Application of econazole in inhibition of conjugational transfer of antibiotic resistance gene plasmid

Abstract

The invention discloses an application of econazole in inhibition of conjugational transfer of antibiotic drug-resistant gene plasmids. The drug econazole can effectively inhibit conjugational transfer of plasmids mcr-1, blaNDM-1, blaNDM-5, blaKPC-2 and tmexCD1-toprJ1 carrying last-line antibiotic drug resistance genes in a low-dose and broad-spectrum manner in bacteria and among bacteria, and the econazole is safe to mammalian cells, has broad-spectrum activity and is relatively clear in action mechanism, so that the econazole can be used for preparing drugs for preventing and treating antibiotic resistance. Econazole can be prepared as a conjugation inhibitor for preventing ARG propagation.

Description

Technical Field

[0001] The invention belongs to the technical field of antibacterial drug development, and particularly relates to an application of econazole in inhibiting the conjugation transfer of antibiotic resistance gene plasmids. Background Art

[0002] Infectious diseases caused by multidrug-resistant (MDR) bacteria have a high mortality rate and pose a serious threat to public health. Horizontal gene transfer (HGT) significantly promotes the spread of antimicrobial resistance genes (ARGs) in resistant bacteria under different environments. The mechanisms of HGT involve conjugation, transformation, transduction and gene transfer mediated by outer membrane vesicles (OMVs), among which plasmid-mediated conjugation transfer is the main one. Conjugation is a universally conserved HGT mechanism in bacterial communities, in which DNA is transferred from donor to recipient bacteria through direct contact. Conjugation can occur under different conditions, allowing bacteria to change their metabolic characteristics by exchanging genetic material. Therefore, conjugation is an important driver of bacterial evolution and environmental adaptation.

[0003] Carbapenems, polymyxins, and tigecycline are the last line of antibiotics for treating severe infections caused by MDR bacteria. However, pathogens resistant to these antibiotics have emerged worldwide, seriously affecting their therapeutic efficacy. The main mechanism of carbapenem resistance is that different types of bacteria carrying carbapenem hydrolase genes can produce carbapenemases, such as bla KPC and bla NDM . In addition, the global spread of plasmid-mediated polymyxin resistance genes (mcr) threatens the clinical application of polymyxins. For tigecycline, it has recently been discovered that the plasmid-mediated high-level tigecycline resistance gene tet (X4) and the plasmid-encoded resistance-nodulation-division type efflux pump gene cluster tmexCD1-topJ1 and its homologous genes can cause tigecycline resistance. Even "super bacteria" that carry different types of "last line of defense" antibiotic resistance genes continue to emerge. The horizontal transfer ability of these resistance genes poses a huge challenge to infection prevention and control.

[0004] Previous studies have shown that non-antimicrobial drugs (including non-antibiotic drugs, heavy metals, ionic liquids, and gases) can affect (promote or inhibit) the conjugation process. The development of conjugation inhibitors may be a promising strategy to address the antimicrobial drug response to the antibiotic resistance crisis. It can slow the spread of resistance genes without affecting the basic biochemical reactions of bacteria and restore the efficiency of existing antibiotics. For example, the neurohormone melatonin can effectively inhibit the conjugation transfer of ARGs mediated by RP4-7 plasmids and plasmids carrying mcr-1. In addition, studies have found that two anti-HIV drugs, abacavir and azidothymidine, can effectively reduce the spread of plasmids carrying extended-spectrum β-lactamase and carbapenemase genes. Despite these efforts, there are currently no reports of inhibitors that can effectively prevent the conjugation transfer of resistance genes to all three last-line antibiotics (carbapenems, polymyxins, and tigecycline). Therefore, further research on safe and effective conjugation inhibitors among non-antimicrobial drugs is of great significance for the prevention and control of MDR.

[0005] Econazole (Econ) is a broad-spectrum imidazole antifungal drug that can be applied topically in creams and sprays to treat superficial fungal infections. Topical or intravaginal use of econazole nitrate is safe and well tolerated in most patients. In addition, Econ exhibits some non-antifungal properties. Previous studies have shown that Econ can inhibit the growth and invasion of cancer cells. In addition, Econ can synergize with polymyxins to fight polymyxin-resistant Enterobacteriaceae by dissipating proton motive force (PMF), and can also work in combination with conventional antibiotics to effectively eliminate bacterial resistant cells. Although the various beneficial effects of Econ have been studied, the potential application of Econ as a binding inhibitor has not been explored. Summary of the invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide an application of econazole in inhibiting the conjugative transfer of antibiotic resistance gene plasmids.

[0007] The present invention is achieved by using econazole or its pharmaceutically acceptable salt in the preparation of an inhibitor for the conjugation transfer of antibiotic resistance gene plasmids within and between bacterial genera.

[0008] Preferably, the antibiotic resistance gene plasmid includes mcr-1, bla NDM-1 ,bla NDM-5 ,bla KPC-2 and tmexCD1-toprJ1.

[0009] Preferably, the concentration range of econazole is 8 to 128 μg / mL, the temperature is 30 to 42° C., the pH value is 6.0 to 8.0, and the donor-acceptor ratio is 1:1.

[0010] Preferably, the bacteria include drug-resistant Escherichia coli, Klebsiella pneumoniae, and Serratia marcescens; the drug resistance includes carbapenem resistance, rifampicin resistance, and polymyxin resistance.

[0011] The present invention overcomes the shortcomings of the prior art and provides an application of econazole in inhibiting the conjugative transfer of antibiotic resistance gene plasmids. The present invention studies the effect of econazole on the conjugative transfer of plasmid-mediated resistance genes, focusing on the last line of antibiotic resistance genes mcr-1, bla KPC-2 ,bla NDM ,tet(X4) and tmexCD1-toprJ1. By constructing a conjugation model, the effect of econazole on the conjugation efficiency of the mcr-1 plasmid under different conjugation conditions (temperature, pH, conjugation ratio and conjugation time) was explored. In addition, the present invention also tested the effect of econazole on the conjugation efficiency of the mcr-1 plasmid. KPC-2 ,bla NDM , tet(X4) and tmexCD1-toprJ1, the inhibitory ability of clinical bacterial plasmid conjugation transfer. The mechanism of its inhibitory effect was studied by detecting the effects of econazole on cell membrane permeability, conjugation bridge-related pathways and PMF. PMF is essential for maintaining a series of key processes of bacteria such as flagellar movement, efflux activity and ATP synthesis. The discovery of the present invention is of great significance for strengthening the clinical application of econazole, especially in preventing the last line of defense ARGs from spreading through conjugation.

[0012] Compared with the shortcomings and deficiencies of the prior art, the present invention has the following beneficial effects: the drug Econ can effectively inhibit the plasmids mcr-1 and bla carrying the last line of antibiotic resistance genes within and between bacterial genera in a low dose and broad spectrum manner. NDM-1 ,bla NDM-5 ,bla KPC-2 and tmexCD1-toprJ1 conjugative transfer, and Econ is safe for mammalian cells, has a broad-spectrum activity, and has a relatively clear mechanism of action. Therefore, Econ can be prepared as a conjugative inhibitor for preventing ARG propagation. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The growth kinetics curves of the donor (A) and the recipient (B) after treatment with different concentrations of Econ; the colony forming units (CFUs) of the donor and the recipient were determined by plate counting method every 4 hours, and the growth curves were drawn using Graphpad8.0; the X-axis is the incubation time, and the Y-axis is the CFU count, the unit is log 10CFU / ml; Donor: Polymyxin-resistant Escherichia coli J53 carrying IncI2 highly transmissible mcr-1 positive plasmid pEC_mcr1; Recipient: Rifampicin-resistant Escherichia coli EC600;

[0014] Figure 2 The statistics of the inhibitory effect of Econ on the conjugative transfer of mcr-1 plasmid; the X-axis represents the Econ concentration and the Y-axis represents the conjugation frequency; unpaired two-sided t-test was used for statistical analysis, and for n=3 biological independent experiments, the data are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001;

[0015] Figure 3 The inhibitory effect of Econ on EC600 conjugation transfer of Escherichia coli carrying mcr-1 plasmid under different conditions is statistically shown; the experimental conditions include (A) conjugation time, (B) conjugation temperature, (C) conjugation pH, and (D) conjugation ratio; the X-axis represents the concentration of Econ under different conjugation conditions, and the Y-axis represents the fold change of conjugation frequency; unpaired two-sided t-test was used for statistical analysis, and for n=3 biological independent experiments, the data are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001;

[0016] Figure 4 is the statistical analysis of the inhibitory effect of Econ on the conjugative transfer of clinically important antibiotic-resistant plasmids; (A) Econ treated plasmids carrying bla NDM-1 The frequency of conjugative transfer of plasmids from E. coli 9 to E. coli EC600; (B) in the presence of Econ, the frequency of conjugative transfer of plasmids from K. pneumoniae 7 carrying bla NDM-5 The frequency of conjugative transfer of plasmids to E. coli EC600; (C) In the presence of Econ, plasmids carrying bla KPC-2 (D) The frequency of conjugative transfer of plasmids from Serratia marcescens P49 to E. coli EC600; (E) 8-128 μg / mL Econ promotes the frequency of conjugative transfer of plasmids carrying tet(X4) from E. coli donors to E. coli recipients; the X-axis represents the Econ concentration and the Y-axis represents the conjugative frequency. Statistical analysis was performed using an unpaired two-sided t-test. Data are presented as mean ± SEM; N = 3 biologically independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001;

[0017] Figure 5Econ affects the membrane permeability and membrane protein gene expression statistics of donor and recipient strains; Among them, the outer membrane permeability of donor (A) and recipient (B); the overall membrane permeability of donor (C) and recipient (D); the relative expression level of membrane protein genes of donor (E) and recipient (F); Donor: polymyxin-resistant Escherichia coli J53 carrying IncI2 highly transmitted mcr-1 positive plasmid pEC_mcr1; Recipient: rifampicin-resistant Escherichia coli EC600; Unpaired two-sided t-test was used for statistical analysis; For n = 3 biological independent experiments, data are mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

[0018] Figure 6 The statistical results of Econ dissipating bacterial membrane potential, inhibiting ATP synthesis, and inhibiting ATP efflux activity; among them, the depolarization effect of Econ treatment on the bacterial membrane of donors (A) and receptors (B); the ATP levels of donors (C) and receptors (D) after Econ treatment, 10μM CCCP as positive control; the inhibitory effect of Econ treatment on the Nile infrared flow of donors (E) and receptors (F) was added 50mM glucose within 135s and the fluorescence was monitored for 4.75 minutes; donor: polymyxin-resistant Escherichia coli J53 carrying IncI2 high-transmission mcr-1 positive plasmid pEC_mcr1; receptor: rifampicin-resistant Escherichia coli EC600; unpaired two-sided t-test was used for statistical analysis, for n=3 biological independent experiments, the data are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001;

[0019] Figure 7 This is a picture of Econ inhibiting the motility of donor and receptor colonies in the flagella experiment; the bacterial suspensions of the donor and receptor strains were adjusted to OD 600 nm=0.5, inoculated in TSB medium containing 0.25% agar; donor: polymyxin-resistant Escherichia coli J53 carrying IncI2 highly transmissible mcr-1 positive plasmid pEC_mcr1; recipient: rifampicin-resistant Escherichia coli EC600;

[0020] Figure 8 It is the statistical result of the relative expression level of the gene virB11 related to the Mpf system of the receptor strain after treatment with Econ (0-64 μg / mL) Receptor: EC600 of rifampicin-resistant Escherichia coli; Statistical analysis was performed using an unpaired two-sided t-test; for n=3 biological independent experiments, data are mean ± SEM; *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001;

[0021] Fig. 9 The above is the mechanism by which Econ inhibits the conjugative transfer of ARGs carried by plasmids; the upper and lower lipid bilayers represent the cell membranes of the donor and recipient strains, respectively; for the donor strain, Econ can dissipate the bacterial membrane PMF (①) and reduce the ATP level (②), both of which are energy sources for T4SS (③④); Econ can also inhibit the expression level of the Mpf system gene virB11 in the donor strain ⑤, resulting in the inhibition of conjugative transfer efficiency; for the recipient strain, Econ can reduce its cell membrane permeability by inhibiting the expression of membrane proteins ⑥. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0023] 1. Methods

[0024] 1. Strains and reagents

[0025] All strains used in the examples of the present invention are shown in Table 1 below:

[0026] Table 1

[0027]

[0028] In short, the present invention uses different combinations of donor and recipient strains to test the inhibitory effect of Econ on the frequency of conjugative plasmid conjugation. Polymyxin-resistant Escherichia coli J53 (donor) carrying IncI2 highly transmissible mcr-1 positive plasmid pEC_mcr1 and rifampicin (RIF)-resistant Escherichia coli EC600 (MIC>1000μg / mL) (recipient) were used as model strains to study the effect of Econ on conjugation under different conditions and explore its mechanism. At the same time, 5 strains carrying different last-line antibiotic resistance genes (bla NDM-1 ,bla NDM-5 ,bla KPC-2 , tet(X4) and tmexCD1-toprJ1) were used as donor strains for conjugation experiments. The strains were cultured overnight in Luria-Bertani (LB) broth or LB agar plates at 37°C. Econazole nitrate was purchased from Sigma-Aldrich (Chemical Co, USA). Econ was first dissolved in dimethyl sulfoxide (DMSO) and then diluted to different working concentrations in culture medium. All experiments used 0.2% DMSO as a negative control, represented by 0μg / mL Econ group.

[0029] 2. Bacterial growth dynamics

[0030] Overnight cultures of E. coli J53 and E. coli EC600 carrying pEC_mcr1 plasmid were diluted 1:1000 with 3 mL of LB broth supplemented with different concentrations of Econ (0-128 μg / mL). The strains were incubated at 37°C with constant temperature shaking at 200 rpm for 12 h. GraphPad 8.0 (San Diego, CA, USA) was used to count the viable bacteria at each time point (0, 2, 4, 6, 8, 10, 12 h) and draw growth curves. The absorbance at 600 nm was measured using a DR3900 spectrophotometer from HACH, USA. Three biological replicates were used in all experiments.

[0031] 3. Bacterial conjugation

[0032] Escherichia coli J53 carrying the pEC_mcr1 plasmid was used as the donor strain, and Escherichia coli EC600 was used as the recipient strain. Overnight cultures of the donor and recipient were diluted 1:100 in 3 mL LB broth and continuously shaken at 200 rpm at 37°C until OD 600 nm reached 0.5. 2 mL of each culture was taken, washed with PBS and resuspended in 2 mL of brain heart infusion broth (BHI). Different concentrations of Econ (0-128 μg / mL) were added to the mixture of donor and recipient strains in a ratio of 1:1. Agar plates containing 1 μg / mL polymyxin and 300 μg / mL rifampicin were used to collect and count the conjugates. In addition, different conjugation conditions were explored, including conjugation time, temperature, pH value and donor-recipient ratio. All experiments were repeated three times. The conjugation frequency was calculated by dividing the number of conjugates by the number of donors. The change fold of the conjugation frequency was calculated by dividing the conjugation frequency after Econ treatment by the conjugation frequency of untreated D.

[0033] Also select IncX3 bla NDM-1 Escherichia coli 9 with positive plasmid, IncX3 bla NDM-5 Klebsiella pneumoniae 7 with positive plasmid, IncP bla KPC-2 Serratia marcescens P49 carrying a positive plasmid, Escherichia coli G5 carrying an IncX1 tet (X4) positive plasmid, and Klebsiella pneumoniae P13 carrying an IncFIA tmexCD1-topJ1 positive plasmid were used as donors for conjugation experiments. NDM-1 ,bla NDM-5 ,bla KPC-2The recipient strains of tet(X4) and tet(X4) were E. coli EC600. The strain P13 carrying tmexCD1-toprJ1 was carbapenem-resistant E. coli eco57. The conjugation frequency of tmexCD1-toprJ1-positive plasmid from P13 to EC600 was low (<1×10 -7 The conjugation conditions of clinical strains were as follows: conjugation time, 24 h; conjugation temperature, 37°C; conjugation pH, 7.0; and a donor-recipient ratio of 1:1.

[0034] 4. Drug sensitivity test and PCR detection

[0035] The minimum inhibitory concentration (MIC) of donors, recipients, and conjugates was determined by broth dilution method. Escherichia coli ATCC 25922 was used as control. The results were interpreted according to the American Clinical and Laboratory Standards Association guidelines. 100 pseudoconjugates were randomly selected for PCR verification and agarose gel electrophoresis identification of the corresponding drug resistance genes.

[0036] 5. Membrane permeability assessment

[0037] The permeability of the bacterial outer membrane was determined using the fluorescent probe 1-N-phenylnaphthylamine (NPN). Briefly, an overnight culture of E. coli carrying pEC_mcr1 (donor) and EC600 (acceptor) was diluted 1:100 in fresh LB broth and incubated until OD 600nm =0.5. The strain was centrifuged and resuspended in sterile PBS (pH = 7.2) to adjust the OD 600nm =0.5. Then stain with 10 μM NPN at 37°C in the dark for 30 min. Treat with Econ at a concentration of 0-128 μg / mL at 37°C in the dark for 2 h. TM The fluorescence of NPN was measured by a microplate reader (PerkinElmer, Waltham, MA, USA) with excitation and emission wavelengths of 350 nm and 420 nm, respectively.

[0038] Bacterial inner membrane permeability was determined using propidium iodide (PI), which was slightly modified from a previously described method. Briefly, overnight cultures of donors and recipients were diluted 1:100 in 3 mL of fresh LB and incubated at 37°C until the exponential phase was reached. The cultures were then shaken continuously at 200 rpm for 3 h at Econ concentrations ranging from 0 to 128 μg / mL. After the incubation period, the cultures were washed with PBS and the bacteria were resuspended in the same volume of PBS. PI (Beyotime, Shanghai, China) at a final concentration of 0.05 μM was added to the bacterial suspension and incubated in the dark for 30 min. The cultures were then washed twice with PBS and resuspended in the same volume of fresh PBS. The fluorescence intensity of the samples was monitored using a PerkinElmer EnSight microplate reader with excitation and emission wavelengths of 535 nm and 615 nm, respectively.

[0039] 6. Determination of bacterial membrane potential

[0040] The voltage-sensitive dye 3,3-dipropylthiodicarbonylidene iodide (Beyotime, Shanghai, China) was used to measure the bacterial membrane potential. Briefly, 0-128 μg / mL of Econ was added to the stained bacterial cells. 5 μM valinomycin was used as a positive control. The fluorescence level of the samples was measured for 19 minutes using a PerkinElmer EnSight microplate reader.

[0041] 7. Flagellar motility experiment

[0042] The method for evaluating flagellar motility was as described previously with slight modifications. Briefly, cultures of donors and recipients were inoculated in LB medium containing 0.3% agar and supplemented with different concentrations of Econ (0, 32, 512 μg / mL). After incubation at 37°C for 48 h, the size of the swimming zone was measured.

[0043] 8. Intracellular ATP determination

[0044] Intracellular ATP was measured using the Enhanced ATP Assay Kit (Beyotime, Shanghai, China) following the manufacturer's instructions with slight modifications. Briefly, after 3 h of treatment with 0-128 μg / mL Econ, the donor and recipient strains were lysed and the supernatants were measured using a PerkinElmer EnSight microplate reader.

[0045] 9. Optimize the Nile infrared emission test

[0046] Efflux activity was measured using the Nile infrared efflux assay as described previously with minor modifications. Donor and recipient bacterial cells were centrifuged and resuspended in PBS buffer containing 1 mM MgCl2, and the OD 600nmAdjust to 1.0. The cells were allowed to stand for about 15 minutes, and CCCP was added at a final concentration of 10 μM. After 15 minutes, different concentrations of Econ were added. After 15 minutes, the cell suspension was stained with 5 μM Nile Red for 1 hour. The cells were centrifuged and resuspended in PBS with the same concentration of Econ as before. Subsequently, the fluorescence of the cell suspension was detected using a PerkinElmer EnSight microplate reader. The fluorescence of the cell suspension was observed for more than 120 seconds, and then 50 mM glucose was added to quickly trigger the Nile infrared emission and monitored for an additional 165 seconds.

[0047] 10. qRT-PCR analysis

[0048] Total RNA from donor and recipient cells was extracted using a bacterial RNA extraction kit (Vazyme, Nanjing, China) according to the manufacturer's instructions with an Econ concentration of 0 to 64 μg / mL. Quantitative real-time PCR (qRT-PCR) was performed on a Roche LightCycler 480 (Roche Diagnostics GmbH, Basel, Switzerland) using the primers reported previously and the HiScript II One-Step RT-PCR Kit (Vazyme). The 16S rRNA gene was used as an internal reference gene. -ΔΔCt The relative expression of genes was obtained by analysis method. The primers used in the examples of the present invention are shown in Table 2 below.

[0049] Table 2

[0050]

[0051]

[0052] 11. Statistical analysis

[0053] All experiments were performed with at least 3 biological replicates, and the data are presented as mean ± SD. Unpaired t-test was used for comparison between two groups, and one-way ANOVA was used for comparison between multiple groups. GraphPad Prism 9.0 was used to calculate p values ​​( * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001).

[0054] 2. Results

[0055] 1. Econ inhibits conjugative transfer of drug-resistant plasmids

[0056] The present invention transfers the naturally mcr-1 IncI2 plasmid pEC_mcr1 from Escherichia coli J53 (donor) to Escherichia coli EC600 (recipient) by conjugation to determine the inhibitory effect of Econ on conjugation. Studies have shown that Econ has no obvious antibacterial effect on Enterobacteriaceae, and the MIC is greater than 128 μg / mL. In order to further eliminate the effect of Econ on bacterial growth, the growth curves of donor bacteria and recipient bacteria were measured after treatment with different concentrations of Econ (0-128 μg / mL). The results showed that all tested concentrations of Econ had no effect on the normal growth of the donor and the recipient ( Figure 1 Therefore, the following experiments used sub-inhibitory concentrations of Econ ranging from 0 to 128 μg / mL.

[0057] In the concentration range of 8 to 128 μg / mL, Econ significantly reduced the conjugation frequency of IncI2 plasmid pEC_mcr1 carrying mcr-1 ( Figure 2 In order to comprehensively study the inhibitory effect of Econ on conjugation transfer under different conditions, the present invention studied four factors, including conjugation time, conjugation temperature, conjugation pH, selection of donor-acceptor conjugation ratio ( Figure 3 ). The conjugate was verified by MIC and PCR experiments. The MICs of the donor, recipient, and conjugate are shown in Table 3 below.

[0058] Table 3

[0059]

[0060] The results showed that all conjugates acquired drug-resistant plasmids from the donor, and the conjugation system was suitable for further experiments. The conjugation results showed that Econ had an inhibitory effect on conjugation under different conjugation conditions. Specifically, Econ inhibited the conjugation transfer of pEC_mcr1 in a time-dependent manner ( Figure 3 A). In terms of binding temperature, Econ can block the binding of pEC_mcr1 at 30℃, 37℃ and 42℃, and the inhibitory effect of Econ is most obvious at 37℃ ( Figure 3 B). In addition, Econ showed a significant inhibitory effect on the conjugation transfer of pEC_mcr1 at pH values ​​of 6.0, 7.0, and 8.0, and the inhibitory effect was strongest at pH 7.0 ( Figure 3 C). In addition, although Econ can inhibit the conjugative transfer of pEC_mcr1 at different donor-acceptor ratios, the inhibitory effect is highest when the donor-acceptor ratio is 1:1 ( Figure 3 D) Econ inhibits the conjugative transfer of IncI2 plasmid carrying mcr-1 under different conjugative conditions, so Econ has the potential for clinical application as a conjugative inhibitor.

[0061] In order to fully study the broad-spectrum inhibitory effect of Econ on conjugation, the present invention uses conjugative drug-resistant plasmids carrying different clinically important NDM-1 (IncX3),bla NDM-5 (IncX3),bla KPC-2 (IncP), tet(X4)(IncX1) and tmexCD1-toprJ1(IncFIA). NDM-1 ,bla NDM-5 ,bla KPC-2 The conjugation of the plasmid tmexCD1-toprJ1 has an inhibitory effect ( Figure 4 ). In brief, 16 μg / mL, 64 μg / mL, 4 μg / mL, and 8 μg / mL of Econ were required to inhibit the expression of bla NDM-1 ,bla NDM-5 ,bla KPC-2 Conjugative transfer of plasmids containing tmexCD1-toprJ1 from Enterobacteriaceae donors to Escherichia coli recipients ( Figure 4 A, B, C, D). Higher concentrations of Econ increase the inhibitory effect on conjugation frequency ( Figure 4 E). In contrast, 8 μg / mL and 128 μg / mL of Econ significantly promoted the conjugative transfer of the plasmid carrying tet(X4). These results indicate that the inhibitory effect of Econ on plasmid conjugation is broad-spectrum and dose-dependent.

[0062] 2. Econ reduces membrane permeability and inhibits the expression of membrane protein genes

[0063] The bacterial cell membrane is considered to be an important barrier to prevent DNA translocation, including conjugative transfer of plasmids. Therefore, the present invention measures the permeability of the outer membrane and inner membrane of the donor Escherichia coli (E. coli J53 carrying pEC_mcr1) and the recipient E. coli (E. coli EC600) to detect cell membrane variations after Econ treatment. NPN is a hydrophobic fluorescent probe that is not rejected by the outer membrane and has weak fluorescence in an aqueous environment. Once the integrity of the outer membrane is disturbed, NPN enters the phospholipid layer of the outer membrane and releases significant fluorescence. In the present embodiment, the fluorescence intensity of the donor and recipient strains treated with 32μg / mL to 128μg / mL Econ increased significantly, indicating that Econ can interfere with the integrity of the bacterial outer membrane ( Figure 5A, B). This finding is consistent with previous data (reference: Xu, C., Chen, K., Chan, KF, Chan, EWC, Guo, X., Chow, HY, Zhao, G., Zeng, P., Wang, M., Zhu, Y. and Li, X., 2020. Imidazole type antifungal drugs are effective colistin adjuvants that resensitize colistin-resistant Enterobacteriaceae. Advanced Therapeutics, 3 (9), p. 2000084.). Econ can bind to the lipid bilayer of the bacterial cell membrane, causing slight dissolution of the liposome bilayer, leading to increased outer membrane permeability. The effect of Econ on bacterial outer membrane permeability occurred when the Econ concentration was higher than that inhibiting conjugative transfer, indicating that there may be other mechanisms for Econ's inhibitory effect on conjugative transfer. The bacterial membrane permeability of the donor strain and recipient strain detected by PI staining did not change and was significantly reduced, respectively ( Figure 5 C, D).

[0064] In order to further study the effect of Econ on membrane permeability, the present invention measured the relative expression of genes encoding membrane pore proteins. In the donor strain, the presence of Econ downregulated the expression levels of the outer membrane genes opmA and ompF by about 2 times. At the same time, 16 μg / mL Econ alone can downregulate the expression level of the inner membrane gene tolA by 1.4 times. At concentrations of 32 and 64 μg / mL, Econ can slightly increase the expression level of tolA by about 1.2 times ( Figure 5 E). In contrast, the expression levels of ompA, ompF, and ompF of the receptor were significantly downregulated by nearly 3-fold, indicating that Econ can reduce the overall membrane permeability of the receptor by inhibiting the expression of important inner membrane porins ( Figure 5 F).

[0065] 3. Econ dissipative bacterial proton motive force

[0066] Since PMF and ATP hydrolysis may be the energy sources for substrate translocation and secretion during conjugation, and PMF is the driving force for ATP synthesis, the present invention assumes that Econ may inhibit plasmid conjugation transfer by dissipating PMF. PMF is the electrochemical potential of protons established by the electron transport chain, resulting in the extrusion of protons from the inside of the bacterial cell to the outside. PMF is the sum of two parameters, namely membrane potential and transmembrane proton gradient. Studies have shown that Econ can restore the antibacterial effect of polymyxin against polymyxin-resistant Enterobacteriaceae by acting as a PMF dissipator. In an embodiment of the present invention, the bacterial membrane potential of donor and recipient strains was measured using the fluorescent probe DiSC3(5), which can accumulate in polarized cells. The accumulation of DiSC3(5) on the bacterial membrane leads to overall fluorescence quenching in the cell suspension. After the bacterial membrane potential is depolarized, DiSC3(5) cannot accumulate and is rapidly released into the culture medium, resulting in a dequenching effect that can be monitored by fluorescence. Using K, which can cause membrane potential dissipation + The carrier valinomycin was used as a positive control. The results showed that 8-128 μg / mL of Econ could dissipate PMF by depolarizing the bacterial membrane potential of both donor and recipient strains due to the release of the dye into the culture medium. Figure 6 A, B).

[0067] Previous studies have shown that PMF can drive many biochemical processes, including ATP production, efflux activity, and bacterial motility. Therefore, the present invention first used an enhanced ATP assay kit to study the effect of Econ on donor and acceptor ATP synthesis. 8-128 μg / mL Econ can cause a significant decrease in ATP levels in both donor and acceptor strains ( Figure 6 C, 6D), indicating that Econ can effectively inhibit the ATP synthesis of the donor strain, and ATP is required for substrate transport and pilus production in the type IV secretion system (T4SS).

[0068] It is known that PMF is the driving force of efflux pumps. In order to further confirm the dissipation effect of Econ on PMF, the present invention measured the efflux activities of the donor (IncI2 (mcr1)) and the acceptor after Econ treatment. Nile red is a lipophilic fluorescent probe that emits weak fluorescence in aqueous solution but strong fluorescence in a non-polar environment, so it is often used to stain intracellular lipids. Nile red is a substrate for various efflux pumps, and it can be pumped in and out immediately in cells that are energized. When the efflux pump is inhibited and Nile red is anchored in the bacterial membrane, stronger fluorescence can be detected. Our results show that Econ can effectively inhibit the efflux of Nile red from the donor strain at 8-128 μg / mL, and in a dose-dependent manner ( Figure 6E). After 135s of Econ treatment, 50mM glucose was added to activate the cells, triggering Nile Red efflux again. Nile Red of the wild-type strain was immediately pumped out. However, the inhibitory effect of Econ on the efflux pump was still obvious in the reactivated donor and recipient cells, and the inhibitory effect of 64μg / mL and 128μg / mL Econ on the Nile Red efflux pump was stronger. The inhibitory effect of Econ on Nile Red efflux was also observed in the recipient ( Figure 6 F), which indicates that Econ, as a PMF dissipator, can effectively inhibit the efflux pump.

[0069] In addition, since flagella formation and flagellar rotation, which affect bacterial motility, are both dependent on PMF, flagellar experiments were used to examine bacterial motility of donor and recipient strains. Figure 7 As shown, the migration distances of the donor and recipient strains inoculated on the semisolid medium containing 32 μg / mL and 512 μg / mL Econ were significantly reduced in a dose-dependent manner compared with the medium without Econ. The migration distances of the donor and recipient were 1.43 ± 0.05 cm and 1.20 cm, respectively. Econ dose-dependent treatment shortened the migration distances to 0.83 ± 0.05 cm and 0.77 ± 0.05 cm, respectively. Therefore, Econ, as a PMF dispersant, can effectively inhibit the movement of PMF donor and recipient strains.

[0070] 4. Econ hinders the formation of the junction pair (Mpf) system

[0071] The formation of conjugative pairs organized by the Mpf gene is an important biological process to ensure DNA mobilization in conjugative gene transfer. The conjugative system of the donor strain pEC_mcr1 plasmid belongs to the Mpf system and is composed of 11 proteins, namely VirB1 to VirB11. These 11 proteins are assembled in a complex called the type IV secretion system (T4SS). By quantitatively detecting the expression level of virB11, the expression of the Mpf gene in the donor strain can be determined. After the addition of Econ (32-64 μg / mL), the expression level of virB11 was significantly downregulated by about 3 times. These results indicate that Econ can inhibit the expression of the Mpf gene, thereby effectively blocking the Mpf system and ultimately inhibiting DNA transport and the generation of pili ( Figure 8 ).

[0072] Discussion

[0073] The global spread of drug-resistant genes has seriously affected the effectiveness of current antibiotics in clinical treatment and has become an urgent threat to public health. HGT is one of the main mechanisms driving the widespread spread of ARGs, among which plasmid-mediated conjugation plays a key role. In addition, ARGs are increasingly frequently detected in a variety of environments such as water, soil, and air, which has attracted widespread attention. To make matters worse, many environmental factors and drugs can promote the conjugation process, such as silver ions, heavy metals, and subinhibitory concentrations of antibiotics, fungicides, non-antibiotic drugs, ionic lipids, non-nutritive sweeteners, and even gases. However, research on preventing or inhibiting the spread of ARGs is still relatively rare. The use of FDA-approved drugs provides a rapid and economical way to identify conjugation inhibitors with acceptably low toxicity to mammalian cells. For example, it has been reported that FDA-approved melatonin can inhibit the conjugative transfer of different types of clinical plasmids carrying mcr-1, but melatonin has no significant effect on plasmids carrying bla. NDM Conjugation with plasmids containing either tet(X4) or tet(X4) did not work.

[0074] So far, only a few promising targets have been identified for new drug development to inhibit the conjugation process. It has been reported that TraE, an important component of T4SS, is the target of some molecules that inhibit conjugation. A recent study showed that PMF dissipators may become inhibitors of bacterial activity and horizontal gene transfer. Econ has been shown to be a highly effective polymyxin adjuvant that can restore the antibacterial effect of polymyxin, but the inhibitory effect of Econ on the plasmid-mediated conjugation process has not been reported. In the present invention, it was found that subinhibitory concentrations of Econ can inhibit the growth of bacteria carrying the last-line antibiotic resistance genes mcr-1 (IncI2), bla NDM-1 (IncX3),bla NDM-5 (IncX3),bla KPC-2 The plasmids carrying these last-line antibiotic resistance genes belong to different groups including IncFIA, IncX1, IncX3, IncI2 and IncP, suggesting that Econ has a broad-spectrum inhibitory effect on plasmid conjugation. In addition, Econ can maintain its inhibitory effect under different conjugation conditions (temperature, time, pH and donor-receptor ratio). In the mechanism study of the present invention, it was found that the inhibitory effect of Econ is related to multiple pathways, including reducing the permeability of the receptor inner membrane, dissipating the bacterial PMF of the donor and the receptor, and blocking the Mpf system of the donor ( Fig. 9). Econ inhibits conjugative transfer of plasmids by inhibiting the expression of inner membrane proteins, such as TolA, which interact with porins on the cell membrane. Most importantly, Econ inhibits T4SS, a macromolecular complex that mediates DNA conjugative transfer and virulence factor transport, by inhibiting the expression of the Mpf gene encoding the T4SS protein. In addition, since both PMF and ATP are energy sources for T4SS, the dissipation of PMF caused by Econ inhibits ATP synthesis, which also inactivates T4SS.

[0075] In summary, the drug Econ can effectively inhibit the plasmids mcr-1 and bla carrying the last line of antibiotic resistance genes within and between bacterial genera in a low-dose and broad-spectrum manner. NDM-1 ,bla NDM-5 ,bla KPC-2 and tmexCD1-toprJ1 conjugative transfer. The inhibitory effect of Econ on conjugation was verified by different conjugation methods, and the application of Econ at working concentrations did not affect the growth of donor and recipient strains. Its mechanism of action includes reducing inner membrane permeability, dissipating PMF, and blocking T4SS. Compared with previously published conjugation inhibitors, Econ is safe for mammalian cells, has a broad spectrum of activity, and has a relatively clear mechanism of action. Therefore, the application of the conjugation inhibitor Econ is a potential effective control measure to prevent the spread of ARG.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of econazole or a pharmaceutically acceptable salt thereof in the preparation of an inhibitor of conjugative transfer of antibiotic resistance gene plasmids within and between bacterial genera.

2. The use according to claim 1, characterized in that The antibiotic resistance gene plasmid includes mcr-1, bla NDM-1 ,bla NDM-5 ,bla KPC-2 and tmexCD1-toprJ1.

3. The use according to claim 2, characterized in that The concentration range of the econazole is 8 to 128 μg / mL, the temperature is 30 to 42° C., the pH value is 6.0 to 8.0, and the donor-acceptor ratio is 1:

1.

4. The use according to claim 1, characterized in that The bacteria include drug-resistant Escherichia coli, Klebsiella pneumoniae, and Serratia marcescens; the drug resistance includes carbapenem resistance, rifampicin resistance, and polymyxin resistance.

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