A composition for preventing and treating Stenotrophomonas maltophilia and its application

Abstract

The present invention discloses a composition for preventing and treating Stenotrophomonas maltophilia and its application, belonging to the technical field of prevention and treatment of Stenotrophomonas maltophilia. The active ingredients of the composition of the present invention contain protocatechuic aldehyde and chlorogenic acid, and the mass ratio of the protocatechuic aldehyde to the chlorogenic acid is 1:8. The composition has a good antibacterial effect on Stenotrophomonas maltophilia, can significantly reduce the usage amount of a single medicament, and has a synergistic effect. In addition, it can also reduce the drug resistance of Stenotrophomonas maltophilia to ampicillin. Therefore, it has important significance in the prevention and treatment of drug-resistant Stenotrophomonas maltophilia.

Description

Technical Field

[0001] The present invention belongs to the technical field of prevention and control of Stenotrophomonas maltophilia, and particularly relates to a composition for preventing and controlling Stenotrophomonas maltophilia and its application. Background Art

[0002] Stenotrophomonas maltophilia (S. maltophilia) is a conditional pathogen, belonging to non-fermenting Gram-negative bacilli, and is likely to infect patients with severe diseases, low immunity, and those using broad-spectrum antibacterial drugs. Moreover, the incidence and fatality rate of the infection show an upward trend every year. Stenotrophomonas maltophilia widely exists in nature such as water, soil, food, animals and plants, and has a complex drug resistance mechanism, with characteristics of multiple drug resistance, extensive drug resistance, and even pan-drug resistance to a variety of antibiotics. In particular, it is naturally resistant to β-lactam antibiotics and aminoglycoside drugs. Moreover, with the extensive application of antibiotics, its drug resistance is continuously increasing. In recent years, the infection caused by Stenotrophomonas maltophilia has attracted much attention, and the treatment of this bacterium in clinical practice is very difficult. Therefore, it is a trend to search for new drugs for treating the infection caused by Stenotrophomonas maltophilia. Summary of the Invention

[0003] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide a composition for preventing and controlling Stenotrophomonas maltophilia and its application.

[0004] To achieve the above purpose, the present invention adopts the following technical solutions:

[0005] A composition for preventing and controlling Stenotrophomonas maltophilia, the active ingredient contains protocatechuic aldehyde and chlorogenic acid, and the mass ratio of the protocatechuic aldehyde to the chlorogenic acid is 1:8.

[0006] In a specific embodiment, the active ingredient of the composition for preventing and controlling Stenotrophomonas maltophilia further contains β-lactam antibiotics.

[0007] In a specific embodiment, the β-lactam antibiotic is ampicillin.

[0008] In a specific embodiment, the mass ratio of the protocatechuic aldehyde, chlorogenic acid and ampicillin is 1:8:1.

[0009] Use of the above composition in the preparation of a drug for preventing and controlling Stenotrophomonas maltophilia.

[0010] Use of the above composition in the preparation of a drug for preventing and controlling diseases caused by Stenotrophomonas maltophilia.

[0011] Use of the above composition in reducing the drug resistance of Stenotrophomonas maltophilia to ampicillin or in the preparation of a drug for reducing the drug resistance of Stenotrophomonas maltophilia to ampicillin.

[0012] A drug for preventing and treating Stenotrophomonas maltophilia and / or diseases caused by Stenotrophomonas maltophilia, characterized in that it contains the above-mentioned composition. The drug for preventing and treating Stenotrophomonas maltophilia and / or diseases caused by Stenotrophomonas maltophilia further comprises a pharmaceutically acceptable carrier, solvent, diluent, excipient or other medium, and the dosage form of the drug is selected from powder, granule, capsule, injection, oral liquid or tablet.

[0013] Advantages of the technical solution of the present invention:

[0014] Protocatechuic aldehyde combined with chlorogenic acid in the composition of the present invention has a good inhibitory effect on Stenotrophomonas maltophilia:

[0015] (1) When protocatechuic aldehyde is combined with chlorogenic acid, the diameter of the inhibition zone is the largest, which proves that the combination of the two substances has a synergistic antibacterial effect. When protocatechuic aldehyde and chlorogenic acid are used alone, the minimum inhibitory concentration reaches 1024 μg / mL and 8192 μg / mL respectively. When protocatechuic aldehyde and chlorogenic acid are used in combination, the minimum inhibitory concentration is 32 μg / mL and 256 μg / mL respectively, and the combined antibacterial index is 0.5, indicating that the combination of the two substances can synergistically inhibit bacteria and improve the antibacterial effect;

[0016] (2) When ampicillin is used alone, the minimum inhibitory concentration reaches 256 μg / mL. When protocatechuic aldehyde and chlorogenic acid are combined with ampicillin, the minimum inhibitory concentrations are 32 μg / mL, 256 μg / mL and 32 μg / mL respectively, and the combined antibacterial index is 0.375, proving that protocatechuic aldehyde and chlorogenic acid can jointly inhibit bacterial growth with ampicillin;

[0017] (3) Protocatechuic aldehyde combined with chlorogenic acid can change cell permeability, and bacterial cell nucleic acid leaks;

[0018] (4) Observed by scanning electron microscopy and transmission electron microscopy, the integrity of the bacterial structure can be significantly damaged at the site where protocatechuic aldehyde is combined with chlorogenic acid;

[0019] (5) The use of protocatechuic aldehyde combined with chlorogenic acid has a certain effect on the formation of the biofilm of Stenotrophomonas maltophilia, but cannot completely prevent the formation of the biofilm, and the use of protocatechuic aldehyde combined with chlorogenic acid has a certain effect on the biofilm already formed by Stenotrophomonas maltophilia, but cannot completely destroy the already formed biofilm.

[0020] In summary, protocatechuic aldehyde combined with chlorogenic acid can effectively inhibit the growth of Stenotrophomonas maltophilia, and at the same time can improve the sensitivity of bacteria to ampicillin, and inhibit the growth of Stenotrophomonas maltophilia through various ways such as affecting cell permeability, the formation and stability of cell membranes, and destroying cell integrity. Description of the drawings

[0021] Figure 1 In vitro antibacterial results of different substances against Stenotrophomonas maltophilia (where 1 is the water treatment group, 2 is the gentamicin treatment group, 3 is the protocatechuic aldehyde combined with chlorogenic acid treatment group, 4 is the chlorogenic acid treatment group, and 5 is the protocatechuic aldehyde treatment group);

[0022] Figure 2 Effect of protocatechuic aldehyde combined with chlorogenic acid on nucleic acid leakage of Stenotrophomonas maltophilia;

[0023] Figure 3 Scanning electron micrographs of Stenotrophomonas maltophilia treated with water (left) and protocatechuic aldehyde combined with chlorogenic acid (right);

[0024] Figure 4 Transmission electron micrographs of Stenotrophomonas maltophilia treated with water (left) and protocatechuic aldehyde combined with chlorogenic acid (right);

[0025] Figure 5 Effect of protocatechuic aldehyde combined with chlorogenic acid on biofilm formation of Stenotrophomonas maltophilia;

[0026] Figure 6 Effect of protocatechuic aldehyde combined with chlorogenic acid on pre - formed biofilm of Stenotrophomonas maltophilia. Detailed implementation manners

[0027] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0028] The present invention will be further described in detail below with reference to specific examples and data. The following examples are only for illustrative purposes of the present invention and do not limit the scope of the present invention in any way.

[0029] Example 1

[0030] A composition for preventing and treating Stenotrophomonas maltophilia, the active ingredients of which contain protocatechuic aldehyde and chlorogenic acid, and the mass ratio of protocatechuic aldehyde to chlorogenic acid is 1:8.

[0031] Example 2

[0032] A composition for preventing and treating Stenotrophomonas maltophilia, the active ingredients of which contain protocatechuic aldehyde, chlorogenic acid and ampicillin, and the mass ratio of protocatechuic aldehyde, chlorogenic acid to ampicillin is 1:8:1.

[0033] Example 3 Bacteriostatic effect of protocatechuic aldehyde combined with chlorogenic acid on Stenotrophomonas maltophilia

[0034] 1. Bacteriostatic circle experiment

[0035] The antibacterial effect of protocatechuic aldehyde combined with chlorogenic acid against Stenotrophomonas maltophilia was determined by the pour plate method and the inhibition zone method. The bacteria were activated, centrifuged at 7000 r / min for 10 min at 4 °C, washed 3 times with PBS, and diluted with PBS to make the concentration of the bacterial suspension 1×10 5 CFU / mL. Take 700 μL of the bacterial suspension and add it to 100 mL of LB agar medium that has been heated and cooled. Mix well. Pour 25 - 30 mL of the medium into each petri dish. After the medium solidifies, use an 8 mm punch to make 5 holes. Add 200 μL of sterile water, 200 μL of gentamicin (80 μg / mL), 200 μL of protocatechuic aldehyde solution (4 mg / mL), 200 μL of chlorogenic acid solution (10 mg / mL), and 100 μL each of the protocatechuic aldehyde solution (4 mg / mL) and chlorogenic acid solution (10 mg / mL) mixed well into each hole. Place it in a 30 °C constant temperature incubator and incubate upright for 24 h. Then measure the diameter of each inhibition zone on the petri dish. Repeat three times for parallel experiments.

[0036] The results are as Figure 1 shown. Obvious inhibition zones appeared after treatment with protocatechuic aldehyde, chlorogenic acid, and protocatechuic aldehyde combined with chlorogenic acid, and no inhibition zone appeared in the water treatment group. This proves that protocatechuic aldehyde and chlorogenic acid can effectively inhibit the growth of Stenotrophomonas maltophilia. At the same time, compared with the groups treated with protocatechuic aldehyde and chlorogenic acid alone, the diameter of the inhibition zone in the group treated with protocatechuic aldehyde combined with chlorogenic acid was the largest, indicating that the antibacterial activity of protocatechuic aldehyde combined with chlorogenic acid against Stenotrophomonas maltophilia is more significant than that of using protocatechuic aldehyde and chlorogenic acid alone.

[0037] 2. Determination of the minimum inhibitory concentration of protocatechuic aldehyde and chlorogenic acid when used alone

[0038] (1) Determination of the minimum inhibitory concentration of protocatechuic aldehyde when used alone

[0039] Weigh an appropriate amount of protocatechuic aldehyde and dissolve it in 1 mL of sterile water to prepare a 4096 μg / mL aqueous solution of protocatechuic aldehyde. Then, use the serial dilution method to adjust the concentration of the aqueous solution of protocatechuic aldehyde to 2048 μg / mL, 1024 μg / mL, 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, and 1 μg / mL with SCDLP liquid medium. Each concentration solution is reserved for use.

[0040] First, add 100 μL of the bacterial suspension to each well of a 96-well microplate. Then, add 100 μL of protocatechuic aldehyde solutions at various concentrations to each well. Additionally, add 200 μL of SCDLP medium as a blank control well, and add a mixture of 100 μL of the bacterial suspension and 100 μL of SCDLP medium as a growth control well. Repeat each concentration three times for parallel experiments. After culturing in a constant temperature incubator at 30 °C for 48 h, place the 96-well microplate in a microplate reader and measure the absorbance value of each well at 600 nm.

[0041] (2) Determination of the minimum inhibitory concentration of chlorogenic acid when used alone

[0042] Weigh an appropriate amount of chlorogenic acid and dissolve it in 1 mL of sterile water to prepare a 65536 μg / mL chlorogenic acid aqueous solution. Then, use the SCDLP liquid medium to adjust the concentrations of the chlorogenic acid solution to 32786 μg / mL, 16384 μg / mL, 8192 μg / mL, 4096 μg / mL, 2048 μg / mL, 1024 μg / mL, 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, and 1 μg / mL respectively by the equal dilution method. Keep each concentration solution for later use.

[0043] First, add 100 μL of the bacterial suspension to each well of a 96-well microplate. Then, add 100 μL of chlorogenic acid solutions at various concentrations to each well. Additionally, add 200 μL of SCDLP medium as a blank control well, and add a mixture of 100 μL of the bacterial suspension and 100 μL of SCDLP medium as a growth control well. Repeat each concentration three times for parallel experiments. After culturing in a constant temperature incubator at 30 °C for 48 h, place the 96-well microplate in a microplate reader and measure the absorbance value of each well at 600 nm.

[0044] 3. Determination of the minimum inhibitory concentration of the combination of protocatechuic aldehyde and chlorogenic acid

[0045] The minimum inhibitory concentration (MIC) was determined by the checkerboard method when protocatechuic aldehyde and chlorogenic acid acted on Stenotrophomonas maltophilia in combination. According to the MICs of protocatechuic aldehyde and chlorogenic acid obtained from the above experiments when used alone, appropriate amounts of protocatechuic aldehyde and chlorogenic acid were weighed and dissolved in 1 mL of sterile water respectively to prepare a 4096 μg / mL aqueous solution of protocatechuic aldehyde and a 32768 μg / mL aqueous solution of chlorogenic acid. Then, by the method of serial dilution, the concentrations of the protocatechuic aldehyde solution were adjusted to 2048 μg / mL, 1024 μg / mL, 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL with SCDLP liquid medium; the concentrations of the chlorogenic acid solution were adjusted to 16384 μg / mL, 8192 μg / mL, 4096 μg / mL, 2048 μg / mL, 1024 μg / mL, 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL.

[0046] First, 100 μL of the bacterial suspension was added to each well of a 96-well microplate, and then 50 μL of the protocatechuic aldehyde solution at each concentration and 50 μL of the chlorogenic acid solution at each concentration were added to each well. Within the above selected range, different concentrations of protocatechuic aldehyde and different concentrations of chlorogenic acid were paired pairwise at arbitrary concentrations in different wells to exhaust all possible combinations. Additionally, 200 μL of SCDLP medium was added as a blank control well, and a mixture of 100 μL of the bacterial suspension and 100 μL of SCDLP medium was added as a growth control well. Each concentration was repeated three times for parallel experiments. After culturing in a constant temperature incubator at 30 °C for 48 h, the 96-well microplate was placed in a microplate reader to measure the absorbance values of each well at 600 nm.

[0047] Percentage of fungal growth = (OD value of each well - OD value of the blank control well) / (OD value of the growth control well - OD value of the blank control well) × 100%.

[0048] Percentage of fungal growth inhibition = 1 - percentage of fungal growth.

[0049] The MIC value was taken as the lowest inhibitory concentration with an inhibition rate of over 80% compared to the growth control well.

[0050] The calculation formula for the fractional inhibitory concentration index (FICI) is as follows:

[0051]

[0052] Where MIC 原儿茶醛(单独1) 、MIC绿原酸(单独1) represent the MIC values when protocatechuic aldehyde and chlorogenic acid are used alone, respectively. MIC 原儿茶醛(联合1) 、MIC 绿原酸(联合1) represent the MIC values when protocatechuic aldehyde and chlorogenic acid are used in combination, respectively.

[0053] The fractional inhibitory concentration index (FICI) is calculated according to formula (1). The criteria for the combined effect of drugs are as follows: when FICI is less than or equal to 0.5, it is a synergistic effect; when FICI is greater than 0.5 and less than or equal to 4, it is an indifferent effect; when FICI is greater than 4, it is an antagonistic effect.

[0054] Table 1 Antibacterial effect of the combination of protocatechuic aldehyde and chlorogenic acid against Stenotrophomonas maltophilia

[0055]

[0056] As can be seen from Table 1, when protocatechuic aldehyde and chlorogenic acid are used alone, the minimum inhibitory concentrations are 1024 μg / mL and 8192 μg / mL, respectively. When protocatechuic aldehyde and chlorogenic acid act on Stenotrophomonas maltophilia in combination, the minimum inhibitory concentrations are 256 μg / mL and 2048 μg / mL, respectively. The FICI calculated by formula (1) is 0.5, which proves that protocatechuic aldehyde combined with chlorogenic acid synergistically inhibits bacterial growth.

[0057] Example 4 Reduction of the resistance of Stenotrophomonas maltophilia to ampicillin by the combination of protocatechuic aldehyde and chlorogenic acid

[0058] 1. Determination of the minimum inhibitory concentration of ampicillin

[0059] Weigh an appropriate amount of ampicillin and dissolve it in 1 mL of sterile water to prepare an aqueous solution of ampicillin at 4096 μg / mL. Then, use the serial dilution method to adjust the concentration of the ampicillin solution to 2048 μg / mL, 1024 μg / mL, 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, and 1 μg / mL with SCDLP liquid medium. Each concentration solution is reserved for use.

[0060] First, add 100 μL of the bacterial suspension to each well of a 96-well microplate, then add 100 μL of each concentration of ampicillin solution to each well. Additionally, add 200 μL of SCDLP medium as a blank control well, and add a mixture of 100 μL of the bacterial suspension and 100 μL of SCDLP medium as a growth control well. Each concentration is repeated three times for parallel experiments. After culturing in a constant temperature incubator at 30 °C for 48 h, place the 96-well microplate in a microplate reader and measure the absorbance value of each well at 600 nm.

[0061] 2. Determination of the minimum inhibitory concentration when protocatechuic aldehyde, chlorogenic acid and ampicillin are used in combination

[0062] The minimum inhibitory concentration of protocatechuic aldehyde, chlorogenic acid and ampicillin in combination against Stenotrophomonas maltophilia was determined by the checkerboard method. Using the combination of protocatechuic aldehyde and chlorogenic acid as a whole and then combining it with ampicillin again, the minimum inhibitory concentrations of protocatechuic aldehyde and chlorogenic acid when combined and the minimum inhibitory concentration of ampicillin when used alone obtained from the above experiments were used. Appropriate amounts of protocatechuic aldehyde, chlorogenic acid and ampicillin were weighed and dissolved in 1 mL of sterile water respectively to prepare 2048 μg / mL protocatechuic aldehyde aqueous solution, 16384 μg / mL chlorogenic acid aqueous solution and 1024 μg / mL ampicillin aqueous solution. Then, by the method of equal dilution, the concentrations of the protocatechuic aldehyde solution were adjusted to 1024 μg / mL, 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL with SCDLP liquid medium; the concentrations of the chlorogenic acid solution were adjusted to 8192 μg / mL, 4096 μg / mL, 2048 μg / mL, 1024 μg / mL, 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL; the concentrations of the ampicillin solution were adjusted to 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, and each concentration was reserved for use.

[0063] First, 100 μL of bacterial suspension was added to each well of a 96-well microplate, then 25 μL of each concentration of protocatechuic aldehyde solution and 25 μL of each concentration of chlorogenic acid solution were added to each well, and finally 50 μL of each concentration of ampicillin solution was added. Within the above selected range, different concentrations of protocatechuic aldehyde, different concentrations of chlorogenic acid and different concentrations of ampicillin were randomly combined in threes in different wells to exhaust all possible combinations. Additionally, 200 μL of SCDLP medium was added as a blank control well, and a mixture of 100 μL of bacterial suspension and 100 μL of SCDLP medium was added as a growth control well. Each concentration was repeated three times for parallel experiments. After culturing in a constant temperature incubator at 30 °C for 48 h, the 96-well microplate was placed in a microplate reader to measure the absorbance value of each well at 600 nm.

[0064] The formula for calculating the fractional inhibitory concentration index (FICI) is as follows:

[0065]

[0066] In the formula, MIC 原儿茶醛(单独2) and MIC 绿原酸(单独2) respectively represent the MIC values when the two drugs, protocatechuic aldehyde and chlorogenic acid, are used in combination, and MIC 氨苄青霉素(单独2) represents the MIC value when ampicillin is used alone, and MIC 原儿茶醛(联合2) and MIC 绿原酸(联合2) and MIC 氨苄青霉素(联合2) respectively represent the MIC values when the three substances, protocatechuic aldehyde, chlorogenic acid, and ampicillin, are used in combination.

[0067] The fractional inhibitory concentration index (FICI) is calculated according to formula (2). Criteria for the results of drug combination: When FICI is less than or equal to 0.5, it is a synergistic effect; when FICI is greater than 0.5 and less than or equal to 4, it is an indifferent effect; when FICI is greater than 4, it is an antagonistic effect.

[0068] The results of the combination experiment are shown in Table 2:

[0069] Table 2 Antibacterial effects of the combination of protocatechuic aldehyde, chlorogenic acid, and ampicillin on Stenotrophomonas maltophilia

[0070]

[0071]

[0072] As can be seen from Table 2, when protocatechuic aldehyde is combined with chlorogenic acid and ampicillin is used alone, the minimum inhibitory concentrations are 256 μg / mL, 2048 μg / mL, and 256 μg / mL respectively. When the three drugs, protocatechuic aldehyde, chlorogenic acid, and ampicillin, are used in combination, the minimum inhibitory concentrations against Stenotrophomonas maltophilia are 32 μg / mL, 256 μg / mL, and 32 μg / mL respectively. The FICI calculated by formula (2) is 0.375, proving that protocatechuic aldehyde and chlorogenic acid can also jointly inhibit bacterial growth with ampicillin synergistically.

[0073] The above results show that the combination of protocatechuic aldehyde and chlorogenic acid not only has in vitro antibacterial activity against Stenotrophomonas maltophilia, but also can improve the sensitivity of Stenotrophomonas maltophilia to ampicillin and reduce the drug resistance of Stenotrophomonas maltophilia to ampicillin.

[0074] Example 5 Antibacterial mechanism of the combination of protocatechuic aldehyde and chlorogenic acid against Stenotrophomonas maltophilia

[0075] 1. Nucleic acid leakage

[0076] Activate Stenotrophomonas maltophilia, then centrifuge at 4°C and 7000 r / min for 10 min, wash three times with PBS, and adjust the bacterial concentration to 1×10 8 CFU / mL to obtain a standby bacterial suspension. According to the minimum inhibitory concentration of protocatechuic aldehyde and chlorogenic acid in combination obtained from the above experiments as MIC, weigh appropriate amounts of protocatechuic aldehyde and chlorogenic acid and dissolve them in 1 mL of sterile water respectively to prepare protocatechuic aldehyde solutions and chlorogenic acid solutions with a concentration of 80×MIC. Then, dilute them with sterile water to 40×MIC, 20×MIC, and 10×MIC protocatechuic aldehyde solutions and chlorogenic acid solutions respectively. Take 900 μL of the bacterial suspension, 50 μL of the protocatechuic aldehyde solution, and 50 μL of the chlorogenic acid solution and mix them evenly. The final concentrations of protocatechuic aldehyde and chlorogenic acid are 4×MIC, 2×MIC, and MIC. Mix 900 μL of the bacterial solution and 100 μL of sterile water as the control group, and set three parallel experiments for each concentration. Incubate with shaking at 30°C for 30 min, and measure the OD value at 260 nm.

[0077] The nucleic acid absorption peak value is generally around 260 nm. Use an ultraviolet spectrophotometer to measure the ultraviolet absorption intensity at 260 nm of the bacterial suspension of Stenotrophomonas maltophilia treated with protocatechuic aldehyde and chlorogenic acid. The results are as Figure 2 shown. The absorption value at 260 nm of Stenotrophomonas maltophilia treated with protocatechuic aldehyde combined with chlorogenic acid increased significantly compared with the water treatment group, and with the increase in concentration, the absorbance value at 260 nm also increased, indicating that after treating Stenotrophomonas maltophilia cells with protocatechuic aldehyde combined with chlorogenic acid, DNA leakage occurred in the bacterial cells, and with the increase in concentration, this effect was more obvious.

[0078] 2. Scanning electron microscope

[0079] Activate Stenotrophomonas maltophilia, and then centrifuge it at 7000 r / min for 10 min at 4°C, wash it three times with PBS, and resuspend it with PBS to form a bacterial suspension. According to the minimum inhibitory concentration of protocatechuic aldehyde and chlorogenic acid obtained from the above experiments as MIC, weigh appropriate amounts of protocatechuic aldehyde and chlorogenic acid, dissolve them separately with sterile water to obtain 20×MIC protocatechuic aldehyde solution and chlorogenic acid solution. Add 4.5 mL of bacterial suspension, 0.25 mL of 20×MIC protocatechuic aldehyde solution and 0.25 mL of chlorogenic acid solution to a 15 mL centrifuge tube respectively, so that the final concentrations of protocatechuic aldehyde and chlorogenic acid become MIC; in another group, add 4.5 mL of bacterial suspension and 0.5 mL of sterile water to a 15 mL centrifuge tube as a control group. After shaking in a water bath at 30°C for 30 min, centrifuge at 7000 r / min for 10 min at 4°C, wash it three times with PBS, discard the supernatant, add 2.5% glutaraldehyde solution, and fix it in the dark at 4°C for 24 h. Also centrifuge at 7000 r / min for 10 min at 4°C, discard the supernatant, and dehydrate it with 30%, 40%, 50%, 70%, 80%, 90%, and 100% ethanol solutions for 5 min at each concentration. Mix 50% ethanol and tert-butanol evenly at a ratio of 1:1, displace the bacterial suspension with the mixed solution and 100% tert-butanol solution for 10 min respectively, freeze-dry, sputter gold, and observe through a scanning electron microscope.

[0080] The results are as Figure 3 shown. The surface of Stenotrophomonas maltophilia treated with sterile water is intact, smooth and without damage. The surface of Stenotrophomonas maltophilia treated with protocatechuic aldehyde combined with chlorogenic acid shows rupture and defect, the surface is relatively rough, and Stenotrophomonas maltophilia appears aggregated, and the structural integrity of the bacteria is damaged.

[0081] 3. Transmission electron microscope

[0082] After pretreatment of Stenotrophomonas maltophilia (the same as that of scanning electron microscope), dehydrate it with 30%, 40%, 50%, 70%, 80%, 90%, and 100% ethanol solutions for 5 min at each concentration, then treat it with pure acetone for 5 min, and then treat it with a 1:1 mixture of embedding agent and acetone for 1 h, a 3:1 mixture of embedding agent and acetone for 3 h, and pure embedding agent for 12 h. Cut the embedded sample with an ultramicrotome, stain the section with lead citrate solution and 50% ethanol saturated solution of uranyl acetate for 5 min each, and observe through a transmission electron microscope.

[0083] As Figure 4 shown, the transmission electron microscope shows the internal microstructure changes of Stenotrophomonas maltophilia treated with protocatechuic aldehyde combined with chlorogenic acid. Stenotrophomonas maltophilia treated with sterile water is rod-shaped, with a smooth and intact surface without damage. However, after treatment with protocatechuic aldehyde combined with chlorogenic acid, the surface of the bacteria is damaged, the cell contents leak, and the integrity of the bacteria is damaged.

[0084] 4. Biofilm Sensitivity Experiment

[0085] (1) Influence on Biofilm Formation

[0086] Activate Stenotrophomonas maltophilia, then centrifuge at 4°C and 7000 r / min for 10 min, wash three times with PBS, and adjust the bacterial concentration to 1×10 8 CFU / mL with PBS. According to the minimum inhibitory concentration (MIC) obtained from the previous experiment when protocatechuic aldehyde and chlorogenic acid are used in combination, weigh appropriate amounts of protocatechuic aldehyde and chlorogenic acid and dissolve them separately in 1 mL of sterile water to prepare protocatechuic aldehyde solutions and chlorogenic acid solutions with concentrations of 40×MIC and 32×MIC, and then dilute them with sterile water to 24×MIC, 16×MIC, 8×MIC, and 4×MIC concentrations of protocatechuic aldehyde solutions and chlorogenic acid solutions

[0087] Add 100 μL of the bacterial suspension to a 96-well microplate, and then add 50 μL of the protocatechuic aldehyde solution and 50 μL of the chlorogenic acid solution to the wells, so that the final concentrations of the protocatechuic aldehyde and chlorogenic acid solutions in the wells become 10×MIC, 8×MIC, 6×MIC, 4×MIC, 2×MIC, and MIC. At the same time, mix 100 μL of the bacterial suspension and 100 μL of sterile water as a negative control, and repeat each concentration three times for parallel experiments. After culturing at 30°C for 48 h, wash three times with sterile water, add 200 μL of methanol solution to each well to fix for 30 min, wash three times with sterile water, invert and blot dry with filter paper, then add crystal violet solution and stain in the dark for 10 min, wash three times with sterile water, add 200 μL of 95% ethanol to each well for decolorization treatment, let stand for 30 min, and then use a microplate reader to measure the absorbance values of each well of the 96-well microplate at 595 nm

[0088] (2) Influence on the Formed Biofilm

[0089] Activate Stenotrophomonas maltophilia, then centrifuge at 4°C and 7000 r / min for 10 min, wash three times with PBS, and adjust the bacterial concentration to 1×10 8CFU / mL. Add 20 μL of the bacterial suspension and 180 μL of SCDLP liquid medium to each well of a 96-well microplate. After culturing in a constant temperature incubator at 30 °C for 48 h, pour out the remaining bacterial suspension in the wells, wash three times with PBS, and then add 100 μL of protocatechuic aldehyde solution and 100 μL of chlorogenic acid solution to each well, so that the concentrations of protocatechuic aldehyde and chlorogenic acid in each well are 10×MIC, 8×MIC, 6×MIC, 4×MIC, 2×MIC, and MIC. At the same time, add 200 μL of sterile water as a control group. Oscillate for 30 min at 30 °C, and set three parallel experiments for each concentration. Subsequently, pour out the solution in the wells, wash three times with sterile water, add 200 μL of methanol solution to each well to fix for 30 min, wash three times with sterile water, invert and blot dry with filter paper, add crystal violet solution and stain in the dark for 10 min, wash three times with sterile water, add 200 μL of 95% ethanol to each well for decolorization treatment, let stand for 30 min, and then use a microplate reader to measure the absorbance value of each well of the 96-well microplate at 595 nm.

[0090] The results are as Figure 5 , 6 shown, Figure 5 indicating that compared with the sterile water treatment group, the combination of protocatechuic aldehyde and chlorogenic acid can effectively reduce the formation amount of Stenotrophomonas maltophilia biofilm, and with the increase of the concentration, the formation amount of Stenotrophomonas maltophilia biofilm gradually decreases; in Figure 6 it is shown that the combination of protocatechuic aldehyde and chlorogenic acid can significantly reduce the amount of Stenotrophomonas maltophilia biofilm that has been formed, and also with the increase of the concentrations of protocatechuic aldehyde and chlorogenic acid, the amount of the formed Stenotrophomonas maltophilia biofilm gradually decreases.

[0091] As described above, it is only a preferred embodiment of the present invention, and it is not a limitation of the present invention in other forms. Any person skilled in the art may use the disclosed technical content to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution content of the present invention still belong to the protection scope of the technical solution of the present invention.

Claims

1. A composition for inhibiting Stenotrophomonas maltophilia, characterized in that, The active ingredient contains protocatechuic aldehyde and chlorogenic acid, and the mass ratio of the protocatechuic aldehyde to the chlorogenic acid is 1:

8.

2. The composition for inhibiting Stenotrophomonas maltophilia according to claim 1, wherein The active ingredient further contains β-lactam antibiotics.

3. The composition for inhibiting Stenotrophomonas maltophilia according to claim 2, wherein The β-lactam antibiotic is ampicillin.

4. The composition for inhibiting Stenotrophomonas maltophilia according to claim 3, wherein The mass ratio of the protocatechuic aldehyde, the chlorogenic acid and the ampicillin is 1:8:

1.

5. Use of the composition according to any one of claims 1-4 in the preparation of a medicament for inhibiting Stenotrophomonas maltophilia.

6. Use of the composition according to any one of claims 1-4 in the preparation of a medicament for preventing and treating diseases caused by Stenotrophomonas maltophilia.

7. Use of the composition of claim 1 in the preparation of a medicament for reducing the drug resistance of Stenotrophomonas maltophilia to ampicillin.

8. A drug for inhibiting Stenotrophomonas maltophilia and / or preventing and treating diseases caused by Stenotrophomonas maltophilia, characterized in that, Containing the composition according to any one of claims 1-4.

9. The drug for inhibiting Stenotrophomonas maltophilia and / or preventing and treating diseases caused by Stenotrophomonas maltophilia according to claim 8, characterized in that, The medicament further contains a pharmaceutically acceptable carrier.

10. The drug for inhibiting Stenotrophomonas maltophilia and / or preventing and treating diseases caused by Stenotrophomonas maltophilia according to claim 8, characterized in that, The dosage form of the medicament is selected from powder, granule, capsule, injection, oral liquid or tablet.

Images