CN115778936A - Application of usnic acid synergistic polymyxin in resisting gram-negative bacterial infection - Google Patents
Application of usnic acid synergistic polymyxin in resisting gram-negative bacterial infection Download PDFInfo
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- CN115778936A CN115778936A CN202211623191.8A CN202211623191A CN115778936A CN 115778936 A CN115778936 A CN 115778936A CN 202211623191 A CN202211623191 A CN 202211623191A CN 115778936 A CN115778936 A CN 115778936A
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- Prior art keywords
- polymyxin
- usnic acid
- antibacterial
- pharmaceutically acceptable
- usnic
- Prior art date
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- WEYVVCKOOFYHRW-UHFFFAOYSA-N usninic acid Natural products CC12C(=O)C(C(=O)C)=C(O)C=C1OC1=C2C(O)=C(C)C(O)=C1C(C)=O WEYVVCKOOFYHRW-UHFFFAOYSA-N 0.000 title claims abstract description 117
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Abstract
The invention discloses application of usnic acid in preparing a medicament for enhancing the antibacterial infection efficacy of polymyxin, and application of a composition of usnic acid and polymyxin in preparing a medicament for enhancing the antibacterial infection efficacy. The invention not only proves the antibacterial activity of the synergestic synergistic polymyxin of usnic acid by a chessboard method minimum inhibitory concentration test and an in vitro bacterial growth curve, but also proves that the validity of the polymyxin in vivo can be effectively enhanced by usnic acid by utilizing a mouse wound infection model. The invention discovers that usnic acid can obviously enhance the antibacterial activity of polymyxin, and provides a new treatment strategy for clinically treating bacterial infectious diseases.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of usnic acid synergistic polymyxin in resistance to gram-negative bacterial infection.
Background
The problem of bacterial resistance has become a global public health problem, and the suppression of bacterial resistance is imminent. In particular, in human medicine and animal clinics in recent years, fewer and fewer clinically available antibiotics are caused by the rapid spread of carbapenem-resistant gram-negative bacteria. In particular, the emergence of multiple drug resistant gram-negative bacteria has resulted in the near clinical unavailability of drugs and a series of public health safety issues. In recent years, various departments of China pay considerable attention to the health sustainable development of livestock and poultry breeding industry, and a series of policies such as ' action plans for suppressing bacterial drug resistance ' (2016-2020), ' action plans for suppressing animal-derived bacterial drug resistance (2017-2020), and the like and ' action plans ' are recently introduced in China to suppress the development of bacterial drug resistance.
The polymyxin antibacterial drugs include polymyxin A, polymyxin B, polymyxin C, polymyxin D and polymyxin E. Among them, polymyxins B and E are clinically used and have strong effects on gram-negative bacteria such as Escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa, etc. Wherein polymyxin E is also called colistin, CAS number 1066-17-7, trade name colistin, colistin and colistin. The clinical application is mainly in the form of sulfate or methanesulfonate, namely polymyxin E sulfate and polymyxin E methanesulfonate. Currently, polymyxin E is considered to be one of the most important antibacterial drugs for clinical treatment of infections such as carbapenem-resistant escherichia coli, multidrug-resistant pseudomonas aeruginosa, and multidrug-resistant klebsiella pneumoniae.
Clinical application of polymyxin often causes multiple toxic side effects including renal toxicity, neurotoxicity, pulmonary toxicity, skin toxicity and the like, and severely limits the application of polymyxin. The toxic and side effects of polymyxin can be obviously reduced by reducing the using dosage of polymyxin, but the sterilization effect of polymyxin in a body is obviously reduced, the clinical output is reduced, and the generation of drug resistance of polymyxin can be increased. In addition, in recent years, the emergence and spread of polymyxin resistance gene (MCR) plasmids carried by pathogenic bacteria such as Escherichia coli have led to the development of polymyxin resistance by some gram-negative bacteria. These factors severely limit the clinical utility of polymyxin. Therefore, the development of effective polymyxin synergists and the development of compound polymyxin preparations have become important strategies for the clinical treatment of multidrug-resistant gram-negative bacterial infections.
Usnic acid, CAS number 125-46-2, molecular formula C 18 H 16 O 7 Molecular weight 344.32, structure shown in figure 1. Usnic acid, also known as usnic acid, is isolated from lichen, either from natural usnea. It has been reported that usnic acid has excellent antibacterial activity against gram-positive bacteria such as Staphylococcus aureus (documents: gupta VK, verma S, gupta S, singh)A, pal A, srivastava SK, srivastava PK, singh SC, darokar MP, membrane-damagingpotiall of natural L- (-) -usnic acid in Staphylococcus aureus, eurJClin Microbiol Infect Dis.2012Dec;31 (12):3375-83.). In addition, usnic acid has a certain inhibitory effect on Porcine Epidemic Diarrhea Virus (PEDV) (literature: zhanjing. Usnic acid effects on PEDV replication and PEDV cell culture genetic stability analysis [ D ]]Nanjing university of agriculture, 2020). Usnic acid also has the function of promoting wound healing (the document: wangxiane, guanifeng, luoyuan. Usnic acid has the functions of healing the skin wound of the rat and hyperplasia of scar through PI3K/Akt pathway [ J]Chinese journal of dermatological disease, 2021,35 (10): 1111-1118+ 1131.). Usnic acid and analogues thereof also have biological functions of anti-inflammation, anti-oxidation, anti-cancer, improving immunity of the organism (documents: sana. Uighur, johnson, yangxin Yao, gao Xiao, quchang sea, zhang Qian, liuchun, tupeng Fei, chaxinyun, development of chemical components and pharmacological activity of usnic acid and analogues thereof [ J Xinyun]Chinese traditional medicine journal, 2018,43 (19): 3811-3821. ). So far, the antibacterial activity of the usnic acid as an antibacterial adjuvant and a synergistic antibacterial medicament is not reported.
Disclosure of Invention
In order to solve the technical problem that multiple drug-resistant gram-negative bacteria are difficult to treat, the invention provides a method for enhancing the antibacterial activity of polymyxin, and particularly adopts usnic acid and polymyxin (preferably polymyxin E), wherein the two are not only simple in functional addition, but also achieve the synergistic antibacterial effect. The invention also discloses an antibacterial combination proportion of usnic acid and polymyxin E, and provides a new treatment strategy for clinical treatment of bacterial infection, particularly multi-drug resistance gram-negative bacterial infection, and specifically bacterial infection with a polymyxin resistance gene (MCR).
The invention provides application of usnic acid or pharmaceutically acceptable salt thereof in preparing an antibacterial synergist for enhancing the antibacterial infection efficacy of polymyxin antibacterial drugs.
The second aspect of the invention provides the application of usnic acid or pharmaceutically acceptable salts thereof in preparing medicines for resisting bacterial infectious diseases.
In a third aspect of the invention, an antimicrobial composition is provided.
The antibacterial composition comprises usnic acid or pharmaceutically acceptable salt thereof and polymyxin antibacterial drugs.
Further, the mass ratio of the usnic acid or the pharmaceutically acceptable salt thereof to the polymyxin antibacterial drug is (20-320): 1, preferably (20-80): 1, and specifically comprises the following components in percentage by mass, 20.
The antibacterial composition can be used for treating bacterial skin infections.
A fourth aspect of the invention provides an antimicrobial product.
The antibacterial product contains the antibacterial composition and a pharmaceutically acceptable carrier.
The dosage form of the antibacterial product can be selected from any one of the following: tablet, cream, capsule, sustained release tablet, controlled release tablet, oral liquid, syrup, dripping pill, injection, and lyophilized powder for injection.
The pharmaceutically acceptable carrier includes, but is not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (such as cellulose acetate phthalate, carboxymethyl cellulose, etc.). The antibacterial product can be made into various dosage forms, including but not limited to tablet, capsule, cream, syrup, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, liposome, transdermal agent, buccal tablet, suppository, lyophilized powder for injection, etc. Can be common preparation, sustained release preparation, controlled release preparation and various microparticle drug delivery systems.
The compositions provided herein are utilized to administer to a subject an effective amount of an antimicrobial composition in the prevention and/or treatment of a bacterial infection.
The dosage and method of use of the antibacterial composition of the present invention depend on a variety of factors including the age, weight, sex, physical health, nutritional status, activity intensity of the compound, administration time, metabolic rate, severity of the disease condition, and the subjective judgment of the treating physician.
Preferably, the final therapeutic doses of polymyxin E and usnic acid are 0.5mg/kg and 20mg/kg body weight, respectively.
In the present invention, the term "effective amount" refers to a dose that achieves treatment, prevention, alleviation and/or alleviation of the disease or disorder described herein in a subject.
In the present invention, the term "subject" may refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., that receives an antibacterial composition of the present invention to treat, prevent, alleviate and/or alleviate a disease or disorder described herein.
In the invention, the usnic acid pharmaceutically acceptable salt can be specifically usnic acid sodium salt.
In the invention, the structural formula of the usnic acid is shown as a formula I:
in the present invention, the polymyxin antibacterial agent is at least one selected from the group consisting of: 1) Colistin (i.e. polymyxin E) or a pharmaceutically acceptable salt thereof (e.g. polymyxin E sulfate); 2) Polymyxin B or a pharmaceutically acceptable salt thereof (e.g., polymyxin B sulfate).
In the present invention, the bacterium is a bacterium having a polymyxin resistance gene.
Further, the bacterium is a gram-negative bacterium.
Further, the gram-negative bacterium is a multidrug resistant gram-negative bacterium.
Further, the bacteria are one or more of escherichia coli, klebsiella pneumoniae, salmonella, shigella and acinetobacter baumannii; preferably, the one or more of escherichia coli, klebsiella pneumoniae, salmonella, shigella, acinetobacter baumannii has a polymyxin resistance gene or multiple resistance genes.
Further, the polymyxin drug resistance gene comprises a polymyxin drug resistance gene MCR-1.
Further, the bacteria are klebsiella pneumoniae; preferably, the klebsiella pneumoniae is klebsiella pneumoniae with a polymyxin resistance gene or multiple drug resistance.
At present, although the prior art discloses that polymyxin is combined with other antibacterial drugs, most of the drugs have poor drug properties, the invention not only provides an in-vitro synergistic experiment of the combination of usnic acid and polymyxin, but also further provides an animal level experiment to prove that the synergistic sterilization effect is better. No research reports exist so far about the application of usnic acid as a polymyxin synergist in enhancing the antibacterial activity of polymyxin.
The novel application of usnic acid provided by the invention, namely the method for enhancing the antibacterial activity of polymyxin, has the following excellent technical effects:
(1) The invention proves the antibacterial activity of the synergetically synergistic polymyxin of usnic acid by a chessboard method minimum inhibitory concentration test and an in vitro sterilization curve.
(2) Different from the existing combination of polymyxin and antibiotics, the invention provides a mouse drug-resistant bacterial infection model experiment, proves that the usnic acid can effectively enhance the polymyxin and the in-vivo effectiveness at the animal level, and has more proving power for the next clinical application.
(3) The invention clarifies that usnic acid can restore the sensitivity of polymyxin-resistant bacteria, further evaluates the effectiveness of the combined use of usnic acid and polymyxin-resistant bacteria in vivo and in vitro, is favorable for developing a novel antibiotic synergist and relieves the problem of bacterial drug resistance with increasingly serious harm.
(4) The invention provides a new application of usnic acid in the synergism of the antibacterial activity of polymyxin antibiotics, and can solve the technical problems of clinical drug resistance, low treatment index and the like of polymyxin.
Drawings
Fig. 1 is a time sterilization curve of usnic acid in combination with polymyxin E against e.coli B2;
FIG. 2 is a time sterilization curve of usnic acid in combination with polymyxin E against E.coli ATCC 25922;
FIG. 3 is a time sterilization curve of usnic acid in combination with polymyxin E against E.coli GZP 080-8;
fig. 4 is an evaluation of the therapeutic effect of polymyxin E and usnic acid alone and in combination on the infection of mouse skin wound with E.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified.
Usnic acid, CAS number 125-46-2, formula C used in the examples below 18 H 16 O 7 Molecular weight 344.32, purchased from Aladdin reagent, cat # U275043, purity>98 percent. Colistin sulfate was purchased from Aladdin reagent, cat # C114323, activity>19000U/mg. Polymyxin B sulfate is purchased from Aladdin reagent company, and the potency is more than or equal to 6000U/mg. A certain amount of colistin sulfate or polymyxin B sulfate is weighed to prepare a mother liquor with the concentration of 16mg/mL. Usnic acid was prepared using DMSO as a mother solution at a mother solution concentration of 40 mg/mL. The prepared mixture was stored in a-20 ℃ refrigerator.
Pathogenic bacteria used in the following examples:
the Escherichia coli standard strain is Escherichia coli (Escherichia coli) ATCC25922 (hereinafter, E.coli 25922) which is purchased from China veterinary institute strain preservation center (China veterinary microbial strain preservation management center);
coli B2 (e.coli B2) carries a plasmid of colistin resistance gene 1 (MCR-1) and a plasmid of new delhi metallo-beta-lactamase 5 (NDM-5), and specific information is described in the following documents: song M, liu Y, huang X, ding S, wang Y, shen J, zhu k. Abroad-spectrum anti-inflammatory adjuvant mutations gains multi-drug-resistant Gram-negative pathogens. Nat microbiol.2020aug;5 (8):1040-1050.
Klebsiella pneumoniae k.pneumoniae 1202 (ST 11, KPC-2-producer) + pHNSHP45 (mcr-1), and specific information has been described in the following documents: liu YY, wang Y, walsh TR, yi LX, zhang R, spencer J, doi Y, tian G, dong B, huang X, yu LF, gu D, ren H, chen X, lv L, he D, zhou H, liang Z, liu JH, shen J. Occupant of plasmid-mediated collagen resistance mechanisms MCR-1in antibodies and human bearings chip; 16 (2):161-8. The strain carries MCR-1 plasmid and is resistant to polymyxin.
Pseudomonas aeruginosa Standard strain ATCC15692 (i.e., pseudomonas aeruginosa PA 01) was purchased from the American Type Culture Collection (ATCC).
Salmonella SH16SF0764 (Salmonella SH16SF 0764), isolated clinically, harbors the MCR-1 plasmid and is maintained and supplied by the national center for veterinary drug safety evaluation, university of agriculture, china.
Acinetobacter baumannii standard strain ATCC 19606 was purchased from the American Type Culture Collection (ATCC).
Coli e.coli 09b19, e.coli13h1, e.coli GZP080-8 are described in: lu Yang, yingbo Shen, junyao Jiang, xueyang Wang, dongyan Shao, margaret M.C.Lam, kathryn E.Holt, bing Shao, congming Wu, jianzhong Shen, timothy R.Walsh, stefan Schwarz, yang Wang & Zhangqi Shen, distingt intake in antibacterial resistance genes, amplifying expression Escherichia coli reducing 50ars of antibacterial use in sexual production in China, nature Fou, 2022,3:197 to 205; both carry the MCR-1 plasmid, polymyxin resistant.
Staphylococcus aureus ATCC29213 was purchased from the American Type Culture Collection (ATCC).
Staphylococcus aureus ATCC33591 was purchased from the American Type Culture Collection (ATCC).
Bacterial culture media were used in the following experiments:
MHB broth medium was purchased from Beijing Luoqiao technology GmbH and prepared as follows: weighing 25.0g of the extract in 1L of distilled water, boiling until the extract is completely dissolved, and autoclaving at 121 deg.C for 15min for use.
MHA culture medium is purchased from Beijing Luqiao technology GmbH, and the preparation method is as follows: weighing 38.0g in 1L distilled water, boiling to dissolve completely, autoclaving at 121 deg.C for 15min, cooling to 55 deg.C, and pouring onto plate.
The brain heart infusion medium (BHI) is purchased from Beijing Luqiao technology GmbH, and the preparation method is as follows: weighing 38.5g of the product, heating and stirring the product to dissolve the product in 1000mL of distilled water, adjusting the pH value to 7.3, and carrying out autoclaving at 121 ℃ for 15 minutes for later use.
The BHI solid culture medium is purchased from Beijing road bridge technology GmbH, and the preparation method is as follows: weighing 50.0g of the product in 1000mL of distilled water, heating and boiling until the product is completely dissolved, autoclaving at 121 ℃ for 20min, cooling to 55 ℃, and pouring into a flat plate for later use.
Example 1 evaluation of synergistic antibacterial Activity of combinations of usnic acid and polymyxin
(1) Determination of Minimum Inhibitory Concentration (MIC) of antimicrobial Activity of usnic acid, polymyxin B and polymyxin E when used alone
The Minimum Inhibitory Concentrations (MICs) of different strains on usnic acid, polymyxin B and polymyxin E were determined according to the standard methods of the American society for Clinical and Laboratory Standards Institute (CLSI). The specific operation is as follows: selecting a single bacterial colony in BHI broth, culturing the bacteria in a shaking table at 37 ℃ until the bacteria reach the logarithmic phase, and adjusting the concentration of the bacterial liquid to 0.5 McLeod turbidity by using a McLeod turbidimeter for standby. Usnic acid, polymyxin B and polymyxin E were diluted multiple concentration gradients in MHB medium, respectively. Taking 100 μ L of bacterial liquid (the final concentration of bacterial liquid in each hole is 1.0x10) 6 CFUs/mL) were added to a 96-well U-plate followed by the addition of different drugs per well. The final concentration of the usnic acid medicine is 160 mu g/mL, 80 mu g/mL, 40 mu g/mL, 20 mu g/mL, 10 mu g/mL, 5 mu g/mL, 2.5 mu g/mL, 1.25 mu g/mL and 0.625 mu g/mL in sequence; the final drug concentrations of polymyxin B and polymyxin E were each set at 16. Mu.g/mL, 8. Mu.g/mL, 4. Mu.g/mL, 2. Mu.g/mL, 1. Mu.g/mL, 0.5. Mu.g/mL, 0.25. Mu.g/mL, 0.125. Mu.g/mL, 0.0625. Mu.g/mL. The negative control group only contains MHB culture medium; the positive control group contained 100. Mu.L of the bacterial suspension to be tested, and 1. Mu.L of DMSO was added to each well. After the drug is added, the culture plate is placed in an incubator at 37 ℃ for constant temperature culture for 18h, and the result is observed, and the Concentration of the drug contained in the macroscopic clear hole in a 96-well plate is the Minimum Inhibitory Concentration (MIC). The test results are shown in Table 1.
TABLE 1 MIC values (μ g/mL) of usnic acid, polymyxin E, polymyxin B for each bacterium
As can be seen from Table 1, the antibacterial activity of usnic acid against gram-negative bacteria such as the tested strains E.coli25922, E.coli B2, ATCC15692, K.pneumoconiae 1202 (ST 11, KPC-2-producer) + pHNSHP45 (mcr-1), E.coli 09B19, E.coli13h1, E.coli GZP080-8, ATCC 19606, salmonella SH16SF0764, etc. is greater than the maximum concentration measured, i.e. > 160. Mu.g/mL; the MIC of polymyxin E to the gram-negative bacteria is 0.25. Mu.g/mL, 8. Mu.g/mL, 1. Mu.g/mL, 8. Mu.g/mL, 4. Mu.g/mL, 8. Mu.g/mL, 0.5. Mu.g/mL, 8. Mu.g/mL, respectively; the MICs of polymyxin B against the gram-negative bacteria were 0.25. Mu.g/mL, 8. Mu.g/mL, 1. Mu.g/mL, 8. Mu.g/mL, 4. Mu.g/mL, 0.5. Mu.g/mL, and 8. Mu.g/mL, respectively. The MIC of usnic acid to gram-positive bacteria ATCC29213 and ATCC33591 is 20 mu g/mL.
Polymyxin B and polymyxin E had MICs for both gram positive bacteria ATCC29213, ATCC33591 greater than the maximum concentration tested, i.e., > 16. Mu.g/mL.
2. Combined application antibacterial activity experiment of usnic acid and antibacterial drug polymyxin
The combined application of usnic acid and the antibacterial polymyxin to pathogenic bacteria is used for determining the combined medication index (FICI): the FICI values of strains E.coli25922, E.coli B2, ATCC15692, K.pneumoniae 1202 (ST 11, KPC-2-producer) + pHNSHP45 (mcr-1), E.coli 09B19, E.coli13h1, E.coli GZP080-8, ATCC 19606, salmonella SH16SF0764 and the like when usnic acid is used in combination with an antibacterial agent were determined by a checkerboard method. The specific operation is as follows:
respectively taking 2MIC of antibacterial agent polymyxin E (as A drug) and usnic acid (as B drug) as maximum concentration, diluting to 8-11 concentrations by MHB broth medium ratio,adding MHB broth culture medium containing two drugs with different concentrations 50 μ L along the horizontal axis and the vertical axis of a 96-microporous culture plate, respectively, and adding bacterial liquid of various pathogenic bacteria 50 μ L to obtain final pathogenic bacteria content of 1 × 10 6 And (4) culturing the cells in a CFU/hole at the constant temperature of 37 ℃ for 18-24 h, and observing the result. The MICs of the two drugs used alone and in combination are recorded, and the FICI value (partial inhibitory concentration index) is calculated according to the following formula.
FICI = MIC first drug combination/MIC first drug combination + MIC second drug combination/MIC second drug combination
And (4) judging the standard: FICI is less than or equal to 0.5, and the synergistic effect is achieved; FICI is more than 0.5 and less than or equal to 1, and the additive effect is achieved; 1 < FICI < 2, irrelevant; FICI > 2, antagonistic action. Namely: FICI of usnic acid and polymyxin E = MIC (polymyxin E combination)/MIC (polymyxin E alone) + MIC (usnic acid combination)/MIC (usnic acid alone).
TABLE 2 FICI values of usnic acid in combination with antibacterial agents for E.coli standard strains E.coli25922, E.coli B2, ATCC15692, K.pneumoniae 1202 (ST 11, KPC-2-producer) + pHNSHP45 (mcr-1), E.coli 09B19, E.coli13h1, E.coli GZP08-8, ATCC 19606, salmonella SH16SF0764, etc
The results for all bacteria tested in this assay were as follows:
the results of the in vitro MIC assay showed that the MIC of usnic acid alone against the gram-negative bacteria was greater than the maximum tested concentration, i.e., > 160. Mu.g/mL. The combination of usnic acid and polymyxin E can improve the antibacterial activity of polymyxin:
when colibacillus standard strain ATCC25922 is combined and used with polymyxin E and usnic acid, the polymyxin E and the usnic acid exert the maximum synergistic effect when the concentration of the usnic acid is 5 mu g/mL and the concentration of the polymyxin E is 0.0625 mu g/mL, and the FICI is 0.28, so that the synergistic effect is judged.
When the clinical escherichia coli E.coli B2 strain and the polymyxin E and the usnic acid are jointly applied, the polymyxin E and the usnic acid exert the maximum synergistic effect when the concentration of the usnic acid is 40 mu g/mL and the concentration of the polymyxin E is 1 mu g/mL, and the FICI is 0.375, the synergistic effect is judged.
When the strain ATCC15692 and polymyxin E and usnic acid were used in combination, the maximum synergistic effect was exhibited when the concentration of usnic acid was 40. Mu.g/mL and the concentration of polymyxin E was 0.5. Mu.g/mL, and the FICI was 0.75, and the result was judged to be additive.
When polymyxin E and usnic acid are applied in combination to K.pneumoconiae 1202 (ST 11, KPC-2-producer) + pHNSHP45 (mcr-1), the maximum synergistic effect is achieved when the concentration of usnic acid is 20 mug/mL and the concentration of polymyxin E is 1 mug/mL, and the FICI is 0.25, the synergistic effect is determined.
When the polymyxin E and the usnic acid are jointly applied aiming at the clinical Escherichia coli E.coli 09b19 strain and the concentration of the usnic acid is 20 mug/mL, the polymyxin E concentration is 1. Mu.g/mL, the two exert the maximum synergistic effect, the FICI is 0.375, and the synergistic effect is judged.
When the polymyxin E and usnic acid are jointly applied to the clinical Escherichia coli E.coli13h1 strain, the polymyxin E and usnic acid exert the maximum synergistic effect when the concentration of usnic acid is 20 mu g/mL and the concentration of polymyxin E is 1 mu g/mL, and the FICI is 0.25, so that the synergistic effect is judged.
When polymyxin E and usnic acid are jointly applied to a clinical escherichia coli E.coli GZP11-15 strain, the polymyxin E and usnic acid exert the maximum synergistic effect when the concentration of usnic acid is 20 mu g/mL and the concentration of polymyxin E is 1 mu g/mL, and the FICI is 0.25, so that the synergistic effect is judged.
When the combination of polymyxin E and usnic acid is applied to ATCC 19606 strain, the maximum synergistic effect is exerted between the concentration of usnic acid 20. Mu.g/mL and the concentration of polymyxin E0.0625. Mu.g/mL, and the FICI is 0.25, the synergistic effect is judged.
When polymyxin E and usnic acid are jointly applied to the clinical Salmonella SH16SF0764 strain, the polymyxin E and usnic acid exert the maximum synergistic effect when the concentration of usnic acid is 20 mu g/mL and the concentration of polymyxin E is 1 mu g/mL, and the FICI is 0.25, so that the synergistic effect is judged.
In conclusion, the usnic acid can remarkably enhance the antibacterial activity of polymyxin against gram-negative bacteria when added in a concentration of 5-40 mug/mL.
Example 2 synergistic bactericidal profile for combination of polymyxin and usnic acid
1. Test materials
Usnic acid and colistin sulfate were the same as in example 1.
2. In vitro bactericidal curve test
After culturing Ecoli B2, ATCC25922 and E.coli GZP080-8 in BHI broth for 6 hours, an equal volume of DMSO (final concentration of 0.1%), polymyxin E sulfate, usnic acid, polymyxin E sulfate and usnic acid mixture was added.
(1) Grouping and dosing for the Ecoli B2 strain were as follows:
control group: 0.1% DMSO;
polymyxin E group: polymyxin E sulfate 4. Mu.g/mL;
usnic acid group: usnic acid 80 mug/mL;
usnic acid and polymyxin E combination: usnic acid 80. Mu.g/mL, polymyxin E sulfate 4. Mu.g/mL.
Then 100. Mu.L of the bacterial liquid was applied to MHA agar plates with a diameter of 10cm for 1h, 3h, 6h, 12h and 24h, respectively, and colony counting was performed after overnight culture.
(2) For E.coli ATCC25922 bacteria, the groupings and dosages were as follows:
control group: 0.1% DMSO;
polymyxin E group: polymyxin E sulfate 0.25. Mu.g/mL;
usnic acid group: usnic acid 20 mu g/mL;
usnic acid and polymyxin E combination: usnic acid 20. Mu.g/mL, polymyxin E sulfate 0.25. Mu.g/mL.
Then 100. Mu.L of the bacterial liquid was applied to MHA agar plates with a diameter of 10cm for 1 hour, 3 hours, 6 hours, 12 hours and 24 hours, and colony counting was performed after overnight culture.
(3) Coli GZP080-8 strain, grouping and dosing were as follows:
control group: 0.1% DMSO;
polymyxin E group: polymyxin E sulfate 4. Mu.g/mL;
usnic acid group: usnic acid 80 mug/mL;
usnic acid and polymyxin E combination: usnic acid 80. Mu.g/mL, polymyxin E sulfate 4. Mu.g/mL.
Then 100. Mu.L of the bacterial liquid was applied to MHA agar plates with a diameter of 10cm for 1 hour, 3 hours, 6 hours, 12 hours and 24 hours, and colony counting was performed after overnight culture.
The results were as follows:
as shown in fig. 1, for the e.coli B2 strain, polymyxin E and usnic acid alone had no significant effect on bacterial colony count at 24h compared to the control group. When usnic acid and polymyxin E were treated in combination, the number of colonies at 3h of treatment was 2.97Log 10 CFU/mL; at 6h, the number of colonies was 0Log 10 CFU/mL, and the colony number is 0Log until 24h 10 CFU/mL。
As shown in FIG. 2, the colony count was reduced to 5.2Log in the polymyxin E-treated group at 24h as compared with the control group against ATCC25922 strain 10 CFU/mL, usnic acid had no significant effect on bacterial colony count. When the usnic acid and the polymyxin E are treated in a combined mode, the colony number is reduced to 3.8Log at 1h, 3h, 6h, 12h and 24h 10 CFU/mL、3.4Log 10 CFU/mL、3.0Log 10 CFU/mL、0Log 10 CFU/mL、0Log 10 CFU/mL。
As shown in fig. 3, with respect to the e.coli GZP080-8 strain, there was no significant change in the number of colonies at 24h in the polymyxin E-treated group and usnic acid-alone treated group, compared to the control group. When the usnic acid and polymyxin E are treated in a combined mode, the colony number is reduced to 4.7Log at 1h, 3h, 6h, 12h and 24h 10 CFU/mL、4.6Log 10 CFU/mL、3.9Log 10 CFU/mL、0Log 10 CFU/mL、0Log 10 CFU/mL。
Example 3 therapeutic Effect of polymyxin E in combination with usnic acid on the bacterial clearance of wound infection in mice
Female BALB/c mice, weighing 18-20 g, were purchased from Beijing Wintonli Hua, inc. Mice were housed in the animal room of the national veterinary drug safety center of the animal medical college of the university of agriculture, china, at room temperature (25 ± 2 ℃, relative humidity 50 ± 10%, using conventional light, day: night =12h, feeding conventional mice to maintain pellet feed, mice were acclimatized in the animal room for 1 week prior to the official experiment.
After the mice are bred adaptively for 1 week in a laboratory, all the mice establish a skin wound model, and the main steps are as follows: the hair on the back was removed with an electric depilator, and the mice were anesthetized with 4% chloral hydrate by intraperitoneal injection, each administered at 10 mg/kg. Disinfecting the depilated part of the back with alcohol, marking round mark with marking pen, cutting off the skin of the back with high-pressure sterilized ophthalmic scissors, making round wound with diameter of 1cm, and cutting off the skin. Subsequently, mice were infected with e.coli B2 and 10 μ L (1 × 10) was added dropwise to the wound using a micropipette gun 9 CFU/mL) of e.coli B2 bacteria, the amount of infected bacteria per mouse was 1 × 10 7 CFU, control group dropped PBS on wound surface, waited skin to absorb the dropped liquid, and the mouse was observed to be awake. Coli B2 infected mice were further divided into 4 groups, namely a solvent-treated control group, a polymyxin E-treated group, an usnic acid-treated group, and a polymyxin E-combined usnic acid-treated group, each of which was 6 mice. mu.L of physiological saline, 10. Mu.L of polymyxin E (1 mg/mL, corresponding to a dose of 0.5mg/kg body weight for polymyxin E administered to each mouse), 10. Mu.L of usnic acid (40 mg/mL, corresponding to a dose of 20mg/kg body weight for each mouse), 10. Mu.L of polymyxin E (1 mg/mL) + usnic acid (40 mg/mL) (corresponding to a dose of 0.5mg/kg body weight for each mouse and a dose of 20mg/kg body weight for each mouse) were directly added dropwise to the skin infection at 1h and 24h, respectively. After 48h post-infection, mice were sacrificed under anesthesia, the wound and surrounding skin tissue were cut with an autoclave surgical instrument, and subcutaneous adherent tissue was peeled off. Putting the skin tissue into a 2mL grinding centrifuge tube, adding 500 mu L PBS and grinding beads, grinding in a high-temperature low-speed tissue grinding instrument, selecting a skin tissue grinding program, and fully grinding at 4 ℃. Tissue homogenate was diluted to appropriate fold (500 fold): 20 μ L of stock solution was placed in a 2mL centrifuge tube and diluted by 1.98mL sterile PBS. 100 mu L of diluted bacterial liquid is taken and dripped into the polymyxin-containing bacterial liquidE (the final concentration is 2 mu g/mL, and the main purpose is to prevent and control other mixed bacteria pollution in the operation process) on the BHI solid culture medium, uniformly coating the bacterial liquid by using an applicator, culturing for 14-16 h in an incubator at 37 ℃, observing the growth condition of bacterial colonies, and counting.
The results are as follows: as shown in FIG. 4, the average value of the bacterial load of the skin wound of the solvent-treated control group was 7.1Log 10 The average bacterial loads of CFU, polymyxin E and usnic acid treatment groups are respectively 6.5Log 10 CFU and 4.5Log 10 The colony number average value of the CFU, polymyxin E and usnic acid combined treatment group is reduced to 0.87Log 10 And (4) CFU. This indicates that the combined treatment of polymyxin E and usnic acid significantly improves the bacterial clearance to skin wounds compared to the treatment with either polymyxin E or usnic acid alone.
Claims (10)
1. The usnic acid or the pharmaceutically acceptable salt thereof is applied to the preparation of an antibacterial synergist for enhancing the antibacterial infection efficacy of polymyxin antibacterial drugs.
2. Application of usnic acid or pharmaceutically acceptable salt thereof in preparing medicines for resisting bacterial infectious diseases.
3. An antibacterial composition comprises usnic acid or pharmaceutically acceptable salts thereof and polymyxin antibacterial drugs.
4. The antimicrobial composition of claim 3, wherein: the mass ratio of the usnic acid or the pharmaceutically acceptable salt thereof to the polymyxin antibacterial drug is (20-320): 1, preferably (20-80): 1.
5. The antimicrobial composition of claim 4, wherein: the antibacterial composition is used for treating bacterial skin infections.
6. The use according to claim 1 or 2 or the antimicrobial composition according to any one of claims 3 to 5, characterized in that: the usnic acid has a structural formula shown in formula I:
the polymyxin antibacterial drug is selected from at least one of the following: 1) Polymyxin E or a pharmaceutically acceptable salt thereof; 2) Polymyxin B or a pharmaceutically acceptable salt thereof.
7. The use according to claim 1 or 2 or the antimicrobial composition according to claim 3 or 4, characterized in that: the bacteria are gram-negative bacteria;
further, the bacterium is a bacterium in which a polymyxin resistance gene exists;
further, the gram-negative bacterium is a multidrug-resistant gram-negative bacterium.
8. The use or antimicrobial composition according to claim 7, characterized in that: the bacteria are one or more of escherichia coli, klebsiella pneumoniae, salmonella, shigella and acinetobacter baumannii; preferably, the one or more of escherichia coli, klebsiella pneumoniae, salmonella, shigella, acinetobacter baumannii has a polymyxin resistance gene or multiple resistance genes.
9. An antibacterial product comprising the antibacterial composition according to any one of claims 3 to 8.
10. The antimicrobial product of claim 9, wherein: the product is a pharmaceutical preparation, and the dosage form of the pharmaceutical preparation is selected from any one of the following: tablet, cream, capsule, sustained release tablet, controlled release tablet, oral liquid, syrup, dripping pill, injection, and lyophilized powder for injection.
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