Use of bio-organic acids for the preparation of plasmid conjugative transfer inhibitors and / or antibacterial agents
By using bio-organic acids such as citric acid, fumaric acid, or α-ketoglutarate as plasmid conjugation and transfer inhibitors, the problem of drug-resistant plasmid transmission has been solved, achieving effective inhibition of multiple drug-resistant plasmids and providing an effective means to control bacterial drug resistance.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- YANGZHOU UNIV
- Filing Date
- 2026-05-08
- Publication Date
- 2026-06-05
AI Technical Summary
Existing technologies have failed to effectively inhibit the conjugation and transfer of drug-resistant plasmids, leading to the spread of drug-resistant bacteria, especially conjugable drug-resistant plasmids such as blaNDM and the spread of a large number of drug-resistant genes, which seriously weakens the effectiveness of antibiotics.
Bio-organic acids such as citric acid, fumaric acid, or α-ketoglutarate were used as plasmid conjugation and transfer inhibitors. In vitro and in vivo experiments were conducted to verify their inhibitory effects on the conjugation and transfer of various drug-resistant plasmids at different concentrations, including the inhibition of plasmids carrying RP4-7, mcr-1, tet(X4), and blaNDM-5.
It significantly inhibited the conjugation and transfer of drug-resistant plasmids, limited the spread of various clinically resistant plasmids, and provided a strategy for controlling bacterial resistance. In vitro and in vivo experimental results showed its effectiveness.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of biological organic acids in the preparation of plasmid conjugation transfer inhibitors and / or antibacterial drugs. Background Technology
[0002] The discovery and application of antibiotics was a milestone in modern medicine, significantly reducing the incidence and mortality of infectious diseases. However, due to the improper use of antibiotics in human and veterinary clinical practice, reports of antibiotic resistance in pathogens are increasing, even leading to the emergence of "superbugs," severely weakening the effectiveness of antibiotics. Statistics show that in 2019, more than 1.2 million people died from infections caused by drug-resistant bacteria. The increasing severity of the drug resistance problem is largely attributed to the horizontal spread of resistance plasmids, especially conjugable resistance plasmids. (e.g., bla...) NDM Most of these genes are located on conjugative plasmids and also carry other drug resistance genes, thus enabling bacteria to develop multidrug resistance. Similarly, the mcr and tet(X)-related genes, which mediate resistance to important antibiotics such as polymyxin and tigecycline, are widely prevalent globally because they are located on conjugable transferable plasmids.
[0003] Conjugation transfer occurs through physical contact between bacteria, via fimbriae bridges or pores, directly transferring DNA. Plasmids, especially transferable plasmids, are the primary carriers for conjugation. Numerous in vitro studies have shown that various compounds, including sub-inhibitory concentrations of antibiotics, nanomaterials, disinfectants, disinfection byproducts, ionic liquids, heavy metals, anticonvulsants, antiepileptic drugs, and non-nutritive sweeteners, can promote plasmid conjugation transfer, revealing factors in clinical drug use and the environment that may promote the spread of drug resistance. Plasmid conjugation transfer in the gut is influenced and regulated by many factors, including those from the bacteria themselves, the host, and the environment. Host stress (immune factors, intestinal hormones, metabolites) and the intestinal environment of animals, such as pH, oxygen levels, osmotic pressure, and the presence or absence of antibiotics, can affect the conjugation transfer of drug-resistant plasmids. Furthermore, studies have shown that bacterial conjugation transfer in the gut, like in vitro, can be interfered with by exogenous substances. Research has found that substances such as acetylshikonin, melatonin, indole, biochar, and unsaturated fatty acids can significantly inhibit the frequency of plasmid conjugation transfer both in vivo and in vitro. These studies suggest that screening and developing appropriate conjugation transfer inhibitors is an effective means of controlling the spread of drug resistance.
[0004] Bio-organic acids such as citric acid, fumaric acid, and α-ketoglutarate are among the most widely used acidulants in the food industry, used to adjust acidity and improve flavor in foods such as carbonated beverages, fruit juices, jams, and candies. They also have applications in the pharmaceutical field: citric acid and its derivatives (such as calcium citrate and ferric citrate) are used as anticoagulants, expectorants, diuretics, and buffers in sustained-release drug formulations; fumaric acid and its derivatives are used as antibacterial agents and pharmaceutical intermediates; and α-ketoglutarate improves hyperglycemia in diabetes treatment by inhibiting hepatic gluconeogenesis. However, their potential application in inhibiting the spread of drug resistance remains undetermined. Summary of the Invention
[0005] Purpose of the invention: The purpose of this invention is to provide the application of biological organic acids in the preparation of plasmid conjugation transfer inhibitors and / or antibacterial drugs.
[0006] The present invention also includes the application of the aforementioned bio-organic acids in the preparation of drugs for preventing the spread of drug resistance.
[0007] Technical solution: In order to solve the above-mentioned technical problems, the present invention provides the application of biological organic acids in the preparation of plasmid conjugation transfer inhibitors and / or antibacterial drugs.
[0008] The drug includes a drug for preparing bacterial resistance transmission caused by conjugation transfer of resistance plasmids.
[0009] The antibacterial properties include bacteriostasis and / or bactericidal effects.
[0010] The antibacterial and / or bactericidal properties include inhibiting the spread of bacteria in the environment and / or within the body and / or killing bacteria in the environment and / or within the body.
[0011] The bio-organic acid includes one or more of citric acid, fumaric acid, or α-ketoglutarate.
[0012] The drug is a single-component or compound preparation.
[0013] The drug resistance plasmids include plasmids carrying RP4-7, plasmids carrying mcr-1, plasmids carrying tet(X4), or plasmids carrying bla. NDM-5 One or more of the plasmids.
[0014] The bacteria in question are bacteria, and the donor bacteria include *Escherichia coli* MG1655 (carrying IncP type plasmid RP4-7, containing ampicillin (AMP) and chloramphenicol (CHL) resistance genes), *Escherichia coli* CSZ4 (carrying mcr-1 gene IncX4 type plasmid), and *Escherichia coli* L65 (carrying bla...). NDM-5The following bacteria were identified: *E. coli* IncX3 plasmid (carrying *mcr-1* gene IncI2 plasmid), *E. coli* RF2-1 (carrying *tet(X4)* gene IncFII plasmid), *E. coli* EC600 (carrying *strA / strB* gene IncI1 plasmid), and *Klebsiella pneumoniae* C12 (carrying *bla* gene). NDM-5 The *IncX3* plasmid and *Klebsiella pneumoniae* YZ6 containing the hygromycin resistance gene were isolated in our laboratory. The recipient bacterium was *Escherichia coli* EC600 carrying the rifampicin resistance gene.
[0015] The concentration of the bio-organic acid is 20~200μg / mL.
[0016] The dosage forms of the drug include tablets, capsules, oral liquids, syrups, pills, injections, and lyophilized powder injections.
[0017] Beneficial effects: Compared with existing technologies, the advantages of this invention are: This invention discovers the possibility of using bio-organic acids as conjugation transfer inhibitors to control the spread of antibiotic resistance levels through in vitro and in vivo conjugation experiments. This invention provides the use of bio-organic acids as conjugation transfer inhibitors, limiting the conjugation transfer of various clinically resistant plasmids, and is one of the strategies for solving the problem of bacterial resistance. Attached Figure Description
[0018] Figure 1 The figure shows the results of the inhibitory effect of citric acid / fumaric acid / α-ketoglutarate on plasmid conjugation and transfer in vitro.
[0019] Figure 2 The figure shows the results of the significant inhibitory effect of citric acid / fumaric acid / α-ketoglutarate on the conjugation and transfer of plasmids carrying important clinical drug resistance genes.
[0020] Figure 3 The figure shows the inhibitory effect of citric acid / fumaric acid / α-ketoglutarate on conjugation transfer in the intraperitoneal cavity of mice.
[0021] Figure 4 The figure shows the inhibitory effect of citric acid / fumaric acid / α-ketoglutarate on conjugation transfer in the mouse intestine. Detailed Implementation
[0022] 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.
[0023] Example 1: Conjugation transfer experiment and determination of conjugation transfer frequency of RP4-7 plasmid
[0024] Donor bacteria: *Escherichia coli* MG1655 with RP4-7 plasmid; recipient bacteria: *Escherichia coli* EC600. All strains were publicly known and used. The sources of *Escherichia coli* MG1655, *Escherichia coli* EC600, and the RP4-7 plasmid can be found in the published article "Acetylshikonin reduces the spread of antibiotic resistance via plasmid conjugation" (International Journal of Antimicrobial Agents, 2024, 64(6):107370). The bacteria were cultured overnight at 37°C and 200 rpm, respectively. They were then collected by centrifugation and resuspended to OD600 = 0.5 using 0.01 M PBS buffer (pH=7.2) or LB broth from Qingdao Haibo Biotechnology Co., Ltd. (product number: HB0128). Donor and recipient bacteria were each mixed in 500 μL batches and then mixed with different concentrations (final concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL) of citric acid (CA-50, CA-100, and CA-200), fumaric acid (FA-50, FA-100, and FA-200), and α-ketoglutarate (α-KG-50, α-KG-100, and α-KG-200), respectively, and incubated for 15 hours. All experiments were performed in triplicate.
[0025] The mixed system was then inoculated onto LB agar selection plates containing ampicillin. Recipient bacteria were screened using LB agar plates containing 100 μg / mL ampicillin and 300 μg / mL rifampin, and single-drug agar plates containing 300 μg / mL rifampin. After 15 h of incubation, the colony count was determined. The conjugation transfer frequency was calculated as follows: Conjugation transfer frequency = number of conjugates (CFU / mL) / number of recipient bacteria (CFU / mL). This ratio is a dimensionless numerical value representing the average number of conjugates per recipient bacterium. Figure 1The vertical axis is on a logarithmic scale, 10 -3 A frequency value of 0.001 indicates that approximately one conjugate is formed per 1000 recipient bacteria. See results below. Figure 1 , Figure 1 The results showed that citric acid, fumaric acid, and α-ketoglutarate could all significantly inhibit the conjugation and transfer of engineered plasmid RP4-7.
[0026] Example 2: Conjugation transfer assay carrying plasmids containing important clinical drug resistance genes
[0027] For those carrying mcr-1, tet(X4) and bla NDM-5 Clinical strains with positive plasmids were tested to elucidate the effect of biogenic organic acids on the conjugation transfer of clinical plasmids carrying important drug resistance genes. Donor bacteria included *E. coli* CSZ4 (carrying the *mcr-1* gene IncX4 plasmid), *E. coli* LD67-1 (carrying the *mcr-1* gene IncI2 plasmid), *E. coli* RF2-1 (carrying the *tet(X4)* gene IncFII plasmid), *E. coli* EC600 (carrying the strA / strB gene IncI1 plasmid), and *E. coli* L65 (carrying the bla...). NDM-5 (IncX3 type plasmid), Klebsiella pneumoniae C12 (carrying bla) NDM-5 The IncX3 gene plasmid was isolated in our laboratory. Recipient bacteria: Escherichia coli EC600 used in the experiment was a strain preserved in our laboratory and carried the rifampicin resistance gene. All of the above strains are well-known and publicly available strains. For the sources of Escherichia coli and Klebsiella pneumoniae and the plasmid, please refer to the published article "Acetylshikonin reduces the spread of antibiotic resistance via plasmid conjugation" (International Journal of Antimicrobial Agents, 2024, 64(6): 107370).
[0028] All studies employed triplet replications. 500 μL of donor bacteria cultured overnight and 500 μL of recipient bacteria cultured overnight (OD600 = 0.5) were mixed and then inoculated with different concentrations (final concentrations of 50 μg / mL and 200 μg / mL) of citric acid (CA-50, CA-200), fumaric acid (FA-50, FA-200), and α-ketoglutarate (α-KG-50, α-KG-200), respectively, and cultured for 15 hours. The mixtures were then inoculated onto LB agar selection plates containing antibiotics (tigecycline, polymyxin, or meropenem, respectively). Transconjugators were screened using two-drug plates with corresponding resistances (LB agar plates containing 300 μg / mL rifampicin and 2 μg / mL tigecycline, 300 μg / mL rifampicin and 2 μg / mL polymyxin, and 300 μg / mL rifampicin and 2 μg / mL meropenem), and recipient bacteria were screened using single-drug plates containing rifampicin (300 mg / L). After 15 h of incubation, colony counts were performed, and the conjugation transfer frequency was calculated using the formula in Example 1. The conjugation transfer frequency was compared using the conjugation transfer frequency of the blank control group (without any organic acid) as a baseline. The lower the frequency, the stronger the inhibition of plasmid conjugation transfer. The strength of the inhibition effect could be determined by comparing the frequencies of different organic acid treatment groups at the same concentration. Results are shown below. Figure 2 Citric acid, fumaric acid, or α-ketoglutarate have a significant inhibitory effect on conjugation transfer of plasmids carrying important clinical drug resistance genes.
[0029] Example 3: Plasmid conjugation transfer assay between bacterial genera
[0030] The donor bacteria was Escherichia coli MG1655 carrying the RP4-7 plasmid, and the recipient bacteria was Klebsiella pneumoniae YZ6. All of the above strains are well-known and publicly available strains. The sources of Escherichia coli MG1655 and Klebsiella pneumoniae YZ6 and the RP4-7 plasmid can be found in the published article "Acetylshikonin reduces the spread of antibiotic resistance viaplasmid conjugation" (International Journal of Antimicrobial Agents, 2024, 64(6): 107370). 500 μL of donor bacteria cultured overnight and 500 μL of recipient bacteria cultured overnight (OD600=0.5) were mixed and then mixed with different concentrations of biological organic acids (final concentrations of 50 μg / mL and 200 μg / mL): citric acid (CA-50, CA-200), fumaric acid (FA-50, FA-200), and α-ketoglutarate (α-KG-50, α-KG-200), respectively, and cultured for 15 hours. The mixtures were then inoculated onto LB agar selection plates containing antibiotics (hygromycin). Transgender zygotes were screened using double-drug plates (100 μg / mL ampicillin and 100 mg / L hygromycin) with the corresponding resistance, and recipient bacteria were screened using single-drug plates containing hygromycin (100 mg / L). After 15 hours of culture, the colony count was performed, and the conjugation transfer frequency was calculated using the formula in Example 1. Results are shown below. Figure 2 In addition, the intergeneric syngenetic transfer of the RP4-7 plasmid from Escherichia coli to Klebsiella pneumoniae was also significantly inhibited by citric acid, fumaric acid, or α-ketoglutarate.
[0031] Example 4 Intraperitoneal conjugation transfer experiment in mice
[0032] Donor bacteria: *Escherichia coli* MG1655 with RP4-7 plasmid; recipient bacteria: *Escherichia coli* EC600. All strains were publicly known and used. The sources of *Escherichia coli* MG1655, *Escherichia coli* EC600, and the RP4-7 plasmid can be found in the published article "Acetylshikonin reduces the spread of antibiotic resistance via plasmid conjugation" (International Journal of Antimicrobial Agents, 2024, 64(6):107370). *Escherichia coli* MG1655 and *Escherichia coli* EC600 were cultured overnight, washed with 0.01 M PBS buffer (pH=7.2), resuspended to OD600 = 0.5, and a mixture of donor and recipient bacteria (100 μL each, volume ratio 1:1) was injected intraperitoneally. Fifteen minutes post-infection, the control group received intraperitoneal injections of PBS buffer (pH=7.2), while the experimental groups received additional organic acids (final concentrations of 50 μg / mL and 200 μg / mL), namely citric acid (CA-50, CA-200), fumaric acid (FA-50, FA-200), and α-ketoglutarate (α-KG-50, α-KG-200). Mice were euthanized 24 hours after infection. Liver, spleen, and kidneys were aseptically collected, homogenized, serially diluted, and cultured on antibiotic-containing agar plates. Colony counts and conjugation transfer frequencies were calculated after 15 hours of incubation. Results are as follows: Figure 3 As shown, the results of the peritoneal conjugation transfer model indicate that citric acid, fumaric acid, or α-ketoglutarate can inhibit plasmid conjugation transfer in peritoneal organs such as the liver, spleen, and kidney.
[0033] Example 5: Intestinal conjugation transfer assay in mice
[0034] Donor bacteria: *Escherichia coli* X7213 (carrying the mcr-1 positive IncX4 plasmid) and *Escherichia coli* (carrying the IncP plasmid RP4-7), recipient bacteria were intestinal flora. All of the above strains were publicly known strains. The sources of *Escherichia coli* X7213, the IncX4 plasmid, and the RP4-7 plasmid can be found in the published article "Acetylshikonin reduces the spread of antibiotic resistance via plasmid conjugation" (International Journal of Antimicrobial Agents, 2024, 64(6): 107370). Donor and recipient bacteria (intestinal flora) were conjugated in the mouse intestine via gavage once daily for 15 consecutive days. The donor bacteria were cultured overnight and adjusted to OD600 = 0.5. Mice were then orally administered 200 μL of the donor bacterial culture. Twenty-four hours after infection, fresh fecal matter was aseptically collected, homogenized, serially diluted, and plated onto agar plates containing antibiotics (polymyxin and ampicillin). Colony counts and conjugation transfer frequencies were calculated after 15 hours of incubation. Results are as follows: Figure 4 As shown, Figure 4 The horizontal axis represents the concentrations (citric acid, fumaric acid, and α-ketoglutarate at 50 μg / mL and 200 μg / mL, respectively) in in vitro experiments. For in vivo animal experiments, the in vitro concentrations need to be converted to mouse gavage doses. The conversion is based on the following: Assuming an average mouse weight of 20 g, the daily gavage volume is 200 μL. For example, at a concentration of 200 μg / mL, the daily dose per mouse = 200 μg / mL × 0.2 mL = 40 μg, which translates to a dose per kilogram of body weight = 40 μg / 0.02 kg = 2000 μg / kg = 2 mg / kg. Similarly, 50 μg / mL corresponds to a dose of 0.5 mg / kg. In the intestinal environment, concentrations of citric acid, fumaric acid, and α-ketoglutarate at 2 mg / kg (corresponding to 200 μg / mL) or 0.5 mg / kg (corresponding to 50 μg / mL) significantly inhibited the conjugation and transfer of resistance plasmids to the intestinal flora. The 200 μg / mL (i.e. 2 mg / kg) group showed the strongest inhibitory effect.
Claims
1. Application of bio-organic acids in the preparation of plasmid conjugation transfer inhibitors and / or antibacterial drugs.
2. The application according to claim 1, characterized in that, The drug includes drugs for preparing bacterial resistance transmission caused by conjugation transfer of resistance plasmids.
3. The application according to claim 1, characterized in that, The antibacterial properties include bacteriostasis and / or bactericidal effects.
4. The application according to claim 3, characterized in that, The antibacterial and / or bactericidal effects include inhibiting the spread of bacteria in the environment and / or within the body and / or killing bacteria in the environment and / or within the body.
5. The application according to any one of claims 1 to 4, characterized in that, The bio-organic acid includes one or more of citric acid, fumaric acid, or α-ketoglutarate.
6. The application according to any one of claims 1 to 4, characterized in that, The drug is a single-component or compound preparation.
7. The application according to any one of claims 1 to 4, characterized in that, The drug resistance plasmids include those carrying RP4-7, mcr-1, tet(X4), or bla. NDM-5 One or more of the plasmids.
8. The application according to any one of claims 1 to 4, characterized in that, The bacteria are bacteria, among which the donor bacteria include *E. coli* MG1655 carrying the IncP type plasmid RP4-7, *E. coli* CSZ4 carrying the mcr-1 gene IncX4 type plasmid, and *E. coli* carrying the bla gene. NDM-5 E. coli L65 carrying the IncX3 gene plasmid, E. coli LD67-1 carrying the IncI2 gene plasmid, E. coli RF2-1 carrying the tet(X4) gene IncFII gene plasmid, E. coli EC600 carrying the IncI1 gene plasmid containing the strA / strB gene, and E. coli carrying the bla gene plasmid... NDM-5 Klebsiella pneumoniae C12 containing the IncX3 gene plasmid and Klebsiella pneumoniae YZ6 containing the hygromycin resistance gene, wherein the recipient bacterium is Escherichia coli EC600 carrying the rifampicin resistance gene.
9. The application according to any one of claims 1 to 4, characterized in that, The concentration of the bio-organic acid is 20~200μg / mL.
10. The application according to any one of claims 1 to 4, characterized in that, The dosage forms of the drug are tablets, capsules, oral liquids, syrups, pills, injections, and lyophilized powder injections.