Use of cladrabine in the preparation of a medicament for inhibiting the growth of multi-drug resistant klebsiella pneumoniae

Abstract

The application belongs to the technical field of non-antibiotic compounds as bacteriostatic agents, and discloses an application of a known non-antibiotic compound, cladrabine, in inhibiting the growth of multi-drug resistant Klebsiella pneumoniae. The application finds that cladrabine can inhibit the growth of multi-drug resistant Klebsiella pneumoniae, and the minimum bacteriostatic concentration reaches 64 mg / L. Further exploration of the bacteriostatic mechanism finds that after exposure to cladrabine, the permeability of the bacterial cell membrane is enhanced, and the macromolecular substances (nucleic acids and proteins) in the cell are excreted, causing the integrity of the bacterial cell membrane to be destroyed, and further causing the death of Klebsiella pneumoniae. The application proposes that cladrabine can destroy the cell membrane of bacteria, change the permeability of the cell membrane, has a good inhibitory effect on multi-drug resistant Klebsiella pneumoniae, and can be further applied to the preparation of related drugs.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial agent technology, specifically relating to the application of the known drug cladribine in the preparation of drugs that inhibit the growth of multidrug-resistant Klebsiella pneumoniae. Background Technology

[0002] Klebsiella pneumoniae is an important opportunistic Gram-negative bacterium and one of the most common pathogens in hospitals, frequently causing serious hospital-acquired infections (including urinary tract infections, lung infections, and bloodstream infections). Studies show that the clinical detection rate of Klebsiella pneumoniae is second only to Escherichia coli, and it is showing an increasing trend year by year. In recent years, with the overuse and abuse of antibiotics in clinical practice, Klebsiella pneumoniae has become increasingly resistant to antimicrobial agents. Infections caused by multidrug-resistant Klebsiella pneumoniae have become an increasingly serious global problem, posing a considerable challenge to clinical treatment. Therefore, the search for effective inhibitors against multidrug-resistant Klebsiella pneumoniae is urgent and of great significance for the clinical treatment of multidrug-resistant Klebsiella pneumoniae infections.

[0003] Cladobine is a chloride analog of deoxyadenosine and is clinically used to treat multiple sclerosis (MS). There is currently no evidence that cladobine can inhibit the growth of multidrug-resistant Klebsiella pneumoniae, and there is a lack of research on the inhibition of multidrug-resistant Klebsiella pneumoniae by cladobine. Summary of the Invention

[0004] To overcome the aforementioned problems in the prior art, this invention provides the application of cladribine in the preparation of drugs that inhibit the growth of multidrug-resistant Klebsiella pneumoniae. Addressing the widespread clinical prevalence of multidrug-resistant Klebsiella pneumoniae, the aim is to provide the application of cladribine (CAS No.: 4291-63-8; English name: Cladribine) in inhibiting the growth of multidrug-resistant Klebsiella pneumoniae, offering broad prospects for the development of novel antibiotic alternatives and providing a scientific basis for controlling the spread of drug-resistant Klebsiella pneumoniae.

[0005] The present invention achieves the above objectives through the following technical solutions:

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] The application of cladribine in the preparation of drugs to inhibit the growth of multidrug-resistant Klebsiella pneumoniae. Furthermore, the multidrug-resistant Klebsiella pneumoniae is resistant to one or more of the following: meropenem, imipenem, ertapenem, cefotaxime, gentamicin, fosfomycin, ciprofloxacin, tetracycline, tigecycline, or colistin.

[0008] Furthermore, the drug-resistant Klebsiella pneumoniae is a migratory Klebsiella pneumoniae. The drug described in this application is a non-antibiotic antibacterial agent.

[0009] This application utilizes the microbroth dilution method to determine the minimum inhibitory concentration (MIC) of cladribine against Klebsiella pneumoniae. The MIC of cladribine against the migratory bird-derived Klebsiella pneumoniae was determined to be 64 μg / mL, exhibiting extremely strong inhibitory activity.

[0010] Furthermore, the results obtained by ultraviolet spectrophotometry in this application indicate that exposure to cladribine leads to the leakage of intracellular macromolecules (nucleic acids and proteins), resulting in damage to the integrity of the bacterial cell membrane. NPN and PI staining results show that cladribine treatment increases the permeability of the Klebsiella pneumoniae cell membrane, damaging the integrity of the bacterial cell membrane and ultimately causing bacterial death.

[0011] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0012] This invention screens non-antibiotic-resistant bacterial inhibitors from existing libraries of natural small molecule compounds. Based on research on the effects of cladribine on multidrug-resistant Klebsiella pneumoniae, it was found that the non-antibiotic compound cladribine has a good antibacterial effect against multidrug-resistant Klebsiella pneumoniae and can effectively inhibit its growth, providing a new approach for the development of novel antibacterial agents against multidrug-resistant Klebsiella pneumoniae. Furthermore, the inhibitory effect of cladribine on multidrug-resistant Klebsiella pneumoniae was clarified, providing a theoretical basis for effectively curbing the spread and prevalence of multidrug-resistant Klebsiella pneumoniae. Attached Figure Description

[0013] Figure 1 Figure A shows the intracellular protein and nucleic acid leakage of bacteria after treatment with different concentrations of cladribine. Figure B shows the intracellular protein leakage of 700603; Figure C shows the intracellular protein leakage of 21QH43K; Figure D shows the intracellular nucleic acid leakage of 700603; Figure E shows the intracellular nucleic acid leakage of 21QH43K; Figure F shows the intracellular nucleic acid leakage of 21QH35K. (Note: Absorbance at 280 nm represents intracellular protein leakage, and absorbance at 260 nm represents intracellular nucleic acid leakage).

[0014] Figure 2 The figures show the changes in the permeability of the inner and outer membranes of bacteria after treatment with different concentrations of cladribine. Figure A shows the changes in the inner membrane permeability of 700603; Figure B shows the changes in the inner membrane permeability of 21QH43K; Figure C shows the changes in the inner membrane permeability of 21QH35K; Figure D shows the changes in the outer membrane permeability of 700603; Figure E shows the changes in the outer membrane permeability of 21QH43K; and Figure F shows the changes in the outer membrane permeability of 21QH35K. Detailed Implementation

[0015] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0016] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0017] Example 1: Antimicrobial susceptibility testing of multidrug-resistant Klebsiella pneumoniae

[0018] This invention uses the standard strain of Klebsiella pneumoniae ATCC700603 and migratory Klebsiella pneumoniae (deposited in the National Veterinary Microbial Resistance Risk Assessment Laboratory of the College of Veterinary Medicine, South China Agricultural University) as test strains. Ten commonly used antibiotics, namely meropenem, imipenem, ertapenem, cefotaxime, gentamicin, fosfomycin, ciprofloxacin, tetracycline, tigecycline and colistin, were selected for drug susceptibility testing using the micro-broth serial dilution method.

[0019] The quality control strain ATCC25922 and the test strain were streaked onto their respective agar media and incubated at 37°C for 16–18 h. Single colonies were picked and inoculated into 500 μL of MH broth and incubated at 37°C with shaking at 180 rpm until the logarithmic growth phase. 180 μL of MH broth was added to the first row of wells in a 96-well plate, and 100 μL to the remaining wells. Then, 20 μL of the appropriate concentration of the drug solution was added to the wells in the first row. The mixture was then pipetted and 100 μL was transferred to the next well. This process was repeated until 100 μL was transferred from the last well. The logarithmic growth phase bacterial culture was diluted 1:100 with MH broth, and 100 μL of the diluted culture was added to each well. Negative and positive controls were added to each 96-well plate. The plates were incubated at 37°C for 16–18 h, and three biological replicates were performed. The results were interpreted in accordance with the Clinical and Laboratory Susceptibility Standards Indication (CLSI) of the U.S. Clinical and Laboratory Susceptibility Standards, and the criteria are shown in Table 1.

[0020] The results of the drug susceptibility test are shown in Table 2. The two strains, 21QH43K and 21QH35K, showed resistance to eight commonly used clinical antibiotics: meropenem, imipenem, ertapenem, cefotaxime, gentamicin, fosfomycin, ciprofloxacin, and tetracycline. These eight drugs belong to the categories of carbapenems, β-lactams, aminoglycosides, fosfomycin, fluoroquinolones, and tetracyclines, respectively. Therefore, these two strains are multidrug-resistant Klebsiella pneumoniae.

[0021] Table 1 CLSI Standard Judgment Results

[0022]

[0023] Table 2. Results of drug susceptibility testing for Klebsiella pneumoniae

[0024]

[0025]

[0026] Note: The MIC values ​​in the table are in mg / L, and the results are the average of three biological replicates.

[0027] Example 2: Inhibitory effect of cladribine on multidrug-resistant Klebsiella pneumoniae

[0028] The MIC of cladribine against multidrug-resistant Klebsiella pneumoniae was determined using the micro-broth two-fold dilution method, following the same procedure as above. The inhibition results are shown in Table 3.

[0029] Table 3. Inhibition results of cladribine against multidrug-resistant Klebsiella pneumoniae.

[0030]

[0031] As shown in Table 3, the MIC of cladribine against multidrug-resistant Klebsiella pneumoniae is 64 mg / L, and it has strong inhibitory activity.

[0032] Example 3: Disruption of cell membrane integrity by cladribine in multidrug-resistant Klebsiella pneumoniae

[0033] Klebsiella pneumoniae was inoculated into 15 mL centrifuge tubes containing 4 mL of LB broth and incubated at 37°C and 180 rpm for approximately 4 hours. The OD of the bacterial culture was measured. 600 Adjust the concentration to approximately 0.5, then centrifuge, resuspend in PBS, and incubate overnight with different concentrations of cladribine. The next day, centrifuge again, collect the supernatant, and transfer it to a cuvette. Finally, use a UV spectrophotometer to measure the absorbance at 260 nm and 280 nm. Figure 1 It can be seen that the leakage of nucleic acids and proteins increases with increasing treatment concentration, which indicates that cladribine can disrupt the integrity of the Klebsiella pneumoniae cell membrane.

[0034] Example 4: Cladribine enhances cell membrane permeability of multidrug-resistant Klebsiella pneumoniae

[0035] Klebsiella pneumoniae was inoculated into 15 mL centrifuge tubes containing 4 mL LB broth and incubated overnight at 37°C and 180 rpm with shaking. The next day, the supernatant was discarded by centrifugation, and the bacterial pellet was resuspended in PBS and adjusted to OD500. 600The concentration was 0.5. 800 μL of PBS was added to a 2 mL EP tube, 100 μL of PBS was added to the control group, and different concentrations of cladribine were added to the experimental groups to achieve final drug concentrations of 1 / 4 MIC, 1 / 2 MIC, MIC, 2 MIC, and 4 MIC. Then, 100 μL of the prepared bacterial solution was added to both the control and experimental groups, and the tubes were incubated at 37°C in the dark for 2 hours. Subsequently, PI and NPN probes were added for staining, achieving final concentrations of 0.5 μM and 10 μM, respectively. The tubes were then incubated at 37°C in the dark for 30 minutes, followed by centrifugation at 5000 rpm for 5 minutes. The supernatant was discarded, and the tubes were washed twice with PBS. 200 μL of the solution was then transferred to a black 96-well plate and placed in a microplate reader for fluorescence detection. The excitation light of the PI probe was measured at 535 nm, and the emission light at 615 nm. The excitation light of the NPN probe was measured at 350 nm, and the emission light at 420 nm. The entire procedure was performed in the dark.

[0036] Depend on Figure 2 It was found that, compared with the control group, the fluorescence intensity of multidrug-resistant Klebsiella pneumoniae increased with the increase of cladribine concentration, indicating that the permeability of the bacterial cell membrane of Klebsiella pneumoniae increased after exposure to cladribine, which may cause the death of multidrug-resistant Klebsiella pneumoniae.

[0037] In summary, the results show that cladribine has a good antibacterial effect against multidrug-resistant Klebsiella pneumoniae, and that it exerts its antibacterial effect by increasing or disrupting the integrity of the cell membrane.

[0038] Obviously, the specific implementation schemes described above are merely a further detailed explanation of the purpose, technical solution and beneficial effects of the present invention. It should be understood that the above descriptions are only specific examples of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of cladribine in the preparation of drugs to inhibit the growth of multidrug-resistant Klebsiella pneumoniae, characterized in that, Cladribine is the only active inhibitory component.

2. The application according to claim 1, characterized in that, Multidrug-resistant Klebsiella pneumoniae is resistant to multiple drugs, including meropenem, imipenem, ertapenem, cefotaxime, gentamicin, fosfomycin, ciprofloxacin, or tetracycline.

3. The application according to claim 1, characterized in that, Drug-resistant Klebsiella pneumoniae is a migratory bird-derived Klebsiella pneumoniae.

4. The application according to claim 1, characterized in that, The drug is a non-antibiotic antibacterial agent.

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