The 1-day-old chickens used in this study were obtained from Shanxi Jiabo Agricultural and Animal Husbandry Development Co., Ltd. The chicks were group-housed and given ad libitum access to food and water until 21 days of age.

Five new AMPs were generated through the incorporation of amino acid substitutions into the CATH-2 (1-15) sequence. CATH-2 (1-15) (RFGRFLRKIRRFRPK), C2-1 (RFGRFLRKIRRFIPK), C2-2 (RFGRFLRKIRRMRLK), C2-3 (RFGRHLRKIIRFRIK), C2-4 (RFGRNLRKIRRFWPK), and C2-5 (RFGRFLRKIRFFRLK) were synthesized by PeptideValley. The purity of the products was determined to be greater than 95% by high-performance liquid chromatography (HPLC) and mass spectrometry.

The physicochemical properties of the AMPs were analyzed using ProtParam software (https://web.expasy.org/protparam/). The spatial structure of the AMPs was constructed using PRBS (https://mobyle2.rpbs.univ-paris-diderot.fr/cgi-bin/portal.py#welcome), while helical wheel models of the resulting peptides were created using HeliQuest (https://heliquest.ipmc.cnrs.fr/).

E. coli (ATCC 8739) and clinical isolates of E. coli (E1, E9, E12, E14, E16, and E18) were stored at the Animal Biochemical Laboratory of Shanxi Agricultural University and grown in Luria-Bertani (LB) broth as previously reported (Zhao et al., 2021). Circular 6 mm paper pieces were prepared with filter paper and autoclaved for sterilization. Solutions of penicillin (PG), florfenicol (FF), ciprofloxacin (CIP), enrofloxacin (ENR), and gentamicin (GM) were prepared according to the Clinical and Laboratory Standards Institute (CLSI). Then, 0.5 mL of antibiotic solution was added to 100 sterilized papers. Subsequently, the paper pieces were fully infiltrated and dried in a vacuum drying oven to produce antibiotic susceptibility papers. The E. coli strain was retrieved from the -20°C freezer and activated by incubating it on LB agar media at 37°C for 24h. Individual colonies were selected and transferred to LB liquid media. Thereafter, the E. coli suspension was obtained by shaking at 37°C and 160 r/min for 18h. The obtained suspension was centrifuged at 5000 r/min for 5min, after which the supernatant was discarded, and the bacteria were resuspended in fresh medium to a concentration of 1×10⁶ CFU/mL. Two hundred microliters of the bacterial suspension mentioned above was spread evenly onto LB agar medium. Then, the antibiotic susceptibility papers were placed on the medium surface using sterile tweezers. After 24h of incubation at 37°C, the diameter of the inhibition zone was measured to determine the susceptibility of E. coli to antibiotics.

The minimum inhibitory concentrations (MICs) of the AMPs were determined using the microbroth dilution technique (van Harten et al., 2021). One hundred microliters of bacterial suspension at a concentration of 1×10⁶ CFU/mL was pipetted into a 96-well plate. Then, 100 μL of AMP or gentamicin at various concentrations was added, while the bacterial suspension without AMPs and blank medium were used as controls. The plate was incubated at a constant temperature of 37°C for 18h, after which the turbidity of the wells was observed to determine the MIC. Fifty microlitres of liquid was extracted and plated on LB agar from wells exhibiting complete bacterial growth inhibition. The minimum bactericidal concentration (MBC) of the AMPs was assessed by counting colonies following a 24h incubation at 37°C.

The sterilization kinetics assay was performed following previous methods (Peng et al., 2022; Yong Fang et al., 2023). Gentamicin, C2-1 and C2-2 at a concentration of 8 μg/mL each were added to a bacterial suspension of 1×10⁵ CFU/mL, and incubated at 37°C for 0, 15, 30, 60, 120, or 240min. At each time point, 10 μL of sample was diluted in 1 mL of PBS, and 100 μL of the dilution was plated on LB agar plates. After incubating at 37°C for 24h, the number of colonies was counted.

The MICs of the AMPs against E. coli (ATCC 8739) at different temperatures and physiological salt concentrations were evaluated. The thermal stability of the AMPs was assessed by incubating them at a concentration of 500 μg/mL for 1h at 0, 37, and 100°C, using untreated AMPs as a control. The salt ion stability of the AMPs was determined by incubating them at a concentration of 500 μg/mL in solutions of NaCl (150 mM), KCl (4.5 mM), MgCl2 (1 mM), and CaCl2 (2.5 mM) for 1h, using untreated samples as controls. The changes in the MICs of the AMPs were assessed following these experimental procedures.

Fresh blood was harvested from chickens, anticoagulated with 0.2% sodium heparin, and centrifuged at 1000 r/min for 10min to remove the supernatant. The lower erythrocytes were collected, gently washed twice with sterile PBS, and subsequently prepared as a 2% erythrocyte suspension. The erythrocyte suspension was mixed with AMPs at different concentrations in equal volumes. Erythrocytes treated with PBS served as a negative control, while erythrocytes treated with 2% Triton X-100 served as a positive control. The mixture was incubated at 37°C for 1h, after which the absorbance at 570 nm was measured using a multifunctional enzyme marker.

Chicken renal cells were trypsinized, seeded at 5000 cells per well in 96-well plates, and incubated for 24h. The cell culture medium was subsequently discarded, and AMPs were added at different concentrations to the wells, which were subsequently filtered through a 0.22 μm filter to eliminate bacteria. The mixture was incubated for another 24h, after which 50 μL of 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl tetrazolium bromide (MTT) reagent (5 mg/mL) was directly added to each well. The mixture was incubated for 4h. Then 50 μL of MTT reagent (5 mg/mL) was added directly to each well and incubated for another 4h. To dissolve the crystals, 150 μL of dimethyl sulfoxide (DMSO) was added, and the mixture was vortexed for 10min. The absorbance was measured at 490 nm using a multifunctional enzyme marker.

Fifty 21-day-old chickens were randomly divided into five groups: blank control (500 μL of normal saline), E. coli infection (500 μL of normal saline), C2-2 treatment (200 μg/mL, 500 μL), gentamicin treatment (200 μg/mL, 500 μL) and enrofloxacin treatment (200 μg/mL, 500 μL). Except for the blank control group injected with 500 μL of normal saline, the other four groups were injected with 500 μL of 3×10⁹ CFU/mL E. coli (E16) into the pectoral muscles. Then, normal saline, C2-2, gentamicin and enrofloxacin were injected through the pectoral muscles 4h after E. coli infection according to the above methods. Clinical symptoms and were observed and documented for the chickens in each group after infection. After 72h, the chickens in each group were euthanized and examined for internal organ damage. Paraffin sections were prepared for microscopic examination of pathological changes. Under sterile conditions, 1 g samples of heart, liver, and spleen were collected, and each sample was supplemented with 20 mL of sterile PBS. After thorough grinding, the sample was diluted to the appropriate multiple, and 100 μL of the diluent was spread onto LB agar medium. The plates were incubated for 24h, after which the colonies were counted.

Statistical analysis was performed using one-way analysis of variance (ANOVA). The data represent three independent experiments and are presented as mean ± SEM. A p-value of< 0.05 was considered to indicate statistical significance.

The sequences and physicochemical properties of the peptides used are listed in Table 1 . CATH-2 (1-15) is an N-terminal truncated peptide of chicken CATH-2 that consists of 15 amino acids and has an α-helical secondary structure and 8 positive charges. Based on the CATH-2 (1-15) sequence, five peptides, named C2-1 to C2-5, were designed by substituting amino acids to modify their charge and hydrophobicity. C2-2 was positively charged with 8 net charges, and the others had 7 net charges. Among them, CATH-2 (1-15) had the lowest hydrophobicity (0.103), and C2-5 had the highest hydrophobicity (0.355). According to the average hydrophilicity, C2-4 had the strongest hydrophilicity, followed by CATH-2 (1-15), and C2-2 ranked third. The predicted secondary structures showed that all five peptides were α-helical ( Figure 1A ).The spiral-wheel model highlighted changes in the hydrophobic moment, as well as the hydrophobic and hydrophilic characteristics of the AMPs ( Figure 1B ). All six peptides had an amphipathic structure that formed a hydrophilic and hydrophobic side.

To comprehensively and accurately evaluate the effects of AMPs on different strains of E. coli, we isolated 6 strains of E. coli (E1, E9, E12, E14, E16, and E18) from poultry farms in Shanxi Province and analyzed their resistance to penicillin, florfenicol, ciprofloxacin, enrofloxacin and gentamicin, which are commonly used in the aquaculture industry for the treatment of E. coli infections. The results indicated that the 6 E. coli strains were all resistant to penicillin, florfenicol and enrofloxacin. Moreover, only the E14 strain of E. coli was sensitive to ciprofloxacin, while three strains (E12, E14, and E16) of E. coli were susceptible to gentamicin. In summary, E. coli strains E1 and E18 were found to have relatively high levels of resistance ( Table 2 ).

The antimicrobial activities of CATH-2 (1-15) and the designed peptides were tested against 7 strains of E. coli. C2-1 and C2-2 exhibited potent antibacterial activity against 6 MDR E. coli strains. The MICs of C2-1 ranged from 0.5 to 16 μg/mL, and 0.5 μg/mL could inhibit the growth of E9. The MICs of C2-2 ranged from 2 to 8 μg/mL. Compared with the CATH-2 (1-15), it increased the inhibitory effect on six MDR E. coli strains by 2 to 32 times. The inhibitory effects of C2-3 and C2-5 were slightly weaker than those of C2-1 and C2-2. The inhibitory effect of C2-4 was lower than that of the CATH-2 (1-15), which had the poorest inhibitory effect. The MICs of gentamicin were between 4- and 64 μg/mL ( Table 3 ). The MBC values of the AMPs further confirmed the above results ( Table 4 ). In brief, the MBC values of CATH-2 (1-15) and the designed peptides were found to be greater than the MIC values. It is worth noting that the MBC values of C2-2 remained within the range of 2-8 μg/mL. The results suggested that C2-1 and C2-2 exhibited potent in vitro inhibitory effects on several MDR E. coli strains.

In this study, the bactericidal kinetics of C2-1, C2-2, and gentamicin were evaluated against E. coli (ATCC8739), indicating their efficacy against bacteria. Gentamicin at a concentration of 8 μg/mL exhibited the most potent bactericidal effect, as it completely eradicated bacteria within 60min. C2-2 showed the second most potent bactericidal activity, while C2-1 had the least potent effect, requiring 240min for complete bacterial eradication ( Figure 2 ).

The thermal stability and salt ion stability of AMP are two important factors limiting its clinical use and were also tested in this study. As shown in Table 5 , after 1h of incubation at 100°C, CATH-2 (1-15) exhibited a 2-fold increase in antimicrobial efficacy, whereas C2-5 experienced a 4-fold reduction under the same conditions, while the MICs of the other four designed peptides remained constant. In addition, antimicrobial activity of CATH-2 (1-15) increased 2-fold in the presence of sodium ions and fourfold in the presence of potassium and magnesium ions. C2-1 demonstrated a 4-fold increase in antimicrobial activity with sodium ions but a fourfold decrease with calcium ions. C2-2 displayed a 16-fold increase in antimicrobial activity with sodium ions, potassium ions, or magnesium ions and a 2-fold increase with calcium ions. C2-3 experienced a 4-fold increase in antimicrobial activity with potassium or magnesium ions. C2-4 exhibited a 2-fold decrease in antimicrobial activity with sodium, potassium, or calcium ions, while C2-5 exhibited a fourfold decrease with sodium, potassium, or magnesium ions and an eightfold decrease with calcium ions. In general, with the exception of C2-4 and C2-5, the other analogs consistently retained potent antimicrobial activity at diverse temperatures or under salt ion conditions.

In this study, we investigated the hemolytic activity of peptides derived from CATH-2 on mature chicken erythrocytes. Except for C2-1, the remaining four peptides exhibited no significant hemolytic activity compared to that of CATH-2 (1-15) ( Figure 3A ). In addition, cytotoxicity is an important indicator of the safety of AMPs. Except for C2-1, no significant cytotoxicity against chicken kidney cells was observed after treatment with the designed peptides in the concentration range of 0-64 μg/mL ( Figure 3B ).

The E16 strain of E. coli was selected for chicken infection based on its resistance and because a previous study reported that E16 exhibited greater toxicity. To determine the role of C2-2 in preventing E. coli infection in vivo, chickens were infected with 1.5× 10⁹ CFU of E16 with or without C2-2 administration, and survival was monitored over time. The blank control group showed no abnormal changes after 3 days of infection and maintained 100% survival. In contrast, the E. coli infection group exhibited the most severe clinical symptoms, such as depression, loss of appetite, drooping wings, sensitivity to cold, and discharge of yellowish-green feces, which were present 12h post infection. Mortality of normal infected chickens occurred at 1 day post infection, whereas infected chickens treated with C2-2 began to die at 2 days post infection. In addition, the survival rate of E. coli infected chickens in the presence of C2-2 was 70% by day 3, which was markedly greater than that of chickens not receiving C2-2 administration (30%) ( Figure 4A ). The 3-day mortality rate of the C2-2 treatment group was intermediate between that of the gentamicin and enrofloxacin treatment groups at 10% and 50%, respectively. There was no difference in survival rate between the enrofloxacin-treated and untreated groups, and the survival rate in both groups was significantly lower than that in the gentamicin-treated group, which was due to the presence of gentamicin-sensitive and enrofloxacin-resistant E16 strains ( Figure 4A ). To investigate whether the attenuation of pathogenesis noted above was due to a decreased burden of E. coli in the chickens administered C2-2, we analyzed the bacterial loads in the heart, liver and spleen after 3 days of infection. The results showed that C2-2 and gentamicin significantly reduced the bacterial loads in the heart, liver and spleen of infected chickens, whereas enrofloxacin affected only the heart bacterial load. The liver bacterial burden in the E. coli infection group was 1.61×10⁷ CFU/g, and the liver bacterial load in the C2-2 treatment group was 2.37×10⁴ CFU/g, which was reduced by approximately 680 times, indicating that C2-2 treatment had the greatest effect on the bacterial load in the liver ( Figure 4B ).

After the chickens died, an autopsy was conducted to examine the pathological changes in the heart, liver, and intestine. Chickens infected with MDR E. coli had a heart surface covered with a yellow fibrinous exudate. The liver was enlarged, tender in texture, yellow in color, and occasionally covered with yellow-white cellulose exudate. In the intestines of the chickens, congestion, hemorrhage and enlargement of the mesenteric lymph nodes were observed to varying degrees. Some of the affected chickens exhibited symptoms of splenomegaly and severe thymic congestion. Overall, in contrast to those in the E. coli infection group, there was a significant reduction in organ lesions in both the C2-2 and gentamicin treatment groups ( Figure 4C ). The histologic sections of the heart, liver and jejunum from the different treatment groups are presented in Figure 4D . Compared with tose in the negative control group, the E. coli infection group and the enrofloxacin treatment group exhibited significant thickening of the external cardiac membrane, extensive fibrous coverage on the surface, swelling, and rupture of myocardial fibers, as well as fibrous exudates in the interstitial spaces. Hepatocytes showed swelling and granular degeneration, accompanied by disordered arrangement of hepatocyte cords and marked infiltration of inflammatory cells. The jejunal villi were observed to be disrupted, and the intestinal mucosa was congested and swollen with infiltration of inflammatory cells. In the C2-2 treatment group and the gentamicin treatment group, the myocardial fibers were arranged neatly without any granular degeneration of cells, with only a small amount of fibrinous exudate on the surface. The liver lobules appeared normal in structure with visible hepatocytes and no granular degeneration. The jejunal villi were relatively structurally intact with a small number of inflammatory cells in the submucosa.