-3 MIC we observed is 2.8 μM for V. parahaemolyticus SAK11 (8 μg/mL), which is only two-fold higher than the tetracycline MIC of 1.2 μM observed for SAK11, and highly similar to the Tetracycline MIC of 2.3 μM for NY477 (Fig. 1). Overall, GATR-3 displayed consistently strong and low-micromolar MIC values across multiple Vibrio species, demonstrating potent antibacterial activity that was comparable to Mastoparan-AF peptide under the same testing conditions.

Antimicrobial activity assay (EC₅₀) for peptides against V. vulnificus and V. parahaemolyticus

To quantify direct bactericidal potency under Vibrio-adapted, low-cation conditions, EC₅₀ values were determined (Fig. 2) after three hours of incubation with each peptide in 10 mM phosphate buffer containing 1 % NaCl, followed by plating for enumeration of surviving bacteria.

Fig. 2.

EC₅₀ results for Vibrio strains against GATR-3, LL-37-NH₂, and Mastoparan-AF-NH₂ peptides.Vibrio vulnificus MO6 EC₅₀ results against A. GATR-3, B. LL-37-NH₂, and C. Mastoparan-AF-NH₂ peptides. Vibrio vulnificus JY1701 EC₅₀ results against D. GATR-3, E. LL-37-NH₂, and F. Mastoparan-AF-NH₂ peptides. Vibrio parahaemolyticus NY477 EC₅₀ results against G. GATR-3, H. LL-37-NH₂, and I. Mastoparan-AF-NH₂ peptides. Vibrio parahaemolyticus SAK11 EC₅₀ results against J. GATR-3, K. LL-37-NH₂, and L. Mastoparan-AF-NH₂ peptides. Escherichia coli EC₅₀ results against M. GATR-3, N. LL-37-NH₂, and O. Mastoparan-AF-NH₂ peptides.

GATR-3 demonstrated nanomolar to low-micromolar EC₅₀ activity across all Vibrio strains tested (Table 2). Against Vibrio vulnificus MO6, GATR-3 was highly potent, with an EC₅₀ of 3.46 × 10⁻³ µM (9.88 × 10⁻³ µg/mL), the lowest value among all Vibrio isolates. The environmental strain V. vulnificus JY1701 showed an EC₅₀ of 1.17 × 10⁻² µM (3.35 × 10⁻² µg/mL). For V. parahaemolyticus NY477, it exhibited an EC₅₀ of 1.19 × 10⁻² µM (3.40 × 10⁻² µg/mL), while SAK11 showed a similar EC₅₀ of 1.75 × 10⁻² µM (4.99 × 10⁻² µg/mL). Against the control strain E. coli ATCC 25922, GATR-3 was extremely potent, with an EC₅₀ of 4.44 × 10⁻⁵ µM (1.27 × 10⁻⁴ µg/mL), confirming its broad-spectrum and potent efficacy.

Table 2.

Antimicrobial (EC₅₀) activities of GATR-3 and other peptides as measured in µM against V. vulnificus and V. parahaemolyticus. Note: the lower the EC₅₀ number, the more potent the antibacterial activity. Peptide molecular weights: GATR-3 2859.354 g/mol, LL-37 4493.37 g/mol, M-AF 1513.922 g/mol.

Species / Strain	GATR-3 (µM) (95 % CI)	LL-37-NH2 (µM) (95 % CI)	Mastoparan-AF-NH2 (µM) (95 % CI)
V. vulnificus MO6	3.46×10⁻³ (4.13×10⁻⁴–5.18×10⁻²)	7.77×10⁻¹ (2.67×10⁻¹–2.17)	8.19×10⁻¹ (1.31×10⁻¹–4.43)
V. vulnificus JY1701	1.17×10⁻² (2.36×10⁻³–4.83×10⁻²)	4.74×10⁻¹ (1.65×10⁻¹–1.36)	7.60×10⁻² (3.75×10⁻²–1.50×10⁻¹)
V. parahaemolyticus NY477	1.19×10⁻² (3.01×10⁻³–4.58×10⁻²)	1.05×10⁻³ (3.14×10⁻⁵–9.64×10⁻³)	6.18×10⁻³ (1.72×10⁻³–1.80×10⁻²)
V. parahaemolyticus SAK11	1.75×10⁻² (4.69×10⁻³–8.01×10⁻²)	3.12×10⁻³ (4.36×10⁻⁴–1.32×10⁻²)	6.67×10⁻³ (5.22×10⁻⁴–8.26×10⁻²)
E. coli	4.44×10⁻⁵ (1.51×10⁻⁵–1.93×10⁻⁴)	3.78×10⁻⁴ (1.17×10⁻⁴–1.63×10⁻³)	8.72×10⁻⁴ (1.35×10⁻⁴–5.95×10⁻³)

The control peptides LL-37 and Mastoparan-AF also displayed bactericidal activity under the same conditions (Fig. 2). LL-37 showed EC₅₀ values of 7.77 × 10⁻¹ µM (3.49 µg/mL) for V. vulnificus MO6, 4.74 × 10⁻¹ µM (2.13 µg/mL) for JY1701; we observed EC₅₀ values of 1.05 × 10⁻³ µM (4.71 × 10⁻³ µg/mL) for V. parahaemolyticus NY477, and 3.12 × 10⁻³ µM (1.40 × 10⁻² µg/mL) for SAK11. Mastoparan-AF exhibited EC₅₀ values of 8.19 × 10⁻¹ µM (1.24 µg/mL) for V. vulnificus MO6, and 7.60 × 10⁻² µM (1.15 × 10⁻¹ µg/mL) for JY1701; while we found 6.18 × 10⁻³ µM (9.36 × 10⁻³ µg/mL) for V. parahaemolyticus NY477, and 6.67 × 10⁻³ µM (1.01 × 10⁻² µg/mL) for SAK11. In E. coli, LL-37 and Mastoparan-AF had EC₅₀s of 3.78 × 10⁻⁴ µM and 8.72 × 10⁻⁴ µM, respectively, both less potent than GATR-3 by roughly one order of magnitude.

The 95 % confidence intervals shown in Table 2 confirmed that GATR-3′s EC₅₀ values overlapped across all the V. parahaemolyticus and V. vulnificus strains, indicating statistically similar EC₅₀ activity. For example, for V. vulnificus MO6, the EC₅₀ was 3.46 × 10⁻³ µM (95 % CI: 4.13 × 10⁻⁴ – 5.18 × 10⁻²), while V. vulnificus JY1701 showed 1.17 × 10⁻² µM (95 % CI: 2.36 × 10⁻³ – 4.83 × 10⁻²), demonstrating overlapping 95 % confidence intervals and thus no statistically significant difference (Fig. 3). LL-37 and Mastoparan-AF were both less active against V. vulnificus (approx. 10^−1 µM) than against V. parahaemolyticus (approx. 10^−3 µM) in these EC₅₀ experiments (p < 0.05), suggesting greater strain-specific sensitivity among these peptides than for GATR-3. Supplemental Table 4 shows the EC₅₀ results in terms of μg/mL for comparison.

Fig. 3.

Comparison of EC₅₀ antibacterial assay of GATR-3 peptide (log μM) against Vibrio strains to LL-37-NH₂ and Mastoparan-AF-NH₂ peptides. The x-axis is the log of the EC₅₀ activity (log μM). The y-axis represents different bacterial strains.

Pairwise statistical analysis in Supplemental Table 5 confirmed that GATR-3′s EC₅₀ values were significantly different from those of both LL-37 and Mastoparan-AF for all tested bacteria by comparing the 95 % confidence intervals. LL-37 and Mastoparan-AF were not significantly different for E. coli, V. vulnificus MO6, or V. parahaemolyticus SAK11 but diverged for V. vulnificus JY1701 and V. parahaemolyticus NY477. GATR-3 was notably more potent than both comparator peptides against E. coli and both V. vulnificus strains, whereas LL-37 and Mastoparan-AF were slightly more active than GATR-3 against V. parahaemolyticus NY477 and SAK11.

Overall, the EC₅₀ analysis demonstrates that GATR-3 retains strong bactericidal efficacy under conditions simulating the ionic environment required for Vibrio growth (low cations, 1 % NaCl). The peptide’s consistent sub-micromolar activity across all strains, combined with its superior potency against V. vulnificus, supports its characterization as a highly active and salt-tolerant antimicrobial candidate.

Comparing activity in MIC conditions to activity in Vibrio-adapted EC₅₀ conditions

Comparison of MIC and Vibrio-adapted EC₅₀ datasets showed that GATR-3 remained highly active under both nutrient-rich and low-cation conditions. In MIC testing with Mueller–Hinton broth (MHB), GATR-3 achieved low-micromolar inhibition across several Vibrio strains (Table 1). In the EC₅₀ assay performed in phosphate buffer lacking divalent cations and containing 1 % NaCl (approximately 171 mM NaCl; Supplemental Table 3), GATR-3 demonstrated bactericidal activity in the nanomolar-to-low-micromolar range (Table 2 (µM) and Supplemental Table 4 (μg/ml)). LL-37 showed marked cation sensitivity, displaying measurable bactericidal activity only in the low-cation EC₅₀ buffer but minimal inhibition in MHB. Mastoparan-AF peptide remained active in both environments, although its efficacy against V. vulnificus was consistently lower than that of GATR-3. These findings indicate that GATR-3 retains potent antibacterial activity across varying ionic strengths and in the presence of cations, supporting its characterization as a salt-tolerant antimicrobial peptide with stable performance in both assay systems.

Biofilm formation by Vibrio vulnificus and Vibrio parahaemolyticus

We performed a quantitative comparison of biofilm formation among Vibrio isolates and E. coli after 24 h incubation. Biofilm biomass was determined by crystal violet staining and measurement of OD₆₀₀^38,40. V. vulnificus MO6 and V. parahaemolyticus NY477 exhibited the highest biofilm-forming capacities (shown in Fig. 4). Among bacterial strains, V. vulnificus MO6 displayed the highest biofilm formation (mean OD₆₀₀ of 0.257 ± 0.021), followed by V. parahaemolyticus NY477 (0.198 ± 0.082), V. parahaemolyticus SAK11 (0.093 ± 0.020), and V. vulnificus JY1701 (0.087 ± 0.004).

Fig. 4.

Biofilm Formation of Vibrio isolates and E. coli. Quantitative comparison of biofilm formation among Vibrio isolates and E. coli after 24 h incubation. Biofilm biomass was determined by crystal violet staining and measurement of OD₆₀₀. V. vulnificus MO6 and V. parahaemolyticus NY477 exhibited the highest biofilm-forming capacities.

We then determined the strength of the biofilms formed by V. vulnificus and V. parahaemolyticus strains after 24 h incubation. The strengths of the biofilm formation of the different strains were classified following Stepanović et al⁴⁰ (Table 3). ODc is defined as the average OD of the negative control plus three times its standard deviation⁴⁰. The mean optical density (OD₆₀₀) values of the solubilized crystal violet biofilm stain were compared to the negative control (NC, ODc = 0.0429). All Vibrio strains produced OD₆₀₀ readings above the ODc threshold, confirming measurable biofilm formation under the tested conditions. E. coli was used as a control, and it formed moderate biofilms under the media conditions used, consistent with prior reports of the strength of E. coli 25,922 biofilm⁴⁵. Based on Stepanović et al’s classification (weak = ODc–2×ODc; moderate = 2×ODc–4×ODc; strong > 4×ODc)⁴⁰, MO6 and NY477 were classified as strong biofilm formers, while JY1701 and SAK11 were moderate formers. The E. coli reference strain (OD₆₀₀ = 0.109 ± 0.010) also fell within the moderate range. Statistical analysis confirmed significant differences among Vibrio strains. One-way ANOVA showed a highly significant difference in mean OD₆₀₀ values (F = 28.86, p = 1.03 × 10⁻⁸). Post hoc Tukey’s HSD tests indicated that V. vulnificus MO6 produced significantly higher biofilm biomass than V. vulnificus JY1701 (p < 0.01) and V. parahaemolyticus SAK11 (p < 0.01), and V. parahaemolyticus NY477 was significantly higher than JY1701 and SAK11 (p < 0.01). The difference between MO6 and NY477 was not statistically significant, suggesting comparable strong biofilm-forming ability between these two clinical isolates. In conclusion, we found that V. vulnificus MO6 and V. parahaemolyticus NY477 form dense, adherent, strong biofilms under laboratory conditions, while JY1701 and SAK11 exhibit weaker but consistent surface attachment, thus moderate biofilms.

Table 3.

Strength of Biofilm Formation of Vibrio isolates after 24 h incubation. The classification of the strength of the biofilm formation of strains was classified following Stepanović et al⁴⁰. The data are expressed as the mean OD₆₀₀ ± SD with fold change relative to the optical density cut-off (ODc = 0.0429).

Species / Strain	Mean OD₆₀₀ ± SD	Fold change (OD/ODc)	Biofilm Classification
V. vulnificus MO6	0.257 ± 0.021	6.21	Strong
V. vulnificus JY1701	0087 ± 0.004	2.10	Moderate
V. parahaemolyticus NY477	0.198 ± 0.082	4.79	Strong
V. parahaemolyticus SAK11	0.093 ± 0.020	2.25	Moderate
E. coli	0.109 ± 0.010	2.63	Moderate
Negative control (NC)	0.041 ± 0.005	1.00	-

Antibiofilm activity of GATR-3 against vibrio vulnificus MO6 and vibrio parahaemolyticus NY477

We next evaluated the ability of GATR-3 to inhibit this biofilm formation using the minimum biofilm inhibitory concentration (MBIC) assay, in which the peptide is present throughout the initial biofilm development period (Fig. 5). The concentration at which peptides inhibit 50 % of biofilm biomass is a useful comparator; thus, we estimated the MBIC₅₀ for each experiment by analyzing the data shown. Against V. vulnificus MO6, GATR-3 MBIC₅₀ is 13.63 µg/mL, whereas for V. parahaemolyticus NY477, the MBIC₅₀ is 37.61 µg/mL (Table 4). For comparison, the MBIC₅₀ of the control peptide Mastoparan-AF for V. vulnificus MO6 biofilm is ∼64 µg/mL, and for V. parahaemolyticus NY477 is 12.47 µg/mL. Complete eradication of preformed biofilms was not achieved under these conditions, likely reflecting partial GATR-3 peptide degradation by bacterial proteases during prolonged incubation. These results are in the same range as the biofilm-inhibitory activity of GATR-3 against A. baumannii, MBIC₅₀= 9.3 µg/mL³². In our prior study, the biofilm-inhibitory activity of LL-37 was evaluated against Pseudomonas aeruginosa, and it was also only able to eradicate 50 % of the biofilms (MBIC₅₀) at 1 µg/mL under similar conditions⁴¹. Thus, GATR-3 exhibits measurable biofilm-inhibition activity against both V. vulnificus and V. parahaemolyticus, supporting its potential as a dual-action peptide with both planktonic and biofilm-inhibitory effects.

Fig. 5.

Minimum Biofilm Inhibitory Concentration (MBIC) of GATR-3 and Mastoparan-AF-NH₂ against Vibrio strains. A. V. vulnificus MO6 against GATR-3, B. V. vulnificus MO6 against Mastoparan-AF-NH₂, C. V. parahaemolyticus NY477 against GATR-3, D. V. parahaemolyticus NY477 against Mastoparan-AF-NH₂.

Table 4.

Estimated MBIC₅₀ values of GATR-3 and Mastoparan-AF against Vibrio vulnificus MO6 and Vibrio parahaemolyticus NY477. The minimum biofilm inhibitory concentration required to inhibit 50% of biofilm biomass (MBIC₅₀) was calculated from the MBIC data, determined using crystal violet staining after 24 h incubation under standard peptide assay conditions. The means of biological replicates and 95% confidence intervals (CI) were calculated by nonlinear regression analysis in GraphPad Prism.

Peptide	Organism	Estimated MBIC50 (μg/mL)	95 % CI (μg/mL)
GATR-3	V. vulnificus MO6	13.63	8.654 to 21.85
	V. parahaemolyticus NY477	37.16	23.49 to 58.20
Mastoparan-AF	V. vulnificus MO6	∼64	N/A
	V. parahaemolyticus NY477	12.47	5.548 to 28.02

Discussion

GATR-3 is a rationally designed synthetic antimicrobial peptide that has exhibited strong bactericidal activity against multiple Gram-negative bacteria, including Pseudomonas aeruginosa and Acinetobacter baumannii³². The objective of this study was to evaluate the GATR-3 antimicrobial peptide for its potential antibacterial activity against V. vulnificus, V. parahaemolyticus, and related marine Vibrio species. A designed antimicrobial peptide requires maximizing bacterial membrane disruption while minimizing cytotoxicity to human cells. GATR-3 peptide meets these core design principles of moderate cationicity (+7), amphipathicity^31,46, hydrophobicity⁶⁵, and relatively short length (22 amino acids), which align well with features that enhance membrane disruption while minimizing host cytotoxicity as we previously demonstrated.

Antimicrobial agents and peptides against Vibrio species

Several antimicrobial peptides have been reported to exhibit activity against Vibrio species, including pleurocidin⁴⁷, magainin-2, pexiganan⁴⁷, and granulysin-derived peptides⁴⁸. Tachyplesin peptide, isolated from horseshoe crabs, has been reported to have excellent activity against V. parahaemolyticus (MICs=0.4–5 μg/mL) and V. vulnificus (MIC ≈ 2.5 µg/mL)^49,50, whereas other peptides such as melittin⁵¹, NKL-24^52,53, and oyster-derived peptides including cgMolluscidin and Cg-defensin⁵⁴ showed limited efficacy under physiological salt conditions. Additional AMPs targeting V. vulnificus have also been reported. For instance, the HPA3P peptide on gold nanoparticles significantly improved survival in V. vulnificus-infected mice⁵⁵. Bovine lactoferrin-derived peptides (LFchimera, LFcin17–30, LFampin5–284)⁴⁶ have been shown to effectively kill multiple Vibrio species, including V. vulnificus, V. cholera, and V. alginolyticus⁵⁶. In this study, we showed that the synthetic peptide GATR-3 shows potent antibacterial and biofilm-inhibition activity against clinically relevant Vibrio species. GATR-3 exhibited low MICs (8–32 µg/mL) and effectively reduced biofilm formation by V. vulnificus and V. parahaemolyticus, demonstrating stronger efficacy than LL-37 and comparable activity to Mastoparan-AF under Vibrio-adapted conditions.

GATR-3 activity and Lipid A-LPS architecture

GATR-3 peptide demonstrated strong antibacterial activity, improved salt/cation tolerance against V. vulnificus and V. parahaemolyticus under CLSI peptide MIC conditions³⁵, compared with previously reported peptides described above. Against V. vulnificus MO6, the MIC was 8 μg/mL (2.8 μM), matching GATR-3′s previously reported potency against P. aeruginosa 2110³². Similar MICs were observed for V. parahaemolyticus SAK11 and V. harveyi ATCC 35084, and moderate activity was observed against V. alginolyticus strains (MIC = 32 µg/mL).

In contrast, GATR-3 resistance was observed for V. mimicus ATCC 33653 and V. cholerae 9064 (MIC ≥ 128 µg/mL). The resistance of V. mimicus and V. cholerae to GATR-3 likely reflects intrinsic differences in these bacterias outer-membrane and LPS architecture. Both of these species possess polymyxin-resistant LPS phenotypes mediated by lipid A remodeling, resulting in l-Ara4N and/or pEtN substitutions that diminish surface negative charge and impede electrostatic binding of cationic peptides ^57,58,59,64. In contrast, V. vulnificus and V. parahaemolyticus typically retain more anionic, unmodified LPS under standard culture conditions, permitting potent GATR-3 activity^25,60,61. These findings parallel known patterns of polymyxin and LL-37 sensitivity among Vibrio species²⁵ and provide a plausible mechanistic basis for the strain-specific MIC differences observed in this study. Given that the activity of cationic peptides depends on electrostatic interactions with negatively charged lipid A, bacteria possessing these modified LPS structures (e.g., V. mimicus and V. cholerae) are expected to exhibit reduced susceptibility. The differences between the bacterial strains used in this study can be explained by GATR-3′s ability to disrupt the bacterial membranes through its LPS-targeting mechanism.

In our previous work, GATR-3 was shown to directly disrupt gram-negative bacterial membranes through depolarization and pore formation, leading to loss of membrane integrity and bacterial death³². Overall, these results extend our previous studies by showing that GATR-3 has potent anti-bacterial activity against V. vulnificus and V. parahaemolyticus.

Mastoparan-AF antimicrobial peptide

Mastoparan-AF is an amphipathic, cationic α-helical peptide isolated from hornet venom (Vespa affinis), which has been shown to possess broad-spectrum antimicrobial activity against gram-negative bacteria, including moderate activity against V. parahaemolyticus^36,37. Acting primarily through membrane disruption, Mastoparan-AF permeabilizes bacterial cell envelopes by forming surface dents, pores, and invaginations that lead to cell lysis; this effect is attributed to its ability to adopt a 3–11 amphipathic helical structure that promotes interaction with lipid bilayers⁶². Direct data for Mastoparan-AF against V. vulnificus is not previously reported in the literature to our knowledge and is reported here for the first time. Mastoparan-AF showed very strong activity against V. harveyi with an MIC of 8 µg/mL (5.28 µM) and was active at 16 µg/mL (10.57 µM) against five strains: V. vulnificus MO6 and JY1701, V. mimicus ATCC 33653, and V. alginolyticus strains 0–04-D1 and 22–08–18. For V. parahaemolyticus, the peptide retained strong activity against strain SAK11 (16 µg/mL) but demonstrated moderate activity against NY477 (32 µg/mL, 21.14 µM). Against V. cholerae 9064 and V. alginolyticus 8653, the MIC increased to 64 µg/mL, indicating reduced susceptibility. These findings align with previous reports describing Mastoparan-AF activity against V. parahaemolyticus CCRC10806 at MIC=16 µg/mL³⁷.

Human cathelicidin peptide LL-37

The human cathelicidin peptide LL-37 is well-known to be cation- and salt-sensitive against gram-negative bacteria^21,22,63. LL-37 acts primarily by binding to negatively charged bacterial membranes through its cationic residues, inserting into the lipid bilayer, and forming transmembrane pores that disrupt membrane integrity and cause cell lysis; it can also penetrate cells to target intracellular molecules while modulating host immune responses⁶⁴. Turner et al. showed that the addition of 1 mM Ca²⁺ significantly increased the MIC of LL-37 against E. coli^24,65 by blocking the ability of the LL-37 peptide to bind to the LPS. This finding was extended in this study against two Vibrio species, V. vulnificus and V. parahaemolyticus. In our study, the LL-37 was ineffective under MIC conditions (MHB) against V. vulnificus, V. parahaemolyticus, and all other Vibrio strains tested, likely due to divalent-cation-mediated inhibition of peptide-LPS interactions⁶⁶. As can be seen in the low-divalent cation EC₅₀ assays, LL-37 has a reasonable antibacterial activity against V. vulnificus and V. parahaemolyticus. This is in agreement with the reported LL-37 MIC ≈ 50 µg/mL against V. vulnificus clinical strain L-180⁶⁷. Interestingly, we noted that for V. harveyi strain 116B, LL-37 MIC is reported to be >512 µg/mL, but this strain is still strongly killed by GATR-3 and Mastoparan-AF at 8 µg/mL.

The antibiotic tetracycline was included as a control, which showed antimicrobial activity against most strains, within a range of effective MICs. As these tetracycline MICs were performed in MHB, following the peptide MIC protocol, the results may be lower than reported MICs performed following the normal CLSI method using CA-MHB, as the Mg²⁺ and Ca²⁺ can influence the MIC assay results.

Biofilm-inhibitory effect of GATR-3

The partial inhibition of Vibrio biofilm formation by GATR-3 is consistent with prior findings for α-helical antimicrobial peptides such as LL-37 ⁴¹. The ∼50 % reduction in biofilm biomass in the range of ∼14–38 µg/mL against V. vulnificus MO6 and V. parahaemolyticus NY477 suggests that GATR-3 interferes with early cell adhesion and biofilm formation via reduced cell viability, rather than directly lysing preformed biofilms. The plateau of inhibition at ∼50 % likely reflects proteolytic degradation of the peptide during prolonged incubation, a limitation also observed with other cationic AMPs in similar assays⁴¹. The MBIC₅₀ for GATR-3 against A. baumannii 5075 in our previous study was 9.3 μg/mL (range 0.18–21.7 μg/mL for all A. baumannii strains tested), suggesting that GATR-3 has similar activity or slightly less biofilm-inhibiting activity against Vibrio biofilms. The moderate antibiofilm activity observed here, in combination with potent planktonic killing, suggests that GATR-3 could act by preventing early Vibrio biofilm formation while providing direct bactericidal effects, particularly in a topical formulation where peptide stability can be optimized. GATR-3 could potentially be used in combination with standard antibiotics for treatment ⁵⁶.

Novelty and limitations of the study

The novelty of this work lies in the application of the rationally designed synthetic antimicrobial peptide GATR-3 against multiple Vibrio species. To our knowledge, this is the first report evaluating GATR-3 or any alligator-derived synthetic peptide against members of the Vibrio genus. In addition to its direct antibacterial activity, this study is also the first to demonstrate biofilm inhibitory activity of GATR-3 against V. vulnificus and V. parahaemolyticus, revealing an additional mechanism by which this peptide may be effective against marine and wound-associated infections. Also, direct data for Mastoparan-AF against V. vulnificus is reported here for the first time. These findings expand the known spectrum of GATR-3 peptide activity and highlight its potential as a salt-tolerant, cation-independent antimicrobial candidate for these marine pathogens, V. vulnificus and V. parahaemolyticus.

This study has several limitations. While the peptide showed potent in vitro bactericidal and biofilm-inhibition activity, in vivo validation in oyster or mammalian wound infection models was beyond the scope of this work and will be addressed in future studies. Another limitation is the number and diversity of Vibrio isolates examined. Although our panel included representative clinical and environmental strains across multiple Vibrio species (V. vulnificus, V. parahaemolyticus, V. cholerae, V. mimicus, and V. alginolyticus), it did not encompass all the clinically relevant or emerging Vibrio isolates. Nevertheless, the consistent activity of GATR-3 against strains that are not inherently resistant to polymyxin B underscores its potential as a broad-spectrum anti-Vibrio agent, warranting further development.

Conclusion

Overall, these findings demonstrate that GATR-3 maintains broad and potent antimicrobial activity across multiple Vibrio species, comparable or superior to known AMPs such as Mastoparan-AF and markedly better than LL-37 under MIC conditions. Low MIC acitivities against V. vulnificus and V. parahaemolyticus suggest that this peptide has therapeutic potential for marine and wound-associated infections, even in moderate-salt environments. Given its favorable therapeutic index and antibacterial activity, GATR-3 may also act synergistically with conventional antibiotics to enhance their treatment efficacy. Collectively, the potent antibacterial and biofilm-inhibitory properties of GATR-3 against V. vulnificus and V. parahaemolyticus highlight its promise for future studies as a next-generation topical therapeutic candidate for preclinical development against wound infections caused by these clinically important marine pathogens.

CRediT authorship contribution statement

Sabrina Hsin-Yin Tsai: Writing – review & editing, Visualization, Formal analysis, Data curation. Brett A. Froelich: Writing – review & editing, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Conceptualization. Monique L. van Hoek: Writing – review & editing, Writing – original draft, Visualization, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no competing interests in this research.

Data availability

No data was used for the research described in the article.