Application of indole compound with electron withdrawing group in inhibition of plasmid conjugation transfer

Abstract

The invention discloses an application of an indole compound with an electron withdrawing group in inhibition of plasmid conjugation transfer. According to the invention, 12 indole compounds are preliminarily screened, and it is found that the indole compounds carrying electron withdrawing side chain groups are an effective plasmid conjugation transfer inhibitor. Subsequently, by taking a clinical strain carrying blaNDM-5, tet (X4) and mcr-1 plasmids as a donor, further verifying the effect of indoleacetic acid (IAA), 7-bromoindole and 5-nitroindole in inhibiting the conjugational metastasis of the clinical plasmids, and finding that the three compounds significantly inhibit the conjugational metastasis of three clinical related plasmids. In addition, an in-vivo conjugational transfer experiment carried out by taking IAA as an example shows that the conjugational transfer frequency of RP4-7 plasmids of livers and spleens in mice in an IAA drug treatment group is obviously reduced. The method is an effective method for preventing and controlling drug resistance spreading and is expected to provide reference for research on drug resistance prevention and control strategies.

Description

Technical Field

[0001] The invention relates to application of indole compounds with electron-withdrawing groups in inhibiting plasmid conjugation transfer, belonging to the technical field of medicine. Background Art

[0002] Antibiotics can effectively kill pathogenic microorganisms and play a pivotal role in modern medicine, saving countless lives. However, in recent years, due to the abuse of antibiotics, bacterial resistance has been generated in large numbers, posing a huge challenge to human health and production safety. Bacterial resistance can be generated in two ways. One is that pathogenic microorganisms are repeatedly exposed to antibiotics, causing resistance gene mutations to acquire resistance, and the other is to acquire and spread resistance through mobile genetic components such as resistance plasmids and transposons. Among them, plasmid-mediated horizontal transfer of resistance genes is the main transmission route of antibiotic resistance genes in humans, animals and the environment. Plasmid conjugative transfer inhibitors are one of the effective strategies to control the spread of resistance genes. Common plasmid conjugative transfer inhibitors include fatty acids, polysaccharide antibiotics and nanomaterials. Most plasmid conjugative transfer inhibitors mainly act on the type IV secretion system of bacteria, and a few inhibitors block the conjugative transfer of plasmids by reprogramming bacterial metabolism.

[0003] Indole and its derivatives are compounds of pyrrole and benzene, which are widely distributed in nature, amino acids (such as tryptophan), animal hormones (such as melatonin) and plant hormones. Indole compounds have a variety of biological activities, such as antiviral, anti-inflammatory, anticancer, antioxidant, antimicrobial, anti-tuberculosis, anti-diabetic and anti-malarial, and show great application potential in clinical treatment. In addition, as an intercellular signaling molecule, indole is involved in multiple bacterial physiological processes, including spore formation, plasmid stability, drug resistance and biofilm formation. The molecular formula of indole is C 8 H 7 N. The chemical structure of indole is shown in Formula 1:

[0004]

[0005] Indole compounds have the advantages of natural origin, easy access and low toxicity, and are undoubtedly an ideal candidate for plasmid conjugative transfer inhibitors. However, there is currently no research on screening effective conjugative transfer inhibitors based on the structure-activity relationship of indole compounds. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide an application of indole compounds having electron-withdrawing groups in inhibiting plasmid conjugation transfer.

[0007] Technical solution: In order to solve the above technical problems, the present invention provides the use of indole compounds with electron-withdrawing groups in inhibiting plasmid conjugation transfer.

[0008] Wherein, the general structural formula of the indole compound having an electron-withdrawing group is as follows:

[0009]

[0010] Wherein, the R1, R2 or R3 is an electron-withdrawing group selected from one of halogen, carboxyl or nitro.

[0011] Among them, the indole compound with an electron-withdrawing group includes one or more of 5-iodoindole, 3-indoleacetic acid, 7-bromoindole, 7-chloroindole, 7-fluoroindole, 5-nitroindole, 3-indolecarboxylic acid, 3-indolecarboxaldehyde, 3-indolepropionic acid, and 3-indolebutyric acid.

[0012] Wherein, the plasmid conjugative transfer is plasmid transfer between bacteria, and the bacteria are Gram-negative bacteria.

[0013] The plasmid conjugation transfer includes an engineering plasmid (RP4-7) and a clinical plasmid (bla NDM-5 , tet(X4) and mcr-1).

[0014] The donor bacteria include bacteria of the same genus (Escherichia coli) and bacteria of different genus (Klebsiella pneumoniae).

[0015] Wherein, the use concentration of the indole compound is 0.1 μg / mL to 10 μg / mL.

[0016] The present invention also includes a method for inhibiting in vitro plasmid conjugative transfer, comprising the following steps: contacting an indole compound having an electron-withdrawing group at an effective concentration with bacteria to inhibit the plasmid conjugative transfer between the bacteria.

[0017] Wherein, the concentration of the indole compound having an electron-withdrawing group is 0.1 μg / mL to 10 μg / mL.

[0018] Among them, the inhibition of plasmid conjugative transfer mentioned in the present invention refers to the inhibition of plasmid conjugative transfer in vitro. The in vitro mentioned in the present invention refers to the environment or space outside the organism, that is, outside the living body, such as outside the human body or animal body.

[0019] The present invention also includes the use of indole compounds with electron-withdrawing groups in the preparation of drugs for inhibiting plasmid conjugation transfer in vivo or blocking the intestinal spread of drug-resistant bacteria.

[0020] The in vivo mentioned in the present invention refers to in vivo of a living organism, specifically refers to in vivo of a human or an animal.

[0021] Wherein, the in vivo plasmid conjugative transfer is the plasmid transfer between bacteria in vivo, and preferably, the bacteria are Gram-negative bacteria.

[0022] The drug-resistant bacteria include drug-resistant bacteria E.coli DH5α carrying RP4-7 plasmid, drug-resistant bacteria E.coli DH5α carrying bla NDM-5 The resistant bacteria K.pneumoniae C12 or E.coli L65 carrying plasmids, the resistant bacteria E.coli RS3-1 or E.coli RF2-1 carrying tet(X4) plasmids, and the resistant bacteria E.coli LD67-1 carrying mcr-1 clinical plasmids.

[0023] The drug for inhibiting conjugative transfer in vivo includes preparing a drug for reducing the frequency of conjugative transfer of plasmids in mouse liver and / or spleen.

[0024] Wherein, the use concentration of the indole compound having an electron-withdrawing group is 0.5 mg / kg mouse.

[0025] Among them, the blocking of the intestinal spread of drug-resistant bacteria described in the present invention includes inhibiting the frequency of conjugative transfer of plasmids and keeping the number of bacteria unchanged to block their spread.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following outstanding advantages: The present invention discloses for the first time the effect of indole compounds with electron-withdrawing groups in inhibiting plasmid conjugation transfer. Specifically, indole compounds with electron-withdrawing groups can effectively inhibit the conjugation transfer of engineering plasmids (RP4-7) and three clinical plasmids (bla NDM-5 , tet(X4) and mcr-1). The in vivo conjugative transfer experiment using IAA as an example showed that the frequency of plasmid conjugative transfer in the liver and spleen of mice treated with IAA drugs was significantly reduced. This invention provides a new perspective for the development of new drug resistance prevention and control strategies, and also provides a new method to block the spread of increasingly serious drug resistance genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Screening of indole compounds for inhibiting plasmid conjugative transfer.

[0028] Figure 2 The effects of 12 different concentrations of indole compounds on the conjugative transfer frequency of RP4-7 plasmid.

[0029] Figure 3 To evaluate the effectiveness of indoleacetic acid, 7-bromoindole and 5-nitroindole in inhibiting clinical plasmid conjugative transfer.

[0030] Figure 4 To evaluate the in vivo effect of indoleacetic acid in inhibiting plasmid conjugative transfer. DETAILED DESCRIPTION

[0031] The technical scheme of the present invention is further described below in conjunction with the accompanying drawings. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several modifications and improvements can also be made, which should also be considered to belong to the protection scope of the present invention. The experimental methods in the following embodiments are conventional methods unless otherwise specified. The experimental materials used in the following embodiments are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative experiments in the following embodiments are all repeated three times, and the results are averaged.

[0032] The LB broth medium used in the examples is an aqueous solution containing 10 g / L tryptone, 5 g / L yeast extract powder and 10 g / L NaCl; the MHB broth medium is an aqueous solution of 2 g / L beef powder, 1.5 g / L soluble starch and 17.5 g / L acid hydrolyzed casein; and the DMEM basal medium is a solution containing 2 mM L-glutamic acid, 1 mM sodium pyruvate, glucose, amino acids, inorganic salts and other basic nutrients.

[0033] CD-1 female mice were purchased from the Center of Comparative Medicine of Yangzhou University.

[0034] The 12 indole compounds were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. with the product numbers S43256 (5-iodoindole), V34639 (3-indoleacetic acid), S49050 (5-methylindole), S47797 (7-bromoindole), S48076 (7-chloroindole), S46713 (4-fluoroindole), S49039 (5-nitroindole), S31552 (3-indolecarboxylic acid), S30763 (3-indolecarboxaldehyde), S18033 (3-indolepropionic acid), T90319 (3-indolebutyric acid), and S30160 (5-hydroxyindoleacetic acid).

[0035] The strains used in the examples are all strains preserved in this laboratory.

[0036] Example 1 Screening of indole compounds that inhibit plasmid conjugative transfer

[0037] Conjugative transfer frequency determination: E. coli DH5α carrying RP4-7 plasmid was used as donor and E. coli C600 was used as recipient to determine the conjugative transfer frequency (donor bacteria and recipient bacteria are from the article Zhang M, Yang B, Shi J, Wang Z, Liu Y. Host defense peptides mitigate the spread of antibiotic resistance inphysiologically relevant condition. Antimicrob Agents Chemother. 2024Apr

[0038] 3;68(4):e0126123). Inoculate a single colony in 1 mL LB medium and culture overnight. Inoculate 10 mL LB medium at a volume ratio of 1:100 and culture in a shaker at 37°C and 200 rpm until the logarithmic phase. Use a microplate reader to adjust the OD of the bacteria. 600nm To 0.25. 1 mL of donor bacteria E. coli DH5α and 1 mL of recipient bacteria E. coli C600 were pipetted into a new 5 mL EP tube, and different concentrations of indole compounds (5-iodo-1H-indole (5-iodoindole, 5 mg / mL): Purify, S43256-100 mg; Indole-3-lactic acid (3-indoleacetic acid, 5 mg / mL): Purify, V34639-100 mg; 5-Methylindole (5-methylindole, 5 mg / mL): Purify, S49050-100 mg; 7-bromo-1H-indole (7-bromoindole, 5 mg / mL): Purify, S47797-100 mg; 7-chloro-1H-indole (7-chloroindole, 5 mg / mL): Purify, S48076-100 mg) were added. mg; 7-fluoro-1H-indole (7-fluoroindole, 5mg / mL): Purify, S46713-100 mg; 5-Nitroindole (5-nitroindole, 5mg / mL): Purify, S49039-100 mg; Indole-3-carboxylic acid (3-indolecarboxylic acid, 5mg / mL): Purify, S31552-100 mg; Indole-3-carboxaldehyde (3-indolecarboxaldehyde, 5mg / mL): Purify, S30763-100 mg; Indole-3-propionicacid (3-indolepropionic acid, 5

[0039] mg / mL): Purify, S18033-100 mg; Indole-3-butyric acid (3-indolebutyric acid, 5 mg / mL): Purify, T90319-100 mg; 5-Hydroxyindole-3-acetic acid (5-hydroxyindoleacetic acid, 5

[0040] mg / mL): Purify, S30160-100 mg;) to a final concentration of 0.1, 1, 10 μg / mL, and cultured in a shaker at 37°C, 200 rpm for 15 hours. After that, the conjugated bacteria were diluted to an appropriate concentration gradient and smeared on a rifampicin single-drug agar plate and an ampicillin, rifampicin double-drug agar plate to screen the recipient bacteria and conjugants. The agar plate was placed in a 37°C incubator for overnight culture. The bacteria on the agar plate were then counted to calculate the conjugation transfer frequency. Conjugation transfer frequency = number of conjugants / number of recipient bacteria.

[0041] Minimum inhibitory concentration (MIC) determination: Take the conjugate, donor bacteria and recipient bacteria monoclonal inoculation in 1mL LB medium and culture overnight. Pipette 100μL MHB medium into a 96-well plate, add rifampicin or ampicillin to the first well to a final concentration of 2048μg / mL, pipette and mix well, pipette 100μL into the second well, dilute in turn to the last well, and discard 100μL. Dilute the bacteria cultured overnight in MHB medium at 1:1000, take 100μL of the diluted bacterial suspension into a 96-well plate. After culturing at 37°C for 18h, the lowest concentration with no obvious bacterial growth is the MIC concentration.

[0042] Polymerase chain reaction (PCR) analysis: The reaction system included 2×Ex Tag Premix (25 μL), 1 μL each of the upstream primer 5'-TCGGACGTTTGACCGGAA-3' and the downstream primer 5'-GCTTCGTGTGTTTCAGCAA-3', 1 μL of DNA template, and ddH 2 O to 50μL; the reaction program is 95℃5min; 95℃30s, 55℃30s, 72℃40s, 30 cycles; 72℃5min. Use agarose gel electrophoresis at 120V constant voltage for 15min to spot-check the PCR reaction amplification product, and determine whether it contains the required target fragment based on the marker band.

[0043] The analysis results are shown in Figure 1 and Figure 2The results showed that, except for 5-methylindole and 5-hydroxyindoleacetic acid, the other 10 indole compounds could effectively inhibit plasmid conjugative transfer. Indole compounds carrying electron-withdrawing groups had a stronger inhibitory effect on plasmid conjugative transfer.

[0044] Example 2 Evaluation of the Effect of Indoleacetic Acid, 7-Bromoindole and 5-Nitroindole in Inhibiting Clinical Plasmid Conjugative Transfer

[0045] Determination of conjugative transfer frequency of clinical plasmids: using plasmids carrying bla NDM-5 , tet(X4) and mcr-1 clinical plasmids were used as donors, and E. coli C600 was used as the recipient to determine the frequency of conjugation transfer (the above clinical strains are all from the article Zhang M, Yang B, Shi J, Wang Z, Liu Y. Host defense peptides mitigate the spread of antibiotic resistance in physiologically relevant condition. Antimicrob Agents Chemother. 2024Apr 3; 68(4): e0126123). Inoculate a single colony in 1 mL LB medium and culture overnight. Inoculate in 10 mL LB medium at a ratio of 1:100 and culture in a shaker at 37°C and 200 rpm until the logarithmic phase. Use an enzyme reader to adjust the OD of the bacteria. 600nm To 0.25. Pipette 1mL of donor bacteria and 1mL of recipient bacteria into a new 5mL EP tube, add different concentrations of drugs (Indole-3-lactic acid (3-indoleacetic acid, 5mg / mL): Purify, V34639-100 mg; 7-bromo-1H-indole (7-bromoindole, 5mg / mL): Purify, S47797-100mg; 5-Nitroindole (5-nitroindole, 5mg / mL): Purify, S49039-100 mg;) to a final concentration of 0.1, 1, and 10μg / mL, and culture in a shaker at 37°C and 200rpm for 15h. After that, dilute the conjugated bacteria to an appropriate concentration gradient and apply them to agar plates containing antibiotics to screen the recipient bacteria and conjugants. Place the agar plates in a 37°C incubator for overnight culture. Then count the bacteria on the agar plates to calculate the frequency of conjugation transfer. Conjugative transfer frequency = number of conjugates / number of recipient bacteria.

[0046] The analysis results are shown in Figure 3 The results showed that indoleacetic acid, 7-bromoindole and 5-nitroindole significantly inhibited the bla NDM-5 , tet(X4) and mcr-1.

[0047] Example 3 Evaluation of the in vivo effect of indoleacetic acid in inhibiting plasmid conjugative transfer

[0048] Mouse in vivo conjugation transfer model: 16 female CD-1 mice (Yangzhou University Comparative Medicine Center, 6-8 weeks old, weight 20±2g) were randomly divided into a control group and an IAA treatment group (IAA, 0.5mg / kg), with 8 mice in each group. The mice were adaptively fed for 3 days, with free access to food and water. The donor bacteria E.coli DH5α and the recipient bacteria E.coli C600 were cultured overnight, and then the bacterial OD was adjusted. 600 nm To 0.5. First, 100 μL of donor and recipient bacteria were injected intraperitoneally. 15 minutes after infection, 100 μL of PBS and IAA (0.5 mg / kg) were injected intraperitoneally again. 24 hours later, the mice were euthanized, and the liver and spleen of the mice were collected and ground. The ground organs were diluted to an appropriate concentration gradient. The diluted grinding solution was applied to the corresponding agar plate containing antibiotics, and the colony count was performed 12 hours later to calculate the frequency of bacterial conjugation transfer in the organs.

[0049] The analysis results are shown in Figure 4 The results showed that the frequency of bacterial conjugative transfer in the liver and spleen of mice treated with IAA was significantly reduced compared with that in the control group. At the same time, the number of recipient bacteria in the liver and spleen did not change significantly in the IAA-treated group, indicating that IAA inhibited the frequency of plasmid conjugative transfer and the number of bacteria remained unchanged.

[0050] In summary, indole compounds with electron-withdrawing groups can significantly inhibit the conjugation and transfer of engineered plasmids and clinical plasmids, and show good inhibitory effects both in vivo and in vitro. This invention provides a new perspective for the development of new drug resistance prevention and control strategies, and also provides a new method for blocking the spread of increasingly serious drug resistance genes.

Claims

1. Application of an indole compound having an electron-withdrawing group in inhibiting plasmid conjugation transfer, wherein the indole compound having an electron-withdrawing group has the following general structural formula: in, The R1, R2 or R3 is an electron-withdrawing group selected from one of halogen, carboxyl or nitro.

2. The use according to claim 1, characterized in that: The indole compound with an electron-withdrawing group includes one or more of 5-iodoindole, 3-indoleacetic acid, 7-bromoindole, 7-chloroindole, 7-fluoroindole, 5-nitroindole, 3-indolecarboxylic acid, 3-indolecarboxaldehyde, 3-indolepropionic acid, and 3-indolebutyric acid.

3. The use according to claim 2, characterized in that: The plasmid conjugative transfer is plasmid transfer between bacteria. Preferably, the bacteria are Gram-negative bacteria.

4. The use according to claim 2, characterized in that: The plasmids include engineering plasmid RP4-7, clinical plasmid bla NDM-5 , tet(X4) or mcr-1.

5. The use according to claim 1, characterized in that: The donor bacteria in the plasmid conjugative transfer include bacteria of the same species or different species. Preferably, the same species includes Escherichia coli, and the different species includes Klebsiella pneumoniae.

6. The use according to claim 1, characterized in that: The use concentration of the indole compound having an electron-withdrawing group is 0.1 μg / mL to 10 μg / mL.

7. A method for inhibiting in vitro plasmid conjugative transfer, characterized in that: The method comprises the following steps: contacting an indole compound with an electron-withdrawing group at an effective concentration with bacteria to inhibit the plasmid conjugation transfer between the bacteria.

8. The method according to claim 7, characterized in that: The concentration of the indole compound having an electron-withdrawing group is 0.1 μg / mL to 10 μg / mL.

9. The use according to claim 7, characterized in that: The bacteria include drug-resistant bacteria E.coli DH5α carrying RP4-7 plasmid, NDM-5 The resistant bacteria K.pneumoniae C12 or E.coli L65 carrying plasmids, the resistant bacteria E.coli RS3-1 or E.coli RF2-1 carrying tet(X4) plasmids, and the resistant bacteria E.coli LD67-1 carrying mcr-1 clinical plasmids.

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