Application of synergistic effect of antibiotic and antibacterial peptide Chicken Cath-3 in the treatment of drug-resistant pseudomonas aeruginosa infection

Abstract

The application belongs to the technical field of biological medicine, and discloses application of an antibacterial peptide Chicken Cath-3 and antibiotics in resisting drug-resistant pseudomonas aeruginosa infection. The application verifies that the antibacterial peptide Chicken Cath-3 and CTX combined drug resistance has obvious synergistic effect on the drug-resistant pseudomonas aeruginosa through the chessboard method minimum inhibitory concentration test and the in-vitro time-kill curve experiment, and the antibacterial peptide Chicken Cath-3 can significantly improve the bactericidal capacity of the CTX antibiotic on the multi-drug-resistant pseudomonas aeruginosa strain. The application provides a new combined drug strategy, and provides a new research idea and direction for solving the increasingly serious drug resistance problem of the pseudomonas aeruginosa.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of the antimicrobial peptide Chicken Cath-3 in synergistic antibiotics against drug-resistant Pseudomonas aeruginosa infection. Background Technology

[0002] Pseudomonas aeruginosa, a pathogen capable of infecting various animals and causing a range of serious diseases, has shown increasing resistance to antibiotics in recent years. This bacterium not only forms biofilms, increasing the difficulty of treatment, but also poses a significant challenge to clinical care, becoming a major threat to global public health. Given the severe situation of Pseudomonas aeruginosa resistance, the development of novel antimicrobial combinations and treatment regimens targeting this pathogen is urgently needed.

[0003] Antibiotics are a class of chemical substances produced by microorganisms that can inhibit or kill other microorganisms at low concentrations. Since their discovery, they have played a significant role in human health. They are used not only to prevent and treat infectious diseases in animals but also exhibit antitumor, antiviral, and insecticidal / weed-controlling functions in agriculture. However, with the widespread use of antibiotics in disease prevention and control, especially their unscientific and irrational misuse, the problems of bacterial resistance and drug residues are becoming increasingly prominent.

[0004] Antimicrobial peptides, as naturally occurring small polypeptides encoded by the organism's genes, are key molecules in the immune system, exhibiting direct killing effects against bacteria, fungi, viruses, and even protozoa and tumor cells. Compared to traditional antibiotic treatment, antimicrobial peptides (AMPs) have several significant advantages: they can not only simultaneously combat multiple pathogens, but also directly kill microorganisms or indirectly kill pathogens by activating the immune system, effectively killing bacteria while also possessing anti-inflammatory and wound-healing functions. Most importantly, compared to traditional antibiotics, AMPs typically induce lower transient resistance. Furthermore, combining antibiotics with antimicrobial peptides can effectively improve treatment outcomes. This means that the simultaneous or sequential use of two or more antibiotics to treat diseases caused by microbial infections offers numerous advantages, including enhanced drug efficacy and reduced drug toxicity. Currently, there are no reports on the application of the antimicrobial peptide Chicken Cath-3 in combination with antibiotics against drug-resistant Pseudomonas aeruginosa infections. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for enhancing the antibacterial activity of antibiotics. This method combines a natural antimicrobial peptide derived from chicken, Chicken Cath-3, with the antibiotic CTX (cefotaxime). The combination is not merely an additive effect, but rather achieves a synergistic antimicrobial effect. This invention also discloses the antimicrobial combination of the antimicrobial peptide Chicken Cath-3 and the antibiotic CTX, providing a new treatment strategy for the clinical treatment of drug-resistant Pseudomonas aeruginosa infections, especially multidrug-resistant Pseudomonas aeruginosa infections.

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

[0007] The first aspect of this invention relates to the use of the antimicrobial peptide Chicken Cath-3 in combination with cefotaxime in the preparation of a medicament against drug-resistant Pseudomonas aeruginosa. The amino acid sequence of the antimicrobial peptide Chicken Cath-3 is shown in SEQ ID NO.1, specifically RVKRFWPLVPVAINTVAAGINLYKAIRRK.

[0008] Preferably, the drug-resistant Pseudomonas aeruginosa is a multidrug-resistant Pseudomonas aeruginosa.

[0009] More preferably, the multidrug resistance refers to resistance to three or more antibiotics or antibacterial drugs, such as penicillins, fosfomycins, penicillins, aminoglycosides, β-lactams, quinolones, tetracyclines, cephalosporins, glycylcyclines, and macrolides.

[0010] More preferably, the multidrug resistance is resistance to any three or more of the following antibiotics: FOS, KAN, AMP, FFC, TET, CTX, TIG, CS, MEM, and CIP.

[0011] In some specific preferred embodiments of the present invention, the standard strain of Pseudomonas aeruginosa is ATCC 27853, and the multidrug-resistant Pseudomonas aeruginosa strains include DYE7 (resistant to antibiotics such as FOS, KAN, AMP, FFC, etc.), DYC7 (resistant to antibiotics such as TET, KAN, AMP, FFC, etc.), DS-C41-1 (resistant to antibiotics such as FOS, CTX, TIG, CS, etc.), DS-C44-1 (resistant to antibiotics such as FOS, CTX, TIG, etc.), JMA210 (resistant to antibiotics such as FOS, MEM, CTX, TIG, etc.), and JMA36 (resistant to antibiotics such as FOS, CIP, CTX, TIG, etc.).

[0012] A second aspect of the present invention is to provide an antibacterial composition comprising the above-mentioned antibacterial peptide Chicken Cath-3 and cefotaxime.

[0013] Preferably, the concentration of the antimicrobial peptide Chicken Cath-3 in the composition is 0.5–4 μg / ml, and the concentration of cefotaxime is 1–32 μg / ml.

[0014] The antibacterial composition can be used to inhibit multidrug-resistant Pseudomonas aeruginosa and has a synergistic effect. In the in vitro bactericidal experiment of the present invention, Pseudomonas aeruginosa is first cultured and incubated to the logarithmic growth phase, and then a certain concentration of the antimicrobial peptide Chicken Cath-3 and cefotaxime are co-incubated with the diluted bacterial solution for combined bactericidal action.

[0015] Preferably, the multidrug-resistant Pseudomonas aeruginosa strains are DYE7, DYC7, DS-C41-1, DS-C44-1, JMA210, and JMA36.

[0016] A third aspect of the present invention is to provide the use of the above-described antibacterial composition in the preparation of a medicament against drug-resistant Pseudomonas aeruginosa infection.

[0017] Preferably, the drug contains the antimicrobial peptide Chicken Cath-3, cefotaxime, and pharmaceutically acceptable carriers and excipients.

[0018] Preferably, the drug dosage form can be selected from any of the following: capsules, sustained-release or controlled-release tablets, oral liquids, injections, lyophilized powder for injection, and creams.

[0019] Experiments have shown that a combination of the antimicrobial peptide Chicken Cath-3 and cefotaxime can be used to treat drug-resistant Pseudomonas aeruginosa infections, with synergistic enhancement of bactericidal effects.

[0020] This invention also provides the application of the above-mentioned antimicrobial peptide Chicken Cath-3 in the preparation of an antimicrobial synergist that enhances the antimicrobial efficacy of the antibiotic CTX against drug-resistant Pseudomonas aeruginosa infection. Compared with the traditional combination of antibiotics for antimicrobial purposes, this invention develops an antibiotic synergistic alternative and provides a new use for the natural antimicrobial peptide Chicken Cath-3 as an antimicrobial synergist for antibiotic drugs.

[0021] Compared with the prior art, the beneficial effects of this application are as follows:

[0022] This invention, through checkerboard analysis and bactericidal curves, reveals that the combined use of antimicrobial peptide Chicken Cath-3 and cefotaxime produces a significant antibacterial effect against drug-resistant Pseudomonas aeruginosa, and the effect is superior to that of either drug alone. This indicates that the combined use of antimicrobial peptide Chicken Cath-3 and cefotaxime has a significant synergistic effect, which can significantly improve the bactericidal ability of antibiotics and has broad application prospects in the field of combating drug-resistant bacterial infections. Attached Figure Description

[0023] Figure 1 The time-bactericidal curve of the combined use of antimicrobial peptide Chicken Cath-3 and cefotaxime against DYE7 is shown.

[0024] Figure 2 The time-bactericidal curve of the combined use of antimicrobial peptide Chicken Cath-3 and cefotaxime against JMA36 is shown.

[0025] Figure 3 The time-bactericidal curve of the combined use of antimicrobial peptide Chicken Cath-3 and cefotaxime against DS-C41-1 is shown. Detailed Implementation

[0026] 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.

[0027] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods; the materials and reagents used are commercially available conventional reagents and materials, unless otherwise specified.

[0028] Example 1: Combined antimicrobial effect of antimicrobial peptide Chicken Cath-3 and CTX against Pseudomonas aeruginosa

[0029] 1. Material preparation

[0030] The bacterial strains ATCC 27853, DYE7, DYC7, DS-C41-1, DS-C44-1, JMA210, and JMA36 used in the experiment were all commercially available. The antimicrobial peptide Chicken Cath-3 was purchased from Hubei Qiangyao Biotechnology Co., Ltd., synthesized using a solid-phase chemical synthesis method, and purified by reversed-phase chromatography to a purity higher than 95%. Its amino acid sequence is RVKRFWPLVPVAINTVAAGINLYKAIRRK. The powdered Chicken Cath-3 was diluted to 2000 μg / mL with sterile ultrapure water, aliquoted into 1 mL tubes, and stored at -80℃ for later use.

[0031] 2. MIC determination of Chicken Cath-3 and CTX against Pseudomonas aeruginosa

[0032] The minimum inhibitory concentration (MIC) of the test strains was determined using the microbroth dilution method. 200 μL of MH broth, 2 μL of *Pseudomonas aeruginosa* cultured for 4–6 h to the logarithmic growth phase, and two-fold serially diluted CTX (maximum total concentration 1024 μg / mL) and two-fold serially diluted Chicken Cath-3 (maximum total concentration 256 μg / mL) were added to 96-well polypropylene microplates. Broth containing only the drug and no bacterial culture was used as a negative control, and broth containing only the drug and no bacterial culture was used as a positive control. Each test drug solution was performed in triplicate and incubated at 37°C for 18 h. The MIC results were recorded. The sensitivity of the test strains to different drugs was determined by referring to the breakpoint range specified by CLSI.

[0033] 3. Checkerboard method analysis to determine the FICI of Chicken Cath-3 combined with CTX against Pseudomonas aeruginosa.

[0034] Standard Pseudomonas aeruginosa ATCC27853 and multidrug-resistant Pseudomonas aeruginosa DYE7, DYC7, DS-C41-1, DS-C44-1, JMA210, and JMA36 were cultured for 4-6 hours. During the logarithmic growth phase, 50 μL of broth was added to columns 1-8 of a 96-well plate using Chicken Cath-3 as drug A and CTX as drug B. The mixed drug solution was diluted to 4 times its average MIC, and drug B was diluted to 4 times its average MIC. 50 μL of Chicken Cath-3 (4 times its MIC) was added to wells 1-8 of column H. Using an 8-channel pipette, the solution in column H was mixed by pipetting, and 50 μL was transferred to column G, mixed by pipetting 15 times. This process was repeated until the solution in column B was reached, and the last 50 μL of solution was discarded. The concentration of drug B was adjusted to 1.5 mL. Seven concentrations were prepared in EP tubes, ranging from 4 MIC to 1 / 4 MIC. Then, following a low-to-high concentration gradient, 50 μL of diluent was added to columns 2 through 8, with 50 μL of broth added to the first column. Finally, 100 μL of diluted bacterial culture was added to each well, and the tubes were incubated at 37°C for 18–22 h. The experiment was repeated three times. Results interpretation: According to Fratini et al., the fractional inhibitory concentration index (FICI) was calculated using the following formula:

[0035]

[0036] Wherein MICA and MICB are the MIC values ​​when A and B are used alone, respectively, and MICAB and MICBA are the MIC values ​​of drug A and drug B when A and B are used in combination, respectively.

[0037] The criteria for determining FICI values ​​are as follows:

[0038] When FICI ≤ 0.5, it indicates a synergistic effect between A and B in antibacterial activity. When 0.5 < FICI ≤ 1.0, it indicates an additive effect between A and B. When 1 < FICI ≤ 2, it indicates irrelevance between A and B in antibacterial activity. When FICI > 2, it indicates an antagonistic relationship between A and B in antibacterial activity. The calculation results are shown in Table 1.

[0039] Table 1

[0040]

[0041] As can be seen from Table 1, for the above-mentioned multidrug-resistant Pseudomonas aeruginosa DYE7, DYC7, DS-C41-1, DS-C44-1, JMA210, and JMA36, there is a synergistic antibacterial effect (FICI ≤ 0.5) when Chicken Cath-3 is combined with CTX, especially for the clinical multidrug-resistant Pseudomonas aeruginosa DYE7, the synergistic effect is significant.

[0042] Example 2 The experimental procedure of the time-killing curve in vitro of Chicken Cath-3 and CTX against multidrug-resistant Pseudomonas aeruginosa is as follows:

[0043] (1) Use a disposable inoculation loop to pick a single colony from the preserved strain into 4 mL of sterilized LB broth in a test tube and incubate at 37°C and 180 rpm overnight.

[0044] (2) Take 200 μL of the overnight culture broth to measure the OD600 value, and adjust it to OD600 in the range of 0.1 - 0.15 by diluting with autoclaved PBS.

[0045] (3) Take 20 μL of the diluted bacterial solution and dilute it 100-fold and transfer it to 2 mL of LB broth. Set three replicates for each group. Take 25 μL for gradient dilution and drop plating. Incubate at 37°C in an inverted position and record it as 0 h, and continue to shake and culture.

[0046] (4) Add drug treatments to the CTX single-drug MIC group, Cath-3 single-drug MIC group, Cath-3-CTX combined drug group, and blank group for different strains.

[0047] (5) At 2, 4, 6, 8, 10, and 12 h after drug treatment, take 25 μL of the bacterial solution for gradient dilution and drop plating. Set three replicates for each group and incubate at 37°C in an inverted position.

[0048] (6) Calculate the bacterial solution concentration based on the average colony count, and use GraphPad Prism V8.3.0 software to make the bacterial growth curve for 12 h and perform data analysis.

[0049] The experimental results are as Figure 1-3 As shown, by Figure 1 It was found that, compared with the control group, the Cath-3 group, and the CTX group, the combined use of Cath-3 and CTX significantly reduced the growth of clinically multidrug-resistant Pseudomonas aeruginosa DYE7. Against multidrug-resistant Pseudomonas aeruginosa JMA 36 and DS-C41-1, the Cath-3 group showed almost no antibacterial effect, but the combined use of Cath-3 and CTX significantly enhanced the antibacterial effect.

[0050] Obviously, the above embodiments of the present invention are merely examples to clearly illustrate the technical solution of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. The use of an antibacterial composition in the preparation of a medicament against multidrug-resistant Pseudomonas aeruginosa infection, characterized in that, The composition comprises the antimicrobial peptide Chicken Cath-3 and cefotaxime, wherein the amino acid sequence of the antimicrobial peptide Chicken Cath-3 is shown in SEQ ID NO. 1, and the multidrug-resistant Pseudomonas aeruginosa strain is DYE7, DYC7, DS-C41-1, DS-C44-1, JMA210, or JMA36; the concentration of the antimicrobial peptide Chicken Cath-3 in the composition is 0.5–4 μg / mL, and the concentration of cefotaxime is 1–32 μg / mL.

2. The application according to claim 1, characterized in that, The drug contains the antimicrobial peptide Chicken Cath-3, cefotaxime, and pharmaceutically acceptable carriers and excipients.

3. The application according to claim 1, characterized in that, The dosage form of the drug is selected from any one of capsules, sustained-release or controlled-release tablets, oral liquids, injections, lyophilized powder for injection, and creams.

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