CN115501214B - Composition of pyrogallol and sulfamethoxazole and application thereof in inhibiting streptococcus suis type 2 - Google Patents
Composition of pyrogallol and sulfamethoxazole and application thereof in inhibiting streptococcus suis type 2 Download PDFInfo
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
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- A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
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Abstract
The invention belongs to the technical field of veterinary medicines, and particularly relates to a composition of pyrogallol and sulfaisoxazole and application thereof in inhibiting streptococcus suis type 2. The pyrogallic acid composition provided by the invention comprises: the optimal proportioning concentration of pyrogallol and sulfaisoxazole for streptococcus suis type 2 (HB strain) is as follows: pyrogallic acid: sulfoisoazole=8 μg/ml:256 μg/ml; the grading bacteriostasis concentration index is 0.5, and the optimal proportioning concentration for streptococcus suis type 2 (SC 19 strain) is as follows: pyrogallic acid: sulfoisoazole=16 μg/ml:256 μg/ml; the graded bacteriostasis concentration index is 0.375, and the pyrogallol and the sulfaisoxazole have synergistic bacteriostasis on the streptococcus suis type 2, and the composition reduces the concentration of the pyrogallol and the sulfaisoxazole for single bacteriostasis, saves the cost, and has wide application prospect and development value in the fields of food processing, medicine and aquaculture.
Description
Technical Field
The invention belongs to the technical field of veterinary medicines, and particularly relates to a composition of pyrogallol and sulfaisoxazole and application thereof in inhibiting streptococcus suis type 2.
Background
Streptococcus Suis (SS) is an important zoonotic pathogen, and can be classified into 33 serotypes according to the difference of capsular polysaccharides, and can infect pigs and humans, causing sepsis, meningitis, pneumonia, arthritis and even death. At present, the focus of bacterial infection control is mainly prevention and antibiotic treatment. However, with the large and unreasonable use of antibiotics, bacterial resistance is very serious.
Clinical trial data shows that different degrees of resistance appear in different countries and regions of isolated streptococcus suis. In the Italian Cucco isolated Streptococcus suis, 61.5% of the strains developed resistance to two antibiotics and 29.5% of the strains developed resistance to three antibiotics. Canadian scholars Arndt tested 379 strains of Streptococcus suis for resistance to tetracycline, tiamulin and spectinomycin, and found that most strains were resistant to tetracycline, tiamulin and spectinomycin. Among 1163 strains of streptococcus suis isolated clinically from vanHout, the netherlands scholars had 78.4% resistance to tetracycline and 48.1% resistance to clindamycin. The clinical 34 strains of streptococcus suis are subjected to drug resistance analysis by Chinese scholars, 91.18 percent of strains are multi-drug resistant, 82.3 to 100.0 percent of strains are drug resistant to sulfonamides, 79.4 to 94.1 percent of strains are drug resistant to aminoglycosides, 88.2 to 97.1 percent of strains are drug resistant to tetracyclines, 79.4 to 85.3 percent of linkeamine drugs are drug resistant, and 50 to 88.2 percent of strains are drug resistant to macrolides. Thus, searching for new antibiotic substitutes, developing new antibacterial agents, or finding compounds that reverse antibiotic resistance are key approaches to solving the antibiotic resistance problem.
Pyrogallol (molecular weight 126.11, molecular formula: C) 6 H 6 O 3 ) Is a natural polyphenol compound extracted from the stems of Cotinus coggygria, has biological functions of resisting infection, resisting fungi and the like, is also a reducing agent, and can generate free radicals, especially superoxide anions. Currently according to the literatureJiaosuan is reported to have antibacterial effect against Acinetobacter baumannii, salmonella, pseudomonas putida, vibrio vulnificus, etc., but is ineffective against fungi such as Fusarium oxysporum, penicillium, candida albicans, etc. In addition, the Escherichia coli can synthesize pyrogallol with high efficiency, and the concentration can reach 1800+/-100 mg/L (Huo YX, ren H, yu H, et al CipA-mediating enzyme self-assembly to enhance the biosynthesis of pyrogallol in Escherichia coll. ApplMicrobiol Biotechnol.2018;102 (23): 10005-10015.) according to the presumption that pyrogallol is insensitive to Escherichia coli, that is, pyrogallol has no inhibition effect on Escherichia coli.
The main antibacterial mechanism of the sulfamethoxazole acts on dihydrofolate synthase in a bacterial body to prevent the synthesis of bacterial folic acid and reduce the amount of metabolic activity of tetrahydrofolate, and the balance of the bacterial essential substances for synthesizing purine, thymidine and deoxyribonucleic acid, thereby inhibiting the growth and reproduction of bacteria. Sulfoisoazole has antibacterial effect on bacteria belonging to family Enterobacteriaceae such as Staphylococcus aureus, streptococcus pyogenes, streptococcus pneumoniae, escherichia coli, klebsiella, salmonella, shigella, etc., gonococcus, meningococcus, and Haemophilus influenzae. However, in recent years, bacteria have extremely high resistance to the product, particularly bacteria of the genera Streptococcus, neisseria and Enterobacteriaceae.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the problem of serious drug resistance of the existing streptococcus suis and provides application of pyrogallol in preparing medicines for treating or preventing streptococcus suis type 2 infection.
It is another object of the present invention to provide a pyrogallol composition comprising: pyrogallol and sulfaisoxazole.
Another problem of the present invention is to provide the use of a pyrogallol composition for the preparation of a medicament for the treatment or prevention of infection with Streptococcus suis (Streptococcus suis) type 2. The composition provided by the invention has good antibacterial and bactericidal effects on streptococcus suis type 2.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
jiaosuan is used for preparing medicines for treating or preventing streptococcus suis type 2 infection by taking pyrogallol as an active ingredient or one of active ingredients, and is used for preparing medicines for treating or preventing streptococcus suis type 2 infection, inhibiting growth of streptococcus suis type 2 in vitro or directly killing streptococcus suis type 2.
A pyrogallol composition, said pyrogallol composition comprising: pyrogallol, sulfaisoxazole; the mass ratio of the pyrogallol to the sulfamethoxazole is 0.03125-0.0625:1, and the structural formula of the pyrogallol is as follows:
the application of the pyrogallol composition in preparing the medicine for treating or preventing the streptococcus suis type 2 infection comprises the step of taking the composition of the invention as an active ingredient or one of active ingredients, preparing the medicine for treating or preventing the streptococcus suis type 2 infection, and inhibiting the growth of the streptococcus suis type 2 in vitro or directly killing the streptococcus suis type 2.
The composition described above, preferably, comprises the following components in the composition in the optimal mass ratio for streptococcus suis type 2 (SC 19 strain): pyrogallic acid: sulfoisoxazole=16: 256; the graded antibacterial concentration index of 0.375 pyrogallol and clindamycin have synergistic antibacterial effect on streptococcus suis type 2.
Compared with the prior art, the invention has the following advantages and effects:
the invention provides that pyrogallol and sulfaisoxazole have synergistic antibacterial effect for the first time, and the composition can be used for resisting streptococcus suis type 2. The invention proves that the composition of pyrogallol and sulfaisoxazole has good antibacterial effect on streptococcus suis type 2 through a plurality of method researches, lays a theoretical foundation for developing novel medicines for resisting streptococcus suis type 2 diseases, provides candidate substances for the development of antibiotic substitutes, and has good application prospect in the aspect of preventing and treating streptococcus suis diseases.
After the sulfamethoxazole and pyrogallol are combined, the antibacterial concentration of the sulfamethoxazole and the antibacterial concentration of the pyrogallol are respectively reduced, the index of the grading antibacterial concentration (FICI) of the combined medicament is 0.5, and the sulfamethoxazole and the pyrogallol have synergistic antibacterial effect (the synergistic antibacterial effect, the FICI is less than or equal to 0.5, the additive effect, the FICI is less than or equal to 0.5 and less than or equal to 1, the irrelevant effect, the FICI is less than or equal to 1 and less than or equal to 2, and the antagonistic effect, the FICI is more than 2). Therefore, the pyrogallol can reverse the drug resistance of the streptococcus suis type 2 to the sulfaisoxazole, the composition of the sulfaisoxazole and the pyrogallol is expected to become a novel bioactive molecule for preventing and treating the streptococcus suis type 2 disease, a solution is provided for preventing and treating the drug-resistant streptococcus suis type 2 disease, the method has a good application prospect in preventing and treating the streptococcus suis type 2 disease, and a theoretical basis is provided for popularization and application of the pyrogallol composition as an antibiotic substitute.
Drawings
FIG. 1 shows the chemical structure of pyrogallol monomers.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified. The embodiment of the invention is illustrated by taking streptococcus suis type 2 HB (self-screened) and SC19 strain (CN 106995489A) as examples, and other streptococcus suis type 2 substances disclosed by the invention also have inhibiting and killing effects.
Statistical analysis of the following examples of the invention: all experiments were repeated at least 3 times independently.
Example 1:
determination of minimum inhibitory concentration and minimum bactericidal concentration of pyrogallol on streptococcus suis type 2 HB, SC19 strains:
1. material
Mueller-Hinton (MH) broth (available from BD company) was used in drug susceptibility experiments to examine the bacteriostatic efficacy of drugs.
2. Test method
The bacterial colony of the purified streptococcus suis type 2 HB and SC19 strain is respectively picked by a sterile inoculating loop and fully separatedDispersed in MH liquid culture medium, each of which is configured to be 5×10 5 Standard bacterial suspension of CFU/ml;
different concentrations of pyrogallod solution diluted with MH broth were added to sterile 96-well polystyrene plates, respectively, with 50. Mu.l of 1 st to 10 th Kong Jiajiao th gallod solution per well, 50. Mu.l of MH broth was added to 11 th well as a growth control, and 100. Mu.l of MH broth alone was added to 12 th well as a negative control.
50 μl of standard bacterial suspension was added to wells 1 to 11, incubated at 37℃for 16-20h, and the MIC value was determined by observing the presence or absence of bacterial growth. The bacterial liquid was aspirated from each well, plated onto MH plates, and MBC was determined without colony growth.
3. Results
As is clear from Table 1, it has an inhibitory effect on the growth of Streptococcus suis type 2 (HB) at a concentration of pyrogallol of 32. Mu.g/ml, and has a bactericidal effect on Streptococcus suis type 2 (HB) at a concentration of pyrogallol of 64. Mu.g/ml; the pyrogallol has an inhibitory effect on the growth of Streptococcus suis type 2 (SC 19) at a concentration of 64. Mu.g/ml, and has a bactericidal effect on Streptococcus suis type 2 (SC 19) at a concentration of 64. Mu.g/ml;
TABLE 1 minimum inhibitory concentration and minimum bactericidal concentration of pyrogallol on Streptococcus suis type 2
Example 2:
minimum inhibitory concentration and minimum bactericidal concentration of Succinum Streptococcus 2 type HB, SC19 strains:
1. material
Mueller-Hinton (MH) broth (available from BD company) was used in drug susceptibility experiments to examine the bacteriostatic efficacy of drugs.
2. Test method
The purified Streptococcus suis type 2 HB and SC19 strain colonies were picked up with sterile inoculating loops, and thoroughly dispersed in MH liquid medium, each configured as 5X 10 5 Standard bacterial suspension of CFU/ml;
sulfoisoazole solutions of different concentrations after dilution with MH broth were added to sterile 96-well polystyrene plates, respectively, with sulfamethoxazole solution added at wells 1 to 10, 50. Mu.l per well, 50. Mu.l of MH broth added at well 11 as growth control, and 100. Mu.l of MH broth alone added at well 12 as negative control.
50 μl of standard bacterial suspension was added to wells 1 to 11, incubated at 37℃for 16-20h, and the MIC value was determined by observing the presence or absence of bacterial growth. The bacterial liquid was aspirated from each well, plated onto MH plates, and MBC was determined without colony growth.
3. Results
As is clear from Table 2, it has an inhibitory effect on the growth of Streptococcus suis type 2 (HB) at a sulfamethoxazole concentration of 1024. Mu.g/ml; when the concentration of the sulfamethoxazole is 2048 mug/ml, the sulfamethoxazole has bactericidal effect on streptococcus suis type 2 (HB). When the concentration of the sulfamethoxazole is 2048 mug/ml, the sulfamethoxazole has an inhibiting effect on the growth of streptococcus suis type 2 (SC 19); when the concentration of the sulfamethoxazole is 4096 mug/ml, the sulfamethoxazole has a bactericidal effect on streptococcus suis type 2 (SC 19).
TABLE 2 minimum inhibitory concentration and minimum bactericidal concentration of Succinum Streptococcus 2 with Sulfoisoazole
Example 3:
pyrogallol and sulfaisoxazole have synergistic antibacterial effect on streptococcus suis type 2:
1. material
Mueller-Hinton (MH) broth (available from BD company) was used in drug susceptibility experiments to examine the bacteriostatic efficacy of drugs.
2. Test method
The bacterial colonies of purified streptococcus suis type 7, streptococcus suis type 9, streptococcus equi subspecies zooepidemicus, streptococcus suis type 2 HB and streptococcus suis SC19 are respectively picked up by a sterile inoculating loop, transferred after shaking overnight, and fully dispersed in MH liquid culture medium to prepare 5 multiplied by 10 5 Standard bacterial suspension of CFU/ml;
50 μl of MH liquid culture medium is added to each row of 2 to 10 wells of a sterile 96-well polystyrene plate by a row gun, pyrogallol solution with 4 times MIC concentration is added to each row of 1 and 2 wells, 50 μl of pyrogallol solution is sucked out of the second well and added to the third well after being blown and mixed by the row gun, and the like, the mixture is blown and mixed by a multiple ratio for dilution, and 50 μl of pyrogallol solution is sucked out of the 11 wells for discarding. 50 μl of MH broth was added to well 12 as a growth control, where the concentration of pyrogallos contained in each column was consistent.
The different concentrations of the sulfamethoxazole solution are diluted in sterile 4ml EP tubes with MH liquid culture medium according to the MIC values of the sulfamethoxazole of different strains, and the maximum concentration is 4 times of the MIC concentration. The concentration of row 2 is half that of row 1, and so on, the concentration of the sulfamethoxazole added in row 8 is calculated and the corresponding solution is prepared, then 50 μl of the solution is added into a 96-well polystyrene plate, and the concentration of antibiotics in each row is consistent.
At this time, each hole of the 96-hole polystyrene plate contains the sulfaisoxazole and pyrogallol solution with different concentrations, the concentration of the sulfaisoxazole in each row of solution is the same, and the concentrations of the 1 st row to the 8 th row are reduced in a multiple ratio; the concentration of pyrogallod solution contained in each column of the solution is the same, and the concentrations from column 1 to column 10 are reduced by a multiple ratio.
Mu.l of standard bacterial suspension was added to each of wells 1 to 10 and 12, and after addition, 50. Mu.l or 100. Mu.l MH medium was added to each of 96 wells, so that the total system per well was 200. Mu.l. Each row of 11 th holes is a negative control, and only pyrogallol and sulfaisoxazole solution are used; wells 12 were growth control, standard bacterial fluid only.
And incubating the 96-well polystyrene plate for 16-20h at 37 ℃, observing whether bacteria grow or not, and calculating the synergistic antibacterial index according to the minimum combined antibacterial concentration of the antibiotics and the compounds.
3. Results
As is clear from Table 3, when pyrogallol is used in combination with sulfamethoxazole, the concentration of pyrogallol is 8. Mu.g/ml, the concentration of sulfamethoxazole is 256. Mu.g/ml, which has an inhibitory effect on the growth of Streptococcus suis type 2 (HB), and the index of the hierarchical inhibitory concentration is 0.5; the concentration of pyrogallol is 16 mug/ml, the concentration of sulfaisoxazole is 256 mug/ml, the pyrogallol has an inhibition effect on the growth of streptococcus suis type 2 (SC 19), and the graded antibacterial concentration index is 0.375, which indicates that pyrogallol and sulfaisoxazole have a synergistic antibacterial effect. FICI calculation formula: MIC at MIC/a alone at MIC/a combination and MIC at MIC/B alone at mic+b combination at fici=a combination.
TABLE 3 minimum inhibitory concentration and graded inhibitory concentration index of pyrogallol in combination with sulfaisoxazole on Streptococcus suis
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (4)
1. Use of a pyrogallol composition for the preparation of a medicament for the treatment or prevention of streptococcus suis type 2 infection, said pyrogallol composition comprising: pyrogallol, sulfaisoxazole; the mass ratio of the pyrogallol to the sulfaisoxazole is 0.03125-0.0625:1.
2. Use of a pyrogallol composition for inhibiting or killing streptococcus suis type 2 in vitro, said pyrogallol composition comprising: pyrogallol, sulfaisoxazole; the mass ratio of the pyrogallol to the sulfaisoxazole is 0.03125-0.0625:1.
3. The use according to claim 1 or 2, wherein said streptococcus suis type 2 is streptococcus suis SC19.
4. The use according to claim 2, wherein the concentration of pyrogallol in the pyrogallol composition is 16 μg/ml and the concentration of sulfaisoxazole is 256 μg/ml when used for inhibiting streptococcus suis type 2 in vitro.
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| CN116350641B (en) * | 2023-02-07 | 2024-09-24 | 湖北省农业科学院畜牧兽医研究所 | Sabina alcohol and sulfamethoxazole composition and application thereof in bacteria inhibition |
| CN116211879B (en) * | 2023-02-07 | 2024-05-31 | 湖北省农业科学院畜牧兽医研究所 | Sabina alcohol and clindamycin composition and application thereof in bacteria inhibition |
| CN116077509B (en) * | 2023-02-07 | 2024-09-24 | 湖北省农业科学院畜牧兽医研究所 | Composition of pyrolin and sulfaisoxazole and application thereof in bacteria inhibition |
| CN116196324B (en) * | 2023-02-07 | 2024-09-24 | 湖北省农业科学院畜牧兽医研究所 | Composition of pyrolin and clindamycin and application thereof in bacteria inhibition |
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