Application of combination of meropenem or aztreonam and paromomycin sulfate in preparation of antibacterial drugs

Abstract

The invention relates to the technical field of medicines, in particular to application of combination of meropenem or aztreonam and paromomycin sulfate in preparation of antibacterial drugs. The experimental result of the in vitro combination effect shows that when meropenem or aztreonam is combined with paromomycin sulfate, the meropenem or aztreonam has a remarkable synergistic effect on carbapenem-resistant klebsiella pneumoniae, and paromomycin sulfate can reduce the bacteriostatic concentration of meropenem or aztreonam, so that the meropenem or aztreonam can be used for preparing the medicine for treating the carbapenem-resistant klebsiella pneumoniae. Therefore, the paromomycin sulfate can be used for improving the inhibition effect of meropenem or aztreonam on carbapenem-resistant klebsiella pneumoniae.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of meropenem or aztreonam in combination with paromomycin sulfate in the preparation of antibacterial drugs. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Elderly patients with heart failure are prone to lung infections, with Klebsiella pneumoniae being one of the main pathogens. Studies show that the detection rate of Klebsiella pneumoniae in elderly patients with heart failure and concurrent lung infection is 20%. Other studies have also confirmed that Klebsiella pneumoniae is a major Gram-negative bacterium in pneumonia complicated by cardiovascular disease (including heart failure) in elderly patients.

[0004] Furthermore, bacterial resistance has become one of the major threats to global public health. The emergence and prevalence of multidrug-resistant (MDR), extensively drug-resistant (XDR), and even pandrug-resistant (PDR) bacteria pose a significant threat to human health. Among the many drug-resistant bacterial infections encountered in clinical practice, carbapenem-resistant Gram-negative bacilli, especially the rapidly increasing carbapenem-resistant Enterobacteriaceae (CRE) in recent years, have attracted widespread attention. Since the first report of carbapenem-resistant Klebsiella pneumoniae (CRKP) in 2001, CRKP has spread rapidly worldwide. CRKP is an important pathogen of hospital-acquired infections, including pneumonia (especially hospital-acquired pneumonia and ventilator-associated pneumonia), urinary tract infections, bloodstream infections, abdominal infections, and purulent liver abscesses.

[0005] For carbapenem-resistant Klebsiella pneumoniae (CRP) infections, there are currently few effective treatments and very limited clinical research data. Antibiotics with good antibacterial activity against CRP generally have limitations when used as monotherapy. For example, polymyxins are prone to heterogeneous resistance and have significant nephrotoxicity and neurotoxicity, as well as low lung tissue permeability; tigecycline has low blood concentrations; and ceftazidime / avibactam is ineffective against metalloenzyme-producing strains. Furthermore, with the widespread clinical use of antibiotics, the resistance rate of CRP to polymyxins, tigecycline, and other drugs is rapidly increasing.

[0006] Therefore, there is an urgent need to explore drugs that can overcome carbapenem-resistant Klebsiella pneumoniae infection. Summary of the Invention

[0007] To overcome the above problems, the present invention provides the application of meropenem or aztreonam in combination with paromomycin sulfate in the preparation of antibacterial drugs.

[0008] To achieve the above technical objectives, the present invention adopts the following technical solution: In a first aspect, the invention provides the use of paromomycin sulfate in the preparation of a medicament that enhances the antibacterial effect of meropenem or aztreonam; wherein the bacterium is Klebsiella pneumoniae.

[0009] In one or more embodiments, the Klebsiella pneumoniae is a carbapenem-resistant Klebsiella pneumoniae.

[0010] Preferably, the types of carbapenem-resistant Klebsiella pneumoniae producing carbapenemases include: oxacillinase-48 (OXA-48), New Delhi metallo-β-lactamase (NDM), Klebsiella pneumoniae carbapenemase (KPC), imipenem metallo-β-lactamase (IMP), and Verona integrin-encoded metallo-β-lactamase (VIM).

[0011] OXA-48 belongs to class D β-lactamases, NDM belongs to class B β-lactamases, KPC belongs to class A β-lactamases, IMP belongs to class B β-lactamases, and VIM belongs to class B β-lactamases.

[0012] In one or more embodiments, the mass ratio of paromomycin sulfate to meropenem is (0.5~64):(0.25~64). The mass ratio of paromomycin sulfate to aztreonam is (2~64):(8~64).

[0013] A second aspect of the present invention provides the use of meropenem or aztreonam in combination with paromomycin sulfate in the preparation of an antibacterial drug; wherein the bacterium is Klebsiella pneumoniae.

[0014] In one or more embodiments, the Klebsiella pneumoniae is a carbapenem-resistant Klebsiella pneumoniae.

[0015] Preferably, the types of carbapenem-resistant Klebsiella pneumoniae producing carbapenemases include: oxacillinase-48 (OXA-48), New Delhi metallo-β-lactamase (NDM), Klebsiella pneumoniae carbapenemase (KPC), imipenem metallo-β-lactamase (IMP), and Verona integrin-encoded metallo-β-lactamase (VIM).

[0016] In one or more embodiments, the mass ratio of paromomycin sulfate to meropenem is (0.5~64):(0.25~64). The mass ratio of paromomycin sulfate to aztreonam is (2~64):(8~64).

[0017] A third aspect of the present invention provides an antibacterial drug comprising meropenem or aztreonam and paromomycin sulfate; wherein the bacterium is Klebsiella pneumoniae.

[0018] In one or more embodiments, the Klebsiella pneumoniae is a carbapenem-resistant Klebsiella pneumoniae.

[0019] Preferably, the types of carbapenem-resistant Klebsiella pneumoniae producing carbapenemases include: oxacillinase-48 (OXA-48), New Delhi metallo-β-lactamase (NDM), Klebsiella pneumoniae carbapenemase (KPC), imipenem metallo-β-lactamase (IMP), and Verona integrin-encoded metallo-β-lactamase (VIM).

[0020] In one or more embodiments, the mass ratio of paromomycin sulfate to meropenem is (0.5~64):(0.25~64). The mass ratio of paromomycin sulfate to aztreonam is (2~64):(8~64).

[0021] When meropenem and paromomycin sulfate were used in combination to combat carbapenem-resistant Klebsiella pneumoniae NCTC13439, the minimum inhibitory concentration of meropenem was 8 μg / mL and the minimum inhibitory concentration of paromomycin sulfate was 1 μg / mL. When meropenem and paromomycin sulfate were used in combination to combat carbapenem-resistant Klebsiella pneumoniae KP26, the minimum inhibitory concentration (MIC) of both meropenem and paromomycin sulfate was 64 μg / mL. When meropenem and paromomycin sulfate were used in combination to combat carbapenem-resistant Klebsiella pneumoniae KP27, the minimum inhibitory concentration (MIC) of meropenem was 16 μg / mL and that of paromomycin sulfate was 1 μg / mL. When meropenem and paromomycin sulfate were used in combination to combat carbapenem-resistant Klebsiella pneumoniae KP29, the minimum inhibitory concentration (MIC) of meropenem was 16 μg / mL and that of paromomycin sulfate was 1 μg / mL. When meropenem and paromomycin sulfate were used in combination to combat carbapenem-resistant Klebsiella pneumoniae KPn24, the minimum inhibitory concentration (MIC) of meropenem was 0.5 μg / mL and the MIC of paromomycin sulfate was 1 μg / mL. When meropenem and paromomycin sulfate were used in combination to combat carbapenem-resistant Klebsiella pneumoniae NCTC13442, the minimum inhibitory concentration (MIC) of meropenem was 0.25 μg / mL, and the MIC of paromomycin sulfate was 0.5 μg / mL. When aztreonam and paromomycin sulfate were used in combination to combat carbapenem-resistant Klebsiella pneumoniae KP26, the minimum inhibitory concentration (MIC) of aztreonam was 8 μg / mL, and the MIC of paromomycin sulfate was 64 μg / mL.

[0022] In one or more embodiments, the drug further includes pharmaceutically acceptable carriers, excipients, and diluents.

[0023] The non-pharmaceutical active ingredients that may be included, such as carriers, excipients, and diluents, are well known in the art, and those skilled in the art can determine that they meet clinical standards.

[0024] Preferably, the carrier, excipients, and diluents include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

[0025] Preferably, the dosage form of the drug is a suspension, emulsion, granules, spray, injection, transdermal absorbent, a dosage form suitable for transfection, tablet, powder, granules or capsule.

[0026] A fourth aspect of the present invention provides a pharmaceutical composition comprising the antibacterial agent described in the third aspect.

[0027] The beneficial effects of this invention are as follows: The experimental results of in vitro combined use in this invention show that meropenem or aztreonam combined with paromomycin sulfate exhibits a significant synergistic effect against carbapenem-resistant Klebsiella pneumoniae. Paromomycin sulfate can reduce the inhibitory concentration of meropenem or aztreonam, thus indicating that paromomycin sulfate can enhance the inhibitory effect of meropenem or aztreonam on carbapenem-resistant Klebsiella pneumoniae. Attached Figure Description

[0028] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0029] Figure 1 The results show the determination of the types of carbapenemases produced by the experimental strains using bacterial immunochromatographic reagent. Among them, a is carbapenem-resistant Klebsiella pneumoniae NCTC13442, b is carbapenem-resistant Klebsiella pneumoniae NCTC13439, c is carbapenem-resistant Klebsiella pneumoniae KP26, d is carbapenem-resistant Klebsiella pneumoniae KP27, e is carbapenem-resistant Klebsiella pneumoniae KP29, and f is carbapenem-resistant Klebsiella pneumoniae KPn24. Figure 2 The results of the in vitro interaction (static) of meropenem and paromomycin sulfate in combination against carbapenem-resistant Klebsiella pneumoniae NCTC13442. Figure 3 The results of the in vitro interaction (static) of meropenem and paromomycin sulfate in combination against carbapenem-resistant Klebsiella pneumoniae KP26. Figure 4 The results of the in vitro interaction (static) of meropenem and paromomycin sulfate in combination against carbapenem-resistant Klebsiella pneumoniae KP27. Figure 5 The results of the in vitro interaction (static) of meropenem and paromomycin sulfate in combination against carbapenem-resistant Klebsiella pneumoniae KP29. Figure 6 The results of the in vitro interaction (static) of meropenem and paromomycin sulfate in combination against carbapenem-resistant Klebsiella pneumoniae KPn24. Figure 7 The results of the in vitro interaction (static) of meropenem and paromomycin sulfate in combination against carbapenem-resistant Klebsiella pneumoniae NCTC13439. Figure 8 The results of the in vitro interaction (static) of aztreonam and paromomycin sulfate in combination against carbapenem-resistant Klebsiella pneumoniae KP26. Figure 9 In vitro (dynamic) interaction of meropenem and paromomycin sulfate against carbapenem-resistant Klebsiella pneumoniae NCTC13442. Figure 10 In vitro (dynamic) interaction of meropenem and paromomycin sulfate against carbapenem-resistant Klebsiella pneumoniae KP26. Figure 11In vitro (dynamic) interaction of meropenem and paromomycin sulfate against carbapenem-resistant Klebsiella pneumoniae KP27. Figure 12 In vitro (dynamic) interaction of meropenem and paromomycin sulfate against carbapenem-resistant Klebsiella pneumoniae KP29. Figure 13 In vitro (dynamic) interaction of meropenem and paromomycin sulfate against carbapenem-resistant Klebsiella pneumoniae KPn24. Figure 14 In vitro (dynamic) interaction of meropenem and paromomycin sulfate against carbapenem-resistant Klebsiella pneumoniae NCTC13439. Figure 15 In vitro (dynamic) interaction between aztreonam and paromomycin sulfate in combination against carbapenem-resistant Klebsiella pneumoniae KP26; Figure 16 Time-survival curves of meropenem combined with paromomycin sulfate for the treatment of carbapenem-resistant Klebsiella pneumoniae KP26 infection with giant wax moth; Figure 17 Time-survival curves for the combined use of aztreonam and paromomycin sulfate in the treatment of carbapenem-resistant Klebsiella pneumoniae KP26 infection in the large wax moth; Figure 18 The histopathological results of meropenem combined with paromomycin sulfate in the treatment of carbapenem-resistant Klebsiella pneumoniae KP26 infection of the large wax moth are shown in the following: a is the control group, b is the meropenem monotherapy group, c is the paromomycin sulfate monotherapy group, and d is the meropenem combined with paromomycin sulfate group. Figure 19 The histopathological results are as follows: a) The combined use of aztreonam and paromomycin sulfate was used to treat carbapenem-resistant Klebsiella pneumoniae KP26 infection of the large wax moth. Among them, a) is the control group, b) is the aztreonam monotherapy group, c) is the paromomycin sulfate monotherapy group, and d) is the aztreonam and paromomycin sulfate combination group. Detailed Implementation

[0030] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0031] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0032] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0033] Quality control strain: Klebsiella pneumoniae ATCC BAA-1705.

[0034] Experimental strains: carbapenem-resistant Klebsiella pneumoniae NCTC13442, carbapenem-resistant Klebsiella pneumoniae KP26, carbapenem-resistant Klebsiella pneumoniae KP27, carbapenem-resistant Klebsiella pneumoniae KP29, carbapenem-resistant Klebsiella pneumoniae KPn24, and carbapenem-resistant Klebsiella pneumoniae NCTC13439.

[0035] Both the quality control strains and the experimental strains were obtained from the strain library previously established by our research group. The types of carbapenemases produced by the experimental strains were determined using bacterial immunochromatographic reagents. The results are as follows: Figure 1 As shown, from Figure 1 It can be seen that strain NCTC13442 produces OXA 48 types of carbapenemases were identified. Strain NCTC13439 produced VIM-type carbapenemases, strain KP26 produced NDM-type carbapenemases, strains KP27 and KP29 both produced KPC-type carbapenemases, and strain KPn24 produced IMP-type carbapenemases.

[0036] Both the quality control strains and the experimental strains are stored in Before use, subculture the bacteria on MH agar (Mueller-Hinton Agar, MHA) at 80 ℃, with at least two subcultures. Pick a single colony from the medium and resuspend it in MH broth (Mueller-Hinton Broth, MHB) liquid medium. Incubate overnight at 35 ℃ using a shaker. Adjust the bacterial suspension concentration to 1 × 10⁻⁶ using a McFarland turbidimetric tube. 9 Quantity / mL, for later use.

[0037] Paromomycin sulfate and meropenem powders were dissolved separately in dimethyl sulfoxide (DMSO) to a final concentration of 10 mg / mL, stored in sterile centrifuge tubes, labeled, and stored at -20 °C for later use. Aztreonam raw material powder was dissolved in ultrapure water to a final concentration of 5 mg / mL, stored in sterile centrifuge tubes, labeled, and stored at -20 °C for later use.

[0038] Example 1 Meropenem or aztreonam in combination with paromomycin sulfate for carbapenem-resistant Klebsiella pneumoniae (static): Follow the CLSI (M100) standards published by the Clinical and Laboratory Standards Institute (CSIS). Prepare sterile 96-well plates (rows AH, columns 1-12), leaving column 12 empty initially. Add 50 μL of serially diluted meropenem or aztreonam solution in cationic-regulated Mueller-Hinton broth (CAMHB) to each well in columns 2-11, with concentrations ranging from 0.5 to 256 μg / mL. Add 50 μL of serially diluted paromomycin sulfate solution in CAMHB to each well in row AG, with concentrations ranging from 256 to 4 μg / mL. Add 50 μL of CAMHB medium to each well in column 1 and row H. Pick colonies of bacteria (NCTC13442, KP26, KP27, KP29, KPn24, NCTC13439) cultured overnight at 35°C on MHA plates using a sterile loop, dissolve them in sterile phosphate-buffered saline (PBS), and adjust the concentration to 1×10⁻⁶ using a turbidity analyzer. 9 CFU / mL, diluted to 2×10⁻⁶ with CAMHB medium. 6 CFU / mL, the above bacterial suspension was added to 100 μL of the drug solution in each well of a 96-well plate (final concentrations of meropenem and aztreonam: 0.125 μg / mL-64 μg / mL, final concentration of paromomycin sulfate: 1 μg / mL-64 μg / mL). Column 12 was used as a blank control (no reagents added to this well), a negative control (only 200 μL of CAMHB medium added), and a high-concentration (128-512 μg / mL) drug monotherapy (100 μL of drug solution and 100 μL of bacterial suspension added). After preparation, the plates were placed in a constant temperature incubator and incubated at 35 ℃ for 24 h before observing the results. No sterile growth was observed in the negative control wells, while significant bacterial growth was observed in the growth control wells (H1). The results of this experiment are valid.

[0039] Referring to the CLSI M100 standard, the readings were compared with the growth control wells. Under natural light, the growth of bacteria in each well was visually assessed to determine the percentage compared to the growth control wells. Combined with the OD values ​​of each well measured by the microplate reader, the minimum inhibitory concentration (MIC) was defined as the drug concentration that could inhibit the growth of more than 90% of bacteria.

[0040] Growth rate calculation method: (OD 待测孔 - OD 空白对照孔 ) / (OD 生长对照孔 - OD 空白对照孔 ) × 100%.

[0041] Synergy evaluation: The results of the combined drug susceptibility test are used to judge the interaction after the combination of two drugs by the fractional inhibitory concentration index.

[0042] The formula for the fractional inhibitory concentration index (FICI) is as follows: ΣFICI = FIC A + FIC B = C A / MIC A + C B / MIC B ; Among them, FICI is the fractional inhibitory concentration index, FIC A and FIC B respectively represent the MIC value of the drug when drugs A and B are combined divided by the MIC value of the drug when drugs A and B are used alone. MIC A and MIC B are the minimum inhibitory concentrations of drugs A and B alone, and C A and C B are the respective concentrations of the two drugs when the same drug effect is achieved when the two drugs are combined. FICI ≤ 0.5 indicates that the two drugs have a synergistic effect; 0.5 < FICI ≤ 1 indicates that the two drugs have an additive effect; 1 < FICI ≤ 2 indicates that the two drugs have no interaction; FICI > 2 indicates that the two drugs have an antagonistic effect.

[0043] Experimental results: (1) The in vitro interaction results (static) of the combination of meropenem and paromomycin sulfate against carbapenem-resistant Klebsiella pneumoniae are shown in Table 1 and Figures 2-7 as follows.

[0044] Table 1 In vitro interaction results (static) of the combination of meropenem and paromomycin sulfate against carbapenem-resistant Klebsiella pneumoniae evaluated by the FICI model

[0045] In Table 1, MIC represents the minimum inhibitory concentration; MIC A represents the minimum inhibitory concentration of meropenem when the drug is used alone; C A represents the minimum inhibitory concentration of meropenem when the drugs are combined; MIC BIndicates the minimum inhibitory concentration of paromomycin sulfate when used alone; C B The minimum inhibitory concentration (MIC) of paromomycin sulfate is indicated when the drugs are used in combination; FICI represents the fractional inhibitory concentration index.

[0046] (2) The in vitro interaction results (static) of aztreonam and paromomycin sulfate in combination against carbapenem-resistant Klebsiella pneumoniae are shown in Table 2 and Figure 8 As shown.

[0047] Table 2. In vitro interaction results (static) of aztreonam and paromomycin sulfate combination against carbapenem-resistant Klebsiella pneumoniae using the FICI model.

[0048] In Table 2, MIC represents the minimum inhibitory concentration; A Indicates the minimum inhibitory concentration of aztreonam when used alone; C A The MIC indicates the minimum inhibitory concentration of aztreonam when used in combination with other drugs. B Indicates the minimum inhibitory concentration of paromomycin sulfate when used alone; C B The minimum inhibitory concentration (MIC) of paromomycin sulfate is indicated when the drugs are used in combination; FICI represents the fractional inhibitory concentration index.

[0049] In vitro interaction (static) experiments showed that meropenem combined with paromomycin sulfate exhibited a significant synergistic effect against carbapenem-resistant Klebsiella pneumoniae producing OXA-48, NDM, KPC, IMP, and VIM (FICI < 0.5); aztreonam combined with paromomycin sulfate also showed a significant synergistic effect against NDM-producing carbapenem-resistant Klebsiella pneumoniae. Paromomycin sulfate could reduce the inhibitory concentration of meropenem or aztreonam, thus indicating that paromomycin sulfate could enhance the inhibitory effect of meropenem or aztreonam against carbapenem-resistant Klebsiella pneumoniae.

[0050] Example 2 The in vitro interaction (dynamic) of meropenem or aztreonam with paromomycin sulfate against carbapenem-resistant Klebsiella pneumoniae was evaluated using the time-killing curve method: The specific procedure is as follows: Using a sterile loop, pick up bacterial colonies (NCTC13442, KP26, KP27, KP29, KPn24, NCTC13439) cultured overnight at 35°C from an MHA plate, dissolve them in sterile PBS, and adjust the concentration to 1×10⁻⁶ using a turbidimetric tube. 9 CFU / mL, diluted to a working concentration of 1×10⁻⁶ CFU / mL with CAMHB medium. 6CFU / mL, and then the bacterial suspension at the above working concentration was divided into four groups: one group as the control group, and the other three groups were meropenem or aztreonam alone group, paromomycin sulfate alone group, and meropenem or aztreonam and paromomycin sulfate combination group. The target concentration was prepared by adding the mother liquor of the active pharmaceutical ingredient to the bacterial suspension at the target concentration (corresponding to CFU / mL in Example 1). A C B C A +C B After preparation, 100 μL of the solution was added to each well of a 96-well plate, with three replicates per well (technical reproducibility). The 96-well plates were sealed with sealing film and placed in a microplate reader. The incubation temperature was set to 35 ℃, and kinetics were monitored at 600 nm. The OD value was measured every 1 h, and the experiment was terminated after 24 h of OD measurements. The OD values ​​at each time point were analyzed and plotted to obtain a time-killing curve.

[0051] In vitro interaction results (dynamic) of meropenem and paromomycin sulfate in combination against carbapenem-resistant Klebsiella pneumoniae. Figures 9-14 As shown.

[0052] Compared with the control group or the drug monotherapy group, the combination of meropenem and paromomycin sulfate synergistically inhibited carbapenem-resistant Klebsiella pneumoniae that produce OXA-48, NDM, KPC, IMP and VIM.

[0053] In vitro interaction results (dynamic) of aztreonam and paromomycin sulfate in combination against carbapenem-resistant Klebsiella pneumoniae. Figure 15 As shown.

[0054] Compared with the control group or the drug-only group, the combination of aztreonam and paromomycin sulfate synergistically inhibits NDM-producing carbapenem-resistant Klebsiella pneumoniae.

[0055] Example 3 In vivo efficacy assay of meropenem or aztreonam in combination with paromomycin sulfate against carbapenem-resistant Klebsiella pneumoniae (using a large wax moth infection model): Larvae of the large wax moth, weighing 200±10 mg, were selected. The experimental strain KP26 was cultured overnight at 35 °C in CAMHB medium, and the bacterial suspension was adjusted to the following concentration using a turbidity analyzer: 1×10⁻⁶. 9CFU / mL. Using a 50 μL microsyringe, 10 μL of bacterial suspension was injected into the peritoneal cavity of the larvae through the first right hind leg. Thirty minutes after infection, the larvae were randomly divided into four groups: a control group, a meropenem or aztreonam monotherapy group, a paromomycin sulfate monotherapy group, and a meropenem or aztreonam and paromomycin sulfate combination group, with 15 larvae in each group. The control group of *Meretrix meretrix* larvae was injected with 10 μL of sterile PBS to reduce the error margin for potential lethality. The other three groups were injected with 10 μL of the drug: 2 μg / larva in the meropenem or aztreonam monotherapy group, 2 μg / larva in the paromomycin sulfate monotherapy group, and 2 μg / larva in both the meropenem or aztreonam and paromomycin sulfate combination group. After injection, the larvae of each group were placed in glass petri dishes and incubated at 37 ℃. The survival of the larvae was recorded daily until the 4th day. Time-survival curves were plotted, the average survival time of each group of large wax moths was calculated, and the differences between groups were statistically analyzed.

[0056] The formula for calculating average survival time is: (D1×1+D2×2+D3×3+S3×4) / 15, where D n S1 is the number of animals that died on day n, and S2 is the number of animals that survived on day three.

[0057] The time-survival curve of meropenem combined with paromomycin sulfate for the treatment of carbapenem-resistant Klebsiella pneumoniae KP26 infection in the large wax moth is as follows: Figure 16 As shown in Table 3, the average survival time results are as follows.

[0058] The time-survival curve of aztreonam combined with paromomycin sulfate for the treatment of carbapenem-resistant Klebsiella pneumoniae KP26 infection in the large wax moth is shown below. Figure 17 As shown in Table 4, the average survival time results are as follows.

[0059] Different batches of large wax moth larvae were treated according to the above experimental procedure. Two days after infection and drug intervention, one larva was randomly selected from each group to prepare frozen sections, which were then stained with glycogen (PAS) for histopathological analysis and observation of the large wax moth.

[0060] Histopathological results of meropenem combined with paromomycin sulfate for the treatment of carbapenem-resistant Klebsiella pneumoniae KP26 infection in the large wax moth are as follows: Figure 18 As shown.

[0061] Table 3. Mean survival time of *Klebsiella pneumoniae* after infection with carbapenem-resistant *Klebsiella pneumoniae* KP26.

[0062] * Indicates comparison with control p =0.294, #Indicates comparison with control p =1.000, a Indicates comparison with control p <0.001, b Indicates a comparison with the paromomycin sulfate group p =0.001, c Compared with the meropenem group p =0.002.

[0063] The results showed that, compared with the control group or the drug-only group, the combination of meropenem and paromomycin sulfate significantly improved the survival rate of carbapenem-resistant Klebsiella pneumoniae KP26 moth. Figure 16 ), and prolong their survival time (Table 3). The combined treatment group showed almost no visible black patches on tissue sections, indicating that the combined use of the two treatments significantly reduced the bacterial load in the body infected with the large wax moth (Table 3). Figure 18 ).

[0064] Histopathological results of the combined use of aztreonam and paromomycin sulfate for the treatment of carbapenem-resistant Klebsiella pneumoniae KP26 infection in the large wax moth are as follows: Figure 19 As shown.

[0065] Table 4. Mean survival time of *Klebsiella pneumoniae* after infection with carbapenem-resistant *Klebsiella pneumoniae* KP26.

[0066] * Indicates comparison with control p =0.526, # Indicates comparison with control p =1.000, a Indicates comparison with control p =0.001, b Indicates a comparison with the paromomycin sulfate group p =0.002, c Compared with the meropenem group p =0.008.

[0067] The results showed that, compared with the control group or the drug-only group, the combination of aztreonam and paromomycin sulfate significantly improved the survival rate of carbapenem-resistant Klebsiella pneumoniae KP26 moth. Figure 17 ), and prolong their survival time (Table 4). The combined treatment group showed almost no visible black patches on tissue sections, indicating that the combined use of the two treatments significantly reduced the bacterial load in the body infected with the large wax moth (Table 4). Figure 19 ).

[0068] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of paromomycin sulfate in the preparation of drugs that enhance the antibacterial effects of meropenem or aztreonam; wherein the bacterium is Klebsiella pneumoniae.

2. The application as described in claim 1, characterized in that, The Klebsiella pneumoniae mentioned is a carbapenem-resistant Klebsiella pneumoniae.

3. The application as described in claim 1, characterized in that, The mass ratio of paromomycin sulfate to meropenem is (0.5~64):(0.25~64). The mass ratio of paromomycin sulfate to aztreonam is (2~64):(8~64).

4. The application of meropenem or aztreonam in combination with paromomycin sulfate in the preparation of antibacterial drugs; the bacteria is Klebsiella pneumoniae.

5. The application as described in claim 4, characterized in that, The Klebsiella pneumoniae mentioned is a carbapenem-resistant Klebsiella pneumoniae.

6. The application as described in claim 4, characterized in that, The mass ratio of paromomycin sulfate to meropenem is (0.5~64):(0.25~64). The mass ratio of paromomycin sulfate to aztreonam is (2~64):(8~64).

7. An antibacterial drug, characterized in that, Including meropenem or aztreonam and paromomycin sulfate; the bacteria is Klebsiella pneumoniae; the Klebsiella pneumoniae is carbapenem-resistant Klebsiella pneumoniae.

8. The antibacterial drug as described in claim 7, characterized in that, The mass ratio of paromomycin sulfate to meropenem is (0.5~64):(0.25~64). The mass ratio of paromomycin sulfate to aztreonam is (2~64):(8~64).

9. The antibacterial drug as described in claim 7, characterized in that, The dosage forms of the antibacterial drugs are suspensions, emulsions, granules, sprays, injections, transdermal absorption agents, dosage forms suitable for transfection, tablets, powders, granules, or capsules.

10. A pharmaceutical composition, characterized in that, Includes the antibacterial drug as described in any one of claims 7 to 9.

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