Pseudomonas aeruginosa flagella inhibiting peptide and synthesis method and application thereof

Abstract

The application discloses a flagellum inhibiting peptide of pseudomonas aeruginosa and a preparation method and application thereof, and the sequence of the flagellum inhibiting peptide is Lys-Ile-Gly-Leu-Phe-Arg-Trp-Arg. The synthetic inhibiting peptide has a time-dependent self-assembly ability, can gradually evolve from scattered granular into filamentous structure, and finally forms a stable net-like morphology. The synthetic inhibiting peptide can significantly inhibit the flagellum motility of PAO1 and other strains of pseudomonas aeruginosa, and has synergistic antibacterial activity when combined with allicin, ciprofloxacin, meropenem, levofloxacin, polymyxin B and the like. Moreover, the synthetic inhibiting peptide has obvious bacteriostatic and healing-promoting effects on burn wound infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical peptides, specifically relating to flagellar inhibitory peptides of Pseudomonas aeruginosa, their synthesis methods, and applications. Background Technology

[0002] *Pseudomonas aeruginosa* is a Gram-negative opportunistic pathogen widely distributed in water, soil, hospital environments, and on human skin. It is a leading cause of morbidity and mortality in patients with cystic fibrosis and immunocompromised individuals. Due to its significant antibiotic resistance, eradication of *P. aeruginosa* is becoming increasingly difficult. *P. aeruginosa* strains are known to utilize their high levels of intrinsic and acquired resistance mechanisms to resist most antibiotics. Treatment of *P. aeruginosa* infections remains a significant challenge. The greatest challenge associated with this bacterium lies in its high capacity to develop antibiotic resistance (AMR) and form biofilms, making it a leading cause of serious healthcare-associated infections (HAIs) and hospital outbreaks worldwide. Antibiotic resistance is a major global health challenge, causing significant morbidity and mortality globally. Antibiotic resistance in *P. aeruginosa* is multifactorial, as it can occur through innate, acquired, or adaptive mechanisms. The diversity of antibiotic resistance mechanisms contributes to the development of multidrug-resistant strains and renders conventional antibiotics ineffective against Pseudomonas aeruginosa infections. In recent years, adaptive resistance, a novel type of resistance, has attracted significant attention, with biofilm-mediated resistance being a prime example and a major cause of infection recurrence. Therefore, developing alternative treatment strategies for Pseudomonas aeruginosa infections has become a research hotspot in this field.

[0003] The pathogenicity of *Pseudomonas aeruginosa* relies primarily on the synergistic effects of multiple virulence factors, including flagella, type IV pili, outer membrane proteins, the secretory system, biofilm formation ability, and various extracellular enzymes and toxins. Flagella are not only a key structure for bacterial motility but also play a crucial role in bacterial chemotaxis, adhesion, colonization, and the early stages of biofilm formation. Studies have shown that downregulation of flagella-related genes (such as fliC, fliD, and motAB) can significantly weaken the swimming ability and virulence expression of *Pseudomonas aeruginosa*, suggesting that the flagellar system could be an ideal target for antiviral intervention. Antiviral strategies do not directly kill bacteria but rather reduce their ability to invade the host by interfering with their pathogenic mechanisms, thereby creating conditions for the host's immune system to clear the pathogen. Compared to traditional antibiotics, antiviral drugs exert less pressure on bacterial survival, are theoretically less likely to induce drug resistance, and have less impact on normal flora, thus possessing high clinical application potential.

[0004] Due to their diverse structures, broad biological activities, and unique mechanisms of action, peptides have attracted considerable attention in the field of anti-infection in recent years. Some peptides can self-assemble into nanostructures, interfering with bacterial membrane structure or motility systems to exert antibacterial or antiviral effects.

[0005] Because Pseudomonas aeruginosa employs a variety of antibiotic resistance strategies, the most effective treatment in the future may require combination therapy, which combines novel treatments with traditional treatments such as conventional antibiotics, to successfully eradicate the pathogen from vulnerable, immunocompromised patients. Summary of the Invention

[0006] The purpose of this invention is to synthesize a novel flagellation inhibitory peptide (Pep) and to identify its purity and molecular weight using high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Furthermore, transmission electron microscopy (TEM) is used to observe the time-dependent self-assembly behavior of Pep and to investigate its morphological evolution at different time points.

[0007] The regulatory mechanism of Pep on flagellar structure and function: 1) Evaluating the effect of Pep on the motility of flagella in multiple strains of Pseudomonas aeruginosa, including PAO1, and clarifying its concentration dependence and strain specificity. Combined with single-bacterial motility trajectory tracking and velocity quantification analysis, the inhibitory effect of Pep on motility was accurately assessed. 2) Observation of ultrastructural damage: Morphological changes in bacterial flagella before and after Pep treatment were observed using transmission electron microscopy to verify the targeting of Pep at the physical structural level. 3) Molecular mechanism analysis: Differentially expressed genes after Pep treatment were analyzed through transcriptome sequencing. Combined with GO functional enrichment analysis and KEGG pathway enrichment analysis, and verified by qRT-PCR, key differentially expressed genes were elucidated to clarify the molecular mechanism by which Pep synergistically weakens pathogenicity through multiple targets (energy metabolism inhibition + structural gene downregulation).

[0008] (3) In vitro antibacterial efficacy and biofilm clearance capacity of combined drugs. Based on the characteristic of Pep as "antiviral rather than bactericidal", this study explored its potential as an adjuvant in combination with antibacterial drugs. 1) Synergistic inhibition: First, the minimum inhibitory concentrations (MICs) of Pep, allicin and each antibiotic were determined. The checkerboard method was used to screen combinations with synergistic effects. 2) Synergistic bactericidal effect: The bactericidal kinetics of Pep and allicin combination drug at specific concentrations on planktonic bacteria were evaluated by time-bactericidal curves. 3) Anti-biofilm activity: A biofilm model was established to evaluate the inhibitory effect of Pep and allicin combination on biofilm formation and the clearance rate of mature biofilm. Combined with morphological observation, the destructive effect of combined drug on bacterial structural integrity was visually photographed.

[0009] (4) Evaluation of in vivo efficacy and biosafety. Based on the in vitro mechanism and efficacy verification, a mouse burn wound infection model was established to simulate the clinical infection environment. 1) Initial safety screening: Before in vivo experiments, the biocompatibility of the combined drugs at effective antibacterial concentrations was evaluated through cytotoxicity and hemolysis experiments to ensure the safety of subsequent in vivo experiments. 2) Evaluation of in vivo efficacy: A burn wound model of PAO1 infection was established. The ability of the combined drugs to promote wound healing and clear infection was comprehensively evaluated by dynamically monitoring the wound healing rate, quantifying the wound bacterial load, and conducting histopathological analysis.

[0010] Technical solution: To achieve the above objectives, the technical solution adopted by this invention is as follows:

[0011] A flagellation inhibitory peptide of Pseudomonas aeruginosa, with the sequence Lys-Ile-Gly-Leu-Phe-Arg-Trp-Arg.

[0012] A method for synthesizing flagellar inhibitory peptides from Pseudomonas aeruginosa, comprising the following steps: (1) Weigh Fmoc-Arg(pbf)-WangResin resin and add it to the solid-phase synthesis reactor. After adding N,N-dimethylformamide DMF to swell the resin, discharge the DMF. (2) Deprotection: Add a deprotection solution consisting of hexahydropyridine and DMF to the reaction column, stir with nitrogen gas, and then dry it. (3) Weighing: Measure the protected amino acid Fmo-Trp(Boc)-OH, and then weigh O-(benzotriazol-1-yl)-N,N,N,N'-tetramethylurea hexafluorophosphate HBTU; (4) Deprotection washing: Add an appropriate amount of DMF to the reaction column, puff with nitrogen, dry the column, and repeat the operation; (5) Feeding: Add the prepared protected amino acid Fmo-Trp(Boc)-OH and O-(benzotriazol-1-yl)-N,N,N,N'-tetramethylurea hexafluorophosphate HBTU to the reaction column, then add N-methylmorpholine NMM, and stir with nitrogen. (6) Washing after reaction: Drain the solution in the reaction column, add an appropriate amount of DMF to wash, agitate with nitrogen, drain again, and repeat the operation. (7) Test: Take the resin into a small test tube, add two drops each of phenol anhydrous ethanol mixture, redistilled pyridine and ninhydrin anhydrous ethanol solution, and heat in a dry heater; if the solution turns blue and the resin is discolored and opaque after taking it out, the reaction is not complete and the reaction is repeated; if the solution is slightly yellow and the resin is colorless and transparent, the reaction is complete. Couple the amino acids Fmoc-Arg(Pbf)-OH, Fmo-Phe-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, and Boc-Lys(BOC)-OH in sequence, following the five steps above (2)-(6), until the last amino acid is coupled; after the last amino acid is coupled and the protective washing has been completed, dry the column, add an appropriate amount of methanol, puff with nitrogen, dry the column, add an appropriate amount of DCM, puff with nitrogen, dry the column; finally, add an appropriate amount of methanol to the reactor, puff with nitrogen, dry the reactor, and place the resin in a container in a vacuum dryer for vacuum drying; (8) Cutting and washing of peptides: Seal the centrifuge tube and put it into the centrifuge. Centrifuge, take it out, pour off the supernatant, add ether, stir evenly with a glass rod, and centrifuge again; repeat the washing operation; drying: put the washed peptides into a vacuum dryer and vacuum dry; to obtain crude peptides; (9) Purification of polypeptides

[0013] The crude product was dissolved in an acetonitrile-water mixture by ultrasonication, filtered through a microporous membrane, and the filtrate was purified by HPLC to obtain the pure polypeptide.

[0014] As a preferred embodiment, the method for synthesizing the flagellar inhibitory peptide of Pseudomonas aeruginosa described above includes the following steps: (1) Weigh Fmoc-Arg(pbf)-WangResin resin and add it to the solid-phase synthesis reactor. After adding N,N-dimethylformamide DMF to swell the resin, discharge the DMF. (2) Deprotection: Add an appropriate amount of deprotection solution consisting of 20% hexahydropyridine + 80% DMF to the reaction column, stir and agitate under nitrogen for 30-60 minutes, and then dry. (3) Weighing: Measure 1 to 3 times the molar amount of the protective amino acid Fmo-Trp(Boc)-OH, and then weigh 1 to 3 times the molar amount of O-(benzotriazol-1-yl)-N,N,N,N'-tetramethylurea hexafluorophosphate HBTU. (4) Deprotection washing: Add an appropriate amount of DMF to the reaction column, agitate with nitrogen for 2-5 minutes, dry the column, and repeat the operation; (5) Feeding: Add the prepared protected amino acid Fmo-Trp(Boc)-OH and O-(benzotriazol-1-yl)-N,N,N,N'-tetramethylurea hexafluorophosphate HBTU to the reaction column, and then add 2 to 6 times the molar amount of N-methylmorpholine NMM resin. Stir and agitate under nitrogen for 30 to 60 minutes. (6) Washing after reaction: Drain the solution in the reaction column, add an appropriate amount of DMF to wash, agitate with nitrogen for 2-5 minutes, drain, and repeat the operation; (7) Test: Take the resin in a small test tube, add two drops each of 80% phenol + 20% anhydrous ethanol, redistilled pyridine and 5% ninhydrin anhydrous ethanol solution, and heat in a dry heater at 110 degrees Celsius for 3-5 minutes; if the solution turns blue and the resin is discolored and opaque after removal, the reaction is not complete and should be repeated; if the solution is slightly yellow and the resin is colorless and transparent, the reaction is complete. Couple Fmoc-Arg(Pbf)-OH, Fmo-Phe-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, and Boc-Lys(BOC)-OH amino acids in sequence, following the five steps above (2)-(6), until the last amino acid is coupled; after the last amino acid is coupled and the protective washing has been completed, dry the column, add methanol to the reaction column, puff with nitrogen for 2-6 minutes, dry the column, add an appropriate amount of DCM, puff with nitrogen for 2-6 minutes, dry the column, repeat the operation 2-3 times; finally, add methanol to the reaction vessel, puff with nitrogen for 2-5 minutes, dry the column, repeat the operation 2-3 times, and place the resin in a container and vacuum dry it in a vacuum dryer for 12-24 hours; (8) Cutting and washing of peptides: Seal the centrifuge tube and place it in a centrifuge. Centrifuge at 4000 rpm for 3-5 minutes. Remove the tube, discard the supernatant, add ether, stir with a glass rod, and centrifuge again. Repeat this washing process 2-5 times. Drying: Place the washed peptides in a vacuum desiccator and vacuum dry for 12-24 hours to obtain crude peptides. (9) Purification of polypeptides The crude product was dissolved in an acetonitrile-water mixture by ultrasonication, then filtered through a 0.45µm microporous membrane. The filtrate was purified by HPLC under the following conditions: C 18 10A Preparative Column: 10cm*25cm, wavelength 220nm; Pump A: acetonitrile solution containing 0.1% trifluoroacetic acid; Pump B: aqueous solution containing 0.1% trifluoroacetic acid; Gradient elution: 0-100 min: A: 21%-31%, 100.01-120 min: A: 31%-70%; Receive the effluent after 24-36 min, and freeze-dry the received solution to obtain pure peptides.

[0015] Beneficial effects:

[0016] This study first identified the flagellar inhibitory peptide Pep from *Pseudomonas aeruginosa* using mass spectrometry and high-performance liquid chromatography (HPLC), confirming its molecular weight of 1075.32 Da and purity of 97%. Transmission electron microscopy revealed that Pep exhibits time-dependent self-assembly, gradually evolving from scattered granules to a filamentous structure, ultimately forming a stable network morphology. Functional experiments demonstrated that Pep significantly inhibited the flagellar motility of several *Pseudomonas aeruginosa* strains, including PAO1, in a concentration-dependent and strain-specific manner. Tracking and quantifying the bacterial migration trajectories further confirmed that Pep dose-dependently reduced the average bacterial motility. Ultrastructural observation showed that Pep-treated bacteria exhibited structural damage including flagella breakage, stiffening, and detachment. Transcriptomic analysis identified 1089 differentially expressed genes. GO enrichment analysis revealed that these genes are mainly involved in energy and biosynthetic processes such as carbohydrate metabolism, ATP synthesis, and nucleoside triphosphate biosynthesis. KEGG pathway enrichment further showed that oxidative phosphorylation, peptidoglycan biosynthesis, and carbon metabolism pathways were significantly affected. Several key genes related to pathogenicity were significantly downregulated, such as the flagella assembly gene fliD, and type IV fimbriae-related genes pilQ and fimV. qRT-PCR validation results showed that the expression trends of these genes were consistent with the transcriptome data. These results indicate that Pep systematically weakens the pathogenic potential of *Pseudomonas aeruginosa* through multi-target synergistic effects.

[0017] Based on this, this invention further investigated the combined antibacterial effects of Pep with allicin and four commonly used clinical antibiotics. The results showed that Pep alone had no significant antibacterial activity, with a MIC greater than 1 mg / mL, while allicin completely inhibited bacterial growth at 1 mg / mL. When Pep and allicin were used in combination, they exhibited a significant synergistic effect, achieving a near 100% inhibition rate at a Pep concentration of 0.6 mg / mL and an allicin concentration of 31.25 μg / mL; the combined group at a Pep concentration of 0.6 mg / mL and an allicin concentration of 0.125 mg / mL completely killed PAO1 within 6 hours. Furthermore, when Pep was used in combination with ciprofloxacin, meropenem, levofloxacin, and polymyxin B, the MICs of each antibiotic decreased to one-quarter of their original values, indicating that Pep can act as an antibacterial potentiator to reduce the dosage of antibiotics. Morphological observation further confirmed that the combined use of Pep and allicin can significantly disrupt the structural integrity of bacteria, causing the bacterial cells to shrink, rupture, and leak their contents. The inhibition rate of PAO1 biofilm formation reached 83.55%, and the clearance rate of mature biofilms reached 90.73%.

[0018] Finally, this study systematically evaluated the in vivo efficacy and biosafety of the combined use of Pep and allicin by establishing a mouse burn wound infection model. Cytotoxicity and hemolysis experiments showed that the combined treatment had no cytotoxicity within the effective antibacterial concentration range, with a hemolysis rate of less than 5%, indicating good biocompatibility. In the wound healing experiment, the combined treatment group achieved a healing rate of 66.897% on day 5 and 99.189% on day 14 in the infection model, with near-complete healing and new hair growth; the healing process was also accelerated in the uninfected model. Bacterial load quantification results showed that the residual bacterial load in the wound tissue of the combined treatment group was significantly reduced to 4.2 × 10⁻⁶. 3 CFU / g, much lower than the model group's 2.8 × 10⁻⁶. 9 CFU / g. Histopathological analysis showed that the wound tissue structure in the combined treatment group was intact, re-epithelialization was good, collagen deposition was increased, and the expression of inflammatory factors was decreased, suggesting that it has the potential to promote tissue repair, regulate inflammatory response, and control infection.

[0019] Pep systematically weakens the pathogenicity of *Pseudomonas aeruginosa* by interfering with flagellar structure and function, inhibiting energy metabolism, and suppressing the expression of related virulence genes. When used in combination with allicin and clinical antibiotics, Pep exhibits a significant synergistic antibacterial effect. The combined use of Pep and allicin effectively promotes wound healing in vivo and demonstrates good biocompatibility. This study provides a new candidate molecule and theoretical basis for antiviral strategies targeting the bacterial motility system, and offers a feasible solution to address the challenge of *Pseudomonas aeruginosa* drug resistance. Attached Figure Description

[0020] Figure 1 This is the mass spectrum of the flagellar inhibitory peptide.

[0021] Figure 2 This is a liquid chromatogram.

[0022] Figure 3 Diagram showing changes in polypeptide morphology.

[0023] Figure 4 The image shows a visual representation of the swimming experiment (A) and the quantitative results (B), n=3.

[0024] Figure 5 Visual representations (A, B, C) and quantitative results (D) of the swimming experiment, n=3, ****P < 0.0001.

[0025] Figure 6 These represent changes in flagella morphology (A, B, C).

[0026] Figure 7 The inhibition zones of each drug against PAO1 are shown.

[0027] Figure 8Inhibitory rates of each drug against PAO1: A: Pep; B: Allicin; C: Polymyxin B; D: Combined drugs, n=4.

[0028] Figure 9 This is a diagram of a bacterial growth experiment, n=4.

[0029] Figure 10 To observe the cell morphology of PAO1 before and after drug treatment using transmission electron microscopy.

[0030] Figure 11 The live / dead fluorescence staining pattern of PAO1 (green: SYTO-9; red: PI).

[0031] Figure 12 The results of plate counting for PAO1 at different time points are shown.

[0032] Figure 13 The crystal violet method was used to determine the inhibition of biofilm by different drugs. A: Pep; B: Allicin; C: Polymyxin B; D: Combined drugs, n=4, ***P < 0.001, ****P < 0.0001.

[0033] Figure 14 To determine the biofilm clearance effect of different drugs using the crystal violet method, A: Pep; B: Allicin; C: Polymyxin B; D: Combined drugs, n=4, ****P < 0.0001.

[0034] Figure 15 Plate count results of PAO1-infected mouse skin, n=3, ****P < 0.0001.

[0035] Figure 16 Summary of stained images. Detailed Implementation

[0036] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims.

[0037] Example 1: Synthesis, identification, and in vitro activity evaluation of flagellate inhibitory peptides

[0038] 1 Materials and Instruments

[0039] 1.1 Reagents and Tests

[0040] Table 1 Experimental Reagents

[0041] 1.2 Experimental strains

[0042] The strains used in the experiment were all clinically collected strains from Nanjing Public Health Medical Center, as shown in Table 2.

[0043] Table 2 Experimental strains

[0044] 2 Experimental Methods

[0045] 2.1 Synthesis and Identification of Polypeptides

[0046] 2.1.1 Synthesis of polypeptides

[0047] The flagellar inhibitory peptide sequence is Lys-Ile-Gly-Leu-Phe-Arg-Trp-Arg. Its sequence listing is shown in Sequence Listing NO1.

[0048] The flagellar inhibitory peptide sequence provided by this invention is Lys-Ile-Gly-Leu-Phe-Arg-Trp-Arg. The synthesis method includes the following steps: (1) Weigh 5g of Fmoc-Arg(pbf)-WangResin resin and add it to the solid-phase synthesis reactor. Add 50ml of N,N-dimethylformamide (DMF) to swell the resin for 30min and then discharge the DMF.

[0049] (2) Deprotection: Add an appropriate amount of deprotection solution (20% hexahydropyridine + 80% DMF) to the reaction column, stir and agitate with nitrogen for 30 minutes, and then dry.

[0050] (3) Weighing: Measure three times the molar amount of the protective amino acid Fmo-Trp(Boc)-OH, and then weigh three times the molar amount of O-(benzotriazol-1-yl)-N,N,N,N'-tetramethylurea hexafluorophosphate (HBTU).

[0051] (4) Deprotection washing: Add an appropriate amount of DMF to the reaction column, agitate with nitrogen for 2 minutes, dry the column, and repeat the operation 6 times.

[0052] (5) Feeding: Add the prepared protected amino acid and HBTU into the reaction column, and then add 6 times the molar amount of N-methylmorpholine (NMM) of the resin. Stir with nitrogen for 30 minutes.

[0053] (6) Washing after reaction: Drain the solution in the reaction column, add an appropriate amount of DMF to wash, agitate with nitrogen for 2 minutes, drain, and repeat the operation 3 times.

[0054] (7) Detection: Take an appropriate amount (10-20 pieces) of resin into a small test tube, add two drops each of solution A (80% phenol + 20% anhydrous ethanol), solution B (re-distilled pyridine), and solution C (5 g ninhydrin + 100 mL anhydrous ethanol). Heat in a dry heater for 3 minutes (110 degrees Celsius). If the solution is blue and the resin is discolored and opaque after removal, the reaction is not complete and needs to be repeated; if the solution is slightly yellow and the resin is colorless and transparent, the reaction is complete and the next amino acid can be linked. Couple Fmoc-Arg(Pbf)-OH, Fmo-Phe-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Boc-Lys(BOC)-OH in sequence. The specific steps are the same as those in (2)-(6) above, until the last amino acid is linked.

[0055] (8) Washing and drying after synthesis: After the last amino acid is added and the protective agent has been removed and washed, the mixture is dried under vacuum. An appropriate amount of methanol is added to the reaction column, and the mixture is agitated with nitrogen for 2 minutes. The mixture is then dried under vacuum, and an appropriate amount of DCM is added. The mixture is agitated with nitrogen for 2 minutes. The mixture is then dried under vacuum, and this process is repeated 3 times. Finally, an appropriate amount of methanol is added to the reaction vessel, and the mixture is agitated with nitrogen for 2 minutes. The mixture is then dried under vacuum, and this process is repeated twice. The resin is then placed in a suitable container and dried under vacuum in a vacuum dryer for 12 hours.

[0056] (9) Peptide cleavage: Washing: Seal the centrifuge tube and centrifuge at 4000 rpm for 3 minutes. Remove the tube, discard the supernatant, add ether, stir well with a glass rod, and centrifuge again. Repeat this washing process 5 times. Drying: Place the washed peptides in a vacuum desiccator and vacuum dry for 24 hours. The resulting white powder is the crude peptide.

[0057] 2.1.2 Purification of peptides

[0058] The crude product was dissolved by sonication in a mixed solvent of H2O and acetonitrile in a ratio of approximately 3:1, and then filtered through a 0.45µm microporous membrane. The filtrate was purified by HPLC. Purification conditions were as follows: C18 10A preparative column: 10cm*25cm, wavelength 220nm, pump A (0.1% Trifluoroacetic Acid in 100% Acetonitrile), pump B (0.1% Trifluoroacetic Acid in 100% Water). Gradient elution: 0-100 min: A: 21%-31%, 100.01-120 min: A: 31%-70%. The eluent after 24-36 min was collected, and the receiving solution was freeze-dried to obtain the purified product.

[0059] 2.1.3 Identification of polypeptides

[0060] This invention utilizes a TSQ Quantis triple quadrupole liquid chromatography-mass spectrometry (LC-MS) system for peptide molecular weight determination. The sample pretreatment steps are as follows: 1.0 mg of the peptide sample is accurately weighed using an analytical balance and completely dissolved in 1.0 mL of ultrapure water. The solution is then vortexed to ensure complete dissolution, preparing a 1.0 mg / mL peptide stock solution. A two-step dilution method is then used, first diluting the stock solution to 10 μg / mL, and then further diluting it to 500 ng / mL as the working solution for direct injection analysis. Mass spectrometry detection employs an electrospray ionization (ESI) source in positive ion mode, with the following parameters: spray voltage 3500 V, capillary temperature 300℃, evaporation temperature 350℃, sheath gas pressure 30 Arb, and auxiliary gas pressure 10 Arb. The mass analyzer is selected in Q1 full scan mode. Before the experiment, the mass axis is calibrated using a standard tuning solution, and an ultrapure water blank control is set up to eliminate background interference.

[0061] The purity of peptides was determined by high performance liquid chromatography (HPLC). Accurately weigh 1.0 mg of the peptide sample to be tested, completely dissolve it in 1.0 mL of ultrapure water, and prepare a 1.0 mg / mL stock solution by vortexing. Transfer 100 μL to a liquid chromatography vial for analysis. A binary mobile phase system was used: Mobile phase A was a 0.1% trifluoroacetic acid in acetonitrile solution (accurately measure 1 L of acetonitrile, add 1 mL of trifluoroacetic acid, mix well, and sonicate for 30 min to degas); Mobile phase B was a 0.1% trifluoroacetic acid in aqueous solution (1 L of ultrapure water, add 1 mL of trifluoroacetic acid, mix well, and sonicate for 30 min to degas). Chromatographic conditions: a Chrom Core 120C18 column (5 μm, 4.6 × 250 mm), a detection wavelength of 220 nm, a flow rate of 1.0 mL / min, a column temperature of 30 ℃, an injection volume of 10 μL, and a gradient elution program: 5% A–95% B at 0.01 min, followed by a linear gradient to 95% A–5% at 25 min. B, rapidly increase to 100% A to rinse the column in 25.1 min, and calculate the peptide purity by peak area normalization.

[0062] 2.1.4 TEM characterization of peptides

[0063] Transmission electron microscopy (TEM) combined with negative staining was used to observe the temporal morphological evolution of peptides. First, a 4 mg / mL peptide solution was prepared and the pH was precisely adjusted to 7.0. Samples were taken at five key time points: 5 min, 30 min, 1 h, 16 h, and 48 h. For TEM sample preparation, tweezers were cleaned with anhydrous ethanol and dried with the smooth side of filter paper. After holding the edge of the copper mesh, 10 μL of peptide solution was added and allowed to stand for 1 min 30 s to adsorb onto the carbon membrane. Excess liquid was then slowly absorbed from the edge with the smooth side of filter paper. When the water stains were almost dry, the illumination was stopped, and 10 μL of 2% uranium acetate was immediately added for negative staining for 2 min. Finally, the dye was quickly absorbed with the rough side of filter paper and the membrane was dried. The copper mesh was stored in a centrifuge tube for later TEM imaging. The structural features of the peptides at different time points were captured by TEM to analyze the peptide self-assembly kinetics.

[0064] 2.2 Preparation of culture medium

[0065] (1) Luria-Bertani (LB) medium (1×LB liquid medium): Liquid LB medium refers to 10 g sodium chloride, 10 g tryptone, and 5 g yeast extract per liter. After autoclaving at 121℃ for 30 min, store at room temperature.

[0066] (2) 2.5% LB solid medium: Weigh 10 g sodium chloride, 10 g tryptone, 5 g yeast extract, and 25 g agar powder, add ultrapure water to make up to 1 L, sterilize at 121℃ for 30 min, and store at room temperature.

[0067] (3) 0.8% LB solid medium: Weigh 10 g sodium chloride, 10 g tryptone, 5 g yeast extract and 8 g agar powder respectively, add ultrapure water to make up to 1 L, sterilize at 121℃ for 30 min and store at room temperature.

[0068] (4) Bacterial migration medium: that is, a semi-solid medium of 0.3% agar, which means weighing 5 g of sodium chloride, 10 g of tryptone and 3 g of agar powder per liter, adding ultrapure water to make up to 1 L, adjusting the pH to 7.2-7.4, sterilizing at 121℃ for 20 min, and the medium should be prepared and used immediately.

[0069] 2.3 Bacterial resuscitation and culture

[0070] (1) Resuscitation of bacterial strains: A small amount of bacterial solution was taken from the glycerol tube of Pseudomonas aeruginosa strain stored at -80℃ and streaked onto LB solid medium. The culture was incubated at 37℃ for 24 h until colonies grew on the surface of the culture dish. The culture was then sealed with sealing film and stored at 4℃ for later use.

[0071] (2) Culture of bacterial strains: single colonies were picked from the Pseudomonas aeruginosa plate and inoculated into 6 mL of LB liquid medium. In order to provide a uniform culture environment, help the suspension of bacterial cells and reduce precipitation, after inoculation, the bacterial tubes were placed in a shaker at 37℃ and 200 rpm / min and cultured overnight (12-16 h). The OD600 value of the bacterial solution was measured.

[0072] 2.4 Effects of peptides on bacterial flagellar motility

[0073] 2.4.1 Swimming Experiment

[0074] First, the culture medium was prepared. Bacterial migration is the movement of bacteria in a liquid environment or a semi-solid culture medium with low density. Generally, a semi-solid medium containing 0.3% agar is used to verify the migratory ability of bacteria. The experimental strains included the standard reference strains PAO1 (BOX29-45) and PA19 (10-19), and two clinical isolates (1-3-4 from pus and 1-3-27 from bronchoalveolar lavage fluid). The strains preserved at -80℃ were first streaked on LB agar plates and incubated at 37℃ for 16 h to activate them. Single colonies were picked and inoculated into 6 mL of LB broth and cultured at 37℃ with shaking at 200 r / min for 12–16 h until the logarithmic growth phase. After centrifugation and washing, the bacterial concentration was adjusted to OD. 600 =0.2. Sterilize the prepared migratory medium and cool to approximately 46-47°C. At this temperature, add different concentrations of peptides according to the experimental design, mix well, and immediately pour into plates. Accurately add 6 mL of medium to each plate and allow to solidify. Take OD... 600 Two µL of the 0.2% bacterial suspension was vertically inoculated into the center of the culture medium. The medium containing the bacterial suspension was left to stand for a period of time, then smoothly transferred to a 37°C incubator and incubated for 20 h to observe the results. After incubation, the migration diameter of the bacteria in the culture medium was observed and measured to evaluate the effect of different concentrations of peptide treatment on bacterial motility.

[0075] 2.4.2 Effect of peptides on flagellar motility

[0076] To investigate the effect of different concentrations of peptides on the motility of Pseudomonas aeruginosa standard strain PAO1. (1) Pick a single colony on a PAO1 plate and inoculate it into LB liquid medium. Incubate overnight at 37°C and 200 r / min on a shaker. (2) The next day, centrifuge and wash the bacterial cells. Transplant to the logarithmic phase with an OD600 of 0.01. (3) Collect the bacterial cells by centrifugation at 5000 rpm for 10 minutes. Remove impurities by washing twice with sterile Q water (each time resuspended in 1 mL of Q water and centrifuged at 12000 rpm for 1 minute). (4) Resuspend the bacterial cells in 1 mL of peptide solutions of different concentrations (0.3, 0.6, 0.9 mg / mL pep) and sterile Q water (control group). Incubate at 37°C for 1 hour. (5) Drop the treated bacterial solution onto an ultrasonically cleaned slide. Use a fluorescence inverted microscope under 60x bright field conditions, with an exposure time of 9 ms and a frame rate of 50 Hz for 8 seconds to record the bacterial movement. Analyze the images to determine the movement speed and evaluate the dose-effect of the peptide on bacterial motility.

[0077] 2.4.3 Observation of flagella morphology using transmission electron microscopy

[0078] The effect of peptides on the flagella morphology of the Pseudomonas aeruginosa standard strain PAO1 was observed using transmission electron microscopy (TEM). First, single colonies were picked from PAO1 solid plates and inoculated into LB liquid medium, incubated overnight at 37°C and 200 r / min with shaking. The next day, the cells were washed by centrifugation. The bacterial concentration was then adjusted to OD600 of 0.01, and two 6 mL tubes of bacterial culture were transferred to each tube and cultured under the same conditions until the logarithmic growth phase. The cells were collected by centrifugation at 5000 rpm for 10 min. The cells were resuspended in 1 mL of Q water in each tube and transferred to 1.5 mL EP tubes, centrifuged at 12000 rpm for 1 min. The supernatant was discarded, and the cells were resuspended in 1 mL of Q water in each tube, centrifuged at 12000 rpm for 1 min. The supernatant was discarded, and 1 mL of 0.6 mg / mL Pep was added to the cell pellet as the treatment group, while 1 mL of Q water was added to the other tube as the control group. The cells were incubated at 37°C for 1 h. After incubation, the samples were prepared using TEM and photographed.

[0079] 3 Experimental Results

[0080] 3.1 Identification of polypeptides

[0081] To determine the physicochemical properties and chemical purity of the peptide used in this study, the molecular weight of the peptide was accurately determined using a triple quadrupole liquid chromatography-mass spectrometry (TSQ Quantis) system, and its purity was analyzed by high-performance liquid chromatography (HPLC). Mass spectrometry analysis showed that the measured molecular weight of the peptide was 1075.32 Da, consistent with the theoretical molecular weight, confirming the accuracy of its chemical structure. Meanwhile, HPLC results showed that the peptide purity was 97%, indicating low impurity content and good homogeneity, meeting the basic purity requirements for subsequent in vitro and in vivo antibacterial experiments. These results provide a reliable material basis for subsequent research on antibacterial activity and mechanism.

[0082] 3.2 Transmission electron microscopy observation of peptide morphology changes

[0083] To investigate the self-assembly behavior and morphological evolution of peptides in solution, this study used TEM to observe the morphology of samples at different incubation time points. The results showed that the morphology of Pep exhibited a clear time-dependent evolution: initially, peptide molecules were scattered as granules or short aggregates; with prolonged incubation, Pep gradually assembled into filamentous structures, which further extended and cross-linked; after sufficient incubation, Pep finally formed a stable network structure, maintaining this network morphology at subsequent observation time points. This morphological evolution suggests that Pep possesses a time-dependent self-assembly capability. Its dynamic behavior, from a dispersed state through a filamentous intermediate state to a network structure, may be related to a multi-level assembly process driven by non-covalent interactions between peptide molecules (such as hydrogen bonds and hydrophobic interactions). The formation of this network structure may provide a structural basis for its subsequent biological functions (such as promoting wound healing).

[0084] 3.3 Bacterial Migration Experiment

[0085] To investigate the effect of peptides on the motility of Pseudomonas aeruginosa flagella, this study employed a bacterial swimming motility assay for phenotypic analysis. Standard strains PAO1 and PA19, as well as two clinical isolates (strain 1-3-4 from pus samples and strain 1-3-27 from bronchoalveolar lavage fluid samples), were inoculated into semi-solid culture media containing different concentrations of Pep (0.1, 0.3, and 0.6 mg / mL). Changes in motility were assessed by measuring the bacterial swimming diameter.

[0086] The experimental results showed that the inhibitory effect of Pep on migration ability exhibited strain-dependent and concentration-dependent characteristics. At a treatment concentration of 0.3 mg / mL, Pep significantly inhibited the migration of PAO1, PA19, and strain 1-3-27 from irrigation fluid, while the inhibitory effect on the migration of strain 1-3-4 from pus was not significant, suggesting that there are differences in the sensitivity of strains from different sources to Pep. Further concentration gradient experiments using PAO1 as the target showed that the 0.6 mg / mL Pep treatment group had the most significant inhibitory effect on migration, while the inhibitory effects of the 0.1 mg / mL and 0.3 mg / mL treatment groups were similar.

[0087] In summary, 0.3 mg / mL Pep generally inhibited the motility of multiple Pseudomonas aeruginosa strains, while 0.6 mg / mL showed the best inhibitory effect on P. aeruginosa PAO1. Therefore, 0.6 mg / mL was selected as the treatment concentration for subsequent studies on the antibacterial mechanism.

[0088] 3.5 Effect of peptides on flagellar motility

[0089] To quantitatively assess the effect of peptides on the motility of *Pseudomonas aeruginosa*, this study used a fluorescence inverted microscope with a high-sensitivity digital camera to monitor and record bacterial motility before and after drug administration in real time. Image analysis software was used to track bacterial trajectories and quantify their velocity. The study found that the inhibitory effect of Pep on bacterial motility exhibited a significant dose-dependent characteristic: as the treatment concentration gradually increased, the average motility of the bacterial population showed a gradient decreasing trend. This phenomenon suggests that Pep may weaken bacterial motility by interfering with the rotational dynamics of the flagellar motor or affecting the proton driving force of the flagella. These results further confirm the regulatory role of Pep on bacterial motility phenotype at the quantitative level, suggesting that it may target the bacterial flagellar system or interfere with related energy metabolism pathways. This study provides kinetic evidence for a deeper understanding of the antibacterial mechanism of Pep.

[0090] Table 3 Bacterial Motion Speed

[0091] 3.6 Observation of flagella morphology using transmission electron microscopy

[0092] To verify the damaging effect of peptides on the flagella of *Pseudomonas aeruginosa* at the ultrastructural level, this study used TEM to observe and photograph the morphological changes of bacterial flagella before and after drug administration. The experiment included a 0.6 mg / mL Pep treatment group and a Q-water control group. After co-incubation with *P. aerosol* strain PAO1 for 1 h, samples were prepared using negative staining and photographed. TEM observations showed that the flagella in the control group exhibited a natural morphology, with typical wavy curvature, intact structure, and firm attachment. In contrast, after treatment with 0.6 mg / mL Pep for 1 h, the bacterial flagella showed significant structural damage, mainly manifested as breakage at the flagella tail, loss of natural curvature of the flagella filaments resulting in a rigid state, and detachment of the flagella from the bacterial cell at the root. These morphological changes indicate that Pep can directly act on the flagellar structure, possibly by disrupting the assembly integrity of flagellar proteins or affecting the stability of the connection between the flagella and the bacterial cell, thereby leading to abnormal flagellar morphology and weakened function. This further confirms the targeted destructive effect of Pep on the bacterial motility system at the ultrastructural level.

[0093] Example 2: In vitro antibacterial performance study

[0094] 1 Materials and Instruments

[0095] 1.1 Reagents and Tests

[0096] Table 4 Experimental Reagents

[0097] 2 Experimental Methods

[0098] 2.1 Evaluation of the antibacterial effect of combined drug administration

[0099] 2.1.1 Antibacterial zone experiment

[0100] To evaluate the antibacterial activity of the test drug against *Pseudomonas aeruginosa* PAO1, an in vitro antibacterial experiment was conducted using the filter paper disc diffusion method (Kirby-Bauer method). Overnight cultures of *Pseudomonas aeruginosa* PAO1 were diffused at OD0.05. 600 0.01 mg was transferred to fresh LB liquid medium and incubated in a 37°C shaker (200 rpm / min). The OD of the bacterial culture was monitored periodically. 600 Value, wait until it grows to the logarithmic growth phase (OD). 600After the initial concentration is 0.5 (approximately 3-4 hours), remove the plate and set aside. Simultaneously, prepare a double-layer agar plate: First, melt 10 mL of 2.5% LB solid medium and pour it into a sterile petri dish as the bottom layer. Let it solidify completely at room temperature. Then, take 6 mL of molten 0.8% LB soft agar medium (cooled to approximately 42 °C), add 500 μL of the aforementioned logarithmic growth phase PAO1 bacterial suspension, mix thoroughly, and quickly pour it onto the solidified bottom agar layer. Gently shake the petri dish to distribute the bacterial suspension evenly. Let it solidify completely at room temperature for 5 minutes to obtain the bacterial double-layer plate. After the top soft agar has solidified, use sterile forceps to pick up a sterile circular filter paper disc and gently place it on the plate surface. Use a micropipette to add 5 μL of the test drug solution of different concentrations to the center of each filter paper disc, ensuring complete absorption of the drug solution. After incubating the plates upside down in a 37℃ incubator for 24 h, the size of the inhibition zone was observed to evaluate the antibacterial effect of the drug. The initial concentration of the polypeptide group was 2.4 mg / mL, the initial concentration of the allicin group was 4 mg / mL, and the initial concentration of the poly B group was 0.1 mg / mL, with subsequent concentrations decreasing by a factor of two. The initial concentration of the combination group was 0.6 mg / mL of pep + 0.5 mg / mL of allicin, with subsequent concentrations of pep at 0.6 mg / mL, but with allicin decreasing by a factor of two.

[0101] 2.1.2 Drug MIC Experiment

[0102] The minimum inhibitory concentration (MIC) of the test drug against PAO1 was determined using the microbroth dilution method. First, the test drug was serially diluted twofold in sterile 96-well plates using LB liquid medium to obtain a series of different concentration gradients. Then, 100 μL of OD was added to each well. 600 A logarithmic phase bacterial suspension with a concentration of 0.02 was used to bring the final volume of each well to 200 μL. A positive control group (containing bacterial suspension but no drug) and a negative control group (containing culture medium but no bacterial suspension) were also included to assess culture medium contamination and the normal growth status of the bacteria. After sample addition, the 96-well plate was incubated at 37°C for 24 h. After incubation, the turbidity of the culture medium in each well was first recorded visually, and the lowest drug concentration at which complete clarity and no visible bacterial growth were defined as the MIC value. Simultaneously, the absorbance (OD) of the bacterial suspension in each well was measured at 600 nm using a microplate reader. 600 The MIC (Minimum Interpretation Value) was quantitatively verified using spectroscopic methods to ensure the objectivity and accuracy of the visual interpretation results. The above experiment was independently repeated three times to guarantee the accuracy and repeatability of the results.

[0103] 2.1.3 Antibacterial experiments of peptides combined with different types of antibiotics

[0104] The checkerboard microdilution assay was used to systematically evaluate the in vitro antibacterial effects of peptides combined with commonly used clinical antibiotics. The tested antibiotics included ciprofloxacin, meropenem, levofloxacin, and polymyxin B. Before the experiment, the test strains were cultured to the logarithmic growth phase, and the bacterial concentration was adjusted to OD600 = 0.02 with fresh culture medium. Based on the determined MICs of each antibiotic against PAO1, working solutions with twice the MIC concentration were prepared as stock solutions. The combined antibacterial assay was performed in 96-well sterile cell culture plates. Each antibiotic was diluted row-by-row using the two-fold dilution method, followed by the addition of the peptide solution to each well, and finally the prepared bacterial culture. The final peptide concentration in each well was 0.6 mg / mL. A positive control containing only bacterial culture and a negative control containing only LB medium were also included in the experiment. After sample addition, the 96-well plate was continuously incubated in a microplate reader, and the OD was measured every 1 hour at 37°C. 600 The values ​​were monitored for a total of 24 hours, and the antibacterial effect of the combined medication was evaluated by plotting growth curves. To ensure the reliability of the experimental results, the above experiments were independently repeated three times.

[0105] 2.1.4 Drug Antibacterial Rate Determination Experiment

[0106] The growth-inhibiting activity of each drug against PAO1 was quantitatively evaluated using a microbroth dilution method combined with spectrophotometry. First, 100 µL of LB broth containing different concentration gradients of the test drug was added to each well of a 96-well microplate, establishing a concentration gradient series using a two-fold dilution method. Then, 100 µL of bacterial suspension (OD200) was added to each well. 600 = 0.02), making the final volume of each well 200 µL. After incubating the 96-well plate at 37°C for 24 h, the absorbance (OD) of each well was measured at 600 nm using a microplate reader. 600 The experiment included multiple control groups: a positive control group (containing only bacterial culture medium without the drug, i.e., blank control) to assess the maximum bacterial growth under experimental conditions; a negative control group (containing only LB medium without bacterial culture medium, i.e., solvent control) to correct the basic absorbance of the culture and monitor aseptic status; and a sample control group (containing only LB medium and the drug, without bacterial culture medium) to eliminate the influence of the drug itself on absorbance. The inhibition rate was calculated using the following formula: Inhibition rate (%) = [1 - (OD sample – OD sample control) / (OD blank control – OD solvent control)] × 100%. The antibacterial efficacy of the drug could be evaluated by the relationship between the inhibition rate and the drug concentration. The above experiment was independently repeated three times.

[0107] 2.1.5 Bacterial Growth Curve Experiment

[0108] LB liquid medium was added to each well of a 96-well plate, followed by different concentrations of the drug. Then, 100 µL of OD was added to each well. 600 Prepare bacterial suspension at a concentration of 0.02 μL to achieve a total volume of 0.2 mL per well. Include positive and negative control columns. After addition, place the wells in a microplate reader and measure OD every 1 hour. 600 The value was continuously monitored for 24 hours.

[0109] The antibacterial activity of the drug against PAO1 was dynamically evaluated using a micro-broth dilution method combined with time-growth curve analysis. In sterile 96-well plates, the test drug was serially diluted twofold using LB medium, with 100 μL of different concentrations added to each well, followed by 100 μL of OD200 per well. 600 The bacterial suspension was prepared at a concentration of 0.02, resulting in a final volume of 200 μL per well. A positive control (100 μL bacterial suspension + 100 μL LB medium, no added drugs) and a negative control (200 μL LB medium, no added bacteria) were also included. The 96-well plate was placed in a multi-functional microplate reader at 37℃. After shaking and mixing every 1 hour, the absorbance (OD) of each well was measured at 600 nm. 600 ), continuously monitored for 24 hours. OD was plotted on the x-axis. 600 The values ​​were plotted on the ordinate to create growth curves of PAO1 at different drug concentrations. The experiment was repeated three times independently.

[0110] 2.1.6 Scanning electron microscopy observation of PAO1 cell morphology after drug treatment

[0111] Overnight cultures of Pseudomonas aeruginosa PAO1 were analyzed using OD2000. 6000.01 μL of the bacterial culture medium was transferred to fresh LB broth and cultured at 37°C with shaking until the logarithmic growth phase. Subsequently, the bacterial cells were collected by centrifugation, resuspended in phosphate-buffered saline (PBS), and washed to remove residual culture medium components. Experimental groupings were as follows: the combined treatment group received a final concentration of 0.6 mg / mL peptide and 0.125 mg / mL allicin, respectively; the polymyxin B treatment group received a final concentration of 12.5 μg / mL polymyxin B; and untreated bacteria served as a negative control. After incubation at 37°C for 1 hour, the bacterial cells were collected by centrifugation, immediately resuspended in electron microscopy fixative (2.5% glutaraldehyde), and fixed overnight at 4°C to ensure adequate preservation of bacterial morphology and structure. After fixation, the samples were dehydrated three times with anhydrous ethanol using a gradient and then freeze-dried. The dried bacterial samples were fixed to the scanning electron microscope stage with carbon conductive tape, and then sputtered with gold to enhance conductivity. Finally, they were observed and images were acquired under a scanning electron microscope.

[0112] 2.1.7 Transmission electron microscopy observation of PAO1 cell morphology after drug treatment

[0113] Overnight cultures of Pseudomonas aeruginosa PAO1 were analyzed using OD2000. 600 The bacterial cells were transferred to fresh LB liquid medium at a concentration of 0.01 and grown to the logarithmic phase at 37 °C. They were then resuspended in PBS. Experimental groups were treated as follows: the combined treatment group received 0.6 mg / mL of peptide and 0.125 mg / mL of allicin; the polymyxin B positive control group received 12.5 μg / mL of polymyxin B; and the negative control group received an equal volume of PBS. All samples were incubated at 37 °C for 1 hour. The cells were collected by centrifugation, resuspended in electron microscopy fixative, and then incubated overnight at 4 °C to ensure complete fixation. After fixation, dehydrated samples were prepared, stained, and spotted onto a copper grid. The copper grid was placed on the TEM stage, and the voltage was adjusted (usually 80-120 kV) for TEM imaging.

[0114] 2.1.8 Observation of the effects of drug treatment on bacteria using laser confocal microscopy

[0115] Live / dead bacteria were stained with Propidium iodide (PI) and SYTO-9 dye, and the effects of different drug treatments on the integrity and survival status of PAO1 bacterial membranes were observed by laser confocal microscopy.

[0116] (1) PAO1 was inoculated into 6 mL LB liquid medium and cultured at 37℃ with shaking at 200 rpm / min for 16 h. The next day, the OD was used as the inoculum. 600 The transfer rate was 0.01, and the cells were incubated at 37°C with shaking for 4 hours until the OD reached its maximum. 600=0.8. (2) Centrifuge to collect bacterial cells, wash with PBS 3 times, and resuspend in PBS. (3) Take 10 mL of bacterial suspension, centrifuge at 12000 rpm for 1 min to collect bacterial cells, and label them respectively. (4) Prepare the control group and the other 4 drug administration groups. The control group is resuspended in PBS; the drug administration concentration of the PEP group is 0.6 mg / mL; the drug administration concentration of the allicin group is 0.125 mg / mL; the drug administration concentration of the combined group is PEP 0.6 mg / mL + allicin 0.125 mg / mL; the poly B concentration is 12.5 μg / mL. The final volume of each group is 200 μL. All are incubated at 37℃ for 1 hour, and gently shaken to mix during the period. (5) After the incubation, add 0.3 μL of PI staining solution (final concentration 30 μM) and 0.45 μL of SYTO-9 staining solution (final concentration 7.5 μM) to the bacterial suspension respectively, and incubate at room temperature in the dark for 30 min. (6) Take 10 μL of the stained bacterial strain and add it to the pre-prepared 1% agarose thin-layer glass slide. Gently cover it with a coverslip and press it gently with the pen cap to distribute the bacterial solution evenly, avoiding the formation of air bubbles. Use absorbent paper to remove excess bacterial solution and ensure the sample is flat. (7) According to the different fluorescent staining of live and dead bacteria, the survival status of bacteria is observed using a Zeiss SLM900 laser confocal microscope. The excitation wavelength of SYTO-9 is 483 nm and the emission wavelength is 503 nm, which is used to label live bacteria; the excitation wavelength of PI is 493 nm and the emission wavelength is 636 nm, which is used to label dead bacteria.

[0117] 2.2 Evaluation of the bactericidal effect of combined drug administration

[0118] 2.2.1 Time-based kill dynamics experiment

[0119] To investigate the bactericidal kinetics of the drug against the Pseudomonas aeruginosa standard strain PAO1, this study used the time-bactericidal curve method. The frozen PAO1 strain was inoculated into LB medium and activated overnight; the next day, the bacterial culture was adjusted to OD0.05. 600 Transfer the culture medium to 0.01 g and continue incubation with shaking until the logarithmic growth phase (OD200). 600≈0.5). Subsequently, the bacterial culture was centrifuged to collect the bacterial cells, and washed three times with phosphate-buffered saline (PBS) to remove residual culture medium components. The experiment included drug treatment groups and negative control groups. The final concentration of the single-drug peptide group was 0.6 mg / mL, the final concentration of the single-drug allicin group was 0.125 mg / mL, the combined drug group was PEP 0.6 mg / mL + allicin 0.125 mg / mL, and the final concentration of the single-drug polymyxin B group was 12.5 μg / mL. Samples were collected at seven time points: 0, 1, 2, 4, 6, 12, and 24 h after drug administration and incubation. The samples were diluted with PBS at appropriate folds using the tenfold dilution method, from 0 to -7. 100 μL of each dilution was aspirated onto agar medium, plated, and placed in an incubator. After 24 h, the samples were observed and photographed, and colony counts were performed. The colony forming units (CFU / mL) were calculated for plates with colony counts between 30 and 300. The results were plotted with time as the x-axis and logarithmic. 10 A bactericidal curve was plotted with (CFU / mL) as the ordinate to evaluate the bactericidal rate and effect of each drug combination. Simultaneously, photographic composites of the 0-gradient plate results at each time point were created to visually present changes in colony growth.

[0120] 2.3 Effects of combined drug administration on biomembranes

[0121] 2.3.1 Establishment of a biofilm model

[0122] A Pseudomonas aeruginosa PAO1 in vitro biofilm model was constructed using 96-well microplates. Overnight cultured PAO1 cells were collected by centrifugation and washed once with LB liquid medium containing 1% glucose to remove residual metabolic products and replenish carbon source. The OD of the bacterial culture was adjusted with fresh LBGT medium (LB supplemented with 1% glucose). 600 The inoculum value was 0.01. To reduce evaporation deviation caused by the edge effect, 200 μL of sterile PBS or culture medium was added to the peripheral wells. 200 μL of inoculum was added to a sterile 96-well plate, with 6 replicates per group; blank control wells containing only culture medium and bacterial control wells without glucose were also included to assess the promoting effect of glucose on biofilm formation and the background value of the culture medium. The plate was placed in a 37°C incubator and incubated statically for 48 h. This duration ensures that PAO1 forms a mature three-dimensional biofilm structure, including microcolonies and extracellular polymeric substances (EPS). Shaking should be avoided during this period to allow the bacteria to form a mature biofilm structure at the bottom of the well and the air-liquid interface through initial adhesion, microcolon formation, and extracellular polysaccharide secretion.

[0123] 2.3.2 Biomembrane Inhibition Experiment

[0124] The inhibitory effect of different concentrations of the drug on biofilm formation of *Pseudomonas aeruginosa* PAO1 was evaluated using crystal violet staining. Overnight activated PAO1 bacterial cultures were collected by centrifugation and washed once with LB liquid medium containing 1% glucose to remove residual metabolites and replenish carbon sources. The OD of the bacterial culture was then adjusted with fresh LBGT medium. 600 A value of 0.02 was used as the inoculum. In sterile 96-well cell culture plates, the test drug was serially diluted twofold using LBGT medium, with 100 μL of different concentrations added to each well, followed by 100 μL of OD200 per well. 600 The bacterial suspension was prepared at a concentration of 0.02, resulting in a final volume of 200 μL per well. Positive control wells (containing only bacterial suspension and culture medium) and negative control wells (containing only culture medium) were also included. The plates were incubated at 37°C for 48 h to allow for biofilm formation. After incubation, floating bacterial suspension was carefully aspirated from each well. Each well was gently washed three times with 200 μL of sterile PBS to remove unattached bacteria. The plates were then inverted and dried at 37°C for 10 min. Next, 200 μL of 0.1% (w / v) crystal violet staining solution was added to each well, and the plates were stained at 37°C for 30 min. The staining solution was discarded, and the plates were slowly rinsed three times with running water until the water was clear. The plates were then inverted and dried for 10 min. Finally, 200 μL of anhydrous ethanol was added to each well, and the crystal violet was dissolved at room temperature for 30 min, gently agitated to ensure even release. The absorbance (OD) of each well at 570 nm was measured using a microplate reader. 570 ), with OD 570 The value reflects the amount of biofilm formation. By comparing the OD values ​​of different treatment groups with the control group... 570 The anti-biofilm activity of each drug under different concentration conditions was evaluated. All experiments were independently repeated three times.

[0125] 2.3.3 Biofilm clearance experiment

[0126] The effect of different drug concentrations on the removal of established biofilms was evaluated using crystal violet staining. Overnight activated *Pseudomonas aeruginosa* PAO1 bacterial culture was centrifuged to collect the cells, washed once with LB liquid medium containing 1% glucose to remove residual metabolites and replenish carbon source, and then the OD of the bacterial culture was adjusted with fresh LBGT medium. 600A value of 0.01 was used as the inoculum. 200 μL of the bacterial culture was added to each well of a sterile 96-well cell culture plate, with blank control wells containing only culture medium. The plate was incubated at 37°C for 48 h to allow mature biofilms to form at the bottom of the wells and the air-liquid interface. After incubation, the floating bacterial culture was carefully aspirated along the well walls, avoiding contact with the biofilm attached to the bottom and walls of the wells. 200 μL of a gradient of different drug solutions prepared with PBS was added to each well (drug concentrations determined based on previous experiments). Positive control wells (containing bacteria, with PBS) and blank culture medium control wells (sterile, with PBS) were also included. The plate was incubated again at 37°C for 24 h to allow the drugs to fully interact with the biofilm. After incubation, the supernatant was carefully aspirated from the wells, and each well was gently washed three times with 200 μL of sterile PBS to remove residual drugs and loose, unattached bacteria. The plates were then inverted and dried at 37°C for 10 min. Then, add 200 μL of 0.1% (w / v) crystal violet staining solution to each well and incubate at 37°C for 30 min. Discard the staining solution, rinse the plate three times slowly with running water until the water is clear, and invert to dry for 10 min. Finally, add 200 μL of anhydrous ethanol to each well, dissolve the crystal violet at room temperature for 30 min, and gently shake to ensure even release. Measure the absorbance (OD) of each well at 570 nm using a microplate reader. 570 ), with OD 570 The value reflects the amount of residual biofilm after drug treatment. The OD values ​​were compared between different treatment groups and the positive control group without drug treatment. 570 Values ​​were used to evaluate the scavenging activity of each drug on mature biofilms. All experiments were independently repeated three times.

[0127] 2.3.4 Observation of PAO1 biofilm using scanning electron microscopy

[0128] The effects of different drug treatments on the morphology and structure of mature biofilms of *Pseudomonas aeruginosa* PAO1 were observed using scanning electron microscopy. The OD of the bacterial culture was adjusted using method 2.3.3. 600A value of 0.01 was used as the inoculum. The bacterial solution was added to 96-well PVC plates and incubated statically at 37°C for 48 h to allow bacteria to form a mature biofilm on the PVC surface. After incubation, the floating bacterial solution was carefully aspirated to avoid disturbing the biofilm. The experimental groups were treated as follows: the combined treatment group was given PBS containing a final concentration of 0.6 mg / mL peptide and 0.125 mg / mL allicin; the polymyxin B positive control group was given PBS containing a final concentration of 12.5 μg / mL polymyxin B; and the untreated biofilm control group was given an equal volume of PBS. The culture plates were incubated statically at 37°C for another 24 h to allow the drugs to fully interact with the biofilm. After incubation, the supernatant was aspirated, and the plates were gently washed three times with sterile PBS to remove residual drugs and loose bacteria. The bottom of each PVC well was carefully cut off with a sterile blade and immediately immersed in pre-cooled 2.5% glutaraldehyde fixative, and fixed overnight at 4°C to ensure that the morphology and structure of the bacteria and biofilm extracellular matrix were fully preserved. After fixation, the samples were dehydrated three times with anhydrous ethanol. The dehydrated samples were then freeze-dried to completely remove moisture and maintain the three-dimensional structure of the biofilm. The dried biofilm samples were fixed to the scanning electron microscope stage with carbon conductive tape, and a gold film was sputtered to enhance conductivity. Finally, the surface morphology of the biofilms in different treatment groups was observed under a scanning electron microscope, and representative images were acquired.

[0129] 3 Experimental Results

[0130] 3.1 Antibacterial zone experiment

[0131] The antibacterial ability was initially evaluated by observing the size of the inhibition zone in each group. The experimental results showed that (1) no obvious inhibition zone was observed in the polypeptide single-action group at any of the test concentrations; (2) in contrast, the allicin group showed a clearly identifiable inhibition zone at a concentration of 4 mg / mL, confirming its antibacterial ability at this concentration; (3) the combined drug group showed a synergistic effect, and inhibition zones were observed in the concentration combinations numbered 1, 2, and 3. After analysis, the minimum concentration combination with inhibition zone in the combined group was Pep 0.6 mg / mL and allicin 0.125 mg / mL. This result suggests that when Pep and allicin are used together, they can produce a significant antibacterial effect even at concentrations far below their respective individual effective concentrations, and the two have the potential for combined application; (4) the polymyxin B group, as a positive control, showed significant antibacterial activity, and inhibition zones were observed at concentrations numbered 1 to 4, that is, the minimum concentration with inhibition zone was 12.5 μg / mL.

[0132] In summary, through qualitative analysis of the inhibition zones, the antibacterial efficacy of each test substance was preliminarily clarified, and the synergistic antibacterial effect of Pep and allicin was revealed.

[0133] 3.2 Drug MIC Experiment

[0134] The minimum inhibitory concentrations (MICs) of each drug are shown in Table 5.

[0135] Table 5. MIC of different drugs against PAO1

[0136] 3.3 Antibacterial Experiments of Peptides Combined with Different Antibiotics

[0137] This study systematically investigated the antibacterial effects of peptides combined with four commonly used clinical antibiotics—ciprofloxacin, meropenem, levofloxacin, and polymyxin B—on PAO1 using the checkerboard microdilution method. The results showed that the combination of Pep with these four antibiotics significantly enhanced the antibacterial activity of each antibiotic, reducing their minimum inhibitory concentration (MIC) to one-quarter of that achieved with either antibiotic alone. This result indicates that Pep, as a potential antibacterial synergist, can effectively reduce the dosage of commonly used clinical antibiotics, and its mechanism may involve the inhibition of bacterial motility and virulence factors. In conclusion, the strategy of combining Pep with antibiotics not only helps reduce the effective inhibitory concentration of antibiotics but also provides a new approach to mitigating the development of bacterial resistance.

[0138] 3.4 Antibacterial rate experiment

[0139] The antibacterial activity of the drugs was evaluated by calculating the inhibition rate. Experimental results showed that, within a 24-hour culture period, the peptide failed to exhibit a significant antibacterial effect even at a concentration as high as 2.4 mg / mL, at which bacterial growth was not significantly inhibited. In contrast, allicin exhibited strong antibacterial activity, with an inhibition rate close to 100% against the test strains at a concentration of 1 mg / mL. Notably, poly B, as a positive control, showed an inhibition rate close to 100% at an extremely low concentration (3.125 μg / mL), demonstrating its highly effective bactericidal properties. Further research revealed a significant synergistic effect when Pep and allicin were used in combination: in the combined treatment group, when the Pep concentration was reduced to 0.6 mg / mL (approximately 1 / 4 of its ineffective dose when used alone), and the allicin concentration was reduced to 31.25 μg / mL (approximately 1 / 32 of its dose when used alone), the inhibition rate remained close to 100%. This result indicates that the combined application of Pep and allicin can significantly reduce their respective effective concentrations, suggesting that they may produce a synergistic antibacterial effect through different mechanisms of action.

[0140] 3.5 Bacterial Growth Curve Experiment

[0141] The antibacterial effect of each group was systematically evaluated by continuously monitoring bacterial growth curves over 24 hours, using OD as the metric. 600The changing trends of the values ​​reflect the bacterial proliferation kinetics. Experimental results showed that when Pep (0.6 mg / mL) was used in combination with allicin (0.125 mg / mL), the growth curve of the treatment group exhibited a significant inhibitory effect; throughout the 24-hour culture period, the bacterial OD values... 600 The values ​​remained at a low level, suggesting that combination therapy could effectively inhibit the growth process of PAO1, but single-drug administration could not inhibit bacterial growth.

[0142] 3.6 Scanning electron microscopy observation of PAO1 cell morphology after drug treatment

[0143] SEM observations showed that the untreated control group PAO1 strain exhibited a typical rod-shaped morphology under electron microscopy, with a smooth, plump surface, intact bacterial structure, and clear boundaries, demonstrating a healthy and active bacterial physiological state. In contrast, after treatment with the combined drugs, the bacterial morphology changed significantly, with obvious shrinkage, twisting, and even collapse, indicating severe damage to the bacterial structural integrity. Polymyxin B treatment also led to drastic changes in bacterial morphology, with widespread shrinkage and flattening of the bacterial cells. These results indicate that both the combined drugs and polymyxin B can significantly disrupt the morphological structure of *Pseudomonas aeruginosa*.

[0144] TEM observations showed that after treatment with the combined drugs, the bacteria exhibited a ruptured state with leakage of contents; after treatment with polymyxin B, the bacteria showed a shrunken and deformed state. TEM results indicated that the combined drugs and polymyxin B significantly altered the structure of *Pseudomonas aeruginosa*, causing the bacteria to gradually collapse, leak contents, and eventually die.

[0145] 3.8 Observation of the effects of drug treatment on bacteria using laser confocal microscopy

[0146] Laser confocal scanning microscopy (CLSM) combined with live / dead bacterial fluorescence staining (SYTO-9 / propidium iodide double staining) can directly reflect the bactericidal effect of drugs and the degree of damage to bacterial membrane structure. Experimental results showed that in the peptide-treated group, the vast majority of bacteria exhibited green fluorescence, with almost no red fluorescently labeled dead cells, indicating that the peptide alone had no significant bactericidal effect, and the bacterial membrane remained intact. In the allicin-only group, some bacteria showed red fluorescence, suggesting that allicin has certain antibacterial activity and can cause the death of a small number of bacteria. However, when Pep (pepper) and allicin were used in combination, a large number of bacteria showed red fluorescence under laser confocal microscopy, and the number of dead strains was significantly higher than in the single-drug group, with obvious disintegration of the bacterial membrane structure. This phenomenon indicates that the combined drug regimen can effectively disrupt the integrity of the bacterial membrane, leading to bacterial rupture and death.

[0147] 3.9 Time-based lethal dynamics

[0148] The time-kill kinetics of PAO1 were systematically investigated using different dosing regimens to examine the bactericidal rate and time required for complete sterilization. The time-kill curves visually reflect the changing trend of bacterial colony count over time under drug action. Results showed that no colony growth was observed on the 0-gradient plates (i.e., the undiluted solution plate) in the combined drug group after 6 hours of treatment, indicating that the combined drug treatment could achieve complete sterilization of PAO1 within 6 hours. In the Poly B group, no colony growth was observed on the 0-gradient plates after 2 hours of treatment, indicating that Poly B has rapid bactericidal ability and can completely eliminate bacteria within 2 hours.

[0149] 3.10 Biomembrane inhibition and clearance experiments

[0150] This study further evaluated the intervention effects of different drug treatments on Pseudomonas aeruginosa PAO1 biofilms, conducting experiments from two dimensions: inhibition of biofilm formation and clearance of existing biofilms. In the biofilm inhibition experiment, the results showed that the peptides had almost no inhibitory effect on PAO1 biofilm formation. In contrast, allicin showed some inhibitory activity within the experimentally defined concentration range, suggesting a certain ability to resist biofilm formation. Polymyxin B exhibited a strong inhibitory effect, especially at a concentration of 25 μg / mL, where its inhibition rate of biofilm formation reached 78%, indicating that it could effectively disrupt the biofilm structure at this concentration. Furthermore, the combined treatment group (Pep 0.6 mg / mL combined with allicin 0.125 mg / mL) showed a synergistic effect, with a particularly outstanding inhibitory effect on biofilm formation, achieving a disruption rate of 83.55%, significantly better than any single drug treatment, suggesting that this combination can effectively inhibit the early formation process of biofilms.

[0151] In the biofilm clearance experiment, all drug-treated groups showed a certain clearance ability against established mature biofilms. Polymyxin B at 25 μg / mL achieved a clearance rate of 87.31%; while the combined drug group (Pep 0.6 mg / mL combined with allicin 0.125 mg / mL) achieved a clearance rate of 90.73%. Considering both inhibition and clearance results, the combined drug treatment not only has a synergistic effect in inhibiting biofilm formation but also demonstrates excellent performance in clearing existing biofilms, showing its potential to solve biofilm-related pollution problems.

[0152] This embodiment systematically evaluated the antibacterial activity and synergistic effect of peptide (Pep), allicin, and commonly used clinical antibiotics against Pseudomonas aeruginosa PAO1 through a series of in vitro experiments. The results showed that Pep alone had no significant antibacterial effect, with a minimum inhibitory concentration (MIC) greater than 1 mg / mL, and could not inhibit bacterial growth or disrupt biofilms. In contrast, allicin at a concentration of 1 mg / mL completely inhibited bacterial growth and had some inhibitory effect on biofilm formation. When Pep and allicin were used in combination, they exhibited a significant synergistic effect: the combined group achieved a near 100% inhibition rate at a Pep concentration of 0.6 mg / mL (approximately 1 / 4 of the ineffective dose alone) and an allicin concentration of 31.25 μg / mL (approximately 1 / 32 of the MIC alone); at a Pep concentration of 0.6 mg / mL and an allicin concentration of 0.125 mg / mL, the combined group completely killed PAO1 within 6 hours. Furthermore, when Pep was used in combination with ciprofloxacin, meropenem, levofloxacin, and polymyxin B, the MICs of each antibiotic decreased to one-quarter of their original values, indicating that Pep can serve as an antibacterial potentiator to reduce the dosage of clinical antibiotics. Morphological observations further confirmed that the combined use significantly disrupted the integrity of bacterial structures: scanning electron microscopy and transmission electron microscopy revealed bacterial shrinkage, distortion, rupture, and leakage of contents; laser confocal microscopy showed the disintegration and death of a large number of bacterial membrane structures. In biofilm-related experiments, the combined use inhibited PAO1 biofilm formation by 83.55% and cleared mature biofilms by 90.73%, significantly better than individual drug treatments. Scanning electron microscopy also showed a loose biofilm structure and reduced thickness. These results indicate that although Pep itself has weak antibacterial activity, its antibacterial effect can be significantly enhanced when used in combination with allicin or multiple antibiotics, effectively reducing the effective concentration, disrupting bacterial structure and biofilm integrity, demonstrating its potential as a novel antibacterial potentiator.

[0153] Example 3: Study on the efficacy of drugs against burn wound infection

[0154] 1 Materials and Instruments

[0155] 1.1 Reagents and Tests

[0156] Table 6 Experimental Reagents

[0157] 1.3 Laboratory Animals

[0158] SPF-grade healthy adult female Kunming (KM) mice, 6-8 weeks old, weighing 30-35 g, were purchased from Jiangsu Qinglongshan Biotechnology Co., Ltd., certificate number: SCXK (Su) 2024-0001.

[0159] 2. Experimental Methods

[0160] 2.1 In vitro safety evaluation

[0161] 2.1.1 Cell safety evaluation

[0162] The in vitro cytotoxicity of the drug was evaluated using mouse fibroblast L929 cells.

[0163] (1) Cell resuscitation: The frozen L929 cells were quickly taken out from the -80℃ freezer and placed in a 37℃ constant temperature water bath. They were gently shaken until only a small amount of ice remained in the cryovial. The cryovial was then transferred to a biosafety cabinet, the tube was wiped with 75% ethanol for disinfection, and the tube was opened. The cell suspension was transferred to a 15 mL sterile centrifuge tube and centrifuged at 1000 rpm for 5 minutes at room temperature to remove the cryopreservation solution. After discarding the supernatant, the cell pellet was resuspended in DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics. The cells were gently pipetted and transferred to a culture dish. The complete medium was added to a final volume of 10 mL. The cells were gently shaken to distribute them evenly and placed in a 37℃, 5% CO2 constant temperature incubator for static culture.

[0164] (2) Cell passage: When the cell density reaches about 90%, passage is performed. The old culture medium is discarded, and the cell monolayer is gently washed twice with sterile phosphate-buffered saline (PBS) to remove residual serum and metabolic waste. 1 mL of trypsin digestion solution is added, and the cells are incubated in a 37°C incubator for 2-4 minutes. During this period, the cell morphology changes are observed under an inverted microscope. When the cells become round and some cells begin to detach, 2 times the volume of complete culture medium is added immediately to stop the trypsin activity. The cells are gently pipetted to completely detach and disperse into a single-cell suspension. The cells are seeded into a new culture dish at a passage ratio of 1:2. The complete culture medium is added to a final volume of 10 mL. The cells are gently shaken to mix and then placed in a 37°C, 5% CO2 incubator for further culture.

[0165] (3) Cell cryopreservation: For cells in good growth condition, the cells are harvested using the digestion method described above. After centrifugation, the supernatant is discarded, and the cells are resuspended in cell cryopreservation solution. The cells are then transferred to 2 mL cryopreservation tubes, sealed with sealing film, and placed in a -80℃ freezer for 6-8 h before being transferred to liquid nitrogen for long-term storage.

[0166] (4) CCK-8 (Cell Counting Kit-8) assay: The CCK-8 assay was used to detect the effect of different concentrations of drugs on the viability of L929 cells to assess their cytotoxicity. L929 cells in logarithmic growth phase were divided into 5 × 10⁶ cells per well. 3Cells were seeded at a density of 1000 μL in 96-well plates and incubated at 37°C with 5% CO2 for 24 h to allow for cell adhesion. After aspirating the supernatant, fresh complete culture medium containing different concentrations of the test drug was added, and incubation continued for another 24 h. A negative control group (cells without drug, used to assess normal cell viability) and a blank control group (wells containing pure culture medium without cell or drug seeding, used to correct for baseline absorbance) were also set up. After incubation, 10 μL of CCK-8 solution was added to each well, and the plates were incubated in the dark for 2 h. The absorbance (OD) of each well was then measured at 450 nm using a microplate reader. 450 Cell viability was calculated as follows: Cell viability (%) = [(OD sample - OD blank) / (OD negative control - OD blank)] × 100%. Cell viability curves were plotted against drug concentration to evaluate the cytotoxic effects of the drug. To ensure the reliability and statistical significance of the results, six technical replicates were set up for each drug concentration, and the entire experiment was independently repeated three times.

[0167] 2.1.2 Blood compatibility evaluation

[0168] The in vitro hemocompatibility of the test drug was assessed using a erythrocyte hemolysis test. Healthy adult Kunming (KM) mice were used as donor animals, and fresh whole blood was collected and placed in anticoagulant tubes. To remove interfering factors such as plasma proteins and platelets, erythrocytes were resuspended in sterile PBS, centrifuged at 1500 rpm for 10 minutes, and washed. Washing was repeated three times until the supernatant was clear and colorless. The washed erythrocytes were diluted with PBS to prepare a 4% (v / v) erythrocyte suspension for later use.

[0169] Take a clean V-bottom 96-well plate and add 100 μL of the test drug solution of different concentrations (serially diluted twofold with PBS) to each well. Then add 100 μL of 4% red blood cell suspension to each well and mix gently. A negative control group (100 μL PBS + 100 μL red blood cell suspension, representing 0% hemolysis) and a positive control group (100 μL 0.1% Triton X-100 solution + 100 μL red blood cell suspension, representing 100% hemolysis) are set up, with 6 replicates for each treatment group. Incubate the 96-well plate at 37℃ for 1 h. After incubation, centrifuge the plate at 1500×g for 10 min to allow unruptured red blood cells to precipitate at the bottom of the wells. Carefully aspirate 100 μL of supernatant from each well and transfer it to a new flat-bottom 96-well plate. Measure the absorbance (OD) of each well at 414 nm using a microplate reader. 414Based on the measured absorbance values, the hemolysis rate is calculated using the following formula: Hemolysis rate (%) = [(OD test sample - OD negative control) / (OD positive control - OD negative control)] × 100%. It is generally considered that a hemolysis rate below 5% indicates that the drug meets safety requirements regarding blood compatibility.

[0170] 2.2 Effect of combined drug administration on the healing of burn wounds infected with Pseudomonas aeruginosa PAO1

[0171] 2.2.1 Establishment of an animal model of PAO1-infected burn wounds

[0172] To evaluate the therapeutic efficacy and safety of flagellate inhibitory peptide, allicin, and their combined application on burn wounds infected with Pseudomonas aeruginosa, a mouse burn infection model was established in this study (refer to Gong Y, Wang P, Cao R, et al. Exudate Absorbing and Antimicrobial Hydrogel Integrated with MultifunctionalCurcumin-Loaded Magnesium Polyphenol Network for Facilitating Burn Wound Healing. ACS Nano. 2023 Nov 28;17(22):22355-22370. You Y, Yu X, Jiang J, et al. Bacterial cell wall-specific nanomedicine for the elimination of Staphylococcus aureus and Pseudomonas aeruginosa through electron-mechanicalintervention. Nat Commun. 2025 Mar 22;16(1):2836). First, the Pseudomonas aeruginosa standard strain PAO1 was inoculated into LB liquid medium and cultured with shaking at 37℃ and 200 rpm / min for 16 h. The culture was then transferred the following day to the logarithmic growth phase. After centrifugation and washing with fresh LB medium, the bacterial culture was adjusted to a concentration of 1×10⁻⁶. 8 CFU / mL was prepared for use. The backs of mice were shaved 24 hours prior to the experiment. On the day of modeling, the mice were weighed and their weight recorded. After anesthesia, a preheated (100°C) cauterization probe was placed directly into the skin on the mouse's back for 10 seconds to create a burn wound. Necrotic tissue was then removed following clinical debridement techniques. Immediately, 50 μL of the above-mentioned bacterial solution (i.e., 5 × 10⁻⁶ CFU / mL) was dripped into the wound. 6(CFU / mouse) After the wound has absorbed the bacterial fluid for 15 minutes, medical sterile petroleum jelly gauze (fine gauze) is applied to the wound, followed by a 3M transparent dressing to fix and close the wound. The four corners are sutured to establish a closed infection model. After 24 hours of routine feeding postoperatively, the successful establishment of the model is confirmed by observing the redness and swelling of the wound, exudation, and the overall condition of the mice.

[0173] Local drug administration began on the 3rd postoperative day. Based on the results of in vitro cytotoxicity experiments (no cytotoxicity was observed at concentrations of PEP 0.6 mg / mL, allicin 0.125 mg / mL, and the combination of PEP 0.6 mg / mL + allicin 0.125 mg / mL and polymyxin B 12.5 μg / mL), the in vivo experimental drug concentration was increased 10-fold, with an administration volume of 100 μL per dose. Mice were randomly divided into 8 groups of 6 each, as follows: (1) Control group (burns without infection); (2) Control + PEP group (burns without infection followed by PEP); (3) Control + combination drug group (burns without infection followed by combination drug); (4) Model group (burns with infection, no medication administered); (5) PEP group (burns with infection followed by PEP); (6) Allicin group (burns with infection followed by allicin); (7) Combination drug group (burns with infection followed by combination drug); (8) Poly B group (burns with infection followed by polymyxin B). Note that uninfected and infected mice should be handled in separate batches and placed in different UV-sterilized experimental rooms. Each group was given medication and wound photographs were taken every other day, and mouse weight changes were recorded. Mice were anesthetized and sacrificed on day 14 after treatment, and skin tissue from the wound was collected for the following tests: plate count method to assess bacterial load in the wound tissue; HE staining to observe inflammatory infiltration and wound repair; Masson staining to assess collagen fiber proliferation and deposition; and immunohistochemical staining to detect the expression levels of TNF-α (inflammatory factor) and CD31 (vascular endothelial marker) in the tissue, so as to comprehensively evaluate the antibacterial efficacy, healing-promoting effect and in vivo safety of each group of drugs.

[0174] 2.2.2 Plate counting experiment

[0175] To accurately assess the bacterial load within wounds, this study employed tissue homogenization combined with plate count to quantitatively analyze surviving bacteria in skin samples. Under aseptic conditions, skin was excised approximately 5 mm from the wound edge, and back skin tissue was collected, weighed, and placed in a 1.5 mL sterile centrifuge tube pre-loaded with three sterile magnetic beads. 1 mL of sterile PBS was added. A cryogenic homogenizer was used, with parameters set to a vibration frequency of 60 Hz and homogenization time of 60 s. This process was repeated six times at appropriate intervals to ensure thorough homogenization. After homogenization, the resulting tissue suspension was considered the stock solution and serially diluted 10-fold with PBS under aseptic conditions to obtain different concentration gradients. These diluted solutions were then plated and incubated at 37°C for 24 hours. Plate counts were then performed to calculate the number of colonies grown. The bacterial count per gram of tissue was calculated by combining the dilution factor with the original weight of the tissue sample.

[0176] 2.2.3 Histological staining analysis

[0177] Back skin tissue was collected from each group and fixed overnight at 4°C with 4% paraformaldehyde. After fixation, the tissue samples were dehydrated using a gradient of ethanol. Subsequently, the dehydrated and cleared tissue blocks were routinely embedded in paraffin and cut into continuous sections with a thickness of 4 μm using a paraffin microtome. Each group of sections was stained with hematoxylin and eosin (H&E) and Masson staining. H&E staining, as a basic histological staining method, can clearly show the overall structure of the wound tissue and the degree of inflammatory cell infiltration, thus being used to assess the intensity of the inflammatory response and the progress of wound healing. Masson staining allows for direct observation of the level of collagen fiber deposition within the wound tissue.

[0178] 2.2.4 Immunohistochemical experiments

[0179] Immunohistochemical staining was used to detect the expression of TNF-α and CD31 in back skin tissue sections. First, the tissue sections were blocked with goat serum working solution to block non-specific binding sites and reduce background staining interference. After blocking, diluted anti-TNF-α and anti-CD31 antibodies were added as primary antibodies, and the sections were incubated overnight in a humidified chamber at 4°C to allow the antibodies to fully bind to the corresponding antigens in the tissue sections. The next day, after the sections were removed and brought to room temperature, horseradish peroxidase (HRP)-labeled secondary antibody was added and incubated at room temperature for 60 minutes. The specific binding of the secondary and primary antibodies amplified the signal. Subsequently, freshly prepared DAB chromogenic solution was added for the colorimetric reaction. DAB, catalyzed by HRP, forms an insoluble brown precipitate, thus marking the location of the target antigen. The colorimetric time was controlled to approximately 10 minutes, during which color changes were observed under a microscope to terminate the reaction as needed. After colorimetric development, the sections underwent routine dehydration, clearing, and mounting. Finally, all stained sections were digitally scanned using a panoramic slide scanning system.

[0180] 3 Experimental Results

[0181] 3.1 Cytotoxicity assay

[0182] Cytotoxicity assays showed that, within the tested concentration range, neither the peptide nor polymyxin B alone caused cytotoxicity. However, allicin exhibited significant cytotoxic effects at concentrations up to 0.5 mg / mL, and in combination with other drugs (Pep 0.6 mg / mL combined with allicin 0.5 mg / mL). Notably, no cytotoxicity was observed in allicin or its combination with the peptide within their effective antibacterial concentration range, indicating good biocompatibility at doses where antibacterial activity is achieved.

[0183] 3.2 Hemolysis test

[0184] The hemolysis test results showed that the positive control group (0.1% Triton X-100) exhibited complete hemolysis, confirming the effectiveness of the experimental system. The hemolysis rates of the peptide and the combined drug were significantly lower than the internationally recognized hemolysis standard (5%) within the detection concentration range. This indicates that they do not cause erythrocyte membrane rupture or hemoglobin release, demonstrating good blood compatibility and biosafety.

[0185] 3.3 Evaluation of wound healing effect and in vivo safety

[0186] Experimental results showed that in the PAO1-infected burn wound model, the Model group (burn wound with added bacteria) exhibited the slowest wound healing, displaying typical characteristics of delayed infection healing. The healing rate was only 9.257% on day 5 post-injury and only 72.924% by day 14. In contrast, all treatment groups showed varying degrees of healing-promoting effects. On day 5 post-injury, the healing rates of the Pep group, allicin group, and Poly B group were 35.239%, 38.198%, and 32.584%, respectively, while the combined treatment group achieved a healing rate of 66.897%, significantly better than other single treatment groups. As the healing process progressed, by day 9, the healing rate of the combined treatment group reached 89.849%, far exceeding the rates of the Pep group (58.222%), allicin group (59.468%), and Poly B group (77.868%) at the same time point. By the 14th day, the wound in the combined treatment group had almost completely closed, with a healing rate of 99.189%, while the rates in the Pep group, allicin group, and Poly B group were 88.192%, 91.622%, and 94.16%, respectively. This dynamic healing curve shows that the combined treatment group not only promoted healing faster than other single treatment groups, but also achieved the best final healing quality.

[0187] In the uninfected, uncomplicated burn wound model, the healing process in all groups was generally faster than in the infected model, providing a reference system for evaluating the in vivo safety and healing-promoting ability of drugs. The healing rates in the Control group (burns without infection) were 44.814%, 85.161%, and 97.915% on days 5, 9, and 14, respectively. Further intervention with peptides (Control+pep) or combination drugs (Control+combination) on top of the Control group further accelerated wound healing. Specifically, the healing rate in the Control+pep group reached 63.207% on day 5 post-injury, and the Control+combination group reached 76.673%, significantly higher than the Control group alone at the same time point. By day 9, the healing rates in the Control+pep group and the Control+combination group increased to 89.534% and 90.635%, respectively, and reached 98.62% and 99.404% on day 14, essentially achieving complete healing. Importantly, throughout the observation period, no irritating reactions such as exudation or necrosis were observed in the wounds of mice in each treatment group, and new hair growth was observed in the later stage of healing. This fully demonstrates that the peptides and combination drugs have good biocompatibility in vivo, no local irritation, and can accelerate wound healing.

[0188] In summary, this study, through quantitative analysis of wound healing rates at consecutive time points, confirms that in the case of wound infection, the combined drug group can significantly overcome the healing delay caused by infection, and its healing speed and final effect are superior to those of other drug treatment groups; in the absence of infection, the peptides and combined drugs also exhibited positive healing-promoting activity and excellent safety.

[0189] 3.4 Evaluation of antibacterial effect on wound

[0190] Fourteen days later, skin tissue from mouse wounds was collected, ground, and plate-counted. The residual bacterial count in the Model group wound tissue reached 2.8 × 10⁹ CFU / g, indicating successful establishment of the infection model. This demonstrated that the body's own defense mechanisms alone were insufficient to effectively eliminate pathogens, leading to persistent bacterial colonization and proliferation. In stark contrast, the combined drug group and the Poly B group exhibited superior in vivo antibacterial activity, with significantly reduced residual bacterial counts in wound tissue to 4.2 × 10³ CFU / g and 3 × 10³ CFU / g, respectively. In conclusion, the combined drug significantly inhibited bacterial proliferation in wounds and effectively controlled local infection, which is one of the important pharmacological mechanisms by which it promotes the healing of infected wounds.

[0191] 3.5 Summary of Staining Analysis

[0192] Based on comprehensive histological and immunohistochemical assessments, the combined drug demonstrated significant therapeutic effects in promoting wound healing in mice. In a bacterial wound infection model, H&E staining revealed intact wound tissue structure, good re-epithelialization, visible new hair follicle structures, and minimal inflammatory cell infiltration in the combined drug group, indicating its effective promotion of tissue repair. Masson staining further showed increased and more tightly packed collagen fiber deposition in this group, suggesting its promotion of skin tissue regeneration and repair in the wound area. Immunohistochemical CD31 staining results showed a reduced number of new blood vessels in the wound in the combined drug group, a phenomenon that may be related to the subsidence of the inflammatory response and wound maturation after infection control. Simultaneously, TNF-α staining results showed decreased expression of inflammatory factors in this group, confirming its anti-inflammatory effect. In addition, in a wound model without bacterial infection, the peptide and the combined drug also showed positive effects in promoting wound repair and reducing inflammation. In summary, the combined drug can effectively promote wound healing through multiple mechanisms, including promoting tissue regeneration, regulating collagen deposition, and reducing inflammatory responses.

Claims

1. A flagellation inhibitory peptide of Pseudomonas aeruginosa, characterized in that, The flagellar inhibitory peptide sequence is Lys-Ile-Gly-Leu-Phe-Arg-Trp-Arg.

2. The method for synthesizing the flagellar inhibitory peptide of Pseudomonas aeruginosa according to claim 1, characterized in that, Includes the following steps: (1) Weigh Fmoc-Arg(pbf)-WangResin resin and add it to the solid-phase synthesis reactor. After adding N,N-dimethylformamide DMF to swell the resin, discharge the DMF. (2) Deprotection: Add a deprotection solution consisting of hexahydropyridine and DMF to the reaction column, stir with nitrogen gas, and then dry it. (3) Weighing: Measure the protected amino acid Fmo-Trp(Boc)-OH, and then weigh O-(benzotriazol-1-yl)-N,N,N,N'-tetramethylurea hexafluorophosphate HBTU; (4) Deprotection washing: Add an appropriate amount of DMF to the reaction column, puff with nitrogen, dry the column, and repeat the operation; (5) Feeding: Add the prepared protected amino acid Fmo-Trp(Boc)-OH and O-(benzotriazol-1-yl)-N,N,N,N'-tetramethylurea hexafluorophosphate HBTU to the reaction column, then add N-methylmorpholine NMM, and stir with nitrogen. (6) Washing after reaction: Drain the solution in the reaction column, add an appropriate amount of DMF to wash, agitate with nitrogen, drain again, and repeat the operation. (7) Detection: Take the resin in a small test tube, add two drops each of phenol-anhydrous ethanol mixture, redistilled pyridine and ninhydrin-anhydrous ethanol solution, and heat in a dry heater; if the solution turns blue and the resin is discolored and opaque after removal, the reaction is not complete, and the reaction is repeated once; if the solution is slightly yellow and the resin is colorless and transparent, the reaction is complete; couple Fmoc-Arg(Pbf)-OH, Fmo-Phe-OH, Fmoc-Leu-OH, Fmoc-Gly-OH in sequence. Fmoc-Ile-OH, Boc-Lys(BOC)-OH amino acids, the specific steps are the same as the above five steps (2)-(6) until the last amino acid is connected; after the last amino acid is connected and the protective washing has been completed, the column is dried, an appropriate amount of methanol is added to the reaction column, nitrogen is purged, the column is dried, an appropriate amount of DCM is added, nitrogen is purged, the column is dried; finally, an appropriate amount of methanol is added to the reaction vessel, nitrogen is purged, the column is dried, and the resin is placed in a container and placed in a vacuum dryer for vacuum drying; (8) Peptide cutting and washing: Seal the centrifuge tube and put it into the centrifuge. Centrifuge, remove the tube, discard the supernatant, add ether, stir evenly with a glass rod, and centrifuge again; repeat the washing process. Drying: The washed polypeptides were placed in a vacuum dryer and dried under vacuum to obtain crude polypeptides; (9) Purification of polypeptides The crude product was dissolved in an acetonitrile-water mixture by ultrasonication, filtered through a microporous membrane, and the filtrate was purified by HPLC to obtain the pure polypeptide.

3. The method for synthesizing the flagellar inhibitory peptide of Pseudomonas aeruginosa according to claim 2, characterized in that, Includes the following steps: (1) Weigh Fmoc-Arg(pbf)-WangResin resin and add it to the solid-phase synthesis reactor. After adding N,N-dimethylformamide DMF to swell the resin, discharge the DMF. (2) Deprotection: Add an appropriate amount of deprotection solution consisting of 20% hexahydropyridine + 80% DMF to the reaction column, stir and agitate under nitrogen for 30-60 minutes, and then dry. (3) Weighing: Measure 1 to 3 times the molar amount of the protective amino acid Fmo-Trp(Boc)-OH, and then weigh 1 to 3 times the molar amount of O-(benzotriazol-1-yl)-N,N,N,N'-tetramethylurea hexafluorophosphate HBTU. (4) Deprotection washing: Add an appropriate amount of DMF to the reaction column, agitate with nitrogen for 2-5 minutes, dry the column, and repeat the operation; (5) Feeding: Add the prepared protected amino acid Fmo-Trp(Boc)-OH and O-(benzotriazol-1-yl)-N,N,N,N'-tetramethylurea hexafluorophosphate HBTU to the reaction column, and then add 2 to 6 times the molar amount of N-methylmorpholine NMM resin. Stir and agitate under nitrogen for 30 to 60 minutes. (6) Washing after reaction: Drain the solution in the reaction column, add an appropriate amount of DMF to wash, agitate with nitrogen for 2-5 minutes, drain, and repeat the operation; (7) Test: Take the resin in a small test tube, add two drops each of 80% phenol + 20% anhydrous ethanol, redistilled pyridine and 5% ninhydrin anhydrous ethanol solution, and heat in a dry heater at 110 degrees Celsius for 3-5 minutes; if the solution turns blue and the resin is discolored and opaque after removal, the reaction is not complete and should be repeated; if the solution is slightly yellow and the resin is colorless and transparent, the reaction is complete. Couple Fmoc-Arg(Pbf)-OH, Fmo-Phe-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, and Boc-Lys(BOC)-OH amino acids in sequence, following the five steps above (2)-(6), until the last amino acid is coupled; after the last amino acid is coupled and the protective washing has been completed, dry the column, add methanol to the reaction column, puff with nitrogen for 2-6 minutes, dry the column, add an appropriate amount of DCM, puff with nitrogen for 2-6 minutes, dry the column, repeat the operation 2-3 times; finally, add methanol to the reaction vessel, puff with nitrogen for 2-5 minutes, dry the column, repeat the operation 2-3 times, and put the resin into a container and place it in a vacuum dryer to dry for 12-24 hours; (8) Cutting and washing of peptides: Seal the centrifuge tube and put it into the centrifuge. Centrifuge at 4000 rpm for 3-5 minutes. Take it out, pour off the supernatant, add ether, stir evenly with a glass rod, and centrifuge again. Repeat this operation and wash 2-5 times. Drying: Place the washed polypeptide in a vacuum dryer and dry under vacuum for 12-24 hours to obtain crude polypeptide; (9) Purification of polypeptides The crude product was dissolved in an acetonitrile-water mixture by ultrasonication, then filtered through a 0.45µm microporous membrane. The filtrate was purified by HPLC under the following conditions: C 18 10A Preparative Column: 10cm*25cm, wavelength 220nm; Pump A: acetonitrile solution containing 0.1% trifluoroacetic acid; Pump B: aqueous solution containing 0.1% trifluoroacetic acid; Gradient elution: 0-100 min: A: 21%-31%, 100.01-120 min: A: 31%-70%; Receive the effluent after 24-36 min, and freeze-dry the received solution to obtain pure peptides.

4. The application of the flagellation inhibitory peptide according to claim 1 in inhibiting the flagellation ability of multiple strains of Pseudomonas aeruginosa.

5. The flagellation inhibitory peptide of claim 1 systematically weakens the pathogenic potential of Pseudomonas aeruginosa by interfering with energy metabolism, disrupting flagellar structure and function, and inhibiting the expression of virulence-related genes.

6. The flagellar inhibitory peptide of claim 1 weakens the pathogenic potential of Pseudomonas aeruginosa by inhibiting flagella assembly gene fliD, type IV fimbrial-related genes pilQ and fimV, global regulatory genes pprB and phoP / phoQ, virulence factor synthesis genes lasA and exoS, biofilm formation-related psl gene cluster, resistance adaptation gene oprH, and energy metabolism gene atpC.

7. The use of the flagellar inhibitory peptide and allicin composition according to claim 1 in the preparation of a drug for inhibiting and killing Pseudomonas aeruginosa.

8. The use of the flagellar inhibitory peptide of claim 1 in combination with ciprofloxacin, meropenem, levofloxacin or polymyxin B in the preparation of a drug for inhibiting and killing Pseudomonas aeruginosa.

9. The use of the flagellar inhibitory peptide and allicin composition according to claim 1 in the preparation of a medicament for treating burn wound infection.

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