Antibiotic preparation containing glucose and application thereof

Abstract

The invention relates to a preparation for resisting persistent bacteria. The preparation comprises glucose and cephalosporin antibiotics. The invention provides a small molecule metabolite capable of improving the capability of the cephalosporin antibiotics to remove the persistent bacteria, the killing effect of the cephalosporin antibiotics on the persistent bacteria is improved, so that the problem of drug resistance of aeromonas veronii is solved, and the small molecule metabolite can be used as a potential auxiliary drug. Compared with the existing method which only uses antibiotics as antibacterial drugs, the method has better effect, higher safety and operability and better application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbiology, in particular to an antibiotic preparation containing glucose and the use thereof. BACKGROUND

[0002] Pathogenic bacteria seriously endanger human health and the sustainable development of the breeding industry, and effective measures need to be taken for prevention and control. Since the discovery of antibiotics, they have played a major role in the treatment of bacterial infectious diseases, which has led to the increasing use of antibiotics. Although the use of antibiotics can effectively kill bacteria, the misuse and abuse of antibiotics can lead to bacterial resistance. Bacteria with drug resistance reduce or lose the effectiveness of otherwise effective antibiotic therapy, leading to treatment failure and chronic, stubborn infections.

[0003] Aeromonas veronii is a zoonotic pathogenic bacterium that can infect humans and animals and is found in livestock meat, aquatic products, vegetables and water bodies. It can infect mammals, including humans, causing gastrointestinal inflammation, peritonitis, meningitis, septicemia and trauma infection, and poses a serious threat to human health. When treating Aeromonas veronii infection with antibiotics, it was found that it was naturally resistant to many antibiotics, such as sulfonamide potentiator trimethoprim and aminoglycoside streptomycin, but was more sensitive to most β-lactam antibiotics, such as cephalothin. Its drug resistance mechanism is complex and evolves rapidly. If a method is not found to enhance the killing effect of antibiotics on persister bacteria, the currently effective β-lactam antibiotics may gradually lose their effectiveness due to the large number of persister bacteria, leading to further evolution of drug resistance.

[0004] Currently, the clinical approach to combating bacterial drug resistance mutations is to replace antibiotics, but there is still a lack of effective means to fundamentally solve antibiotic-resistant persister bacteria. Therefore, it is particularly urgent to develop a new strategy for clearing persister bacteria, which is of great significance to the clinic and the breeding industry. SUMMARY

[0005] To solve the technical problems in the prior art, the present application provides a preparation for combating pathogenic persister bacteria, comprising glucose and a cephalosporin antibiotic.

[0006] The preparation described above, wherein the cephalosporin antibiotic is cephalothin, cefoxitin or cefalexin.

[0007] The preparation described above, wherein the content of glucose is 5-15 mM; preferably, the content of glucose is 10 mM.

[0008] The preparation described above, wherein the use concentration of the cephalosporin antibiotic is not less than 0.2 times the minimum inhibitory concentration; preferably, the use concentration of the cephalosporin antibiotic is not less than 0.25 times the minimum inhibitory concentration.

[0009] The preparation as described above, wherein the pathogenic persistent bacteria is Aerococcus viridans.

[0010] The preparation as described above, wherein the pathogenic persistent bacteria is Aerococcus viridans that is resistant to at least 50 times the minimum inhibitory concentration of a cephalosporin antibiotic; preferably, the pathogenic persistent bacteria is Aerococcus viridans that is resistant to at least 50 times the minimum inhibitory concentration of cephalothin.

[0011] Use of glucose in the preparation of a preparation for increasing the activity of a cephalosporin antibiotic in killing persistent bacteria.

[0012] A method for increasing the activity of a cephalosporin antibiotic in killing persistent bacteria, comprising: using a cephalosporin antibiotic in combination with glucose.

[0013] A method for increasing the sensitivity of persistent bacteria to a cephalosporin antibiotic, comprising: administering a cephalosporin antibiotic and glucose to a carrier containing the persistent bacteria.

[0014] An activity enhancer of a cephalosporin antibiotic comprising glucose, the enhancer being configured to increase the sensitivity of Aerococcus viridans to a cephalosporin antibiotic; preferably, the enhancer is configured to increase the sensitivity of Aerococcus viridans that is resistant to a cephalosporin antibiotic to the cephalosporin antibiotic.

[0015] The present application provides a small molecule metabolite for increasing the activity of a cephalosporin antibiotic in killing persistent bacteria, and increasing the killing effect of the cephalosporin antibiotic on the persistent bacteria, thereby overcoming the problem of drug resistance of Aerococcus viridans, and can be used as a potential adjuvant drug. The use of glucose in combination with an antibiotic can significantly improve the bactericidal effect of the antibiotic, and compared with the existing use of only an antibiotic as an antibacterial drug, has better effect, higher safety and operability, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0016] Hereinafter, preferred embodiments of the present application will be described in further detail with reference to the accompanying drawings, in which:

[0017] Figure 1 is the survival condition of the persistent bacteria after 2 hours of treatment with different antibiotics in the presence of added glucose according to an embodiment of the present application; and

[0018] Figure 2 is the metabolic activity of the persistent bacteria after 2 hours of treatment with different antibiotics in the presence of added glucose according to an embodiment of the present application. DETAILED DESCRIPTION

[0019] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present application with reference to the drawings accompanying the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0020] In the following detailed description, reference will be made to the accompanying drawings, which form a part of this description, illustrating certain embodiments of the present application. In the drawings, like numerals describe similar components throughout the several views of the drawings. Various embodiments of the present application are described in sufficient detail to enable those of ordinary skill in the art to make and use the technical solutions of the present application. It is to be understood that other embodiments can be utilized and that changes can be made without departing from the scope of the present application.

[0021] The experimental methods used herein are conventional methods in the art unless otherwise specified; the ingredients or materials used are commercially available unless otherwise specified.

[0022] The current focus of research on persister bacteria is to improve the efficiency of antibiotic killing of drug-resistant bacteria or their sensitivity to antibiotics by adding metabolites, so that the originally ineffective antibiotics become effective, and the formed persister bacteria are further killed. In recent years, it has been found that the exogenous addition of some small molecule metabolites can synergize with antibiotics to promote the bactericidal effect, and can be prepared into a compound preparation with antibiotics, which has important significance for controlling bacteria, especially repeated infections of drug-resistant bacteria.

[0023] Glucose is a monosaccharide widely distributed in nature and has a wide range of uses. For most microorganisms, glucose is an ideal carbon source and energy source. In the cultivation process of bacteria, glucose can be decomposed by bacteria to produce energy, and at the same time provide carbon skeleton for the growth and reproduction of bacteria. In addition, some products of glucose metabolism can participate in the synthesis of hormones. For example, in the synthesis process of steroid hormones, acetyl coenzyme A produced by glucose decomposition is the raw material for the synthesis of cholesterol. And the signal molecules produced in the process of glucose metabolism can participate in the signal transduction process in cells. Thus, the metabolic activity, gene expression, etc. of cells are regulated. However, there is no relevant report on whether exogenous addition of glucose can improve the clearance of persister bacteria in cephalosporin antibiotics against Veillonella dispar.

[0024] In order to make the technical solutions of the present application clearer, some terms appearing in the present application have the following explanations:

[0025] The pathogenic bacteria as used in the present application refer to bacteria that can cause diseases in organisms in nature, especially in humans, which include gram-negative bacteria and gram-positive bacteria. Different pathogenic bacteria have different resistance to the same antibiotic; even the same pathogenic bacteria have different resistance to different antibiotics. Those skilled in the art should understand that bacteria resistant to other drugs can be killed by other different bacteria. For example, pseudomonas aeruginosa resistant to colistin can be resistant to some types of cephalosporin antibiotics. In the present application, the pathogenic bacteria are aeromonas veronii.

[0026] The persister as used in the present application is a phenotypic variant with transient resistance to antibiotics and plays a major role in the development of chronic infection and antibiotic resistance. When antibiotics are used to treat bacterial infections, the persister form is an important way for bacteria to cope with antibiotic killing. Persister bacteria can tolerate lethal concentrations of antibiotics when they are in a state of temporary dormancy or slow growth inside tissue cells and in biofilms; when the concentration of antibacterial drugs decreases or the body's immunity is low, the persister bacteria will recover from dormancy and cause a recurrence of infection.

[0027] The OD value as used in the present application is the abbreviation of optical density, which represents the optical density of the detected object. The optical density of the bacterial culture solution at 600 nm (indicated by OD 600 ) is measured, the concentration of the bacterial culture solution can be measured, and the growth of the bacteria can be estimated, so the optical density at 600 nm can be used to represent the cell density of the bacteria, wherein the optical density is proportional to the concentration of the bacteria in the culture solution.

[0028] The MIC as used in the present application is the abbreviation of Minimum Inhibitory Concentration, which refers to the minimum drug concentration that can inhibit the growth of pathogenic bacteria in the culture medium after in vitro culture of bacteria for 18 to 24 hours. It is usually determined by observing the growth of bacteria in the culture medium containing gradient concentration of drugs after 18 to 24 hours, and is an index for measuring the antibacterial activity of antibacterial drugs.

[0029] The MBC as used in the present application is the abbreviation of Minimum Bactericidal Concentration, which is a core index for measuring the ability of antibacterial drugs to completely kill microorganisms. It is defined as the minimum drug concentration required to kill 99.9% of test microorganisms. Its determination needs to be carried out after the minimum inhibitory concentration (MIC) is determined, and the lowest concentration of sterile growth is observed by gradient dilution culture method

[0030] The sub-MIC (sub-inhibitory concentration) as described herein refers to an antibacterial drug concentration lower than the minimum inhibitory concentration (MIC), only 1 / 4 to 1 / 230 (different for drugs and strains) of the MIC.

[0031] The M9 medium as described herein is a minimum medium with a clear chemical composition. The M9 medium is a basic synthetic medium for culturing bacteria in microbiology, with mineral salts and glucose as the main components, and is particularly suitable for experiments that require precise control of nutritional components.

[0032] The PBS buffer as described herein is a phosphate buffered saline solution, which is a solution simulating the in vivo environment of an organism. The PBS buffer has salt balance and adjustable pH buffering effect, and is mainly used to maintain the stability of the liquid acidity and alkalinity during experimental operation, and to create a stable experimental environment. The PBS buffer is a commonly used reagent in the art, and the composition, method of obtaining, etc. are not limited.

[0033] Glucose, as a small molecule metabolic substance for improving antibiotic clearance of persister bacteria, can control bacterial, especially drug-resistant bacterial infection. The present application found that the bactericidal effect of cephalothin on Veillonella dispar was significantly improved after adding glucose. The results showed that glucose can enhance the sensitivity of Veillonella dispar to cephalothin. As a control group without the addition of glucose, the sensitivity to cephalothin did not significantly improve.

[0034] The present application found that the survival rate of persister bacteria was significantly decreased when treated with other antibiotics (such as cefoxitin and cefalexin) after the addition of glucose. These results showed that the sensitivity of Veillonella dispar to cephalosporin antibiotics was improved to different degrees after the addition of glucose, indicating that this effect can be applied to cephalosporin antibiotics.

[0035] Therefore, the present application provides a preparation for resisting pathogenic persister bacteria, comprising glucose and a cephalosporin antibiotic.

[0036] In some embodiments, the cephalosporin antibiotic is selected from the group consisting of cephalothin, cefazolin, cefuroxime, cephapirin, cephradine, cefalonium, cefaclom, cefamandole, cefonicid, cefuroxime, cefamandole, cefotiam, cefonicid, ceforanide, ceftazidime, cefoperazone, cefpiramide, cefmenoxime, cefsulodin, cefixime, cefteram, cefetamet, ceftibuten, cefdinir, cefpodoxime, cefditoren, cefpirome, cefepime, ceftaroline, cefepime.

[0037] The skilled in the art should understand that there are various clinical uses or new categories of cephalosporin antibiotics, which cannot be listed one by one in the present application, and it is more difficult for the skilled in the art to verify the technical effect of each cephalosporin antibiotic combined with glucose through experiments. However, as a large category of antibiotics, cephalosporin antibiotics are similar in structure and mechanism of action: they are all β-lactam antibiotics, which play a bactericidal role by inhibiting bacterial cell wall synthesis. The present application selects three of them for verification to illustrate the gain effect of cephalosporin antibiotics when used in combination with glucose.

[0038] In some embodiments, the content of glucose in the preparation is 5-15 mM; preferably, the content of glucose is 10 mM. In some embodiments, the use concentration of cephalosporin antibiotic in the preparation is not less than 0.2 times the minimum inhibitory concentration; preferably, the use concentration of cephalosporin antibiotic is not less than 0.25 times the minimum inhibitory concentration; more preferably, the use concentration of cephalosporin antibiotic is not less than 0.25 times the minimum inhibitory concentration.

[0039] In some embodiments, the pathogenic retainer referred to in the present application is A. wautersii. In some embodiments, the retainer is A. wautersii which can tolerate at least 50 times the minimum inhibitory concentration of cephalosporin antibiotic. Thus, the retainer can also be referred to as a drug-resistant bacterium.

[0040] Thus, the present application proposes the use of glucose in the preparation of a preparation for improving the activity of cephalosporin antibiotic in killing retainers.

[0041] At the same time, a method for improving the activity of cephalosporin antibiotic in killing retainers is proposed, which comprises: using cephalosporin antibiotic in combination with glucose.

[0042] Further, a method for improving the sensitivity of retainers to cephalosporin antibiotic is proposed, which comprises: applying cephalosporin antibiotic and glucose to a carrier containing retainers.

[0043] Therefore, according to one embodiment of the present application, glucose can be used to prepare an activity enhancer of cephalosporin antibiotic, which is configured to improve the sensitivity of A. wautersii to cephalosporin antibiotic; preferably, the enhancer is configured to improve the sensitivity of A. wautersii resistant to cephalosporin antibiotic to cephalosporin antibiotic.

[0044] Thus, the addition of glucose to cephalosporin antibiotic can significantly improve the sensitivity of drug-resistant bacteria to it, thereby achieving the purpose of further killing retainers and providing a brand-new technical method for the treatment of drug-resistant bacteria.

[0045] The technical solutions of the present application will be illustrated below through the following specific embodiments. The skilled in the art should understand that the following embodiments are only for illustrating the technical solutions of the present application, but not limiting the technical solutions of the present application.

[0046] Example 1 Determination of MIC / MBC values of A. wautsii

[0047] The MIC / MBC of A. wautsii to different antibiotics was determined by micro broth dilution method. The antibiotics used in this application were cephalosporins, such as cephalothin, cefoxitin and cefalexin, respectively.

[0048] Step 1, preparation of antibiotic stock solution. The concentration of the antibiotic stock solution was 1280 μg / mL. The antibiotic was directly purchased from the manufacturer or relevant institution. The required amount of antibiotic solution or powder can be calculated by formula. For example: 100 mL of antibiotic stock solution with a concentration of 1280 μg / mL is required, and the antibiotic used is a powder with a molecular weight of 396. According to the formula, the amount of antibiotic powder required is: (1280 μg / ml x 100 ml x 10 -3 ) / 396 = 0.347 mg, then 0.347 mg of antibiotic is accurately weighed on an analytical balance and dissolved in 100 mL of diluent. The prepared antibiotic stock solution should be stored in an environment below -60°C, and the storage period should not exceed 6 months.

[0049] Step 2, culture of bacteria and preparation of samples. A. wautsii C4 stored at -80°C in the laboratory was streaked on LB solid medium plates and incubated in a 30°C constant temperature incubator for 24 hours; a single colony was picked from the plate and inoculated in 5 mL of LB medium, incubated at 30°C, 150 rpm to the stationary phase; an appropriate amount of saturated bacteria was collected and centrifuged at 8000 rpm for 2 min. Then the bacteria were washed with sterile PBS buffer for 3 times; the washed bacteria were adjusted to OD 600 value of 0.01 with LB medium, then 5 mL of bacterial solution was aliquoted in test tubes for standby.

[0050] Step 3, preparation of MIC plates and inoculation of bacterial solution. Under sterile conditions, the antibiotic to be tested was diluted by two-fold dilution method. Taking cephalothin as an example, 5 μL of Cep was gradually diluted at concentrations of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625 μg / mL and added to a sterile 96-well polystyrene plate, the 12th well was not added with drug as a growth control, three parallel. Then 95 μL of the prepared bacterial solution was added to each well, sealed and placed in a 30°C constant temperature incubator for incubation, and the results were judged after 16-20 h.

[0051] Step 4, determination of MIC values. The OD 600 value of the 96-well plate was determined by a microplate reader. OD 600The antibiotic concentration corresponding to the value that is stable or changes little is the minimum inhibitory concentration (MIC) of the drug to the bacteria. The test is meaningful only when the bacteria in the positive control well (i.e., without the antibiotic) grow obviously. When a single jump well appears in the micro-broth dilution method, the highest drug concentration that inhibits the growth of the bacteria should be recorded. If multiple jump wells appear, the result should not be reported and the test should be repeated.

[0052] Step 5, plate coating. Take 100 μL of the bacterial solution determined as the minimum inhibitory concentration with no growth phenomenon in a 1.5 mL centrifuge tube, centrifuge at 8000 rpm for 2 min, discard the residual culture medium and collect the bacterial bodies, resuspend the bacterial bodies with 1 mL of PBS, take 50 μL of the resuspended solution and uniformly coat on an antibiotic-free LB agar plate, and invert culture for 24 h.

[0053] Step 6, MBC value result judgment. After the culture is completed, the number of surviving bacteria is analyzed. The minimum bactericidal concentration (MBC) is the lowest antibiotic concentration at which the bactericidal rate is ≥99.99%. The specific experimental results are shown in Table 1.

[0054] Table 1 MBC / MIC measurement results of Veillonella dispar under different antibiotics

[0055]

[0056]

[0057] Example 2, Glucose can improve the killing ability of cephalosporin antibiotics on persister bacteria

[0058] In order to understand whether the addition of glucose can improve the killing ability of cephalosporin antibiotics on persister bacteria, the test is divided into 6 groups: 3 control groups (only adding the corresponding antibiotic) and 3 experimental groups (adding different antibiotics and 10 mM glucose, respectively). The added antibiotics and the acting concentrations are as follows: 0.125 μg / mL cephalothin, 0.5 μg / mL cefoxitin and 4 μg / mL cefalexin.

[0059] Step 1, culture of bacteria and preparation of samples. Veillonella dispar C4 single colony was picked from an LB plate and inoculated into 5 mL of LB medium, which was cultured at 30°C and 150 rpm for overnight to the stationary phase. The bacterial solution was collected by centrifugation at 8000 rpm for 2 min, the supernatant was removed and the bacterial bodies were washed with sterile PBS buffer, and finally the bacterial bodies were suspended with LB liquid medium, the OD value of the bacterial solution was adjusted to 0.2, and then 5 mL was aliquoted in a test tube for standby. 600

[0060] Step 2, preparation of persister bacteria. 50xMIC cephalothin was added to the above test tube to treat the bacterial solution for 6 h, which was used to kill the non-persister bacteria in the stationary phase. The bacterial bodies were collected by centrifugation and washed twice with PBS buffer.​

[0061] Step 3, the bacteria were resuspended in M9 medium containing 10 mM glucose (to provide 60 mM of carbon) and different antibiotics (cephalothin, cefoxitin and cefalexin) and incubated at 30 °C. After about 2 hours, 1 mL of bacterial culture was collected, gradient diluted in PBS, and 100 μL of the bacterial solution was spread on LB agar to plate and detect the viable bacteria number, and then the survival rate of persister bacteria under different antibiotic concentrations was calculated. The formula was: survival rate (%) = (the number of persister bacteria at a certain time point after adding glucose / the number of persister bacteria at a certain time point without adding glucose) x 100%. The results are shown in Table 1. Figure 1 Figure 1 It can be seen that, compared with the control of adding only antibiotics, the addition of glucose can induce rapid killing of persister bacteria by antibiotics, and the survival rate of persister bacteria is reduced by about 90%. The stimulation of glucose can significantly enhance the bactericidal effect of cephalosporin antibiotics on persister bacteria.

[0062] Example 3, metabolic activity of persister bacteria after glucose-antibiotic combination treatment

[0063] In order to further verify that the addition of glucose improves the killing ability of cephalosporin antibiotics on persister bacteria, the present application uses WST-1 cell proliferation and cytotoxicity assay kit (catalog number C0036; Beyotime) to evaluate the number of active bacteria of Aeromonas veronii C4 after glucose-antibiotic combination treatment.

[0064] The persister bacteria sample was prepared according to the method of steps 1 and 2 of Example 2.

[0065] Step 1, the persister bacteria treated with 10 mM glucose and different antibiotics for about 2 hours in Example 2 step 3 were collected and the bacterial cells were washed with PBS buffer for 2-3 times to remove glucose and antibiotics.

[0066] Step 2, the bacterial cells were resuspended in 1 mL of LB medium, and then inoculated into a 96-well plate at a bacterial density of 2 x 10 7 CFU / mL per well.

[0067] Step 3, 10% WST-1 was added to each well, and incubated at 30 °C for 5 h. The absorbance at 450 nm wavelength was detected by a microplate reader.

[0068] The results are shown in Table 2. Figure 2 Figure 2 It can be seen that, after the addition of glucose, the number of viable bacteria is significantly reduced, and the metabolic activity of persister bacteria is significantly reduced by about 80%, indicating that the addition of glucose can further kill persister bacteria by cephalosporin antibiotics.

[0069] ​​Based on the description of Examples 2 and 3, the present application demonstrates that stimulation of glucose can significantly enhance the killing effect of cephalosporin antibiotics on A. veronii persisters.

[0070] Finally, it should be noted that the above detailed description of the embodiments of the present application is only a preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. A preparation for combating pathogenic persister bacteria, comprising glucose and cephalosporin antibiotics.

2. The preparation according to claim 1, wherein the cephalosporin antibiotic is cephalothin, cefoxitin or cephalexin.

3. The preparation according to claim 1, wherein the content of glucose is 5-15 mM; preferably, the content of glucose is 10 mM.

4. The preparation according to claim 1, wherein the concentration of the cephalosporin antibiotic is not less than 0.2 times the minimum inhibitory concentration; preferably, the concentration of the cephalosporin antibiotic is not less than 0.25 times the minimum inhibitory concentration. The preparation according to claim 1 , wherein the pathogenic persister is Aeromonas welchii persister.

6. The preparation according to claim 5, wherein the pathogenic persister is Aeromonas welchii that can tolerate at least 50 times the minimum inhibitory concentration of cephalosporin antibiotics; preferably, the pathogenic persister is Aeromonas welchii that can tolerate at least 50 times the minimum inhibitory concentration of cephalothin.

7. The use of glucose in the preparation of preparations for improving the activity of cephalosporin antibiotics in killing pathogenic bacteria.

8. A method for improving the activity of cephalosporin antibiotics in killing pathogenic bacteria, comprising: Combine cephalosporin antibiotics with glucose.

9. A method for increasing the sensitivity of pathogenic bacteria to cephalosporin antibiotics, comprising: Cephalosporin antibiotics and glucose are administered to a vector containing the pathogenic bacteria.

10. An activity enhancer for cephalosporin antibiotics comprising glucose, wherein the enhancer is configured to increase the sensitivity of Aeromonas vermiformis to cephalosporin antibiotics; preferably, the enhancer is configured to increase the sensitivity of Aeromonas vermiformis that is resistant to cephalosporin antibiotics to cephalosporin antibiotics.

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