Application of Trichostatin A in reversing bacterial drug resistance

Abstract

The invention relates to application of TrichostatinA serving as a metal beta-lactamase inhibitor combined with beta-lactam antibiotics in preparation of drugs for resisting drug-resistant bacteria. The invention further relates to a combined medicine for resisting the drug-resistant bacteria, the active ingredients of the combined medicine comprise the Trichostatin A and the beta-lactam antibiotics, the Trichostatin A and the beta-lactam antibiotics are respectively used as independent drug delivery units, or the Trichostatin A and the beta-lactam antibiotics jointly form a combined drug delivery unit. The invention discloses that the histone deacetylase inhibitor TrichostatinA has good inhibitory activity on MBLs (NDM-1 and VIM-2) for the first time, and has excellent synergistic antibacterial activity on drug-resistant strains for expressing MBLs (NDM-1 and VIM-2) when being combined with beta-lactam antibiotics for use.

Description

Technical Field

[0001] The invention relates to the technical field of biological medicines, and in particular to the application of Trichostatin A in reversing bacterial drug resistance. Background Art

[0002] β-lactam antibiotics (including penicillins, cephalosporins, carbapenems, etc.) are currently widely used anti-infective drugs in clinical practice, but due to their overuse and abuse, bacterial resistance is becoming increasingly serious. Metallo-β-lactamases (MBLs) are key enzymes for bacterial resistance to β-lactam antibiotics. They contain one or two metal Zn ions in their active sites. They can be divided into B1, B2, and B3 classes according to the similarity of their gene coding sequences. Class B1 is the most common and widely spread MBLs in clinical practice, including NDM, IMP, VIM, etc. The active site contains two metal Zn ions, which can hydrolyze almost all β-lactam antibiotics. They are becoming more and more common in clinical practice, and clinical treatment is extremely difficult. Metallo-β-lactamase inhibitors (MBLIs) can inhibit the activity of MBLs, thereby restoring the effectiveness of β-lactam antibiotics against resistant bacteria expressing MBLs. So far, no MBLIs have been marketed for clinical use, which is an unmet clinical need. Therefore, the development of MBLIs is an important breakthrough in the development of new antibacterial drugs, with very good clinical application prospects and social and economic benefits.

[0003] There have been many reports on MBLIs, but most of the reported MBLIs have the following problems, which make their drug development prospects slim. First, some of the metalloproteins in humans are members of the MBLs superfamily, and overly broad-spectrum and non-selective MBLIs, such as metal chelators EDTA and thioglycolic acid, can act on these proteins and cause toxic side effects; second, some MBLIs have strong inhibitory effects on enzymes in enzyme inhibition experiments, but the effect is not ideal when combined with β-lactam antibiotics on Gram-negative bacteria expressing MBLs. Therefore, it is crucial to find new MBLIs that are effective, safe and non-toxic.

[0004] Trichostatin A, referred to as TSA, is a potent and specific inhibitor of histone deacetylase types I and II. 50 The value is 1.8nM, contains a hydroxamic acid group, and its chemical structure is as follows:

[0005] Summary of the invention

[0006] Based on this, the purpose of the present invention is to provide a new application of Trichostatin A in medicine, in particular, the application of Trichostatin A in reversing bacterial resistance.

[0007] The technical solution for achieving the above-mentioned purpose is as follows.

[0008] The first aspect of the present invention is to provide the use of Trichostatin A as a metallo-β-lactamase inhibitor in combination with β-lactam antibiotics in the preparation of a drug against drug-resistant bacteria, wherein the bacteria are drug-resistant bacteria expressing metallo-β-lactamase.

[0009] The second aspect of the present invention is to provide a combination drug for combating drug-resistant bacteria, wherein the active ingredients include Trichostatin A and β-lactam antibiotics, wherein the Trichostatin A and β-lactam antibiotics are respectively independent dosing units, or the Trichostatin A and β-lactam antibiotics together form a combined dosing unit, and the bacteria are drug-resistant bacteria expressing metallo-β-lactamase.

[0010] In some embodiments, the drug-resistant bacteria expressing metallo-β-lactamase is a drug-resistant Gram-negative bacteria expressing metallo-β-lactamase.

[0011] In some embodiments, the drug-resistant bacteria expressing metallo-β-lactamase is a drug-resistant Gram-negative bacillus expressing metallo-β-lactamase.

[0012] In some embodiments, the drug-resistant bacteria expressing metallo-β-lactamase is drug-resistant Escherichia coli expressing metallo-β-lactamase.

[0013] In some embodiments, the metallo-β-lactamase is of NDM-1 or VIM-2 type.

[0014] In some embodiments, the β-lactam antibiotic is a carbapenem antibiotic.

[0015] In some embodiments, the carbapenem antibiotic is meropenem.

[0016] The present invention discloses for the first time that the histone deacetylase inhibitor Trichostatin A has good inhibitory activity (IC 50 value), and combined with the carbapenem antibiotic meropenem, it has excellent synergistic antibacterial activity (FICI value) against MBLs (NDM-1, VIM-2)-resistant strains, and can provide a new and effective treatment option for diseases caused by MBLs (NDM-1, VIM-2)-resistant bacteria in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Time-kill curves of trichostatin A and meropenem alone or in combination against E. coli (NDM-1) constructed strains. DETAILED DESCRIPTION

[0018] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0019] The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, such as the fourth edition of Molecular Cloning: A Laboratory Manual edited by Green and Sambrook, published in 2013, or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0020] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0021] Definitions To facilitate understanding of this technology, certain terms and phrases are defined below.

[0022] Escherichia coli (E. coli), Escherichia coli, is a Gram-negative bacillus.

[0023] MEM: Meropenem, chemical formula is C 17 H 25 N3O5S, a broad-spectrum carbapenem antibiotic, is a β-lactam antibiotic.

[0024] (1) Study on the inhibitory activity of Trichostatin A on MBLs in vitro

[0025] 1) Construction of recombinant engineered bacteria

[0026] 2) Expression and purification of MBLs

[0027] 3) Enzyme inhibitory activity IC of Trichostatin A 50 Determination

[0028] (2) Antibacterial experiment of Trichostatin A combined with β-lactam antibiotics

[0029] The MIC (minimum inhibitory concentration) value of meropenem alone or in combination with Trichostatin A against MBLs-producing Escherichia coli was determined by the microbroth dilution method. The meropenem dilution was 128-0.25μg / mL, and the concentration of Trichostatin A was 128-2μg / mL. At the same time, meropenem combined with captopril (classic MBLI) was set as a positive control, and meropenem combined with DMSO was set as a negative control. Incubate at 37°C for 16-20h and record the MIC value.

[0030] The present invention is further described in detail below through specific examples.

[0031] Example 1: Study on the in vitro MBLs inhibitory activity of Trichostatin A

[0032] 1. Construction of recombinant engineering bacteria:

[0033] Recombinant plasmids (NDM-1, VIM-2 and IMP-1 recombinant plasmids) were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0034] Take out the BL21(DE3) competent cells from the -80℃ ultra-low temperature refrigerator and put them on ice to melt. Take 1μL of recombinant plasmids (NDM-1, VIM-2 and IMP-1 recombinant plasmids) in the clean bench and add them to 100μL BL21(DE3) competent cells, and mix them with a pipette. Then, ice-bathe the competent cells for 30 minutes, heat-shock at 42℃ for 90s, and then ice-bathe for 2 minutes. Then, add 900μL LB liquid culture medium to the competent cells and culture them in a constant temperature shaking incubator at 37℃ and 220rpm for 1 hour. Spread the competent cell liquid evenly on the LB solid culture medium containing 25μg / mL kanamycin and invert and culture overnight at 37℃.

[0035] 2. Isolation and purification of MBLs:

[0036] 1) Preparation of solution:

[0037] Lysis buffer: Weigh 5.95 g of HEPES and dissolve it in 500 mL of sterile distilled water to make the final concentration of the buffer 50 mM. Then add NaCl and 0.1% Triton X-100 to the buffer at a final concentration of 300 mM, and adjust the pH to about 8.0 with 1 M NaOH solution.

[0038] Binding buffer: NaCl and 0.2% TritonX-100 were added to 50 mM HEPES buffer to a final concentration of 300 mM, and the pH was adjusted to about 8.0.

[0039] Washing buffer: Add 300 mM NaCl, 50 mM imidazole and 100 μM ZnCl2 to a final concentration of 50 mM HEPES buffer, and adjust the pH to about 8.0.

[0040] Elution buffer: Add NaCl to a final concentration of 300 mM, imidazole to 250 mM, and ZnCl2 to 100 μM in a 50 mM HEPES buffer, and adjust the pH to about 8.0.

[0041] Replacement buffer 1: Weigh 2.98 g of HEPES and dissolve it in 500 mL of sterile distilled water to make the final concentration of the buffer solution 25 mM, and add NaCl and ZnCl2 to the buffer solution with final concentrations of 300 mM and 100 μM, respectively, and adjust the pH to about 7.0.

[0042] Replacement buffer 2: Add NaCl and ZnCl2 to a final concentration of 200 mM and 100 μM, respectively, to 25 mM HEPES buffer and adjust the pH to about 7.0.

[0043] Replacement buffer 3: Add NaCl and ZnCl2 to a final concentration of 100 mM and 100 μM respectively to 25 mM HEPES buffer and adjust the pH to about 7.0.

[0044] Expression and purification of three common clinical metallo-β-lactamases (NDM-1, VIM-2, IMP-1):

[0045] 1) Expression and purification of NDM-1:

[0046] Recovery: Use an inoculation loop to take a loop of glycerol bacteria carrying the NDM-1 gene plasmid, inoculate it onto an LB agar plate containing 25 μg / mL kanamycin, and invert it in a 37°C constant temperature shaking incubator for overnight culture for 12-16 hours.

[0047] Activation: Use an inoculation loop to pick a single colony of E. coli expressing NDM-1 and transfer it to 10 mL of LB liquid culture medium containing 25 μg / mL kanamycin. Incubate overnight at 37°C, 150 rpm, on a shaking incubator for 12-16 h.

[0048] Amplification culture: Take 2.5 mL of the bacterial solution from the previous step and add it to 250 mL of LB liquid medium containing 25 μg / mL kanamycin (two copies), and culture at 37°C, 150 rpm, on a shaking table for about 4 hours until the bacterial solution concentration reaches OD 600 The value is 0.6-0.8.

[0049] Low-temperature induction culture: Add 250 μL 0.1 M IPTG to the bacterial solution prepared in the previous step, and induce culture at low temperature at 18°C ​​and 150 rpm for about 20 hours.

[0050] Bacteria collection: centrifuge the bacterial solution after low temperature induction at 10,000 rpm, 4°C, 5 min. Discard the supernatant culture medium, resuspend the bacterial precipitate with an appropriate amount of sterile PBS and centrifuge (10,000 rpm, 4°C, 5 min), remove the supernatant PBS, and store the bacterial precipitate at -80°C.

[0051] Disrupt bacteria: resuspend the frozen bacterial pellet in an appropriate amount of lysis buffer and add a corresponding volume of protease inhibitor (x100). Then, place the resuspended bacterial solution in an ice bath and use a cell ultrasonic disruptor for ultrasonic treatment. Under 400 W conditions, ultrasonicate for 5 s with an interval of 5 s, for a total of 15 min.

[0052] Centrifugation: The broken bacterial solution is centrifuged at 12,000 rpm, 4°C, 3 min. The supernatant is collected and filtered through a 0.45 μM microporous filter membrane to obtain crude protein. In addition, a small amount of bacterial precipitate is collected for subsequent detection.

[0053] Nickel column affinity purification: First, wash the equilibrated nickel column with 5 mL of Binding buffer, then load the crude protein sample collected in the previous step onto the nickel column and repeat the flow once. Then, wash the column with Washing buffer containing 50 mM imidazole to wash away the impurities, and wash 6 times in total; then elute with Elution buffer containing 250 mM imidazole, collect the eluate, and finally treat the nickel column again and store it in 20% ethanol.

[0054] SDS-PAGE gel electrophoresis: The crude protein, eluate and other samples collected in the previous step were subjected to SDS-PAGE gel electrophoresis and stained with Coomassie Brilliant Blue Rapid Stain to identify the protein purification status and collect them.

[0055] Dialysis: The purified protein is placed in a dialysis bag and first dialyzed with replacement buffer 1 for 2-4 hours, then replaced with replacement buffer 2 for continued dialysis, and replaced with replacement buffer 3 after 6-8 hours for 12 hours. After 12 hours of dialysis, replacement buffer 3 is replaced again for continued dialysis for 2 hours. The dialyzed protein sample is collected and filtered through a 0.22μM microporous membrane to collect the target protein. The collected target protein is subjected to subsequent enzyme activity detection and stored at -20℃ after aliquoting.

[0056] 2) Expression and purification of VIM-2:

[0057] Recovery: Use an inoculation loop to take a loop of glycerol bacteria carrying the VIM-2 gene plasmid, inoculate it onto an LB agar plate containing 25 μg / mL kanamycin, and invert it in a 37°C constant temperature shaking incubator for overnight culture for 12-16 hours.

[0058] Activation: Use an inoculation loop to pick a single colony of E. coli expressing VIM-2 and transfer it to 10 mL of LB liquid medium containing 25 μg / mL kanamycin. Incubate overnight at 37°C, 150 rpm, on a shaking incubator for 12-16 h.

[0059] Amplification culture: Take 2.5 mL of the bacterial solution from the previous step and add it to 250 mL of LB liquid medium containing 25 μg / mL kanamycin (two copies), and culture at 37°C, 150 rpm, on a shaking table for about 4 hours until the bacterial solution concentration reaches OD 600 The value is 0.6-0.8.

[0060] Low-temperature induction culture: Add 250 μL 0.1 M IPTG to the bacterial solution prepared in the previous step, and induce culture at low temperature at 18°C ​​and 150 rpm for about 20 hours.

[0061] Bacteria collection: centrifuge the bacterial solution after low temperature induction at 10,000 rpm, 4°C, 5 min. Discard the supernatant culture medium, resuspend the bacterial precipitate with an appropriate amount of sterile PBS and centrifuge (10,000 rpm, 4°C, 5 min), remove the supernatant PBS, and store the bacterial precipitate at -80°C.

[0062] Disrupt bacteria: resuspend the frozen bacterial pellet in an appropriate amount of lysis buffer and add a corresponding volume of protease inhibitor (x100). Then, place the resuspended bacterial solution in an ice bath and use a cell ultrasonic disruptor for ultrasonic treatment. Under 400 W conditions, ultrasonicate for 5 s with an interval of 5 s, for a total of 15 min.

[0063] Centrifugation: The broken bacterial solution is centrifuged at 12,000 rpm, 4°C, 3 min. The supernatant is collected and filtered through a 0.45 μM microporous filter membrane to obtain crude protein. In addition, a small amount of bacterial precipitate is collected for subsequent detection.

[0064] Nickel column affinity purification: First, wash the equilibrated nickel column with 5 mL of Binding buffer, then load the crude protein sample collected in the previous step onto the nickel column and repeat the flow once. Then, wash the column with Washing buffer containing 50 mM imidazole to wash away the impurities, and wash 6 times in total; then elute with Elution buffer containing 250 mM imidazole, collect the eluate, and finally treat the nickel column again and store it in 20% ethanol.

[0065] SDS-PAGE gel electrophoresis: The crude protein, eluate and other samples collected in the previous step were subjected to SDS-PAGE gel electrophoresis and stained with Coomassie Brilliant Blue Rapid Stain to identify the protein purification status and collect them.

[0066] Dialysis: The purified protein is placed in a dialysis bag and first dialyzed with replacement buffer 1 for 2-4 hours, then replaced with replacement buffer 2 for continued dialysis, and replaced with replacement buffer 3 after 6-8 hours for 12 hours. After 12 hours of dialysis, replacement buffer 3 is replaced again for continued dialysis for 2 hours. The dialyzed protein sample is collected and filtered through a 0.22μM microporous membrane to collect the target protein. The collected target protein is subjected to subsequent enzyme activity detection and stored at -20℃ after aliquoting.

[0067] 3) Expression and purification of IMP-1:

[0068] Recovery: Use an inoculation loop to take a loop of glycerol bacteria carrying the IMP-1 gene plasmid, inoculate it onto an LB agar plate containing 25 μg / mL kanamycin, and invert it in a 37°C constant temperature shaking incubator for overnight culture for 12-16 hours.

[0069] Activation: Use an inoculation loop to pick a single colony of E. coli expressing IMP-1 and transfer it to 10 mL of LB liquid culture medium containing 25 μg / mL kanamycin. Incubate overnight at 37°C, 150 rpm, on a shaking incubator for 12-16 hours.

[0070] Amplification culture: Take 2.5 mL of the bacterial solution from the previous step and add it to 250 mL of LB liquid medium containing 25 μg / mL kanamycin (two copies), and culture at 37°C, 150 rpm, on a shaking table for about 4 hours until the bacterial solution concentration reaches OD 600 The value is 0.6-0.8.

[0071] Low-temperature induction culture: Add 250 μL 0.1 M IPTG to the bacterial solution prepared in the previous step, and induce culture at low temperature at 18°C ​​and 150 rpm for about 20 hours.

[0072] Bacteria collection: centrifuge the bacterial solution after low temperature induction at 10,000 rpm, 4°C, 5 min. Discard the supernatant culture medium, resuspend the bacterial precipitate with an appropriate amount of sterile PBS and centrifuge (10,000 rpm, 4°C, 5 min), remove the supernatant PBS, and store the bacterial precipitate at -80°C.

[0073] Disrupt bacteria: resuspend the frozen bacterial pellet in an appropriate amount of lysis buffer and add a corresponding volume of protease inhibitor (x100). Then, place the resuspended bacterial solution in an ice bath and use a cell ultrasonic disruptor for ultrasonic treatment. Under 400 W conditions, ultrasonicate for 5 s with an interval of 5 s, for a total of 15 min.

[0074] Centrifugation: The broken bacterial solution is centrifuged at 12,000 rpm, 4°C, 3 min. The supernatant is collected and filtered through a 0.45 μM microporous filter membrane to obtain crude protein. In addition, a small amount of bacterial precipitate is collected for subsequent detection.

[0075] Nickel column affinity purification: First, wash the equilibrated nickel column with 5 mL of Binding buffer, then load the crude protein sample collected in the previous step onto the nickel column and repeat the flow once. Then, wash the column with Washing buffer containing 50 mM imidazole to wash away the impurities, and wash 6 times in total; then elute with Elution buffer containing 250 mM imidazole, collect the eluate, and finally treat the nickel column again and store it in 20% ethanol.

[0076] SDS-PAGE gel electrophoresis: The crude protein, eluate and other samples collected in the previous step were subjected to SDS-PAGE gel electrophoresis and stained with Coomassie Brilliant Blue Rapid Stain to identify the protein purification status and collect them.

[0077] Dialysis: The purified protein is placed in a dialysis bag and first dialyzed with replacement buffer 1 for 2-4 hours, then replaced with replacement buffer 2 for continued dialysis, and replaced with replacement buffer 3 after 6-8 hours for 12 hours. After 12 hours of dialysis, replacement buffer 3 is replaced again for continued dialysis for 2 hours. The dialyzed protein sample is collected and filtered through a 0.22μM microporous membrane to collect the target protein. The collected target protein is subjected to subsequent enzyme activity detection and stored at -20℃ after aliquoting.

[0078] 3. Enzyme Inhibition Activity IC of Trichostatin A 50 Determination:

[0079] 1) Enzyme kinetics experiment:

[0080] 1. Experimental methods

[0081] The enzyme kinetics experiment was carried out in a 96-well plate. The hydrolysis substrate was selected as nitrocefin, and the detection wavelength was 492nm. The final concentration of nitrocefin was 1-30μM, and then the MBLs solution (the final concentration of NDM-1, VIM-2 and IMP-1 was 10nM) was added. After mixing, the absorbance change was immediately detected. The detection cycle was once every 10s, and the detection was 3min in total. Each group of experiments was paralleled at least three times. The hydrolysis rate in the first 30s of the reaction was used for the calculation of Km.

[0082] (II) Experimental results

[0083] The Km values ​​of three MBLs (NDM-1, VIM-2 and IMP-1) are shown in Table 1.

[0084] Table 1 Km values ​​of three MBLs

[0085]

[0086] 2) IC 50 Determination of:

[0087] 1. Experimental methods

[0088] Dissolve the inhibitor (Trichostatin A or positive control drug Captopril) with DMSO (dimethyl sulfoxide), then dilute with the corresponding buffer, and ensure that the final DMSO concentration is below 2%. The inhibitor concentration range is 0-300μM, and the concentration of the substrate nitrocefin is 10μM. First, incubate the inhibitor with the enzyme at different concentrations for 15 minutes in advance, then add the substrate to initiate the reaction, mix by blowing, and immediately measure the change in absorbance at 492nm. Measure once every 10s, continue to detect for 1min, and take the increase in the hydrolysis product in the first 30s to calculate the initial reaction rate. Each group of experiments should be measured at least three times in parallel.

[0089] The residual activity of MBLs after adding different concentrations of inhibitors was calculated. The residual activity of the enzyme R (100%) = (Vi / V0) x 100% (Vi: the rate at which the enzyme hydrolyzes the substrate in the presence of the inhibitor; V0: the rate at which the enzyme hydrolyzes the substrate in the absence of the inhibitor). Graphpad Prism 8.0 was used to draw a nonlinear fitting graph of the inhibitor concentration and the residual activity of the enzyme to obtain the IC 50 value.

[0090] (II) Experimental results

[0091] IC of Trichostatin A against three MBLs (NDM-1, VIM-2 and IMP-1) 50 The results are shown in Table 2: IC values ​​of Trichostatin A for NDM-1, VIM-2 and IMP-1 50The values ​​were 179.71 μM, 105.32 μM and 236.62 μM, respectively, indicating that Trichostatin A has good inhibitory activity against the three MBLs.

[0092] Table 2 IC of Trichostatin A against three MBLs 50 and K i value

[0093]

[0094] Example 2 Antibacterial experiment of Trichostatin A combined with β-lactam antibiotics:

[0095] 1) Chessboard experiment:

[0096] 1. Experimental methods

[0097] The experimental strain was constructed according to the method described in Example 1:

[0098] E.coliBL21(DE3) / pET26b-NDM-1 is abbreviated as E.coli(NDM-1)

[0099] E.coliBL21(DE3) / pET26b-VIM-2 is abbreviated as E.coli(VIM-2)

[0100] E.coliBL21(DE3) / pET26b-IMP-1 is abbreviated as E.coli(IMP-1)

[0101] Determine the MIC values of meropenem / inhibitor (Trichostatin A or positive control captopril or negative control DMSO) alone and in combination against three constructed strains (Escherichia coli) according to the microbroth dilution method established by the Clinical and Laboratory Standards Institute (CLSI) of the United States. The combined antibacterial experiment is achieved through a checkerboard experiment, that is, by preparing dilution plates for the combined use of two drugs: Prepare a 5,120 μg / mL meropenem (MEM) stock solution with sterile distilled water, and prepare a 12,800 μg / mL inhibitor stock solution with DMSO, and store it at -20 °C for later use. Under sterile conditions, the horizontal rows of the 96-well plate are used for gradient dilution of MEM. Add 100 μL of MEM solution (512 μg / mL) to the first column of the 96-well plate, add 50 μL of culture medium to the 2nd - 11th columns. Pipette 50 μL of the solution from the first column and transfer it to the second column, mix well by pipetting. In the same way, pipette 50 μL of the mixed solution from the second column to the third column and mix well again. Operate continuously like this until the 11th column (0.5 - 512 μg / mL), and finally pipette 50 μL of the mixed solution and discard it. In the vertical columns, add 50 μL of the inhibitor diluted to different concentrations in advance with the culture medium (512 - 8 μg / mL). Adjust the culture solution concentration of the above Escherichia coli to 0.5 McFarland concentration and then dilute it 100 times, and add IPTG with a final concentration of 0.5 mM. Pipette 100 or 150 μL of the bacterial solution into the 96-well plate so that the final concentration of the bacterial solution is about 5x10 5 CFU / mL, and the final concentrations of MEM and the inhibitor are both 0 - 128 μg / mL, and the final volume of each well is 200 μL. Place the 96-well plate added with the bacterial solution in a constant temperature incubator at 37 °C and incubate overnight for 16 - 20 h, and then record the MIC value. The MIC value is the minimum concentration of the drug where no obvious bacterial growth is observed with the naked eye.

[0102] Calculation of the combined antibacterial index (FICI): The FICI value can be calculated according to a specific equation, that is, FICI = FIC A +FIC B , FIC A is the MIC value of drug A when used in combination / the MIC value of drug A when used alone, FIC B is the MIC value of drug B when used in combination / the MIC value of drug B when used alone, which is used to evaluate the interaction between two different drugs when used in combination. If FICI ≤ 0.5, it indicates that the combination of the two drugs has a synergistic effect. If 0.5 < FICI ≤ 4, there is no relevant effect when the two drugs are used in combination. If FICI > 4, it indicates that there is an antagonistic effect when the two drugs are used in combination. The smaller the FICI index, the stronger the synergistic effect of the drug.

[0103] (II) Experimental results

[0104] The MIC values ​​(Table 3, Table 4, Table 5, respectively) and FICI values ​​(Table 6) of the inhibitor (Trichostatin A or positive control drug captopril or negative control DMSO) and meropenem alone or in combination against resistant strains expressing NDM-1, VIM-2 and IMP-1 showed that the FICI of the combination of Trichostatin A and meropenem against resistant strains expressing NDM-1 and VIM-2 were both <0.50, indicating that this combination has good synergistic antibacterial activity against both resistant strains, but does not have synergistic activity against resistant strains expressing IMP-1.

[0105] Table 3 MIC values ​​(μg / mL) of meropenem alone and in combination with inhibitors at different concentrations (μg / mL) against E. coli (NDM-1)

[0106]

[0107] a The MIC of Trichostatin A against E. coli (NDM-1) is >1024 μg / mL. b The MIC of captopril against E. coli (NDM-1) is >1024 μg / mL, and the MIC of MEM against E. coli (NDM-1) is 128 μg / mL

[0108] Table 4 MIC values ​​(μg / mL) of meropenem alone and in combination with inhibitors at different concentrations (μg / mL) against E. coli (VIM-2)

[0109]

[0110] a The MIC of Trichostatin A against E. coli (VIM-2) is >1024 μg / mL. b The MIC of captopril against E. coli (VIM-2) was >1024 μg / mL, and the MIC of MEM against E. coli (VIM-2) was 32 μg / mL.

[0111] Table 5 MIC values ​​(μg / mL) of meropenem alone and in combination with inhibitors of different concentrations (μg / mL) against E. coli (IMP-1)

[0112]

[0113] a The MIC of Trichostatin A against E. coli (IMP-1) is >1024 μg / mL. bThe MIC of captopril against E. coli (IMP-1) was >1024 μg / mL, and the MIC of MEM against E. coli (IMP-1) was 32 μg / mL.

[0114] Table 6 Minimum FICI for the combined use of meropenem and trichostatin A

[0115]

[0116] 2) Time-sterilization curve:

[0117] 1. Experimental methods

[0118] The constructed strain E.coli BL21(DE3) / pET26b-NDM-1 was used as the experimental strain, and the meropenem group (32μg / mL), the Trichostatin A group (64μg / mL), the combined drug group (32μg / mLMEM+64μg / mL TrichostatinA) and the blank control group (containing only culture medium and bacterial solution) were set up. Take four sterile shake tubes, and add 10mL of LB liquid culture medium containing the corresponding drug concentration to each shake tube. Take 100μL of the bacterial solution with a concentration of 0.5 McFarland and add it to 10mL LB liquid culture medium (25μg / mL kanamycin), and culture at 37°C until the logarithmic growth phase (about 4-6h). Then adjust the concentration of the bacterial solution in the logarithmic growth phase to 1.0 McFarland concentration with LB liquid culture medium, and add 100μL of bacterial solution to each shake tube. In a 96-well plate, LB liquid medium was used to dilute each group of bacterial liquid by 10 times. 10 μL of each dilution was dripped onto the MH agar plate. After the plate was placed for about 30 minutes, it was inverted and incubated overnight at 37°C for 16-20 hours. The number of colonies on the plate was observed and recorded. 10 The curve was drawn with CFU / mL as the ordinate. All experiments were repeated three times independently.

[0119] (II) Experimental results

[0120] The results of the time-kill curve experiment are as follows Figure 1 As shown: In the absence of meropenem or Trichostatin A, the resistant strain of E. coli (NDM-1) in the growth control group grew normally; the addition of 32μg / mL meropenem alone had inhibitory activity on the growth of the strain within 2 hours, but the number of bacteria began to increase rapidly after 2 hours; the addition of 64μg / mL Trichostatin A alone could not inhibit the growth of resistant bacteria, and the growth of the strain was almost the same as that of the growth control group; however, when meropenem was used in combination with Trichostatin A, the number of bacteria decreased by more than 2log in 8 hours 10CFU / mL, indicating that the combination of the two has excellent synergistic antibacterial effect.

[0121] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of Trichostatin A as a metallo-β-lactamase inhibitor in the preparation of a drug for reversing bacterial resistance, or use of Trichostatin A as a metallo-β-lactamase inhibitor combined with β-lactam antibiotics in the preparation of a drug against resistant bacteria, wherein the bacteria are resistant bacteria expressing metallo-β-lactamase.

2. The use according to claim 1, characterized in that: The drug-resistant bacteria expressing metal β-lactamase are drug-resistant Gram-negative bacteria expressing metal β-lactamase; preferably, the drug-resistant bacteria expressing metal β-lactamase are drug-resistant Gram-negative brevicaurus expressing metal β-lactamase; more preferably, the drug-resistant bacteria expressing metal β-lactamase are drug-resistant Escherichia coli expressing metal β-lactamase.

3. The use according to claim 1, characterized in that: The metallo-β-lactamase is of NDM-1 or VIM-2 type.

4. The use according to claim 1, characterized in that: The β-lactam antibiotic is a carbapenem antibiotic.

5. The use according to claim 4, characterized in that: The carbapenem antibiotic is meropenem.

6. A combined drug for combating drug-resistant bacteria, characterized in that: Its active ingredients include Trichostatin A and beta-lactam antibiotics, wherein the Trichostatin A and beta-lactam antibiotics respectively form independent dosing units, or the Trichostatin A and beta-lactam antibiotics together form a combined dosing unit, and the bacteria are drug-resistant bacteria expressing metal beta-lactamase.

7. The combined drug according to claim 6, characterized in that: The drug-resistant bacteria expressing metal β-lactamase are drug-resistant Gram-negative bacteria expressing metal β-lactamase; preferably, the drug-resistant bacteria expressing metal β-lactamase are drug-resistant Gram-negative brevicaurus expressing metal β-lactamase; more preferably, the drug-resistant bacteria expressing metal β-lactamase are drug-resistant Escherichia coli expressing metal β-lactamase.

8. The combined drug according to claim 6, characterized in that: The metallo-β-lactamase is of NDM-1 or VIM-2 type.

9. The combined drug according to any one of claims 6 to 8, characterized in that: The β-lactam antibiotic is a carbapenem antibiotic.

10. The use according to claim 9, characterized in that: The carbapenem antibiotic is meropenem.

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