A tetracycline antibiotic synergist and its application in drug-resistant Escherichia coli

Abstract

The present invention provides a tetracycline antibiotic synergist and its application in drug-resistant Escherichia coli, belonging to the field of medical technology. Specifically, α-terpineol is used as a synergist in combination with tetracycline antibiotics to fight against drug-resistant Enterobacter. Experiments show that α-terpineol can effectively enhance the sensitivity of tetracycline antibiotics tigecycline / doxycycline to drug-resistant Enterobacter. The combined use of α-terpineol and tigecycline / doxycycline can significantly reduce the minimum inhibitory concentration of tigecycline / doxycycline in the combined combination, which can effectively improve the efficacy of the above-mentioned antibiotics against drug-resistant Enterobacter, and achieve the purpose of reducing the dosage of antibiotics, which is of great significance for solving clinical drug-resistant Escherichia coli infections. The present invention provides technical support for the treatment and control of drug-resistant Enterobacter in clinical practice.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, specifically to the field of prevention and treatment of bacterial infectious diseases in animals, and relates to the application of a natural plant extract alpha-pinene alcohol in combination with tetracycline antibiotics to combat drug-resistant Escherichia coli. Background Art

[0002] Tigecycline, as a new tetracycline antibiotic, is one of the last lines of defense for the treatment of carbapenemase-producing multidrug-resistant Enterobacteriaceae infections. In recent years, the emergence of tigecycline-resistant strains has severely restricted the clinical use of tigecycline. Plasmid-mediated tet(X) variants can cause strains to develop high-level resistance to tigecycline and spread between different species of bacteria through conjugative plasmids. High-level resistance to tigecycline can not only lead to the failure of first- and second-generation tetracycline drugs, but may also form cross-resistance to new tetracycline antibiotics. The spread of resistance will pose a serious threat to ecological security and public health.

[0003] Because my country's livestock and poultry breeding volume ranks among the highest in the world and the breeding density is high, even though a policy of reducing the use of animal antibiotics has been implemented in recent years, my country's use of antibiotics still ranks first in the world. With the increasing use of antibiotics, improper use has become more and more common, leading to the emergence of drug resistance in many Enterobacteriaceae. At present, various types of drug-resistant bacteria have spread widely around the world, and even super-resistant bacteria containing multi-drug resistance genes have appeared. Antibiotics often cause bacteria to become resistant after 3-12 years of clinical use, and at the same time, it will take some time to develop new antibiotics. Therefore, it is particularly important to develop safe and effective new antimicrobial enhancers for the prevention and control of drug-resistant bacteria.

[0004] my country has a very rich variety of plants, and many natural compounds have certain bactericidal effects and are weakly toxic or non-toxic, which are very suitable as antibiotic synergists. Therefore, the present invention aims to explore a new antibiotic synergist based on natural compounds, so as to achieve the effect of not increasing the dosage of tetracycline drugs or even reducing the dosage, and explore its clinical application value. Summary of the invention

[0005] The purpose of this invention is to find a compound as a synergist to solve the current problem of difficulty in treating infectious diseases caused by tetracycline-resistant strains. This study found that the natural compound α-terpineol can be used as a synergist for tetracycline antibiotics.

[0006] α-Terpineol is a monoterpene compound with the molecular formula C 10 H 18O, has high optical activity, and has good effects in agricultural insecticide, sterilization, sanitary mite removal, and insect repellent. The present invention found that α-terpineol can effectively reduce the minimum inhibitory concentration of tigecycline and doxycycline, enhance the sensitivity of the two to important drug-resistant Escherichia coli, and reduce the dosage of the two drugs, thereby effectively solving the drug resistance problem of drug-resistant Escherichia coli. This invention has great clinical significance for the future treatment or prevention of the above-mentioned drug-resistant Enterobacter infection.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides the use of α-terpineol as a tetracycline antibiotic synergist in drug-resistant Escherichia coli. The tetracycline antibiotic is tigecycline or doxycycline; and the drug-resistant Escherichia coli includes tet(X) Escherichia coli.

[0009] The present invention also provides a pharmaceutical composition for drug-resistant Escherichia coli, the composition comprising α-terpineol and a tetracycline antibiotic, the tetracycline antibiotic being tigecycline or doxycycline; the weight ratio of the α-terpineol to tigecycline is in the range of 256:1 to 128:1; the weight ratio of the α-terpineol to doxycycline is in the range of 64:1 to 16:1.

[0010] Furthermore, the composition of the present invention also includes quercetin; the weight ratio of the α-terpineol, quercetin and tigecycline includes but is not limited to the following ratios: 64:64:1 or 512:128:5 or 128:512:5; the weight ratio of the α-terpineol, quercetin and doxycycline includes but is not limited to the following ratios: 16:16:1 or 128:32:5 or 32:128:5.

[0011] Preferably, the weight ratio of α-terpineol, quercetin and tigecycline is 128:512:5; the weight ratio of α-terpineol, quercetin and doxycycline is 32:128:5.

[0012] The present invention found that compared with the use of α-terpineol alone, the addition of quercetin can reduce the concentration and amount of α-terpineol used, and can also reduce the MIC of tigecycline by 4 to 16 times. Since α-terpineol is expensive, the combination of quercetin and α-terpineol can greatly reduce the cost of medication, and can reduce the use and cost of α-terpineol by at least 5 times.

[0013] The present invention also provides a pharmaceutical preparation for resisting drug-resistant Enterobacteriaceae, wherein the pharmaceutical preparation is a preparation prepared with a drug combination of α-terpineol, tigecycline or doxycycline as an active ingredient; or a preparation prepared with a pharmaceutical combination of quercetin, α-terpineol, tigecycline or doxycycline as an active ingredient.

[0014] The present invention has the following effects:

[0015] 1. The present invention explores the feasibility of using α-terpineol as a synergist for tetracycline antibiotics. Currently, there are few studies on the application of α-terpineol in the treatment of bacterial diseases. Therefore, the present invention studies α-terpineol and uses it for the first time to treat drug-resistant Enterobacter infection. This provides technical support for the treatment and control of drug-resistant Enterobacter in veterinary clinics.

[0016] 2. After research, this invention found that α-pineneol can effectively enhance the sensitivity of tetracycline antibiotics tigecycline / doxycycline to drug-resistant Enterobacteriaceae, and effectively improve the efficacy of the above antibiotics against drug-resistant Enterobacteriaceae, which is of great significance for solving clinical drug-resistant Escherichia coli infections.

[0017] 3. The present invention has been tested and shown that the combined use of α-terpineol and tigecycline / doxycycline can significantly reduce the minimum inhibitory concentration of tigecycline / doxycycline in the combined combination, thereby achieving the purpose of reducing the amount of antibiotics used, confirming that α-terpineol is a synergist that can be used in combination with tetracycline antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the effect of the combination of tigecycline and α-terpineol on the MIC of tigecycline in animal-derived tet(X) Escherichia coli in Example 1.

[0019] Figure 2 This is Example 2, the effect of the combination of doxycycline and α-terpineol on the MIC of doxycycline in animal-derived tet(X) Escherichia coli.

[0020] Figure 3 This is a line graph of the bactericidal effects of α-terpineol and doxycycline alone and in combination against Escherichia coli JMAT114 in Example 3.

[0021] Figure 4 This is a line graph of the chick survival rate after using α-terpineol and doxycycline alone or in combination in a chick infection model according to Example 5. DETAILED DESCRIPTION

[0022] In order to explain the technical solution in more detail, the beneficial effects of the present invention are described below in combination with specific embodiments and experiments. The effects of the present invention are not limited to the description of the embodiments of the present invention. For ordinary technicians in this field, other experimental results can be obtained based on these without creative work.

[0023] The present invention has no special limitation on α-terpineol, tigecycline, doxycycline, etc., and any conventional method used in the art can be used. Tigecycline is a third-generation tetracycline antibiotic, a broad-spectrum active intravenous antibiotic, and is also active against methicillin-resistant Staphylococcus aureus, and is the first drug in the glycyltetracycline class. Doxycycline hydrochloride is a broad-spectrum antibiotic that has an inhibitory effect on rickettsia, chlamydia, mycoplasma, etc. in addition to its effects on gram-negative and gram-positive bacteria. It is a light yellow or yellow crystalline powder, easily soluble in water, and slightly soluble in ethanol.

[0024] Example 1: Application of α-terpineol as a synergist of tigecycline in Escherichia coli carrying tet(X)

[0025] 1. Test consumables

[0026] 1. Reagents and drugs

[0027] LB broth medium, MH broth medium, and MacConkey agar medium were all purchased from Qingdao Haibo Biotechnology Co., Ltd. and were prepared according to the indicated proportions when used.

[0028] α-Terpineol: produced by Shanghai McLean Biochemical Technology Co., Ltd., content: 98%, batch number: C14311906.

[0029] α-Terpineol was prepared with DMSO to 20480 μg / mL as a stock solution, filtered using a 0.22 μm filter head, and stored frozen in a -20°C refrigerator.

[0030] Tigecycline: produced by Beijing Solebow Technology Co., Ltd., content: 98%, batch number: 2230815001.

[0031] Tigecycline was prepared with sterile distilled water to a concentration of 1280 μg / mL as a stock solution, filtered using a 0.22 μm filter, and stored frozen in a -20°C refrigerator away from light.

[0032] 2. Strains

[0033] Experimental strains: The 8 strains of animal-derived tet(X) Escherichia coli used in the present invention were preserved in the Veterinary Pharmacology Laboratory of the College of Veterinary Medicine of Qingdao Agricultural University.

[0034] Quality control strain: Escherichia coli standard strain ATCC25922 was purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0035] 3. Instruments

[0036] Single-person single-sided vertical clean bench (Shanghai Longyue Instrument Equipment Co., Ltd.); steam sterilizer (Tomy Digital Biology, Japan); electronic analytical balance (Ohaus Instrument (Changzhou) Co., Ltd.); micro-adjustable pipette (Transferpette); desktop full-temperature oscillating incubator (Tianjin Labote Instrument Equipment Co., Ltd.), UV-visible spectrophotometer (Shanghai Yuanxi Instrument Co., Ltd.); electric constant temperature incubator (Shanghai Senxin Experimental Instrument Co., Ltd.); electric constant temperature blast drying oven (Shanghai Jinghong Experimental Equipment Co., Ltd.).

[0037] 2. Test methods

[0038] 1. Strain revival and cryopreservation

[0039] The ATCC25922 strain stored in -80°C glycerol was streaked on MacConkey medium for activation, and a typical single colony was picked out and inoculated into LB broth, cultured overnight in a 37°C incubator, and stored in a 4°C refrigerator.

[0040] The drug-resistant Escherichia coli strain stored in glycerol at -80°C was streaked and activated in MacConkey medium with tigecycline added, and a typical single colony was picked out and inoculated into LB broth, cultured in a 37°C incubator overnight, and stored in a 4°C refrigerator.

[0041] All strains used must be frozen and stored in 30% glycerol broth for backup for subsequent experiments.

[0042] 2. Preparation of test bacterial solution

[0043] Take part of the activated bacterial solution and add it to MH broth medium to adjust the bacterial solution to 0.5 McFarland turbidity standard (concentration of about 10 8 CFU / mL), and then diluted 100 times with MH broth, at which time the bacterial solution concentration was 10 6 CFU / mL, as the test bacterial solution.

[0044] 3. Determination of single drug susceptibility MIC

[0045] During MIC and subsequent FIC determination, the operation must be carried out in the dark due to the characteristics of tigecycline.

[0046] Eight avian-derived Escherichia coli strains carrying tet(X) from different sources were selected as test strains, with ATCC 25922 as the quality control strain. The MIC of tigecycline and α-terpineol was determined by the microbroth dilution method in a 96-well plate. Tigecycline / α-terpineol was added to the first column and serially diluted to the eighth column. A total of 50 μL of drug and bacterial suspension was fixed in each well. The ninth and tenth columns were set as positive (without drug) and negative (without bacteria) controls, respectively. 50 μL of bacterial suspension was added successively from the ninth column forward, and the total volume in each well was 100 μL. At this time, tigecycline / α-terpineol was diluted to 1 / 10, 1 / 20, 1 / 40, 1 / 80, 1 / 160, 1 / 320, 1 / 640, and 1 / 1280 times the concentration of the stock solution.

[0047] Using ATCC 25922 as the quality control strain, the quality control of tigecycline was carried out according to the above steps.

[0048] 4. Combined drug susceptibility test

[0049] Taking avian-derived JMAT114 and LXAT23 as examples, referring to the MIC measurement results, the microbroth checkerboard dilution method was used to design the combination of the two drugs at 1, 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, and 1 / 128 times the MIC. α-Terpineol was added to the first vertical column and serially diluted 2-fold horizontally to the eighth column. Tigecycline was added to the first horizontal row and serially diluted vertically to the eighth row. The volume of drug + broth in each well was fixed at 50 μL. 50 μL of bacterial suspension (10 6 CFU / mL) was added successively from the eighth column forward, and the final volume in each well was 100 μL. At the same time, the single MIC of α-terpineol, the single MIC of tigecycline, negative (without bacteria), and positive (without drug) controls were set. It was incubated in an incubator at 37 °C for 16 - 20 h, and the results were observed and recorded.

[0050] The calculation of the FICI index (fractional inhibitory concentration index FICI) and the result judgment criteria are as follows:

[0051] FICI = (α-terpineol combined MIC / α-terpineol alone MIC) + (tigecycline combined MIC / tigecycline alone MIC) FIC criteria: When FIC ≤ 0.5, the drugs show a synergistic effect; when 0.5 < FIC ≤ 1, the two drugs show an additive effect; when 1 < FIC ≤ 2, the two drugs show an indifferent effect; when FIC > 2, the two drugs show an antagonistic effect.

[0052] III. Test results

[0053] For the combined application test of avian-derived JMAT114 and LXAT23, the strains were collected and isolated from chicken farms in Jimo District and Laixi Chicken Farm by the laboratory.

[0054] The quality control result of tigecycline in ATCC25922 was 0.0625 μg / mL, which was within the accused range of 0.03-0.25 μg / mL.

[0055] The combined drug sensitivity results are shown in Table 1

[0056] Table 1 MIC determination results of α-terpineol and tigecycline against animal-derived Escherichia coli strains carrying tet(X)

[0057]

[0058] It can be seen from Table 1 that the MIC of tigecycline against the two strains of tet(X)-containing Escherichia coli was 8 μg / mL, and the MIC of α-pineneol against the two strains was 1024 μg / mL. When the JMAT114 strain was added with sub-inhibitory concentrations, i.e., 1 / 2MIC and 1 / 4MIC, α-pineneol, the MIC of tigecycline was 0.125 and 1 μg / mL, indicating that the added α-pineneol can reduce the MIC of tigecycline by 64 and 8 times, respectively, when used in combination, and the two drugs can show a synergistic effect in this range. When the LXAT23 strain was added with 1 / 2MIC, 1 / 4MIC, and 1 / 8MIC α-pineneol, the MIC of tigecycline was 0.0625, 0.25, and 1 μg / mL. Under the sub-concentration gradient, α-pineneol can reduce the MIC of tigecycline by 128, 32, and 8 times, respectively, when used in combination. The two drugs can show a synergistic effect in this range.

[0059] Among the 8 selected tet(X) Escherichia coli strains from different poultry sources, when 1 / 2MIC α-pineneol was added, the MIC of tigecycline could be reduced by 128 times, at this time FIC=0.5078, and the two drugs had an additive effect; when 1 / 4MIC α-pineneol was added, the MIC of tigecycline could be reduced by 4-128 times, at this time FIC≤0.5, and the two drugs had a synergistic effect; among them, when 1 / 8MIC concentration of α-pineneol was added, the MIC of tigecycline could be reduced by 4-8 times, at this time FIC<0.5, and the two drugs had a synergistic effect.

[0060] like Figure 1 The results showed that when sub-inhibitory concentration (≤1 / 2MIC) of α-terpineol was selected, the growth inhibition of tigecycline against tet(X)-resistant Escherichia coli was effectively enhanced, and when 1 / 4MIC concentration (i.e., 128 μg / mL) of α-terpineol was added, the two drugs showed a synergistic effect, indicating that α-terpineol at this concentration restored the sensitivity of JMAT114 strain to tigecycline.

[0061] Note:

[0062] JMAT114:

[0063] When tigecycline was used in combination with 1 / 2MIC α-terpineol, FIC = [512÷1024+0.125÷8] = 0.5156;

[0064] When tigecycline was used in combination with 1 / 4MIC α-terpineol, FIC = [256÷1024+1÷8] = 0.375;

[0065] LXAT23:

[0066] When tigecycline was used in combination with 1 / 2MIC α-terpineol, FIC = [512÷1024+0.0625÷8] = 0.5078;

[0067] When tigecycline was used in combination with 1 / 4MIC α-terpineol, FIC = [256÷1024+0.25÷8] = 0.2813;

[0068] When tigecycline was used in combination with 1 / 8 MIC α-terpineol, FIC = [128÷1024+1÷8] = 0.25;

[0069] The unit of drug dosage is μg / mL.

[0070] Example 2: Use of α-terpineol as a synergist of doxycycline in Escherichia coli carrying tet(X)

[0071] 1. Test consumables

[0072] 1. Reagents and drugs

[0073] The culture medium was the same as that in Example 1.

[0074] The α-terpineol is the same as that in Example 1.

[0075] Doxycycline hydrochloride: produced by Beijing Solebow Technology Co., Ltd., content: 97%, batch number: 3230830001.

[0076] Doxycycline was prepared with sterile distilled water to 1280 μg / mL as a stock solution, filtered using a 0.22 μm filter, and stored frozen in a -20°C refrigerator away from light.

[0077] 2. Strains

[0078] Experimental strains: The 12 strains of animal-derived tet(X)-carrying Escherichia coli used in the present invention were preserved in the Veterinary Pharmacology Laboratory of the College of Veterinary Medicine of Qingdao Agricultural University.

[0079] Quality control strain: Escherichia coli standard strain ATCC25922 was purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0080] 3. Instruments

[0081] Same as in the first embodiment.

[0082] 2. Test methods

[0083] 1. Strain revival and cryopreservation

[0084] The ATCC25922 strain stored in -80°C glycerol was streaked on MacConkey medium for activation, and a typical single colony was picked out and inoculated into LB broth, cultured overnight in a 37°C incubator, and stored in a 4°C refrigerator.

[0085] The drug-resistant Escherichia coli strain stored in -80°C glycerol was streaked and activated in MacConkey medium with doxycycline added, and a typical single colony was picked out and inoculated into LB broth, cultured in a 37°C incubator overnight, and stored in a 4°C refrigerator.

[0086] All strains used must be frozen and stored in 30% glycerol broth for backup for subsequent experiments.

[0087] 2. Preparation of test bacterial solution

[0088] The steps are the same as those in Example 1.

[0089] 3. Determination of single drug susceptibility MIC

[0090] During MIC and subsequent FIC determination, the test should be performed in the dark due to the characteristics of doxycycline.

[0091] Taking avian JMAT114 and fly DSF41-1 as test strains, ATCC 25922 as quality control strain, the MIC of doxycycline and α-terpineol was determined in a 96-well plate by microbroth dilution method. Doxycycline / α-terpineol was added to the first column, and gradient dilution was performed backward to 8 columns. The drug-added bacterial solution in each well was fixed to 50 μL, and the ninth and tenth columns were set as (no drug) positive and (no bacteria) negative wells as controls. 50 μL of bacterial solution was added from the ninth column forward, and the total volume of each well was 100 μL. At this time, doxycycline / α-terpineol was diluted to 1 / 10, 1 / 20, 1 / 40, 1 / 80, 1 / 160, 1 / 320, 1 / 640, and 1 / 1280 times the mother solution.

[0092] ATCC 25922 was used as the quality control strain and doxycycline was quality controlled in the same manner as above.

[0093] 4. Combined drug sensitivity test

[0094] Avian-derived JMAT114 and fly-derived DSF41-1 were used as test strains. Referring to the MIC measurement results, the microbroth checkerboard dilution method was adopted to design the combination of the two drugs at 1-fold, 1 / 2-fold, 1 / 4-fold, 1 / 8-fold, 1 / 16-fold, 1 / 32-fold, 1 / 64-fold, and 1 / 128-fold MIC. α-Terpineol was added to the first vertical column and serially diluted 2-fold horizontally to the eighth column. Doxycycline was added to the first horizontal row and serially diluted vertically to the eighth row. The fixed volume of drug + broth in each well was 50 μL. 50 μL of bacterial suspension (10 6 CFU / mL) was added successively from the eighth column forward, and the final volume in each well was 100 μL. At the same time, the single MIC of α-terpineol, the single MIC of doxycycline, (without bacteria) negative, and (without drugs) positive wells were set as controls. It was placed in an incubator at 37 °C for 16 - 20 h, and the results were observed and recorded.

[0095] The calculation of the FICI index (fractional inhibitory concentration index FICI) and the result judgment criteria are as follows:

[0096] FICI = (α-terpineol combined MIC / α-terpineol alone MIC) + (doxycycline combined MIC / doxycycline alone MIC) FIC standard: When FIC ≤ 0.5, the drugs show a synergistic effect; when 0.5 < FIC ≤ 1, the two drugs show an additive effect; when 1 < FIC ≤ 2, the two drugs show an irrelevant effect; when FIC > 2, the two drugs show an antagonistic effect.

[0097] III. Test Results

[0098] For the combined application test of avian-derived JMAT114 and fly-derived DSF41-1, the strains were collected and isolated from a poultry farm in Jimo District and a pig farm in Qingdao by the laboratory.

[0099] The result of the quality control of doxycycline with ATCC25922 was 1 μg / mL, which was within the control range of 0.5 - 2 μg / mL.

[0100] The combined drug sensitivity results are shown in Table 2

[0101] Table 2 MIC determination results of α-terpineol and doxycycline against animal-derived Escherichia coli strains carrying tet(X)

[0102]

[0103] It can be seen from Table 2 that the MIC of doxycycline against the two strains of Escherichia coli containing tet(X) is 32μg / mL, and the MIC of α-terpineol against the two strains of Escherichia coli containing tet(X) is 1024μg / mL. When α-terpineol is added at sub-inhibitory concentrations, i.e., 1 / 2MIC and 1 / 4MIC, the MIC of doxycycline is 0.25 and 1μg / mL, indicating that the addition of α-terpineol can reduce the MIC of doxycycline by 128 times and 32 times respectively when used in combination. In this range, the two drugs can have a synergistic effect.

[0104] Among the 8 selected tet(X) Escherichia coli strains from different poultry sources, the two drugs showed synergistic effects in 6 strains, and additive effects in the remaining 2 strains. When 1 / 2MIC α-terpineol was added to the 6 strains with synergistic effects, the MIC of doxycycline decreased by 128 times, at which time FIC = 0.5078, and the two drugs showed additive effects; when 1 / 4MIC α-terpineol was added, the MIC of doxycycline could be reduced by 4 to 32 times, at which time FIC ≤ 0.5, and the two drugs showed synergistic effects.

[0105] like Figure 2 As shown in the data, when sub-inhibitory concentration (≤1 / 2MIC) of α-terpineol was selected, the growth inhibition of doxycycline against tet(X)-resistant Escherichia coli was effectively enhanced, and when 1 / 4MIC concentration (i.e., 128 μg / mL) of α-terpineol was added, the two drugs exhibited a synergistic effect, indicating that α-terpineol at this concentration restored the sensitivity of the JMAT114 strain to doxycycline.

[0106] Note:

[0107] JMAT114:

[0108] When doxycycline was used in combination with 1 / 2MIC α-terpineol, FIC = [512÷1024+0.25÷32] = 0.5078;

[0109] When doxycycline was used in combination with 1 / 4MIC α-terpineol, FIC = [256÷1024+1÷32] = 0.2813;

[0110] DSF41-1:

[0111] When doxycycline was used in combination with 1 / 2MIC α-terpineol, FIC = [512÷1024+0.25÷32] = 0.5078;

[0112] When doxycycline was used in combination with 1 / 4MIC α-terpineol, FIC = [256÷1024+4÷32] = 0.375;

[0113] The unit of drug dosage is μg / mL.

[0114] Example 3: Bactericidal effect of α-terpineol combined with tetracycline antibiotics on tet(X)-resistant Escherichia coli

[0115] 1. Test consumables

[0116] 1. Reagents and drugs

[0117] The culture medium was the same as that in Example 1.

[0118] The α-terpineol is the same as that in Example 1.

[0119] Doxycycline hydrochloride: produced by Beijing Solebow Technology Co., Ltd., content: 97%, batch number: 3230830001.

[0120] Doxycycline was prepared with sterile distilled water to 1280 μg / mL as a stock solution, filtered using a 0.22 μm filter, and stored frozen in a -20°C refrigerator away from light.

[0121] 2. Strains

[0122] Experimental strains: The 12 strains of animal-derived tet(X)-carrying Escherichia coli used in the present invention were preserved in the Veterinary Pharmacology Laboratory of the College of Veterinary Medicine of Qingdao Agricultural University.

[0123] Quality control strain: Escherichia coli standard strain ATCC25922 was purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0124] 3. Instruments

[0125] Same as in the first embodiment.

[0126] 2. Test methods

[0127] 1. Strain revival and cryopreservation

[0128] The ATCC25922 strain stored in -80°C glycerol was streaked on MacConkey medium for activation, and a typical single colony was picked out and inoculated into LB broth, cultured overnight in a 37°C incubator, and stored in a 4°C refrigerator.

[0129] The drug-resistant Escherichia coli strain stored in -80°C glycerol was streaked and activated in MacConkey medium with doxycycline added, and a typical single colony was picked out and inoculated into LB broth, cultured in a 37°C incubator overnight, and stored in a 4°C refrigerator.

[0130] All strains used must be frozen and stored in 30% glycerol broth for backup for subsequent experiments.

[0131] 2. Preparation of test bacterial solution

[0132] The steps are the same as those in Example 1.

[0133] 3. Time-killing curve experiment

[0134] (1) Inoculate 100 μL of the experimental strain culture in a centrifuge tube containing 8 mL of MH broth and culture in a shaker at 37°C and 180 rpm until the McFarland turbidity reaches 0.5.

[0135] (2) Dilute 10-fold with MH broth to obtain a concentration of 10 7 CFU / mL of experimental bacterial solution.

[0136] (3) Preparation of drug and broth mixture:

[0137] a. Control group: 900 μL MH broth + 100 μL bacterial solution.

[0138] b. Single drug group: 875μL MH broth + 25μL A / B drug + 100μL bacterial solution.

[0139] c. Combined group: 850μL MH broth + 25μL A drug + 25μL B drug + 100μL bacterial solution.

[0140] As shown in Example 2, the MICs of α-terpineol and doxycycline against Escherichia coli JMAT114 are 1024 μg / mL and 32 μg / mL, respectively. According to this result, 6 groups of experiments were set up: ① α-terpineol group alone: ​​1 / 2MIC (512 μg / mL); ② doxycycline group alone: ​​MIC (32 μg / mL); ③ doxycycline group alone: ​​1 / 2MIC (16 μg / mL); ④ α-terpineol combined with doxycycline group: 1 / 2MIC (512 μg / mL) + MIC (32 μg / mL); ⑤ α-terpineol combined with doxycycline group: 1 / 2MIC (512 μg / mL) + 1 / 2MIC (16 μg / mL); ⑥ control group.

[0141] (4) Place six sets of centrifuge tubes in a 37°C shaker and culture at 180 rpm for 24 h.

[0142] (5) Samples were taken at 0 h, 3 h, 6 h, 9 h, 12 h, and 24 h, and the samples were added to the MH agar plate after gradient dilution. After evenly spreading, the plate was placed in a 37 °C incubator for incubation for 16-18 h, and the colonies were counted. Three biological replicates were set for this step, and the final result was the average of the three counts.

[0143] 3. Test results

[0144] The results are as follows Figure 3As shown in Table 3, Escherichia coli can grow normally in the control group, indicating that the activity of the strain is normal; among the three groups using single drugs, only the MIC doxycycline group has a slight inhibition on the growth of bacteria, but it does not have a strong bactericidal ability; in the combination drug group, the MIC doxycycline + 1 / 2MIC α-pineneol group has a significant bactericidal effect, showing a bactericidal effect on bacteria at 3 hours, and can kill all the bacteria in the centrifuge tube at 9 hours; although the 1 / 2MIC doxycycline + 1 / 2MIC α-pineneol group did not show obvious bactericidal effect, it showed better antibacterial effect than the single drug group.

[0145] Table 3 Bactericidal effect of α-terpineol and doxycycline alone and in combination against Escherichia coli JMAT114

[0146]

[0147]

[0148] Example 4: Application of a mixture of α-terpineol and quercetin as a synergist for tetracycline antibiotics

[0149] 1. Test consumables

[0150] 1. Reagents and drugs

[0151] α-Terpineol and tigecycline are the same as those in Example 1.

[0152] Quercetin: Produced by Shanghai McLean Biochemical Technology Co., Ltd., content: 97%.

[0153] Quercetin was dissolved in DMSO and prepared to 20480 μg / mL as a stock solution, filtered using a 0.22 μm filter head, and stored frozen in a -20°C refrigerator.

[0154] Quercetin and α-terpineol are mixed in three ratios of 2:8, 1:1 and 8:2 for later use.

[0155] 2. Strains

[0156] Experimental strains: The 6 strains of animal-derived tet(X)-carrying Escherichia coli used in the present invention were preserved in the Veterinary Pharmacology Laboratory of the College of Veterinary Medicine of Qingdao Agricultural University.

[0157] Quality control strain: Escherichia coli standard strain ATCC25922 was purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0158] 3. Instruments

[0159] Same as in Example 1.

[0160] 2. Test methods

[0161] 1. Strain revival and cryopreservation

[0162] The ATCC25922 strain stored in -80°C glycerol was streaked on MacConkey medium for activation, and a typical single colony was picked out and inoculated into LB broth, cultured overnight in a 37°C incubator, and stored in a 4°C refrigerator.

[0163] The drug-resistant Escherichia coli strain stored in -80°C glycerol was streaked and activated in MacConkey medium supplemented with tigecycline, and a typical single colony was picked out and inoculated into LB broth, cultured overnight in a 37°C incubator, and stored in a 4°C refrigerator.

[0164] All strains used must be frozen and stored in 30% glycerol broth for backup for subsequent experiments.

[0165] 2. Preparation of test bacterial solution

[0166] The steps are the same as those in Example 1.

[0167] 3. Determination of single drug susceptibility MIC

[0168] During MIC and subsequent FIC determination, the operation must be carried out in the dark due to the characteristics of tigecycline.

[0169] Taking JMAT114 and LXAT23 as test strains, ATCC25922 as quality control strain, the MIC of tigecycline and α-terpineol / quercetin mixture was determined in 96-well plates by microbroth dilution method. Tigecycline or α-terpineol / quercetin mixture was added to the first column, and gradient dilution was performed backward to 8 columns. The drug-added bacterial solution in each well was fixed to 50 μL, and the ninth and tenth columns were set as (no drug) positive and (no bacteria) negative wells as controls. 50 μL of bacterial solution was added from the ninth column forward, and the total volume of each well was 100 μL. At this time, the tigecycline or α-terpineol / quercetin mixture was diluted to 1 / 10, 1 / 20, 1 / 40, 1 / 80, 1 / 160, 1 / 320, 1 / 640, and 1 / 1280 times the concentration of the mother solution.

[0170] 4. Combined drug sensitivity test

[0171] Referring to the MIC measurement results, the microbroth chessboard dilution method was used to design the combination of the two drugs at 1, 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64 and 1 / 128 times the MIC. The α-pineneol / quercetin mixture was added to the first vertical column, and the horizontal gradient was diluted 2 times to the eighth column. Tigecycline was added to the first horizontal row, and the vertical gradient was diluted to the eighth row. The volume of the drug + broth in each well was fixed at 50 μL. In the eighth column, 50 μL of bacterial solution (10 6(CFU / mL), and the final volume in each well is 100 μL. At the same time, set the single MIC of α-terpineol / quercetin mixture, the single MIC of tigecycline, (without bacteria) negative and (without drugs) positive wells as controls. And place them in an incubator at 37 °C for 16 - 20 h, observe and record the results.

[0172] The calculation of the FICI index (fractional inhibitory concentration index FICI) and the result judgment criteria are as follows:

[0173] FICI = (combined MIC of the mixture / single MIC of the mixture) + (combined MIC of tigecycline / single MIC of tigecycline)

[0174] FIC criteria: When FIC ≤ 0.5, the drugs show a synergistic effect; when 0.5 < FIC ≤ 1, the two drugs show an additive effect; when 1 < FIC ≤ 2, the two drugs show an irrelevant effect; when FIC > 2, the two drugs show an antagonistic effect.

[0175] III. Test results

[0176] The result of tigecycline quality control for ATCC25922 is 0.0625 μg / mL, which meets the accusation range of 0.03 - 0.25 μg / mL.

[0177] The drug sensitivity results are shown in Table 4

[0178] After mixing α-terpineol and quercetin in different proportions, the MIC for both experimental strains is 1024 μg / mL. In the combined drug sensitivity experiment, it can maintain a synergistic effect with tigecycline (FIC ≤ 0.5). Compared with using α-terpineol alone, adding quercetin can reduce the usage concentration and amount of α-terpineol. Since α-terpineol is expensive, choosing the combination of quercetin and α-terpineol can reduce the drug cost. It can reduce the usage amount of α-terpineol by up to 5 times at the same time, and also reduce the MIC of tigecycline by 4 - 16 times.

[0179] Table 4 Drug sensitivity test results of α-terpineol / quercetin mixture and tigecycline against Escherichia coli strains carrying tet(X)

[0180]

[0181]

[0182] Example 5. Application of α-terpineol combined with doxycycline in a chicken infection model

[0183] I. Experimental consumables

[0184] 1. Reagents and drugs

[0185] Doxycycline hydrochloride: produced by Beijing Solebow Technology Co., Ltd., content: 97%, batch number: 3230830001.

[0186] Doxycycline was prepared with sterile distilled water to 2000 μg / mL as a stock solution, filtered using a 0.22 μm filter, and stored frozen in a -20°C refrigerator away from light.

[0187] α-Terpineol: produced by Shanghai McLean Biochemical Technology Co., Ltd., content: 98%, batch number: C14311906.

[0188] α-Terpineol was prepared with DMSO to 400,000 μg / mL as a stock solution, filtered using a 0.22 μm filter head, and stored frozen in a -20°C refrigerator.

[0189] Because DMSO is toxic, when used for therapeutic purposes, it is necessary to ensure that DMSO does not exceed 5% of the dosage. Therefore, before using the drug, it is necessary to take some of the reserve mother solution and mix it in a ratio of mother solution: water = 1:11.5 for administration. At this time, DMSO accounts for about 4.5% < 5% of the dosage.

[0190] 2. Strains

[0191] Experimental strains: The chicken-derived tet(X) Escherichia coli used in the present invention was preserved in the Veterinary Pharmacology Laboratory of the College of Veterinary Medicine of Qingdao Agricultural University.

[0192] Quality control strain: Escherichia coli standard strain ATCC25922 was purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0193] 3. Instruments

[0194] Single-person single-sided vertical clean bench (Shanghai Longyue Instrument Equipment Co., Ltd.); steam sterilizer (Tomy Digital Biology, Japan); electronic analytical balance (Ohaus Instrument (Changzhou) Co., Ltd.); micro-adjustable pipette (Transferpette); desktop full-temperature oscillating incubator (Tianjin Labteri Instrument Equipment Co., Ltd.), UV-visible spectrophotometer (Shanghai Yuanxi Instrument Co., Ltd.); electric constant temperature incubator (Shanghai Senxin Experimental Instrument Co., Ltd.); electric constant temperature blast drying oven (Shanghai Jinghong Experimental Equipment Co., Ltd.).

[0195] 2. Test methods

[0196] 1. Establishment of chick infection model

[0197] The avian Escherichia coli LXAT23 in Example 2 was used to conduct a challenge test on 14-day-old broilers.

[0198] The bacterial solution was inoculated into 50 mL of LB broth and cultured in a constant temperature shaker at 37°C and 180 rpm for 4 h to obtain a concentration of 10 9 The test bacterial solution was centrifuged at 4°C and 4000 rpm, the supernatant was removed, and the solution was resuspended with physiological saline to obtain the challenge bacterial solution.

[0199] Thirty-two 14-day-old chicks were taken as the infection control group, α-pineneol treatment group, doxycycline treatment group and combined treatment group, with 6 chicks in each group for the first three groups and 14 chicks in the combined treatment group. After fasting and deprivation of water for 3 hours, each chicken was intraperitoneally injected with 1 mL of the challenge bacteria solution. The drug was administered 1 hour after the challenge, and the administration method was oral gavage. The dosage of doxycycline was 10 mg / kg body weight, and the dosage of α-pineneol was 160 mg / kg body weight. After administration, the chickens were fed and watered normally, and the drug was administered once every 24 hours for 3 consecutive days. After stopping the drug, the chicks were observed for 2 days, and the survival of the chicks was recorded and the survival rate was calculated.

[0200] 3. Test results

[0201] The survival rate of the chick infection model is shown in Table 5 and Figure 4 Shown

[0202] The mortality rate of the test strain in the control group chicks within one day was about 83.3%. After three consecutive days of administration, the therapeutic effect was combined group > doxycycline group > α-terpineol group. It can be seen that the combined drug use improved the survival rate of the chicks, and there was no recurrence of infection two days after drug withdrawal, which was consistent with the results of the in vitro drug sensitivity test and time-bactericidal test. The combined drug use can enhance the antibacterial and bactericidal effect of doxycycline.

[0203] Table 5 Results of the application test of α-terpineol, doxycycline and their combination in the chick infection model

[0204]

[0205] Example 6: A pharmaceutical preparation for use against drug-resistant Enterobacteriaceae

[0206] The pharmaceutical preparation is prepared by mixing alpha-terpineol and doxycycline in a weight ratio of 16:1.

[0207] Example 7: A pharmaceutical preparation for use against drug-resistant Enterobacteriaceae

[0208] The pharmaceutical preparation is prepared by mixing alpha-pineol, quercetin and tigecycline in a weight ratio of 128:5 to 12:5.

Claims

1. The use of α-terpineol as a tetracycline antibiotic synergist in the preparation of an anti-drug resistant Escherichia coli drug, characterized in that: The tetracycline antibiotic is tigecycline or doxycycline; the drug-resistant Escherichia coli is tet(X) Escherichia coli; the weight ratio of α-terpineol to tigecycline is 256:1 to 128:1; the weight ratio of α-terpineol to doxycycline is 64:1 to 16:

1.

2. A pharmaceutical composition for tet(X) Escherichia coli, characterized in that: The composition comprises α-terpineol and a tetracycline antibiotic, wherein the tetracycline antibiotic is tigecycline or doxycycline; the weight ratio of the α-terpineol to tigecycline is 256:1 to 128:1; and the weight ratio of the α-terpineol to doxycycline is 64:1 to 16:

1.

3. The pharmaceutical composition according to claim 2, characterized in that: The composition further comprises quercetin; in the composition, 128 μg / mL of α-terpineol, 128 μg / mL of quercetin, and 1 μg / mL of tigecycline; or 51.2 μg / mL of α-terpineol, 204.8 μg / mL of quercetin, and 1 μg / mL of tigecycline; or 204.8 μg / mL of α-terpineol, 51.2 μg / mL of quercetin, and 1 μg / mL of tigecycline. Tigecycline 1μg / mL; or α-terpineol 128μg / mL, quercetin 128μg / mL, tigecycline 0.5μg / mL; or α-terpineol 6.4μg / mL, quercetin 25.6μg / mL, tigecycline 2μg / mL; or α-terpineol 204.8μg / mL, quercetin 51.2μg / mL, tigecycline 0.5μg / mL.

4. A pharmaceutical preparation for use against drug-resistant Enterobacteriaceae, characterized in that: The pharmaceutical preparation is a preparation prepared with the pharmaceutical composition according to claim 2 or 3 as an active ingredient; the drug-resistant Enterobacter is tet(X) Escherichia coli.

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