Scorpion-Venom-Derived Antimicrobial Peptide Css54 Exerts Potent Antimicrobial Activity by Disrupting Bacterial Membrane of Zoonotic Bacteria

Antibiotic resistance is an important issue affecting humans and livestock. Antimicrobial peptides are promising alternatives to antibiotics. In this study, the antimicrobial peptide Css54, isolated from the venom of C. suffuses, was found to exhibit antimicrobial activity against bacteria such as Listeria monocytogenes, Streptococcus suis, Campylobacter jejuni, and Salmonella typhimurium that cause zoonotic diseases. Moreover, the cytotoxicity and hemolytic activity of Css54 was lower than that of melittin isolated from bee venom. Circular dichroism assays showed that Css54 has an α-helix structure in an environment mimicking that of bacterial cell membranes. We examined the effect of Css54 on bacterial membranes using N-phenyl-1-naphthylamine, 3,3′-dipropylthiadicarbbocyanine iodides, SYTOX green, and propidium iodide. Our findings suggest that the Css54 peptide kills bacteria by disrupting the bacterial membrane. Moreover, Css54 exhibited antibiofilm activity against L. monocytogenes. Thus, Css54 may be useful as an alternative to antibiotics in humans and animal husbandry.


Introduction
The development and emergence of multidrug-resistant bacteria are a serious crisis for humans and livestock worldwide [1]. Many countries have monitored antibiotic use to decrease antibiotic resistance [2]. Because of antibiotic-resistant infections, annual global deaths are expected to increase from 700,000 in 2014 to 10 million by 2050. Most antibiotics are used in animal husbandry to protect against infection, treat ill animals, and promote growth [3,4]. Antibiotic-resistant bacteria used in animal husbandry spread to humans in various ways, such as through food, contaminated water, and soil [5]. Zoonotic diseases are those transferred from animals to infect humans, threatening the life and health of people worldwide [6]. The World Health Organization (WHO) reports the number of diseases originating from contaminated food. The Interagency Food Safety Analytics Collaboration described Salmonella typhimurium and Listeria monocytogenes as priority foodborne pathogens [7]. Listeria monocytogenes and Salmonella typhimurium that originate in animals can cause bacterial diseases such as listeriosis and salmonellosis, which are major bacterial zoonosis diseases [8].
Listeria monocytogenes is a Gram-positive bacterium found in various environments such as water, soil, and extreme environments, including low and high temperatures, high salt concentrations, and a broad pH range. Listeria monocytogenes infects immunocompromised persons, pregnant women, and neonates, resulting in abortion, meningitis, and blood poisoning [9]. Salmonella typhimurium is a

Peptide Structure and Characterization
Css54 was isolated from C. suffusus venom. The amino acid sequence, molecular weight, and net charge of Css54 are listed in Table 1. Css54 consists of 25 amino acids. The net charge value and hydrophobicity were 5 and 0.532, respectively. A wheel diagram and three-dimensional structure analysis showed that the hydrophobic and hydrophilic regions formed an α-helix structure ( Figure 1A,B). Css54 synthesis and molecular weights were confirmed by reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 column and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS; Figure S1A,B).

Circular Dichroism Measurements
The secondary structure of Css54 was measured by circular dichroism (CD) spectroscopy in various concentrations of sodium dodecyl sulfate (SDS) to mimic the negative charge of the bacterial membrane and trifluoroethanol (TFE) solution to mimic the hydrophobic environment of the bacterial membrane [21,22]. In 10 mM sodium phosphate, mimicking an aqueous environment, Css54 displayed a random coil (Figure 2A). In contrast, Css54 exhibited an α-helix conformation in membrane-mimicking environments, depending on the TFE and SDS concentrations ( Figure 2B,C). These results indicate that Css54 can affect the bacterial membrane through electrostatic and hydrophobic interactions.

Circular Dichroism Measurements
The secondary structure of Css54 was measured by circular dichroism (CD) spectroscopy in various concentrations of sodium dodecyl sulfate (SDS) to mimic the negative charge of the bacterial membrane and trifluoroethanol (TFE) solution to mimic the hydrophobic environment of the bacterial membrane [21,22]. In 10 mM sodium phosphate, mimicking an aqueous environment, Css54 displayed a random coil (Figure 2A). In contrast, Css54 exhibited an α-helix conformation in membrane-mimicking environments, depending on the TFE and SDS concentrations ( Figure 2B,C). These results indicate that Css54 can affect the bacterial membrane through electrostatic and hydrophobic interactions.

Circular Dichroism Measurements
The secondary structure of Css54 was measured by circular dichroism (CD) spectroscopy in various concentrations of sodium dodecyl sulfate (SDS) to mimic the negative charge of the bacterial membrane and trifluoroethanol (TFE) solution to mimic the hydrophobic environment of the bacterial membrane [21,22]. In 10 mM sodium phosphate, mimicking an aqueous environment, Css54 displayed a random coil (Figure 2A). In contrast, Css54 exhibited an α-helix conformation in membrane-mimicking environments, depending on the TFE and SDS concentrations ( Figure 2B,C). These results indicate that Css54 can affect the bacterial membrane through electrostatic and hydrophobic interactions.

Antimicrobial Activities against Zoonotic Pathogens
The antimicrobial activities of peptides and antibiotics against zoonotic pathogens are summarized in Table 2. We used melittin isolated from bee venom as a control, which is known as a lytic peptide with strong antibacterial activity and used in various antibiotics for animals. Css54 showed broad-spectrum antimicrobial activity against L. monocytogenes, S. typhimurium, Campylobacter jejuni, and Streptococcus suis, with minimum inhibitory concentrations (MICs) ranging from 2 to 4 µM. These MIC values are similar to those of melittin. The highest MIC values for cefotaxime, vancomycin, and colistin were more than 64 µM. These results suggest that Css 54 effectively inhibited zoonotic bacterial growth.

Cytotoxicity and Hemolytic Activities of Antimicrobial Peptides
To measure the toxicity of Css54, we tested hemolytic activity by measuring hemoglobin, which appears red in the supernatant of sheep red blood cells (sRBCs) after treatment with the peptide. Melittin, which was used as a control, induced more than 80% hemolysis at 16 µM. However, the hemolytic percentage of Css54 was approximately 40% at 16 µM ( Figure 3A). To investigate the cytotoxicity of AMPs against mammalian cells, we performed an MTT assay. Css54 exhibited lower cytotoxicity than melittin at a concentration of 2.5 µM; melittin treatment resulted in cell survival rates of approximately 10%. Css54 displayed a cell viability rate of approximately 50% at 8 µM. These results indicate that Css54 exhibited lower cytotoxicity than melittin ( Figure 3B).

Antimicrobial Activities in Various Environments and Stability Against Heat
To assess the antimicrobial activity of Css54 under various L. monocytogenes growth conditions, Css54 was tested by MIC assay at pH values of 4, 6, 8, and 10, temperatures of 4-40 °C, and salt concentrations of 0-8%. At different pH values, Css54 at 0.5× and 1× MIC inhibited the growth of L. monocytogenes compared to the control ( Figure 4A). Moreover, Css54 showed antimicrobial activity at various temperatures ( Figure 4B). In the salt sensitivity test, the antimicrobial activity of Css54 was maintained in the presence of different salt concentrations ( Figure 4C). Moreover, Css54 retained its antimicrobial activity after heating at 100 °C for 80 min ( Figure 4D). These results suggest that the antimicrobial activity of Css54 can be maintained under the growth conditions of L. monocytogenes and showed thermal stability for 80 min.

Antimicrobial Activities in Various Environments and Stability against Heat
To assess the antimicrobial activity of Css54 under various L. monocytogenes growth conditions, Css54 was tested by MIC assay at pH values of 4, 6, 8, and 10, temperatures of 4-40 • C, and salt concentrations of 0-8%. At different pH values, Css54 at 0.5× and 1× MIC inhibited the growth of L. monocytogenes compared to the control ( Figure 4A). Moreover, Css54 showed antimicrobial activity at various temperatures ( Figure 4B). In the salt sensitivity test, the antimicrobial activity of Css54 was maintained in the presence of different salt concentrations ( Figure 4C). Moreover, Css54 retained its antimicrobial activity after heating at 100 • C for 80 min ( Figure 4D). These results suggest that the antimicrobial activity of Css54 can be maintained under the growth conditions of L. monocytogenes and showed thermal stability for 80 min.

Antimicrobial Activities in Various Environments and Stability Against Heat
To assess the antimicrobial activity of Css54 under various L. monocytogenes growth conditions, Css54 was tested by MIC assay at pH values of 4, 6, 8, and 10, temperatures of 4-40 °C, and salt concentrations of 0-8%. At different pH values, Css54 at 0.5× and 1× MIC inhibited the growth of L. monocytogenes compared to the control ( Figure 4A). Moreover, Css54 showed antimicrobial activity at various temperatures ( Figure 4B). In the salt sensitivity test, the antimicrobial activity of Css54 was maintained in the presence of different salt concentrations ( Figure 4C). Moreover, Css54 retained its antimicrobial activity after heating at 100 °C for 80 min ( Figure 4D). These results suggest that the antimicrobial activity of Css54 can be maintained under the growth conditions of L. monocytogenes and showed thermal stability for 80 min.
To visualize bacteria in the biofilm, we used SYTO9, a green dye that stains live cells. After treatment with Css54 and melittin at 1, 2, and 4 μM, L. monocytogenes (KCTC 3710), L. monocytogenes (KCCM 43155), and L. monocytogenes were viable within the biofilm (KCCM 40307), respectively, compared to the control ( Figure 5D-F). These images were consistent with the quantitative measurement of biofilms by crystal violet staining and indicate that Css54 has a strong antibiofilm activity, similar to melittin against L. monocytogenes.  To visualize bacteria in the biofilm, we used SYTO9, a green dye that stains live cells. After treatment with Css54 and melittin at 1, 2, and 4 µM, L. monocytogenes (KCTC 3710), L. monocytogenes (KCCM 43155), and L. monocytogenes were viable within the biofilm (KCCM 40307), respectively, compared to the control ( Figure 5D-F). These images were consistent with the quantitative measurement of biofilms by crystal violet staining and indicate that Css54 has a strong antibiofilm activity, similar to melittin against L. monocytogenes.

Outer Membrane Permeability and Membrane Depolarization Assay
We examined the disruption of the outer membrane of S. typhimurium after treatment with Css54 at 0.5×, 1×, and 2× MIC in an N-phenyl-1-naphthylamine (NPN) uptake assay. NPN shows increased fluorescence intensity in hydrophobic environments [26]. The fluorescence intensity increased in a dose-dependent manner after treatment with Css54 ( Figure 6A). These data suggest that the hydrophobic environment increased as the outer membranes were disrupted by Css54, resulting in an increase in fluorescence intensity. at 0.5×, 1×, and 2× MIC in an N-phenyl-1-naphthylamine (NPN) uptake assay. NPN shows increased fluorescence intensity in hydrophobic environments [26]. The fluorescence intensity increased in a dose-dependent manner after treatment with Css54 ( Figure 6A). These data suggest that the hydrophobic environment increased as the outer membranes were disrupted by Css54, resulting in an increase in fluorescence intensity.
The effect of Css54 on membrane potential was investigated using DiSC3 (5). DiSC3 (5) is distributed in the cytoplasmic membrane of bacteria, where self-quenching occurs. When the bacterial membrane is damaged by antimicrobial agents, the dye is released into the medium, resulting in increased fluorescence intensity [27]. After completely stabilizing the fluorescence intensity of DiSC3(5), L. monocytogenes and S. typhimurium were treated with Css54. In S. typhimurium, Css54 MIC showed a fluorescence intensity of approximately 30 min after 30 min ( Figure 6B). In L. monocytogenes, Css54 at 2× MIC showed a fluorescence intensity of approximately 110 after 30 min ( Figure 6C). The fluorescence intensity increased in a dose-dependent manner, indicating that Css54 induced membrane depolarization of S. typhimurium and L. monocytogenes.

Effect of Peptides on Membrane Integrity
The integrity of the bacterial membrane was determined using SYTOX green and propidium iodide (PI). Neither dye can pass through the intact membrane. However, if the bacterial membrane is disrupted by antimicrobial agents, these dyes bind to DNA, resulting in an increase in fluorescence intensity. Css54 at 0.5, 1, and 2× MIC at 30 min showed fluorescence intensities of approximately 31, 65, and 93 in S. typhimurium ( Figure 7A). However, the control group of S. typhimurium showed a The effect of Css54 on membrane potential was investigated using DiSC 3 (5). DiSC 3 (5) is distributed in the cytoplasmic membrane of bacteria, where self-quenching occurs. When the bacterial membrane is damaged by antimicrobial agents, the dye is released into the medium, resulting in increased fluorescence intensity [27]. After completely stabilizing the fluorescence intensity of DiSC 3 (5), L. monocytogenes and S. typhimurium were treated with Css54. In S. typhimurium, Css54 MIC showed a fluorescence intensity of approximately 30 min after 30 min ( Figure 6B). In L. monocytogenes, Css54 at 2× MIC showed a fluorescence intensity of approximately 110 after 30 min ( Figure 6C). The fluorescence intensity increased in a dose-dependent manner, indicating that Css54 induced membrane depolarization of S. typhimurium and L. monocytogenes.

Effect of Peptides on Membrane Integrity
The integrity of the bacterial membrane was determined using SYTOX green and propidium iodide (PI). Neither dye can pass through the intact membrane. However, if the bacterial membrane is disrupted by antimicrobial agents, these dyes bind to DNA, resulting in an increase in fluorescence Antibiotics 2020, 9, 831 8 of 16 intensity. Css54 at 0.5, 1, and 2× MIC at 30 min showed fluorescence intensities of approximately 31, 65, and 93 in S. typhimurium ( Figure 7A). However, the control group of S. typhimurium showed a fluorescence intensity of approximately 4. Css54 at 0.5, 1, and 2× MIC increased fluorescence intensity by approximately 17, 23, and 43 at 30 min in L. monocytogenes, whereas this value in the control group of L. monocytogenes was approximately 11 ( Figure 7B). In S. typhimurium and L. monocytogenes, treatment with Css54 immediately increased the fluorescence intensity in a dose-dependent manner compared to the control sample in both strains. fluorescence intensity of approximately 4. Css54 at 0.5, 1, and 2× MIC increased fluorescence intensity by approximately 17, 23, and 43 at 30 min in L. monocytogenes, whereas this value in the control group of L. monocytogenes was approximately 11 ( Figure 7B). In S. typhimurium and L. monocytogenes, treatment with Css54 immediately increased the fluorescence intensity in a dose-dependent manner compared to the control sample in both strains.
We further analyzed the integrity of the bacterial membranes by PI staining and flow cytometry. In S. typhimurium, the percentage of PI staining was approximately 16% in the control group. After treatment with Css54 at 0.5× and 1× MIC, approximately 21% and 75% of bacteria were stained with PI ( Figure 7C). In L. monocytogenes, the percentage of PI staining was approximately 6% in the control group. Following treatment with Css54 at 0.5× and 1× MIC, around 57% and 77% of bacteria were stained with PI ( Figure 7D). Css54 dose-dependently induced an increase in PI-positive bacteria. Collectively, these data show that Css54 killed bacteria by disrupting the membranes of L. monocytogenes and S. typhimurium.

Discussion
The overuse of antibiotics has rapidly increased the development of antibiotic-resistant bacteria and become a serious concern worldwide [28]. Many antibiotics are globally used on animals used as food, which is a major source of antibiotic-resistant bacteria development [29]. Thus, new We further analyzed the integrity of the bacterial membranes by PI staining and flow cytometry. In S. typhimurium, the percentage of PI staining was approximately 16% in the control group. After treatment with Css54 at 0.5× and 1× MIC, approximately 21% and 75% of bacteria were stained with PI ( Figure 7C). In L. monocytogenes, the percentage of PI staining was approximately 6% in the control group. Following treatment with Css54 at 0.5× and 1× MIC, around 57% and 77% of bacteria were stained with PI ( Figure 7D). Css54 dose-dependently induced an increase in PI-positive bacteria. Collectively, these data show that Css54 killed bacteria by disrupting the membranes of L. monocytogenes and S. typhimurium.

Discussion
The overuse of antibiotics has rapidly increased the development of antibiotic-resistant bacteria and become a serious concern worldwide [28]. Many antibiotics are globally used on animals used as food, which is a major source of antibiotic-resistant bacteria development [29]. Thus, new antibacterial agents have been developed to kill antibiotic-resistant bacteria without causing resistance. AMPs have been considered as substitutes for antibiotics. AMPs play important roles as host defense molecules against infection in all living organisms [30]. AMPs show broad-spectrum activity against many strains of Gram-positive and Gram-negative bacteria through different modes of action, such as membrane disruption and via the intracellular target model [31,32]. AMPs have been reported to be less likely to develop drug resistance and have an advantageous effect on nutrient sources and the gut microbiota in animals [29,33]. Therefore, AMPs are good alternatives to the antibiotics used in humans and animal husbandry.
Venoms of various species are beneficial sources of bioactive molecules, such as AMPs [14]. In this study, we used melittin isolated from bee venom as a control peptide, as it is a representative lytic peptide known to have strong antimicrobial activity [34,35]. In a previous study, Css54 isolated from C. suffusus showed antimicrobial activity and synergistic effects with rifampicin, which is used to treat tuberculosis. Moreover, Css54 displayed an α-helical structure in 60% TFE [20]. However, how Css54 kills bacteria and whether it has antimicrobial activity against other bacteria, except for S. aureus and E. coli, remains unclear. Therefore, we focused on the antimicrobial activity and mechanism of action of Css54 against bacterial pathogens in zoonosis.
Zoonotic diseases are caused by pathogens such as bacteria, fungi, and viruses that transmit between animals and humans [36]. The WHO report in 2015 warned of the number of diseases caused by food contaminated with pathogenic bacteria such as Salmonella sp., Listeria Sp., Campylobacter sp., and the Enterobacteriaceae family [37]. We examined the antimicrobial activity of Css54 against bacteria on the zoonosis announced by the WHO; S. suis is a zoonotic pathogen that is related to swine infection [38]. Antimicrobial assays showed that Css54 exhibited antimicrobial activity against four strains similar to melittin. Moreover, the antimicrobial activity of conventional antibiotics such as cefotaxime, vancomycin, colistin, and ampicillin was evaluated. Css 54 exerted antimicrobial activity with low MIC compared to antibiotics against some strains. In a previous study, treatment with melittin showed no adverse effects on stomach tissue, including its function, and melittin was confirmed to have a protective effect against gastric inflammation and antitumor effects in vivo [39][40][41]. These reports indicate that Css54 can be developed as an effective treatment because of its lower toxicity (versus melittin). We confirmed that hemolytic activity towards sRBCs and cytotoxicity against pig kidney cells PK (15) was lower than that of melittin. The MIC value of Css54 ranged from 2 to 4 µM, and hemolytic activity and cytotoxicity following treatment with 16 µM Css54 (approximately 4-fold of the MIC value) were approximately 10% and 40%, respectively. Therefore, Css54 has the potential for treating bacterial diseases in animals and humans.
Listeria monocytogenes can survive in various typical environments, such as food processing and preservation [42]. This species can grow over a wide pH range of 4.5-9.6 and a temperature range of 2-45 • C. Moreover, L. monocytogenes can survive in a high-salt environment, with up to 10% NaCl [43]. Thus, L. monocytogenes is frequently present in animal foods and can contaminate foods. In addition to the biofilm inhibitory effect of Css54, one of the requirements for food and feed additives is stability under these conditions. Interestingly, Css54 maintained antimicrobial activity over wide pH, temperature, and NaCl ranges. Additionally, the processing of many foods and feeds involves a heating step, and thus antimicrobial agents must exhibit thermal stability [44]. Css54 showed antimicrobial activity after incubation for 80 min at 100 • C. These data demonstrate the potential of Css54 in applications such as food and feed additives.
Biofilm is a self-produced matrix of extracellular polymeric substances produced by microorganisms. Biofilms are important for the growth and survival of bacteria under various conditions, including low temperature, high salt concentrations, and low pH [45]. Listeria monocytogenes is a problem in food safety because it can form biofilms upon contact with food surfaces and persist in food processing environments [23]. Therefore, food additives used in the food industry should have antibiofilm activity. Recently, food additives have gradually become one of the most important antimicrobial agents in the food industry. Our data showed that Css54 can inhibit biofilm formation by L. monocytogenes, similar to melittin.
AMPs display secondary structures such as α-helices, β-sheets, loop types, and mixed structures [46]. CD spectra were determined to analyze the secondary structure of the peptides. AMPs showed a random coil structure in aqueous solution and formed a secondary structure in membrane-mimicking environments. We evaluated the secondary structure of Css54 in aqueous solution as well as bacterial membrane-mimicking environments such as SDS and TFE solutions. Css54 formed a random coil in an aqueous solution. In contrast, Css54 has an α-helix structure in SDS, which mimics the negatively charged bacterial membrane, and in TFE, which can help AMPs induce secondary structure and mimic the hydrophobic environment [47]. These results suggest that Css54 exerts its antimicrobial activity by interacting with the bacterial membrane.
To determine the antimicrobial mechanism, we examined the effect of Css54 on bacterial membranes in an outer membrane permeability assay using NPN dye and a cytoplasmic membrane depolarization assay using DiSC 3 (5); we also examined membrane integrity using SYTOX green and PI. Css54 significantly increased NPN fluorescence intensity and DiSC 3 (5) fluorescence intensity. Our results demonstrate that Css54 disrupts the outer membrane of S. typhimurium and induces membrane depolarization of L. monocytogenes and S. typhimurium. We examined the integrity of the bacterial membrane after treatment with Css54 using SYTOX green, PI, and SYTOX green. Fluorescence intensity increased when Css54 was added to the bacteria. This result was confirmed by flow cytometry using PI. Treatment with Css54 increased the PI fluorescence intensity, indicating that the bacterial membrane was disrupted by Css54. Our data show that Css54 has antimicrobial activity and a mechanism similar to melittin, but with lower cytotoxicity than melittin.

Antimicrobial Activity Assay
To investigate the antimicrobial activity of AMPs and antibiotics, the minimum inhibitory concentrations (MICs) of peptides and antibiotics were determined using the broth dilution method [50]. Listeria monocytogenes (KCTC 3710), L. monocytogenes (KCCM 40307), L. monocytogenes (KCCM 43155), and S. suis (KCTC 3557) were cultured overnight at 37 • C in brain heart infusion (BHI). Salmonella typhimurium (KCTC 14028), S. typhimurium (CCARM 8009), S. typhimurium (CCARM 8013), and C. jejuni (KCTC 5327 were cultured overnight at 37 • C in Mueller Hinton broth (MHB). These bacteria were diluted in appropriate media to a final concentration of 2 × 10 5 CFU/mL. AMPs and antibiotics were diluted at concentrations of 0.5.64 µM in 10 mM sodium phosphate buffer (Sp buffer, pH 7.2) in 96-well plates. Next, a 50-µL aliquot of bacteria was added to 50 µL of diluted peptide and antibiotics and incubated at 37 • C for 16-24 h. The growth of bacteria was measured as the absorbance at 600 nm using a microplate reader. MIC values were determined as the lowest concentration that inhibited bacterial growth compared to the control without AMPs and antibiotics.

Hemolytic Activity
The hemolytic activity of the peptides was determined using sRBCs. sRBCs were centrifuged at 2000× g for 10 min at 4 • C and washed three times with phosphate-buffered saline (PBS, pH 7.4). The washed sRBCs were diluted in PBS to a final concentration of 8%. AMPs (100 µL) were serially diluted from 1 to 16 µM in 96-well plates, and then sRBCs were subsequently mixed for 1 h at 37 • C. The reacted samples in the 96-well plate were centrifuged at 1500× g for 10 min. The supernatant was transferred to a new 96-well plate, and the absorbance was measured at 414 nm (A 414 ). Next, 0.1% TritonX-100 and PBS were used as negative and positive controls, respectively. The percentage of hemolysis was calculated as follows [51]:

Cytotoxicity Activity
The cytotoxicity of AMPs towards PK(15)-isolated porcine kidney cells was determined by MTT assay. PK(15) cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin at 37 • C with 5% CO 2 . The cells were seeded into 96-well plates at 2 × 10 4 cells/well and incubated for 24 h. AMPs were added to the wells at concentrations ranging from 0.5 to 16 µM and incubated for 24 h. Next, 20 µL of 0.5 mg/mL MTT was added to the wells and incubated for 4 h. The supernatant was removed, and 100 µL DMSO was added to dissolve the formazan crystals. The absorbance was measured at 570 nm (A570) using a microplate reader. The positive control was incubated only with DMEM. The percent cell viability was calculated according to the following formula: % Cell viability = (A 570 in peptides solution/A 570 in positive control) × 100

Stability of Peptides
The stability of Css54 was determined in different environments with varying pH values, temperatures, and salt concentrations. To confirm the effect of the antimicrobial activity of Css54 at various pH values, Sp buffer was adjusted with NaOH and HCl to pH values of 4, 6, 8, and 10. Css54 at 0.5× and 1× MIC was diluted in the prepared buffer and incubated with L. monocytogenes at 2 × 10 5 CFU/mL in BHI medium for 12 h. The growth of L. monocytogenes was determined by measuring the absorbance at 600 nm. To evaluate the stability of Css54 at various temperatures, L. monocytogenes at 2 × 10 5 CFU/mL in BHI medium was incubated with Css54 at 1× MIC at 4, 20, 30, and 40 • C for 30 min. The suspensions were plated on BHI agar plates. Colonies were counted after overnight incubation.
To determine the effect of Css54 at various salt concentrations, L. monocytogenes at 2 × 10 5 CFU/mL in BHI medium supplemented with 2%, 4%, 6%, and 8% NaCl was incubated with Css54 at 1× MIC overnight. The growth of L. monocytogenes was determined by measuring the absorbance at 600 nm. To confirm heat resistance, Css54 at a final concentration of 1× MIC was incubated for different times (20,40,60, and 80 min) at 100 • C and cooled on ice. Thereafter, L. monocytogenes at 2 × 10 5 CFU/mL was added to the suspension and incubated overnight. The growth of L. monocytogenes was determined by measuring the absorbance at 600 nm.

Biofilm Inhibition Assay
To confirm biofilm inhibition by AMPs, L. monocytogenes was cultured in BHI at 37 • C. Bacterial suspensions at a final concentration of 5 × 10 5 CFU/mL were diluted in BHI supplemented with 0.2% glucose. Bacterial suspensions (90 µL) and peptides (10 µL) at concentrations of 1-8 µM were mixed in a 96-well tissue culture plate. The mixture with bacteria and peptides was incubated for 24 h at 37 • C. After incubation, the supernatant was gently discarded, and the biofilms were fixed with 100% methanol for 10 min. The methanol was discarded and dried. The dried biofilms were stained with 0.1% crystal violet for 10 min and then rinsed with distilled water until the BHI medium supplemented with 0.2% glucose appeared colorless. Finally, the stained biofilms were dissolved in 95% ethanol and measured at an absorbance of 595 nm (A 595 ). The percentage of biofilm mass was calculated using the following equation: Biofilm mass (%) = (A 595 of treated AMPs/A 595 of untreated biofilm) × 100

Outer Membrane Permeability Assay
Outer membrane permeability was measured using an NPN uptake assay. Salmonella typhimurium (ATCC 14028) was cultured in MHB medium. The bacteria were washed three times with 5 mM HEPES buffer (pH 7.2) and resuspended to an OD 600 of 0.4 in 5 mM HEPES buffer (pH7.2). The bacteria were placed in a black 96-well plate, and NPN at 10 µM was added. Additionally, Css54 and melittin at 0.5×, 1×, and 2× MIC were added to each well. NPN fluorescence intensity was measured at excitation and emission wavelengths of 350 and 420 nm, respectively, every 5 min for 30 min.

Membrane Depolarization Assay
Membrane depolarization by the peptides in L. monocytogenes (KCTC 3710) and S. typhimurium (ATCC 14028) was measured using DiSC 3 (5). Listeria monocytogenes (KCTC 3710) and Salmonella typhimurium (ATCC 14028) were cultured in BHI and MHB media, respectively. The bacteria were washed three times with 5 mM HEPES buffer (pH 7.2) supplemented with 20 mM glucose. The bacteria were resuspended to an OD 600 of 0.2 in 5 mM HEPES buffer (pH 7.2) supplemented with 20 mM glucose and 5 mM KCl. DiSC 3 (5) at 0.1 µM was added and placed in a black 96-well plate. The mixture was incubated for 1 h to stabilize the fluorescence intensity. Css54 and melittin at 0.5×, 1×, and 2× MIC were added to the mixture. The fluorescence intensity was measured at excitation and emission wavelengths of 622 and 670 nm, respectively [53].

SYTOX Green Uptake Assay
Listeria monocytogenes (KCTC 3710) and Salmonella typhimurium (ATCC 14028) were cultured in BHI and MHB media, respectively. The bacteria were washed three times with 10 mM Sp buffer (pH 7.2). The bacteria were resuspended to a final concentration of 2 × 10 7 CFU/mL in 10 mM Sp buffer. The bacteria were incubated with 1 µM SYTOX Green for 15 min in a black 96-well plate. Css54 and melittin at 0.5×, 1×, and 2× MIC were added to the mixture. The fluorescence intensity was measured at excitation and emission wavelengths of 485 and 520 nm, respectively [54].

Flow Cytometry
The integrity of the bacterial membranes was analyzed by flow cytometry. Listeria monocytogenes (KCTC 3710) and Salmonella typhimurium (ATCC 14028) were cultured in BHI and MHB media, respectively. The bacteria were washed three times with 10 mM Sp buffer and then resuspended to an OD 600 of 0.4 in 10 mM Sp buffer. The bacterial suspension was mixed with 2 µg/mL PI and incubated with Css54 at 0.5× and 1× MIC for 10 min. The suspension was centrifuged and washed to remove unbound dye. The data were measured by CytoFLEX flow cytometry (Beckman Coulter, Brea, CA, USA) [55].

Conclusions
In conclusion, Css54 has antimicrobial activity against bacteria in zoonotic disease. Css54 showed lower cytotoxicity than melittin. Moreover, Css54 maintained antimicrobial activity under the growth conditions of L. monocytogenes and showed thermal stability. Css54 effectively inhibited biofilm formation by L. monocytogenes. Css54 displayed an α-helix structure in bacterial membrane-mimicking environments. Css54 showed membrane lytic mechanisms similar to melittin. Our results suggest that Css54 can be used as an antibiotic and feed additive.