Use of monoterpenic phenolic compounds for the preparation of an inhibitor of conjugative transfer of drug resistance plasmids

Abstract

The application discloses application of monoterpene phenolic compounds in preparation of drug-resistant plasmid conjugative transfer inhibitors, belongs to the technical field of biological medicine, and the monoterpene phenolic compounds are carvacrol and / or thymol; the drug-resistant plasmid includes carbapenem drug-resistant plasmid. The application provides application of carvacrol and thymol in preparation of medicines for inhibiting conjugative transfer of carbapenem drug-resistant plasmid in bacteria species and between species. Experiments show that carvacrol and thymol significantly inhibit the conjugative transfer of the commonly seen carbapenem drug-resistant gene bla NDM The conjugative transfer of positive IncX3 type drug-resistant plasmid to animal source and human source strains in species and between species is proved, so that it is proved that carvacrol and thymol can be used as inhibitors of conjugative transfer of drug-resistant plasmid, so as to reduce the spread of the level of carbapenem drug resistance in feed, drinking water and organisms and even in wound infection. The inhibitor can replace antibiotics and has important popularization and application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of monoterpenoid phenolic compounds in the preparation of drug-resistant plasmid conjugation transfer inhibitors. Background Technology

[0002] In recent years, due to the irrational use of antimicrobial drugs worldwide, major clinical pathogens have developed serious resistance to commonly used drugs. The emergence and prevalence of antibiotic resistance pose a serious threat to human and animal health, and plasmid-mediated horizontal gene transfer (HGT) plays a crucial role in this process. The main pathways of HGT in bacteria include conjugation, transformation, and transduction. Among these three pathways, conjugation transfer is generally considered the most important; therefore, inhibiting bacterial plasmid conjugation transfer has become one of the most important means of controlling the spread of resistance genes.

[0003] Carbapenems are a class of β-lactam antibiotics, crucial for treating multidrug-resistant bacteria and severe bacterial infections in humans. In recent years, carbapenem-resistant Enterobacteriaceae have spread rapidly globally, and NDM-type carbapenemases have been widely found in both animal and human bacteria. Notably, although carbapenems are not approved for use in livestock and poultry farming, NDM-producing carbapenem-resistant strains have already emerged and become prevalent in the livestock and poultry farming chain. The IncX3 plasmid carries the carbapenem resistance gene bla... NDM The most prevalent plasmid type plays a crucial mediating role, potentially spreading horizontally into pathogenic *Escherichia coli* and highly virulent *Klebsiella pneumoniae* (HvKP), which poses a significant clinical threat to humans, and remaining stable there. Furthermore, if carrying *bla*... NDM The spread of the IncX3 plasmid into highly virulent carbapenem-resistant Klebsiella pneumoniae (Hv-CRKP) producing the KPC-2 enzyme will render avibactam, a β-lactamase inhibitor recently introduced for human clinical use, ineffective, posing a serious threat to human life. Currently, controlling the spread of carbapenem-resistant Enterobacteriaceae in human clinical practice and livestock farming has attracted high attention from various countries; however, there are currently no reports on carbapenem-resistant plasmid conjugation transfer inhibitors, and no conjugation transfer inhibitors have been explicitly approved for clinical use.

[0004] Natural plants, as a rich resource, provide bioactive compounds for the treatment of various diseases. Carvacrol and thymol belong to the monoterpenoid phenolic class and are isomers of each other. They have similar aromas, hence carvacrol is also known as isothymol. Both possess antibacterial and anti-inflammatory properties, and their addition to animal feed has positive effects on growth performance, gut microbiota, and animal welfare. Long-term, repeated inhalation of high doses has not revealed chronic toxicity. Based on this, this invention explores the potential value of carvacrol and thymol as plasmid conjugation transfer inhibitors. Summary of the Invention

[0005] The purpose of this invention is to provide the application of monoterpenoid phenolic compounds in the preparation of drug-resistant plasmid conjugation transfer inhibitors, thereby solving the problems existing in the prior art. The plant-derived natural monoterpenoid phenolic compounds (carvacrol and thymol) provided by this invention significantly inhibit the clinically common carbapenem resistance gene bla. NDM The positive IncX3 type drug-resistant plasmid was detected to be conjugated and transferred within and between species of animal and human strains, thus confirming that carvacrol and thymol can act as inhibitors of drug-resistant plasmid conjugation and transfer, thereby reducing the spread of carbapenem resistance levels in feed, drinking water, organisms, and even wound infections.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the application of monoterpenoid phenolic compounds in the preparation of drug-resistant plasmid conjugation transfer inhibitors, wherein the monoterpenoid phenolic compounds are carvacrol and / or thymol.

[0008] Furthermore, the resistance plasmid includes a carbapenem resistance plasmid.

[0009] Furthermore, the carbapenem resistance plasmid is bla NDM Positive IncX3 plasmid.

[0010] Furthermore, the carvacrol and / or thymol inhibit the intra- and inter-species synergistic transfer of carbapenem resistance plasmids.

[0011] Furthermore, the carvacrol and / or thymol are used as additives in feed, drinking water, and topical ointments to prevent the spread of antibiotic resistance genes to intraspecific and interspecific bacteria.

[0012] Furthermore, the intraspecific bacteria are Escherichia coli; the interspecific bacteria are Klebsiella pneumoniae.

[0013] Furthermore, the concentration of carvacrol and / or thymol in the inhibitor is 16-64 μg / mL.

[0014] Furthermore, the concentration of carvacrol and / or thymol in the inhibitor is 64 μg / mL.

[0015] Furthermore, the inhibitor is in the form of powder, soluble powder, granules, water dispersant, suspension, emulsifiable concentrate, microemulsion, or aqueous solution.

[0016] The present invention also provides a drug for inhibiting the intra- and inter-species transfer of carbapenem resistance plasmids, comprising carvacrol and / or thymol.

[0017] The present invention discloses the following technical effects:

[0018] This invention provides the application of plant-derived natural monoterpenoid phenolic compounds (carvacrol and thymol) in the preparation of drugs that inhibit the intra- and inter-species fusion and transfer of carbapenem resistance plasmids in bacteria. Experiments have shown that carvacrol and thymol significantly inhibit the clinically common carbapenem resistance gene bla. NDM Positive IncX3 type drug-resistant plasmids were observed to co-transfer between animal and human strains, both intraspecifically and interspecifically. This confirms that carvacrol and thymol can act as inhibitors of drug-resistant plasmid conjugation transfer, thereby reducing the spread of carbapenem resistance in feed, drinking water, and even wound infections, and can replace antibiotics. Furthermore, carvacrol and thymol exhibit more significant inhibitory effects at 37°C, which is closer to the body temperature of humans and animals, indicating that these two compounds have the potential to be used to prepare in vivo conjugation transfer inhibitors. Simultaneously, the plant-derived natural monoterpenoid phenolic compounds provided by this invention have advantages such as good stability, low cost, environmental friendliness, and easy availability. Therefore, the drug-resistant plasmid conjugation transfer inhibitors provided by this invention have significant value for widespread application. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Line graph showing the stability of plasmids after different strains were transformed into the IncX3 type plasmid;

[0021] Figure 2 The effect of different antibacterial concentrations of carvacrol at 37℃ (a) and 25℃ (b) on intraspecific conjugation frequency of IncX3 type plasmids;

[0022] Figure 3 The effect of different antibacterial concentrations of carvacrol at 37℃ (a) and 25℃ (b) on the interspecific synaptic frequency of the IncX3 type plasmid;

[0023] Figure 4 The effect of different antibacterial concentrations of thymol at 37℃ (a) and 25℃ (b) on intraspecific conjugation frequency of IncX3 type plasmids;

[0024] Figure 5 The effect of different antibacterial concentrations of thymol at 37℃ (a) and 25℃ (b) on the interspecific synaptic frequency of the IncX3 type plasmid;

[0025] Figure 6 The effect of different antibacterial concentrations of carvacrol and thymol combined drugs on interspecies synergistic transfer of IncX3 plasmid at 37℃ was investigated. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] Unless otherwise specified, all materials, instruments and reagents used in this invention are commercially available; unless otherwise specified, all experimental methods used are conventional experimental methods in the field.

[0032] The IncX3 type bla used in the following embodiments NDM The plasmid-producing Escherichia coli donors, environmentally sourced KPC-2 enzyme-producing Klebsiella pneumoniae, and avian pathogenic Escherichia coli were preserved by the Veterinary Pharmacology Laboratory of the College of Veterinary Medicine, Qingdao Agricultural University, while the human-derived KPC-2 enzyme-producing highly virulent Klebsiella pneumoniae was provided by Qingdao Municipal Hospital.

[0033] Example 1

[0034] The IncX3-containing bla used in this embodiment NDM The plasmid-derived *E. coli* donor bacteria were successfully transferred to three recipient bacteria—environmentally sourced *Klebsiella pneumoniae*, human highly virulent *Klebsiella pneumoniae*, and avian pathogenic *E. coli*—using a broth-based conjugation transfer assay. The donor bacteria were resistant to meropenem and ceftazidime / avibactam, but sensitive to amikacin. All recipient bacteria were resistant to amikacin; avian pathogenic *E. coli* was sensitive to meropenem, and *Klebsiella pneumoniae* was sensitive to ceftazidime / avibactam. The specific steps included:

[0035] (1) Preparation of bacterial culture

[0036] The purified donor and recipient bacteria were inoculated onto LB agar medium and cultured overnight. Single colonies from the medium were picked and inoculated into 10 mL centrifuge tubes containing 4 mL of sterile LB broth. The tubes were incubated at 37°C for 4 h, and the OD values ​​of the donor and recipient bacteria were measured using a UV spectrophotometer. 600 Around 0.5.

[0037] (2) Conjugation culture

[0038] The donor bacteria were mixed with environmental Klebsiella pneumoniae, human highly virulent Klebsiella pneumoniae, and avian pathogenic Escherichia coli in a 1:1 ratio and incubated at 37°C overnight for conjugation culture.

[0039] (3) Conjugate screening

[0040] After the conjugation culture is completed, the mixed bacterial culture is centrifuged at 5000 r / min for 5 min, the supernatant is discarded, and physiological saline is added and mixed well for later use.

[0041] Pathogenic Escherichia coli + IncX3 plasmid: Spread 100 μL of the mixed bacterial suspension onto LB agar plates (1 μg / mL meropenem + 32 μg / mL amikacin), incubate at 37°C for 20 h, pick colonies and culture in LB broth with shaking, and determine the presence of blavin in the conjugates by PCR. NDMGene.

[0042] Klebsiella pneumoniae + IncX3 plasmid (environmentally sourced Klebsiella pneumoniae + IncX3 plasmid, human highly virulent Klebsiella pneumoniae + IncX3 plasmid): Spread 100 μL of the mixed bacterial suspension onto LB agar plates (16 μg / mL ceftazidime / avibactam sodium + 32 μg / mL amikacin), incubate at 37°C for 20 h, pick colonies and culture in LB broth with shaking, and determine the presence of bla in the conjugates by PCR. NDM Gene.

[0043] Example 2

[0044] The IncX3 plasmid stability test was performed on the conjugates screened in Example 1. The specific operation steps are as follows:

[0045] (1) The three purified conjugates were inoculated into 3 mL LB broth (three in parallel), cultured at 37°C for 24 h, and then passaged for a total of 14 days.

[0046] (2) Dilute the daily subculture to a suitable gradient, take 100 μL and spread it on an LB agar plate, and incubate overnight at 37°C.

[0047] (3) Pick 95 single colonies into a 96-well plate containing MH broth containing 16 μg / mL ceftazidime / avibactam sodium, and set up a negative control.

[0048] (4) Perform PCR identification on the turbid bacterial culture to amplify bla NDM Genes are used to verify whether the turbid bacterial solution contains the target gene. For example, if the well contains bla... NDM Genes, then, prove bla NDM The plasmid was not lost.

[0049] (5) Calculate plasmid stability and plot the plasmid stability curve. The results are as follows: Figure 1 As shown, this indicates that the plasmid can remain stable for a long time after being transferred to the recipient bacteria.

[0050] Example 3

[0051] The determination of the minimum inhibitory concentrations (MICs) of carvacrol and thymol against *Escherichia coli* ATCC 25922, *E. coli* donor bacteria, *E. coli* recipient bacteria, and *Klebsiella pneumoniae* recipient bacteria specifically includes the following steps:

[0052] (1) Drug preparation

[0053] Carvacrol is an organic compound named 2-methyl-5-isopropylphenol, with the chemical formula C60-2000. 10 H 14O, weigh carvacrol and add it to anhydrous ethanol to make its concentration 5120 μg / mL. The resulting solution must be filtered through a 0.22 μm filter membrane before use; the chemical formula of thymol is C 10 H 14 O, named 5-methyl-2-isopropylphenol, is prepared by weighing thymol and adding a certain amount of anhydrous ethanol to make its concentration 5120 μg / mL. The resulting solution must be filtered through a 0.22 μm filter membrane before use.

[0054] (2) Preparation of bacterial culture

[0055] Using an inoculation loop, purified *E. coli* ATCC 25922, *E. coli* donor bacteria, *E. coli* recipient bacteria, and *Klebsiella pneumoniae* recipient bacteria were streaked onto LB agar plates and incubated overnight. Single colonies from the culture medium were then inoculated into 10 mL centrifuge tubes containing 4 mL of sterile MH liquid culture medium and incubated at 37°C for 4 hours until the turbidity reached 0.5 McFarland turbidity. The culture was then diluted 1:100 to achieve a bacterial count of approximately 1 × 10⁻⁶. 6 CFU / mL;

[0056] (3) Determination of MIC by broth dilution method

[0057] In a 96-well plate, add 180 μL of MH broth to the first column, and 100 μL of broth to the second through last columns. Add 20 μL of the prepared drug to each well in the first column, mix well, and then transfer 100 μL to the second column for further mixing. Repeat this serial dilution until the last column, discarding 100 μL (two parallel concentrations for each drug). Add 100 μL of the diluted bacterial culture to each well, and incubate the 96-well plate at 37°C for 16–18 h. Read the results.

[0058] The minimum inhibitory concentrations (MICs) of carvacrol and thymol against Escherichia coli ATCC 25922, Escherichia coli donor bacteria, Escherichia coli recipient bacteria, and Klebsiella pneumoniae recipient bacteria are shown in Table 1.

[0059] Table 1. Minimum inhibitory concentrations of the tested strains against carvacrol and thymol.

[0060]

[0061] Example 4

[0062] Effects of carvacrol on intraspecies conjugation transfer of carbapenem resistance plasmids at different temperatures

[0063] The specific experimental procedure is as follows:

[0064] a) Preparation before the experiment: Prepare several 50mL centrifuge tubes, several 5mL centrifuge tubes, LB liquid medium, LB agar medium, and phosphate buffer solution. All of them should be autoclaved at 121℃ for 25 minutes and set aside. Amikacin and meropenem solutions should be filtered and sterilized and set aside.

[0065] b) Culturing: Using two 50mL centrifuge tubes, add 40mL of LB liquid medium. Add meropenem and amikacin to the centrifuge tubes inoculated with the E. coli donor and recipient strains, respectively. Incubate the centrifuge tubes overnight at 37°C and 180rpm to obtain the donor strain—E. coli carrying a meropenem resistance plasmid, and the recipient strain—E. coli resistant to amikacin.

[0066] c) Centrifuge the obtained donor and recipient bacterial cultures, wash three times with phosphate buffer, and resuspend in phosphate buffer to allow the donor and recipient bacteria to reach OD500. 600 =0.5;

[0067] d) Add 1 mL of the bacterial suspension obtained in the previous step to a 5 mL centrifuge tube at a donor-recipient ratio of 1:1. Determine the MIC of the conjugation system based on the smaller MIC of the donor and recipient bacteria for carvacrol, which is 128 μg / mL. Add a certain volume of carvacrol to achieve carvacrol concentrations of 1 / 2 MIC, 1 / 4 MIC, 1 / 8 MIC, and 0 μg / mL (control group) (three replicates are set up for each).

[0068] e) The experimental and control groups with different concentrations of carvacrol were incubated at 37℃ and 25℃ for 4 hours each (three replicates at different temperatures). After incubation, the bacterial cells were collected by centrifugation and diluted with phosphate buffer. 75 μL of the bacterial solution was spread on LB solid medium containing meropenem and amikacin, as well as on single-drug plates containing amikacin and meropenem. The plates were incubated at 37℃ for 16 hours. The number of colonies on LB agar was counted, the conjugation transfer frequency was calculated, and the effect of different doses of carvacrol on plasmid conjugation transfer was analyzed.

[0069] The results are as follows Figure 2 As shown, at 37℃, carvacrol compounds at concentrations of 1 / 2 MIC, 1 / 4 MIC, and 1 / 8 MIC can inhibit bla. NDM The conjugation transfer frequency of positive IncX3 plasmids within the species (P < 0.05) was 1.7-2 times lower than that of the control group. Figure 2(a) At 25℃, carvacrol concentrations of 1 / 2 MIC and 1 / 4 MIC inhibited the intraspecific conjugation transfer frequency of IncX3 type plasmids; carvacrol at 1 / 2 MIC significantly inhibited the intraspecific conjugation transfer of drug-resistant plasmids (P < 0.01), with a conjugation frequency 5.25 times lower than the control group. Figure 2 (b)

[0070] Example 5

[0071] Effects of carvacrol on interspecies synergistic transfer of carbapenem resistance plasmids at different temperatures

[0072] The specific experimental procedure is similar to that in Example 4, except that the recipient bacterium is human highly virulent Klebsiella pneumoniae, and meropenem in e) is replaced with ceftazidime / avibactam sodium.

[0073] The results are as follows Figure 3 As shown, at 37°C, when the carvacrol concentration is 1 / 2 MIC, carvacrol compounds can significantly reduce bla. NDM The conjugation transfer frequency of positive IncX3 plasmids between species (P < 0.001) was 6.1 times lower than that of the control group. At 1 / 4 MIC, the conjugation frequency was significantly reduced (P < 0.05). Figure 3 (a) At 25℃, the conjugation frequency of different concentrations of carvacrol in the experimental groups was lower than that in the control group, but the difference was not statistically significant. Figure 3 (b)

[0074] Example 6

[0075] Effects of thymol on intraspecies conjugation transfer of drug-resistant plasmids at different temperatures

[0076] The specific experimental procedure is similar to that in Example 4, except that carvacrol is replaced with thymol.

[0077] The results are as follows Figure 4 As shown, at 37°C, when the concentration of thymol was 1 / 2 MIC, the thymol compound concentration significantly decreased. NDM The intraspecific conjugation transfer frequency of positive IncX3 plasmids (P < 0.01) Figure 4 (a) At 25℃, the conjugation frequency of different concentrations of thymol in the test groups was lower than that in the control group, but the difference was not statistically significant. Figure 4 (b)

[0078] Example 7

[0079] Effects of thymol on interspecies synergistic transfer of drug-resistant plasmids at different temperatures

[0080] The specific experimental procedure is similar to that in Example 5, except that carvacrol is replaced with thymol.

[0081] The results are as follows Figure 5 As shown, at 37°C, when the concentration of thymol is 1 / 2 MIC, the thymol content decreases extremely significantly. NDM The conjugation transfer frequency of positive IncX3 plasmids between species was significantly reduced at 1 / 4 MIC (P < 0.0001), with the frequency decreasing by 25.8-fold and 2.4-fold respectively compared to the control group. Figure 5 (a) At 25°C, 1 / 2MIC, 1 / 4MIC, and 1 / 8MIC thymol all significantly reduced bla. NDM The frequency of conjugation and transfer of positive IncX3 plasmids between bacterial species (P < 0.01) Figure 5 (b)

[0082] Example 8: Effect of the combination drug of carvacrol and thymol on interspecies synergistic transfer of drug-resistant plasmids

[0083] When carvacrol and thymol are combined in a 1:1 ratio to form a drug combination, the MIC of the composition against both the donor bacteria and the recipient Klebsiella pneumoniae was determined to be 128 μg / mL according to the method in Example 3.

[0084] The specific experimental procedure is similar to that in Example 5, except that carvacrol is replaced with a 1:1 mixture of carvacrol and thymol, and the temperature is set at 37°C, which has the most significant inhibitory effect in the above examples.

[0085] The results are as follows Figure 6 As shown, at 37°C, the combination of carvacrol and thymol can significantly reduce bla at 1 / 2 MIC. NDM The conjugation transfer frequency of positive IncX3 plasmids between species was significantly reduced at 1 / 4 MIC (P < 0.0001), with the frequency decreasing by 1.8-fold and 1.56-fold respectively compared to the control group. Figure 6 ).

[0086] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of carvacrol and / or thymol in the preparation of carbapenem resistance plasmid conjugation transfer inhibitors, characterized in that, The carbapenem resistance plasmid is bla NDM Positive IncX3 plasmid; The concentration of carvacrol and / or thymol in the inhibitor is 32-64 μg / mL; The inhibitor is used to prevent the spread of bacterial resistance genes to intraspecific and interspecific bacteria. The intraspecific bacteria are Escherichia coli; the interspecific bacteria are Klebsiella pneumoniae.

2. The application according to claim 1, characterized in that, The inhibitor is available in the form of powder, granules, water dispersibles, suspensions, emulsifiable concentrates, microemulsions, or aqueous solutions.

3. The application according to claim 2, characterized in that, The powder is a soluble powder.

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