Peptide composition compounded with antibiotics and application thereof

Abstract

The present application relates to the technical field of biotechnology, in particular to a peptide composition compounded with antibiotics and application thereof, wherein the peptide composition compounded with antibiotics comprises antibacterial peptides and antibiotics, and can also comprise other pharmaceutical effective components, adjuvants, carriers or auxiliary components. The present application also discloses application of the peptide composition in antibacterial aspect, wherein the peptide composition can effectively improve the bactericidal effect of antibiotics, ensure that the antibacterial effect is not affected, and reduce the use of antibiotics. Compared with the prior art, the bactericidal effect of the peptide composition is superior to that of either one of the peptide or the antibiotic alone, especially for drug-resistant pseudomonas aeruginosa and acinetobacter, and the peptide composition has high safety, no hormone, and has broad-spectrum, high-efficiency and safe antibacterial activity, so that the peptide composition has good commercial application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a peptide composition in combination with antibiotics and its application. Background Technology

[0002] Since penicillin was first used clinically in 1943, antibiotics have played a significant role in treating infectious diseases and protecting human health. However, with the increase in their use, the emergence of irrational applications such as overuse and abuse has not only resulted in a large waste of antibiotic drugs, but also led to an increase in bacterial resistance. Antibiotic abuse and the resulting large number of antibiotic-resistant bacteria are among the greatest threats to global public health, food safety and economic development today (Li Xingxing, Tao Liang. Clostridium difficile infection - a global public health threat caused by antibiotic abuse [J]. Science, 2021, 73(03):40-42+49+4.).

[0003] In 2017, the World Health Organization published its first list of antibiotic-resistant "priority pathogens," listing 12 bacterial species that pose the greatest threat to human health. These included bacteria such as Acinetobacter baumannii and Pseudomonas aeruginosa, which are resistant to carbapenems. Both Pseudomonas aeruginosa and Acinetobacter baumannii have natural resistance genes, which makes them prone to multidrug resistance (MDR) (Liu Guiqin, Jiang Cuixia. Analysis of the changes in drug resistance of Acinetobacter baumannii and Pseudomonas aeruginosa from 2017 to 2019 [J]. Community Medicine Journal, 2021, 19(13): 807-810.). Some strains have even become insensitive to all antimicrobial drugs, which brings great difficulty to clinical treatment (Zhang Lingyan. Analysis of drug resistance and prognostic risk factors of Acinetobacter baumannii bloodstream infection [D]. Anhui Medical University, 2021.).

[0004] Acinetobacter baumannii and Pseudomonas aeruginosa have become the most serious bacterial species with antibiotic resistance problems. They are defined as non-fermenting Gram-negative bacilli with the highest drug resistance rate and the highest hospital infection rate, posing a huge risk of infection to immunocompromised patients in hospitals (Cheng Xiangqun, Guo Qinglian, Huang Jin, Chen Wei. Drug resistance analysis of clinical strains of Pseudomonas aeruginosa and Acinetobacter baumannii [J]. Journal of Clinical Hematology, 2016, 29(12):998-999.).

[0005] Among the many drugs for treating Acinetobacter baumannii and Pseudomonas aeruginosa, fluoroquinolone antibiotics, represented by levofloxacin, are widely used due to their broad-spectrum antibacterial activity, strong antibacterial effect, good oral absorption, high tissue concentration, and other pharmacokinetic characteristics and pharmacodynamic properties. They also have strong antibacterial activity against most Gram-negative bacteria (Gao Yun, Wang Jie, Ding Hongzi, Yan Hong, Lei Lili. Correlation between frequency of antibacterial drug use and drug resistance of Pseudomonas aeruginosa [J]. Northwest Pharmaceutical Journal, 2021, 36(03):504-507.). However, with the increase in its usage, the occurrence of unreasonable applications such as overuse and abuse has led to a decrease in the sensitivity of fluoroquinolone antibacterial drugs to bacteria and an increase in bacterial resistance (He Xiaojing, Song Fang, Li Xiaobing, Guan Lingyan. Pharmacodynamics and resistance mechanism of levofloxacin to rats with chronic lung infection of Pseudomonas aeruginosa [J]. Chinese Journal of Hospital Pharmacy, 2019, 39(17):1734-1740.).

[0006] For these multidrug-resistant strains, carbapenem antibiotics are generally considered to be the "trump card" for humans in the fight against bacteria. Meropenem is the first 1β-methylcarbapenem antibiotic, which is stable against β-lactamases and has the characteristics of strong antibacterial activity and broad antibacterial spectrum. Meropenem, a synthetic broad-spectrum carbapenem antibiotic, has significant anti-infective and antibacterial effects. Studies have reported that meropenem is effective in treating Acinetobacter baumannii and Pseudomonas aeruginosa infections, and is one of the effective antibiotics currently available for the treatment of Acinetobacter baumannii (Tang Qiufeng, Liu Fang, Liu Yu, Xue Genshan. Effects of different concentrations of meropenem on biofilm formation ability of clinically isolated Acinetobacter baumannii [J / OL]. Chinese Journal of Nosocomial Infection, 2022(01):6-10.). It is also a commonly used and effective anti-Pseudomonas aeruginosa drug in clinical practice (Pang Cailian, Liu Minjuan, Lin Jingtao, Chen Kaini, Huang Jiaying, Wu Pin, Zhou Zhengwei, Xu Yanru. Enzyme production and drug resistance analysis of carbapenem-resistant Pseudomonas aeruginosa in Dongguan area [J]. Hainan Medical Journal, 2021, 32(21):2797-2799.).

[0007] With the increasing use of carbapenem antibiotics, according to the CHINET bacterial resistance monitoring report in China, the resistance rate of Acinetobacter spp. to meropenem in my country rose from 39.0% in 2010 to 70.5% in 2021.

[0008] The increasingly serious drug resistance problem of Acinetobacter baumannii and Pseudomonas aeruginosa has aroused high vigilance from all sectors (Xia Fangyuan, Jin Ou, Tao Zhen, Pan Yu, Xu Xuemei, Tong Aijiao. Correlation analysis between drug resistance rate of Pseudomonas aeruginosa and Acinetobacter baumannii and frequency of antimicrobial drug use [J]. Chinese Journal of Clinical Pharmacy, 2021, 30(04): 263-267.). It is well known that the use of antimicrobial drugs is closely related to bacterial drug resistance. Long-term and large-scale use of antimicrobial drugs will inevitably lead to an increase in bacterial drug resistance. For example, the increased frequency of levofloxacin use is significantly positively correlated with the drug resistance of Pseudomonas aeruginosa (Gao Yun, Wang Jie, Ding Hongzi, Yan Hong, Lei Lili. Correlation between frequency of antimicrobial drug use and drug resistance of Pseudomonas aeruginosa [J]. Northwest Pharmaceutical Journal, 2021, 36(03): 504-507.).

[0009] Existing research data suggests that bacterial resistance is becoming increasingly serious. Foreign studies have confirmed that the more antibiotics are used, the more likely it is to induce bacterial resistance and colonization of drug-resistant strains, and even cause outbreaks of drug-resistant bacterial infections (Qing Changchun. Analysis of related factors and prevention and control strategies of drug resistance of Gram-negative bacteria in hospitals from 2016 to 2019 [J]. Journal of Rational Clinical Drug Use, 2021, 14(19):37-41.).

[0010] Therefore, addressing the problem of antibiotic resistance in pathogens is of paramount importance. Besides strictly controlling antibiotic use, the rational selection and use of antimicrobial drugs is the most effective way to prevent and treat infectious diseases. Consequently, the combined use of antimicrobial drugs is increasingly widely applied in clinical practice to address cross-infection caused by multidrug-resistant pathogens, reduce drug toxicity, and slow the emergence of drug-resistant strains (Feng Qi. Study on the synergistic antimicrobial effects of α-spiral antimicrobial peptides with traditional antibiotics in vivo and in vitro [D]. Jilin University, 2014.).

[0011] Antimicrobial peptides are a class of bioactive polypeptides encoded by specific genes within organisms. They possess broad-spectrum antimicrobial activity, inhibiting or killing bacteria, fungi, viruses, and parasites. They are also mild and non-irritating, and have unparalleled advantages in terms of drug resistance. Antimicrobial peptides interact with microbial cell membranes, forming pores and affecting the osmotic pressure inside and outside the cell, thereby causing cell death. Since the structure of microbial cell membranes has evolved over millions of years, it is unlikely to undergo significant changes in a short period. Therefore, this antimicrobial mechanism of antimicrobial peptides does not easily induce drug resistance in bacteria. Thus, combining antimicrobial peptides with antibiotics can not only improve therapeutic efficacy but also reduce the dosage of traditional antibiotics, avoiding antibiotic abuse and mitigating the frequency of drug-resistant strains to some extent.

[0012] Existing literature has reported the use of antimicrobial peptides in combination with antibiotics as antibacterial drugs, but not all antimicrobial peptides and antibiotics have a synergistic relationship. Some show additive or unrelated effects, and some even show antagonistic effects.

[0013] He Ping disclosed that the combined drug effect of antimicrobial peptide IDR-1018 and meropenem against Acinetobacter baumannii ATCC BAA-1605 was irrelevant (He Ping. Study on antibacterial and antibiofilm activity of IDR-1018 combined with meropenem or sulbactam [D]. Dalian Medical University, 2018). Rossolini et al. disclosed that the combined use of peptide SET-M33 and meropenem has a synergistic bactericidal effect against Pseudomonas aeruginosa 854 and Acinetobacter baumannii VA566 / 00, while showing an additive effect against Pseudomonas aeruginosa AV65 and Acinetobacter baumannii N50 (Rossolini, Gian, Maria, et al. Synergistic activity profile of an antimicrobial peptide against multidrug-resistant and extensively drug-resistant strains of Gram-negative bacterial pathogens [J]. Journal of Peptide Science An Official Publication of the European Peptide Society, 2017.); Ammar A et al. disclosed that the combined use of peptide H4 and levofloxacin had a synergistic effect against Pseudomonas aeruginosa ATCC 27853 and an additive effect against Pseudomonas aeruginosa ATCC BAA2114 (Ammar A, Mohammed TQ, Ahmad A, et al. Hybridization and antibiotic synergism as a tool for reducing the cytotoxicity of antimicrobial peptides[J]. Infection & Drug Resistance, 2018, Volume 11:835-847.); Feng, Q. et al. disclosed that the combined use of antimicrobial peptides PL-5, PL-18, and PL-26 with levofloxacin had a synergistic effect against Pseudomonas aeruginosa (Feng, Q., Huang, Y., Chen, M. et al. Functional synergy of α-helical antimicrobial peptides and traditional antibiotics against Gram-negative and Gram-positive bacteria in vitro and in vivo.Eur J Clin MicrobiolInfect Dis 34,197–204(2015).). .

[0014] Therefore, when different peptides are used in combination with the same antibiotic, or when the same peptide is used in combination with the same antibiotic, their antibacterial effects on different strains of the same bacterial species can have synergistic, additive, irrelevant, or antagonistic effects.

[0015] Meanwhile, Zharkova MS disclosed that the antimicrobial peptide protegrin 1, when used in combination with meropenem, has a synergistic effect against Acinetobacter 7226 / 16 and an additive effect against Pseudomonas aeruginosa MDR522 / 17. When protegrin 1 is used in combination with levofloxacin, it also has an additive effect against Acinetobacter 7226 / 16 and an additive effect against Pseudomonas aeruginosa MDR522 / 17. Zharkova MS also disclosed that the antimicrobial peptide ChBac3.4, when used in combination with meropenem, has a synergistic effect against Acinetobacter 7226 / 16 and an additive effect against Pseudomonas aeruginosa MDR522 / 17. When ChBac3.4 is used in combination with levofloxacin, it also has a synergistic effect against Acinetobacter 7226 / 16 and an additive effect against Pseudomonas aeruginosa MDR522 / 17 (Zharkova MS, Orlov DS, Golubeva OY, et al. Application of Antimicrobial Peptides of the Innate Immune System inCombination With Conventional Antibiotics—A Novel Way to Combat AntibioticResistance[J]. Frontiers in Cellular and Infection Microbiology, 2019, 9.).

[0016] Therefore, it can be seen that when the same antimicrobial peptide is used in combination with the same antibiotic, even if there is a synergistic effect in the bactericidal effect against Acinetobacter baumannii and Pseudomonas aeruginosa, the synergistic effect cannot be achieved in all cases.

[0017] During their research, the inventors unexpectedly discovered that the combined use of antibiotics and the antimicrobial peptides described in this invention can effectively improve the bactericidal effect of antibiotics and reduce the dosage. Furthermore, it can simultaneously exert a strong synergistic bactericidal effect against Pseudomonas aeruginosa and Acinetobacter baumannii, effectively alleviating the drug resistance of resistant strains and showing good market application prospects. Summary of the Invention

[0018] To address the shortcomings of existing technologies, one objective of this invention is to provide a peptide composition in combination with an antibiotic, and a second objective is to provide an application of the peptide composition in combination with an antibiotic, characterized in that it comprises: an antimicrobial peptide and an antibiotic, wherein the amino acid sequence of the antimicrobial peptide is SEQ ID NO.1, and the combination of the antimicrobial peptide and the antibiotic provides a stronger bactericidal effect than either of them alone.

[0019] Preferably, the antibiotic includes levofloxacin.

[0020] Preferably, the antibiotic includes meropenem.

[0021] Preferably, the bacteria are Gram-negative bacteria.

[0022] Preferably, the Gram-negative bacterium is Pseudomonas aeruginosa.

[0023] Preferably, the Gram-negative bacterium is Acinetobacter baumannii.

[0024] In particular, the bactericidal effect provided by the combination of the antimicrobial peptide and the antibiotic is a synergistic effect.

[0025] Specifically, the peptide composition includes other pharmaceutical active ingredients, excipients, carriers, or auxiliary ingredients.

[0026] The application of a peptide composition in combination with an antibiotic for the treatment of bacterial infections.

[0027] Specifically, the diseases mentioned include pneumonia, urinary tract infections, gynecological infections, skin infections, soft tissue infections, meningitis, and various cavity infections.

[0028] Preferably, the pneumonia is hospital-acquired pneumonia.

[0029] Preferably, the cavity infections include genitourinary system infections, respiratory tract infections, intestinal infections, abdominal cavity, biliary tract and pelvic cavity infections, etc.

[0030] This invention provides a peptide composition for use with antibiotics and its application. The peptide composition for use with antibiotics of this invention comprises antimicrobial peptides and antibiotics, and may also include other active pharmaceutical ingredients, excipients, carriers, or auxiliary components. Preferably, the antibiotic is levofloxacin or meropenem. The antimicrobial peptide of this invention can be used in combination with levofloxacin or meropenem and has a synergistic bactericidal effect against drug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii. The peptide composition can effectively improve the bactericidal effect of antibiotics, ensuring that the antimicrobial effect is not affected while reducing the use of antibiotics. It is also highly safe, does not easily induce drug resistance, and has broad-spectrum, highly efficient, and safe bactericidal activity. Detailed Implementation

[0031] To address the shortcomings of existing technologies, one objective of this invention is to provide a peptide composition in combination with an antibiotic, and a second objective is to provide an application of the peptide composition in combination with an antibiotic, characterized in that it comprises: an antimicrobial peptide and an antibiotic, wherein the amino acid sequence of the antimicrobial peptide is SEQ ID NO.1, and the combination of the antimicrobial peptide and the antibiotic provides a stronger bactericidal effect than either of them alone.

[0032] Preferably, the antibiotic includes levofloxacin.

[0033] Preferably, the antibiotic includes meropenem.

[0034] Preferably, the bacteria are Gram-negative bacteria.

[0035] Preferably, the Gram-negative bacterium is Pseudomonas aeruginosa.

[0036] Preferably, the Gram-negative bacterium is Acinetobacter baumannii.

[0037] In particular, the bactericidal effect provided by the combination of the antimicrobial peptide and the antibiotic is a synergistic effect.

[0038] Specifically, the peptide composition includes other pharmaceutical active ingredients, excipients, carriers, or auxiliary ingredients.

[0039] The application of a peptide composition in combination with an antibiotic for the treatment of bacterial infections.

[0040] Specifically, the diseases mentioned include pneumonia, urinary tract infections, gynecological infections, skin infections, soft tissue infections, meningitis, and various cavity infections.

[0041] Preferably, the pneumonia is hospital-acquired pneumonia.

[0042] Preferably, the cavity infections include genitourinary system infections, respiratory tract infections, intestinal infections, abdominal cavity, biliary tract and pelvic cavity infections, etc.

[0043] This invention provides a peptide composition for use with antibiotics and its application. The peptide composition for use with antibiotics of this invention comprises antimicrobial peptides and antibiotics, and may also include other active pharmaceutical ingredients, excipients, carriers, or auxiliary components. Preferably, the antibiotic is levofloxacin or meropenem. The antimicrobial peptide of this invention can be used in combination with levofloxacin or meropenem and has a synergistic bactericidal effect against drug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii. The peptide composition can effectively improve the bactericidal effect of antibiotics, ensuring that the antimicrobial effect is not affected while reducing the use of antibiotics. It is also highly safe, does not easily induce drug resistance, and has broad-spectrum, highly efficient, and safe bactericidal activity. Detailed Implementation

[0044] One object of the present invention is to provide a peptide composition in combination with an antibiotic, and a second object is to provide an application of the peptide composition in combination with an antibiotic, characterized in that it comprises: an antimicrobial peptide and an antibiotic, wherein the amino acid sequence of the antimicrobial peptide is SEQ ID NO.1, and the combination of the peptide and the antibiotic provides a stronger bactericidal effect than either of them alone.

[0045] Preferably, the antibiotic includes levofloxacin.

[0046] Preferably, the antibiotic includes meropenem.

[0047] Preferably, the bacteria are Gram-negative bacteria.

[0048] Preferably, the Gram-negative bacterium is Pseudomonas aeruginosa.

[0049] Preferably, the Gram-negative bacterium is Acinetobacter baumannii.

[0050] In particular, the bactericidal effect provided by the combination of the antimicrobial peptide and the antibiotic is a synergistic effect.

[0051] In particular, the peptide composition includes other pharmaceutical active ingredients, excipients, carriers or auxiliary ingredients, and may contain formulation materials for improving the bactericidal effect of the composition, reducing the amount of antibiotics used, or altering, maintaining or preserving, for example, the pH, color, odor, or stability of the composition.

[0052] Other active pharmaceutical ingredients, excipients, carriers, or auxiliary ingredients are not listed here. Those skilled in the art can make specific selections based on their general knowledge.

[0053] The application of a peptide composition in combination with an antibiotic for the treatment of bacterial infections.

[0054] Specifically, the diseases mentioned include pneumonia, urinary tract infections, gynecological infections, skin infections, soft tissue infections, meningitis, and various cavity infections.

[0055] Preferably, the pneumonia is hospital-acquired pneumonia.

[0056] Preferably, the cavity infections include genitourinary system infections, respiratory tract infections, intestinal infections, abdominal cavity, biliary tract and pelvic cavity infections, etc.

[0057] Preferably, the main medium or carrier of the composition can be aqueous or non-aqueous in nature, such as purified water if suitable.

[0058] Those skilled in the art can select appropriate excipients, carriers, or auxiliary components based on the dosage form to be prepared, in accordance with the general technical knowledge and requirements of such dosage form in the art, and add appropriate excipients and additives to the peptide, and prepare it in accordance with conventional formulation techniques.

[0059] The formulations of the present invention can be prepared by any suitable method known in the art, and can be reduced or adjusted according to the needs of practical applications.

[0060] Those skilled in the art can determine the preferred concentration suitable for a given situation using conventional methods.

[0061] The antimicrobial peptides described in this invention can be obtained through chemical synthesis or through expression, separation and purification using genetic engineering techniques (for specific methods, please refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, ColdSpring Harbor, NY, 2001).

[0062] Unless otherwise specified, the terms "peptide composition" and "composition" used in this invention are interchangeable and refer to products containing both antibiotics and the antimicrobial peptides described in this invention as active ingredients. They may also contain other active pharmaceutical ingredients, excipients, carriers, or auxiliary ingredients.

[0063] The term "excipient, carrier or auxiliary ingredient" used in this invention refers to non-toxic liquid diluents or other pharmaceutical excipients or carriers, which can be used to formulate the composition according to actual needs.

[0064] The term "multidrug resistance (MDR)" used in this invention refers to strains that are resistant to three series of drugs: aminoglycosides, fluoroquinolones, and carbapenems.

[0065] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the disclosure as defined solely by the claims and the specification.

[0066] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and all reagents added in the embodiments are commercially available unless otherwise specified.

[0067] The embodiments described herein further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the present invention are within the scope of the present invention.

[0068] Since Pseudomonas aeruginosa and Acinetobacter baumannii have become the most serious bacterial species with antibiotic resistance problems, and are defined as non-fermenting Gram-negative bacilli with the highest drug resistance rate and the highest hospital infection rate, multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii were selected as test strains in the examples.

[0069] Example 1: In vitro antimicrobial test of antimicrobial peptides, levofloxacin and meropenem. Source of test strains: All strains were clinically isolated multidrug-resistant pathogens collected from Sichuan, Guangdong and Beijing from May 2015 to December 2020.

[0070] Strains and strain numbers: Pseudomonas aeruginosa PAE 20-1 (MDR), Pseudomonas aeruginosa PAE 20-2 (MDR), Acinetobacter baumannii ABA 17-2 (MDR), Acinetobacter baumannii ABA 17-3 (MDR), Acinetobacter baumannii ABA 17-5 (MDR), Acinetobacter baumannii ABA 17-6 (MDR).

[0071] Levofloxacin: Batch number 130455-201607, white powder, specification 100mg / vial, content 97.3%, store at 4℃ protected from light, manufactured by China National Institutes for Food and Drug Control, preparation method is to prepare with sterile ultrapure water and dilute stepwise with the corresponding culture medium to the required solution, and use immediately after preparation.

[0072] Meropenem: Batch number D0715A, white powder, specification 1g / vial, content 98.06%, store at 4℃ protected from light, manufacturer is Dalian Meilun Biotechnology Co., Ltd., preparation method is to prepare with sterile ultrapure water and dilute stepwise with culture medium to the required solution, and use immediately after preparation.

[0073] Culture medium and culture conditions: CAMHB (Cation Adjusted Mueller-Hinton) medium, incubated in air at 35-37℃ for 16-20 hours.

[0074] CAMHB formula: acid-hydrolyzed casein (17.5g / L), beef powder (3.0g / L), soluble starch (1.5g / L), calcium ions (20-25mg / L), magnesium ions (10-12.5mg / L).

[0075] Experimental methods: The MIC (Minimal Inhibitory Concentration) values ​​of each test sample against the tested bacterial strain were determined using the microbroth dilution method recommended by the Clinical and Laboratory Standards Institute (CLSI) [Methods for Dilution Antimicrobial Succeptibility Tests for Bacteria That Grow Aerobically; Approved Standard-Tenth Edition, M07-A10, 2015].

[0076] Specific procedures: Add 100 μL of solutions of different concentrations of antimicrobial peptide, levofloxacin, and meropenem to the wells of a sterilized 96-well polystyrene plate, respectively, to achieve final concentrations of 64 mg / L, 32 mg / L, 16 mg / L, 8 mg / L, 4 mg / L, 2 mg / L, and 1 mg / L for the antimicrobial peptide; and final concentrations of levofloxacin, respectively, to achieve final concentrations of 64 mg / L, 32 mg / L, 16 mg / L, 8 mg / L, and 4 mg / L. The final concentrations of meropenem were 2 mg / L, 1 mg / L, 0.5 mg / L, 0.25 mg / L, 0.125 mg / L, 0.1 mg / L, and 0.05 mg / L, respectively. Then, 100 μL of the test bacterial culture (200 μL per well) was added to each well, resulting in a final bacterial concentration of approximately 10. 5 CFU / mL. After sealing, incubate at 37°C for 24 hours, then determine the results. The lowest drug concentration that completely inhibits bacterial growth in the well is the minimum inhibitory concentration (MIC).

[0077] The experimental results are shown in Table 1.

[0078] Table 1. In vitro antibacterial activity of each drug group when used alone The criteria for judging bacterial susceptibility and resistance to levofloxacin and meropenem (CLSI 2020) are shown in Table 2.

[0079] Table 2. Criteria for judging susceptibility and resistance to levofloxacin and meropenem (CLSI 2020) Tables 1 and 2 show that levofloxacin alone had MIC values ​​of 8-64 mg / L against the two tested Pseudomonas aeruginosa strains and four tested Acinetobacter baumannii strains, falling within the resistance range. Meropenem alone had MIC values ​​of 4-32 mg / L against the two tested Pseudomonas aeruginosa strains and four tested Acinetobacter baumannii strains, also falling within the resistance range. Antimicrobial peptides had MIC values ​​of 8-64 mg / L against the two tested Pseudomonas aeruginosa strains and four tested Acinetobacter baumannii strains.

[0080] Example 2: In vitro antimicrobial activity test of antimicrobial peptides combined with levofloxacin. Fractional inhibitory concentration (FIC) index is one of the pharmacodynamic (PD) parameters of antimicrobial drugs. It is a combined drug sensitivity test of two antimicrobial drugs. That is, when two antimicrobial drugs are used at the same time, four situations can occur: synergistic, antagonistic, irrelevant and additive.

[0081] Calculation of FIC index: FIC index = MIC (drug A in combination) / MIC (drug A alone) + MIC (drug B in combination) / MIC (drug B alone).

[0082] FIC index interpretation criteria: When the FIC index < 0.5, the two drugs have a synergistic effect; when the FIC index is 0.5-1, the two drugs have an additive effect; when the FIC index is 1-2, the two drugs have no effect; when the FIC index > 2, the two drugs have an antagonistic effect.

[0083] Since both antimicrobial peptides and antibiotics have antibacterial effects, the FIC index was selected to evaluate the effect of their combined use.

[0084] 50 μL of antimicrobial peptide and levofloxacin solutions of different concentrations were added to the wells of a sterile 96-well polystyrene plate, respectively, to achieve final concentrations of 64 mg / L, 32 mg / L, 16 mg / L, 8 mg / L, 4 mg / L, 2 mg / L, and 1 mg / L for the antimicrobial peptides, and 64 mg / L, 32 mg / L, 16 mg / L, 8 mg / L, 4 mg / L, 2 mg / L, 1 mg / L, 0.5 mg / L, 0.25 mg / L, 0.125 mg / L, 0.1 mg / L, and 0.05 mg / L for the levofloxacin. Then, 100 μL of the test bacterial culture (200 μL per well) was added to each well, resulting in a final bacterial concentration of approximately 10. 5 CFU / mL. After sealing, incubate at 37℃ for 24 hours, then interpret the results.

[0085] The specific values ​​are shown in Table 3 based on the results of the 96-well plate.

[0086] Table 3. In vitro antibacterial activity of antimicrobial peptides in combination with levofloxacin As shown in Table 3, the FIC index of the two tested Pseudomonas aeruginosa strains and four Acinetobacter baumannii strains was <0.5 after the combination of antimicrobial peptide and levofloxacin, indicating a synergistic effect.

[0087] Example 3: In vitro antibacterial activity test of antimicrobial peptide combined with meropenem. 50 μL of antimicrobial peptide and meropenem solutions of different concentrations were added to the wells of a sterilized 96-well polystyrene plate, resulting in final concentrations of antimicrobial peptides of 64 mg / L, 32 mg / L, 16 mg / L, 8 mg / L, 4 mg / L, 2 mg / L, and 1 mg / L; and final concentrations of meropenem of 64 mg / L, 32 mg / L, 16 mg / L, 8 mg / L, 4 mg / L, 2 mg / L, 1 mg / L, 0.5 mg / L, 0.25 mg / L, 0.125 mg / L, 0.1 mg / L, and 0.05 mg / L. Then, 100 μL of the test bacterial solution (200 μL per well) was added to each well, resulting in a final bacterial concentration of approximately 10%. 5 CFU / mL. After sealing, incubate at 37℃ for 24 hours, then interpret the results.

[0088] The specific values ​​are shown in Table 4 based on the results of the 96-well plate.

[0089] Table 4. In vitro antimicrobial activity of antimicrobial peptides in combination with meropenem As shown in Table 4, the combination of antimicrobial peptide and meropenem resulted in FIC indices of <0.5 against the two tested Pseudomonas aeruginosa strains and four Acinetobacter baumannii strains, indicating a synergistic effect.

[0090] In summary, the antimicrobial peptides described in this invention, when combined with antibiotics, exhibit a synergistic antibacterial effect against multidrug-resistant Pseudomonas aeruginosa and Acinetobacter bacilli. Furthermore, the combined use demonstrates good bactericidal activity against both Pseudomonas aeruginosa and Acinetobacter bacilli, reducing the required antibiotic concentration. The polypeptides described in this invention are composed of 19 amino acids, are non-cytotoxic and non-irritating, and their degradation products are natural amino acids, producing no drug residues and causing no irritation. The combination of antimicrobial peptides and antibiotics demonstrates high safety, is unlikely to induce drug resistance, and shows promising application prospects.

[0091] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention. sequence list <110> Zhejiang Yingkang Biomedical Co., Ltd. Nanning Jirui Biomedical Co., Ltd. <120> A peptide composition in combination with antibiotics and its application <141> 2022-03-10 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> PRT <213> Artificial Sequence <400> 1 Met Gly Arg Phe Lys Arg Phe Arg Lys Lys Phe Lys Lys Leu Phe Lys 1 5 10 15 Lys Leu Ser

Claims

1. A peptide composition in combination with an antibiotic, characterized in that... It comprises: an antimicrobial peptide and an antibiotic, wherein the amino acid sequence of the antimicrobial peptide is SEQ ID NO.1, and the antibiotic is levofloxacin or meropenem, and the combination of the antimicrobial peptide and the antibiotic provides a stronger bactericidal effect than either of them alone.

2. The peptide composition according to claim 1, characterized in that... The bacteria in question are Gram-negative.

3. The peptide composition according to claim 2, characterized in that... The Gram-negative bacterium mentioned is Pseudomonas aeruginosa.

4. The peptide composition according to claim 2, characterized in that... The Gram-negative bacterium mentioned is Acinetobacter baumannii.

5. The peptide composition according to any one of claims 1-4, characterized in that... The combination of the antimicrobial peptide and the antibiotic provides a synergistic bactericidal effect.

6. The peptide composition according to any one of claims 1-4, characterized in that... The peptide composition includes excipients, carriers, or auxiliary components.

7. The peptide composition according to claim 5, characterized in that... The peptide composition includes excipients, carriers, or auxiliary components.

8. The use of the peptide composition according to any one of the preceding claims in the preparation of a medicament for treating diseases caused by Pseudomonas aeruginosa or Acinetobacter baumannii infection.

9. The application according to claim 8, characterized in that... The bacterial infections mentioned include pneumonia, gynecological infections, skin infections, soft tissue infections, meningitis, and various cavity infections.

10. The application according to claim 9, characterized in that... The pneumonia in question is hospital-acquired pneumonia.

11. The application according to claim 9, characterized in that... The cavity infections include genitourinary system infections, respiratory tract infections, intestinal infections, abdominal cavity, biliary tract, and pelvic cavity infections.

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