Bacteriostatic agent for yersinia enterocolitica and application

Abstract

The invention provides a bacteriostatic agent aiming at yersinia enterocolitica and application, the bacteriostatic agent comprises a compound solution of lipoic acid and protocatechualdehyde, and when the lipoic acid and the protocatechualdehyde act synergistically, the minimum inhibitory concentrations to the yersinia enterocolitica are 0.625 mg / mL and 0.3125 mg / mL respectively. The yersinia enterocolitica biofilm is inhibited and / or removed through the lipoic acid and protocatechuic aldehyde compound liquid, and the problem that drugs for treating the yersinia enterocolitica infection are prone to drug resistance is solved.

Description

Technical Field

[0001] This invention belongs to the fields of food and pharmaceutical technology, and relates to an antibacterial agent against Yersinia enterocolitica and its application. Background Technology

[0002] Under natural conditions, bacteria can attach to surfaces and form highly structured and community-like biofilms. Compared to planktonic growth, the bacterial cells within biofilms can adjust physiological metabolism, signal responses, stress resistance traits, and developmental dynamics, transforming them into "higher" organisms distinct from single-celled organisms. As the preferred protective survival mode for prokaryotes, biofilms are not only crucial for bacterial survival in the environment but also play a significant role in many infectious diseases. Existing research indicates that approximately 80% of human bacterial infections are related to biofilm formation; more seriously, once bacteria form biofilms, their antibiotic resistance can increase by 10-1000 times, posing a significant challenge to the treatment of related diseases. This is because the extracellular polysaccharide matrix within bacterial biofilms also prevents antibiotics from reaching the bacteria encapsulated within the biofilm. Therefore, antibiotic treatment can suppress infection symptoms by killing planktonic bacteria released from the adherent colony, but it cannot eliminate bacteria still buried in the biofilm. When antibiotic chemotherapy is stopped, the biofilm will become the "source of infection" for bacterial recurrence, thus exhibiting the characteristics of alternating periods of relative quiescence and acute exacerbation, as well as easy recurrence.

[0003] Yersinia enterocolitica (Y. enterocolitica) is a psychrophilic foodborne pathogen widely distributed in food. It can directly cause various digestive system diseases such as chronic gastroenteritis and enterocolitis, posing a threat to human health. It can also cause respiratory diseases, cardiovascular diseases, and bone marrow connective tissue lesions, and even sepsis, ultimately leading to death. Furthermore, Yersinia enterocolitica responds to environmental and host stresses through multicellular collaborative group behavior. Biofilms or biofilm-like communities are considered one of the reasons why Yersinia enterocolitica infections are difficult to treat. Studies have shown that Yersinia enterocolitica biofilms not only adhere to food containers but also participate in macrophage escape; and compared to free-floating cells, cells within biofilms exhibit stronger drug resistance. After ingestion of Yersinia enterocolitica, the bacteria colonize the intestines, secreting extracellular polysaccharides that adhere to the host surface. This biofilm not only protects the bacteria from the host's immune system but also provides nutrients and survival aids, promoting bacterial growth and reproduction, leading to infectious diseases such as acute gastroenteritis, acute appendicitis, and sepsis. Furthermore, the presence of the biofilm causes chronic inflammatory responses in the host tissues, and the bacterial community within the biofilm gradually releases a series of inflammatory mediators, leading not only to tissue damage and dysfunction but also being associated with the occurrence and progression of various chronic diseases. Therefore, clearing the biofilm from both inside and outside the body is far more difficult than combating planktonic bacterial infections caused by Yersinia enterocolitica.

[0004] Synergistic effects between natural compounds from different plant sources have shown significant antibacterial potential. For example, the combination of epigallocatechin gallate (EGCG) from green tea extract and curcumin significantly enhances the antibacterial effect against *Pseudomonas aeruginosa* through multi-target intervention (such as inhibiting the bacterial quorum sensing system and disrupting cell membrane integrity). The combination of plant essential oils (such as thyme and tea tree oil) with phenolic compounds (such as eugenol) also shows significant synergistic antibacterial effects, the mechanism of which may be related to the essential oil components enhancing the cell membrane permeability of phenolic compounds. The mechanisms of synergistic antibacterial action typically involve multi-target intervention, including disrupting bacterial cell membranes, inhibiting biofilm formation, interfering with metabolic pathways, and regulating host immune responses. These multi-target intervention mechanisms not only enhance antibacterial effects but also reduce the risk of bacterial resistance. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides an antibacterial agent against Yersinia enterocolitica and its application. By using a compound solution of thioctic acid and protocatechuic aldehyde to inhibit and / or remove the biofilm of Yersinia enterocolitica, this invention solves the problem of drug resistance easily developed in drugs used to treat Yersinia enterocolitica infections.

[0006] To achieve the above objectives, the present invention provides the following technical solution: an antibacterial agent against Yersinia enterocolitica, comprising a compound solution of lipoic acid and protocatechuic aldehyde, wherein the minimum inhibitory concentrations against Yersinia enterocolitica when lipoic acid and protocatechuic aldehyde act synergistically are 0.625 mg / mL and 0.3125 mg / mL, respectively.

[0007] Furthermore, the lipoic acid and protocatechuic aldehyde compound solution can inhibit and / or remove the biofilm of Yersinia enterocolitica.

[0008] Furthermore, the compound solution of lipoic acid and protocatechuic aldehyde inhibits and / or removes the biofilm of Yersinia enterocolitica by blocking the synthesis of extracellular polysaccharides in Yersinia enterocolitica.

[0009] Furthermore, the compound solution of lipoic acid and protocatechuic aldehyde inhibits and / or removes the biofilm of Yersinia enterocolitica by suppressing the quorum sensing system of Yersinia enterocolitica.

[0010] Furthermore, the compound solution of lipoic acid and protocatechuic aldehyde inhibits and / or clears the biofilm of Yersinia enterocolitica by inhibiting the metabolic activity of the biofilm of Yersinia enterocolitica.

[0011] Furthermore, the compound solution of lipoic acid and protocatechuic aldehyde inhibits and / or removes the biofilm of Yersinia enterocolitica by disrupting the cell membrane integrity of Yersinia enterocolitica.

[0012] Furthermore, the Yersinia enterocolitica strain is selected from one or more foodborne pathogenic strains, which are Yersinia enterocolitica strains that can cause digestive system diseases.

[0013] The present invention also provides the application of the above-mentioned antibacterial agent against Yersinia enterocolitica in the preparation of medicines for treating digestive system diseases.

[0014] Furthermore, the digestive system diseases include chronic gastroenteritis and enterocolitis.

[0015] The present invention also provides the application of the above-mentioned antibacterial agent against Yersinia enterocolitica in the preparation of a medicine for treating systemic lesions caused by digestive system diseases.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects:

[0017] Starting from the natural medicinal plant resource library, the present invention searches for natural antibacterial substances. Based on the research on the effect of lipoic acid - protocatechuic aldehyde on Yersinia enterocolitica, it is found that lipoic acid - protocatechuic aldehyde has a definite effect on inhibiting and removing the biofilm of Yersinia enterocolitica. At the same time, protocatechuic aldehyde, as a natural compound derived from plants, is not likely to produce drug resistance. The experimental results of the present invention show that the combined action of lipoic acid and protocatechuic aldehyde has the potential application value of being developed into a drug against Yersinia enterocolitica, thus providing certain help for solving the problems of Yersinia enterocolitica infectious diseases (such as great treatment difficulty and being difficult to eradicate).

[0018] Furthermore, the present invention determines the minimum inhibitory concentrations (MIC) of LA and PCA after the combined use of lipoic acid - protocatechuic aldehyde against Yersinia enterocolitica by the broth microdilution method and checkerboard microdilution method, which are respectively reduced to 0.63 mg / mL and 0.31 mg / mL, significantly lower than those of the single components (LA MIC = 2.50 mg / mL, PCA MIC = 1.25 mg / mL). The synergy index (FICI = 0.5) confirms its strong synergistic effect. At the same time, the crystal violet staining method and scanning electron microscope are used to detect the inhibitory effect of lipoic acid - protocatechuic aldehyde on the biofilm of Yersinia enterocolitica, revealing the way of inhibiting the growth of Yersinia enterocolitica by lipoic acid - protocatechuic aldehyde (that is, lipoic acid - protocatechuic aldehyde can inhibit the growth of Yersinia enterocolitica by inhibiting the formation of the biofilm of Yersinia enterocolitica and removing the biofilm), and the inhibitory effect on the biofilm is enhanced with the increase of the concentration of lipoic acid - protocatechuic aldehyde, thus providing new ideas for the research and development of natural bacteriostatic agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Graph for determining the minimum inhibitory concentration (MIC) of different concentration compound solutions used in the broth microdilution method against Yersinia enterocolitica; where, **P<0.01, *P<0.05 VS. negative control, minimum inhibitory concentrations of LA (A) and PCA (B) against Y. enterocolitica.

[0020] Figure 2 Results of the checkerboard method synergistic antibacterial experiment of lipoic acid and protocatechuic aldehyde against Yersinia enterocolitica; where FIC≤0.5 is synergy, 0.5<FIC≤1 is additive, 1<FIC≤4 is irrelevant, and FIC>4 is antagonistic.

[0021] Figure 3The growth curves of Yersinia enterocolitica were determined by different concentration gradients of LA, PCA and their compound solutions (1 / 2MIC LA, 1 / 2MIC PCA, 1 / 2MIC LA+1 / 2MIC PCA, 1 / 4MIC LA+1 / 4MIC PCA).

[0022] Figure 4 The effects of LA+PCA on the cell membrane of Y. enterocolitica were observed using laser confocal microscopy (A) and scanning electron microscopy (B) (SYTO 9 was a green fluorescent nucleic acid dye, and propidium iodide (PI) was a red fluorescent nucleic acid dye).

[0023] Figure 5 The effects of different concentration gradients of LA, PCA, and their compound solutions (concentration gradients of 0, 1 / 4MIC LA, 1 / 4MIC PCA, 1 / 4MIC LA+1 / 4MIC PCA, and 1 / 8MIC LA+1 / 8MIC PCA) on the biofilm metabolic activity of Yersinia enterocolitica.

[0024] Figure 6 The inhibitory effect of different concentration gradients of LA, PCA, and their compound solutions (concentration gradients of 0, 1 / 4MIC LA, 1 / 4MIC PCA, 1 / 4MIC LA+1 / 4MIC PCA, and 1 / 8MIC LA+1 / 8MIC PCA) on the synthesis of extracellular polysaccharides (EPS) in Yersinia enterocolitica was determined by ruthenium red binding method.

[0025] Figure 7 The graph shows the inhibitory effect of different concentration gradients of LA and PCA and their compound solutions (concentration gradients of 0, 1 / 4MIC LA, 1 / 4MIC PCA, 1 / 4MIC LA+1 / 4MIC PCA, 1 / 8MIC LA+1 / 8MIC PCA) on the quorum sensing (QS) system of Yersinia enterocolitica.

[0026] Figure 8 To observe the inhibitory effect of LA-PCA compound solution on the biofilm of Y. enterocolitica using optical microscopy (A), laser confocal microscopy (B), and scanning electron microscopy (C). Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0028] Lipoic acid (LA) has the molecular formula C8H10. 14O2S2 is an organic compound that acts as a coenzyme in acyl transfer during metabolism, eliminating free radicals that accelerate aging and cause disease. Lipoic acid, absorbed through the intestines, enters cells and, possessing both lipid and water solubility, serves as an endogenous antioxidant. In recent years, its antibacterial effects and mechanisms have received increasing attention. Studies have shown that lipoic acid not only inhibits Gram-positive bacteria but also exerts a synergistic antibacterial effect by interfering with bacterial metabolic pathways and enhancing antibiotic sensitivity.

[0029] Protocatechualdehyde (PCA) is a natural phenolic compound commonly found in the root of *Salvia miltiorrhiza*, a plant in the Lamiaceae family, and is also widely found in plants of the Aquifoliaceae family and some other Chinese medicinal herbs. It possesses broad-spectrum antibacterial and anti-inflammatory effects. PCA effectively reduces the expression levels of inflammatory factors such as IL-1α and CXCL9 by inhibiting the phosphorylation of STAT3, thereby preventing and / or treating psoriasis (Deng Yu, Li Fulun, Yang Xiaoxuan, Zhao Qi, Yang Jie, Zhou Qian, Li Rui. Use of protocatechualdehyde in the preparation of topical drugs for the prevention and / or treatment of psoriasis [P]. Sichuan Province: CN119345163A, 2025-01-24.).

[0030] Therefore, this invention has conducted certain experimental evaluations on the antibacterial and biofilm removal effects of lipoic acid and protocatechuic aldehyde. In the first aspect, this invention found that lipoic acid-protocatechuic aldehyde can inhibit the formation of Yersinia enterocolitica biofilm and / or remove Yersinia enterocolitica biofilm.

[0031] The minimum inhibitory concentration (MIC) of lipoic acid against *Yersinia enterocolitica* is 2.5 mg / mL; the MIC of protocatechuic aldehyde against *Yersinia enterocolitica* is 1.25 mg / mL. When lipoic acid and protocatechuic aldehyde are used in combination, the MIC of lipoic acid against *Yersinia enterocolitica* is 0.625 mg / mL, and the MIC of protocatechuic aldehyde against *Yersinia enterocolitica* is 0.3125 mg / mL.

[0032] Among them, protocatechuic aldehyde is extracted from plants, including but not limited to: Aquifoliaceae plants, Salvia miltiorrhiza, Lilac leaves, Guava, etc.

[0033] Secondly, the present invention provides the application of lipoic acid-protocatechuic aldehyde in the preparation of antibacterial agents, wherein the lipoic acid-protocatechuic aldehyde inhibits the formation of Yersinia enterocolitica biofilm and / or removes Yersinia enterocolitica biofilm.

[0034] Corresponding to the second aspect, the present invention also provides the use of lipoic acid-protocatechuic aldehyde in the preparation of preservatives.

[0035] Preferably, the protocatechuic aldehyde is obtained by extracting from the above-mentioned plant.

[0036] Thirdly, the present invention provides the use of lipoic acid-protocatechuic aldehyde in the preparation of a medicament against Yersinia enterocolitica infection, wherein the lipoic acid-protocatechuic aldehyde inhibits the formation of Yersinia enterocolitica biofilm and / or removes Yersinia enterocolitica biofilm.

[0037] To better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments are merely explanations of the present invention and are not intended to limit the scope of protection of the present invention.

[0038] (I) Analysis of the reasons why Yersinia enterocolitica easily develops resistance to antimicrobial substances

[0039] The study, "Drug Resistance Study of Yersinia enterocolitica from Fish," points out that Yersinia enterocolitica, as a Gram-negative bacterium, uses its outer membrane as an antibiotic barrier, restricting drug entry. Yersinia enterocolitica can also produce enzymes that destroy antibiotics, rendering them ineffective, and uses efficient excretion pumps to remove antibiotics from cells. Simultaneously, through gene mutation and horizontal gene transfer, Yersinia enterocolitica acquires new drug-resistant genes. Furthermore, Yersinia enterocolitica can form biofilms on various surfaces (such as food processing equipment, medical devices, and biological tissues), providing conditions for its long-term survival and spread in the environment, and resisting attacks from antibiotics and the host's immune system.

[0040] (II) Experimental verification of the inhibitory effect of lipoic acid-protocatechuic aldehyde on Yersinia enterocolitica and Yersinia enterocolitica biofilm.

[0041] 1. Materials, reagents and instruments

[0042] 1.1 Experimental strains

[0043] The strain of Yersinia enterocolitis (Y. enterocolitis CICC 21669) was obtained from the China Industrial Microbial Culture Collection Center (CICC, Beijing, China) and stored in a glycerol bottle at -80°C.

[0044] 1.2 Reagent Source

[0045] Physiological saline, methanol, yeast extract, glucose, peptone, glutaraldehyde, anhydrous ethanol, and isoamyl acetate were all purchased from Shanghai Sigma-Aldrich Trading Co., Ltd.; lipoic acid (HPLC≥98%, CAS:1077-28-7) was provided by Yuanye Biotechnology Co., Ltd. (Shanghai, China), and protocatechuic aldehyde (PCA) (HPLC≥98%, CAS:139-85-5) was provided by Bioengineering Co., Ltd. (Shanghai).

[0046] 1.3 Preparation of main reagents

[0047] LB liquid medium: Weigh 20g of LB broth and dissolve it completely in 1L of distilled water. Sterilize at 121℃ for 15min and store at 4℃ for later use.

[0048] LB solid medium: Weigh 32g of LB nutrient agar powder and dissolve it completely in 1L of distilled water. Sterilize at 121℃ for 15min and store in a 4℃ refrigerator for later use.

[0049] PBS solution: Weigh 8g NaCl, 0.2g KCl, 1.42g Na₂HPO₄, and 0.24g KH₂PO₄. Add approximately 800mL of distilled water to dissolve the components completely. Once the pH reaches 7.4, transfer the solution to a 1L volumetric flask, mix well, and bring to a final volume. Finally, sterilize at 121°C for 15 minutes and store at 4°C for later use.

[0050] Potassium phosphate buffer solution (containing 10 mM MgCl2, pH = 7.0): Weigh 6.8 g K2HPO4 and 0.95 g MgCl2, then add approximately 800 mL of distilled water to fully dissolve them. Adjust the pH and bring the volume to 1 L. Finally, sterilize at 121°C for 15 min and store at 4°C for later use.

[0051] Ampicillin sodium aqueous solution: Weigh 0.4g of ampicillin sodium powder and dissolve it completely in 10mL of sterile distilled water. Filter the solution through a 0.22μm sterile filter and store it in a -20℃ refrigerator for later use.

[0052] 2.5% glutaraldehyde solution: Pipette 1 mL of 25% glutaraldehyde solution into 9 mL of PBS buffer to prepare a 2.5% glutaraldehyde solution and store at room temperature.

[0053] 1.4 Experimental Apparatus

[0054] Autoclave (Tianjin Tester Instrument Co., Ltd.); DH6000A electric thermostatic incubator (Tianjin Tester Instrument Co., Ltd.); Clean bench (Suzhou Sujie Chemical Equipment Co., Ltd.); 96-well plate, 24-well plate (Thermo Fisher Scientific, Denmark); Electronic balance (Shanghai Jinke Tianmei Co., Ltd.); Microplate reader (Shenzhen Mindray Bio-Medical Electronics Co., Ltd.); Field emission scanning electron microscope (FEI Ltd., USA).

[0055] 2. Experimental Methods

[0056] 2.1 Preparation of the medicinal solution

[0057] LA / PCA solution: Weigh LA / PCA powder, prepare LA / PCA stock solution using ethanol as solvent, dissolve it in Mueller-Hinton II broth (MHI), Luria-Bertani medium (LB), and PBS, and filter through a 0.22 μm filter. The final reaction system contains 5% ethanol. All LA / PCA solutions were prepared fresh before use.

[0058] 2.2 Preparation of bacterial suspension

[0059] The bacterial strain was stored in glycerol tubes at -80℃. Upon resuscitation, it was inoculated onto LB solid medium using the streak plate method and incubated upside down at 37℃ (humidity ≥80%) for 18 hours. Single colonies were picked and inoculated onto LB liquid medium, and cultured at 37℃ with shaking at 180 rpm for 12 hours to obtain the initial bacterial suspension. To obtain cells in the logarithmic growth phase, the strain was transferred to 50 mL of fresh LB broth at a 1:50 (v / v) inoculation ratio and cultured until OD (occurrence limit). 600 =0.5 (corresponding to 1×10⁸ CFU / mL).

[0060] 2.3 Determination of the minimum inhibitory concentration (MIC) of lipoic acid and protocatechuic aldehyde against Yersinia enterocolitica

[0061] The minimum inhibitory concentration (MIC) was determined using a modified broth microdilution method. Yersinia enterocolitica was inoculated onto TSB medium and incubated at 37°C with shaking at 180 rpm until OD500 was reached. 600 =0.5 (approximately 10) 8 (CFU / mL). The concentration gradient of the compound solution was set to 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078, and 0.039 mg / mL (solvent: TSB containing 5% ethanol). The negative control was TSB containing 5% ethanol, and the positive control was ampicillin (final concentration 2 mg / mL). 100 μL of bacterial suspension and 100 μL of drug solution were added to each well of a 96-well plate. After incubation at 37°C for 8 h, the absorbance at 600 nm was measured using a microplate reader.

[0062] 2.4 Determination of the synergistic antibacterial activity of LA, PCA, and their compound solutions against Yersinia enterocolitica

[0063] The checkerboard microdilution method was used to evaluate the combined antibacterial effect of lipoic acid and protocatechuic aldehyde against Yersinia enterocolitica. In a 96-well plate, gradient concentration solutions of lipoic acid (2×MIC to 1 / 64×MIC, based on the single MIC value of lipoic acid, 2.5 mg / mL) were added successively in the X-axis direction (columns 1-8), and gradient concentration solutions of protocatechuic aldehyde (2×MIC to 1 / 64×MIC, based on the single MIC value of protocatechuic aldehyde, 1.25 mg / mL) were added in the Y-axis direction (rows A-H). 100 μL of the drug mixture and 100 μL of the bacterial suspension (1×10 8 CFU / mL) were added to each well. After culturing at 37°C for 8 h, the absorbance at 600 nm was measured, and the fractional inhibitory concentration index (FIC) was calculated.

[0064]

[0065] Judgment criteria: FIC ≤ 0.5 is synergy, 0.5 < FIC ≤ 1 is additive, 1 < FIC ≤ 4 is irrelevant, and FIC > 4 is antagonistic.

[0066] 2.5 Determination of the growth curve of LA, PCA and their compound solutions against Yersinia enterocolitica

[0067] Referring to the method for determining the dynamic growth curve, the lipoic acid-protocatechuic aldehyde compound solution was prepared according to the method in 2.1. The growth inhibitory effect of the LA-PCA compound solution against Yersinia enterocolitica was evaluated. The drug solutions with concentrations of 1 / 2MIC LA, 1 / 2MIC PCA, 1 / 2MIC LA + 1 / 2MIC PCA, and 1 / 4MIC LA + 1 / 4MIC PCA were prepared with TSB medium containing 5% ethanol. The negative control was TSB containing 5% ethanol with an equal volume. 100 μL of the bacterial suspension (OD 600 = 0.5) and 100 μL of the gradient compound solution were added to each well of the 96-well plate, and the mixture was continuously cultured in a 37°C constant temperature incubator (humidity ≥ 80%) for 24 h. The absorbance value at 600 nm was measured every 1 h using a full wavelength microplate reader, and the growth curve was generated after processing the data with origin2021.

[0068] 2.6 Effects of the LA-PCA compound solution on the cell membrane integrity and cell morphology of Y. enterocolitica

[0069] The bacterial solution was prepared according to the steps in 2.2, and the drug solution was prepared according to the steps in 2.1.

[0070] 2.6.1 Laser confocal observation

[0071] The effect of LA-PCA compound solutions (concentration gradients of 1 / 2MIC LA, 1 / 2MIC PCA, 1 / 2MIC LA + 1 / 2MIC PCA, and 1 / 4MIC LA + 1 / 4MIC PCA) on the cell membrane permeability of Y. enterocolitica was evaluated using the SYTO 9 / PI double staining method.

[0072] Bacterial cells were washed three times with 0.85% NaCl, then resuspended in physiological saline (containing 5% ethanol) with a gradient of mixed solutions, and treated at 37°C in the dark for 2 h. The cells were then collected by centrifugation (4°C, 5000g, 5 min) and resuspended. 3 μL of a SYTO 9 / PI mixed dye (1:1 ratio) was added to each mL of the bacterial suspension, and the mixture was incubated in the dark for 15 min. 10 μL of the sample was then added to a glass slide and observed using a laser confocal microscope (LSM 900, Carl Zeiss). The excitation wavelengths were 488 nm / 561 nm, and the emission filters were 500-550 nm / 570-620 nm. Image analysis was performed using ZEN 3.4 software (Carl Zeiss).

[0073] 2.6.2 Field Emission Scanning Electron Microscopy Observation

[0074] Y. enterocolitica cells were treated with LA-PCA compound solutions (concentration gradients of 1 / 2MIC LA, 1 / 2MIC PCA, 1 / 2MIC LA + 1 / 2MIC PCA, and 1 / 4MIC LA + 1 / 4MIC PCA) for 24 hours, and then morphological analysis was performed according to the standard SEM sample preparation procedure.

[0075] The bacterial cells were washed three times with PBS (containing 5% ethanol), fixed with 2.5% glutaraldehyde (fixed at 4℃ for 12 h), dehydrated with a gradient of ethanol (30%-100%) (10 min per stage), replaced with isoamyl acetate for 30 min, dried, and then sputter-coated with gold (10 nm thickness). The samples were observed using a field emission scanning electron microscope (FEI MLA650, accelerating voltage 5 kV).

[0076] 2.7 Synergistic inhibition of metabolic activity within biofilm-covered Yersinia enterocolitica by LA-PCA compound solution

[0077] Yersinia enterocolitica was activated according to the method in Section 2.2 and inoculated into TSB medium containing LA and PCA or their compound solutions (concentration gradients of 1 / 4 MIC LA, 1 / 4 MIC PCA, 1 / 4 MIC LA + 1 / 4 MIC PCA, and 1 / 8 MIC LA + 1 / 8 MIC PCA). A negative control was TSB containing 5% ethanol. After incubation at 37°C for 24 h, 200 μL of the bacterial suspension was inoculated into 96-well plates and incubated at 37°C for 48 h to form a biofilm. After discarding the supernatant, the wells were gently washed three times with sterile PBS. 100 μL of PBS and 10 μL of CCK-8 reagent were added to each well, and the plates were incubated at 37°C in the dark for 1 h. The absorbance at 450 nm was measured using a microplate reader.

[0078] Synergistic inhibition of Yersinia enterocolitica quorum sensing by 2.8LA-PCA compound solution

[0079] The inhibitory effect of LA-PCA compound solutions (concentration gradients of 1 / 4MIC LA, 1 / 4MIC PCA, 1 / 4MIC LA + 1 / 4MIC PCA, and 1 / 8MIC LA + 1 / 8MIC PCA) on the secretion of acylhomoserine lactone (AHL) in *Y. enterocolitica* was quantitatively determined using *C. violaceum* (AHL-sensitizing defective reporter strain). *Yersinia enterocolitica* was inoculated into TSB medium containing different drug treatment groups (negative control was TSB containing 5% ethanol), incubated at 37°C for 24 h, and then centrifuged to collect the supernatant. This supernatant was then mixed with an equal volume of *C. violaceum* CV026 bacterial suspension (OD200). 600 =0.5 (containing 0.5 μg / mL kanamycin) was co-cultured in 96-well plates at 30°C for 24 h. The diameter of the purple kanamycin was determined by measuring the colony migration diameter using vernier calipers.

[0080] Synergistic regulation of extracellular polysaccharide content on biofilm of Yersinia enterocolitica by 2.9LA-PCA compound solution

[0081] The inhibitory effect of LA-PCA compound solutions (concentration gradients of 1 / 4MIC LA, 1 / 4MIC PCA, 1 / 4MIC LA + 1 / 4MIC PCA, and 1 / 8MIC LA + 1 / 8MIC PCA) on the extracellular polysaccharide (EPS) of *Y. enterocolitica* biofilm was quantitatively analyzed using ruthenium red staining. Bacterial suspensions were prepared according to the method described in Section 2.2, and 100 μL was inoculated into 96-well plates. Different concentrations of drug treatment groups were mixed with equal volumes of bacterial suspension. A negative control was TSB containing 5% ethanol. After incubation at 37°C for 48 h, the supernatant was discarded, and the plates were gently washed three times with PBS. 2 mL of 0.01% (m / v) ruthenium red staining solution was added to each well, and the plates were incubated at 37°C in the dark for 1 h. The absorbance at 450 nm was measured using a microplate reader.

[0082] 2.10 LA-PCA compound solution synergistically inhibits the biofilm formation of Yersinia enterocolitica using multimodal microscopic imaging analysis

[0083] 2.10.1 Biofilm Samples

[0084] Place a sterile circular glass slide in each well of a 24-well plate, add 2 mL of *Y. enterocolitica* bacterial suspension and different concentrations of the drug (concentration gradient: 1 / 4 MIC LA, 1 / 4 MIC PCA, 1 / 4 MIC LA + 1 / 4 MIC PCA, 1 / 8 MIC LA + 1 / 8 MIC PCA). The negative control is TSB containing 5% ethanol. After incubation at 37°C for 48 h, discard the culture medium and gently rinse the slides twice with sterile PBS to remove any unattached bacteria.

[0085] 2.10.2 Optical Microscope Observation

[0086] After washing the sample twice with PBS buffer, 200 μL of crystal violet (0.1%, v / v) was added, and the sample was incubated at room temperature for 20 min. Crystal violet staining is based on its specific binding to extracellular polysaccharides and proteins in the biofilm, allowing for a direct assessment of the inhibitory effects of LA, PCA, and their compound solutions on the biomass of Yersinia enterocolitica biofilms. After incubation, the sample was rinsed twice with PBS to remove unbound dye, and the biofilm structure was observed under an optical microscope.

[0087] 2.10.3 Observation using laser confocal microscopy

[0088] The distribution of biofilm activity was analyzed using a double staining method with SYTO9 and PI. Biofilm samples treated with the compound solution were stained for 15 min in the dark, washed with PBS, and then subjected to z-axis tomography (step size 1 μm) using an LSM800 microscope (excitation wavelength 488 nm / 561 nm, emission bandpass 500-550 nm / 570-620 nm). The three-dimensional structure was reconstructed using Imaris 9.7 software, and the average thickness of the biofilm was calculated.

[0089] 2.10.4 Scanning Electron Microscopy Observation

[0090] After dehydration with 2.5% glutaraldehyde (fixed at 4℃ for 4h) and gradient ethanol, the samples were critically dried (Leica EMCPD300), gold-plated (10nm thickness) using an ion sputtering instrument (Hitachi E-1045), and the ultrastructure of the biofilm was observed using a MLASEM (accelerating voltage 5kV, working distance 8mm).

[0091] 2.11 Data Analysis

[0092] SPSS 20.0 software was used for statistical processing and analysis. Data are expressed as ±SD. Multiple comparisons and one-way ANOVA were used to analyze and compare the data of each group (P<0.05 was considered statistically significant, and P<0.01 was considered highly statistically significant).

[0093] Experimental results

[0094] 3.1 Minimum inhibitory concentrations of LA and PCA against Y. enterocolitica

[0095] The results of the determination of the minimum inhibitory concentrations (MICs) of LA and PCA against Y. enterocolitica can be found in [link to relevant documentation]. Figure 1 The results showed that the MIC of LA was 2.5 mg / mL. Figure 1 A), the MIC of PCA is 1.25 mg / mL ( Figure 1 B).

[0096] 3.2 Synergistic antibacterial activity of LA, PCA and their compound solutions against Yersinia enterocolitica

[0097] The results of the checkerboard method synergistic antibacterial experiment of LA and PCA against Yersinia enterocolitica are as follows: Figure 2 As shown, the MIC values ​​of LA and PCA when used alone were 2.5 mg / mL and 1.25 mg / mL, respectively. After combination therapy, the MIC values ​​of the two drugs decreased to 0.625 mg / mL and 0.3125 mg / mL, respectively, corresponding to a partial inhibitory concentration index (FIC) of 0.5. When LA concentration ≥ 0.625 mg / mL (1 / 4 MIC) and PCA concentration ≥ 0.3125 mg / mL (1 / 4 MIC) were used in combination, the micropore areas covered by this concentration combination all showed complete inhibition (OD). 600 ≤0.1), take MIC combined with LA at 0.625 mg / mL, and MIC combined with PCA at 0.3125 mg / mL.

[0098] 3.3 Growth curves of LA, PCA, and their compound solutions against Yersinia enterocolitica

[0099] The inhibitory effects of LA and PCA, and their mixtures (concentration gradients of 1 / 2MIC LA, 1 / 2MIC PCA, 1 / 2MIC LA + 1 / 2MIC PCA, 1 / 4MIC LA + 1 / 4MIC PCA) on the growth of Yersinia enterocolitica were studied. Figure 3As shown, the inhibitory effect of 1 / 2MIC LA or 1 / 2MIC PCA alone on the growth of *Y. enterocolitica* was limited, while the low-concentration combined treatment group (1 / 4MIC LA + 1 / 4MIC PCA) significantly inhibited bacterial proliferation, and the high-concentration combined treatment group (1 / 2MIC LA + 1 / 2MIC PCA) reduced the viable bacterial count below the detection limit within 8 hours. In the combined treatment, LA may compensate for the insufficient permeability of PCA to Gram-negative bacteria by disrupting the bacterial outer membrane permeability, thus producing a synergistic effect.

[0100] 3.4 Effects of LA-PCA compound solution on cell membrane integrity and cell morphology of Yersinia enterocolitica

[0101] 3.4.1 Effects of LA and PCA and their compound solutions (concentration gradients of 1 / 2MIC LA, 1 / 2MIC PCA, 1 / 2MIC LA + 1 / 2MIC PCA, 1 / 4MIC LA + 1 / 4MIC PCA) on the cell membrane integrity and cell morphology of Yersinia enterocolitica. Figure 4 As shown. Laser confocal observation. Figure 4 As shown in Figure A, the SYTO9 / PI double staining in the single treatment groups (1 / 2MIC LA, 1 / 2MIC PCA) only showed local red fluorescence, while the proportion of red fluorescence in the low-concentration combined treatment group (1 / 4MIC LA + 1 / 4MIC PCA) was significantly increased, and the green fluorescence in the high-concentration combined treatment group (1 / 2MIC LA + 1 / 2MIC PCA) was completely quenched, indicating that the membrane integrity was extensively damaged.

[0102] 3.4.2 Scanning electron microscopy observation of LA and PCA and their compound solutions (concentration gradients of 1 / 2MIC LA, 1 / 2MIC PCA, 1 / 2MIC LA + 1 / 2MIC PCA, 1 / 4MIC LA + 1 / 4MIC PCA) against Yersinia enterocolitica. Figure 4 B shows that the untreated control group (Control cells) had intact short rod-shaped cells with smooth surfaces. The single-treatment groups (1 / 2MIC LA, 1 / 2MIC PCA) showed localized depressions (LA group) or slight shrinkage (PCA group) on the bacterial surface, respectively. In contrast, the low-concentration combined treatment group (1 / 4MIC LA + 1 / 4MIC PCA) showed large-area cell membrane collapse, and the high-concentration combined treatment group (1 / 2MIC LA + 1 / 2MIC PCA) showed obvious pores and leakage of contents. Cell rupture in the combined group was significantly more pronounced than in the single-drug groups.

[0103] Synergistic inhibition of metabolic activity within biofilm-covered Yersinia enterocolitica by 3,5-LA-PCA compound solution.

[0104] The effects of LA, PCA, and their mixtures (concentration gradients of 1 / 4MIC LA, 1 / 4MIC PCA, 1 / 4MIC LA + 1 / 4MIC PCA, and 1 / 8MIC LA + 1 / 8MIC PCA) on the biofilm metabolic activity of Yersinia enterocolitica were detected by the CCK-8 assay. Figure 5 ), OD of the separate treatment group 450 Compared with the control group, the absorbance values ​​decreased by 16% and 14%, respectively. The absorbance value of the low-concentration combined treatment group of 1 / 8MIC LA + 1 / 8MIC PCA decreased by 34%, and the metabolic inhibition rate increased by 1.9 times (vs LA) and 2.2 times (vs PCA) compared with the single drug group. The absorbance value of the high-concentration combined treatment group of 1 / 4MIC LA + 1 / 4MIC PCA decreased by 44% compared with the control group.

[0105] Synergistic inhibition of Yersinia enterocolitica quorum sensing by 3.6LA-PCA compound solution

[0106] The inhibitory effects of LA and PCA, and their compound solutions (concentration gradients of 1 / 4MIC LA, 1 / 4MIC PCA, 1 / 4MIC LA + 1 / 4MIC PCA, 1 / 8MIC LA + 1 / 8MIC PCA) on the quorum sensing (QS) system of Yersinia enterocolitica were assessed by measuring the diameter of violaceae CV026 after co-culturing with Yersinia enterocolitica treated with different concentrations of the drugs. Figure 7 In the individual treatment groups, LA and PCA reduced the diameter of violetin by 21% and 40%, respectively, while the low-concentration combined treatment group of 1 / 8MIC LA + 1 / 8MIC PCA reduced it by 63%, and the high-concentration combined treatment group of 1 / 4MIC LA + 1 / 4MIC PCA reduced it by 85% compared to the control group. This confirms that it disrupts the QS pathway through dual targets (signal molecule synthesis and receptor response).

[0107] 3.7 Synergistic Regulation of Extracellular Polysaccharide Content in the Biofilm of Yersinia enterocolitica by LA-PCA Compound Solution

[0108] The inhibitory effects of LA, PCA, and their mixtures (concentration gradients of 1 / 4MIC LA, 1 / 4MIC PCA, 1 / 4MIC LA + 1 / 4MIC PCA, and 1 / 8MIC LA + 1 / 8MIC PCA) on the synthesis of extracellular polysaccharides (EPS) in *Yersinia enterocolitica* were determined by the ruthenium red binding method. Figure 6The results showed that the EPS content in the low-concentration combined treatment group (1 / 8MIC LA + 1 / 8MIC PCA) decreased by 40% compared to the control group, significantly higher than the single-drug group (LA: 19%; PCA: 23%). Studies have shown that LA competitively inhibits UDP-glucose dehydrogenase activity, blocking the synthesis of EPS precursor glyconucleotides, while PCA interferes with glycan elongation by binding to the catalytic pocket of glycosyltransferase GTF2. The synergistic effect of these two treatments leads to dual blockade of key nodes in the EPS biosynthetic pathway.

[0109] Multimodal microscopic imaging analysis of the synergistic inhibitory effect of 3.8LA-PCA compound solution on the biofilm of Yersinia enterocolitica.

[0110] The inhibitory effects of LA, PCA, and their compound solutions (concentration gradients of 1 / 4MIC LA, 1 / 4MIC PCA, 1 / 4MIC LA + 1 / 4MIC PCA, and 1 / 8MIC LA + 1 / 8MIC PCA) on the formation of Yersinia enterocolitica biofilm were verified by optical microscopy. Figure 8 A). Biofilms in 24-well plates were treated with crystal violet staining. The untreated control group showed a dense and continuous biofilm structure. In the single-treatment groups, LA and PCA-treated samples still had relatively dense biofilm coverage. However, in the low-concentration combined treatment group of 1 / 8MIC LA + 1 / 8MIC PCA, the biofilm was distributed in a discrete island pattern, and the coverage was significantly reduced. In the high-concentration combined treatment group of 1 / 4MIC LA + 1 / 4MIC PCA, cells rarely aggregated, and the biofilm structure was loose, indicating that the combined solutions synergistically disrupted the integrity of the biofilm structure.

[0111] The inhibitory effect of LA-PCA compound solutions (concentration gradients of 1 / 4 MIC LA, 1 / 4 MIC PCA, 1 / 4 MIC LA + 1 / 4 MIC PCA, and 1 / 8 MIC LA + 1 / 8 MIC PCA) on the biofilm of *Y. enterocolitica* was evaluated using laser confocal microscopy (CLSM) combined with SYTO9 / PI double staining. Figure 8 B). In the control group, the biofilm exhibited continuous and dense green fluorescence (over 90% of viable bacteria were stained with SYTO9). In the single-treatment groups, LA and PCA-treated samples showed mostly green fluorescence and a small portion of red fluorescence (PI-positive dead bacteria). However, in the 1 / 8 MIC LA low-concentration combined treatment group, the proportion of red fluorescence was significantly increased, and the green fluorescence area was fragmented. In the 1 / 4 MIC LA high-concentration combined treatment group, the fluorescence was almost entirely red, and biofilm structure could not form. Three-dimensional reconstruction analysis showed that the biofilm thickness in the combined treatment group decreased from 22.3 μm in the control group to 8.6 μm.

[0112] The inhibitory effects of LA-PCA complexes (1 / 4MIC LA, 1 / 4MIC PCA, 1 / 4MIC LA + 1 / 4MIC PCA, 1 / 8MIC LA + 1 / 8MIC PCA) on the biofilm of *Y. enterocolitica* were evaluated by scanning electron microscopy (SEM). Figure 8 C). In the control group, the biofilm exhibited a complete three-dimensional network structure with a large number of extracellular polymers encapsulating the bacteria. In the single-treatment groups, the LA and PCA-treated samples still had relatively dense biofilm structures with extracellular polymer adhesion. However, in the low-concentration combined treatment group of 1 / 8MIC LA + 1 / 8MIC PCA, the number of biofilm layers decreased and the structure was significantly loose. In the high-concentration combined treatment group of 1 / 4MIC LA + 1 / 4MIC PCA, the biofilm structure was severely disintegrated.

[0113] The above results indicate that the LA-PCA compound solution synergistically disrupts biofilms through multiple pathways, including interfering with bacterial colonization, metabolic homeostasis, signal transduction, and matrix stability. In other words, LA-PCA has a good inhibitory effect on Yersinia enterocolitica biofilms.

[0114] In summary, this invention systematically studies the synergistic antibacterial effects and mechanisms of LA and PCA, discovering the intervention effect of LA-PCA combined use on Yersinia enterocolitica biofilm. It demonstrates that LA-PCA has a definite efficacy in inhibiting and clearing Yersinia enterocolitica biofilm, thereby effectively alleviating bacterial infections caused by Yersinia enterocolitica with low toxicity. This invention not only contributes to promoting the application of natural drugs in the field of antibacterial agents but also provides new ideas for the development of new drugs against Yersinia enterocolitica infection.

Claims

1. An antibacterial agent against Yersinia enterocolitica, characterized in that, The mixture includes a combination of lipoic acid and protocatechuic aldehyde. When lipoic acid and protocatechuic aldehyde work synergistically, the minimum inhibitory concentrations against Yersinia enterocolitica are 0.625 mg / mL and 0.3125 mg / mL, respectively.

2. The antibacterial agent against Yersinia enterocolitica according to claim 1, characterized in that, The lipoic acid and protocatechuic aldehyde compound solution can inhibit and / or remove the biofilm of Yersinia enterocolitica.

3. The antibacterial agent against Yersinia enterocolitica according to claim 1, characterized in that, The compound solution of lipoic acid and protocatechuic aldehyde inhibits and / or removes the biofilm of Yersinia enterocolitica by blocking the synthesis of extracellular polysaccharides in Yersinia enterocolitica.

4. The antibacterial agent against Yersinia enterocolitica according to claim 1, characterized in that, The compound solution of lipoic acid and protocatechuic aldehyde inhibits and / or removes the biofilm of Yersinia enterocolitica by suppressing the quorum sensing system of Yersinia enterocolitica.

5. The antibacterial agent against Yersinia enterocolitica according to claim 1, characterized in that, The compound solution of lipoic acid and protocatechuic aldehyde inhibits and / or removes the biofilm of Yersinia enterocolitica by inhibiting the metabolic activity of the biofilm of Yersinia enterocolitica.

6. The antibacterial agent against Yersinia enterocolitica according to claim 1, characterized in that, The compound solution of lipoic acid and protocatechuic aldehyde inhibits and / or removes the biofilm of Yersinia enterocolitica by disrupting the cell membrane integrity of Yersinia enterocolitica.

7. The antibacterial agent against Yersinia enterocolitica according to claim 1, characterized in that, The Yersinia enterocolitica strain is selected from one or more foodborne pathogenic strains, which are Yersinia enterocolitica strains that can cause digestive system diseases.

8. The use of any one of the antibacterial agents against Yersinia enterocolitica according to claims 1 to 7 in the preparation of a medicine for treating digestive system diseases.

9. The application according to claim 8, characterized in that, The digestive system diseases mentioned include chronic gastroenteritis and enterocolitis.

10. The use of any one of the antibacterial agents against Yersinia enterocolitica according to claims 1 to 7 in the preparation of a medicament for treating systemic lesions caused by digestive system diseases.

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