Application of indometacin combined with polymyxin in resisting salmonella infection

Abstract

The invention belongs to the technical field of biological medicines, and discloses application of indometacin combined with polymyxin in resisting salmonella infection. A checkerboard method minimum inhibition concentration test and an in-vitro time-sterilization curve test prove that the indometacin and colistin combined drug has an obvious synergistic effect on salmonella resistance, and the indometacin can enhance the antibacterial effect of the colistin drugs on salmonella standard strains and colistin-resistant salmonella. The invention provides a new drug combination strategy, and provides a new research thought and research and development direction for solving the increasingly serious drug resistance problem of polymyxin.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of indomethacin combined with polymyxin in resisting salmonella infection. Background Art

[0002] Salmonella is a Gram-negative rod-shaped facultative anaerobic bacterium of the Enterobacteriaceae family. It is one of the most common foodborne pathogens and has developed resistance to multiple antibiotics such as quinolones. Polymyxin is a cationic polypeptide antibacterial drug and is the last line of defense against serious infections caused by multidrug-resistant Gram-negative bacteria. Polymyxin B and polymyxin E (also called colistin) are mainly used clinically. The main mechanism is to destroy the integrity of the bacterial outer membrane by acting on the lipopolysaccharide of the bacterial outer membrane, thereby causing bacterial death. However, with the emergence of the plasmid-mediated polymyxin resistance gene mcr-1, the problem of bacterial colistin resistance has become imminent and poses a threat to human health.

[0003] In recent years, the development of new target antimicrobial drugs has been difficult. In contrast, finding antibiotic adjuvants in combination with antimicrobial drugs (colistin) is a more cost-effective strategy. Therefore, screening effective colistin enhancers and effectively restoring the sensitivity of colistin-resistant bacteria to colistin, thereby treating colistin-resistant bacterial infections, deserves further research and development. Traditional MH broth is a rich laboratory culture medium that cannot summarize most aspects of the host environment. Incorporating environmental factors that are commonly present during host-pathogen interactions may improve the predictive value of standard in vitro antimicrobial susceptibility testing (AST) in determining effective antibiotics for the treatment of microbial infections. Macrophages are one of the main host cell types for the replication and systemic spread of Salmonella. By simulating host conditions, adjuvants that can enhance the antimicrobial efficacy of colistin can be more accurately selected. LPM broth is a culture medium that simulates the macrophage environment, with a weakly acidic pH, low phosphorus and low magnesium. Studies have shown that in low Mg 2+ The inner membrane sensor PhoQ can be activated by the environment or cationic antimicrobial peptides, while under acidic pH conditions, Zn 2+ 、Al 3+ , high concentration Fe 3+ Or PmrB can be activated by the action of cationic antimicrobial peptides.

[0004] Indomethacin, also known as indomethacin, is a widely used nonsteroidal anti-inflammatory drug, often used as an antipyretic, anti-inflammatory and analgesic to treat a variety of inflammatory diseases such as rheumatoid arthritis and osteoarthritis. The mechanism of action is to reduce the synthesis of prostaglandins by inhibiting cyclooxygenase, stop the formation of pain nerve impulses in inflammatory tissues, and inhibit inflammatory reactions. Currently, there is no report on the use of indomethacin as a synergist to enhance the effect of polymyxins against Salmonella and / or polymyxin-resistant Salmonella infections. Summary of the invention

[0005] In order to solve the deficiencies of the prior art, the present invention provides a method for enhancing the antibacterial activity of polymyxin, specifically using indomethacin in combination with polymyxin, the two are not just a simple addition of functions, but achieve a synergistic antibacterial effect. The present invention also discloses an antibacterial combination of indomethacin and polymyxin, which provides a new treatment strategy for clinical treatment of bacterial infections, especially multidrug-resistant Salmonella infections, specifically bacterial infections with polymyxin resistance genes (MCR).

[0006] The first aspect of the present invention is to provide the use of indomethacin in the preparation of an antibacterial synergist for enhancing the anti-Salmonella infection of polymyxin drugs. The structural formula of indomethacin is as follows:

[0007]

[0008] Preferably, the polymyxin drug is polymyxin E.

[0009] Preferably, the Salmonella includes the Salmonella standard strain ATCC 14028 and the polymyxin-resistant Salmonella. The polymyxin-resistant Salmonella has the polymyxin resistance gene MCR-1.

[0010] In some specific embodiments of the present invention, the polymyxin-resistant Salmonella includes clinical Salmonella typhimurium strain 17ES (MCR-1).

[0011] The second aspect of the present invention is to provide an antibacterial composition comprising indomethacin and polymyxin antibiotics.

[0012] Preferably, the polymyxin antibiotic is polymyxin E.

[0013] Preferably, the concentration of indomethacin in the composition is 10-40 μg / mL, and the concentration of polymyxin is 10-40 μg / mL.

[0014] The antibacterial composition can be used to inhibit Salmonella and polymyxin-resistant Salmonella. In the in vitro sterilization experiment of the present invention, the Salmonella is first cultured and incubated to the logarithmic growth phase, and then a certain concentration of colistin and indomethacin are co-incubated with the diluted bacterial solution in LPM broth for combined sterilization. Preferably, the Salmonella includes the Salmonella standard strain ATCC 14028 and the polymyxin-resistant Salmonella S.typhimurium (17ES).

[0015] The third aspect of the present invention is to provide the use of the above antibacterial composition in the preparation of drugs for resisting Salmonella infection.

[0016] The fourth aspect of the present invention is to provide the use of the above antibacterial composition in the preparation of a drug for resisting polymyxin-resistant Salmonella infection.

[0017] Preferably, the drug contains indomethacin, polymyxin antibiotics, and a pharmaceutically acceptable carrier.

[0018] Preferably, the drug dosage form can be selected from any one of the following: capsules, sustained-release or controlled-release tablets, oral liquid dosage forms, injection liquid dosage forms, lyophilized powder injections, and creams.

[0019] Experiments have shown that the combination of indomethacin and colistin can be used to fight salmonella infection and has a significant bactericidal effect, which is significantly enhanced compared to the effect of colistin alone.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention finds through the chessboard method and sterilization curve that the combination of indomethacin and colistin produces a significant antibacterial effect on Salmonella, and the effect is better than that of each drug alone, indicating that colistin and indomethacin have a significant synergistic effect when used in combination. Compared with the traditional antibiotics and antibiotics used in combination for antibacterial, an antibiotic synergistic alternative drug is developed, and a new use of indomethacin as a synergist for polymyxin drugs is provided, which can solve the increasingly serious problem of clinical drug resistance of polymyxins. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Checkerboard test of colistin combined with indomethacin in MH broth and combined inhibitory index (FICI) against ATCC 14028.

[0023] Figure 2 Checkerboard assay of colistin combined with indomethacin in LPM broth and the combined inhibitory index (FICI) against ATCC 14028 and colistin-resistant Salmonella typhimurium (17ES).

[0024] Figure 3 Time-kill curves of colistin combined with indomethacin against ATCC 14028 and colistin-resistant Salmonella typhimurium (17ES). DETAILED DESCRIPTION

[0025] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods; the materials and reagents used are conventional reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0027] The polymyxin antibiotics used in the following examples are all polymyxin E (colistin); the commercially available indomethacin has a purity of 99%, the solvent is DMSO, the CAS registration number is 53-86-1, and the molecular formula is C 19 H 16 ClNO4, molecular mass is 357.79.

[0028] The structural formula of indomethacin is as follows:

[0029]

[0030] Example 1 Synergistic effect of indomethacin and colistin against Salmonella in vitro

[0031] 1. Bacteria and antibiotics

[0032] The standard strain of Escherichia coli ATCC 25922 is preserved in this laboratory and used for the quality control of polymyxin drugs. The standard strain of Salmonella ATCC 14028 and polymyxin-resistant Salmonella S.typhimurium (17ES) (MCR-1) are both preserved in the laboratory and are commercially available. The commercially available indomethacin has a purity of 99%, the solvent is DMSO, and the concentration can be configured to 5120μg / ml, which is diluted to the corresponding concentration when used; the purity of polymyxin E is 95%, the solvent is ddH2O, and the concentration can be configured to 5120μg / ml, which is diluted to the corresponding concentration when used.

[0033] 2. MIC and FICI of indomethacin and colistin against Salmonella

[0034] 2.1 MIC of indomethacin and colistin against Salmonella

[0035] Experimental steps:

[0036] (1) Salmonella (ATCC 14028) and polymyxin-resistant Salmonella typhimurium (17ES) were selected and placed in MH broth, incubated in a shaker at 37°C until the logarithmic growth phase, and then diluted 100 times with MH and LPM broth to obtain bacterial dilution solution for use;

[0037] (2) In a 96-well plate, 180 μL of MH broth was added to A1-C1, 100 μL of MH broth was added to A2-C12, A11-C11 was a positive control, and A12-C12 was a negative control. 180 μL of LPM broth was added to D1-F1, 100 μL of LPM broth was added to D2-F12, D11-F11 was a positive control, and D12-F12 was a negative control;

[0038] (3) Add 20 μL of colistin or indomethacin (concentration 5120 μg / mL) to wells A1-F6, dilute to column 10, mix well, aspirate 100 μL from column 10 and discard;

[0039] (4) Add 100 μL of diluted bacterial solution to A1-F11. Add 100 μL of MH or LPM broth to A12-F12;

[0040] (5) After the drug and bacterial solution are loaded, incubate in a 37°C constant temperature incubator for 16-18 hours and observe the results.

[0041] Table 1 shows the MIC values ​​of selected Salmonella ATCC 14028 and polymyxin-resistant Salmonella typhimurium (17ES) in MH or LPM broth.

[0042] Table 1

[0043]

[0044] 2.2 Chessboard method drug sensitivity test

[0045] The chessboard method was used to combine the drug sensitivity test to determine the FICI of indomethacin combined with colistin against ATCC 14028 and colistin-resistant Salmonella typhimurium (17ES). The specific operation is as follows:

[0046] (1) Pick a single colony of ATCC 14028 and polymyxin-resistant Salmonella typhimurium (17ES) in MH broth, incubate in a shaker at 37°C until the logarithmic growth phase, and then dilute 100 times with MH or LPM broth to obtain a bacterial dilution for use;

[0047] (2) Add 50 μL of MH or LPM broth to 96-well plates A1-H8;

[0048] (3) Add 50 μL of the diluted indomethacin single drug (concentration: 2048 μg / mL) to H1-H8, and serially dilute it to row B. After mixing row B, aspirate and discard 50 μL;

[0049] (4) For ATCC 14028, add the corresponding concentration of colistin single drug to each column. In MH broth, the final concentrations of colistin in each well of each column in the 96-well microplate from left to right are 0 μg / mL, 0.03125 μg / mL, 0.0625 μg / mL, 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, and 2 μg / mL. In LPM broth, the final concentrations of colistin in each well of each column in the 96-well microplate from left to right are 0 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, and 128 μg / mL;

[0050] (5) For polymyxin-resistant Salmonella S. typhimurium (17ES), add the corresponding concentration of colistin single drug to each column. In LPM broth, the final concentrations of colistin in each well of each column in the 96-well microplate from left to right are 0 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, and 128 μg / mL;

[0051] (6) Add 100 μL of the diluted bacterial solution to A1-H8. The inoculum size is 1×10 6 CFU / mL, and each group of experiments is repeated at least three times;

[0052] (7) After loading the drugs and the bacterial solution, incubate in a 37°C constant temperature incubator for 16 - 18 h and then observe the results.

[0053] Use the calculated result of FICI as the judgment basis for the combined drug sensitivity test: FICI = FICa + FICb = MICab / MICa + MICba / MICb, where MICa and MICb are the MIC values of a and b when used alone, and MICab and MICba are the MIC values of drug a and drug b when a and b are used in combination. Evaluation criteria for drug synergy: FICI ≤ 0.5, synergistic effect; 0.5 < FICI ≤ 1, additive effect; 1 < FICI ≤ 2, no interaction; FICI > 2, antagonistic effect.

[0054] The experimental results are as Figure 1 and 2 shown. It can be seen from Figure 1 that there is a synergistic effect (FICI = 0.5) between indomethacin and colistin in MH broth. It can be seen from Figure 2It can be seen that indomethacin combined with colistin has a synergistic effect in LPM broth (FICI=0.09375), and the synergistic effect is significant. For colistin-resistant Salmonella typhimurium (17ES), the FICI value is also less than 0.5, showing a good synergistic effect.

[0055] Example 2 In vitro time-kill curve of indomethacin and colistin against Salmonella

[0056] (1) Single colonies of Salmonella ATCC 14028 and polymyxin-resistant Salmonella S.typhimurium (17ES) were picked and cultured overnight in blank MH broth. The overnight culture was diluted 1:100 into 4 mL of LPM broth for later use. For Salmonella ATCC 14028, the experiment was divided into four groups: control group, no drug treatment group, colistin (16 μg / mL) group, indomethacin group (32 μg / mL) and colistin (16 μg / mL) + indomethacin (32 μg / mL) group, and the system was 5 mL. For S.typhimurium (17ES), the experiment was divided into four groups: control group, no drug treatment group, colistin (32 μg / mL) group, indomethacin group (32 μg / mL) and colistin (32 μg / mL) + indomethacin (32 μg / mL) group, and the system was 5 mL;

[0057] (2) Place the above four groups of centrifuge tubes in a 37°C shaker and incubate them. Take samples at 0h, 3h, 6h, 9h, 12h, and 24h for counting. The method is as follows: Take a 96-well deep-well plate and number it 10 in sequence. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , add 900 μl of sterile saline to each deep-space plate, take 100 μl of bacterial solution at 0 h and 3 h and add it to the 10 -1 tubes, and dilute them in multiples to 10 -4 Then, 100 μL of bacterial solution was sampled at 6 h, 9 h, 12 h, and 24 h and added to 10 -1 tubes, and dilute them in multiples to 10 -8 Then, take 25 μL of each dilution and drop it on LB agar plate, repeat the process 3 times;

[0058] (3) After inverted culture in a 37°C constant temperature incubator for 16-18 hours, count the number of colonies on the plates with 30 to 300. Data processing: Calculate the average number of colonies on the three plates with the same dilution, and calculate the concentration of the bacterial solution based on the average number of colonies.

[0059] Original bacterial solution concentration (CFU / ml) = average colony count × dilution × 40

[0060] The experimental results are as follows Figure 3 As shown by Figure 3 It can be seen that colistin alone has little antibacterial activity, and indomethacin alone has almost no antibacterial activity. Compared with the control group, colistin group, and indomethacin group, the combined use of colistin and indomethacin can always reduce the number of bacteria with CFU greater than 2log10 from 0 to 24 hours, indicating that the combination of indomethacin and colistin can significantly reduce bacterial growth.

[0061] In summary, indomethacin and colistin have a significant combined synergistic antibacterial effect.

[0062] Obviously, the above embodiments of the present invention are only examples for clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the claims of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. The use of indomethacin in the preparation of an antibacterial synergist for enhancing the anti-Salmonella infection of polymyxin drugs, characterized in that: The structural formula of indomethacin is shown below:

2. The use according to claim 1, characterized in that: The polymyxin drug is polymyxin E.

3. The use according to claim 1, characterized in that: The Salmonella includes the Salmonella standard strain ATCC14028 and / or polymyxin-resistant Salmonella.

4. An antibacterial composition, characterized in that Including indomethacin and polymyxin drugs, the structural formula of indomethacin is as follows:

5. The composition according to claim 4, characterized in that The polymyxin drug is polymyxin E.

6. The composition according to claim 4, characterized in that The concentration of indomethacin in the composition is 10-40 μg / mL, and the concentration of polymyxin drugs is 10-40 μg / mL.

7. Use of the composition according to claim 4 in the preparation of a drug for resisting Salmonella infection.

8. Use of the composition according to claim 4 in the preparation of a medicament for resisting polymyxin-resistant Salmonella infection.

9. The use according to claim 7 or 8, characterized in that: The medicine contains indomethacin, polymyxin drugs, and a pharmaceutically acceptable carrier.

10. The use according to claim 9, characterized in that: The dosage form of the drug can be selected from any one of the following: capsules, sustained-release or controlled-release tablets, oral liquid dosage forms, injection liquid dosage forms, lyophilized powder injections, and creams.

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