Use of shikimic acid for the preparation of antibacterial preparations for inhibiting staphyloxin synthesis

Abstract

The application belongs to the technical field of microorganisms, and discloses application of shikimic acid in preparation of antibacterial preparations for inhibiting staphyloxin synthesis. It is found that shikimic acid can inhibit synthesis of staphyloxin in methicillin-resistant Staphylococcus aureus (MRSA), reduce tolerance of the bacteria to oxidative stress, induce accumulation of intracellular active oxygen, and damage cell membrane structure and function, including reducing cell membrane order, inducing membrane depolarization, increasing membrane permeability, and leading to ATP leakage and energy metabolism disorder. A new strategy and preparation direction are provided for clinical treatment of multi-drug resistant Staphylococcus aureus infection, and the application has important theoretical value and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and more specifically, relates to the application of shikimic acid in the preparation of antibacterial agents for inhibiting the synthesis of staphylococcal flavin. Background Technology

[0002] The problem of drug resistance in Staphylococcus aureus is becoming increasingly serious. In recent years, anti-infection strategies targeting bacterial virulence factors have received widespread attention. Staphylococcal flavin is a carotenoid pigment unique to Staphylococcus aureus, which gives the colonies a golden-yellow appearance and plays a key role in bacterial anti-oxidative stress, resistance to host immune killing, and maintenance of cell membrane stability.

[0003] Previous studies have shown that staphylococcal flavins can enhance bacterial survival by scavenging reactive oxygen species (ROS) and reduce the efficiency of antimicrobial drug entry by regulating membrane fluidity. Existing staphylococcal flavin synthesis inhibitors (such as nalidixic acid and brassinolide) suffer from significant cytotoxicity or high synthesis costs, limiting their clinical application.

[0004] Shikimic acid is a natural compound found in various plants and serves as a precursor for the synthesis of many important biomolecules. Recent studies have shown that shikimic acid possesses significant antibacterial, anti-inflammatory, and immunomodulatory effects. However, its influence on staphylococcal flavin synthesis and cell membrane function remains unclear, and its potential in enhancing antibacterial susceptibility needs further exploration. Therefore, developing readily available, safe, and inhibitory natural small-molecule compounds for staphylococcal flavin synthesis has significant clinical application value. Summary of the Invention

[0005] Based on the aforementioned deficiencies in the existing technology, the present invention first provides the application of shikimic acid in the preparation of antibacterial agents for inhibiting the synthesis of staphylococcal flavin.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] Application of shikimic acid in inhibiting the synthesis of staphylococcal flavin in methicillin-resistant Staphylococcus aureus JE2 for non-disease treatment purposes without affecting the viability of methicillin-resistant Staphylococcus aureus.

[0008] Visual observation revealed a significant decrease in the production of staphylococcal flavin in JE2 under shikimic acid concentrations below the bacterial inhibitory concentration. Specifically, the OD in the untreated group... 470nm The value was 0.15, while the OD values ​​of different concentrations of shikimic acid treatment groups were... 470nmThe concentrations decreased to 0.13, 0.09, and 0.09, respectively. This clearly demonstrates that shikimic acid can inhibit the synthesis of MRSA staphylococcal flavin without inhibiting bacterial growth.

[0009] Therefore, the present invention also provides the use of shikimic acid in the preparation of antibacterial agents that inhibit the synthesis of staphylococcal flavin in methicillin-resistant Staphylococcus aureus JE2 without affecting its viability.

[0010] Preferably, in the above applications, the concentration of shikimic acid is 1000~3000 mg / L.

[0011] The expression levels of genes associated with methicillin-resistant Staphylococcus aureus (MRSA) JE2 and its synthetic staphylococcal yellow color were also investigated. The results showed that, compared with the untreated group, the JE2 strain treated with shikimic acid showed significantly higher expression levels. crtM, crtN, crtO, crtP, crtQ The gene expression was significantly downregulated, suggesting that shikimic acid may enhance its anti-MRSA activity by inhibiting the synthesis pathway of staphylococcal flavin.

[0012] Therefore, preferably, in the above applications, the shikimic acid downregulates the JE2 strain. crtM, crtN, crtO, crtP, crtQ Gene expression levels.

[0013] To confirm whether shikimic acid-induced oxidative damage stemmed from an intracellular ROS surge, intracellular reactive oxygen species (ROS) levels were subsequently measured in the treated bacteria. The results showed that shikimic acid treatment significantly increased intracellular ROS accumulation, with the high-concentration shikimic acid group even reaching the levels of the positive control hydrogen peroxide. Given that previous studies have shown that shikimic acid can inhibit the production of staphylococcal flavin, and the downregulation of this pigment weakens the bacteria's ability to scavenge ROS, the significant increase in ROS levels and the pigment detection results corroborate each other, jointly revealing that shikimic acid reduces bacterial tolerance to oxidative stress by inhibiting the synthesis of antioxidant pigments, leading to excessive intracellular ROS accumulation.

[0014] Therefore, the present invention also provides the application of shikimic acid in improving bacterial cell membrane permeability and fluidity without affecting the viability of methicillin-resistant Staphylococcus aureus JE2.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] This invention is the first to discover that shikimic acid can inhibit staphylococcal flavin synthesis, disrupt cell membrane function, interfere with energy metabolism, and promote antibiotic penetration through multiple pathways. This invention provides new targets, new strategies, and new formulation directions for the clinical treatment of multidrug-resistant Staphylococcus aureus infections, and has important theoretical value and broad application prospects. Attached Figure Description

[0017] Figure 1Figure 1 shows the STX production in MRSA strain JE2 after treatment with different concentrations of SA; the left side shows the quantitative analysis of STX extracted by methanol extraction, and the right side shows the colony pigment phenotypes after treatment with different concentrations of shikimic acid.

[0018] Figure 2 Relative expression levels of key genes for STX biosynthesis in MRSA strain JE2 after treatment with different concentrations of SA;

[0019] Figure 3 Growth curves of MRSA strain JE2 under different concentrations of hydrogen peroxide (H2O2) stress after treatment with different concentrations of SA;

[0020] Figure 4 Fluorescence intensity of intracellular reactive oxygen species (ROS) levels in MRSA strain JE2 after treatment with different concentrations of SA;

[0021] Figure 5 The molecular order of lipid bilayer in the cell membrane of MRSA strain JE2 after treatment with different concentrations of SA (GP value).

[0022] Figure 6 Fluorescence intensity of cell membrane depolarization in MRSA strain JE2 after treatment with different concentrations of SA;

[0023] Figure 7 Cell membrane permeability of MRSA strain JE2 after treatment with different concentrations of SA;

[0024] Figure 8 Intracellular and extracellular ATP content of MRSA strain JE2 after treatment with different concentrations of shikimic acid (SA). Detailed Implementation

[0025] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific drawings and embodiments. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0026] Example 1: Determination of the MIC of shikimic acid against MRSA strain JE2

[0027] 1. Experimental materials:

[0028] The autoclaved CAMH broth was cooled and stored for later use. MRSA bacteria JE2 (laboratory preservation). Shikimic acid stock solution was prepared at 100 mg / mL and stored at -20°C.

[0029] 2. Preparations before the experiment: Incubate MRSA strain JE2 on brain heart extract medium until it reaches a suitable size.

[0030] 3. Experiment evaluating the bactericidal effect of shikimic acid:

[0031] (1) The test strain was inoculated into a 50 mL sterile centrifuge tube containing 10 mL of CAMH broth and incubated at 37 °C for 180 rpm until the logarithmic growth phase. The centrifuge tube was then removed. The test MRSA cells were collected by centrifugation at 3000 rpm for 10 min, washed twice with PBS solution, and resuspended. The resuspended bacterial solution was adjusted to OD using a spectrophotometer. 600 =0.5, that is, 1~5×10 8 CFU / mL;

[0032] (2) Dilute the incubated JE2 strain 100 times with CAMH broth, approximately 1~5×10⁻⁶. 6 CFU / mL, for later use;

[0033] (3) Take a sterile 96-well plate, add 180 μL of CAMH broth medium to well 1, and add 100 μL of CAMH broth medium to wells 2-11;

[0034] (4) Add 20 μL of shikimic acid to the first well, blow it evenly, and then take 100 μL into the second well, and so on. Take 100 μL from the tenth well and discard it.

[0035] (5) Add 100 μL of diluted bacterial solution to wells 1 to 10, and add 200 μL of CAMH broth to well 12;

[0036] (6) Repeat steps (3) to (5) three times in parallel;

[0037] (7) Place in a 37℃ incubator and incubate for 18-24 hours. Determine the MIC value based on the turbidity.

[0038] The minimum inhibitory concentration of shikimic acid against MRSA strain JE2 is shown in Table 1.

[0039] Table 1. MIC values ​​of shikimic acid against JE2.

[0040] Example 2: Determination of Staphylococcal Flavin Production

[0041] 1. Experimental materials: MH broth that has been autoclaved and cooled for later use.

[0042] 2. Preparations before the experiment: Single colonies of MRSA were picked from BHI agar plates using a sterile inoculation loop and transferred to MH liquid medium. The culture was incubated at 37°C with shaking at 220 rpm until the logarithmic growth phase. The bacterial suspension was centrifuged at 3500 rpm for 10 minutes to obtain the bacterial pellet, which was then washed twice with sterile PBS buffer. The concentration of the bacterial suspension was calibrated to OD using spectrophotometry. 600nm=0.5, at this point the bacterial count is approximately 10. 8 CFU / mL, prepared by diluting with MH broth to obtain 10 6 CFU / mL working bacterial solution is available for later use.

[0043] 3. Experiment on the determination of flavin production in JE2 staphylococci under the action of shikimic acid

[0044] An antibiotic-free control group was set up: the pretreated bacterial suspension was serially diluted 10-fold using sterile MH medium to adjust the final concentration to 10. 5 CFU / mL; Shikimic acid group: Shikimic acid (1000~3000 mg / L) was added to sterile fresh MH broth to dilute the bacterial solution 10 to 10 times. 5 CFU / mL. The above experimental groups were incubated in a shaker at 37℃ for 24 h.

[0045] The cultures in each group were centrifuged at 13,000 rpm for 1 min, the supernatant was discarded, the bacterial pellet was collected, washed twice with sterile PBS, and then calibrated to uniform turbidity by spectrophotometry. The pellet was then collected by centrifugation again.

[0046] The bacterial pellet was resuspended in 700 µL of methanol and incubated in a 55 °C water bath for 10 min to extract staphylococcal flavin. The extract was centrifuged at 13000 rpm for 2 min, and the supernatant was collected. 200 µL of the supernatant was added to a 96-well transparent plate, with 3 replicates per sample group, and the absorbance (OD) was measured at 470 nm. 470nm ).

[0047] The results are as follows Figure 1 As shown in the figure. Visual observation revealed that, under the action of shikimic acid at concentrations below the bacterial inhibitory concentration (1000~3000 mg / mL), the production of staphylococcal flavin in JE2 significantly decreased. The OD of the untreated group was... 470nm The value was 0.15, while the OD values ​​of different concentrations of shikimic acid treatment groups were... 470nm The concentrations decreased to 0.13, 0.09, and 0.09, respectively. This clearly demonstrates that shikimic acid can inhibit the synthesis of MRSA staphylococcal flavin without inhibiting bacterial growth.

[0048] Example 3: Detection of expression levels of genes related to staphylococcal flavin synthesis

[0049] (1) Sample pretreatment: The concentration of the test strain JE2 was calibrated according to the "Example 1" section to obtain a bacterial count of 10. 8 CFU / mL and diluted to 10 with MH broth. 7 CFU / mL was prepared for use. Two experimental systems were constructed: (i) No drug intervention group: the pretreated bacterial suspension was serially diluted to 10⁻⁶ using sterile BHI medium. 6(i) Shikimic acid treatment group: The concentration of the bacterial suspension was adjusted proportionally using BHI liquid medium containing shikimic acid (2000 mg / L), and the final concentration of the bacterial suspension was diluted to 10. 6 CFU / mL. The standardized bacterial suspension was transferred to a constant temperature shaking incubator (37℃, 220rpm) for 5 hours of dynamic culture, and then bacterial RNA was extracted.

[0050] (2) Total RNA extraction from the test strain JE2: Bacterial RNA was extracted according to the instructions of the OMAGABacteria RNA Kit. The specific procedures are as follows:

[0051] Cell pretreatment: Transfer 2 mL of the pretreated bacterial suspension to an RNase-free microcentrifuge tube and centrifuge at 4°C (4000×g, 10 min) to collect the bacterial pellet. After removing the supernatant, resuspend the pellet using a lysozyme mixture of 100 μL and 10 μL, vortex for 30 seconds to mix, and then digest in a 30°C metal bath for 10 minutes (with 20-second shaking every 2 minutes to promote dissolution).

[0052] Lysis and purification: Add lysis buffer BRK (350 μL, containing 20 μL / mL β-mercaptoethanol) and glass beads (GlassPowder, 30 mg) to the lysis system; vortex at high intensity (5 min) and then centrifuge (13000×g, 5 min); transfer the supernatant (400 μL) to a new centrifuge tube, add an equal volume of pre-cooled 70% ethanol (stored at -20℃) and mix well; transfer the mixture to a HiBind® RNA adsorption column, centrifuge for enrichment (10000×g, 45 s), and then discard the flow-through.

[0053] DNA removal: Prepare the DNase treatment system (DNase Digestion Buffer 73.5 μL; RNase-free DNase I 1.5 μL; total volume 75 μL), and mix gently. Transfer the treatment solution evenly to the surface of the adsorption column membrane and incubate at room temperature (25℃) for 15 min.

[0054] RNA purification: Initial washing was performed with RNA washing buffer I (500 μL), followed by centrifugation (10000 × g, 45 s) after 5 min incubation. Washing was repeated twice with RNA washing buffer II (500 μL). The column was then centrifuged (13000 × g, 2 min) to remove residual ethanol and allow the column to dry. Preheated DEPC water (70 °C, 40 μL) was added, and the column was incubated at room temperature for 5 min. The column was then centrifuged (10000 × g, 1 min) to collect the eluent. This process was repeated once to increase the yield. Purified RNA samples were immediately stored at -80 °C in a cryogenic freezer. Three independent biological replicates were set up for the experiment.

[0055] (3) Quality control of total RNA and preparation of cDNA for the tested strain JE2: RNA purity and concentration were quantitatively determined by spectrophotometry. The A260 / A280 absorbance ratio of the qualified samples should be stable in the range of 1.8-2.1. The integrity of nucleic acid was verified by electrophoresis using a 2% agarose gel (prepared with 1×TAE buffer). The sample loading volume was 100-200 ng / μL (5μL). After electrophoresis at 80V for 30 minutes, the integrity of the 28S / 18S rRNA bands was observed by a gel imaging system. The qualified samples should show clear bands and no signs of genomic DNA contamination or RNA degradation.

[0056] (4) Select qualified RNA samples for cDNA synthesis and perform reverse transcription according to the operating procedures of HiScript III RT SuperMix for qPCR (Vazyme R323-01). First, prepare the gDNA removal system: 4×gDNAwiperMix 4μL, total RNA ranging from 1pg to 1μg (adjust volume according to quantification results), and RNase-free ddH2O to 16μL. The mixture is incubated precisely in a 42℃ metal bath for 2 minutes to complete genomic DNA removal. Then, construct the reverse transcription reaction system: add 5×HiScript III qRT SuperMix 4μL to the above treatment solution, for a total volume of 20μL. The program is set to perform reverse transcription at 37℃ for 15 minutes, and the reaction is terminated by heat inactivation at 85℃ for 5 seconds. The final product cDNA is aliquoted and frozen in an ultra-low temperature freezer at -80℃ for later use. Three independent biological replicates are set up for the experiment and a negative control (without template control) is implemented.

[0057] (5) The mRNA expression level of JE2-related genes after shikimic acid treatment was detected by RT-qPCR:

[0058] Primer design and synthesis: Specific amplification primers were constructed using SnapGene software based on the NCBi GenBank database. The Staphylococcus aureus housekeeping gene gyrB was selected as an endogenous reference gene to standardize the difference in relative mRNA expression levels between the treatment and control groups. All primers were synthesized and purified by HPLC at Guangzhou Qingke Biotechnology Co., Ltd. The primer powder was reconstituted with DEPC water to a final concentration of 10 μmol / L for later use.

[0059] The RT-qPCR detection system was constructed using the SYBR Green fluorescence quantitative method, with a final reaction volume of 20 μL.

[0060] (6) Accurately add 5 μL of cDNA template (20 ng / μL) and 10 μL of SYBR Green Premix ExTaq. TM1 μL each of forward and reverse primers (10 μmol / L) were added, and the volume was made up with RNase-free water. The initial pre-denaturation phase was set at 95°C for 30 seconds to fully open the DNA double strand; subsequently, 40 cycles of amplification were performed, each cycle consisting of 10 seconds of denaturation at 95°C, followed by 30 seconds of annealing / extension at 57°C, with simultaneous fluorescence signal acquisition. After amplification, a melting curve analysis program was executed, sequentially subjecting the cells to a gradient temperature increase of 0.5°C / sec to 95°C for 10 seconds, 57°C for 30 seconds, and 72°C for 30 seconds to verify primer specificity. Target gene expression was measured using relative quantification. -ΔΔCt Processing and analysis.

[0061] The results are as follows Figure 2 As shown, compared with the untreated group, the JE2 strain treated with shikimic acid... crtM, crtN, crtO crtP, crtQ Gene expression was significantly downregulated ( Figure 2 The average relative expression levels decreased to 0.26, 0.34, 0.42, 0.30 and 0.32, respectively. These results suggest that shikimic acid may enhance its anti-MRSA activity by inhibiting the synthesis pathway of staphylococcal flavin.

[0062] Example 4 Oxidative stress tolerance test

[0063] 1. Experimental materials: MRSA standard strain JE2, shikimic acid, sterile PBS, antibiotic-free culture medium, hydrogen peroxide, TSB culture medium, 96-well culture plate, and fully automated growth curve analyzer.

[0064] 2. Preparation before the experiment: Prepare sterile PBS and antibiotic-free culture medium; prepare hydrogen peroxide fresh for use; preheat the fully automated growth curve analyzer to 37°C.

[0065] 3. Experimental steps:

[0066] The concentration of the tested strain JE2 was calibrated according to the "Example 1" section, and the bacterial count was 10. 8 CFU / mL available for use.

[0067] The test strain JE2 was co-cultured with different concentrations of SA (0, 1000, 2000, 3000 mg / L) for 20 h. After culturing, MH broth containing 0.012% H2O2 was added to the deep-well plates, and the cultured bacterial solution was inoculated into the broth at a ratio of 1:100, making the final volume of each well 1 mL. Each group had 3 replicates. Bacterial growth was monitored in real time at 37℃ using a fully automated growth curve analyzer (Tianjin Jieling Instrument Manufacturing Co., Ltd., China), and OD was automatically recorded every 30 min. 600 The growth curves of the SA pretreatment group and the control group were compared to evaluate the effect of SA pretreatment on the growth ability of MRSA under H2O2 stress.

[0068] Given that staphylococcal flavin is an important antioxidant pigment in Staphylococcus aureus, changes in its production may directly affect the bacteria's defense against oxidative stress. Therefore, we further examined the bacteria's tolerance to hydrogen peroxide stimulation after drug treatment to determine whether the drug weakens the bacteria's antioxidant defense by inhibiting pigment synthesis. The results are as follows... Figure 3 As shown, shikimic acid pretreatment reduced the bacteria's tolerance to exogenous oxidative stress, and this effect was concentration-dependent. This indicates that shikimic acid can dose-dependently weaken the bacteria's antioxidant defense capabilities, further supporting the possibility that it may increase the bacteria's sensitivity to oxidative stress by inhibiting the synthesis of antioxidant pigments such as staphylococcal flavin.

[0069] Example 5 Cell Membrane Function Detection

[0070] 1. Experimental materials:

[0071] MRSA standard strain JE2, shikimic acid, Laurdan probe, DiSC3(5) probe, PI dye, HEPES buffer, glucose, PBS, black 96-well plate, microplate reader.

[0072] 2. Pre-experimental preparation: Prepare fluorescent probes in the dark; prepare HEPES buffer containing 5mM glucose; preset the microplate reader to the corresponding wavelength; adjust bacterial suspensions to the same OD value. 600 .

[0073] 3. Experimental steps:

[0074] The test strain JE2 was incubated with different concentrations of SA (0, 1000, 2000, 3000 mg / L) in a shaker at 37°C for 12 h. After incubation, the bacterial cells were collected by centrifugation and washed twice with HRPES buffer to remove residual SA. The bacterial suspension was adjusted to OD200. 600 =0.5, ensuring consistent biomass across groups. The following tests were then performed.

[0075] (1) Cell membrane orderliness detection: Add Laurdan probe (final concentration 10 μM) to the adjusted bacterial suspension, vortex to mix, and incubate at 37℃ in the dark for 20 min. After incubation, transfer 200 μL to a black 96-well plate, with 3 replicates per group. Detect fluorescence at dual emission wavelengths (excitation wavelength 350 nm; emission wavelength 440 / 490 nm). Calculate GP value: GP = (I 440 -I 490 ) / (I 440 +I 490 ).

[0076] (2) Cell membrane depolarization detection: DiSC3(5) fluorescent probe (final concentration 0.5 μM) was added to the adjusted bacterial suspension and incubated at 37℃ in the dark for 30 min. After incubation, 200 μL was transferred to black 96-well plates, with 3 replicates per group. The fluorescence values ​​at excitation wavelength of 622 nm and emission wavelength of 670 nm were measured using a multi-functional microplate reader.

[0077] (3) Cell membrane permeability detection: PI fluorescent probe (final concentration 10 nM) was added to the adjusted bacterial suspension and incubated at 37°C in the dark for 30 min. After incubation, 200 μL was transferred to black 96-well plates, with 3 replicates per group. The fluorescence values ​​at excitation wavelength of 535 nm and emission wavelength of 615 nm were measured using a multi-functional microplate reader.

[0078] Staphylococcal flavin is a carotenoid pigment located on the cell membrane of Staphylococcus aureus. It not only possesses antioxidant activity but also participates in maintaining membrane structural stability. Given that shikimic acid can significantly inhibit the synthesis of this pigment and induce intracellular ROS accumulation, it is speculated that pigment reduction may further damage the physical state of the membrane. Therefore, the membrane order, membrane depolarization, and membrane permeability of bacteria treated with shikimic acid were detected using Laurdan, DiSC3(5), and PI probes, respectively. Figure 4-7 As shown, the results indicate that shikimic acid treatment significantly affected the physical state of the bacterial cell membrane in a dose-dependent manner. Laurdan staining results showed that the GP value gradually decreased with increasing shikimic acid concentration, indicating decreased membrane orderliness and increased membrane fluidity; enhanced DiSC3(5) fluorescence suggested increased membrane depolarization, reflecting impaired proton gradient; the significantly increased proportion of PI-positive cells directly proved that the membrane permeability barrier was disrupted and the bacterial membrane integrity was lost. The above results indicate that shikimic acid treatment caused systemic damage to the bacterial cell membrane, manifested as decreased orderliness, intensified depolarization, and increased permeability. Staphylococcal flavin is embedded in the lipid bilayer of the cell membrane, which can regulate the lipid arrangement order, maintain the low permeability of the membrane, and protect the membrane potential stability. Therefore, the decrease in the production of this pigment directly weakens the rigidity and barrier function of the membrane, leading to increased membrane fluidity, depolarization, and leakage of contents.

[0079] Example 6: Quantitative Detection of Intracellular and Extracellular ATP

[0080] 1. Experimental materials:

[0081] MRSA standard strain JE2, shikimic acid, ATP assay kit (S0026, Shanghai Beyotime Biotechnology Co., Ltd.), lysis buffer, centrifuge tubes, and chemiluminescent microplate reader.

[0082] 2. Preparation before the experiment: Equilibrate the reagent kit to room temperature; prepare fresh bacterial samples; preheat the chemiluminescent microplate reader for 30 minutes.

[0083] 3. Experimental steps:

[0084] (1) Co-culture the test strain with SA for 24 h. Resuspend in PBS three times and then resuspend at OD. 600 =0.5.

[0085] (2) Extracellular ATP detection: Centrifuge at 12000×g, 4℃ for 5 min to collect the supernatant, and add 100 μL of ATP detection working solution to a 96-well microplate. Let it stand at room temperature for 3-5 minutes to allow all ATP to be consumed, add 100 μL of the collected supernatant, mix quickly with a pipette, and then measure the RLU value using a chemiluminescence analyzer.

[0086] (3) Intracellular ATP detection: Collect bacterial pellets by centrifugation, add 200 μL of lysis buffer to each tube, vortex thoroughly, centrifuge at 12000×g, 4℃ for 5 min, and collect the supernatant. Add 100 μL of ATP detection working solution to a 96-well microplate. Incubate at room temperature for 3-5 minutes to allow all ATP to be consumed, add 100 μL of the collected supernatant, mix quickly with a pipette, and then measure the RLU value using a chemiluminescence analyzer.

[0087] (4) Normalize the bacterial biomass. Calculate the ATP content based on the standard curve plotted with known ATP concentrations.

[0088] The PI staining results above confirmed that shikimic acid significantly increases bacterial membrane permeability, and the loss of membrane barrier function is usually accompanied by the leakage of intracellular small molecule metabolites (such as ATP). To assess the impact of membrane damage on bacterial energy metabolism, changes in intracellular and extracellular ATP levels after shikimic acid treatment were further examined. The results are as follows: Figure 8 As shown, shikimic acid primarily affects the physical barrier function of bacterial cell membranes rather than their energy synthesis capacity. The unchanged intracellular ATP level suggests that the bacterial metabolic activity and ATP synthesis system were not severely impaired; however, the significant increase in extracellular ATP directly reflects leakage caused by increased membrane permeability. This result corroborates the aforementioned membrane depolarization and increased PI uptake, and is consistent with the decreasing trend of staphylococcal flavin production, indicating that this pigment plays a crucial role in maintaining the low-permeability state of the membrane.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Application of shikimic acid in inhibiting the synthesis of staphylococcal flavin in methicillin-resistant Staphylococcus aureus JE2 for non-disease treatment purposes without affecting the viability of methicillin-resistant Staphylococcus aureus.

2. Application of shikimic acid in the preparation of antibacterial agents that inhibit staphylococcal flavin synthesis in methicillin-resistant Staphylococcus aureus (S. aureus) JE2 without affecting its viability.

3. The application according to claim 1 or 2, characterized in that, The concentration of shikimic acid is 1000~3000 mg / L.

4. The application according to claim 3, characterized in that, Shikimic acid downregulates JE2 strain crtM, crtN, crtO crtP, crtQ Gene expression levels.

5. Application of shikimic acid in improving bacterial cell membrane permeability and fluidity without affecting the viability of methicillin-resistant Staphylococcus aureus JE2.

Images