Application of α-terpineol as a synergist in combating drug-resistant Enterobacteriaceae

Abstract

The present invention provides the use of α-pineneol as a synergist in combating drug-resistant Enterobacteriaceae, and belongs to the field of medical technology. Specifically, α-pineneol is used as a synergist in combination with colistin or β-lactam antibiotic meropenem to combat drug-resistant Enterobacteriaceae; the drug-resistant Enterobacteriaceae include drug-resistant Salmonella, drug-resistant Escherichia coli, and drug-resistant Klebsiella pneumoniae. The present invention provides a new use of the natural compound α-pineneol: α-pineneol can effectively enhance the sensitivity of drug-resistant Enterobacteriaceae to carbapenem antibiotics meropenem and colistin, and effectively improve the efficacy of the above-mentioned antibiotics on drug-resistant Enterobacteriaceae, which is of great significance for solving clinical drug-resistant Salmonella, drug-resistant Escherichia coli and drug-resistant Klebsiella pneumoniae infections. Provide safe and effective auxiliary drugs for the treatment of drug-resistant Enterobacteriaceae infection diseases and provide technical support for the control of bacterial resistance.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, specifically to antibiotic synergist technology, and relates to the application of a natural compound α-pineneol in combination with β-lactam antibiotics and colistin to combat important drug-resistant Enterobacteriaceae. Background Art

[0002] Colistin is the last antibacterial drug used by humans to treat multidrug-resistant Gram-negative bacteria and is known as the last resort of antimicrobial treatment. The overuse of colistin in animal husbandry has increased the selection pressure of antimicrobial drugs, leading to the emergence of the plasmid-mediated colistin resistance gene mcr. The mcr gene is located on the mobile genetic element of plasmid DNA, so it has the ability to spread horizontally rapidly. Escherichia coli and Salmonella strains carrying mcr currently dominate the world. At present, there is still a lack of new drugs that are effective against colistin-resistant Salmonella and others.

[0003] β-lactam antibiotics are often used to treat diseases caused by Enterobacter infections, among which carbapenems have strong broad-spectrum activity and are not affected by common resistance mechanisms. Clinically, meropenem is called the last-line carbapenem antibiotic for the treatment of multidrug-resistant Gram-negative bacterial infections. Despite this, carbapenem resistance mechanisms inevitably emerge. Carbapenem resistance is usually caused by the expression of carbapenemases, which can hydrolyze almost all β-lactam antibiotics, including carbapenems. The most common in Enterobacteriaceae is class A (bla KPC ), class B metallo-β-lactamases MBLs (bla NDM ,bla VIM ,bla IMP ) and D class (bla OXA-48 ). Among them, class A enzymes and class B enzymes are the main causes of carbapenem resistance.

[0004] In recent years, the improper use of antibiotics in animal husbandry and clinical treatment has led to the emergence of drug resistance in a variety of Enterobacteriaceae. At present, for example, colistin-resistant Salmonella carrying mcr-1, colistin-resistant Escherichia coli with mcr-1 positive, NDM-producing Escherichia coli, and KPC-2-producing Klebsiella pneumoniae have been widely spread around the world. They are resistant to various drugs for treating bacterial infections, which leads to the selection of effective drugs in the treatment of bacterial infectious diseases becoming very limited, and even in severe cases, there is no drug to treat them. The spread of these drug-resistant Enterobacteriaceae and the rapid development of their resistance also seriously threaten global public health security. In response to this situation, we urgently need to find new means to achieve the purpose of antibacterial synergy.

[0005] At present, it is very important to study new synergists to achieve the purpose of antibacterial synergism and solve the current problem of difficulty in treating drug-resistant Enterobacteriaceae infections. my country has rich plant resources and many natural compounds can be extracted and utilized. Among them, α-pineneol (molecular formula C 10 H 18 α-Terpineol is a type of monoterpene compound that exists in more than 150 kinds of plant leaves, flowers, and grass stems. It can be extracted from cardamom oil, pine needle oil, and rosewood oil. It is very rich and has a clove aroma. It is widely used in the manufacture of edible flavors, fragrances, and air fresheners. Like other monoterpene compounds and derivatives, α-terpineol has multiple biological activities such as antibacterial, herbicidal, and anti-tumor. It can also be used to kill insects, repel mosquitoes, and has the effect of treating diabetes and anti-depression.

[0006] Therefore, this invention aims to use α-pineneol as a synergist to effectively treat bacterial infections, especially drug-resistant Enterobacter infections, without increasing the dosage of antibiotics or even reducing the dosage, and to explore its clinical application value. Summary of the invention

[0007] The present invention has discovered a new synergist, namely α-terpineol: α-terpineol can effectively reduce the minimum inhibitory concentration of colistin and meropenem, enhance the sensitivity of the above antibiotics to important drug-resistant Enterobacteriaceae, and reduce the dosage of antibiotics, thereby solving the drug resistance problem of drug-resistant Salmonella, drug-resistant Escherichia coli and drug-resistant Klebsiella pneumoniae. This invention has great clinical significance for the future treatment or prevention of the above-mentioned drug-resistant Enterobacteriaceae infections.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides application of alpha-pinene alcohol in combating drug-resistant Enterobacteriaceae. The alpha-pinene alcohol is used as a synergist in combination with colistin or meropenem, a beta-lactam antibiotic, to combat drug-resistant Enterobacteriaceae. The drug-resistant Enterobacteriaceae include drug-resistant Salmonella, drug-resistant Escherichia coli, and drug-resistant Klebsiella pneumoniae.

[0010] The application also includes the application of α-pineneol combined with colistin in the preparation of an anti-mcr-1 positive strain synergist, wherein the mcr-1 positive strain is mcr-1 Escherichia coli or mcr-1 positive Salmonella; the weight ratio of the α-pineneol to colistin is in the range of 512:1 to 128:1.

[0011] The application includes the application of α-terpineol combined with meropenem in preparing a synergist against avian-derived NDM Escherichia coli or in preparing a synergist against Klebsiella pneumoniae carrying KPC-2; the weight ratio of the α-terpineol to meropenem is in the range of 16:1 to 4:1.

[0012] The present invention also provides a pharmaceutical composition for drug-resistant Enterobacteriaceae, characterized in that: the composition comprises α-terpineol and colistin, and the weight ratio of the α-terpineol to colistin is in the range of 512:1 to 128:1; the composition may also comprise α-terpineol and meropenem; the weight ratio of the α-terpineol to meropenem is in the range of 16:1 to 4:1.

[0013] The preparation method of the pharmaceutical composition of the present invention comprises the following steps: taking α-terpineol, colistin or meropenem according to a proportion, preparing a solution, and mixing.

[0014] The present invention also provides a pharmaceutical preparation for resisting drug-resistant Enterobacteriaceae, wherein the pharmaceutical preparation is prepared with a pharmaceutical composition of α-terpineol and colistin (or meropenem) as active ingredients. The preparation may be an oral preparation or a non-oral preparation, and the non-oral preparation includes an injection.

[0015] Preferably, in the pharmaceutical combination or pharmaceutical preparation of the present invention, each dose of α-terpineol is 8 g, each dose of meropenem is 1 g, and each dose of colistin is 0.03 g.

[0016] The present invention has the following effects:

[0017] 1. The present invention explores a new use of α-terpineol, that is, as a synergist of carbapenem antibiotics and colistin. There are few clinical studies on α-terpineol. The present invention studies α-terpineol and uses it for drug-resistant Enterobacter infection for the first time. It provides early technical support for providing safe and effective auxiliary drugs for the treatment of important drug-resistant Enterobacter infection diseases in veterinary and human medicine and for controlling bacterial resistance.

[0018] 2. Through research, this invention found that α-pineneol can effectively enhance the sensitivity of drug-resistant Enterobacteriaceae to carbapenem antibiotics meropenem and colistin, and effectively improve the efficacy of the above antibiotics against drug-resistant Enterobacteriaceae, which is of great significance for solving clinical drug-resistant Salmonella, drug-resistant Escherichia coli and drug-resistant Klebsiella pneumoniae infections.

[0019] 3. The present invention has been tested and shown that the combined use of α-terpineol with meropenem and colistin, respectively, significantly reduced the minimum inhibitory concentration when meropenem and colistin were used in combination, and reduced the amount of antibiotics used, thus verifying that the combination of α-terpineol and meropenem, and α-terpineol and colistin are synergistic combinations. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effect of meropenem combined with α-terpineol on the MIC of meropenem in Escherichia coli carrying NDM and Klebsiella pneumoniae carrying KPC-2.

[0021] Figure 2 The effect of colistin and α-terpineol combination on the MIC of colistin in mcr-1 Escherichia coli and mcr-1 positive Salmonella from animal sources. DETAILED DESCRIPTION

[0022] In order to more intuitively illustrate the embodiments or technical solutions of this invention, the experiments involved in the embodiments of this invention are described below to illustrate the beneficial effects of the present invention. 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 does not specifically limit α-terpineol, meropenem, colistin, etc., and any conventional method used in the art can be used. Meropenem is a carbapenem antibacterial drug in the β-lactam class. It mainly exerts its antibacterial effect by binding to penicillin protein with a covalent bond to inhibit cell wall synthesis. It has the advantages of a wide antibacterial spectrum and strong antibacterial activity. It is the first choice for clinical treatment of multidrug-resistant bacterial infections. Meropenem is a white to light yellow crystalline powder that is soluble in 0.01 mol / L phosphate buffer. Colistin has strong antibacterial activity against Gram-negative bacteria and is a narrow-spectrum bactericide. It is currently widely used in poultry, livestock and other livestock and human clinical practice. It is an off-white to light yellow powder, odorless, and hygroscopic. It is easily soluble in water, slightly soluble in ethanol, and almost insoluble in acetone, chloroform or ether.

[0024] Experimental Example 1: Combined drug sensitivity test of α-terpineol and meropenem against Escherichia coli carrying NDM

[0025] 1. Test consumables

[0026] 1. Reagents and drugs

[0027] α-Terpineol: produced by Shanghai McLean Biochemical Technology Co., Ltd., content: 98%.

[0028] Meropenem trihydrate: produced by Shanghai McLean Biochemical Technology Co., Ltd., content: 98%, batch number: C15105948.

[0029] α-Terpineol was dissolved in DMSO to prepare a stock solution of 20480 μg / mL and stored in a -20°C refrigerator.

[0030] Meropenem was prepared with 0.01 mol / L phosphate buffer to 5120 μg / mL as a stock solution and stored frozen in a -20°C refrigerator.

[0031] 2. Strains

[0032] Escherichia coli standard strain ATCC 25922: purchased from Qingdao Haibo Biotechnology Co., Ltd. The avian Escherichia coli carrying NDM used in the present invention was stored in the Veterinary Pharmacology Laboratory of the College of Veterinary Medicine of Qingdao Agricultural University. The Escherichia coli was activated by streaking on MacConkey medium, cultured overnight in a 37°C incubator, and stored in a 4°C refrigerator for later use.

[0033] 3. Culture medium

[0034] LB broth medium: Qingdao Haibo Biotechnology Co., Ltd., prepared according to the instruction manual.

[0035] MH broth medium: Qingdao Haibo Biotechnology Co., Ltd., prepared according to the instruction manual.

[0036] MacConkey agar medium: Qingdao Haibo Biotechnology Co., Ltd., prepared according to the instruction manual.

[0037] 4. Instruments

[0038] Steam sterilizer (Tomy Digital Biology, Japan); single-person single-sided vertical clean bench (Shanghai Longyue Instrument Equipment Co., Ltd.); micro-adjustable pipette (Transferpette); electronic analytical balance (Ohaus Instrument (Changzhou) Co., Ltd.); desktop full-temperature oscillating incubator (Tianjin Labteri Instrument Equipment Co., Ltd.), electric constant temperature incubator (Shanghai Senxin Experimental Instrument Co., Ltd.); UV-visible spectrophotometer (Shanghai Yuanxi Instrument Co., Ltd.); electric constant temperature blast drying oven (Shanghai Jinghong Experimental Equipment Co., Ltd.).

[0039] 2. Test methods

[0040] 1. Preparation of test bacterial solution

[0041] Dissolve the E. coli standard strain ATCC 25922 and the isolated avian NDM E. coli strain preserved in -80℃ glycerol at room temperature, resuscitate the bacterial solution and inoculate it on MacConkey agar using the 6-streak method in a clean bench, incubate it inverted at 37℃ in an incubator for 18-24h, pick a typical single colony and inoculate it in LB broth, and incubate it in a 37℃ incubator overnight. Take the bacterial solution into MH broth medium and adjust the bacterial solution to 0.5 McFarland turbidity standard (concentration of about 10 8 CFU / mL), and the bacterial solution was diluted 100 times with MH broth to make the bacterial solution concentration 10 6 CFU / mL, as the test bacterial solution.

[0042] 2. Determination of single drug susceptibility MIC

[0043] Using ATCC 25922 as the quality control strain, for 9 avian-derived Escherichia coli strains carrying NDM, the MIC of meropenem and α-terpineol was determined by the microbroth dilution method specified by CLSI. A 96-well sterile microplate was used, and MIC determination was designed with concentration gradients of 1 / 10, 1 / 20, 1 / 40, 1 / 80, 1 / 160, 1 / 320, and 1 / 640 times the stock solution. The drugs were serially diluted in the 96-well plate. Meropenem / α-terpineol was added to the first column and serially diluted to the seventh column, with 50 μL of broth and drug fixed in each well. After the test bacterial suspension was mixed evenly, it was added to each well of the 96-well plate using an eight-channel pipette, 50 μL per well, and the total volume was 100 μL. At the same time, negative (without bacteria) and positive (without drugs) wells were set as controls. It was then incubated at 37 °C for 18 h, and the results were observed and recorded. Each test was repeated twice. (The DMSO content in each well was less than or equal to 5% to eliminate the influence of DMSO on bacteria).

[0044] 3. Combined drug sensitivity test

[0045] Taking avian-derived Escherichia coli strains LXAMER234 and JMAM270 as examples, with ATCC25922 as the quality control strain, based on the MIC results as a reference, the microbroth dilution checkerboard method was used with a 96-well sterile microplate. The combination was designed with 1-fold, 1 / 2-fold, 1 / 4-fold, 1 / 8-fold, 1 / 16-fold, 1 / 32-fold, and 1 / 64-fold MIC of the two drugs. The two drugs were serially diluted in the 96-well plate respectively. α-Terpineol was added to the first vertical column and serially diluted horizontally to the seventh column, and meropenem was added to the first horizontal row and serially diluted vertically to the seventh row, with 50 μL of broth and drug fixed in each well. After the test bacterial suspension was mixed evenly, it was added to each well of the 96-well plate using an eight-channel pipette, 50 μL per well, and the total volume was 100 μL. At the same time, the individual MIC of α-terpineol, the individual MIC of meropenem, negative (without bacteria), and positive (without drugs) wells were set as controls. It was then incubated at 37 °C for 18 h, and the results were observed and recorded. Each test was repeated twice. (The DMSO content in each well was less than or equal to 5% to eliminate the influence of DMSO on bacteria).

[0046] Calculation and result judgment of the FICI index (fractional inhibitory concentration index FICI):

[0047] FIC index = (MIC of combined use of drug A / MIC of drug A used alone) + (MIC of combined use of drug B / MIC of drug B used alone)

[0048] FIC index criteria: When FIC ≤ 0.5, it is a synergistic effect; when 0.5 < FIC ≤ 1, it is an additive effect;

[0049] When 1 < FIC ≤ 2, it is an indifferent effect; when FIC > 2, it is an antagonistic effect.

[0050] III. Test results

[0051] The MIC value of the drug sensitivity test was determined based on the minimum concentration at which no bacterial growth was observed with the naked eye. The MIC of meropenem against the Escherichia coli quality control bacteria ATCC25922 was 0.03125 μg / mL, which was within the quality control range of 0.008-0.06 μg / mL.

[0052] The combined drug sensitivity results are shown in Table 1

[0053] Table 1 MIC test results of α-terpineol and meropenem against poultry-derived NDM Escherichia coli strains

[0054]

[0055]

[0056] It can be seen from Table 1 that the MIC of meropenem against NDM Escherichia coli strains from poultry is 128 μg / mL, and the MIC of α-terpineol against NDM Escherichia coli strains from poultry is 1024 μg / mL. When 1 / 4 MIC α-terpineol was added, the MIC of meropenem decreased by 64 times and 32 times, respectively, and as the concentration of α-terpineol decreased, the MIC value of meropenem also decreased.

[0057] In the 9 selected NDM Escherichia coli strains from different poultry sources, after adding sub-inhibitory concentration (≤1 / 2MIC) α-terpineol, the MIC of meropenem decreased to varying degrees. Among them, when 1 / 2MIC was added, the MIC of meropenem decreased by 64 times, at which time FIC = 0.5156, and the two drugs had an additive effect; when 1 / 4MIC was added, the MIC of meropenem could be reduced by 16 to 64 times, at which time FIC ≤ 0.5, and the two drugs had a synergistic effect.

[0058] like Figure 1 As shown in A, taking the JZAM270 strain as an example, the use of 1 / 4 MIC concentration of α-terpineol (i.e., 128 μg / mL) in combination with meropenem reduced the meropenem MIC of the JZAM270 Escherichia coli strain from 128 μg / mL to 4 μg / mL, and the FIC was 0.2813, which had a significant synergistic antibacterial effect. The results showed that 1 / 4 MIC concentration (i.e., 128 μg / mL) of α-terpineol restored the sensitivity of the JZAM270 Escherichia coli strain to meropenem.

[0059] Note: LXAMER234: When meropenem and 1 / 2MIC α-terpineol are used together, FIC = [512÷1024+2÷128] = 0.5156;

[0060] When meropenem was used in combination with 1 / 4MIC α-terpineol, FIC = [256÷1024+2÷128] = 0.2656;

[0061] MYA14-1: When meropenem and 1 / 2MIC α-terpineol were used together, FIC = [512÷1024+2÷128] = 0.5156;

[0062] When meropenem was used in combination with 1 / 4MIC α-terpineol, FIC = [256÷1024+4÷128] = 0.2813;

[0063] The unit of drug dosage is μg / mL.

[0064] Experimental Example 2: Combined drug sensitivity test of α-terpineol and meropenem against human Klebsiella pneumoniae carrying KPC-2

[0065] 1. Test consumables

[0066] 1. Reagents and drugs

[0067] α-Terpineol: produced by Shanghai McLean Biochemical Technology Co., Ltd., content: 98%.

[0068] Meropenem trihydrate: produced by Shanghai McLean Biochemical Technology Co., Ltd., content: 98%, batch number: C15105948.

[0069] α-Terpineol was dissolved in DMSO to prepare a stock solution of 20480 μg / mL and stored in a -20°C refrigerator.

[0070] Meropenem was prepared with 0.01 mol / L phosphate buffer to 5120 μg / mL as a stock solution and stored frozen in a -20°C refrigerator.

[0071] 2. Strains

[0072] Escherichia coli standard strain ATCC 25922: purchased from Qingdao Haibo Biotechnology Co., Ltd. The human KPC-2 Klebsiella pneumoniae strain used in the present invention was provided by Qingdao Municipal Hospital. Klebsiella pneumoniae was activated by streaking on MacConkey medium, cultured overnight in a 37°C incubator, and stored in a 4°C refrigerator for later use.

[0073] 3. Culture medium

[0074] Same as the first embodiment.

[0075] 4. Instruments

[0076] Same as the first embodiment.

[0077] 2. Test methods

[0078] 1. Preparation of test bacterial solution

[0079] The standard strain of Escherichia coli ATCC 25922 and the isolated Klebsiella pneumoniae strain carrying KPC stored in -80℃ glycerol were dissolved at room temperature, and the bacterial solution was revived and inoculated on MacConkey agar using the 6-streak method in a clean bench. The culture was inverted and cultured at 37℃ in an incubator for 18-24h. A typical single colony was picked and inoculated into LB broth and cultured in a 37℃ incubator overnight. The bacterial solution was added to the MH broth medium and adjusted to 0.5 McFarland turbidity standard (concentration of about 10 8 CFU / mL), and the bacterial solution was diluted 100 times with MH broth to make the bacterial solution concentration 10 6 CFU / mL, as the test bacterial solution.

[0080] 3. Determination of single drug susceptibility MIC

[0081] ATCC 25922 was used as the quality control strain, and three strains of Klebsiella pneumoniae carrying KPC from humans were used as the test strains. The MIC of meropenem and α-terpineol was determined by the microbroth dilution method specified by CLSI. A 96-well sterile microplate was used, and the MIC was determined at a concentration gradient of 1 / 10, 1 / 20, 1 / 40, 1 / 80, 1 / 160, 1 / 320, and 1 / 640 times the mother solution. The drug was gradiently diluted in the 96-well plate, and meropenem / α-terpineol was added to the first column, and the gradient was diluted to the seventh column, with 50 μL of broth and drug fixed in each well. After the test bacterial solution was mixed, it was added to each well of the 96-well plate with an eight-channel pipette, 50 μL per well, and the total volume was 100 μL. At the same time, negative wells (without bacteria) and positive wells (without drug) were set as controls. The plates were cultured at 37°C for 18 hours, and the results were observed and recorded. Each test was repeated twice. When reading the sample, the lowest concentration gradient in the well with no turbidity visible to the naked eye was taken as the MIC.

[0082] 3. Combined drug sensitivity test

[0083] Human Klebsiella pneumoniae 17254 and CRKP20027697 were used as examples, ATCC 25922 was used as a quality control strain, and the MIC results were used as a reference. The microbroth dilution chessboard method used a 96-well sterile microplate, and the two drugs were designed to be combined at 1 times, 1 / 2 times, 1 / 4 times, 1 / 8 times, 1 / 16 times, 1 / 32 times and 1 / 64 times MIC. The two drugs were gradiently diluted in a 96-well plate, α-terpineol was added to the first column, and the horizontal gradient was diluted to the seventh column. Meropenem was added to the first row, and the vertical gradient was diluted to the seventh row. Each well was fixed with 50 μL of broth and drug. After the test bacterial solution was mixed, it was added to each well of the 96-well plate with an eight-channel pipette, 50 μL per well, and the total volume was 100 μL. At the same time, α-terpineol alone MIC, meropenem alone MIC, (no bacteria) negative and (no drug) positive wells were set as controls. The cells were incubated at 37°C for 18 h, and the results were observed and recorded. Each test was repeated twice.

[0084] The calculation of FICI index (graded inhibition concentration index FICI) and the result judgment are the same as those in Example 1.

[0085] 3. Test results

[0086] The MIC value of the drug sensitivity test was determined based on the minimum concentration at which no bacterial growth was observed with the naked eye. The MIC of meropenem against the Escherichia coli quality control bacteria ATCC25922 was 0.03125 μg / mL, which was within the quality control range of 0.008-0.06 μg / mL.

[0087] The combined drug sensitivity results are shown in Table 2

[0088] Table 2 MIC determination results of α-terpineol and meropenem against human KPC-2 Klebsiella pneumoniae strains

[0089]

[0090] It can be seen from Table 2 that the MICs of meropenem for human KPC Klebsiella pneumoniae are 64μg / mL and 256μg / mL, respectively, and the MIC of α-terpineol for avian Escherichia coli is 1024μg / mL. When 1 / 2MIC (512μg / mL) and 1 / 4MIC (256μg / mL) α-terpineol are added to the 17254 strain, the MIC of meropenem can be reduced to 1μg / mL, which is 64 times lower than the MIC value when meropenem is used alone. When 1 / 2MIC (512μg / mL), 1 / 4MIC (256μg / mL), and 1 / 8MIC (128μg / mL) α-terpineol are added to the CRKP20027697 strain, the MICs of meropenem are 4, 4, and 128μg / mL, which are 64, 64, and 2 times lower than the MIC values ​​when meropenem is used alone. As the concentration of α-terpineol decreased, the MIC value of meropenem decreased.

[0091] In the three selected strains of Klebsiella pneumoniae carrying KPC from humans, the MIC of meropenem decreased to varying degrees after the addition of subinhibitory concentration (≤1 / 2MIC) α-pineneol. When 1 / 2MIC was added, the MIC of meropenem decreased by 64 times, at which time FIC = 0.5156, and the two drugs had an additive effect; when 1 / 4MIC was added, the MIC of meropenem could decrease by 8 to 64 times, at which time FIC ≤ 0.5, and the two drugs had a synergistic effect.

[0092] like Figure 1 As shown in Figure B, taking the 17254 Pneumonia strain as an example, the use of 1 / 4MIC concentration of α-terpineol (i.e., 128μg / mL) in combination with meropenem reduced the MIC of meropenem in the 17254 strain from 64μg / mL to 1μg / mL, and the FIC was 0.2656, which had a synergistic antibacterial effect. The results showed that 1 / 4MIC concentration (i.e., 128μg / mL) of α-terpineol restored the sensitivity of the 17254 strain to meropenem.

[0093] Note: 17254: When meropenem and 1 / 2MIC α-terpineol are used together, FIC = [512÷1024+1÷64] = 0.5156;

[0094] When meropenem was used in combination with 1 / 4MIC α-terpineol, FIC = [256÷1024+1÷64] = 0.2656;

[0095] CRKP20027697: When meropenem and 1 / 2MIC α-terpineol were used together, FIC = [512÷1024+4÷256] = 0.5156;

[0096] When meropenem was used in combination with 1 / 4MIC α-terpineol, FIC = [256÷1024+4÷256] = 0.2656;

[0097] When meropenem was used in combination with 1 / 8 MIC α-terpineol, FIC = [128÷1024+128÷256] = 0.625;

[0098] The unit of drug dosage is μg / mL.

[0099] Experimental Example 3: Combined drug sensitivity test of α-terpineol and colistin against mcr-1 Escherichia coli from animals

[0100] 1. Test consumables

[0101] 1. Reagents and drugs

[0102] α-Terpineol: produced by Shanghai McLean Biochemical Technology Co., Ltd., content: 98%.

[0103] Colistin sulfate (Colistin): produced by Beijing Solebow Technology Co., Ltd., content: 63%, batch number: 1117A0211.

[0104] Colistin was prepared into a 1280 μg / mL stock solution with sterile distilled water and stored frozen in a -20°C refrigerator.

[0105] 2. Strains

[0106] Escherichia coli standard strain ATCC 25922: purchased from Qingdao Haibo Biotechnology Co., Ltd. The 4 avian Escherichia coli containing mcr-1 used in the present invention were stored in the Veterinary Pharmacology Laboratory of the College of Veterinary Medicine of Qingdao Agricultural University. Escherichia coli was activated by streaking on eosin-methylene blue medium, cultured in a 37°C incubator overnight, and stored in a 4°C refrigerator for later use.

[0107] 3. Culture medium

[0108] LB broth medium: Qingdao Haibo Biotechnology Co., Ltd., prepared according to the instruction manual.

[0109] MH broth medium: Qingdao Haibo Biotechnology Co., Ltd., prepared according to the instruction manual.

[0110] MacConkey agar medium: Qingdao Haibo Biotechnology Co., Ltd., prepared according to the instruction manual.

[0111] Eosin-methylene blue agar medium: Qingdao Haibo Biotechnology Co., Ltd., prepared according to the instruction manual.

[0112] 4. Instruments

[0113] Same as the first embodiment.

[0114] 2. Test methods

[0115] 1. Preparation of test bacterial solution

[0116] Dissolve the standard strain of E. coli ATCC 25922 preserved in glycerol at -80℃ and the isolated avian E. coli strain containing mcr-1 at room temperature, take 100μL and inoculate it in LB broth, and culture it in a constant temperature incubator at 37℃ overnight. In the clean bench, use the streak method to inoculate the 25922 strain culture liquid on MacConkey agar and eosin-methylene blue agar containing colistin, and inoculate the mcr-1 E. coli on eosin-methylene blue agar containing colistin. Incubate the culture inverted at 37℃ in an incubator for 18 to 24 hours, pick a typical single colony and inoculate it in LB broth, and culture it in a constant temperature incubator at 37℃ overnight. Take the bacterial solution into MH broth culture medium and adjust the bacterial solution to 0.5 McFarland turbidity standard (concentration of about 10 8 CFU / mL), and the bacterial solution was diluted 100 times with MH broth to make the bacterial solution concentration 10 6 CFU / mL, as the test bacterial solution.

[0117] 2. Determination of single drug susceptibility MIC

[0118] ATCC 25922 was used as the quality control strain, and 4 strains of Escherichia coli containing mcr-1 from poultry were used as experimental strains. The MIC of colistin and α-terpineol was determined by the microbroth dilution method specified by CLSI. A 96-well sterile microplate was used, and the MIC was determined at a concentration gradient of 1 / 10, 1 / 20, 1 / 40, 1 / 80, 1 / 160, 1 / 320, and 1 / 640 times the mother solution. The drug was gradiently diluted in the 96-well plate, colistin / α-terpineol was added to the first column, and the gradient was diluted to the seventh column, with 50 μL of broth and drug fixed in each well. After the test bacterial solution was mixed, it was added to each well of the 96-well plate with an eight-channel pipette, 50 μL per well, and the total volume was 100 μL. At the same time, negative wells (without bacteria) and positive wells (without drug) were set as controls. The plates were cultured at 37°C for 18 hours, and the results were observed and recorded. Each test was repeated twice.

[0119] 3. Combined drug sensitivity test

[0120] Taking MY197-1 and MY2-1 of Escherichia coli containing mcr-1 from poultry as examples, ATCC 25922 was used as a quality control strain, and the MIC results were used as a reference. The microbroth dilution chessboard method used a 96-well sterile microplate, and designed 1 times, 1 / 2 times, 1 / 4 times, 1 / 8 times, 1 / 16 times, 1 / 32 times and 1 / 64 times MIC of the two drugs for combination. The two drugs were gradiently diluted in the 96-well plate, α-terpineol was added to the first column, and the horizontal gradient was diluted to the seventh column. Colistin was added to the first row, and the vertical gradient was diluted to the seventh row. The broth and drug were fixed at 50μL in each well. After the test bacterial solution was mixed, it was added to each well of the 96-well plate with an eight-channel pipette, 50μL per well, and the total volume was 100μL. At the same time, α-terpineol alone MIC, colistin alone MIC, (no bacteria) negative and (no drug) positive wells were set as controls. The cells were incubated at 37°C for 18 h, and the results were observed and recorded. Each test was repeated twice.

[0121] The calculation of FICI index (graded inhibition concentration index FICI) and the result judgment are the same as those in Example 1.

[0122] 3. Test results

[0123] The MIC value of the drug sensitivity test is determined by the minimum concentration at which no bacterial growth is observed with the naked eye as the MIC of the drug. The MIC of colistin against the Escherichia coli quality control bacteria ATCC25922 is 1μg / mL, which is in line with the quality control range of 0.25-2μg / mL.

[0124] The combined drug sensitivity results are shown in Table 3

[0125] Table 3 MIC test results of α-terpineol and colistin against poultry-derived mcr-containing Escherichia coli

[0126]

[0127] As shown in Table 3, the MIC of colistin against avian mcr-1 Escherichia coli MY197-1 and MY2-1 was 4 μg / mL, and the MIC of α-terpineol against MY197-1 and MY2-1 was 1024 μg / mL. When 1 / 2MIC (512 μg / mL), 1 / 4MIC (256 μg / mL), and 1 / 8MIC (128 μg / mL) of α-terpineol were added to the MY197-1 strain, the MIC of colistin was 0.0625, 0.0625, and 2 μg / mL, which were 64, 64, and 2 times lower than the MIC value when colistin was used alone. As the concentration of α-terpineol decreased, the reduction factor of the MIC value of colistin also decreased. When 1 / 2MIC (512μg / mL), 1 / 4MIC (256μg / mL), and 1 / 8MIC (128μg / mL) of α-terpineol were added to the MY2-1 strain, the MIC of colistin was 0.0625, 0.0625, and 1μg / mL, respectively, which were 64, 64, and 4 times lower than the MIC value of colistin alone. As the concentration of α-terpineol decreased, the reduction factor of the MIC value of colistin also decreased.

[0128] In the 4 selected mcr-1 Escherichia coli strains from different sources, after adding sub-inhibitory concentration (≤1 / 2MIC) α-pineneol, the MIC of colistin decreased to varying degrees. When 1 / 2MIC α-pineneol was added, the MIC of colistin decreased by 64 times, at this time FIC=0.55156, and the two drugs had an additive effect; when 1 / 4MIC α-pineneol was added, the MIC of colistin could be reduced by 32 to 64 times, at this time FIC<0.5, and the two drugs had a synergistic effect; among them, when 1 / 8MIC α-pineneol was added to the MY2-1 strain, the MIC of colistin could be reduced by 4 times, FIC=0.375, and the two drugs had a synergistic effect.

[0129] like Figure 2 As shown in A, taking the Escherichia coli MY197-1 strain as an example, the use of α-terpineol at a concentration of 1 / 4 MIC (i.e., 128 μg / mL) in combination with colistin reduced the MIC of colistin in the MY197-1 strain from 4 μg / mL to 0.0625 μg / mL, and the FIC was 0.2656, which had a synergistic antibacterial effect. The results showed that α-terpineol at a concentration of 1 / 4 MIC (i.e., 128 μg / mL) restored the sensitivity of the MY197-1 strain to colistin.

[0130] Note: MY197-1: When colistin and 1 / 2MIC α-terpineol were used together, FIC = [512÷1024+0.0625÷4] = 0.5156;

[0131] When colistin and 1 / 4MIC α-terpineol were used together, FIC = [256÷1024+0.0625÷4] = 0.2656;

[0132] When colistin and 1 / 8MIC α-terpineol were used together, FIC = [128÷1024+2÷4] = 0.625;

[0133] MY2-1: When colistin and 1 / 2MIC α-terpineol were used together, FIC = [512÷1024+0.0625÷4] = 0.5156;

[0134] When colistin and 1 / 4MIC α-terpineol were used together, FIC = [256÷1024+0.0625÷4] = 0.2656;

[0135] When colistin and 1 / 8MIC α-terpineol were used together, FIC = [128÷1024+1÷4] = 0.375;

[0136] The unit of drug dosage is μg / mL.

[0137] Experimental Example 4: Combined drug sensitivity test of α-terpineol and colistin against MCR Salmonella carried by animals

[0138] 1. Test consumables

[0139] 1. Reagents and drugs

[0140] Same as Example 3

[0141] 2. Strains

[0142] Escherichia coli standard strain ATCC 25922: purchased from Qingdao Haibo Biotechnology Co., Ltd. The 4 strains of animal-derived Salmonella containing mcr-1 used in the present invention were stored in the Veterinary Pharmacology Laboratory of the College of Veterinary Medicine of Qingdao Agricultural University. Salmonella was activated by streaking on SS agar medium, cultured in a 37°C incubator overnight, and stored in a 4°C refrigerator for later use.

[0143] 3. Culture medium

[0144] LB broth medium: Qingdao Haibo Biotechnology Co., Ltd., prepared according to the instruction manual.

[0145] MH broth medium: Qingdao Haibo Biotechnology Co., Ltd., prepared according to the instruction manual.

[0146] MacConkey agar medium: Qingdao Haibo Biotechnology Co., Ltd., prepared according to the instruction manual.

[0147] SS agar medium: Qingdao Haibo Biotechnology Co., Ltd., prepared according to the instruction manual.

[0148] 4. Instruments

[0149] Same as the first embodiment.

[0150] 2. Test methods

[0151] 1. Preparation of test bacterial solution

[0152] Dissolve the standard strain of Escherichia coli ATCC 25922 preserved in -80℃ glycerol and the isolated animal-derived mcr-1 Salmonella strain at room temperature, take 100μL and inoculate it in LB broth, and culture it in a 37℃ constant temperature incubator overnight. In the clean bench, use the streak method to inoculate the 25922 strain culture liquid on MacConkey agar and eosin-methylene blue agar containing colistin, and inoculate the mcr Salmonella on SS agar containing colistin. Incubate the culture inverted at 37℃ in an incubator for 18 to 24 hours, pick a typical single colony and inoculate it in LB broth, and culture it in a 37℃ constant temperature incubator overnight. Take the bacterial solution into MH broth culture medium and adjust the bacterial solution to 0.5 McFarland turbidity standard (concentration of about 10 8 CFU / mL), and the bacterial solution was diluted 100 times with MH broth to make the bacterial solution concentration 10 6 CFU / mL, as the test bacterial solution.

[0153] 2. Determination of single drug susceptibility MIC

[0154] ATCC 25922 was used as the quality control strain, and 4 strains of Salmonella containing mcr-1 from animals were used as experimental strains. The MIC of colistin and α-terpineol was determined by the microbroth dilution method specified by CLSI. A 96-well sterile microplate was used, and the MIC was determined at a concentration gradient of 1 / 10, 1 / 20, 1 / 40, 1 / 80, 1 / 160, 1 / 320, and 1 / 640 times the mother solution. The drug was gradiently diluted in the 96-well plate, colistin / α-terpineol was added to the first column, and the gradient was diluted to the seventh column, with 50 μL of broth and drug fixed in each well. After the test bacterial solution was mixed, it was added to each well of the 96-well plate with an eight-channel pipette, 50 μL per well, and the total volume was 100 μL. At the same time, negative wells (without bacteria) and positive wells (without drugs) were set as controls. The wells were cultured at 37°C for 18 hours, and the results were observed and recorded. Each test was repeated twice.

[0155] 3. Combined drug sensitivity test

[0156] Taking the Salmonella SD27 containing mcr-1 from poultry and the Salmonella S8-25-1 containing mcr-1 from pigeon as examples, ATCC 25922 was used as the quality control strain, and the MIC results were used as a reference. The microbroth dilution chessboard method used a 96-well sterile microplate, and designed two drugs at 1 times, 1 / 2 times, 1 / 4 times, 1 / 8 times, 1 / 16 times, 1 / 32 times and 1 / 64 times MIC for combination. The two drugs were gradiently diluted in the 96-well plate, α-pineneol was added to the first column, and the gradient was diluted horizontally to the seventh column. Colistin was added to the first row, and the gradient was diluted vertically to the seventh row. The broth and drug were fixed at 50μL in each well. After the test bacterial solution was mixed, it was added to each well of the 96-well plate with an eight-channel pipette, 50μL per well, and the total volume was 100μL. At the same time, set up α-terpineol single MIC, colistin single MIC, (no bacteria) negative and (no drug) positive wells as controls. Incubate at 37℃ for 18h, observe and record the results. Repeat each test twice.

[0157] The calculation of FICI index (graded inhibition concentration index FICI) and the result judgment are the same as those in Example 1.

[0158] 3. Test results

[0159] The MIC value of the drug sensitivity test is determined by the minimum concentration at which no bacterial growth is observed with the naked eye as the MIC of the drug. The MIC of colistin against the Escherichia coli quality control bacteria ATCC25922 is 1μg / mL, which is in line with the quality control range of 0.25-2μg / mL.

[0160] The combined drug sensitivity results are shown in Table 4

[0161] Table 4 MIC test results of α-terpineol and colistin against mcr-1 Salmonella from animal sources

[0162]

[0163] It can be seen from Table 4 that the MIC of colistin against the pigeon-derived Salmonella S8-25-1 strain containing mcr-1 and the poultry-derived Salmonella SD27 containing mcr-1 was 4 μg / mL, and the MIC of α-pineneol against the S8-25-1 and SD27 strains was 1024 μg / mL. When 1 / 2MIC (512 μg / mL), 1 / 4MIC (256 μg / mL), and 1 / 8MIC (128 μg / mL) of α-pineneol were added to the S8-25-1 strain, the MIC of colistin was 0.0625 μg / mL, 0.5 μg / mL, and 2 μg / mL, respectively, which was 64, 8, and 2 times lower than the MIC value when colistin was used alone. When SD27 was treated with 1 / 2MIC (512μg / mL), 1 / 4MIC (256μg / mL), and 1 / 8MIC (128μg / mL) of α-terpineol, the MIC of colistin was 0.0625μg / mL, 0.5μg / mL, and 1μg / mL, which were 64, 8, and 4 times lower than the MIC values ​​of colistin alone. As the concentration of α-terpineol decreased, the reduction factor of the MIC value of colistin also decreased.

[0164] In the 4 selected mcr-1 Salmonella strains from different sources, after adding sub-inhibitory concentration (≤1 / 2MIC) α-pineneol, the MIC of colistin decreased to varying degrees. When 1 / 2MIC α-pineneol was added, the MIC of colistin decreased by 64 times, and the FIC was 0.5156 at this time, and the two drugs had an additive effect; when 1 / 4MIC α-pineneol was added, the MIC of colistin decreased by 4 to 8 times, and the FIC was ≤0.5 at this time, and the two drugs had a synergistic effect; among them, when 1 / 8MIC α-pineneol was added to the SD27 strain, the MIC of colistin decreased by 4 times, and the FIC was 0.375, and the two drugs had a synergistic effect.

[0165] like Figure 2 As shown in Figure B, taking the Salmonella SD27 strain as an example, the use of 1 / 4 MIC concentration of α-terpineol (i.e., 128 μg / mL) in combination with colistin reduced the MIC of colistin in the SD27 strain from 4 μg / mL to 1 μg / mL, and the FIC was 0.375, which had a synergistic antibacterial effect. The results showed that 1 / 4 MIC concentration (i.e., 128 μg / mL) of α-terpineol restored the sensitivity of the Salmonella SD27 strain to colistin.

[0166] Note: SD27: When colistin and 1 / 2MIC α-terpineol were used together, FIC = [512÷1024+0.0625÷4] = 0.5156;

[0167] When colistin and 1 / 4MIC α-terpineol were used together, FIC = [256÷1024+0.5÷4] = 0.375;

[0168] When colistin and 1 / 8MIC α-terpineol were used together, FIC = [128÷1024+2÷4] = 0.625;

[0169] S8-25-1: When colistin and 1 / 2MIC α-terpineol were used together, FIC = [512÷1024+0.0625÷4] = 0.5156;

[0170] When colistin and 1 / 4MIC α-terpineol were used together, FIC = [256÷1024+0.5÷4] = 0.375;

[0171] When colistin and 1 / 8MIC α-terpineol were used together, FIC = [128÷1024+1÷4] = 0.375;

[0172] The unit of drug dosage is μg / mL.

[0173] Example 1, medication example

[0174] α-Terpineol and meropenem are used together in a weight ratio of 8:1; the administration method is the same as that of meropenem alone. It is used to inhibit NDM Escherichia coli carried by poultry or KPC-2 Klebsiella pneumoniae carried by poultry.

[0175] Example 2, medication example

[0176] α-terpineol and colistin are used in combination at a weight ratio of 8:0.03; the administration method is the same as that of colistin alone. It is used to inhibit mcr-1 positive strains, such as mcr-1 Escherichia coli or mcr-1 positive Salmonella.

[0177] Example 3: An injection for carbapenem-resistant Enterobacteriaceae infection

[0178] The injection is prepared by mixing α-terpineol and meropenem in a weight ratio of 8:1.

[0179] Example 4: An injection for infection with mcr-1 positive strains

[0180] The injection is prepared by mixing α-pineneol and colistin in a weight ratio of 8:0.03.

Claims

1. Use of α-terpineol and colistin in a weight ratio of 128:1 in combination for preparing a drug against Escherichia coli strain MY2-1.

2. Use of α-terpineol and colistin in a weight ratio of 512:1 in combination for preparing a drug for combating Salmonella strain S8-25-1.

3. Use of α-terpineol and colistin in a weight ratio of 512:1 or 128:1 in combination for preparing a drug against Salmonella strain SD27.

4. A pharmaceutical composition for use against Escherichia coli strain MY2-1 or against Salmonella strain SD27, characterized in that: The composition consists of alpha-terpineol and colistin in a weight ratio of 128:

1.

5. A pharmaceutical composition for use against Salmonella strain S8-25-1 or against Salmonella strain SD27, characterized in that: The composition consists of alpha-terpineol and colistin in a weight ratio of 512:

1.

6. The method for preparing the pharmaceutical composition according to any one of claims 4 to 5, characterized in that: The following steps are involved: Take α-pineneol and colistin in proportion, make a solution, and mix them.

7. A pharmaceutical preparation for combating Escherichia coli strain MY2-1 or against Salmonella strain SD27, characterized in that: The pharmaceutical preparation is prepared using the pharmaceutical composition according to claim 4 as an active ingredient.

8. A pharmaceutical preparation for combating Salmonella strain S8-25-1 or combating Salmonella strain SD27, characterized in that: The pharmaceutical preparation is prepared using the pharmaceutical composition according to claim 5 as an active ingredient.

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