Bacteriostatic composition and application thereof in control of staphylococcus aureus pollution

Abstract

The invention discloses a bacteriostatic composition which comprises a synergist and a bacteriostatic agent, the synergist is ethyl phenylacetate or a derivative thereof, and the bacteriostatic agent is selected from hydrogen peroxide, beta-lactam antibiotics, macrolide antibiotics and aminoglycoside antibiotics. The synergist disclosed by the invention is wide in synergistic effect spectrum range, and has a remarkable synergistic bacteriostatic effect when being combined with hydrogen peroxide, amoxicillin, ampicillin, benzylpenicillin potassium, erythromycin, azithromycin and amikacin; the antibacterial effect is weak when the compound is independently used, but the virulence of staphylococcus aureus can be remarkably reduced, the formation of hemolysin, staphylococcus flavin, enterotoxin and a biofilm can be inhibited, and meanwhile, the selective pressure on bacteria is favorably reduced due to the relatively low antibacterial activity, so that the occurrence of drug resistance is delayed; the method is suitable for various application scenes such as food processing industry.

Description

Technical Field

[0001] The present invention belongs to the technical field of bacteriostasis, and particularly relates to a bacteriostatic composition and its application in controlling Staphylococcus aureus contamination. Background Art

[0002] Staphylococcus aureus has the characteristics of being resistant to low temperature, high osmotic pressure and heat, and is easy to survive in various food environments. It is easy to contaminate foods rich in protein or starch, such as milk, meat, eggs, fish and their products, etc., and secrete enterotoxin under suitable conditions, causing food poisoning in consumers, with symptoms such as nausea, vomiting, diarrhea, etc. In severe cases, it may lead to dehydration, shock and even death. In the food supply chain, effectively controlling the contamination of Staphylococcus aureus is beneficial to preventing food poisoning incidents and improving food safety.

[0003] Methicillin-resistant Staphylococcus aureus (MRSA) acquires resistance genes (such as blaZ, mecA genes) encoding β-lactamase and penicillin-binding protein (PBP2a), enabling the hydrolysis of antibiotics or preventing them from binding to the key enzymes for bacterial cell wall synthesis, thereby achieving resistance to β-lactam antibiotics. In addition, MRSA strains can also develop tolerance to various disinfectants and antibiotics through the action of biofilms or efflux pumps. Therefore, effectively inhibiting the tolerance of MRSA strains to disinfectants or antibiotics is of great significance for preventing foodborne MRSA contamination.

[0004] A synergist is a compound used in combination with other bactericides or bacteriostatic agents. Synergists usually do not have direct bacteriostatic activity, but enhance the efficacy of bacteriostatic agents by blocking the mechanisms by which bacteria tolerate bacteriostatic agents. So far, only a few synergists have been approved for use. For example, a composite disinfectant composed of soluble colloidal silver and hydrogen peroxide can enhance the killing effect on bacterial spores and is widely used in drinking water and swimming pool water disinfection; clavulanic acid, as a β-lactamase inhibitor, can inhibit the hydrolysis of the β-lactam ring in penicillin, thereby reversing bacterial drug resistance. Most synergists still have problems such as high toxicity to humans, narrow synergistic spectrum, and easy induction of bacterial drug resistance, thus being restricted in use. Synergists can enhance the activity of traditional antibiotics or disinfectants and reduce their dosage. Therefore, developing more effective bacteriostatic synergists has important application value.

[0005] The virulence factors of Staphylococcus aureus play a key role in its pathogenicity. These virulence factors help the bacteria attach to host cells and evade the host immune response. For example, hemolysin is an exotoxin secreted by Staphylococcus aureus, which can not only lyse red blood cells, but also damage epithelial cells, lymphocytes and monocytes, thus disrupting the immune system; staphyloxanthin has a highly conjugated isoprene structure, which can reduce the oxidative stress of disinfectants on Staphylococcus aureus; biofilms can help Staphylococcus aureus attach to various surfaces and resist the harsh surrounding environment, hindering the penetration of disinfectants to enhance the survival ability of bacteria. Inhibiting the virulence factors of Staphylococcus aureus can not only reduce the attachment of Staphylococcus aureus in the food processing industry and its resistance to disinfectants, but also reduce the harm to the human body. Therefore, screening for virulence inhibitors of Staphylococcus aureus is an effective strategy to control foodborne Staphylococcus aureus contamination. Summary of the Invention

[0006] Through small molecule screening, the inventors of the present application found that the parent ring structure of ethyl phenylacetate (EPA) has a wide spectrum of bacteriostatic synergistic effects and can inhibit the formation of hemolysin, enterotoxin, staphyloxanthin and biofilms of Staphylococcus aureus, thereby reducing its pathogenicity, and thus completed the present invention.

[0007] In a first aspect, the present invention provides a bacteriostatic composition comprising a synergist and a bacteriostatic agent, wherein the synergist is ethyl phenylacetate or a derivative thereof, and the bacteriostatic agent is selected from hydrogen peroxide, β-lactam antibiotics, macrolide antibiotics and aminoglycoside antibiotics.

[0008] In some embodiments, the bacteriostatic agent is selected from at least one of hydrogen peroxide, amoxicillin, ampicillin, potassium penicillin, erythromycin, azithromycin and amikacin.

[0009] Ethyl phenylacetate naturally exists in honey, cocoa and bread, and can also be synthesized by chemical methods. It has a strong aromatic odor and is widely used in tobacco, soap, cosmetics and food flavors. According to the safety assessment report of the Research Institute for Fragrance Materials (RIFM), ethyl phenylacetate is classified as a Cramer I compound (low toxicity) and has high safety. In some embodiments, the ethyl phenylacetate derivatives include ethyl 4-hydroxyphenylacetate, methyl 3,4-dihydroxyphenylacetate, ethyl 4-aminophenylacetate, and ethyl 3,5-dimethoxyphenylacetate.

[0010] In a second aspect, the present invention provides the use of the bacteriostatic composition in controlling foodborne Staphylococcus aureus contamination.

[0011] In some embodiments, the Staphylococcus aureus is ATCC BAA1717. Further, in the antibacterial composition, the concentration of ethyl phenylacetate is 0.32 to 1.28 mg / mL.

[0012] In some embodiments, the antibacterial composition comprises any one of the following:

[0013] 1) Ethyl phenylacetate at a concentration of 1.28 mg / mL and hydrogen peroxide at a concentration of 0.21 mmol / L;

[0014] 2) Ethyl phenylacetate at a concentration of 0.64 to 1.28 mg / mL and amoxicillin at a concentration of 1024 μg / mL;

[0015] 3) Ethyl phenylacetate at a concentration of 0.64 to 1.28 mg / mL and ampicillin at a concentration of 256 μg / mL;

[0016] 4) Ethyl phenylacetate at a concentration of 0.64 to 1.28 mg / mL and potassium penicillin at a concentration of 256 μg / mL;

[0017] 5) Ethyl phenylacetate at a concentration of 0.64 to 1.28 mg / mL and amikacin at a concentration of 4 μg / mL;

[0018] 6) Ethyl phenylacetate at a concentration of 0.32 mg / mL and erythromycin at a concentration of 8 μg / mL;

[0019] 7) Ethyl phenylacetate at a concentration of 0.32 mg / mL and azithromycin at a concentration of 16 μg / mL.

[0020] In a third aspect, the present invention provides a method for attenuating the production of virulence factors of Staphylococcus aureus, comprising: co-incubating a synergist with a bacterial solution, the initial concentration of the bacterial solution being 10 6 CFU / mL, incubating with shaking at 180 r / min, the incubation temperature being 37 °C, and the incubation time being 8 to 30 h, effectively reducing the production of virulence factors of Staphylococcus aureus.

[0021] In some embodiments, the synergist is ethyl phenylacetate.

[0022] In some embodiments, the virulence factors include hemolysin, staphyloxanthin, biofilm, and enterotoxin, and the enterotoxin includes enterotoxin proteins SeQ, SeK, and SelX.

[0023] In some embodiments, the required synergist concentration and incubation time vary depending on the purpose of virulence factor inhibition. For example, when Staphylococcus aureus is ATCC BAA1717, the ethyl phenylacetate concentration required to reduce the hemolytic activity of the culture supernatant by 65.8% is 0.32 mg / mL, and the incubation time is 16 hours; the ethyl phenylacetate concentration required to reduce staphyloxanthin synthesis by 86.7% is 0.256 mg / mL, and the incubation time is 30 hours; the ethyl phenylacetate concentration required to reduce biofilm formation by 32.5% is 0.16 mg / mL, and the incubation time is 24 hours. Furthermore, the ethyl phenylacetate concentration required to reduce the expression of enterotoxin proteins SeQ, SeK, and SelX by 50.3%, 44.2%, and 26.3%, respectively, is 0.32 mg / mL, and the incubation time is 8 hours.

[0024] Compared with the prior art, the present invention has the following technical effects:

[0025] The present invention provides a lead compound, ethyl phenylacetate, which has a dual inhibitory effect on the resistance and pathogenicity of Staphylococcus aureus to antibacterial agents. Its derivatives have similar biological activities and can be used for molecular structure modification to optimize activity. In addition, ethyl phenylacetate is easy to obtain and has a low cost.

[0026] The synergist provided by the present invention for controlling foodborne Staphylococcus aureus contamination has a wide synergistic action spectrum and exhibits a significant synergistic antibacterial effect when used in combination with hydrogen peroxide, amoxicillin, ampicillin, penicillin potassium, erythromycin, azithromycin and amikacin.

[0027] The synergist provided by the present invention has a weak antibacterial effect when acting alone, but can reduce the virulence of Staphylococcus aureus and inhibit the formation of hemolytic toxins, staphyloxanthin, enterotoxins, and biofilms. At the same time, the low antibacterial activity helps reduce the selective pressure on bacteria, thereby delaying the emergence of drug resistance. In addition, ethyl phenylacetate has almost no toxicity and is highly safe, making it suitable for various application scenarios such as the food processing industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The structure of ethyl phenylacetate is shown ( Figure 1 Effects of different concentrations of ethyl phenylacetate (EPA) on the growth of Staphylococcus aureus in A) and Example 1;

[0029] Figure 2 The time-kill curve of ethyl phenylacetate and antibiotics synergistically inhibiting Staphylococcus aureus in Example 3 is shown;

[0030] Figure 3 The checkerboard method heat map results of the synergistic inhibition of Staphylococcus aureus by ethyl phenylacetate and antibiotics in Example 3 are shown. DETAILED DESCRIPTION

[0031] The specific implementation manners of the present invention will be clearly described below in conjunction with embodiments. It is clarified here that the described embodiments are only a part of the present invention. Without departing from the core molecular structure of ethyl phenylacetate, other non-creative embodiments obtained by those of ordinary skill in the art all fall within the protection scope of the present invention. In the following embodiments, three biological replicate experiments are set. Unless otherwise specified, the experimental methods are all conventional experimental methods, and the experimental materials are also purchased from conventional biochemical reagent companies.

[0032] Example 1

[0033] Effect of ethyl phenylacetate on the growth of Staphylococcus aureus.

[0034] Experimental materials: Ethyl phenylacetate, purchased from Shanghai Jiji Biochemical Technology Co., Ltd., with a purity of 99%.

[0035] Experimental strain: Staphylococcus aureus ATCC BAA1717, purchased from the American Type Culture Collection.

[0036] Experimental method: The minimum inhibitory concentration (MIC) of ethyl phenylacetate against Staphylococcus aureus was determined using the two-fold broth dilution method and the growth curve.

[0037] Two-fold broth dilution method: After the ATCC BAA1717 strain was cultured overnight in TSB medium at 37°C, the bacterial suspension was diluted with TSB to an OD600nm of 0.5 ± 0.02. The stock solution of ethyl phenylacetate (dissolved in 1% DMSO) was filtered and sterilized with a 0.22 μm filter membrane, and then serially diluted with TSB to the required working concentrations (5.12, 2.56, 1.28, 0.64, ½ 0.32, 0.16 mg / mL). 198 μL of TSB solution and 2 μL of the diluted bacterial solution were added to each well of a 96-well plate. Subsequently, the 96-well plate was placed in a constant temperature incubator at 37°C for 24 hours. TSB containing 1% DMSO was used as a blank control. The OD600nm values of each well were measured using a spectrophotometer before and after incubation. The minimum inhibitory concentration (MIC) was defined as the lowest drug concentration at which no bacterial growth was visually determined. All experiments were performed in triplicate biologically.

[0038] Growth curve determination: The overnight culture of ATCC BAA1717 strain was diluted to an OD600nm of 0.50 ± 0.02 and then inoculated into TSB medium with or without ethyl phenylacetate (final concentrations of 0.16 mg / mL, 0.32 mg / mL, 0.64 mg / mL, 1.28 mg / mL, and 2.56 mg / mL respectively). The culture solution was placed in a growth curve analyzer and shaken at 180 r / min at 37°C for 36 hours, and the OD600nm value was measured every 30 minutes to monitor the growth of bacteria. TSB containing 1% DMSO was used as a blank control, and each group had 5 parallel replicates. The entire experiment was repeated 3 times.

[0039] The experimental results are as Figure 1 shown. The results of the two-fold broth dilution method showed that the MIC value of ethyl phenylacetate was 5.12 mg / mL. Figure 1 Figure B shows the growth curves of ATCC BAA1717 in an environment with different concentrations of ethyl phenylacetate. The results of the growth curve determination showed that ethyl phenylacetate had no direct antibacterial activity against Staphylococcus aureus at a concentration of 2.56 mg / mL.

[0040] The results showed that ethyl phenylacetate did not directly inhibit the growth of Staphylococcus aureus. Reducing the selective pressure on bacteria can avoid over-concentrating the survival advantages on a few drug-resistant strains, thereby reducing the reproduction rate of drug-resistant bacteria, delaying their spread in the population, and ultimately slowing down the emergence and development of drug resistance. Therefore, ethyl phenylacetate has the potential to be used as a synergist.

[0041] Example 2

[0042] Synergistic antibacterial effect of ethyl phenylacetate and hydrogen peroxide on Staphylococcus aureus.

[0043] Experimental materials: Ethyl phenylacetate, purchased from Shanghai Jizhi Biochemical Technology Co., Ltd., with a purity of 99%; hydrogen peroxide, with a concentration of 30%, purchased from Nanjing Chemical Reagent Co., Ltd.

[0044] Experimental strain: Staphylococcus aureus ATCC BAA1717, purchased from the American Type Culture Collection.

[0045] Experimental method: The synergistic activity of ethyl phenylacetate and hydrogen peroxide was evaluated by the two-fold broth dilution method and the checkerboard assay.

[0046] Two-fold broth dilution method: After culturing the ATCC BAA1717 strain overnight at 37 °C in TSB medium, the bacterial suspension was diluted with TSB to an OD600nm of 0.5 ± 0.02. Hydrogen peroxide was filtered and sterilized through a 0.22 μm filter membrane, and then serially diluted with TSB to the required working concentrations (3.36 mM, 1.68 mM, 0.84 mM, 0.42 mM, 0.21 mM, 0.105 mM). 198 μL of the TSB solution containing hydrogen peroxide and 2 μL of the diluted bacterial solution were added to each well of a 96-well plate. Subsequently, the 96-well plate was incubated in a 37 °C constant temperature incubator for 24 hours. TSB containing 1% DMSO was used as a blank control. The OD600nm values of each well were measured using a spectrophotometer before and after incubation. The minimum inhibitory concentration (MIC) was defined as the lowest drug concentration at which no bacterial growth was visually observed. All experiments were performed with three biological replicates.

[0047] Checkerboard assay: The checkerboard assay was used to evaluate the combined antibacterial effect between hydrogen peroxide and ethyl phenylacetate. In the experiment, ethyl phenylacetate and hydrogen peroxide were first serially diluted in a two-fold decreasing gradient and arranged in a 96-well plate in a row-column manner, so that each well contained different combined concentrations of the two drugs. Then, the same volume of the bacterial suspension (OD600nm = 0.5 ± 0.02) was added to each well to make the final volume consistent. After incubating the 96-well plate at 37 °C for 24 hours, the bacterial growth was observed, and it was recorded whether each well was antibacterial. The FICI (Fractional Inhibitory Concentration Index) value was calculated based on the MIC values to determine the interaction relationship between the two drugs. The calculation and determination criteria of the FICI index are as follows:

[0048] FICI = (MIC of drug A in combination) / (MIC of drug A alone) + (MIC of drug B in combination) / (MIC of drug B alone)

[0049] FICI ≤ 0.5 indicates a synergistic effect; 0.5 < FICI ≤ 1 indicates an additive effect; 1 < FICI ≤ 2 indicates no interaction; FICI > 2 indicates an antagonistic effect.

[0050] The experimental results are shown in Table 1.

[0051] Table 1

[0052] Synergist MICa (mmol / L) FICI MICb (mmol / L) Synergistic multiple Ethyl phenylacetate 0.84 0.5 0.21 4

[0053] In Table 1, MICa represents the MIC value of hydrogen peroxide against ATCC BAA1717 in the absence of ethyl phenylacetate; MICb represents the MIC value of hydrogen peroxide against ATCC BAA1717 in the presence of 1.28 mg / mL of ethyl phenylacetate. The FICI value represents the synergistic antibacterial index of hydrogen peroxide and ethyl phenylacetate. It can be seen that 1.28 mg / mL of ethyl phenylacetate can reduce the MIC value of hydrogen peroxide against ATCC BAA1717 by 4 times.

[0054] Example 3

[0055] Synergistic antibacterial effects of ethyl phenylacetate and its derivatives with β-lactam antibiotics, macrolide antibiotics, and amikacin against Staphylococcus aureus.

[0056] Experimental materials: Ethyl phenylacetate was purchased from Shanghai Jizhi Biochemical Technology Co., Ltd., with a purity of 99%.

[0057] Experimental strain: Staphylococcus aureus ATCC BAA1717 was purchased from the American Type Culture Collection.

[0058] Experimental method: The synergistic antibacterial activities of ethyl phenylacetate and its derivatives with β-lactam antibiotics, macrolide antibiotics, and amikacin were evaluated by the two-fold broth dilution method, checkerboard assay, and time-kill curve.

[0059] The specific steps of the two-fold concentration gradient dilution method and the checkerboard assay were the same as those in Example 2.

[0060] Time-kill curve: After the ATCC BAA1717 strain was cultured overnight in TSB medium at 37 °C, the bacterial suspension was diluted with TSB to an OD600nm of 0.5 ± 0.02. Then the bacterial suspension was added to TSB media containing ethyl phenylacetate, 1 / 4 MIC antibiotic, and the combination (combo) of ethyl phenylacetate and antibiotic, respectively. Each group was cultured at 37 °C, and samples were taken at 0, 3, 6, 12, and 24 hours. The colony-forming units (CFU / mL) were counted by plate spreading, and the bactericidal curve of the number of colonies changing with time was plotted to compare the effects of different treatments on bacterial growth. Three biological replicates were performed for each combination.

[0061] Table 2

[0062]

[0063] In Table 2, MIC is the minimum inhibitory concentration of the antibiotic against ATCC BAA1717, obtained by the two-fold broth dilution method, and FICI is the synergistic antibacterial index of the antibiotic and ethyl phenylacetate, obtained by the checkerboard assay.

[0064] Figure 2 The time-kill curves of ethyl phenylacetate against ATCC BAA1717 strain under the treatment of amoxicillin, ampicillin, penicillin potassium, amikacin, erythromycin and azithromycin alone or in combination were shown respectively. The results showed that the combination of 1.28 mg / mL ethyl phenylacetate and 1 / 4 MIC of amoxicillin or ampicillin could completely inhibit the growth of ATCC BAA1717 within 12 h; the combination of 0.64 mg / mL ethyl phenylacetate, 1 / 4 MIC of penicillin potassium and azithromycin reduced the bacterial count in the combined treatment group by 4 logarithmic values compared with the antibiotic alone treatment group; the combination of 0.32 mg / mL ethyl phenylacetate and 1 / 4 MIC of erythromycin reduced the bacterial count in the combined treatment group by 4 logarithmic values compared with the antibiotic alone treatment group.

[0065] Figure 3 The checkerboard heat map results of the combined antibacterial effects of ethyl phenylacetate with amoxicillin, ampicillin, penicillin potassium, amikacin, erythromycin and azithromycin were shown respectively. The results showed that when combined with 0.64 mg / mL ethyl phenylacetate, the MIC values of macrolide antibiotics (erythromycin and azithromycin) decreased by 4-fold, and the MIC values of β-lactam antibiotics (amoxicillin, ampicillin and penicillin potassium) decreased by 4 - 8-fold. When combined with 1.28 mg / mL ethyl phenylacetate, the MIC value of amikacin decreased by 8-fold.

[0066] This example also evaluated the antibiotic synergistic effect of ethyl phenylacetate derivatives, and the effects of ethyl 4-hydroxyphenylacetate, methyl 3,4-dihydroxybenzoate, ethyl 4-aminophenylacetate and ethyl 3,5-dimethoxybenzoate combined with antibiotics against ATCC BAA1717 were tested in vitro. As shown in Table 3, the ethyl phenylacetate derivatives had a weak effect on the growth of Staphylococcus aureus (MIC ≥ 2.048 mg / mL).

[0067] Table 3

[0068]

[0069] When used in combination with specific antibiotics, these derivatives exhibit significant synergistic effects. In particular, ethyl 4-hydroxyphenylacetate and methyl 3,4-dihydroxyphenylacetate show fractional inhibitory concentration index (FICI) values of 0.125 - 0.5 against β-lactam antibiotics (amoxicillin, ampicillin, potassium penicillin), macrolide antibiotics (erythromycin, azithromycin), and aminoglycoside antibiotics (amikacin, kanamycin sulfate) against ATCC BAA1717. This indicates that ethyl 4-hydroxyphenylacetate and methyl 3,4-dihydroxyphenylacetate can significantly reverse the resistance of ATCC BAA1717 to the above antibiotics, and the mechanism may be to block the bacterial resistance mechanism to these antibiotics.

[0070] Example 4

[0071] Inhibitory effect of ethyl phenylacetate on virulence factors of Staphylococcus aureus in vitro.

[0072] Experimental materials: Ethyl phenylacetate was purchased from Shanghai Jiji Biochemical Technology Co., Ltd. with a purity of 99%; 2% sheep red blood cells were purchased from Nanjing Senbeijia Biotechnology Co., Ltd.; methanol was purchased from Yonghua Chemical Co., Ltd. with a content of ≥99.8%; 1,6-bis(diphenylphosphino)hexane (DPPH) was purchased from Sigma-Aldrich Trading Co., Ltd. with a superior grade of purity; crystal violet was purchased from Shanghai Macklin Biochemical Co., Ltd. with an analytical grade of purity.

[0073] Experimental strain: Staphylococcus aureus ATCC BAA1717 was purchased from the American Type Culture Collection.

[0074] Bacterial culture conditions: The initial concentration of the bacterial solution was 10 6 CFU / mL, incubated with shaking at 180 r / min, and the incubation temperature was 37°C.

[0075] Experimental method:

[0076] Hemolytic activity assay: ATCC BAA1717 was cultured in TSB without or with 0.32 mg / mL ethyl phenylacetate for 16 hours. After incubation, the culture was centrifuged at 8000 r / min for 5 min to obtain the bacterial supernatant. 500 μL of the supernatant was gently mixed with 500 μL of 2% sheep red blood cells and incubated at 37°C for 1 h. After incubation, the mixture was centrifuged at 3000 r / min for 10 min, and the supernatant was collected. The hemolytic activity was quantified by measuring the absorbance of the supernatant at 543 nm. A 1% Triton X-100 solution was used as a positive control, and PBS containing 0.16 mg / mL ethyl phenylacetate was used as a blank control. The experiment was performed with three biological replicates.

[0077] Staphyloxanthin determination: ATCC BAA1717 was inoculated into BHI medium with or without 0.256 mg / mL ethyl phenylacetate and cultured for 30 h. After incubation, 2 mL of the bacterial culture was centrifuged at 6000 r / min for 5 min, and the bacterial pellet was washed three times with PBS buffer, and the OD600nm of the bacterial suspension was adjusted to 1.00 ± 0.02. The bacterial pellet was resuspended in 800 μL of 99% methanol solution and incubated at 55 °C for 2 h in the dark. Then the mixture was centrifuged at 6000 r / min for 5 min, and 200 μL of the supernatant was collected. The absorbance was measured at 462 nm using a microplate reader. Three biological replicate experiments were performed.

[0078] Antioxidant capacity determination: The antioxidant capacity of the bacteria was evaluated using the DPPH free radical scavenging rate assay. ATCC BAA1717 was inoculated into BHI medium with or without 0.32 mg / mL ethyl phenylacetate and cultured for 30 h. After incubation, 2 mL of the bacterial culture was centrifuged at 6000 r / min for 5 min, and the bacterial pellet was washed three times with PBS buffer. Subsequently, ice bath sonication (200 W, on for 3 s and off for 10 s, 60 cycles) was performed. The resulting lysate was centrifuged to collect the supernatant. 30 μL of the sample supernatant and 170 μL of 0.1 mM DPPH solution were added to a 96-well plate and incubated at 37 °C for 30 min in the dark. PBS was used as the blank control. The absorbance was measured at 490 nm using a microplate reader. Three biological replicate experiments were performed.

[0079] Quantification of biofilm by crystal violet staining: On a 96-well plate, ATCC BAA1717 was inoculated into TSB medium with or without 0.16 mg / mL ethyl phenylacetate and cultured for 24 h. After the culture, the culture medium in each well was aspirated using a multi-channel pipette, and washed slowly 3 times with PBS to remove the residual culture medium and free bacteria in the wells. Subsequently, the 96-well plate was placed in a 50 °C oven for fixation for 15 min. 200 μL of 0.1% crystal violet solution was added to each well and incubated for 15 min for staining. Then the excess dye was washed away with 200 μL of PBS buffer. Then 200 μL of absolute ethanol was added to each well and incubated for 5 min for decolorization. After decolorization, the absorbance at 595 nm was measured using a microplate reader. Three biological replicate experiments were performed.

[0080] Detection of enterotoxin protein expression level: ATCC BAA1717 was inoculated into TSB medium with or without 0.32 mg / mL ethyl phenylacetate and cultured for 8 h. Subsequently, total bacterial proteins were extracted, quantified by the BCA method, and digested with trypsin. The resulting peptide segments were desalted, dried, and redissolved in iRT solution for subsequent mass spectrometry analysis. Approximately 500 ng of peptide segments were taken from each sample, separated by a Vanquish neo ultra-high performance liquid chromatography system coupled with an Astral mass spectrometer, and detected in the DIA mode. The obtained mass spectrometry data were analyzed using DIA-NN software against the Staphylococcus aureus UniProt database, and protein quantification was performed based on a 2 m / z isolation window and a 1% FDR threshold. After screening for differentially expressed proteins, the difference in enterotoxin protein expression of ATCC BAA1717 after treatment with ethyl phenylacetate was further evaluated.

[0081] The experimental results are shown in Table 4. Ethyl phenylacetate inhibits the hemolytic activity of the supernatant of Staphylococcus aureus cultures. When 0.32 mg / mL of ethyl phenylacetate was co-cultured with ATCC BAA1717 for 16 h, the hemolytic activity of the culture supernatant was reduced by 65.8%. The blank control showed that 0.32 mg / mL of ethyl phenylacetate itself had no hemolytic activity and had a certain degree of biosafety. Subsequently, the inhibitory effect of ethyl phenylacetate on staphyloxanthin in vitro was determined. The results showed that when 0.256 mg / mL of ethyl phenylacetate was incubated with the bacterial solution for 30 h, the formation of staphyloxanthin in ATCC BAA1717 was reduced by 86.7%. Since staphyloxanthin has antioxidant properties, this example further explored whether the inhibition of staphyloxanthin synthesis by ethyl phenylacetate would also reduce the overall antioxidant capacity of Staphylococcus aureus. The results showed that after co-culturing 0.32 mg / mL of ethyl phenylacetate with the bacterial solution for 30 h, the DPPH radical scavenging rate of ATCC BAA1717 decreased by 37.6% compared with the control group. This indicates that ethyl phenylacetate may inhibit the total antioxidant capacity of bacteria by inhibiting the synthesis of staphyloxanthin, thereby reducing the tolerance of Staphylococcus aureus to hydrogen peroxide. This example also determined the inhibitory effect of ethyl phenylacetate on the biofilm of Staphylococcus aureus in vitro. After co-culturing 0.16 mg / mL of ethyl phenylacetate with the bacterial solution for 24 h, the biofilm formation of ATCC BAA1717 decreased by 32.5% compared with the control group. Finally, this example analyzed the differentially expressed proteins of ATCC BAA1717 after treatment with ethyl phenylacetate by liquid chromatography-mass spectrometry. The results showed that after co-incubating 0.32 mg / mL of ethyl phenylacetate with ATCC BAA1717 for 8 h, the expression levels of enterotoxin proteins SeQ, SeK, and SelX were reduced by 50.3%, 44.2%, and 26.3%, respectively. In summary, co-incubation of ethyl phenylacetate with Staphylococcus aureus can inhibit the pathogenicity of the bacteria.

[0082] Table 4

[0083]

[0084] In Table 4, the inhibition rate is expressed as the mean ± standard deviation, and the p-value represents the significance probability value between the ethyl phenylacetate treatment group and the control group. p < 0.05 indicates that the results are statistically significant.

[0085] Example 5

[0086] Safety assessment of ethyl phenylacetate on A549 cells.

[0087] Experimental materials: Ethyl phenylacetate, purchased from Shanghai Jizhi Biochemical Technology Co., Ltd., with a purity of 99%; MTT cell proliferation and cytotoxicity detection kit, purchased from Beyotime Biotechnology Co., Ltd.; Trypan blue dye, with a concentration of 0.4%, purchased from Thermo Fisher Scientific Co., Ltd.; A549 cell complete medium (Ham's F-12K + 10% FBS + 1% P / S), purchased from Wuhan Punosai Life Science Co., Ltd.

[0088] Experimental cell line: Human non-small cell lung cancer cell line A549, purchased from Wuhan Punosai Life Science Co., Ltd.

[0089] Experimental method for cell viability detection: The cells were seeded in 96-well plates at a density of 2×10 4 cells / well. After the cells adhered, the medium was removed, and the cells were incubated with 200 μL of A549 complete medium containing 0.16 mg / mL, 0.32 mg / mL, 0.64 mg / mL, 1.28 mg / mL, and 2.56 mg / mL of ethyl phenylacetate at 37 °C for 24 h. Subsequently, the MTT cell proliferation and cytotoxicity detection kit was used to detect the cell viability. A549 cell complete medium was used as the control group, and the experiment was repeated three times biologically.

[0090] Experimental method for cell survival rate: The cells were seeded in 96-well plates at a density of 2×10 4 cells / well. After the cells adhered, the medium was removed, and the cells were incubated with 200 μL of A549 complete medium containing 0.16 mg / mL, 0.32 mg / mL, 0.64 mg / mL, 1.28 mg / mL, and 2.56 mg / mL of ethyl phenylacetate at 37 °C for 24 h. Subsequently, the 96-well plates were centrifuged at 500 xg for 5 min, and after carefully removing the supernatant, 50 μL of 0.025% trypsin solution was added to digest the adherent cells, and the cells were incubated in a 37 °C carbon dioxide incubator for 2 min. After the incubation, 150 μL of complete medium was added to terminate the digestion, and the cells were gently pipetted to mix evenly. 5 μL of the cell suspension was mixed with 5 μL of 0.4% trypan blue solution, added to the cell counting chamber, and the cell survival rate was calculated using the trypan blue mode of an automatic cell counter. A549 cell complete medium was used as the control group, and the experiment was repeated three times biologically.

[0091] The experimental results are shown in Table 5. Ethyl phenylacetate at 2.56 mg / mL did not cause a significant effect on the cell viability and survival rate of A549, indicating that ethyl phenylacetate has a certain degree of safety.

[0092] Table 5

[0093]

[0094] The p-value in Table 5 represents the significance probability value between the ethyl phenylacetate treatment group and the control group. A p < 0.05 indicates that the results are statistically significant.

[0095] The specific implementation and verification results of the present invention have been described in detail above to help understand the core methods and ideas of the present invention. However, the present invention is not limited to the above implementation. These implementation methods can also be subject to various changes and modifications. Without departing from the principles of the present invention and the core molecular structure of the compound, they still fall within the protection scope of the present invention.

[0096] In summary, without affecting the growth of Staphylococcus aureus, ethyl phenylacetate can enhance the antibacterial effects of hydrogen peroxide, β-lactam antibiotics, macrolide antibiotics, and amikacin against Staphylococcus aureus, while inhibiting the biological formation of hemolysin, staphyloxanthin, biofilm, and enterotoxin of Staphylococcus aureus, and has certain biological safety. Therefore, ethyl phenylacetate has a dual inhibitory effect on the antibacterial tolerance and virulence of Staphylococcus aureus, and can be used as a synergist to control the contamination of foodborne Staphylococcus aureus.

Claims

1. An antibacterial composition comprising a synergist and an antibacterial agent, wherein the synergist is ethyl phenylacetate or its derivative, and the antibacterial agent is selected from hydrogen peroxide, β-lactam antibiotics, macrolide antibiotics, and aminoglycoside antibiotics.

2. The antibacterial composition according to claim 1, wherein The antibacterial agent is selected from at least one of hydrogen peroxide, amoxicillin, ampicillin, potassium penicillin, erythromycin, azithromycin, and amikacin.

3. The antibacterial composition according to claim 1 or 2, wherein Ethyl phenylacetate derivatives include ethyl 4-hydroxyphenylacetate, methyl 3,4-dihydroxyphenylacetate, ethyl 4-aminophenylacetate, and ethyl 3,5-dimethoxyphenylacetate.

4. Use of the antibacterial composition according to any one of claims 1-3 in controlling foodborne Staphylococcus aureus contamination.

5. The application according to claim 4, wherein, The Staphylococcus aureus is ATCC BAA1717.

6. The use according to claim 5, wherein: In the antibacterial composition, the concentration of ethyl phenylacetate is 0.32-1.28 mg / mL.

7. The application according to claim 5, wherein, The antibacterial composition comprises any one of the following: 1) Ethyl phenylacetate at a concentration of 1.28 mg / mL and hydrogen peroxide at a concentration of 0.21 mmol / L; 2) Ethyl phenylacetate at a concentration of 0.64-1.28 mg / mL and amoxicillin at a concentration of 1024 μg / mL; 3) Ethyl phenylacetate at a concentration of 0.64-1.28 mg / mL and ampicillin at a concentration of 256 μg / mL; 4) Ethyl phenylacetate at a concentration of 0.64-1.28 mg / mL and potassium penicillin at a concentration of 256 μg / mL; 5) Ethyl phenylacetate at a concentration of 0.64-1.28 mg / mL and amikacin at a concentration of 4 μg / mL; 6) Ethyl phenylacetate at a concentration of 0.32 mg / mL and erythromycin at a concentration of 8 μg / mL; 7) Ethyl phenylacetate at a concentration of 0.32 mg / mL and azithromycin at a concentration of 16 μg / mL.

8. A method for attenuating the production of virulence factors of Staphylococcus aureus, comprising: Co-incubate the synergist with the bacterial solution, with the initial concentration of the bacterial solution being 10 6 CFU / mL, incubate with shaking at 180 r / min, the incubation temperature is 37 °C, and the incubation time is 8 - 30 h, effectively reducing the generation of virulence factors of Staphylococcus aureus.

9. The method according to claim 8, wherein The synergist is ethyl phenylacetate or its derivative, and the derivatives include ethyl 4-hydroxyphenylacetate, methyl 3,4-dihydroxyphenylacetate, ethyl 4-aminophenylacetate, and ethyl 3,5-dimethoxyphenylacetate.

10. The method according to claim 8 or 9, wherein The virulence factors include hemolysin, staphyloxanthin, biofilm, and enterotoxin.

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