Antibiotic preparation containing succinic acid and application thereof

Abstract

The invention relates to a preparation for resisting persistent bacteria. The preparation comprises succinic acid 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 invention relates to the field of biotechnology, in particular to an antibiotic preparation containing succinic acid and application thereof. Background Art

[0002] Cephalosporins are a class of β-lactam antibiotics that exert their bactericidal effects by inhibiting bacterial cell wall synthesis and possess broad-spectrum antimicrobial activity. However, bacterial β-lactamases (such as ESBLs) can hydrolyze cephalosporins, leading to the proliferation of drug-resistant bacteria when used indiscriminately. These resistant bacteria can reduce or even eliminate the effectiveness of previously effective antibiotic treatments, leading to treatment failure and persistent recurrence of chronic, recalcitrant infections.

[0003] Aeromonas veronii is a pathogen common to humans, livestock, and aquatic organisms. It is found in livestock and poultry meat, aquatic products, vegetables, and water bodies. It can infect mammals, including humans, causing gastroenteritis, peritonitis, meningitis, sepsis, and traumatic infections, posing a serious threat to human health. During antibiotic treatment of A. veronii infections, it has been found to exhibit natural resistance to multiple antibiotics (such as the sulfonamide potentiator trimethoprim and the aminoglycoside streptomycin) but is relatively sensitive to most β-lactam antibiotics (such as cephalothin). Its resistance mechanisms are complex and evolve rapidly, necessitating the development of methods to enhance antibiotic efficacy against persister bacteria. Summary of the Invention

[0004] In view of the technical problems existing in the prior art, the present invention proposes a preparation for combating pathogenic persister bacteria, comprising succinic acid and cephalosporin antibiotics.

[0005] In the preparation as described above, the cephalosporin antibiotic is cephalothin, cefoxitin or cephalexin.

[0006] In the above-mentioned preparation, the content of succinic acid is 10-20 mM; preferably, the content of succinic acid is 15 mM.

[0007] In the preparation as described above, the concentration of the cephalosporin antibiotic used is not less than 0.2 times the minimum inhibitory concentration; preferably, the concentration of the cephalosporin antibiotic used is not less than 0.25 times the minimum inhibitory concentration.

[0008] In the preparation as described above, the pathogenic persister bacteria are Aeromonas welchii persisters.

[0009] In the preparation as described above, 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.

[0010] The present application relates to the use of succinic acid in the preparation of a preparation for improving the activity of cephalosporin antibiotics in killing pathogenic bacteria.

[0011] The present application further relates to a method for improving the activity of cephalosporin antibiotics in killing pathogenic bacteria, comprising: using the cephalosporin antibiotics in combination with succinic acid.

[0012] The present application also relates to a method for increasing the sensitivity of pathogenic bacteria to cephalosporin antibiotics, comprising: administering cephalosporin antibiotics and succinic acid to a carrier containing the pathogenic bacteria.

[0013] The present application also relates to an activity enhancer for cephalosporin antibiotics containing succinic acid, wherein the enhancer is configured to increase the sensitivity of Aeromonas welchii to cephalosporin antibiotics; preferably, the enhancer is configured to increase the sensitivity of Aeromonas welchii resistant to cephalosporin antibiotics to cephalosporin antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Below, the preferred embodiments of the present invention will be further described in detail with reference to the accompanying drawings, in which:

[0015] Figure 1 is the survival of persister bacteria after 2 hours of treatment with different antibiotics when succinic acid is added according to one embodiment of the present invention; and

[0016] Figure 2 FIG1 is the metabolic activity of persister bacteria after treatment with different antibiotics for 2 hours with the addition of succinic acid according to one embodiment of the present invention. DETAILED DESCRIPTION

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0018] In the detailed description that follows, reference is made to the accompanying drawings that form part of this application and illustrate specific embodiments of the present application. In the accompanying drawings, similar reference numerals describe substantially similar components in different figures. Each specific embodiment of the present application is described below in sufficient detail to enable a person of ordinary skill in the art to implement the technical solutions of the present application. It should be understood that other embodiments may also be utilized or that the embodiments of the present application may be modified.

[0019] As pathogenic bacteria become increasingly resistant to antibiotics, this application aims to enhance the efficiency of antibiotics in killing resistant bacteria or their sensitivity to antibiotics by adding succinate metabolites, thereby rendering previously ineffective antibiotics effective and further killing established persisters. In recent years, it has been discovered that exogenously added small molecule metabolites can synergize with antibiotics to promote bactericidal effects. These metabolites can be prepared together with antibiotics into compound preparations, which are of great significance for controlling recurrent bacterial infections, particularly those caused by drug-resistant bacteria.

[0020] To more clearly illustrate the technical solution of this application, some terms appearing in this application have the following meanings:

[0021] Succinic acid, also known as butanedioic acid, is a dicarboxylic acid and an intermediate metabolite of the tricarboxylic acid cycle (TCA), a central metabolic process in microbial cells. It is also a metabolic end product of many facultative and strict anaerobes. Many microorganisms can produce succinic acid through optimization and metabolic engineering. In medicine, succinic acid has antispasmodic, expectorant, and diuretic properties. It is widely used in detergents, surfactants, food additives, antimicrobials, and the pharmaceutical industry, and is used to synthesize a variety of important chemicals. Notably, studies have shown that succinic acid has antimicrobial properties. At a concentration of 2 mg / ml, succinic acid has inhibitory effects against Staphylococcus aureus, Coccus catarrhalis, and Pseudomonas aeruginosa, Proteus, and Shigella dysenteriae. However, there are no reports on whether exogenous addition of succinic acid can enhance the ability of cephalosporin antibiotics to eliminate persisters in Aeromonas veseri.

[0022] The pathogenic bacteria referred to in this application refer to bacteria that can cause disease in organisms in nature, especially humans, and include Gram-negative bacteria and Gram-positive bacteria. Different pathogens have different tolerances to the same antibiotic; even the same pathogens have different tolerances to different antibiotics. Those skilled in the art should understand that bacteria that are tolerant to other drugs may be killed by other different types of bacteria. For example, Pseudomonas aeruginosa that is resistant to colistin may be intolerant to certain types of cephalosporin antibiotics. In this application, the pathogenic bacteria is Aeromonas vermiformis.

[0023] Persisters, as described herein, are phenotypic variants that exhibit transient resistance to antibiotics and play a major role in the development of chronic infections and antibiotic resistance. When antibiotics are used to treat bacterial infections, persisters are an important way for bacteria to resist antibiotic damage. Persisters, while temporarily dormant or slowly growing within tissue cells and biofilms, can tolerate lethal concentrations of antibiotics. When antibiotic concentrations decrease or the immune system weakens, persisters can resuscitate from dormancy, causing recurrence of infection.

[0024] The OD value used in this application stands for optical density, which indicates the light density absorbed by the substance being tested. Measuring the absorbance of a bacterial culture at 600 nm (expressed as OD600) can be used to measure the concentration of the culture and, therefore, estimate bacterial growth. Therefore, the optical density at 600 nm can be used to indicate bacterial cell density, as the absorbance is proportional to the bacterial concentration in the culture.

[0025] The MIC mentioned in this application is the abbreviation of Minimum Inhibitory Concentration, which refers to the lowest drug concentration that can inhibit the growth of pathogens in the culture medium after 18 to 24 hours of in vitro bacterial culture. It is usually determined by observing the growth of bacteria in a culture medium containing gradient drug concentrations after 18 to 24 hours. It is an indicator for measuring the antibacterial activity of antibacterial drugs.

[0026] The MBC mentioned in this application is the abbreviation of Minimum Bactericidal Concentration (MBC), which is the core indicator for measuring the ability of antimicrobial drugs to completely kill microorganisms. It is defined as the lowest drug concentration required to kill 99.9% of the test microorganisms. Its determination should be carried out after determining the minimum inhibitory concentration (MIC). The lowest concentration of sterile growth is observed by gradient dilution culture method.

[0027] The sub-inhibitory concentration (sub-MIC) described in this application refers to an antibacterial drug concentration lower than the minimum inhibitory concentration (MIC), which is only 1 / 4 to 1 / 230 of the MIC (depending on the drug and strain).

[0028] The M9 medium described in this application is a minimal medium with a well-defined chemical composition. M9 medium is a basic synthetic medium used in microbiology to cultivate bacteria. It is composed mainly of mineral salts and glucose and is particularly suitable for experiments that require precise control of nutrient composition.

[0029] The PBS buffer described herein is a phosphate-buffered saline solution, a solution that simulates the internal environment of a living organism. PBS buffer provides salt balance and an adjustable pH buffering effect, primarily used to maintain the stability of liquid pH during experimental procedures and create a stable experimental environment. PBS buffer is a commonly used reagent in this field, and this application does not limit its composition or method of obtaining it.

[0030] The present application relates to a preparation for combating pathogenic persister bacteria, comprising succinic acid and cephalosporin antibiotics.

[0031] In some embodiments, the cephalosporin antibiotic in the formulation is preferably cephalothin, cefoxitin, or cephalexin. The cephalosporin antibiotic is used at a concentration of no less than 0.2 times the minimum inhibitory concentration; preferably, the cephalosporin antibiotic is used at a concentration of no less than 0.25 times the minimum inhibitory concentration. In some embodiments, the succinic acid content in the formulation is 10-20 mM; preferably, the succinic acid content is 15 mM.

[0032] In some embodiments, the pathogenic persister is an Aeromonas vernix persister. In some embodiments, the pathogenic persister is an Aeromonas vernix that can tolerate at least 50 times the minimum inhibitory concentration of a cephalosporin antibiotic; preferably, the pathogenic persister is an Aeromonas vernix that can tolerate at least 50 times the minimum inhibitory concentration of cephalothin.

[0033] Therefore, the present application proposes the use of succinic acid in the preparation of a preparation for improving the activity of cephalosporin antibiotics in killing pathogenic bacteria.

[0034] This application further relates to a method for increasing the pathogenic bacterial activity of a cephalosporin antibiotic, comprising: administering a cephalosporin antibiotic in combination with succinic acid. The concentrations of the cephalosporin antibiotic and succinic acid are as described above. This method, which can increase the sensitivity of pathogenic bacteria to cephalosporin antibiotics, comprises: administering a cephalosporin antibiotic and succinic acid to a carrier containing the pathogenic bacteria.

[0035] An activity enhancer for cephalosporin antibiotics comprising succinic acid, 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 resistant to cephalosporin antibiotics to cephalosporin antibiotics.

[0036] The present invention found that after adding succinic acid, the bactericidal effect of cephalothin and the like on Aeromonas vermiformis was significantly improved. The results of the examples of the present application showed that succinic acid can enhance the sensitivity of Aeromonas vermiformis to cephalothin. After adding succinic acid, the survival rate of the persisters was significantly reduced when treated with other antibiotics (such as cefoxitin and cephalexin). These results show that the addition of succinic acid increases the sensitivity of Aeromonas vermiformis to cephalosporin antibiotics to varying degrees, indicating that this effect can be applied to cephalosporin antibiotics.

[0037] Therefore, adding succinic acid to cephalosporin antibiotics can significantly increase the sensitivity of drug-resistant bacteria to them, thereby further killing persister bacteria and providing a new technical method for the treatment of drug-resistant bacteria. Furthermore, the antibiotics used in this application to verify the function of succinic acid are cephalosporin antibiotics, such as cephalothin, cefoxitin, and cefalexin.

[0038] Example 1 Determination of MIC / MBC Values ​​of Aeromonas vernix

[0039] The MIC / MBC of different antibiotics against Aeromonas vermiformis was determined by the microbroth dilution method. The antibiotics used in this application are cephalosporin antibiotics, such as cephalothin, cefoxitin, and cefalexin.

[0040] Step 1: Preparation of antibiotic stock solution. The concentration of the antibiotic stock solution was 1280 μg / mL. Antibiotics were purchased directly from manufacturers or relevant institutions.

[0041] Step 2, bacterial culture and sample preparation. Streak Aeromonas vickers C4 stored at -80℃ in the laboratory on an LB solid culture medium plate and place it in a 30℃ constant temperature incubator for 24 hours; pick a single colony from the plate and inoculate it into 5mL LB culture medium, and culture it at 30℃ and 150rpm until the stable period; collect an appropriate amount of saturated bacteria and centrifuge it at 8000rpm for 2min. Then wash the bacteria three times with sterile PBS buffer; adjust the washed bacteria to OD 600 The value is 0.01, and then 5 mL of bacterial solution is dispensed into test tubes for later use.

[0042] Step 3: Prepare the MIC plate and inoculate the bacterial solution. Aseptically dilute the test antibiotic using the two-fold dilution method. For example, for cephalothin, gradually dilute 5 μL of Cep to a concentration gradient of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 μg / mL. Add the diluted solution to a sterile 96-well polystyrene plate, leaving well 12 untreated as a growth control. Repeat the plate three times. Then, add 95 μL of the prepared bacterial solution to each well. Seal the plate and incubate in a 30°C incubator for 16–20 hours. Assess the results.

[0043] Step 4: MIC value determination. Measure the OD value of the 96-well plate using a microplate reader. 600 Value.OD 600 The antibiotic concentration at which the value remains stable or fluctuates little is the minimum inhibitory concentration (MIC) of the drug against the bacteria. The test is only meaningful when there is significant bacterial growth in the positive control well (i.e., without antibiotic). If a single jump well occurs during the broth microdilution test, the highest drug concentration that inhibits bacterial growth should be recorded. If multiple jump wells occur, the result should not be reported and the test should be repeated.

[0044] Step 5: Plate. Transfer 100 μL of each bacterial suspension (those with no growth above the minimum inhibitory concentration) to a 1.5 mL centrifuge tube and centrifuge at 8000 rpm for 2 minutes. Discard the remaining medium and collect the cells. Resuspend in 1 mL of PBS. Spread 50 μL of the suspension evenly on an antibiotic-free LB agar plate and incubate inverted for 24 hours.

[0045] Step 6: Determine the MBC value. After the incubation period, analyze the number of surviving colonies. The minimum bactericidal concentration (MBC) is the lowest antibiotic concentration that achieves a bactericidal rate of ≥99.99%. The specific experimental results are shown in Table 1.

[0046] Table 1 Measurement results of MBC / MIC of Aeromonas verticillata under different antibiotics

[0047] antibiotic MIC (μg / mL) MBC (μg / mL) Cefalotin 0.5 1 Cefoxitin 2 8 Cephalexin 16 32

[0048] Example 2 Succinic acid can enhance the killing ability of cephalosporin antibiotics against persister bacteria

[0049] To determine whether the addition of succinic acid could enhance the ability of cephalosporin antibiotics to kill persister bacteria, six groups were tested: three control groups (adding only the corresponding antibiotic) and three experimental groups (adding different antibiotics and 15 mM succinic acid). The antibiotics added and their effective concentrations were: 0.125 μg / mL cephalothin, 0.5 μg / mL cefoxitin, and 4 μg / mL cephalexin.

[0050] Step 1. Bacterial culture and sample preparation. Pick a single colony of Aeromonas virens C4 from a pre-prepared LB plate and inoculate it into 5 mL of LB medium. Cultivate the culture overnight at 30°C and 150 rpm until the stationary phase. Centrifuge the culture solution, centrifuge at 8000 rpm for 2 minutes, remove the supernatant and wash the cells with sterile PBS buffer. Finally, suspend the cells in LB liquid medium and adjust the OD value of the culture solution. 600 The value was adjusted to 0.2, and then 5 mL was dispensed into test tubes for later use.

[0051] Step 2: Preparation of persister bacteria. Add 50×MIC antibiotic-treated bacteria to the above test tube for 6 hours to kill non-persistent bacteria in the stable phase. Collect the cells by centrifugation and wash twice with PBS buffer.

[0052] Step 3. The bacterial culture was resuspended in M9 medium containing 15 mM succinate (to provide 60 mM carbon) and different antibiotics (cephalothin, cefoxitin, and cephalexin) and incubated at 30°C. After approximately 2 hours, 1 mL of the bacterial culture was collected and serially diluted in PBS. 100 μL of the bacterial solution was plated onto LB agar. The viable count was determined using a plate, and the survival rate of persisters at different antibiotic concentrations was calculated.

[0053] The survival rate calculation formula is: survival rate (%) = (number of persisting bacteria at a certain time point after adding succinic acid / number of persisting bacteria at a certain time point without adding succinic acid) × 100%. Figure 1 .Depend on Figure 1 As can be seen, compared with the control group with only antibiotics, the addition of succinic acid induced the antibiotics to rapidly kill the persister bacteria, reducing the survival rate of the persister bacteria by about 90%. The stimulation of succinic acid can significantly enhance the bactericidal effect of cephalosporins on persister bacteria.

[0054] Example 3 Metabolic activity of persister bacteria after treatment with succinate-antibiotic combination

[0055] To further confirm that the addition of succinic acid improves the killing ability of cephalosporin antibiotics against persister bacteria, the present invention uses a WST-1 cell proliferation and cytotoxicity assay kit (Catalog No. C0036; Beyotime) to evaluate the number of active bacteria of Aeromonas welchii C4 after treatment with the succinic acid-antibiotic combination.

[0056] The persister bacteria sample was prepared according to the method of steps 1-2 of Example 2.

[0057] Step 1: The persister bacteria treated with 15 mM succinic acid and different antibiotic combinations for about 2 hours in Step 3 of Example 2 were collected and washed 2 to 3 times with PBS buffer to remove the succinic acid and antibiotics.

[0058] Step 2: Resuspend the cells in 1 mL of LB medium and plate 2 × 10 7 The bacteria were inoculated into 96-well plates at a density of 800 CFU / mL.

[0059] Step 3: Add 10% WST-1 to each well and incubate at 30°C for 5 h. Detect the absorbance at 450 nm using a microplate reader.

[0060] See the results Figure 2 .Depend on Figure 2 As can be seen, the addition of succinic acid significantly reduced the number of viable bacteria and the metabolic activity of persisters, with a maximum reduction of approximately 60%. This indicates that the addition of succinic acid can indeed further enhance the killing of persisters by cephalosporins. Based on Examples 2 and 3, the present invention demonstrates that succinic acid stimulation can significantly enhance the killing effect of cephalosporins against A. veronii persisters.

[0061] Finally, it should be noted that the above detailed description of the embodiments of the present invention is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention are intended to be included within the scope of protection of the claims.

Claims

1. A preparation for combating pathogenic persister bacteria, comprising succinic acid 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 succinic acid is 10-20 mM; preferably, the content of succinic acid is 15 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. Application of succinic acid 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 succinic acid.

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

10. An activity enhancer for cephalosporin antibiotics comprising succinic acid, 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 cephalosporin antibiotic-resistant Aeromonas vermiformis to cephalosporin antibiotics.

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