Bacteriostatic composition for controlling staphylococcus aureus pollution and application thereof

Abstract

The invention discloses a bacteriostatic composition for controlling staphylococcus aureus pollution and application thereof, and relates to the field of food safety, the bacteriostatic composition comprises a bacteriostatic part and a synergist; the antibacterial part is aminoglycoside antibiotics and / or disinfectants, and the synergist is 4-nitrobenzaldehyde. The 4-nitrobenzaldehyde provided by the invention can obviously enhance the bacteriostatic activity of various aminoglycoside antibiotics (such as gentamicin, kanamycin, tobramycin, neomycin and streptomycin) and common disinfectants (such as hydrogen peroxide, nisin and sodium hypochlorite) on staphylococcus aureus, and has broad-spectrum synergistic capacity.

Description

Technical Field

[0001] This invention relates to the field of food safety, and more particularly to an antibacterial composition for controlling Staphylococcus aureus contamination and its application. Background Technology

[0002] With the widespread use of antimicrobial agents in the medical, livestock, and food industries, the problem of bacterial resistance to antimicrobial agents has become increasingly prominent, posing a significant threat to global public health security. Among these, the problem of antimicrobial resistance in foodborne pathogens is particularly serious, not only affecting food quality and safety control but also potentially leading to decreased treatment effectiveness, prolonged illness, and increased medical costs for foodborne bacterial infections. Staphylococcus aureus (Staphylococcus aureus) Staphylococcus aureus As a common opportunistic pathogen, it is widely present in the food processing environment. It has strong environmental adaptability and multiple tolerance characteristics, and is an important pathogenic source of foodborne bacterial contamination. It has become one of the key bacterial species in food safety control research.

[0003] Aminoglycoside antibacterial agents play a crucial role in the treatment of severe infections due to their excellent bactericidal ability and broad-spectrum activity. Their main mechanism of action involves binding to the 16S rRNA in the 30S subunit of bacterial ribosomes, interfering with protein synthesis, causing mistranslation, and ultimately leading to bacterial death. However, Staphylococcus aureus can reduce the influx efficiency of aminoglycoside antibacterial agents by regulating membrane potential and altering membrane structure. This intrinsic resistance mechanism, dependent on membrane potential barriers, results in persistent adaptive tolerance to aminoglycoside antibacterial agents, posing a significant challenge to overcome in current treatment.

[0004] To address the problem of antibacterial agent tolerance, a "synergistic antibacterial" strategy is considered a promising approach. Benzaldehyde compounds are typical aromatic aldehydes found in traditional Chinese medicine and plant essential oils, exhibiting good antibacterial and antioxidant biological activities. Among them, 4-nitrobenzaldehyde (NIT) is a functionalized derivative of benzaldehyde. The nitro group introduced into its molecular structure has a stronger electron absorption capacity, which helps to enhance its interaction with the cell membrane. However, research on the synergistic effect of 4-nitrobenzaldehyde and antibacterial agents is still lacking. Therefore, this invention provides a synergistic antibacterial strategy based on the combined application of 4-nitrobenzaldehyde and antibacterial agents. The introduction of 4-nitrobenzaldehyde can effectively enhance the antibacterial efficacy of antibacterial agents and effectively reduce their dosage, thereby delaying the development of bacterial tolerance.

[0005] Therefore, those skilled in the art are dedicated to developing an antibacterial composition for controlling Staphylococcus aureus contamination using 4-nitrobenzaldehyde as a synergist, thereby reducing the dosage of the antibacterial agent and delaying the development of bacterial tolerance. Summary of the Invention

[0006] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to develop an antibacterial composition for controlling Staphylococcus aureus contamination with 4-nitrobenzaldehyde as a synergist, thereby reducing the dosage of the antibacterial agent and delaying the development of bacterial tolerance.

[0007] To achieve the above objectives, the present invention provides an antibacterial composition for controlling Staphylococcus aureus contamination, comprising an antibacterial component and a synergist; the antibacterial component is an aminoglycoside antibiotic and / or a disinfectant, and the synergist is 4-nitrobenzaldehyde.

[0008] Furthermore, aminoglycoside antibiotics include gentamicin (GEN), kanamycin, tobramycin, and / or streptomycin.

[0009] Furthermore, the Staphylococcus aureus was the Newman strain.

[0010] Furthermore, the concentration of gentamicin was 0.12 μg / mL, and the concentration of 4-nitrobenzaldehyde was 128 μg / mL.

[0011] Furthermore, the concentration of kanamycin was 2 μg / mL, and the concentration of 4-nitrobenzaldehyde was 128 μg / mL.

[0012] Furthermore, the concentration of tobramycin was 0.12 μg / mL, and the concentration of 4-nitrobenzaldehyde was 128 μg / mL.

[0013] Furthermore, the concentration of neomycin was 0.5 μg / mL, and the concentration of 4-nitrobenzaldehyde was 128 μg / mL.

[0014] Furthermore, when the streptomycin concentration was 2 μg / mL, the 4-nitrobenzaldehyde concentration was 128 μg / mL.

[0015] Furthermore, the disinfectant is hydrogen peroxide, nisin, and / or sodium hypochlorite.

[0016] Furthermore, the Staphylococcus aureus was the Newman strain.

[0017] Further, the concentration of 4-nitrobenzaldehyde is 128 μg / mL; the concentration of hydrogen peroxide is 0.39 mmol / mL, or the concentration of nisin is 32 μg / mL, or the volume ratio of sodium hypochlorite is 0.03%.

[0018] Furthermore, 4-nitrobenzaldehyde is used as a synergist in the food processing industry. It can be compounded with hydrogen peroxide, nisin, or sodium hypochlorite and used as a spray for sterilization.

[0019] Furthermore, using 4-nitrobenzaldehyde as an auxiliary component, experiments verified that it does not readily induce adaptive tolerance in Staphylococcus aureus under continuous exposure. It exhibits good safety and low resistance risk, and can be used as a stable auxiliary component for broad-spectrum antibacterial agents in food protection scenarios requiring long-term antibacterial action, such as coatings, packaging, or environmental spraying.

[0020] Furthermore, 4-nitrobenzaldehyde exhibits high biocompatibility; treatment of the A549 cell line at a concentration of 300 μg / mL for 24 h did not show significant inhibition of A549 cell viability. The high biocompatibility of 4-nitrobenzaldehyde is beneficial for applications in the food processing industry and other similar sectors.

[0021] Furthermore, the combined use of 4-nitrobenzaldehyde and gentamicin can significantly eliminate persistent Staphylococcus aureus cells. This antibacterial composition significantly enhances the elimination of persistent bacteria, reduces the risk of recurrence, and is suitable for chronic infection control or high-risk food microbial control scenarios.

[0022] In a preferred embodiment 1 of the present invention, the determination of the effect of 4-nitrobenzaldehyde on the growth of foodborne pathogens is described in detail. In another preferred embodiment 2 of the present invention, the synergistic antibacterial effect of 4-nitrobenzaldehyde and aminoglycoside antibacterial agents on foodborne pathogens is described in detail. In another preferred embodiment 3 of the present invention, the synergistic antibacterial effect of 4-nitrobenzaldehyde and common disinfectants against Staphylococcus aureus is described in detail. In another preferred embodiment 4 of the present invention, the adaptive development assessment of the tolerance of 4-nitrobenzaldehyde to Staphylococcus aureus antibacterial agents is described in detail. In another preferred embodiment 5 of the present invention, the inhibitory effect of the combination of 4-nitrobenzaldehyde and gentamicin on Staphylococcus aureus cells is described in detail. In another preferred embodiment 6 of the present invention, the biosafety assessment of 4-nitrobenzaldehyde is described in detail; In another preferred embodiment 7 of the present invention, the inhibitory effect of the combination of 4-nitrobenzaldehyde and gentamicin on the cytotoxicity of Staphylococcus aureus is described in detail.

[0023] The beneficial technical effects of this invention are as follows: 1. The 4-nitrobenzaldehyde provided by this invention can significantly enhance the antibacterial activity of various aminoglycoside antibiotics (such as gentamicin, kanamycin, tobramycin, and streptomycin) and commonly used disinfectants (such as hydrogen peroxide, lactobacillus, and sodium hypochlorite) against Staphylococcus aureus, and has a broad-spectrum synergistic effect.

[0024] 2. The antibacterial composition of 4-nitrobenzaldehyde and gentamicin provided by the present invention can effectively eliminate ciprofloxacin-induced Staphylococcus aureus residual cells, solving the problem of weak elimination ability of traditional antibacterial agents against residual bacteria.

[0025] 3. The 4-nitrobenzaldehyde provided by this invention does not induce adaptive resistance in Staphylococcus aureus at sub-inhibitory concentrations and has good long-term safety.

[0026] 4. The 4-nitrobenzaldehyde provided by this invention has low cytotoxicity within the effective concentration range and good biological safety, making it suitable for various application scenarios such as the food processing industry.

[0027] 5. The antibacterial composition composed of 4-nitrobenzaldehyde and gentamicin provided by the present invention, in addition to having a significant synergistic bactericidal effect against Staphylococcus aureus, also exhibits excellent synergistic antibacterial effects against a variety of other foodborne pathogens, and has broad-spectrum antibacterial properties.

[0028] 6. The 4-nitrobenzaldehyde provided by this invention has advantages such as simple molecular structure, wide availability of raw materials, and low cost.

[0029] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0030] Figure 1 This is an adaptive development assessment of the tolerance of 4-nitrobenzaldehyde to Staphylococcus aureus antibacterial agents, which is a preferred embodiment of the present invention (4). Figure 2 This is the result of an evaluation experiment on the inhibitory effect of the combination of 4-nitrobenzaldehyde and gentamicin on Staphylococcus aureus cells in a preferred embodiment 5 of the present invention; Figure 3 This is the result of a biosafety assessment experiment of 4-nitrobenzaldehyde in a preferred embodiment 6 of the present invention; Figure 4 This is the experimental result of the inhibitory effect of the combination of 4-nitrobenzaldehyde and gentamicin on the cytotoxicity of Staphylococcus aureus in a preferred embodiment 7 of the present invention. Detailed Implementation

[0031] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0032] In the embodiments described in this invention, all experiments were performed in triplicate. Unless otherwise stated, the experimental methods followed standard operating procedures in the art, and all reagents and materials used were purchased from common biochemical reagent suppliers. The strains and their sources used in the embodiments are shown in Table 1.

[0033] Table 1. Strains and sources used in the examples

[0034] Example 1: Determination of the effect of 4-nitrobenzaldehyde on the growth of foodborne pathogens.

[0035] Experimental materials: 4-Nitrobenzaldehyde, purchased from Sigma Aldrich, India, 99% purity; tryptone soybean broth (TSB); Luria-Bertani broth (LB); 96-well sterile flat-bottomed microtiter plate; PBS buffer; sterile distilled water.

[0036] Experimental strains: Staphylococcus aureus ATCC BAA1717, Staphylococcus aureus Newman, Staphylococcus aureus ATCC 29213, Listeria monocytogenes ATCC BAA679, Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028.

[0037] Experimental Methods: The minimum inhibitory concentration (MIC) of 4-nitrobenzaldehyde against foodborne pathogens was determined using a two-fold serial dilution method in broth. First, bacteria were inoculated into their respective broth media (TSB for Gram-positive bacteria and LB for Gram-negative bacteria) and incubated overnight at 37°C with shaking. The following day, the bacterial suspension was diluted to 0.5 McFarland standard, and then further diluted to 1×10⁻⁶. 6 CFU / mL. The 4-nitrobenzaldehyde stock solution was prepared with methanol (10 mg / mL) and serially diluted twice with sterile broth to achieve concentrations ranging from 1 to 1024 μg / mL. 100 μL of each concentration of 4-nitrobenzaldehyde solution was mixed with 100 μL of standardized bacterial suspension and added to a 96-well microtiter plate. The plates were then incubated at 37°C for 24 h. The lowest concentration at which no bacterial growth was observed visually was recorded as the MIC (micronizable concentration). All experiments were performed in triplicate.

[0038] The experimental results are shown in Table 2. 4-Nitrobenzaldehyde exhibited broad-spectrum antibacterial activity against a variety of foodborne pathogens, with MICs ranging from 256 to 512 μg / mL. These results indicate that 4-nitrobenzaldehyde, as a single antibacterial agent, possesses a relatively broad basic antibacterial spectrum, especially showing strong antibacterial activity against Staphylococcus aureus, making it suitable as a core active ingredient in synergistic antibacterial compositions.

[0039] Table 2. Experimental results of the effect of 4-nitrobenzaldehyde on the growth of foodborne pathogens.

[0040] Example 2: Synergistic antibacterial effect of 4-nitrobenzaldehyde and aminoglycoside antibacterial agents against foodborne pathogens.

[0041] Experimental materials: 4-nitrobenzaldehyde, purchased from Sigma Aldrich, India, 99% purity; gentamicin, kanamycin, tobramycin, neomycin, and streptomycin, purchased from Shanghai Maclean Biochemical Technology Co., Ltd., 98% purity; tryptone soybean broth; sterile distilled water.

[0042] Experimental strains: Staphylococcus aureus ATCC BAA1717, Staphylococcus aureus Newman, Staphylococcus aureus ATCC 29213, Listeria monocytogenes ATCC BAA679, Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028.

[0043] Experimental Methods: The synergistic activity of 4-nitrobenzaldehyde with aminoglycoside antibacterial agents was evaluated using a two-fold serial dilution method in broth. Aminoglycoside antibacterial agents (gentamicin, kanamycin, tobramycin, neomycin, and streptomycin) were selected and prepared into two-fold serial dilutions using sterile tryptone soybean broth. The experiment was divided into two groups: one group added 4-nitrobenzaldehyde to the dilutions at a final concentration of 128 μg / mL (co-treatment group), and the other group did not add 4-nitrobenzaldehyde (control group). The two dilutions were aliquoted into 96-well microtiter plates, with 100 μL of the antibacterial agent solution and 100 μL of standardized bacterial suspension added to each well (final inoculum concentration 1×10⁻⁶). 6 (CFU / mL). After incubation at 37°C for 24 h, the MICs of each treatment against foodborne pathogens were detected and recorded using a microplate reader. Each experiment was performed in triplicate. The synergistic effect of 4-nitrobenzaldehyde on the activity of aminoglycoside antibacterial agents was evaluated by comparing the differences in MICs between the two groups and calculating the fold increase.

[0044] Experimental Results: As shown in Table 3, 128 μg / mL 4-nitrobenzaldehyde significantly enhanced the antibacterial activity of gentamicin against a variety of foodborne pathogens, especially Staphylococcus aureus and Salmonella typhimurium, with MIC reductions ranging from 8 to over 64 times. In other strains such as Listeria and Escherichia coli, the synergistic effect of 4-nitrobenzaldehyde on gentamicin also reached 8 to 32 times. These results indicate that the combination of 4-nitrobenzaldehyde and gentamicin possesses excellent broad-spectrum synergistic antibacterial activity, suitable for the effective control of various foodborne pathogens.

[0045] Table 3. Experimental results of the synergistic antibacterial effect of 4-nitrobenzaldehyde and gentamicin against foodborne pathogens.

[0046] In the table, MIC represents the minimum inhibitory concentration of gentamicin against various foodborne pathogens; "0 μg / mL NIT" represents the control group without the addition of 4-nitrobenzaldehyde, and "128 μg / mL NIT" represents the combined treatment group with the addition of 128 μg / mL 4-nitrobenzaldehyde.

[0047] To further verify the potential of 4-nitrobenzaldehyde as a broad-spectrum synergist, its antibacterial effect against Staphylococcus aureus was evaluated when used in combination with other aminoglycoside antibacterial agents. The results are shown in Table 4. 4-nitrobenzaldehyde significantly enhanced the antibacterial activity of various aminoglycoside antibacterial agents against Staphylococcus aureus at a concentration of 128 μg / mL. S. aureus The Newman strain showed that 4-nitrobenzaldehyde enhanced kanamycin, cyclophosphamide, and neomycin by 16, 32, and 32 times, respectively; for S. aureus In strain BAA1717, 4-nitrobenzaldehyde reduced the MICs of brucellosis and streptomycin by 16-fold and 8-fold, respectively. These results further demonstrate that 4-nitrobenzaldehyde can act as a synergistic enhancer for various aminoglycoside antibacterial agents, effectively improving their antibacterial activity against Staphylococcus aureus.

[0048] Table 4. Combined use of 4-nitrobenzaldehyde with other aminoglycoside antibacterial agents

[0049] Example 3: Synergistic antibacterial effect of 4-nitrobenzaldehyde and common disinfectants against Staphylococcus aureus.

[0050] Experimental materials: 4-nitrobenzaldehyde, purchased from Sigma Aldrich, India, purity 99%; hydrogen peroxide, purchased from Sinopharm Chemical Reagent Co., Ltd., purity 30%; nisin, purchased from TargetMol Chemicals, USA; sodium hypochlorite, purchased from Shanghai Maclean Biochemical Technology Co., Ltd., available chlorine ≥30%.

[0051] Experimental strains: Staphylococcus aureus ATCC BAA1717, Staphylococcus aureus Newman.

[0052] Experimental Methods: The synergistic activity of 4-nitrobenzaldehyde with commonly used disinfectants was evaluated using a two-fold serial dilution method in broth. Common disinfectants (hydrogen peroxide, nisin, and sodium hypochlorite) were selected and prepared into two-fold serial dilutions using sterile tryptone soybean broth. The experiment was divided into two groups: one group had 4-nitrobenzaldehyde added to the dilutions at a final concentration of 128 μg / mL (co-treatment group), and the other group did not add 4-nitrobenzaldehyde (control group). The two dilutions were aliquoted into 96-well microtiter plates, with 100 μL of disinfectant solution and 100 μL of standardized bacterial suspension added to each well (final inoculum concentration 1×10⁻⁶). 6 (CFU / mL). After incubation at 37°C for 24 h, the MIC of each treatment against Staphylococcus aureus was detected and recorded using a microplate reader. Each experiment was performed in triplicate. The synergistic effect of 4-nitrobenzaldehyde on the antibacterial activity of the disinfectant was evaluated by comparing the differences in MIC between the two groups and calculating the synergistic effect.

[0053] Experimental Results: As shown in Table 5, at a concentration of 128 μg / mL, 4-nitrobenzaldehyde significantly enhanced the antibacterial activity of various commonly used disinfectants against Staphylococcus aureus. When used in combination with 4-nitrobenzaldehyde, the MICs of hydrogen peroxide, nisin, and sodium hypochlorite were significantly reduced, with synergistic effects against BAA1717 and Newman strains ranging from 2 to 4 times. These results indicate that 4-nitrobenzaldehyde can significantly enhance the antibacterial effects of hydrogen peroxide, nisin, and sodium hypochlorite, demonstrating broad potential for application in food safety control.

[0054] Table 5 Synergistic antibacterial effect of 4-nitrobenzaldehyde and common disinfectants against Staphylococcus aureus

[0055] Example 4: Evaluation of the adaptive development of tolerance of 4-nitrobenzaldehyde to Staphylococcus aureus antibacterial agents.

[0056] Experimental materials: 4-nitrobenzaldehyde, purchased from Sigma Aldrich, India, 99% purity; gentamicin, purchased from Shanghai Maclean Biochemical Technology Co., Ltd., 98% purity; tryptone soybean broth; sterile distilled water.

[0057] Experimental strains: Staphylococcus aureus ATCC BAA1717, Staphylococcus aureus Newman.

[0058] Experimental Methods: To assess whether long-term exposure to 4-nitrobenzaldehyde induces antimicrobial resistance in Staphylococcus aureus, BAA1717 and Newman strains were inoculated into tryptone soybean broth containing 25 μg / mL 4-nitrobenzaldehyde. After static incubation at 37°C for 24 h, the inoculum was subcultured daily at a 1% inoculum volume into fresh medium containing the same concentration of 4-nitrobenzaldehyde for 3 consecutive days. From day 4 onwards, the concentration was gradually increased by 25 µg / mL each time until the sub-minimum inhibitory concentration (sub-MIC, 125 µg / mL) was reached, and the bacteria were cultured at this concentration for another 3 days. The control group was treated in the same manner in medium without 4-nitrobenzaldehyde. After the experiment ended on day 6, bacterial cultures were collected from both the experimental and control groups. The bacterial concentrations were uniformly adjusted, and the cultures were then exposed to gentamicin at a concentration of 1×MIC and to untreated control conditions, respectively. Samples were taken at 0, 1, 2, and 3 hours, diluted with PBS, and spread onto TSA plates. After incubation at 37°C for 24 hours, colony-forming units were counted to assess the development of the strain's antimicrobial tolerance to 4-nitrobenzaldehyde. All experiments were performed in triplicate to ensure data accuracy and reproducibility.

[0059] Experimental results: such as Figure 1 As shown, S. aureus Both BAA1717 and Newman strains showed significantly enhanced sensitivity to gentamicin at the MIC concentration after continuous exposure to 4-nitrobenzaldehyde. Compared with the control group, the Log CFU / mL values ​​at 2 and 3 h in the 4-nitrobenzaldehyde treatment group were significantly lower (P<0.05), and the two strains showed consistent performance. These results indicate that Staphylococcus aureus is less likely to develop adaptive tolerance to antimicrobial agents under long-term exposure to 4-nitrobenzaldehyde, and instead shows increased sensitivity to gentamicin. This fully demonstrates that 4-nitrobenzaldehyde has good application safety and is suitable for long-term use as a disinfectant in food industry environments.

[0060] Example 5: Inhibitory effect of the combination of 4-nitrobenzaldehyde and gentamicin on Staphylococcus aureus persistent cells.

[0061] Experimental materials: 4-nitrobenzaldehyde, purchased from Sigma Aldrich, India, 99% purity; gentamicin, purchased from Shanghai Maclean Biochemical Technology Co., Ltd., 98% purity; tryptone soybean broth; phosphate buffered saline (PBS, pH=7.4); sterile distilled water.

[0062] Experimental strains: Staphylococcus aureus ATCC BAA1717, Staphylococcus aureus Newman.

[0063] Experimental Methods: To evaluate the inhibitory effect of the combination of 4-nitrobenzaldehyde and gentamicin on Staphylococcus aureus cells. S. aureus BAA1717 and Newman strains were inoculated into tryptone soybean broth and cultured for 6 h until the late logarithmic growth phase. The bacterial concentration was then adjusted to 0.5 McFarland standard (final volume 4 mL). Ciprofloxacin at a final concentration of 10×MIC was added to the bacterial culture, and the culture was incubated at 37°C for 18 h to induce the formation of persistent bacteria. After treatment, 1 mL of the bacterial culture was used as the persistent bacteria control group. The remaining bacterial cultures were washed twice with phosphate-buffered saline (PBS, pH=7.4) to remove residual drugs. Then, 1 mL of the bacterial culture was added to each of the following groups: 1×MIC gentamicin (GEN treatment group), 1×MIC 4-nitrobenzaldehyde (NIT treatment group), and a combination of 1×MIC gentamicin and 4-nitrobenzaldehyde (combined treatment group). The cultures were then incubated at 37°C for another 18 h. Subsequently, the bacterial cultures of each group were serially diluted, and 5 μL of each culture was spread onto tryptone soybean agar plates. After incubation at 37°C for 24 h, colony-forming units (CFU) were counted to compare the clearance effects of different treatment groups on persistent bacteria. All experiments were performed in triplicate.

[0064] Experimental results: such as Figure 2 As shown in Part A, the survival of Staphylococcus aureus cells induced by ciprofloxacin showed significant differences under different treatment conditions. The colony count in the control group remained consistently high. In contrast, while the use of 1×MIC gentamicin (GEN-1X) or 1×MIC 4-nitrobenzaldehyde (NIT-1X) alone could reduce the colony count to some extent, the antibacterial effect was limited. However, when both were used in combination (GEN-NIT), the colony count decreased significantly, by more than 2 log compared to the control group (P<0.05), demonstrating a significantly better bactericidal effect than either single treatment. Part B presents the visualization results of the experiment. A dilution droplet inoculation method was used, with the bacterial suspension diluted 10-fold from top to bottom (10... 0 Up to 10 5In the figure, "1×" indicates the antibiotic concentration used is its minimum inhibitory concentration (MIC), and "-" indicates no addition. The figure shows the inhibitory effect of different treatment groups on bacterial growth, and the results are consistent with the CFU count in Part A. The experimental results fully demonstrate that the combination of 4-nitrobenzaldehyde and gentamicin has a significant synergistic antibacterial effect on persistent Staphylococcus aureus cells.

[0065] Example 6: Biosafety assessment of 4-nitrobenzaldehyde.

[0066] Experimental materials: 4-nitrobenzaldehyde, purchased from Sigma Aldrich, India, 99% purity; MTT cell proliferation and cytotoxicity assay kit, purchased from Shanghai Beyotime Biotechnology Co., Ltd.; A549 cell complete culture medium, purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0067] Experimental cell line: Human non-small cell lung cancer cell line A549, purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0068] Experimental method: A549 cells were cultured at a rate of 2 × 10⁻⁶. 4 Cells were seeded at a density of [number] cells / well in 96-well plates. After full adhesion, the original culture medium was discarded, and 200 μL of complete culture medium containing 0, 50, 100, 200, and 300 μg / mL 4-nitrobenzaldehyde was added. The plates were incubated at 37°C in a 5% CO2 incubator for 24 h. Subsequently, the cells were treated according to the MTT assay kit instructions, and cell viability was characterized by absorbance at 570 nm.

[0069] Experimental results: such as Figure 3 As shown, within the effective concentration range, 4-nitrobenzaldehyde had minimal impact on the viability of A549 cells. At low concentrations, the cell state was similar to the control group; even at higher concentrations, the cells maintained good viability without significant toxic reactions. These results indicate that 4-nitrobenzaldehyde exhibits good biocompatibility within the experimental concentration range, which is beneficial for its safe use in applications such as the food processing industry.

[0070] Example 7: Inhibitory effect of the combination of 4-nitrobenzaldehyde and gentamicin on Staphylococcus aureus cytotoxicity.

[0071] Experimental materials: 4-Nitrobenzaldehyde, purchased from Sigma Aldrich, India, 99% purity; Gentamicin, purchased from Shanghai Maclean Biotechnology Co., Ltd., 98% purity; Dulbecco's Modified Eagle basal medium (DMEM), purchased from Shanghai Maclean Biotechnology Co., Ltd.; Fetal bovine serum (FBS), purchased from Shanghai Yuanye Biotechnology Co., Ltd.; A549 cell complete culture medium, purchased from Wuhan Pronosai Life Science Co., Ltd.; Calcein / PI cell viability and cytotoxicity assay kit, purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0072] Experimental strains and cell lines: Staphylococcus aureus ATCC BAA1717; human non-small cell lung cancer cell line A549, purchased from Wuhan Pronosei Biotechnology Co., Ltd.

[0073] Experimental method: A549 cells were cultured at a rate of 2 × 10⁻⁶. 4 Cells were seeded at a density of cells / well in 96-well plates containing complete culture medium. After the cells had fully adhered, the original culture medium was discarded and replaced with DMEM medium containing 10% FBS. Staphylococcus aureus BAA1717 was then inoculated at a multiplicity of infection (MOI) of 100. According to the experimental design, three treatment conditions were set: treatment with 128 μg / mL 4-nitrobenzaldehyde alone for 6 h, treatment with 1 μg / mL gentamicin alone for 6 h, and treatment with a combination of 128 μg / mL 4-nitrobenzaldehyde and 1 μg / mL gentamicin for 6 h. The untreated group served as a control. After treatment, A549 cells were stained using the Calcein / PI cell viability and cytotoxicity assay kit, and their survival was observed using a laser confocal microscope.

[0074] Experimental results: Fluorescence microscopy observation results are as follows Figure 4 As shown, 4-nitrobenzaldehyde, alone or in combination with gentamicin, significantly improved cell viability (enhanced green fluorescence), while a large number of red fluorescent cells were observed in the infected control group, indicating cell necrosis. The results indicate that treatment with 4-nitrobenzaldehyde alone or in combination with gentamicin can effectively protect A549 cells from damage caused by Staphylococcus aureus infection.

[0075] The above embodiments and verification results fully illustrate the core ideas and methods of the present invention, aiming to help those skilled in the art better understand the present invention. However, the present invention is not limited to the above embodiments. Equivalent substitutions or non-substantial modifications made to the implementation methods without departing from the principles of the present invention and the core molecular structure of 4-nitrobenzaldehyde should all be covered within the protection scope of the present invention.

[0076] In summary, this invention combines 4-nitrobenzaldehyde with aminoglycoside antibacterial agents or commonly used disinfectants, which significantly enhances the antibacterial activity against Staphylococcus aureus, effectively eliminates its persistent cells, and reduces the risk of bacterial resistance. This antibacterial composition exhibits low cytotoxicity to host cells, good biosafety and application stability, and is suitable for food safety control, industrial disinfection, and other fields, providing an efficient, safe, and widely applicable solution for the prevention and control of foodborne pathogens.

[0077] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. An antibacterial composition for controlling Staphylococcus aureus contamination, characterized in that, The antibacterial composition comprises an antibacterial component and a synergist; the antibacterial component is an aminoglycoside antibiotic and / or a disinfectant, and the synergist is 4-nitrobenzaldehyde.

2. The antibacterial composition according to claim 1, characterized in that, The aminoglycoside antibiotics are gentamicin, kanamycin, tobramycin, neomycin, and / or streptomycin.

3. The antibacterial composition according to claim 2, characterized in that, The Staphylococcus aureus strain in question is the Newman strain.

4. The antibacterial composition according to claim 3, characterized in that, The concentration of gentamicin is 0.12 μg / mL, and the concentration of 4-nitrobenzaldehyde is 128 μg / mL.

5. The antibacterial composition according to claim 3, characterized in that, The concentration of kanamycin is 2 μg / mL, and the concentration of 4-nitrobenzaldehyde is 128 μg / mL.

6. The antibacterial composition according to claim 3, characterized in that, The concentration of tobramycin is 0.12 μg / mL, and the concentration of 4-nitrobenzaldehyde is 128 μg / mL.

7. The antibacterial composition according to claim 3, characterized in that, The concentration of neomycin is 0.5 μg / mL, and the concentration of 4-nitrobenzaldehyde is 128 μg / mL.

8. The antibacterial composition according to claim 3, characterized in that, When the streptomycin concentration is 2 μg / mL, the 4-nitrobenzaldehyde concentration is 128 μg / mL.

9. The antibacterial composition according to claim 1, characterized in that, The disinfectant is hydrogen peroxide, nisin, and / or sodium hypochlorite.

10. The antibacterial composition according to claim 9, characterized in that, The Staphylococcus aureus is the Newman strain; the concentration of 4-nitrobenzaldehyde is 128 μg / mL; the concentration of hydrogen peroxide is 0.39 mmol / mL, or the concentration of nisin is 32 μg / mL, or the volume ratio of sodium hypochlorite is 0.03%.

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