Use of flavonoids in preparation of drugs for inhibiting conjugative transfer of drug-resistant plasmid

Abstract

The application discloses application of a natural flavonoid compound of a plant source in preparation of a medicine for inhibiting conjugative transfer of a drug-resistant plasmid, and drug-resistant plasmid conjugative transfer tests show that a plurality of flavonoid compounds can significantly inhibit conjugative transfer of a multidrug-resistant RP4-7 plasmid and a plurality of clinical plasmids carrying different drug-resistant genes, and the inhibiting ability on mcr-1 positive plasmid is more significant. The natural flavonoid compound provided by the application can simultaneously inhibit conjugative transfer of a drug-resistant plasmid in vitro and in vivo, and provides a new idea for blocking the spread and diffusion of drug resistance.

Description

Application of flavonoids in the preparation of drugs that inhibit conjugation transfer of drug-resistant plasmids Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of flavonoids in inhibiting the conjugation and transfer of drug-resistant plasmids. Background Technology

[0002] Background content

[0003] The overuse of antibiotics has led to a worsening problem of antibiotic resistance, with various resistance genes and their variants constantly emerging, severely impacting all aspects of human life and livestock farming. The long development cycle, high cost, and low efficiency of novel antibiotic research have prompted us to consider feasible solutions to antibiotic resistance from different perspectives. Conjugation transfer (CGT) is an adaptive behavior in bacteria, where bacteria share genetic material, transport effector proteins, and release toxins through direct contact. It occurs with bacterial evolution and provides additional opportunities for bacterial population expansion and gene pool renewal. The large-scale occurrence of CGT events exacerbates the global public health crisis caused by antibiotic resistance, especially in Gram-negative bacterial infections. Therefore, there is an urgent need to develop effective strategies to address the ongoing spread of bacterial resistance. Plasmid elimination and anti-plasmid measures, such as the development of CGT inhibitors, can reduce the prevalence of antibiotic resistance genes (ARGs), maintain bacterial sensitivity to antibiotics, and thus provide a unique approach to preventing the spread of resistance. In recent years, aminocoumarins, quinolones, acridine dyes, surfactant alkyl sulfates, and detergents have been reported as potential plasmid elimination agents. However, no true conjugation transfer inhibitors have yet been approved for clinical use, possibly due to factors such as high toxicity and poor bioavailability.

[0004] Natural plants, as a rich resource, provide many bioactive compounds for the treatment of various diseases. Previous studies have shown that some plant-derived unsaturated fatty acids can induce the loss of drug-resistant plasmids or the inhibition of the type IV secretion system (T4SS) through direct interactions. Therefore, exploring the natural plant field to find safe and effective conjugation metastasis inhibitors holds promise. Natural flavonoids are a class of secondary plant metabolites, widely found in fruits, vegetables, flowers, tea, and honey. Flavonoid formulations possess excellent properties such as inhibiting cancer cell proliferation and stimulating immune function, and have been clinically applied to treat human immunodeficiency virus (HIV), hepatitis, tumors, and cardiovascular diseases. For example, quinine and artemisinin, natural products used to combat malaria, have entered the field of anti-infectives and have made some progress in antifungal, antiviral, and antibacterial activities. Therefore, it is necessary to explore the potential chemical diversity of natural flavonoids from different perspectives to provide new ideas for the development of novel conjugation metastasis inhibitors. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide the application of natural flavonoids in the preparation of drugs for bacterial infectious diseases and / or drugs for inhibiting the conjugation and transfer of drug-resistant plasmids.

[0006] This invention discovers that flavonoids can significantly inhibit the conjugation and transfer of drug-resistant plasmids to combat increasingly serious multidrug-resistant bacterial infections.

[0007] Technical solution: In order to solve the above-mentioned technical problems, the present invention provides the use of flavonoids in the preparation of drugs for bacterial infectious diseases and / or drugs for inhibiting the conjugation and transfer of drug-resistant plasmids.

[0008] This invention also includes the application of flavonoids in the preparation of products or drugs with in vivo immunomodulatory activity. Here, "organisms" refers to livestock and poultry.

[0009] The flavonoids include one or more of the following: luteolin, vitexin, irisin, puerarin, saurolophytin, rhodioloside, sophorol, geraniol, styracil, psoralen, or baicalin.

[0010] The bacterial infectious disease drugs and / or drugs that inhibit the conjugation and transfer of drug-resistant plasmids are single-component or compound preparations.

[0011] The bacteria in question are Gram-negative bacteria.

[0012] The drug-resistant plasmids include those resistant to antibiotics.

[0013] The antibiotics mentioned include one or more of ampicillin, polymyxin, meropenem, or tigecycline.

[0014] The drug resistance plasmids include multidrug-resistant RP4-7 plasmids and bla NDM-1 Positive plasmids, tet(X4) positive plasmids, or mcr-1 positive plasmids.

[0015] The dosage forms of the drugs or conjugation transfer inhibitors for bacterial infectious diseases include tablets, capsules, sustained-release tablets, oral liquids, syrups, pills, injections, and lyophilized powder injections.

[0016] This invention relates to the inhibitory effect and application of flavonoid compounds on the conjugation transfer of drug resistance genes. Conjugation transfer experiments show that various natural flavonoid compounds can significantly inhibit the conjugation transfer of multidrug-resistant RP4-7 plasmids within *Escherichia coli*. Additionally, plasmids carrying three clinically important drug resistance genes (bla...)... NDM-1Conjugation transfer assays using clinical strains with different plasmid types (e.g., tet(X4) and mcr-1) as donor bacteria also demonstrated that various natural flavonoids possess the ability to inhibit conjugation transfer of clinical plasmids, with the mcr-1 resistant plasmid showing the most significant effect. Furthermore, in vivo conjugation assays also verified the in vivo effectiveness of baicalin in inhibiting conjugation transfer. These studies indicate the potential of natural flavonoids as novel conjugation transfer inhibitors, providing a new strategy for the prevention and control of increasingly serious Gram-negative bacterial infections.

[0017] Beneficial Effects: Compared with existing technologies, the advantages of this invention are: In the face of the increasingly serious problem of drug resistance, the natural flavonoids (such as baicalin) provided by this invention can inhibit the conjugation and transfer of multiple drug-resistant plasmids in vitro and in vivo, thereby effectively limiting the spread of bacterial drug resistance. Furthermore, as a safe and effective traditional Chinese medicine monomer, the findings of this research will contribute to the development of... NDM-1 This provides new insights into the clinical prevention and control of important drug-resistant pathogens mediated by tet(X4) and mcr-1. Attached Figure Description

[0018] Figure 1 illustrates the ability of the studied flavonoids to inhibit RP4-7 plasmid conjugation and transfer.

[0019] Figure 2 shows the distribution of side chain groups outside the skeletal structure of the studied flavonoids;

[0020] Figure 3 shows the classification of the studied flavonoids according to their characteristic side chain groups and a comparison of their inhibitory binding abilities.

[0021] Figure 4 shows a comparison of the chemical properties of three groups of flavonoids containing different side chain groups;

[0022] Figure 5 shows the linear regression analysis between the ability of the three groups of flavonoids to inhibit binding and their chemical properties;

[0023] Figure 6 illustrates the ability of some flavonoids to inhibit the conjugation and transfer of various clinically resistant plasmids.

[0024] Figure 7 shows the in vivo conjugation assay. It can be seen that baicalin can inhibit the conjugation and transfer of multiple drug-resistant plasmids in vivo via intraperitoneal injection;

[0025] Figure 8 shows the in vivo conjugation assay. It can be seen that baicalin can inhibit the conjugation and transfer of multiple drug-resistant plasmids in vivo by intraperitoneal injection. Detailed implementation method:

[0026] The present invention will be further illustrated below with specific embodiments. It should be noted that those skilled in the art can make various modifications and improvements without departing from the principles of the present invention, and these should also be considered within the scope of protection of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores. All quantitative experiments in the following embodiments were performed in triplicate, and the results were averaged.

[0027] Table 1

[0028] Test strain sources or literature: Escherichia coli DH5α, Escherichia coli EC600, Escherichia coli LD67-1, Escherichia coli L65, Escherichia coli RW7-1, Escherichia coli RS3-1, Klebsiella pneumoniae C12, Klebsiella pneumoniae C12. surface

[0029] Note: Document 1 is Jia Y, Wang Z, Fang D, Yang B, Liu Y. Acetaminophen promotes shorizontal transfer of plasmid-borne multiple antibiotic resistance genes. SciTotal Environ 2021;782:146916.

[0030] Document 2 is Yang B, Wang Z, Jia Y, Fang D, Li R, Liu Y. Paclitaxel and its derivative facilitate the transmission of plasmid-mediated antibioticresistance genes through conjugative transfer. Sci Total Environ 2022;81 0:152245.

[0031] Reference 3: Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J, et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis 2016;16:161-8.

[0032] Reference 4: Liu Z, Xiao X, Li Y, Liu Y, Li R, Wang Z. Emergence of IncX3 plasmid-harboring bla NDM-5 dominated by Escherichia coli ST48 in a goose farm in Jiangsu, China. Front Microbiol 2019;10:2002.

[0033] Reference 5: Li R, Peng K, Li Y, Liu Y, Wang Z. Exploring tet(X)-bearing tigecycline-resistant bacteria of swine farming environments. Sci Total Environ 2020;733:139306.

[0034] Reference 6: Li R, Lu X, Peng K, Liu Z, Li Y, Liu Y, et al. Deciphering the structural diversity and classification of the mobile tigecycline resistance gene tet(X)-bearing plasmidome among bacteria. mSystems 2020;5:e00134-20.

[0035] Document 7 is Jia Y, Yang B, Shi J, Fang D, Wang Z, Liu Y. Melatonin prevents conjugative transfer of plasmid-mediated antibiotic resistance genes by disrupting proton motive force. Pharmacol Res 2022;175:105978.

[0036] Example 1: Investigation into the function of flavonoids in inhibiting the conjugation and transfer of drug-resistant plasmids

[0037] The ability of various flavonoids to inhibit the conjugation and transfer of the RP4-7 plasmid was determined by conjugation transfer assay. Two known and commonly used laboratory strains were used as donor and recipient strains carrying the drug-resistant plasmid. The donor strain was Escherichia coli DH5α carrying the RP4-7 plasmid, and the recipient strain was Escherichia coli EC600.

[0038] Single colonies of donor and recipient bacteria were picked from LB agar plates containing ampicillin (100 mg / L) and rifampin (300 mg / L), respectively, and inoculated into 1 mL of LB broth. The cultures were incubated overnight at 37°C with a shaker at 200 rpm. The LB broth formulation (per liter) consisted of 5 g yeast extract, 10 g trypsin, and 10 g sodium chloride, adjusted to pH 7.0 with NaOH, and autoclaved at 121°C for 20 min. After overnight incubation, the donor and recipient bacteria were expanded by inoculating them at a 1:100 ratio into larger volumes of LB broth, and incubated for another 5 hours with a shaker until OD (dose-to-volume) reached. 600=0.5. Then, centrifuge at 5000 rpm, resuspend in fresh LB broth, and provide 1 mL of each recipient bacterium to form a conjugation system. Next, add different concentrations of flavonoids (most flavonoids, except for morusin and licorice flavonoid B, have the ability to inhibit RP4-7 plasmid conjugation transfer) to each conjugation system at concentrations of 0.1, 1, 10, and 100 μg / mL. After a conjugation process at 37°C and 200 rpm for 15 h, the conjugation system is removed and diluted for droplet application. The conjugation system after 15 hours of conjugation was considered as a zero gradient. Then, 50 μL of the bacterial culture from the previous gradient was added to 450 μL of physiological saline for continuous 10-fold dilutions, from gradient 1 to gradient 8. After 12 hours, conjugates and recipient bacteria grew. Conjugates were screened using a double-drug plate containing both ampicillin (100 mg / L) and rifampin (300 mg / L), with gradients of 1, 2, 3, and 4. Recipient bacteria were screened using a single-drug plate containing rifampin (300 mg / L), with gradients of 5, 6, 7, and 8. The conjugation frequency was calculated by dividing the number of conjugates by the number of recipient bacteria. Conjugation frequency = (N... 接合子a / N 受体菌b )*10^(ab), where N refers to the number of single colonies, a refers to the gradient of conjugates, and b refers to the gradient of recipient bacteria.

[0039] The experimental results are shown in Figure 1. The results indicate that at the tested sub-inhibitory concentrations, most flavonoids, except for morusin and kicoflavone B, possess the ability to inhibit RP4-7 plasmid conjugation and transfer. Among them, neobavaisoflavone, kysionotin, diosmetin, rhodiosin, vitexin, and scutellarein showed the most significant effects.

[0040] Next, we classified the tested flavonoids into three groups based on their characteristic side chain groups: hydroxy, methoxy, and isopentenyl groups, and compared their ability to inhibit binding. In addition, we compared six chemical properties (molecular weight, polar surface area, rotatable bonds, number of hydrogen bond donors, number of hydrogen bond acceptors, and lipophilicity), and finally performed linear regression analysis between the binding inhibition ability and different chemical properties. The experimental results are shown in Figures 2, 3, 4, and 5. The results show that among the three groups of flavonoids, the methoxy group has the strongest ability to inhibit binding transfer. The chemical property analysis results indicate that the methoxy group has the fewest hydrogen bond donors and the most hydrogen bond acceptors. Furthermore, the linear regression analysis shows a significant correlation between the number of hydrogen bond acceptors and the ability to inhibit binding frequency; that is, the more hydrogen bond acceptors, the stronger the binding inhibition ability. This explains why the methoxy group has a stronger effect in inhibiting binding frequency.

[0041] Then, the effects of flavonoids on clinical plasmid conjugation and transfer of different plasmid types were further investigated. E. coli LD67-1 carrying the mcr-1 positive IncI2 plasmid and E. coli carrying the bla... NDM-1 E. coli L65 carrying the IncX3 positive plasmid and E. coli RW7-1 carrying the tet(X4) positive IncFII plasmid were used as donor bacteria for conjugation experiments. The flavonoids tested included luteolin, casticin, irigenin, puerarin, pectolinarigenin, rhodiosin, maackiain, diosmetin, lysionotin, neobavaisoflavone, and scutellarein, all of which showed good inhibitory effects in previous conjugation experiments. The results are shown in Figure 6. The results indicate that the selected flavonoids generally inhibited the conjugation transfer of clinical plasmids carrying different drug resistance genes, with the effect being more significant for the mcr-1 positive plasmid. For tet(X4) positive plasmids, the inhibitory effect of flavonoids is more significant at low concentrations, such as maackiain, diosmetin, and lysionotin. However, for bla... NDM-1 For positive plasmids, this inhibitory effect is not widespread; for example, maackiain and lysionotin showed inhibitory effects at higher concentrations.

[0042] Example 2: In vivo efficacy of baicalin in inhibiting drug resistance plasmid binding.

[0043] In vivo conjugation assay: Six-week-old female ICR mice provided by the Institute of Comparative Medicine, Yangzhou University, were used for in vivo conjugation and divided into two groups of at least six mice each. Donor and recipient bacteria were shaken to 10^8 CFUs / mL. 1 mL of each bacteria was then injected intraperitoneally into both groups of mice. 15 min later, the control group was injected with 0.01M PBS (7.2-7.4), while the drug-treated group was injected with 0.5 mg / kg of baicalein. 24 h later, the mice were sacrificed, and their spleens and livers were harvested, ground, and plated to calculate the conjugation frequency. The donor bacteria tested were E. coli DH5α carrying the multidrug-resistant RP4-7 plasmid, E. coli LD67-1 carrying the mcr-1 positive IncI2 plasmid, and E. coli carrying the bla... NDM-1 K. pneumoniae C12 carrying the IncX3 plasmid and E. coli RS3-1 carrying the tet(X4) positive IncFI plasmid.

[0044] The experimental results are shown in Figures 7 and 8. The results indicate that, under the action of baicalin, the binding frequency of the RP4-7 plasmid in the liver and spleen was inhibited by 39.5% (P = 0.0007) and 47.1% (P = 0.0001) respectively, compared with the control group. For the mcr-1 positive plasmid, the binding frequency in the liver and spleen was inhibited by 52.6% (P = 0.0386) and 80.9% (P < 0.0001) respectively. For the tet(X4) positive plasmid and bla... NDM-1 Positive plasmids showed that their conjugation frequency in the liver was inhibited by 50.1% (P = 0.001) and 62.6% (P = 0.0096), respectively. These results indicate that baicalin also has the ability to inhibit the conjugation and transfer of multiple drug-resistant plasmids in vivo.

Claims

1. The application of flavonoids as the sole active ingredient in the preparation of drugs for bacterial infectious diseases, characterized in that, The bacteria is E. coli LD67-1 carrying the mgr-1 positive IncI2 type plasmid, and the flavonoids are one or more of luteolin, vitexin, irisin, puerarin, saurolophytin, rhodioloside, sophorol, geraniol, or baicalin.

2. The application of flavonoids as the sole active ingredient in the preparation of drugs for bacterial infectious diseases, characterized in that, The bacteria is E. coli RW7-1 carrying the tet(X4) positive IncFII type plasmid, and the flavonoids are kosmolide, geraniol, scutellarin or baicalin.

3. The application of flavonoids as the sole active ingredient in the preparation of drugs for bacterial infectious diseases, characterized in that, The bacteria are carriers of bla NDM-1 The positive IncX3 plasmid is E. coli L65, and the flavonoid compound is geraniol or baicalin.

4. The application according to claim 1, characterized in that, The bacterial infectious disease drug is a single-component formulation.

5. The application according to claim 1, characterized in that, The dosage forms of the drugs for bacterial infectious diseases are tablets, capsules, oral liquids, pills, injections, and lyophilized powder injections.

6. The application according to claim 1, characterized in that, The drug for bacterial infectious diseases is in the form of sustained-release tablets or syrup.

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