A compound for increasing the sensitivity of klebsiella pneumoniae to antibiotics, composition and application thereof

Abstract

The application discloses a compound and a composition for increasing the sensitivity of Klebsiella pneumoniae to antibiotics, wherein the compound is a biguanide compound, and the structural formula of the biguanide compound is shown in the following formula: wherein R1 is a saturated alkyl group with 4 carbon atoms, R2 is a para-substituted aromatic ring group, and the substituent group is a trifluoromethoxy group; and the composition is the biguanide compound and tigecycline. The compound and the composition can improve the sensitivity of antibiotics and have a significant antibacterial effect.

Description

Technical Field

[0001] This invention relates to a compound, composition, and application of Klebsiella pneumoniae for increasing its antibiotic sensitivity, and belongs to the field of biomedical technology. Background Technology

[0002] Klebsiella pneumoniae is a common opportunistic pathogen that can cause a variety of clinical infections, including pneumonia, skin infections, bloodstream infections, urinary tract infections, and abdominal infections. It has a particularly high incidence in immunocompromised individuals, elderly patients, and patients in intensive care units (ICUs), making it one of the most threatening pathogens in clinical infections.

[0003] In recent years, with the widespread and even irrational use of antimicrobial drugs in clinical diagnosis and treatment, livestock and poultry farming, the detection rate of multidrug-resistant Klebsiella pneumoniae (MDR-KP) has shown a continuous upward trend, gradually becoming a core pathogen of hospital-acquired infections, posing a serious threat to public health and clinical care. Compared with common Klebsiella pneumoniae strains, multidrug-resistant strains exhibit resistance or low sensitivity to many commonly used clinical antibiotics, resulting in a significantly narrowed range of effective treatment options and a substantial increase in the difficulty of treating infections and the risk of treatment failure. Multiple clinical studies have confirmed that multidrug-resistant Klebsiella pneumoniae infection can significantly prolong hospital stays and increase medical costs. The in-hospital mortality rate associated with this infection is typically 30%-40%, and in patients with bloodstream infections or severe infections, the mortality rate can even exceed 50%. Simultaneously, this bacterium has strong environmental adaptability and human-to-human transmission capabilities, easily leading to sustained transmission and even outbreaks within medical institutions, posing a significant challenge to hospital infection control efforts.

[0004] Tigecycline (TGC), the first glycyl tetracycline antibiotic derived from minocycline, exhibits excellent antibacterial activity against a variety of Gram-negative and Gram-positive bacteria. Its main mechanism of action involves specific binding to the 30S subunit of the bacterial ribosome, blocking the entry of aminoacyl-tRNA into the ribosomal A site, thereby inhibiting bacterial protein synthesis and ultimately suppressing bacterial growth. Due to the specific side-chain modifications introduced into its molecular structure, tigecycline can overcome common resistance mechanisms of traditional tetracycline antibiotics to some extent, and has been widely used clinically in the treatment of complicated intra-abdominal infections, skin and soft tissue infections, and other diseases. In the treatment of multidrug-resistant Klebsiella pneumoniae infections, due to the increasing scarcity of effective antibacterial drugs available, tigecycline has become an important clinical treatment, even considered one of the "last lines of defense" against such drug-resistant infections, occupying an irreplaceable position in clinical anti-infective therapy.

[0005] However, with the increasingly widespread clinical application of tigecycline, resistance to tigecycline in Klebsiella pneumoniae has gradually emerged and worsened, severely limiting its clinical efficacy and applicability. Existing research indicates that resistance to tigecycline in Klebsiella pneumoniae is related to multiple molecular mechanisms, the most significant being the overexpression of the multidrug efflux pump system. The efflux pump system, represented by AcrAB-TolC, actively expels intracellular tigecycline, reducing intracellular drug concentration and thus weakening its antibacterial activity. Furthermore, abnormal expression of globally regulatory genes such as ramA, marA, and soxS can further enhance bacterial resistance to tigecycline by regulating the expression of the efflux pump system. In recent years, plasmid-mediated tigecycline resistance genes have been discovered, with the tet(X) family genes being the most typical. These genes encode monooxygenases that can inactivate tigecycline through oxidative modification, leading to high levels of tigecycline resistance in bacteria. The existence of the aforementioned drug resistance mechanisms presents a new bottleneck for the clinical treatment of multidrug-resistant Klebsiella pneumoniae infection with tigecycline, and there is an urgent need to explore new treatment strategies to enhance the antibacterial activity of tigecycline and delay the development of drug resistance.

[0006] In summary, the clinical harm caused by multidrug-resistant Klebsiella pneumoniae infection is becoming increasingly serious. Tigecycline, as a key drug in clinical treatment, faces significant challenges due to its resistance, which has become a major bottleneck restricting treatment efficacy. Existing tigecycline-enhancing regimens have many shortcomings and cannot meet the actual needs of clinical treatment. Therefore, developing a novel and highly effective composition to increase the sensitivity of Klebsiella pneumoniae to antibiotics (especially tigecycline), optimizing combination therapy strategies, significantly improving the drug sensitivity of multidrug-resistant Klebsiella pneumoniae to tigecycline, and reducing medication risks have become urgent technical problems to be solved in the field of combating drug-resistant bacterial infections. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the present invention aims to provide a composition and application for increasing the antibiotic sensitivity of Klebsiella pneumoniae, which specifically solves the technical problem of insufficient sensitivity of multidrug-resistant Klebsiella pneumoniae to tigecycline, and has important clinical significance and application value for improving the treatment effect of drug-resistant bacterial infections.

[0008] This invention provides a compound for increasing the antibiotic sensitivity of Klebsiella pneumoniae. The compound is a biguanide compound with the following structural formula: R1 is a C4 saturated alkyl group, R2 is a para-substituted aromatic cyclic group, and the substituent is trifluoromethoxy. Antibacterial activity studies show that the new biguanide derivative 4C exhibits approximately 78 times higher antibacterial activity than metformin.

[0009] As a further embodiment of the present invention, the structural formula of the compound is as follows: The above compounds were synthesized as follows: Commercially available compound 4-(trifluoromethoxyaniline) was first reacted with sodium dihydrodiimide at 80°C to give an intermediate. Subsequently, it was reacted with n-butylamine in tetrahydrofuran at 40°C until the intermediate was completely reacted. Finally, hydrochloric acid solution was added and stirred for 30 minutes. Then, ethylenediaminetetraacetic acid (EDTA) solution was added dropwise to the reaction mixture and filtered to obtain the above biguanide compound, also known as neobiguanide 4C.

[0010] The present invention also provides the application of the above-mentioned compounds, which are used to inhibit the proliferation of multidrug-resistant Klebsiella pneumoniae.

[0011] Furthermore, the antibiotic used in the application is tigecycline.

[0012] The present invention also provides a composition for increasing the antibiotic sensitivity of Klebsiella pneumoniae, comprising the above-mentioned compounds.

[0013] Furthermore, the composition also includes tigecycline.

[0014] Furthermore, the concentration ratio of the biguanide compound to tigecycline is (4-32):1.

[0015] Furthermore, the composition is used externally.

[0016] The present invention also provides the application of the above composition for increasing the antibiotic susceptibility of Klebsiella pneumoniae.

[0017] Furthermore, the bacteria are clinically isolated multidrug-resistant Klebsiella pneumoniae.

[0018] Compared with the prior art, the technical solution of this application has the following beneficial effects: 1. Precise and clear dosage, significantly improving drug safety and efficacy: This invention clarifies the human equivalent doses of metformin 4C and tigecycline. By accurately converting the human dosage of metformin 4C to 0.486 mg / kg and tigecycline to 0.0811 mg / kg using the body surface area method, it effectively avoids the drug safety risks caused by inappropriate dosage in existing synergistic regimens, while also avoiding the problem of poor efficacy caused by insufficient dosage. It provides a precise dosage basis for safe and effective clinical drug use and solves the technical defects of existing metformin and tigecycline combination therapy, which have high effective doses and significant safety risks.

[0019] 2. In vitro and in vivo studies confirm significant synergistic antibacterial effects, providing a practical and feasible clinical treatment plan: Both in vitro and in vivo studies fully validated the significant synergistic antibacterial effect of the combination of new biguanide 4C and tigecycline. The in vitro checkerboard assay showed that the combination therapy exhibited stable synergistic antibacterial activity against multiple strains of Klebsiella pneumoniae, with partial inhibitory concentration indices (FICIs) ≤0.5. Time-bactericidal experiments further confirmed that the combination therapy not only effectively inhibited the proliferation of multidrug-resistant Klebsiella pneumoniae but also achieved a sustained bactericidal effect, completely suppressing bacterial growth. In vivo mouse studies confirmed that the combination therapy group was significantly more effective than the single-drug group against multidrug-resistant Klebsiella pneumoniae infection, effectively overcoming the treatment bottleneck caused by tigecycline resistance and providing a practical and directly applicable combination therapy plan for the clinical treatment of multidrug-resistant Klebsiella pneumoniae infection.

[0020] 3. Excellent antibacterial effect, effectively solving the problem of multidrug-resistant bacterial infections: This invention uses a combination of biguanide compounds (new biguanide 4C) and tigecycline, which can effectively increase the sensitivity of Klebsiella pneumoniae to antibiotics such as tigecycline, thereby efficiently inhibiting the proliferation of multidrug-resistant Klebsiella pneumoniae, rapidly controlling bacterial infections, significantly improving the treatment effect of multidrug-resistant Klebsiella pneumoniae infections, reducing infection mortality, shortening the length of hospital stay, and reducing medical costs. At the same time, it helps to delay the development of resistance to tigecycline in Klebsiella pneumoniae, alleviate the pressure of hospital infection control, and provide a new technical path in the field of anti-drug-resistant bacterial infections. Attached Figure Description

[0021] Figure 1 Diagram showing the synergistic antibacterial activity of new biguanide 4C combined with tigecycline in different Klebsiella pneumoniae strains; Figure 2 Time-bactericidal curve of new biguanide 4C combined with tigecycline against multidrug-resistant Klebsiella pneumoniae; Figure 3The embodiments of this application provide a comparison chart of mouse survival rate and skin tissue bacterial load after administration of new biguanide 4C, tigecycline and the combination of the two drugs to mice with bacterial infection of skin wounds; Figure 4 The embodiments of this application provide H&E staining images of mouse skin tissue after administration of new biguanide 4C, tigecycline, and the combination of the two drugs to mice with bacterial infection of skin wounds; Figure 5 The embodiments of this application provide a comparative diagram of the levels of blood inflammatory factors in mice with bacterial skin wound infection after administration of new biguanide 4C, tigecycline, and the combination of the two drugs; Figure 6 Comparative diagram of the effects of new biguanide 4C combined with tigecycline on the cell membrane of multidrug-resistant Klebsiella pneumoniae; Figure 7 Comparative images of transmission electron microscopy observations of the effects of the combination of new biguanide 4C and tigecycline on bacterial cell morphology. Detailed Implementation

[0022] To enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. Unless otherwise specified, all raw materials, reagents, instruments, and equipment used in this invention can be purchased on the market or prepared by existing methods.

[0023] This invention discloses a compound for increasing antibiotic sensitivity. The compound is a biguanide compound with the following structural formula: wherein R1 is a C4 saturated alkyl group, R2 is a para-substituted aromatic cyclic group, and the substituent is trifluoromethoxy.

[0024] As a further embodiment of the present invention, the structural formula of the compound is as follows: The above compounds were synthesized as follows: Commercially available compound 4-(trifluoromethoxyaniline) was first reacted with sodium dihydrodiimide at 80°C to obtain an intermediate. Subsequently, it was reacted with n-butylamine in tetrahydrofuran at 40°C until the intermediate was completely reacted. Finally, hydrochloric acid solution was added and stirred for 30 minutes. Then, ethylenediaminetetraacetic acid (EDTA) solution was added dropwise to the reaction mixture and filtered to obtain the above biguanide compound, also known as neobiguanide 4C.

[0025] Embodiments of the present invention also provide applications of the above-mentioned compounds, which are used to inhibit the proliferation of multidrug-resistant Klebsiella pneumoniae.

[0026] As a further embodiment of the present invention, the antibiotic used in the application is tigecycline, which was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0027] Embodiments of the present invention also provide a composition for increasing the antibiotic sensitivity of Klebsiella pneumoniae, comprising the above-described compounds.

[0028] As a further embodiment of the present invention, the composition further includes tigecycline.

[0029] As a further embodiment of the present invention, the concentration ratio of the biguanide compound to tigecycline is (4-32):1.

[0030] As a further embodiment of the present invention, the composition is used externally.

[0031] Embodiments of the present invention also provide applications of the above-described composition for increasing the antibiotic susceptibility of Klebsiella pneumoniae.

[0032] As a further embodiment of the present invention, the bacteria is clinically isolated multidrug-resistant Klebsiella pneumoniae.

[0033] Example 1 In vitro antibacterial efficacy test of biguanide 4C and tigecycline against multidrug-resistant Klebsiella pneumoniae: The checkerboard method was used to combine tigecycline and biguanide 4C on multiple strains of Klebsiella pneumoniae, and the partial inhibitory concentration index (FICI) was used as the criterion for judging the efficacy of the two drugs in combination.

[0034] The specific steps are as follows: S101. Remove the glycerol-preserved strain from the -80°C freezer and inoculate it onto LB solid medium using the streak plating method in a clean bench. Incubate upside down at 37°C for 18-24 hours. Then, select a single colony and inoculate it into 1 mL of LB liquid medium, and incubate overnight on a shaker at 180 rpm and 37°C. Subsequently, dilute the culture 1:100 to the logarithmic growth phase (0.5 McFarland concentration). S102. Dilute different concentrations of the drug with MHB liquid medium. Perform serial dilutions of 4C along the x-axis and tigecycline along the y-axis, with a final drug volume of 100 μL per well, forming an 8×8 checkerboard distribution. Dilute the logarithmic growth phase bacterial culture medium 100-fold to 1×10⁻⁶. 6CFU / mL, and 100 μL is inoculated into each well. A control group (with bacteria but no drug) and a blank group (with neither bacteria nor drug, only containing MHB liquid medium) are additionally set. Incubate in a 37°C bacterial incubator for 18-24 h; S103. Detect the OD value of each well with a microplate reader at a detection wavelength of 600 nm and calculate the FICI value.

[0035] Wherein, MIC ab represents the MIC of 4C after the combination of 4C and tigecycline, MIC a represents the MIC of 4C as a single agent, MIC ba represents the MIC of tigecycline after the combination of 4C and tigecycline, MIC b represents the MIC of the single antibiotic. FICI ≤ 0.5 indicates synergistic effect, 0.5 < FICI ≤ 1 indicates additive effect, 1 < FICI ≤ 2 indicates indifferent effect, and FICI > 2 indicates antagonistic effect; The results show that tigecycline and the novel biguanide 4C exhibit significant synergistic antibacterial activity against multiple strains of Klebsiella pneumoniae (FICI ≤ 0.5), and the results are shown in Figure 1 , Table 1 shows the key proportional data of the synergistic antibacterial effect of 4C and tigecycline. It can be seen that the technical solution of the present embodiment acts on different Klebsiella pneumoniae strains, and all exhibit synergistic antibacterial effect.

[0036] Table 1 Key proportional data of synergistic antibacterial effect of 4C and tigecycline in checkerboard results Example 2 In clinically isolated multidrug-resistant Klebsiella pneumoniae 1115-125 , the bactericidal effects of the novel biguanide 4C and tigecycline administered alone and in combination were compared.

[0037] The specific operation is as follows: S201. Mix drugs of different concentrations and bacterial suspension in logarithmic growth phase at equal volume (100 μL), the combination group is: 4C (16 μg / mL) + tigecycline (1 μg / mL), the single-agent groups are: 4C single agent (16 μg / mL) and tigecycline single agent (1 μg / mL). A control group (containing only bacteria) is set S202. Incubate in a 37°C bacterial incubator, and sample at time points 0, 4, 8, 12 and 24 h respectively. Three replicates are set for each time point.

[0038] S203. Perform 10-fold gradient dilution with sterile PBS buffer solution, and take 100 μL to evenly spread on LB agar plates.

[0039] After overnight incubation at 37°C, plates with colony counts between 30 and 300 were selected for counting, and time-kill curves were plotted. The combined use of the two drugs reduced colony counts by ≥2 Log. 10 When the concentration is (CFU / mL), it is defined as having a synergistic bactericidal effect.

[0040] The results are as follows Figure 2 The results showed that, compared with the single administration, the combined use of the two drugs resulted in a significant decrease in bacterial count, which was significantly lower than the initial bacterial inoculation level, indicating that the combined use of tigecycline and biguanide 4C can achieve a synergistic bactericidal effect.

[0041] Example 3 Animal model experiments were conducted to construct a mouse skin wound infection model and to explore the effect of the combination of new biguanide 4C and tigecycline against bacterial infection at the in vivo level.

[0042] The specific steps are as follows: S401. Collect clinically isolated multidrug-resistant Klebsiella pneumoniae. 1115-125 After centrifuging the bacterial culture in the logarithmic growth phase at 5000 rpm for 5 min, wash the bacteria twice with sterile PBS buffer, resuspend, and adjust to a final concentration of 1×10⁻⁶. 8 CFU / mL bacterial suspension.

[0043] S402. After anesthetizing mice with 30 μL / g tribromoethanol, a circular skin wound (5 mm in diameter) was prepared on the back of the mouse. Then, 50 μL of the prepared Klebsiella pneumoniae was applied. 1115-125 When a bacterial suspension is applied to a circular wound, bacterial infection occurs in about one hour.

[0044] S403. Following bacterial infection, 50 μL of the corresponding drug was applied to the circular wounds of mice in each group. The combination group received 4C (6 mg / kg) + tigecycline (1 mg / kg), while the single-drug groups received 4C (6 mg / kg) and tigecycline (1 mg / kg) alone. A model group (containing bacteria only) and a blank control group (containing neither bacteria nor the drug) were established. S404. Observe the mice continuously for 7 days and record the survival rate; use sterile surgical scissors to cut approximately 1cm × 1cm skin tissue from the constructed mouse skin wound as the center; samples for histopathological analysis were fixed with tissue fixative; blood was collected from the retro-orbital venous plexus, and the collected whole blood was placed in a non-anticoagulant blood collection tube, allowed to stand at room temperature until completely coagulated, and then centrifuged at 4°C to separate the serum. The supernatant serum was aliquoted into sterile cryovials and stored at -20°C.

[0045] S405. After homogenizing the collected, fixed skin tissue samples, dilute them with sterile PBS buffer and spread them evenly on LB agar plates. Then, count the colonies using plate counting and plot the results.

[0046] The results are as follows Figure 3 The results showed that the combination of tigecycline and biguanide 4C significantly improved the survival rate of mice after bacterial infection and significantly reduced the bacterial load in the infected part.

[0047] Example 4 The changes in skin tissue structure in different groups of mice were observed using the hematoxylin-eosin staining method. The specific steps are as follows: S401. Mouse skin tissue was labeled and embedded in groups before being sectioned.

[0048] S402. Dewaxing: Place the sections on a metal rack in sequence, immerse them in xylene I and xylene II for 10 min, and then place them on a shaker to shake slowly.

[0049] S403. After soaking, soak the slices in anhydrous ethanol, 95% alcohol and 75% alcohol for 5 minutes each. Then place the slices in a 1000mL beaker and rinse slowly with running water for 5 minutes, and then rinse twice with distilled water.

[0050] S404. Stain the sections with hematoxylin for 5 minutes, rinse the sections with running water, place the sections in hematoxylin for 20 seconds, rinse with tap water for 1 minute, and then place the sections at 45℃ for 7 minutes to re-blue.

[0051] S405. Immerse the slide in eosin for 2 minutes, then rinse with tap water.

[0052] S406. Soak the slices in 75% alcohol, 95% alcohol and anhydrous ethanol for 3 minutes each, and in xylene II and xylene I for 5 minutes each. Then place them in a fume hood to air dry slightly.

[0053] S407. Add neutral resin to the slide, cover with a coverslip, and then place the slide under an inverted microscope to collect sample images.

[0054] The results are as follows Figure 4The control group showed intact skin tissue structure with clear epidermal and dermal structures and no obvious inflammatory cell infiltration. In contrast, the model group exhibited a significant inflammatory response, characterized by epidermal structure destruction, extensive inflammatory cell infiltration in the dermis, and tissue edema, accompanied by damage to some hair follicle structures. The combined treatment group of 4C and tigecycline showed the most significant recovery of skin tissue structure, with an intact epidermis, a significant reduction in inflammatory cells in the dermis, and a marked reduction in tissue edema. The overall tissue morphology was close to that of the normal control group, indicating that the combined medication can significantly improve skin tissue damage caused by infection.

[0055] Example 5 The levels of inflammatory factors in the blood of mice in different groups were detected using ELISA.

[0056] The specific steps are as follows: S501. The mouse inflammatory factor ELISA kit (TNF-α, IL-6, IL-18) from Hunan Aifang Biotechnology Co., Ltd. was used. Washing buffer, standard gradient concentrations and related reagents were prepared according to the instructions.

[0057] S502. Add 100 μL of standard and serum sample to a 96-well plate pre-coated with antibody, with 3 replicate wells for each sample. Add 100 μL of sample diluent to the blank control well.

[0058] S503. After incubation according to the kit instructions, discard the solution and wash the plate thoroughly with washing solution.

[0059] S504. Add biotinylated detection antibody, incubate, and wash the plate.

[0060] S504. Incubate with the enzyme conjugate and wash the plate.

[0061] S505. Add the chromogenic substrate TMB and react in the dark until a clear color gradient appears. Then add the stop solution to terminate the reaction.

[0062] S506. Read the absorbance at 450 nm using an ELISA reader. Fit a standard curve using the standard data, and calculate the concentration of inflammatory factors for each sample. The results can be converted to pg / mL after adjusting for serum stock solution or dilution.

[0063] See results Figure 5 The group treated with 4C combined with tigecycline showed the most significant decrease in the levels of pro-inflammatory factors (TNF-α, IL-6, IL-18), indicating that 4C combined with tigecycline can effectively inhibit the inflammatory response caused by multidrug-resistant Klebsiella pneumoniae infection.

[0064] Example 6 The effects of biguanide 4C and tigecycline alone and in combination on the cell membrane of multidrug-resistant Klebsiella pneumoniae were detected using NPN fluorescent probes and the ONPG method. The specific steps are as follows: S601. Klebsiella pneumoniae 1115-125 After culturing to the logarithmic growth phase, centrifuge at 4°C and 5000 rpm for 5 min, wash 2-3 times with sterile PBS buffer, and resuspend the bacterial culture to OD200. 600 = 0.5, spare.

[0065] S602. Add NPN and ONPG to the bacterial suspension to a final concentration of 10 μmol / L and 3 mmol / L respectively, and incubate at 37°C in the dark for 30 min, shaking to mix every 5 min.

[0066] S603. Add different concentrations of drug and incubate at 37°C for 1 hour. The combination therapy groups were: 4C (8 μg / mL, 16 μg / mL, 32 μg / mL) + tigecycline (1 μg / mL), and the single-drug groups were: 4C alone (8 μg / mL, 16 μg / mL, 32 μg / mL) and tigecycline alone (1 μg / mL). A blank control group (no drug treatment) was set up. Three replicates were set up.

[0067] S604. After treatment, 200 μL was added to a 96-well black reaction plate, and the fluorescence intensity and absorbance were measured at 420 nm using a microplate reader. The NPN excitation wavelength was 350 nm, and the emission wavelength was 420 nm.

[0068] like Figure 6 As shown, compared with the control group, the bacterial fluorescence intensity and OD values ​​after treatment with 4C single drug and combination drug were significantly different. 420 Significantly increased, and showing a clear dose-dependent trend. Fluorescence intensity and OD in the tigecycline monotherapy group... 420 The changes were not significant. These results indicate that 4C can enhance the outer membrane permeability of Klebsiella pneumoniae and disrupt the integrity of the inner membrane.

[0069] Example 7 The effects of combined use of new biguanide 4C and tigecycline on bacterial morphology were observed using transmission electron microscopy.

[0070] The specific steps are as follows: S701. Klebsiella pneumoniae 1115-125 After culturing to the logarithmic growth phase, centrifuge at 4°C and 5000 rpm for 5 min, wash 2-3 times with sterile PBS buffer, and resuspend the bacterial culture to OD200. 600 = 0.5, spare.

[0071] S702. Add different concentrations of the drug and incubate at 37°C for 4 hours. The combination group consisted of 4C (16 μg / mL) + tigecycline (1 μg / mL), and the single-drug group consisted of 4C (16 μg / mL) and tigecycline (1 μg / mL) alone. A blank control group (no drug treatment) was set up.

[0072] S703. After incubation, centrifuge at 4°C for 5 min, discard the supernatant, wash the bacterial cells 2-3 times with sterile PBS buffer, collect the bacterial cells, and fix them at room temperature for 2 h with electron microscopy fixative.

[0073] S704. Gradual dehydration was performed using ethanol at concentrations of 30%, 50%, 70%, 80%, 90%, 95%, and 100% for 20 minutes each time, followed by treatment with 100% acetone twice for 15 minutes each time.

[0074] S705. After fixing with a 1:1 mixture of acetone and 812 embedding agent at room temperature for 2 hours, the sample was infiltrated with a 1:2 mixture of acetone and 812 embedding agent at room temperature for 8 hours. Then, the sample was placed in a pure 812 embedding plate and incubated overnight at 37°C. Finally, the embedding plate was placed in a 60°C oven for polymerization for 48 hours, and the resin block was removed for later use.

[0075] S706. Use an ultramicrotome to cut the resin-embedded tissue into ultrathin sections of 60-80 nm thickness, and spread the sections flat on a copper or nickel mesh for later use.

[0076] S707. Stain the sections in a 2% uranium acetate solution and an alcohol solution for 8 minutes in the dark, then rinse three times with ultrapure water. Next, stain with a 2.6% lead citrate solution for 10 minutes. After staining, rinse and dry the sections for later use.

[0077] S708. Observe and photograph using a JEM-1230 transmission electron microscope.

[0078] Transmission electron microscopy results as follows Figure 7 As shown, the control group bacteria had intact outlines, clear cell membrane boundaries, and uniform and dense cytoplasm distribution. After treatment with tigecycline alone, the overall morphology of the bacteria was not significantly different from the control group, with only slight structural changes. After treatment with 4C alone, the cell membrane structure was disordered, plasmolysis occurred, cytoplasmic density decreased, and the internal structure tended to be loose. The combined drug group showed more obvious damage to the membrane structure, leakage of contents, disordered distribution of substances inside the bacteria, and vacuolar changes in some areas.

[0079] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A compound for increasing the antibiotic sensitivity of Klebsiella pneumoniae, characterized in that, The compound is a biguanide compound, and the biguanide compound has the following structural formula: wherein R1 is a C4 saturated alkyl group, R2 is a para-substituted aromatic cyclic group, and the substituent is trifluoromethoxy.

2. The compound according to claim 1 for increasing the antibiotic sensitivity of Klebsiella pneumoniae, characterized in that: The structural formula of the compound is as follows: 。 3. An application of the compound as described in claims 1-2, characterized in that: The compound is used to inhibit the proliferation of multidrug-resistant Klebsiella pneumoniae.

4. The application of the compound according to claim 3, characterized in that: The antibiotic used in this application is tigecycline.

5. A composition for increasing the antibiotic sensitivity of Klebsiella pneumoniae, characterized in that, Includes the compounds as described in claims 1-2.

6. The composition for increasing the antibiotic sensitivity of Klebsiella pneumoniae according to claim 5, characterized in that: The composition also includes tigecycline.

7. The composition for increasing the antibiotic sensitivity of Klebsiella pneumoniae according to claim 6, characterized in that: The concentration ratio of the biguanide compound to tigecycline is (4-32):

1.

8. The composition for increasing the antibiotic sensitivity of Klebsiella pneumoniae according to claim 7, characterized in that: The composition is for external use only.

9. An application of the composition as described in claims 5-8, characterized in that: The composition is used to increase the susceptibility of Klebsiella pneumoniae to antibiotics.

Images