CN114652716B - Application of dimetridazole in preparation of beta-lactam drug-resistant escherichia coli drug - Google Patents
Application of dimetridazole in preparation of beta-lactam drug-resistant escherichia coli drug Download PDFInfo
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- CN114652716B CN114652716B CN202210332764.5A CN202210332764A CN114652716B CN 114652716 B CN114652716 B CN 114652716B CN 202210332764 A CN202210332764 A CN 202210332764A CN 114652716 B CN114652716 B CN 114652716B
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- A61K31/545—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine
- A61K31/546—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine containing further heterocyclic rings, e.g. cephalothin
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Abstract
The invention relates to the field of biological medicine, in particular to the field of prevention and treatment of animal-derived bacterial diseases. The invention discloses application of dimetridazole in preparation of a beta-lactam drug resistance escherichia coli resistant drug. The invention also provides an escherichia coli drug composition for resisting the beta-lactam drug resistance, which comprises dimetridazole and cephalosporin antibiotics such as cefotaxime. The dimetridazole and cefotaxime combined application has a synergistic effect, can improve the sensitivity of antibiotics and reduce the using amount of the antibiotics, achieves the aim of better synergistic antibacterial, and simultaneously provides technical support for veterinary clinical treatment of diseases caused by beta-lactam drug-resistant escherichia coli.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to the field of prevention and treatment of animal-derived bacterial diseases.
Background
Escherichia coli is a main pathogenic bacterium causing urinary tract infection and blood infection, and after the Escherichia coli disease is caused, drug therapy is mostly adopted, but due to improper use or abuse of antibacterial drugs, the treatment cost is increased, high drug resistance of human and animals is caused, and the serious threat to public health is caused. In the last 10 years, the detection rate of beta-lactam drug-resistant escherichia coli is continuously increased in the global range, the mortality rate of infection caused by the beta-lactam drug-resistant escherichia coli is higher, the beta-lactam drug-resistant escherichia coli is separated from livestock such as chicken, duck, pig, cattle and the like, poultry and wastewater and the environment, drug-resistant strains can exist in the environment and water for a long time, the risk of human infection is increased, and the beta-lactam drug-resistant escherichia coli poses serious threat to the global public health. Colistin is the last line of defense for treating gram-negative bacteria, however, there are reports of colistin resistance in recent years, and the treatment for infections caused by beta-lactam resistant bacteria is in a situation of no medicine cure, so that the development of new effective medicines or the search of new pharmaceutical compositions for solving the problem of drug resistance is urgently needed.
Disclosure of Invention
The invention aims to provide the application of dimetridazole in preparing a beta-lactam drug-resistant escherichia coli resistant medicament.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an application of dimetridazole in preparation of a beta-lactam drug resistance escherichia coli resistant drug.
Preferably, the concentration of the dimetridazole is 2-2000 mug/mL.
The invention also provides a pharmaceutical composition for resisting the beta-lactam drug resistance escherichia coli, which comprises dimetridazole and cephalosporin antibiotics.
Preferably, the cephalosporin antibiotics include cefotaxime.
Preferably, the concentration of the dimetridazole in the pharmaceutical composition is 120-130 mug/mL, and the concentration of the cefotaxime is 60-70 mug/mL.
Preferably, the concentration of dimetridazole in the pharmaceutical composition is 60-65 mug/mL, and the concentration of cefotaxime is 125-130 mug/mL.
Preferably, the concentration of dimetridazole in the pharmaceutical composition is 30-35 mug/mL, and the concentration of cefotaxime is 125-130 mug/mL.
By adopting the technical scheme, the invention has the following beneficial effects:
the dimetridazole and cefotaxime are used together, the combination can improve the sensitivity of antibiotics and reduce the dosage of the antibiotics, and the composition has a synergistic effect. The dimetridazole with the main effect of expelling parasites is used as a synergist of cephalosporin antibiotics, and the dimetridazole has a synergistic effect. The invention provides technical support for the veterinary clinical treatment of diseases caused by beta-lactam drug-resistant escherichia coli.
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FIG. 1 shows the MIC measurements of dimetridazole and cefotaxime;
FIG. 2 shows the results of the combined use of dimetridazole and cefotaxime.
Detailed Description
The invention provides an application of dimetridazole in preparation of a beta-lactam drug resistance escherichia coli resistant drug.
In the invention, the dimetridazole solution takes a TSA culture medium as a solvent and dimetridazole as a solute; the concentration of the dimetridazole solution is 2-2000 mug/mL, and the preferable concentration is 3.9-2000 mug/mL.
The invention also provides a pharmaceutical composition for resisting the beta-lactam drug resistance escherichia coli.
In the invention, the pharmaceutical composition comprises an anti-beta-lactam drug-resistant escherichia coli pharmaceutical composition.
In the present invention, the cephalosporin antibiotics include cefotaxime.
In one embodiment of the present invention, the concentration of dimetridazole in the pharmaceutical composition is 120 to 130 μ g/mL, the concentration of cefotaxime is 60 to 70 μ g/mL, and more preferably, the concentration of dimetridazole is 125 μ g/mL, and the concentration of cefotaxime is 64 μ g/mL.
In another embodiment of the present invention, the concentration of dimetridazole in the pharmaceutical composition is 60-65 μ g/mL, the concentration of cefotaxime is 125-130 μ g/mL, more preferably, the concentration of dimetridazole is 62.5 μ g/mL, and the concentration of cefotaxime is 128 μ g/mL.
In another embodiment of the present invention, the concentration of dimetridazole in the pharmaceutical composition is 30 to 35 μ g/mL, the concentration of cefotaxime is 125 to 130 μ g/mL, more preferably, the concentration of dimetridazole is 31.25 μ g/mL, and the concentration of cefotaxime is 128 μ g/mL.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Description of the Experimental materials
Experimental strains: escherichia coli BNCC358242, purchased from North Nay organisms.
Preparation of a cefotaxime solution of 1024 μ g/mL: accurately weighing 10.24mg of cefotaxime, adding into 10ml of LTSB culture medium, and shaking to dissolve.
Preparation of a dimetridazole solution of 2000 mug/mL: accurately weighing 20.00mg dimetridazole, adding into 5ml LTSB culture medium +5ml NaOH mixed solution, performing ultrasonic treatment for 4h, and working frequency is 40KHz.
Example 1: respectively determining Minimum Inhibitory Concentration (MIC) of cefotaxime and dimetridazole
1) Taking out the Escherichia coli drug-resistant strain frozen at-80 deg.C, inoculating the strain to TSA culture medium with disposable inoculating loop dipped bacterial liquid, culturing overnight at 37 deg.C, selecting single colony to TSB culture medium, culturing at 37 deg.C under shaking at 200rpm for 10-16 h, adjusting to 0.5 McLeod with bacterial turbidimeter to maintain the bacterial concentration at 1 × 10 8 CFU/mL。
2) Respectively measuring MIC of cefotaxime and dimetridazole by trace broth dilution method, and diluting the obtained bacterial solution by 100 times to make its concentration be 1 × 10 6 CFU/mL。
3) One row is selected optionally in a 96-well plate, 100 mu L of cefotaxime solution and 100 mu L of bacteria solution which are diluted according to the multiple proportion are sequentially added into 1 st to 10 th wells, the concentrations of the cefotaxime solution after the dilution according to the multiple proportion are respectively 1024 mu g/mL, 512 mu g/mL, 256 mu g/mL, 128 mu g/mL, 64 mu g/mL, 32 mu g/mL, 16 mu g/mL, 8 mu g/mL, 4 mu g/mL and 2 mu g/mL, the 11 th well is a positive control group, 200 mu g/mL is addedL of the culture solution, and the 12 th well as a negative control group, 200. Mu.L of TSB medium was added. Culturing at 37 deg.C for 12-16 h, observing the result, and determining OD 600 ；
And selecting another row in a 96-well plate, sequentially adding 100 mu L dimetridazole solution and 100 mu L bacterial liquid diluted according to the multiple ratio into the 1 st to 10 th wells, wherein the concentration of the dimetridazole solution after the multiple ratio dilution is 2000 mu g/mL, 1000 mu g/mL, 500 mu g/mL, 250 mu g/mL, 125 mu g/mL, 62.5 mu g/mL, 31.3 mu g/mL, 15.6 mu g/mL, 7.8 mu g/mL and 3.9 mu g/mL respectively, the 11 th well is a positive control group, 200 mu L bacterial liquid is added, the 12 th well is a negative control group, and 200 mu L TSB culture medium is added. Culturing at 37 deg.C for 12-16 h, observing the result, and determining OD 600 。
As a result:
OD measured by cefotaxime solution under different concentrations 600 As shown in table 1:
TABLE 1 OD of treated bacteria solutions of cefotaxime in different concentrations 600
OD measured by dimetridazole solution under different concentrations 600 As shown in table 2:
TABLE 2 OD of bacteria solution treated with dimetridazole solution of different concentrations 600
Considering the two tables together, the MIC values of the available sporozoxime and dimetridazole are respectively as follows: MIC Cefotaxime ＝512μg/mL，MIC Dimetridazole ＝500μg/mL。
Example 2: determination of minimum inhibitory concentration of cefotaxime and dimetridazole
According to the above measurement results, the minimum inhibitory concentration values of the combination were determined by the checkerboard method in 96-well plates, and 50. Mu.L of cefotaxime solution was added to each well, depending on the concentration of the drugThe times are 1MIC, 1/2MIC, 1/4MIC, 1/8MIC, 1/16MIC and 1/32MIC, then 50 muL of dimetridazole solution is added, the concentration of the drug is 2MIC, 1/2MIC, 1/4MIC, 1/8MIC, 1/16MIC and 1/32MIC in sequence, and then 100 muL of 1 multiplied by 10 is added 6 CFU/mL bacterial dilution, and set up only containing 200 u L bacterial dilution of the positive control and only containing 200 u L TSB medium of the negative control. Culturing at 37 deg.C for 12-16 h, observing, and determining OD 600 And calculating FIC (antibacterial concentration fraction index), and evaluating the combined administration result according to the FIC index.
The FIC index calculation method is as follows:
if the FIC is less than or equal to 0.5, a synergistic effect is shown; if 0.5< -FIC is less than or equal to 1, additive effect is shown; if 1-straw FIC-straw-bundle 2, then it appears to be irrelevant: if the FIC is more than or equal to 2, antagonism is shown, and the smaller the FIC index is, the stronger the synergistic effect of the two medicines is.
OD measured when the two drugs are used in combination 600 As shown in table 3:
TABLE 3 OD of bacterial liquid in combination of two drugs 600
( 1-6 cefotaxime with 1MIC, 1/2MIC, 1/4MIC, 1/8MIC, 1/16MIC and 1/32MIC in sequence; dimetridazole with A to G sequentially being 2MIC, 1/2MIC, 1/4MIC, 1/8MIC, 1/16MIC and 1/32MIC )
As can be seen from the above examples, the present invention provides the inhibitory effect of dimetridazole and cefotaxime against the drug-resistant Escherichia coli at different concentrations, wherein MIC was found Dimetridazole ＝500μg/mL，MIC Cefotaxime =512 μ g/mL. The invention also provides OD for the drug-resistant escherichia coli when the dimetridazole and the cefotaxime are used together 600 The combination of dimetridazole and cefotaxime is found to be resistant to multiple drugsThe medicinal escherichia coli isolate has obvious synergistic antibacterial action.
When the FIC index of the two medicines in combination is calculated, the FIC of three combinations is found to be less than 0.5:1/4MIC dimetridazole is combined with 1/8MIC cefotaxime, and the FIC index of the combination is 125/500+64/512=0.375;1/8MIC dimetridazole is combined with 1/4MIC cefotaxime, and the FIC index of the combination is 62.5/500+128/512=0.375;1/16MIC dimetridazole is combined with 1/4MIC cefotaxime, and the FIC index of the combination is 31.25/500+128/512=0.3125. The optimal antibacterial effect is comprehensively considered, and the effect is optimal when 1/16MIC dimetridazole and 1/4MIC cefotaxime are selected for combination.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (1)
1. The application of the pharmaceutical composition of dimetridazole and cefotaxime in preparing an anti-beta-lactam drug-resistant escherichia coli drug is characterized in that the concentration of dimetridazole in the pharmaceutical composition is 30 to 35 mu g/mL, and the concentration of cefotaxime is 125 to 130 mu g/mL.
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