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CN115192593B - Application of daunorubicin in treating multi-drug resistant bacteria infection diseases - Google Patents

️Tue Jan 23 2024

Application of daunorubicin in treating multi-drug resistant bacteria infection diseases Download PDF

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Publication number

CN115192593B

CN115192593B CN202210820471.1A CN202210820471A CN115192593B CN 115192593 B CN115192593 B CN 115192593B CN 202210820471 A CN202210820471 A CN 202210820471A CN 115192593 B CN115192593 B CN 115192593B Authority

China

Prior art keywords

daunorubicin

polymyxin

tigecycline

drug

bacteria

Prior art date

2022-07-13

Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)

Active

Application number

CN202210820471.1A

Other languages

Chinese (zh)

Other versions

CN115192593A (en

Inventor

刘源

王志强

邓天

蔡金菊

史静茹

Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

Yangzhou University

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Yangzhou University

Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)

2022-07-13

Filing date

2022-07-13

Publication date

2024-01-23

2022-07-13 Application filed by Yangzhou University filed Critical Yangzhou University

2022-07-13 Priority to CN202210820471.1A priority Critical patent/CN115192593B/en

2022-10-18 Publication of CN115192593A publication Critical patent/CN115192593A/en

2024-01-23 Application granted granted Critical

2024-01-23 Publication of CN115192593B publication Critical patent/CN115192593B/en

Status Active legal-status Critical Current

2042-07-13 Anticipated expiration legal-status Critical

Links

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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y02A40/70—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in livestock or poultry
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract

本发明公开了柔红霉素或其可接受的盐在制备细菌感染性疾病药物或制备抗生素增效剂中的应用。柔红霉素恢复耐药革兰阴性菌对多粘菌素和替加环素敏感性的作用及应用，协同抗菌活性表明柔红霉素与多粘菌素/替加环素具有显著的协同作用，且对耐药菌的协同作用强于敏感菌。本发明提供的抗肿瘤药物柔红霉素能够与抗生素联合应用在体内外均有较强的抗菌效果，并系统评价了体内外有效性，稳定性以及安全性，以及对细菌耐药性进化和流行传播的影响，该研究的发现将为MCR和Tet(X)介导的耐药病原菌的防控提供新思路。

The invention discloses the use of daunorubicin or an acceptable salt thereof in the preparation of drugs for bacterial infectious diseases or the preparation of antibiotic synergists. The role and application of daunorubicin in restoring the sensitivity of drug-resistant Gram-negative bacteria to polymyxin and tigecycline. The synergistic antibacterial activity shows that daunorubicin has significant synergy with polymyxin/tigecycline. effect, and the synergistic effect on drug-resistant bacteria is stronger than that on sensitive bacteria. The anti-tumor drug daunorubicin provided by the invention can be used in combination with antibiotics to have strong antibacterial effects both in vivo and in vitro. The effectiveness, stability and safety in vivo and in vitro are systematically evaluated, as well as the evolution and development of bacterial resistance. The findings of this study will provide new ideas for the prevention and control of MCR- and Tet(X)-mediated drug-resistant pathogens.

Description

柔红霉素在治疗多重耐药菌感染疾病中的应用Application of daunorubicin in the treatment of multidrug-resistant bacterial infections

技术领域Technical field

本发明属于医药技术领域，具体涉及柔红霉素在治疗多重耐药菌感染疾病中的应用。The invention belongs to the field of medical technology, and specifically relates to the application of daunorubicin in treating multi-drug resistant bacterial infections.

背景内容Background content

1928年英国微生物学家弗莱明首先发现了世界上第一种抗生素—青霉素。当前，抗生素被广泛应用于人医临床中，治疗多种细菌感染，拯救了无数生命。在畜禽养殖中，抗生素不仅同样用于治疗细菌感染，而且还被添加到饲料中促进动物生长和预防疾病。然而抗生素的不合理使用，导致细菌耐药性问题越来越严重，严重限制了抗生素的临床使用。抗生素耐药性被公认为是21世纪对公共卫生最严重的全球威胁之一。目前抗生素治疗在应对细菌感染方面占据主导地位。然而目前新型抗生素的研发能力严重不足，具有独特靶点的新型抗生素的研发越来越困难，近几十年没有一种专门针对革兰阴性菌的新型抗生素被批准。新投入市场的大多数药物只是现有抗生素的改进版本，这显然难以满足当今迫切的需求。而且尽管新型抗生素最初在临床中应用效果良好，但是当细菌逐渐对其适应后，容易产生相应的耐药性，并不断进化。为应对日益严重的多重耐药革兰阴性菌感染，人们试图探索其它的解决办法，目前一种公认的有效的对策是开发抗菌增效剂。这些化合物一般本身没有或只具备微弱的抗菌活性，但是与现有抗生素联合使用时，可减少细菌耐药性的产生，增强抗生素的作用，恢复或增强常用抗生素对多重耐药的革兰阴性菌的活性。相比于研发新药，其优势在于成本相对较低，且安全高效。在临床上，抗生素与相应的抗菌增效剂已得到应用，并取得显著效果。其中，具有代表性的增效剂甲氧苄啶能阻断二氢叶酸还原成四氢叶酸，与能阻断二氢叶酸合成的磺胺类药物联合使用，可产生协同抗菌作用，使体内的叶酸代谢受到双重阻断，抗菌作用增强数倍至数十倍。In 1928, British microbiologist Fleming first discovered the world's first antibiotic, penicillin. Currently, antibiotics are widely used in clinical human medicine to treat a variety of bacterial infections and save countless lives. In livestock and poultry breeding, antibiotics are not only used to treat bacterial infections, but are also added to feed to promote animal growth and prevent disease. However, the irrational use of antibiotics has led to an increasingly serious problem of bacterial resistance, severely limiting the clinical use of antibiotics. Antibiotic resistance is recognized as one of the most serious global threats to public health in the 21st century. Antibiotic treatment currently dominates the response to bacterial infections. However, the current research and development capabilities of new antibiotics are seriously insufficient, and the development of new antibiotics with unique targets is becoming more and more difficult. In recent decades, no new antibiotics specifically targeting Gram-negative bacteria have been approved. Most of the new drugs coming to the market are simply improved versions of existing antibiotics, which clearly cannot meet today's urgent needs. And although new antibiotics initially work well in clinical applications, when bacteria gradually adapt to them, they are prone to develop corresponding resistance and continue to evolve. In order to deal with the increasingly serious infections caused by multidrug-resistant Gram-negative bacteria, people are trying to explore other solutions. Currently, a recognized and effective countermeasure is to develop antibacterial synergists. These compounds generally have no or only weak antibacterial activity on their own, but when used in combination with existing antibiotics, they can reduce the occurrence of bacterial resistance, enhance the effect of antibiotics, and restore or enhance the effectiveness of commonly used antibiotics against multi-resistant Gram-negative bacteria. activity. Compared with the development of new drugs, its advantages are that it is relatively low-cost, safe and efficient. In clinical practice, antibiotics and corresponding antibacterial synergists have been used and achieved remarkable results. Among them, the representative synergist trimethoprim can block the reduction of dihydrofolate into tetrahydrofolate. When used in combination with sulfa drugs that block the synthesis of dihydrofolate, it can produce a synergistic antibacterial effect and increase the concentration of folic acid in the body. Metabolism is double blocked, and the antibacterial effect is enhanced several times to dozens of times.

多粘菌素和替加环素被认为是是治疗MDR革兰阴性菌感染的“最后一道防线”。然而由于这两种药物的大量使用甚至滥用，不可避免地产生了相应的耐药性。近年来相继被发现的新型耐药基因mcr-1和tet(X4)造成了多粘菌素和替加环素耐药性在全球的广泛传播，使得这两种药物的临床应用受到极大挑战。因此，迫切需要开发新的抗菌增效剂来治疗多重耐药菌引起的感染性疾病。Polymyxin and tigecycline are considered the "last line of defense" in the treatment of MDR Gram-negative bacterial infections. However, due to the extensive use and even abuse of these two drugs, corresponding drug resistance has inevitably developed. The new drug resistance genes mcr-1 and tet(X4) that have been discovered in recent years have caused the widespread spread of polymyxin and tigecycline resistance around the world, posing great challenges to the clinical application of these two drugs. . Therefore, there is an urgent need to develop new antibacterial synergists to treat infectious diseases caused by multidrug-resistant bacteria.

发明内容Contents of the invention

发明目的：为了解决现有技术存在的问题，本发明系统探究了抗肿瘤药物柔红霉素作为新型抗菌增效剂的潜力。本发明发现抗菌增效剂柔红霉素和抗生素联合应用以对抗日益严重的多重耐药菌感染。Purpose of the invention: In order to solve the problems existing in the existing technology, the present invention systematically explores the potential of the anti-tumor drug daunorubicin as a new antibacterial synergist. The present invention finds that the antibacterial synergist daunorubicin and antibiotics can be used in combination to combat increasingly serious multi-drug resistant bacterial infections.

技术方案：为了解决上述技术问题，本发明提供了柔红霉素或其可接受的盐在制备细菌感染性疾病药物或制备抗生素增效剂中的应用。Technical solution: In order to solve the above technical problems, the present invention provides the application of daunorubicin or its acceptable salt in the preparation of bacterial infectious disease drugs or the preparation of antibiotic synergists.

本发明的内容还包括所述的柔红霉素作为抗菌增效剂在制备相关具有生物体内免疫调节活性产品或药物中的应用。其中，所述生物指畜禽。The content of the present invention also includes the application of the daunorubicin as an antibacterial synergist in the preparation of related products or drugs with immunomodulatory activity in vivo. Wherein, the living creatures refer to livestock and poultry.

其中，所述细菌感染性疾病药物或抗生素增效剂为单组分或复方制剂。Wherein, the bacterial infectious disease drug or antibiotic synergist is a single component or a compound preparation.

其中，所述细菌为革兰氏阳性菌或革兰氏阴性菌。Wherein, the bacteria are Gram-positive bacteria or Gram-negative bacteria.

其中，所述抗生素为多粘菌素或替加环素。Wherein, the antibiotic is polymyxin or tigecycline.

本发明内容还包括一种药物组合物，所述药物组合物包括柔红霉素和多粘菌素；或柔红霉素和替加环素。The present invention also includes a pharmaceutical composition comprising daunorubicin and polymyxin; or daunorubicin and tigecycline.

其中，所述的药物组合物中，所述柔红霉素的浓度为0.5～64μg/mL。Wherein, in the pharmaceutical composition, the concentration of daunorubicin is 0.5-64 μg/mL.

其中，所述的药物组合物中，所述多粘菌素的浓度为0.0625-16μg/mL，所述替加环素的浓度为0.5-32μg/mL。Wherein, in the pharmaceutical composition, the concentration of polymyxin is 0.0625-16 μg/mL, and the concentration of tigecycline is 0.5-32 μg/mL.

其中，所述柔红霉素和多粘菌素质量比为0.2～1024:1，所述柔红霉素和替加环素质量比为1～128:1。Wherein, the mass ratio of daunorubicin and polymyxin is 0.2-1024:1, and the mass ratio of daunorubicin and tigecycline is 1-128:1.

本发明内容还包括所述的组合物在制备细菌感染性疾病的药物或抗生素增效剂中的应用。The present invention also includes the application of the composition in preparing drugs or antibiotic synergists for bacterial infectious diseases.

其中，所述细菌感染性疾病的药物或抗生素增效剂的剂型为片剂、胶囊、缓释片、控释片、口服液、糖浆、滴丸、注射液剂型、冻干粉针剂型。Wherein, the dosage forms of the drugs for bacterial infectious diseases or antibiotic synergists are tablets, capsules, sustained-release tablets, controlled-release tablets, oral liquids, syrups, dripping pills, injection dosage forms, and freeze-dried powder dosage forms.

本发明内容还包括柔红霉素作为抗菌增效剂对耐药菌的进化、突变、耐药基因水平传播的抑制潜力。The content of the present invention also includes the potential of daunorubicin as an antibacterial synergist to inhibit the evolution, mutation and horizontal spread of drug-resistant bacteria.

本发明的柔红霉素恢复耐药革兰阴性菌对多粘菌素或替加环素敏感性的作用及应用，协同抗菌活性表明柔红霉素与多粘菌素或替加环素具有显著的协同作用，且对耐药菌的协同作用强于敏感菌。时间杀菌曲线结果表明，柔红霉素与多粘菌素或替加环素的联合使用可以有效降低对数期和稳定期耐药菌的细菌量。生物膜试验表明，柔红霉素和多粘菌素或替加环素联合使用不仅可以抑制生物膜的形成，还可以有效清除已经成熟的生物膜细菌，柔红霉素与抗生素的组合最高可使生物膜细菌的存活率降至1％以下。红细胞溶血性试验表明，柔红霉素的加入并不会增加抗生素的溶血活性。耐药菌传代和 MPC检测表明耐药菌的进化和突变受到限制。体外和体内接合的结果表明柔红霉素具有抑制耐药基因水平传播的潜力。体内有效性试验表明，柔红霉素和多粘菌素的联合使用，可以显著提高大蜡螟和小鼠的存活率。以上研究表明柔红霉素作为新型抗菌增效剂的潜力，为日益严重的多重耐药革兰阴性菌感染的防控提供了新策略。The effect and application of daunorubicin of the present invention in restoring the sensitivity of drug-resistant Gram-negative bacteria to polymyxin or tigecycline. The synergistic antibacterial activity shows that daunorubicin and polymyxin or tigecycline have the same Significant synergistic effect, and the synergistic effect on drug-resistant bacteria is stronger than that on sensitive bacteria. The time-killing curve results showed that the combined use of daunorubicin and polymyxin or tigecycline can effectively reduce the bacterial load of drug-resistant bacteria in the logarithmic phase and stationary phase. Biofilm experiments show that the combined use of daunorubicin and polymyxin or tigecycline can not only inhibit the formation of biofilm, but also effectively remove mature biofilm bacteria. The combination of daunorubicin and antibiotics can achieve the highest Reduce the survival rate of biofilm bacteria to less than 1%. The red blood cell hemolytic test showed that the addition of daunorubicin did not increase the hemolytic activity of the antibiotic. Passaging and MPC detection of drug-resistant bacteria indicate that the evolution and mutation of drug-resistant bacteria are restricted. In vitro and in vivo conjugation results indicate that daunorubicin has the potential to inhibit the horizontal spread of resistance genes. In vivo effectiveness tests show that the combined use of daunorubicin and polymyxin can significantly improve the survival rate of Galleria mellonella and mice. The above studies demonstrate the potential of daunorubicin as a new antibacterial synergist and provide a new strategy for the prevention and control of increasingly serious multi-drug-resistant Gram-negative bacterial infections.

有益效果：与现有技术相比，本发明的优点是：对于现阶段耐药性问题日益严重的局面，本发明提供的抗肿瘤药物柔红霉素能够与抗生素联合应用在体内外均有较强的抗菌效果，并系统评价了体内外有效性，稳定性以及安全性，以及对细菌耐药性进化和流行传播的影响，该研究的发现将为MCR和Tet(X)介导的耐药病原菌的防控提供新思路。Beneficial effects: Compared with the prior art, the advantage of the present invention is: in view of the increasingly serious problem of drug resistance at this stage, the anti-tumor drug daunorubicin provided by the present invention can be used in combination with antibiotics to achieve better results both in vivo and in vitro. Strong antibacterial effect, and systematically evaluated the effectiveness, stability and safety in vivo and in vitro, as well as the impact on the evolution and epidemic spread of bacterial resistance. The findings of this study will provide new insights into MCR and Tet(X)-mediated resistance. Provide new ideas for the prevention and control of pathogenic bacteria.

附图说明Description of the drawings

图1为柔红霉素(DNR)与各种抗生素的协同抗菌活性探究；Figure 1 shows the study of the synergistic antibacterial activity of daunorubicin (DNR) and various antibiotics;

图2为柔红霉素(DNR)与各种抗生素的协同抗菌活性探究；Figure 2 shows the study of the synergistic antibacterial activity of daunorubicin (DNR) and various antibiotics;

图3为柔红霉素(DNR)与各种抗生素的协同抗菌活性探究；Figure 3 shows the study of the synergistic antibacterial activity of daunorubicin (DNR) and various antibiotics;

图4为柔红霉素(DNR)与各种抗生素的协同抗菌活性探究；Figure 4 shows the study of the synergistic antibacterial activity of daunorubicin (DNR) and various antibiotics;

图5为柔红霉素(DNR)和多粘菌素/替加环素在体外对处于对数期(A)和稳定期(B)的耐药菌的时间杀菌曲线，多粘菌素的浓度为4μg/mL，替加环素的浓度为32 μg/mL，柔红霉素的浓度为32μg/mL；可知柔红霉素与多粘菌素/替加环素对生长迅速或趋于稳定的耐药菌都具备良好的协同杀菌活性。Figure 5 shows the time-killing curves of daunorubicin (DNR) and polymyxin/tigecycline against drug-resistant bacteria in logarithmic phase (A) and stable phase (B) in vitro. The concentration is 4 μg/mL, the concentration of tigecycline is 32 μg/mL, and the concentration of daunorubicin is 32 μg/mL; it can be seen that the growth rate of daunorubicin and polymyxin/tigecycline is rapid or tend to be Stable drug-resistant bacteria all have good synergistic bactericidal activity.

图6为离子稳定性和溶血性实验，可知柔红霉素与多粘菌素/替加环素协同作用的离子稳定性良好，并且柔红霉素几乎不会增强多粘菌素/替加环素对红细胞的溶血性。Figure 6 shows the ion stability and hemolysis experiments. It can be seen that the ion stability of daunorubicin synergistically with polymyxin/tigecycline is good, and daunorubicin hardly enhances polymyxin/tigecycline. Hemolysis of erythrocytes by cyclins.

图7为抗生物膜及持留菌实验，可知柔红霉素可增强多粘菌素/替加环素对生物膜形成的抑制作用和对已成熟生物膜细菌的清除作用。Figure 7 shows the anti-biofilm and persister experiments. It can be seen that daunorubicin can enhance the inhibitory effect of polymyxin/tigecycline on biofilm formation and the elimination of mature biofilm bacteria.

图8为耐药传代试验和MPC试验，可知柔红霉素可以抑制多粘菌素/替加环素耐药基因的进化，且柔红霉素可降低多粘菌素/替加环素对耐药菌的MPC，且呈现出浓度依赖性。Figure 8 shows the resistance passage test and MPC test. It can be seen that daunorubicin can inhibit the evolution of polymyxin/tigecycline resistance genes, and daunorubicin can reduce the resistance of polymyxin/tigecycline to MPC of drug-resistant bacteria, and showed concentration dependence.

图9为体外和体内接合试验，可知柔红霉素能抑制多粘菌素/替加环素耐药基因的水平传播。Figure 9 shows the in vitro and in vivo conjugation tests, which show that daunorubicin can inhibit the horizontal spread of polymyxin/tigecycline resistance genes.

图10为柔红霉素与多粘菌素的急性毒性实验。可知柔红霉素在测试剂量下不存在毒副作用。Figure 10 shows the acute toxicity experiment of daunorubicin and polymyxin. It can be seen that daunorubicin has no toxic side effects at the test dose.

图11为柔红霉素与多粘菌素/替加环素治疗大蜡螟感染、小鼠腹膜炎-败血症感染和小鼠嗜中性粒细胞减少的大腿感染。可知柔红霉素可以在体内恢复多粘菌素对耐药菌的杀菌活性，包括减少体内菌量和提高动物的存活率。其中，Vehicle的含义是空白对照。Figure 11 shows the treatment of Galleria mellonella infection, peritonitis-septicemia infection in mice and neutropenic thigh infection in mice with daunorubicin and polymyxin/tigecycline. It can be seen that daunorubicin can restore the bactericidal activity of polymyxin against drug-resistant bacteria in the body, including reducing the amount of bacteria in the body and improving the survival rate of animals. Among them, Vehicle means blank control.

具体实施方式Detailed ways

下面通过具体的实施例对本发明进一步说明，应当指出，对于本领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干变型和改进，这些也应视为属于本发明的保护范围。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的实验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量实验，均设置三次重复实验，结果取平均值。The present invention will be further described below through specific embodiments. It should be pointed out that for those of ordinary skill in the art, several modifications and improvements can be made without departing from the principles of the present invention, and these should also be regarded as belonging to this invention. protection scope of the invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative experiments in the following examples were repeated three times, and the results were averaged.

MHB肉汤培养基为含牛肉粉1.5g/L、可溶性淀粉1.5g/L和酸水解酪蛋白17.5g/L的水溶液。MHB broth medium is an aqueous solution containing 1.5g/L beef powder, 1.5g/L soluble starch and 17.5g/L acid-hydrolyzed casein.

表1Table 1

注：文献1为Meirong Song,Yuan Liu,Xiaoyong Huang,Shuangyang Ding,YangWang, Jianzhong Shen and Kui Zhu.A broad-spectrum antibiotic adjuvantreverses multidrug-resistant Gram-negative pathogens.Nat.Microbiol.,2020,5(8):1040-1050.Note: Document 1 is Meirong Song, Yuan Liu, Xiaoyong Huang, Shuangyang Ding, YangWang, Jianzhong Shen and Kui Zhu. A broad-spectrum antibiotic adjuvantreverses multidrug-resistant Gram-negative pathogens. Nat. Microbiol., 2020, 5(8) :1040-1050.

文献2为Yuan Liu,Yuqian Jia,Kangni Yang,Ziwen Tong,Jingru Shi,RuichaoLi,Xia Xiao,Wenkai Ren,Rüdiger Hardeland,Russel J Reiter,ZhiqiangWang.Melatonin overcomes MCR-mediated colistin resistance in Gram-negativepathogens.Theranostics.2020, 10(23):10697-10711.Document 2 is Yuan Liu, Yuqian Jia, Kangni Yang, Ziwen Tong, Jingru Shi, RuichaoLi, Xia Xiao, Wenkai Ren, Rüdiger Hardeland, Russel J Reiter, ZhiqiangWang. Melatonin overcomes MCR-mediated colistin resistance in Gram-negativepathogens.Theranostics.2020 , 10(23):10697-10711.

文献3为Yuan Liu,Yuqian Jia,Kangni Yang,Ruichao Li,Xia Xiao,ZhiqiangWang. Anti-HIV agent azidothymidine decreases Tet(X)-mediated bacterialresistance to tigecycline in Escherichia coli.Commun.Biol.2020,3(1):162.Document 3 is Yuan Liu, Yuqian Jia, Kangni Yang, Ruichao Li, Xia Xiao, ZhiqiangWang. Anti-HIV agent azidothymidine decreases Tet(X)-mediated bacterialresistance to tigecycline in Escherichia coli.Commun.Biol.2020,3(1): 162.

文献4为Haijie Zhang,Feiyu Yu,Xiaoyu Lu,Yan Li,Daxin Peng,ZhiqiangWang,Yuan Liu.Rapid detection of MCR-mediated colistin resistance inEscherichia coli.Microbiology Spectrum,2022,10,e0092022.Document 4 is Haijie Zhang, Feiyu Yu, Xiaoyu Lu, Yan Li, Daxin Peng, ZhiqiangWang, Yuan Liu. Rapid detection of MCR-mediated colistin resistance in Escherichia coli. Microbiology Spectrum, 2022, 10, e0092022.

文献5为Rapid and accurate antibiotic susceptibility determination oftet(X)-positive E. coli using RNA biomarkers.Microbiology Spectrum,2021,9,e00648-21.Document 5 is Rapid and accurate antibiotic susceptibility determination oftet(X)-positive E. coli using RNA biomarkers. Microbiology Spectrum, 2021, 9, e00648-21.

文献6为Yuqian Jia,Bingqing Yang,Jingru Shi,Dan Fang,Zhiqiang Wang,Yuan Liu. Melatonin prevents conjugative transfer of plasmid-mediatedantibiotic resistance genes by disrupting proton motive force.PharmacologicalResearch,2022,175,105978.Document 6 is Yuqian Jia, Bingqing Yang, Jingru Shi, Dan Fang, Zhiqiang Wang, Yuan Liu. Melatonin prevents conjugative transfer of plasmid-mediatedantibiotic resistance genes by disrupting proton motive force. Pharmacological Research, 2022, 175, 105978.

实施例1、柔红霉素与不同种类抗生素的协同抗菌活性Example 1. Synergistic antibacterial activity of daunorubicin and different types of antibiotics

采用棋盘肉汤稀释法测定多种抗生素和柔红霉素之间的协同作用，使用了两株MDR菌株，分别为E.coli B2(bla_NDM-5+mcr-1)和E.coli B3-1(tet(X4))。The checkerboard broth dilution method was used to determine the synergy between multiple antibiotics and daunorubicin, using two MDR strains, E.coli B2 (bla _NDM-5 +mcr-1) and E.coli B3- 1(tet(X4)).

96孔平底板的每孔中各加入100μL MHB肉汤培养基，以构建8×8的体系。最后一行前8孔加入100μL不同浓度不同种类的抗生素，向上倍比稀释，到第二行弃去。在第一列加入100μL柔红霉素，向右倍比稀释，到第七列弃去。在孔中分别加入100μL的 1.5×10⁶CFU上述两种菌液。在37℃恒温箱中培养18h后，使用酶标仪测定600nm处的OD值(OD大于0.1，有细菌生长)。设置三个生物学重复。分级抑菌浓度FIC指数按照以下公式进行计算：Add 100 μL of MHB broth medium to each well of a 96-well flat-bottomed plate to construct an 8×8 system. Add 100 μL of different concentrations of different types of antibiotics to the first 8 wells of the last row, dilute upwards, and discard in the second row. Add 100 μL daunorubicin to the first column, dilute to the right, and discard in the seventh column. Add 100 μL of 1.5×10 ⁶ CFU of the above two bacterial solutions to the wells. After culturing for 18 hours in a 37°C incubator, use a microplate reader to measure the OD value at 600 nm (OD is greater than 0.1, indicating bacterial growth). Three biological replicates were set up. The graded inhibitory concentration FIC index is calculated according to the following formula:

FIC＝MIC(A联合应用)/MIC(A单用)+MIC(B联合应用)/MIC(B单用)FIC=MIC(A combined application)/MIC(A single use)+MIC(B combined application)/MIC(B single use)

试验结果见图1。结果表明发现柔红霉素对美罗培南、多粘菌素、替加环素(三种对抗MDR革兰阴性菌感染的“最后一道防线”抗生素)均有协同作用，与多粘菌素的协同活性最强，替加环素次之(FICI分别为0.375、0.125、0.188)，与另外5种抗生素只具有微弱的协同活性或者无活性(FICI＝2)。The test results are shown in Figure 1. The results showed that daunorubicin was found to have a synergistic effect on meropenem, polymyxin, and tigecycline (three "last line of defense" antibiotics against MDR Gram-negative bacterial infections), and the synergy with polymyxin It has the strongest activity, followed by tigecycline (FICI are 0.375, 0.125, and 0.188 respectively), and has only weak synergistic activity or no activity with the other five antibiotics (FICI=2).

接下来使用了本课题组构建的耐药菌E.coli DH5α(PUC19-bla_NDM-5/mcr-1/tet(X4)) 和空载E.coli DH5α(PUC19)，进行棋盘试验。如图2所示，发现柔红霉素分别与这三种抗生素合用对各自的耐药菌都有协同作用，对敏感菌只具有微弱的协同活性或者无活性，这表明柔红霉素对抗生素的协同作用主要针对耐药菌。联系图1的结果，接下来重点研究了柔红霉素与多粘菌素或替加环素的协同作用，各选择了2株常见的耐药临床菌株，分别为大肠杆菌(1株)、肺炎克雷伯菌(2株)、鲍曼不动杆菌(1株)，进行棋盘试验。如图3所示，发现该协同作用对这些MDR革兰阴性菌仍有效。上述结果初步表明了柔红霉素与多粘菌素或替加环素对MDR革兰阴性菌有良好的协同活性。Next, the drug-resistant bacteria E.coli DH5α (PUC19-bla _NDM-5 /mcr-1/tet(X4)) and empty E.coli DH5α (PUC19) constructed by our research group were used to conduct a checkerboard test. As shown in Figure 2, it was found that daunorubicin combined with these three antibiotics has a synergistic effect on their respective drug-resistant bacteria, but has only weak synergistic activity or no activity on sensitive bacteria, which shows that daunorubicin has a synergistic effect on antibiotics. The synergistic effect mainly targets drug-resistant bacteria. Based on the results in Figure 1, we next focused on the synergistic effect of daunorubicin and polymyxin or tigecycline, and selected two common drug-resistant clinical strains, namely E. coli (1 strain), Klebsiella pneumoniae (2 strains) and Acinetobacter baumannii (1 strain) were tested in a checkerboard test. As shown in Figure 3, this synergistic effect was found to be still effective against these MDR Gram-negative bacteria. The above results preliminarily indicate that daunorubicin and polymyxin or tigecycline have good synergistic activity against MDR Gram-negative bacteria.

然后进一步研究了柔红霉素对多粘菌素或替加环素的协同活性。由于上述测试菌株数量较少，所以各使用了多株不同来源的mcr-1/tet(X4)阳性临床菌株，同样通过棋盘肉汤稀释法测定FICI值，结果表明柔红霉素也能恢复这些临床耐药菌株对多粘菌素/替加环素的敏感性，柔红霉素与多粘菌素的协同活性更强(FICI均小于0.125，柔红霉素对多粘菌素的增效倍数均超过16倍)。The synergistic activity of daunorubicin against polymyxin or tigecycline was then further investigated. Due to the small number of the above-mentioned test strains, multiple mcr-1/tet(X4)-positive clinical strains from different sources were used. The FICI value was also determined by the checkerboard broth dilution method. The results showed that daunorubicin can also restore these strains. The sensitivity of clinical drug-resistant strains to polymyxin/tigecycline, the synergistic activity of daunorubicin and polymyxin is stronger (FICI are both less than 0.125, the synergistic effect of daunorubicin on polymyxin The multiples are more than 16 times).

实施例2、柔红霉素和抗生素联合对指数期和稳定期细菌的杀菌曲线。Example 2. Bactericidal curve of daunorubicin and antibiotic combination against bacteria in exponential phase and stationary phase.

把过夜培养的多粘菌素耐药菌E.coli G92和替加环素耐药菌E.coli B3-1分别在MHB中以1:100稀释。在37℃，200r/min摇床中继续培养，把足够菌液分别培养2小时(至指数生长期)和4小时(至稳定期)。然后取出菌液，分别用柔红霉素(32μg/mL) 和多粘菌素(4μg/mL)、柔红霉素(32μg/mL)和替加环素(32μg/mL)单独或联合处理细菌，并设置无药对照组。设置0，4，8，24h四个时间点，在每个时间点，分别吸取100μL菌液，重新悬浮于900μL无菌磷酸盐缓冲液(PBS)。然后在LB琼脂板的每个区域内分别均匀滴50μL连续十倍倍比稀释的悬浮液，并在37℃恒温箱中培养过夜。 18h后，数菌落数，计算各时间点的菌量。设置三个生物学重复。The polymyxin-resistant bacteria E.coli G92 and tigecycline-resistant bacteria E.coli B3-1 cultured overnight were diluted 1:100 in MHB. Continue culturing in a shaker at 37°C and 200r/min, and incubate enough bacterial liquid for 2 hours (to the exponential growth phase) and 4 hours (to the stable phase). Then take out the bacterial liquid and treat it with daunorubicin (32μg/mL) and polymyxin (4μg/mL), daunorubicin (32μg/mL) and tigecycline (32μg/mL) alone or in combination. bacteria, and set up a drug-free control group. Four time points were set at 0, 4, 8, and 24 hours. At each time point, 100 μL of bacterial solution was taken and resuspended in 900 μL sterile phosphate buffer saline (PBS). Then, 50 μL of the continuously ten-fold diluted suspension was evenly dropped into each area of the LB agar plate, and incubated overnight in a 37°C incubator. After 18 hours, count the number of bacterial colonies and calculate the bacterial load at each time point. Three biological replicates were set up.

实验结果见图5。结果表明，无论对处于对数期还是稳定期的耐药菌，4μg/mL的多粘菌素或32μg/mL替加环素均只具备轻微的杀菌活性，32μg/mL的柔红霉素同样作用微弱。然而，其与多粘菌素或替加环素的联用使mcr/tet(X)阳性菌株的菌量大幅减少，例如多粘菌素与柔红霉素的组合使对数期菌量减少约6-log₁₀。这表明柔红霉素与多粘菌素或替加环素对生长迅速或趋于稳定的耐药菌都具备良好的协同杀菌活性。The experimental results are shown in Figure 5. The results showed that 4 μg/mL polymyxin or 32 μg/mL tigecycline only had slight bactericidal activity against drug-resistant bacteria in the logarithmic phase or the stable phase, while 32 μg/mL daunorubicin also had slight bactericidal activity. The effect is weak. However, its combination with polymyxin or tigecycline significantly reduced the bacterial load of mcr/tet(X)-positive strains. For example, the combination of polymyxin and daunorubicin reduced the bacterial load in the logarithmic phase. About 6-log ₁₀ . This shows that daunorubicin and polymyxin or tigecycline have good synergistic bactericidal activity against rapidly growing or stable drug-resistant bacteria.

实施例3、柔红霉素和抗生素联合的离子稳定性和溶血性Example 3. Ionic stability and hemolytic properties of daunorubicin and antibiotic combination

离子稳定性实验：先在MH肉汤培养基(MHB)中分别添加Na⁺、K⁺、Ca²⁺、Mg²⁺、 EDTA(终浓度均为10mM)、10％血清和10％DMEM。然后在96孔平底板中分别加入 100μL上述MHB肉汤培养基，后续棋盘试验具体方法同上。Ion stability experiment: First, add Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , EDTA (the final concentration is 10mM), 10% serum and 10% DMEM respectively in MH broth medium (MHB). Then, 100 μL of the above-mentioned MHB broth culture medium was added to the 96-well flat-bottom plate, and the specific method of the subsequent checkerboard test was the same as above.

溶血性实验：先将柔红霉素加入到磷酸盐缓冲液(PBS)(10mM，pH＝7.4)中稀释，终浓度分别为0，16，32，64μg/mL。然后在96孔平底板前11列加100μL上述磷酸盐缓冲液(PBS)(10mM，pH＝7.4)，第12列加水。在第1列分别加入100μL多粘菌素或替加环素，向右倍比稀释到第10列弃去。设置三个重复。然后准备新鲜羊血，将10mL 羊血于4℃，3000×g离心10min，弃去上清，管底的沉淀用磷酸盐缓冲液(PBS)吹打两次，然后用磷酸盐缓冲液(PBS)重悬。从上述悬液中吸取3.2mL，加入到36.8mL磷酸盐缓冲液(PBS)(10mM，pH＝7.4)中，配置浓度为8％的红细胞悬液。吸取100μL红细胞悬液加入到96孔板中，置于37℃恒温箱中培养1小时。1h后，从中吸取120μL 上清液，加入到新的2mL离心管中，3000×g离心3-5分钟后，吸取100μL加入到新的96孔板中，使用M200型酶标仪测定OD_576。设置三个生物学重复。溶血率的计算公式为：Hemolysis test: First add daunorubicin to phosphate buffer saline (PBS) (10mM, pH=7.4) and dilute it to final concentrations of 0, 16, 32, and 64 μg/mL respectively. Then add 100 μL of the above phosphate buffer solution (PBS) (10 mM, pH=7.4) to the first 11 columns of the 96-well flat bottom plate, and add water to the 12th column. Add 100 μL of polymyxin or tigecycline to column 1, dilute to the right and discard to column 10. Set up three repetitions. Then prepare fresh sheep blood. Centrifuge 10 mL of sheep blood at 4°C and 3000×g for 10 min. Discard the supernatant. Pipette the precipitate at the bottom of the tube twice with phosphate buffer saline (PBS), and then rinse with phosphate buffer saline (PBS). Resuspended. Pipette 3.2 mL from the above suspension and add it to 36.8 mL of phosphate buffer saline (PBS) (10 mM, pH=7.4) to prepare a red blood cell suspension with a concentration of 8%. Pipette 100 μL of red blood cell suspension into a 96-well plate and place it in a 37°C incubator for 1 hour. After 1 hour, take 120 μL of the supernatant and add it to a new 2 mL centrifuge tube. After centrifugation at 3000×g for 3-5 minutes, take 100 μL and add it to a new 96-well plate. Use an M200 microplate reader to measure OD _576. Three biological replicates were set up. The calculation formula for hemolysis rate is:

溶血率(％)＝[(OD_576样品-OD_576空白)/(OD_576水-OD_576空白)]×100％。Hemolysis rate (%) = [(OD _{576 sample} -OD _{576 blank} )/(OD _{576 water} -OD _{576 blank} )] × 100%.

实验结果见表2和图6。结果表明，在MHB肉汤培养基中分别添加两种一价盐离子Na⁺、K⁺、EDTA、10％血清或10％营养培养基DMEM后，柔红霉素分别与两种抗生素的协同活性仍旧存在，并且在EDTA和血清中的活性更强(FICI值小于对照)；然而两种二价离子使柔红霉素分别与抗生素的协同活性丧失(FICI＝2)。溶血性实验结果表明多粘菌素或替加环素单独作用下几乎不会引起溶血。相比于抗生素的单独作用，柔红霉素与多粘菌素或替加环素的联合作用并没有显著提高溶血率，仍旧维持在较低水平。溶血率最高也仅达到2％左右，甚至有的浓度组合还降低了溶血率。这些结果表明柔红霉素几乎不会增强多粘菌素或替加环素对红细胞的溶血性。The experimental results are shown in Table 2 and Figure 6. The results show that after adding two monovalent salt ions Na ⁺ , K ⁺ , EDTA, 10% serum or 10% nutrient medium DMEM to the MHB broth medium, the synergistic activity of daunorubicin and two antibiotics respectively It still exists and is more active in EDTA and serum (FICI value is less than the control); however, the two divalent ions make daunorubicin lose its synergistic activity with antibiotics respectively (FICI=2). Hemolysis test results show that polymyxin or tigecycline alone will hardly cause hemolysis. Compared with the effect of antibiotics alone, the combined effect of daunorubicin and polymyxin or tigecycline did not significantly increase the hemolysis rate and remained at a low level. The highest hemolysis rate is only about 2%, and some concentration combinations even reduce the hemolysis rate. These results indicate that daunorubicin does little to enhance the hemolytic activity of polymyxin or tigecycline on erythrocytes.

表2Table 2

实施例4、柔红霉素和抗生素联合对生物膜的抑制以及清除、杀死持留菌的活性Example 4. The activity of daunorubicin and antibiotics combined to inhibit biofilm and remove and kill persistent bacteria

生物膜抑制实验：把过夜培养的E.coli G92和E.coli B3-1分别以1:100稀释至LB肉汤中，分别向其中加入多粘菌素(0.008、0.016、0.032、0.063μg/mL)、替加环素(0.25、0.5、1、2μg/mL)或多粘菌素与柔红霉素(8μg/mL)的组合；替加环素与柔红霉素(8 μg/mL)的组合，阴性对照不含药。在37℃，200r/min摇床中培养，8h后，取出，用 300μL磷酸盐缓冲液(PBS)洗涤细菌三次，吸出孔内的液体。然后加入200μL甲醇固定15min，吸出液体，自然风干。加入100μL 0.1％结晶紫染色15min。吸出染料溶液，用磷酸盐缓冲液(PBS)洗涤已染色的生物膜2-3次，自然风干。最后，用100μL 33％的冰醋酸溶解被结晶紫染色的生物膜，并在37℃恒温箱中孵育30min。在570nm处测定冰醋酸的吸光度，测定不同药物浓度作用下生物膜的形成量，设置三个生物学重复。Biofilm inhibition experiment: Dilute overnight cultured E.coli G92 and E.coli B3-1 into LB broth at 1:100, and add polymyxin (0.008, 0.016, 0.032, 0.063μg/ mL), tigecycline (0.25, 0.5, 1, 2 μg/mL) or the combination of polymyxin and daunorubicin (8 μg/mL); tigecycline and daunorubicin (8 μg/mL ), the negative control contains no drug. Cultivate in a shaker at 37°C and 200r/min. After 8 hours, take out the bacteria, wash the bacteria three times with 300μL phosphate buffer saline (PBS), and aspirate the liquid in the well. Then add 200 μL methanol to fix for 15 min, aspirate the liquid, and air dry naturally. Add 100 μL of 0.1% crystal violet for staining for 15 min. Aspirate the dye solution, wash the stained biofilm 2-3 times with phosphate buffer saline (PBS), and air dry naturally. Finally, the biofilm stained by crystal violet was dissolved with 100 μL of 33% glacial acetic acid and incubated in a 37°C incubator for 30 min. The absorbance of glacial acetic acid was measured at 570 nm, and the amount of biofilm formation under different drug concentrations was measured. Three biological replicates were set up.

生物膜清除实验：把过夜培养的E.coli G92和E.coli B3-1分别以1:100稀释至LB肉汤中，37℃，200r/min孵育4h。然后将100μL细菌悬液与等体积的MHB肉汤培养基先后加到96孔平底板内。在37℃下孵育48h后，吸去细菌悬液。用多粘菌素(4-32 μg/mL)、替加环素(32-256μg/mL)单独或与8μg/mL柔红霉素联合作用于已形成的生物膜。在37℃下孵育2h后，超声处理20min，将剩余的细胞分散。最后，将混合液在无菌磷酸盐缓冲液(PBS)中重悬，向后十倍倍比稀释，均匀滴在LB琼脂板上，在37℃下孵育过夜，18h后数菌落数，设置三个生物学重复。Biofilm removal experiment: Dilute the overnight cultured E.coli G92 and E.coli B3-1 into LB broth at 1:100, and incubate at 37°C and 200r/min for 4 hours. Then 100 μL of bacterial suspension and an equal volume of MHB broth medium were added to the 96-well flat-bottomed plate. After incubation at 37°C for 48 h, the bacterial suspension was aspirated. Use polymyxin (4-32 μg/mL) and tigecycline (32-256 μg/mL) alone or in combination with 8 μg/mL daunorubicin to act on the formed biofilm. After incubation at 37°C for 2 h, the remaining cells were dispersed by sonication for 20 min. Finally, resuspend the mixture in sterile phosphate buffer saline (PBS), dilute it ten times backward, drop it evenly on the LB agar plate, incubate at 37°C overnight, count the number of colonies after 18 hours, and set three biological replicates.

实验结果见图7，结果表明，相较于多粘菌素或替加环素的单独作用，柔红霉素的加入显著增强了对耐药菌生物膜形成的抑制作用。然后待生物膜形成后，加入药物，以探究柔红霉素对包裹生物膜的细菌的作用。在清除生物膜的过程中，多粘菌素/替加环素和柔红霉素同样表现出协同作用。其中，柔红霉素与本试验中最高浓度的多粘菌素或替加环素的组合使生物膜细菌的存活率降至1％以下。综上结果表明，柔红霉素可增强多粘菌素或替加环素对生物膜形成的抑制作用和对已成熟生物膜细菌的清除作用。The experimental results are shown in Figure 7. The results show that compared with the separate effects of polymyxin or tigecycline, the addition of daunorubicin significantly enhanced the inhibitory effect on the formation of biofilms of drug-resistant bacteria. Then, after the biofilm is formed, the drug is added to explore the effect of daunorubicin on the bacteria surrounding the biofilm. Polymyxin/tigecycline and daunorubicin also showed a synergistic effect in clearing biofilms. Among them, the combination of daunorubicin and the highest concentration of polymyxin or tigecycline in this trial reduced the survival rate of biofilm bacteria to less than 1%. In summary, the above results show that daunorubicin can enhance the inhibitory effect of polymyxin or tigecycline on biofilm formation and the elimination of mature biofilm bacteria.

实施例5、柔红霉素耐药传代和MPC实验Example 5. Daunorubicin resistance passage and MPC experiment

耐药传代试验：把过夜培养的E.coli G92和E.coli B3-1分别在LB肉汤中以1:100稀释至含药的LB肉汤(0.5×MIC的多粘菌素、0.5×MIC的替加环素或其分别与0.25× MIC柔红霉素的组合)。在37℃，200r/min摇床中培养12小时后，将细菌培养物以1:100 稀释，加入到新的含药培养基中，继续下一代培养。每隔四代，测定培养物的MIC。根据测得的MIC，增大两种抗生素和柔红霉素的浓度，分别维持在0.5×MIC和0.25×MIC。连续传代培养24天。Resistance passage test: E.coli G92 and E.coli B3-1 cultured overnight were diluted 1:100 in LB broth to drug-containing LB broth (0.5×MIC of polymyxin, 0.5× MIC of tigecycline or its combination separately with 0.25× MIC of daunorubicin). After culturing for 12 hours in a shaker at 37°C and 200r/min, dilute the bacterial culture at 1:100 and add it to a new drug-containing medium to continue the next generation of culture. Every fourth passage, the MIC of the cultures was determined. According to the measured MIC, the concentrations of the two antibiotics and daunorubicin were increased and maintained at 0.5×MIC and 0.25×MIC respectively. Continuously subculture for 24 days.

MPC测定：配制含不同浓度的多粘菌素、替加环素、或其分别与柔红霉素组合的 LB琼脂板。将100μL浓度为1.0×10¹⁰CFU的E.coli G92和E.coli B3-1分别涂布在各自抗性的琼脂板上，然后在37℃恒温箱中培养。72h后，观察细菌的生长情况，能抑制细菌生长(突变菌落)的最低浓度被定为药物的MPC。MPC assay: Prepare LB agar plates containing different concentrations of polymyxin, tigecycline, or their respective combinations with daunorubicin. 100 μL of E.coli G92 and E.coli B3-1 with a concentration of 1.0 × 10 ¹⁰ CFU were spread on respective resistant agar plates, and then cultured in a 37°C incubator. After 72 hours, observe the growth of the bacteria, and the lowest concentration that can inhibit bacterial growth (mutated colonies) is determined as the MPC of the drug.

实验结果见图8，结果表明，传代结束后，柔红霉素减缓了多粘菌素或替加环素对传代菌的MIC的增长速度。在亚抑菌浓度的抗生素压力下，多粘菌素或替加环素对耐药菌的MIC分别增大8倍和4倍，而加入柔红霉素后，MIC仅分别增长2倍。这表明柔红霉素可以抑制多粘菌素或替加环素耐药基因的进化，对多粘菌素耐药性进化的抑制作用更强。另外MPC测定结果显示，64μg/mL的柔红霉素使多粘菌素对E.coli G92 (mcr-1)的MPC降为原来的1/16；256μg/mL的柔红霉素使替加环素对E.coli B3-1(tet(X4))的MPC同样降为原来的1/16。这表明柔红霉素可降低多粘菌素或替加环素对耐药菌的MPC，且呈现出浓度依赖性。The experimental results are shown in Figure 8. The results show that after the passage, daunorubicin slows down the growth rate of the MIC of polymyxin or tigecycline on the passaged bacteria. Under the pressure of antibiotics at subinhibitory concentrations, the MICs of polymyxin or tigecycline against drug-resistant bacteria increased 8-fold and 4-fold respectively, while after adding daunorubicin, the MICs only increased 2-fold. This suggests that daunorubicin can inhibit the evolution of polymyxin or tigecycline resistance genes, and has a stronger inhibitory effect on the evolution of polymyxin resistance. In addition, the MPC measurement results showed that 64 μg/mL daunorubicin reduced the MPC of polymyxin to E.coli G92 (mcr-1) to 1/16 of the original value; 256 μg/mL daunorubicin reduced the MPC of tiga The MPC of E.coli B3-1(tet(X4)) by cycline was also reduced to 1/16 of the original value. This shows that daunorubicin can reduce the MPC of polymyxin or tigecycline against drug-resistant bacteria in a concentration-dependent manner.

实施例6、体内体外接合实验Example 6. In vivo and in vitro bonding experiments

体外接合试验：根据本课题组以往的研究，选择了几株能稳定接合的临床菌株。mcr-1供体菌为E.coli LD93-1和E.coli LD67-1，tet(X4)供体菌为E.coli RS3-1和E.coliRF2-1。受体菌均为具有利福平抗性基因的E.coli EC600。过夜培养的供体菌和受体菌分别用LB肉汤以1:100稀释，然后在37℃，200r/min摇床中培养到OD₆₀₀为0.5。菌液4℃，5000×g离心6min后，用磷酸盐缓冲液(PBS)重悬。供体菌和受体菌以1:1的比例混合，总体积为2mL。柔红霉素的终浓度设为0，4，8，16，32μg/mL，分别加入到供受体菌的混合物中，在37℃，200r/min摇床中培养18h。提前配制只含利福平(300 μg/mL)的LB琼脂板，以及利福平(300μg/mL)与多粘菌素(2μg/mL)或替加环素 (4μg/mL)的双药板(单药板筛选受体菌，双药板筛选接合子)。菌液在磷酸盐缓冲液 (PBS)中连续10倍稀释，把50μL稀释液滴在各自抗性的含药琼脂板上。在37℃恒温箱培养18h后，分别计数接合子数和受体菌数，计算接合频率(接合频率＝接合子数/ 受体菌数)。设置三个生物学重复。In vitro conjugation test: Based on the previous research of this research group, several clinical strains that can conjugate stably were selected. The mcr-1 donor bacteria are E.coli LD93-1 and E.coli LD67-1, and the tet(X4) donor bacteria are E.coli RS3-1 and E.coliRF2-1. The recipient bacteria were all E.coli EC600 with rifampicin resistance gene. The donor bacteria and recipient bacteria cultured overnight were diluted 1:100 with LB broth, and then cultured in a shaker at 37°C and 200r/min until the OD ₆₀₀ was 0.5. The bacterial solution was centrifuged at 5000×g for 6 min at 4°C and resuspended in phosphate buffer saline (PBS). Donor bacteria and recipient bacteria were mixed at a ratio of 1:1, with a total volume of 2 mL. The final concentrations of daunorubicin were set to 0, 4, 8, 16, and 32 μg/mL, and were added to the mixture of donor and recipient bacteria respectively, and cultured in a shaker at 37°C and 200 r/min for 18 h. Prepare in advance LB agar plates containing only rifampicin (300 μg/mL) and doublets of rifampin (300 μg/mL) and polymyxin (2 μg/mL) or tigecycline (4 μg/mL) Plates (single-drug plate to screen recipient bacteria, double-drug plate to screen zygotes). The bacterial solution was serially diluted 10 times in phosphate buffer saline (PBS), and 50 μL of the diluted solution was dropped onto the respective drug-containing agar plates of the resistance. After culturing in a 37°C incubator for 18 hours, count the number of zygotes and the number of receptor bacteria, and calculate the ligation frequency (ligation frequency = number of zygotes/number of receptor bacteria). Three biological replicates were set up.

体内接合试验：参照体外接合试验结果，mcr-1供体菌选择E.coli LD67-1，tet(X4) 供体菌选择E.coli RF2-1。受体菌仍旧为具有利福平抗性基因的E.coli EC600。供体菌和受体菌以1:1的比例混合。准备足够的雌性ICR小鼠，每组8只。在小鼠腹腔右侧注射200μL供受体菌的混合物。感染30min后，分别腹腔注射200μL磷酸盐缓冲液(PBS) 或柔红霉素(2mg/kg)。给药24小时后，通过颈椎脱臼法处死小鼠。然后取肝脏，研磨充分，在磷酸盐缓冲液(PBS)中连续10倍稀释，把50μL稀释液均匀滴在各自抗性的含药琼脂板上。分别计数接合子数和受体菌数，计算接合频率。设置三个生物学重复。In vivo conjugation test: Referring to the in vitro conjugation test results, E.coli LD67-1 was selected as the mcr-1 donor bacterium, and E.coli RF2-1 was selected as the tet(X4) donor bacterium. The recipient bacterium is still E.coli EC600 with rifampicin resistance gene. Donor and recipient bacteria were mixed in a 1:1 ratio. Prepare enough female ICR mice, 8 in each group. Inject 200 μL of the mixture of donor bacteria into the right side of the mouse's abdominal cavity. 30 minutes after infection, 200 μL of phosphate buffered saline (PBS) or daunorubicin (2 mg/kg) were injected intraperitoneally. 24 hours after administration, the mice were sacrificed by cervical dislocation. Then take the liver, grind it thoroughly, dilute it continuously 10 times in phosphate buffer saline (PBS), and drop 50 μL of the dilution evenly on the respective resistant drug-containing agar plates. Count the number of conjugants and recipient bacteria respectively, and calculate the conjugation frequency. Three biological replicates were set up.

实验结果见图9，结果表明，体外接合试验发现柔红霉素显著降低了mcr-1/tet(X4) 供体菌与受体菌E.coli EC600的接合频率，且呈现浓度依赖性。考虑到柔红霉素在体外良好的抑制作用，通过构建动物模型进行了体内接合试验。体内接合实验表明在小鼠体内，柔红霉素显著降低了mcr-1/tet(X4)供体菌与受体菌E.coli EC600的接合频率，同样呈现浓度依赖性。体外和体内接合的结果充分表明柔红霉素具有抑制耐药基因水平传播的潜力。The experimental results are shown in Figure 9. The results show that the in vitro conjugation test found that daunorubicin significantly reduced the conjugation frequency of the mcr-1/tet(X4) donor strain and the recipient strain E.coli EC600 in a concentration-dependent manner. Considering the good inhibitory effect of daunorubicin in vitro, an in vivo conjugation test was conducted by constructing an animal model. In vivo conjugation experiments showed that in mice, daunorubicin significantly reduced the conjugation frequency of mcr-1/tet(X4) donor bacteria and recipient bacteria E.coli EC600, also in a concentration-dependent manner. The results of in vitro and in vivo conjugation fully demonstrate the potential of daunorubicin to inhibit the horizontal spread of resistance genes.

实施例7、柔红霉素在CD-1小鼠体内急性毒性实验Example 7. Acute toxicity experiment of daunorubicin in CD-1 mice

BALB/c小鼠随机分为两组，每组8只。小鼠腹腔右侧注射单剂量的多粘菌素(10mg/kg)以及多粘菌素与柔红霉素的组合(10+5mg/kg)。在给药后连续7天观察小鼠有无死亡，并记录体重变化。第7天，每组随机取3只小鼠，摘除右眼球，取全血测定血常规，血清测生化指标。最终结果显示为平均值±标准偏差(mean±SD)。BALB/c mice were randomly divided into two groups, with 8 mice in each group. Mice were injected intraperitoneally on the right side with a single dose of polymyxin (10 mg/kg) and a combination of polymyxin and daunorubicin (10+5 mg/kg). The mice were observed for death for 7 consecutive days after administration, and body weight changes were recorded. On the 7th day, 3 mice from each group were randomly selected, the right eyeballs were removed, whole blood was taken to measure blood routine, and serum was used to measure biochemical indicators. Final results are shown as mean±standard deviation (mean±SD).

实验结果见图10，表3和表4，结果表明给药7天后，多粘菌素组的小鼠体重仅轻微上升，而多粘菌素与柔红霉素联合组的小鼠体重轻微下降。但是两个组的小鼠都没有死亡。在第7天取小鼠的右眼球静脉血，测定了血常规和血生化指标，发现柔红霉素的存在仅使血常规指标发生细微的变化。由于多粘菌素的肝肾毒性，部分与肝肾功能有关的指标发生显著变化，比如谷草转氨酶(AST)、乳酸脱氢酶(LDH)、肌酐(CREA)。同时，柔红霉素与多粘菌素的组合几乎不会使这些指标再发生变化，表明柔红霉素的存在并不会增强多粘菌素的肝肾毒性。The experimental results are shown in Figure 10, Table 3 and Table 4. The results show that after 7 days of administration, the weight of mice in the polymyxin group only increased slightly, while the weight of mice in the combination group of polymyxin and daunorubicin decreased slightly. . But no mice in either group died. On the 7th day, the venous blood of the right eyeball of the mouse was taken, and the routine blood and blood biochemical indicators were measured. It was found that the presence of daunorubicin only caused slight changes in the routine blood indicators. Due to the liver and kidney toxicity of polymyxin, some indicators related to liver and kidney function have significant changes, such as aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatinine (CREA). At the same time, the combination of daunorubicin and polymyxin hardly changes these indicators, indicating that the presence of daunorubicin does not enhance the liver and kidney toxicity of polymyxin.

表3table 3

表4Table 4

实施例8、动物感染细菌模型Example 8. Animal infection bacterial model

大蜡螟感染模型：准备足够的大蜡螟幼虫，随机分组，每组8只。分别于右侧尾足注射1.0×10⁵CFU的E.coli G92或K.pneumoniae D126菌液(每只幼虫10μL)。感染一小时后，在幼虫的左侧尾足分别注射10μL磷酸盐缓冲液(PBS)、多粘菌素(1mg/kg 或5mg/kg)、柔红霉素(1mg/kg)或者多粘菌素和柔红霉素的组合(1mg/kg+1mg/kg 或5mg/kg+1mg/kg)。连续观察记录7天大蜡螟幼虫的存活率变化。Galleria mellonella infection model: Prepare enough Galleria mellonella larvae and randomly group them into 8 larvae in each group. Inject 1.0×10 ⁵ CFU of E.coli G92 or K.pneumoniae D126 bacterial solution into the right tail foot respectively (10 μL per larvae). One hour after infection, 10 μL of phosphate buffered saline (PBS), polymyxin (1 mg/kg or 5 mg/kg), daunorubicin (1 mg/kg) or polymyxin were injected into the left tail foot of the larvae. Combination of daunorubicin and daunorubicin (1mg/kg+1mg/kg or 5mg/kg+1mg/kg). Continuously observe and record changes in the survival rate of Galleria mellonella larvae for 7 days.

小鼠腹膜炎-败血症模型：准备足够的雌性BALB/c小鼠，随机分组，每组8只。在小鼠腹腔右侧注射1.0×10⁸CFU的E.coli G92(每只小鼠200μL)。感染一小时后，分别腹腔注射100μL磷酸盐缓冲液(PBS)、多粘菌素(2mg/kg)、柔红霉素(1mg/kg) 或者多粘菌素和柔红霉素的组合(2+0.5mg/kg或2+1mg/kg)。连续观察记录7天小鼠的存活情况。Mouse peritonitis-sepsis model: Prepare enough female BALB/c mice and randomly group them into groups of 8 mice each. Inject 1.0×10 ⁸ CFU of E.coli G92 into the right side of the abdominal cavity of mice (200 μL per mouse). One hour after infection, 100 μL of phosphate buffered saline (PBS), polymyxin (2 mg/kg), daunorubicin (1 mg/kg) or a combination of polymyxin and daunorubicin (2 +0.5mg/kg or 2+1mg/kg). The survival of mice was continuously observed and recorded for 7 days.

小鼠嗜中性粒细胞减少的大腿感染模型：雌性BALB/c小鼠，随机分组，每组6只。为了减少嗜中性粒细胞，在感染前4天和前1天分别腹腔注射150和100mg/kg环磷酰胺。在小鼠右侧大腿内侧肌肉注射1.0×10⁵CFU的E.coli G92(每只小鼠100μL)。感染后一小时，分别腹腔注射100μL磷酸盐缓冲液(PBS)、多粘菌素(2mg/kg)、柔红霉素(1mg/kg)或者多粘菌素和柔红霉素的组合(2mg/kg+0.5mg/kg或2mg/kg+1 mg/kg)。感染48小时后，通过颈椎脱臼法处死小鼠，分离大腿肌肉，置于PBS中研磨，并在磷酸盐缓冲液(PBS)中连续10倍稀释，然后在LB琼脂板上均匀滴菌液。在37℃恒温箱培养18h后，数菌落数。Mouse neutropenic thigh infection model: female BALB/c mice, randomly divided into groups, 6 mice in each group. To reduce neutrophils, 150 and 100 mg/kg cyclophosphamide were injected intraperitoneally 4 days and 1 day before infection, respectively. The mice were intramuscularly injected with 1.0×10 ⁵ CFU of E.coli G92 (100 μL per mouse) in the right inner thigh. One hour after infection, 100 μL of phosphate buffered saline (PBS), polymyxin (2 mg/kg), daunorubicin (1 mg/kg), or a combination of polymyxin and daunorubicin (2 mg) were injected intraperitoneally. /kg+0.5mg/kg or 2mg/kg+1 mg/kg). 48 hours after infection, the mice were sacrificed by cervical dislocation, the thigh muscles were separated, ground in PBS, and serially diluted 10 times in phosphate buffered saline (PBS), and then evenly dripped on LB agar plates. After culturing for 18 hours in a 37°C incubator, count the number of colonies.

实验结果见图11，结果表明，注射磷酸盐缓冲液(PBS)的大蜡螟幼虫均在4天内死亡，而多粘菌素不能有效保护大蜡螟。其中，注射5mg/kg的多粘菌素后，E.coli G92 感染的幼虫在第四天仍全部死亡，K.pneumoniae D126感染的幼虫也仅存活一只(存活率为12.5％)。然而，柔红霉素与多粘菌素的联合治疗显著提高了大蜡螟的存活率。两种浓度组合分别将感染了E.coli G92的大蜡螟的存活率提升至50％和75％；将感染了K.pneumoniaeD126的大蜡螟的存活率提升至37.5％和62.5％。这种体内协同作用在由E. coli G92引起的小鼠腹膜炎—败血症模型中同样存在。The experimental results are shown in Figure 11. The results showed that the Galleria mellonella larvae injected with phosphate buffer saline (PBS) died within 4 days, and polymyxin could not effectively protect the Galleria mellonella larvae. Among them, after injection of 5 mg/kg polymyxin, all E.coli G92-infected larvae died on the fourth day, and only one K.pneumoniae D126-infected larvae survived (survival rate 12.5%). However, combined treatment with daunorubicin and polymyxin significantly improved the survival rate of Galleria mellonella. The two concentration combinations increased the survival rates of Galleria mellonella infected with E.coli G92 to 50% and 75% respectively; the survival rates of Galleria mellonella infected with K.pneumoniaeD126 increased to 37.5% and 62.5%. This in vivo synergistic effect also exists in the mouse peritonitis-sepsis model caused by E. coli G92.

小鼠腹膜炎-败血症模型结果表明，单独使用多粘菌素或柔红霉素并不能有效治疗 MCR-1阳性大肠杆菌引起的严重感染，但单次剂量的联合用药提高了感染后7天内小鼠的存活率。多粘菌素和柔红霉素的两种组合(2mg/kg+0.5mg/kg，2mg/kg+1mg/kg) 分别将被感染小鼠的存活率提升至50％和75％。其中，2mg/kg的多粘菌素和1mg/kg的柔红霉素的组合协同效果显著(P＝0.0345)。Results from a mouse peritonitis-sepsis model showed that polymyxin or daunorubicin alone were not effective in treating severe infections caused by MCR-1-positive E. coli, but a single dose of the combination improved the efficacy of the combination in mice within 7 days of infection. survival rate. Two combinations of polymyxin and daunorubicin (2 mg/kg + 0.5 mg/kg, 2 mg/kg + 1 mg/kg) increased the survival rate of infected mice to 50% and 75% respectively. Among them, the combination of 2 mg/kg polymyxin and 1 mg/kg daunorubicin had a significant synergistic effect (P=0.0345).

小鼠嗜中性粒细胞减少的大腿感染模型结果表明，在嗜中性粒细胞减少的小鼠大腿感染模型中同样发现了这种协同作用。相比于多粘菌素单独使用(2mg/kg)，多粘菌素和柔红霉素的两种组合显著降低了小鼠大腿肌肉中的耐药菌菌量(P＝0.0022，P<0.001)。这些结果充分表明柔红霉素可以在体内恢复多粘菌素对耐药菌的杀菌活性，包括减少体内菌量和提高动物的存活率。Results from the neutropenic mouse thigh infection model showed that this synergistic effect was also found in the neutropenic mouse thigh infection model. Compared with polymyxin alone (2mg/kg), the two combinations of polymyxin and daunorubicin significantly reduced the amount of drug-resistant bacteria in the thigh muscles of mice (P=0.0022, P<0.001 ). These results fully demonstrate that daunorubicin can restore the bactericidal activity of polymyxin against drug-resistant bacteria in vivo, including reducing the bacterial load in the body and improving the survival rate of animals.

Claims (2)

1. Use of a pharmaceutical composition for the manufacture of a medicament for the treatment of a disease with multiple drug resistant gram negative bacterial infections, characterized in that the pharmaceutical composition comprises daunorubicin and polymyxin; or daunorubicin and tigecycline, wherein the concentration of daunorubicin is 0.5-64 mug/mL, the concentration of polymyxin is 0.0625-16 mug/mL, the concentration of tigecycline is 0.5-32 mug/mL, the mass ratio of daunorubicin to polymyxin is 0.2-1024:1, and the mass ratio of daunorubicin to tigecycline is 1-128:1.

2. The use according to claim 1, wherein the medicament for treating the diseases with multiple drug-resistant gram-negative bacteria infection is in the form of tablets, capsules, oral liquid, syrup, dripping pills, injection and freeze-dried powder injection.

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