A soil antibiotic resistance gene transmission inhibitor and its preparation method and application

Abstract

The present invention relates to a soil antibiotic resistance gene transmission inhibitor, its preparation method and application. The preparation method of the antibiotic resistance gene inhibitor comprises: air-drying lavender, oregano and pine oil branches and leaves, crushing them, sieving them, distilling them according to a certain liquid-to-solid ratio, extracting them, collecting plant essential oils and plant residues; then uniformly mixing the plant residues with artificial root secretions and plant essential oils according to a proportion to obtain the antibiotic resistance gene inhibitor. The antibiotic resistance gene inhibitor is directly applied to soil contaminated with antibiotic resistance genes and stirred with the soil. After 28 days of action, the spread of antibiotic resistance genes can be significantly inhibited. It has good application in inhibiting the spread of antibiotic resistance genes in soil, can significantly reduce the abundance of multiple antibiotic resistance genes, and provides an ideal strategy for preventing and controlling the spread of antibiotic resistance genes in soil.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental pollution control, and in particular relates to a soil antibiotic resistance gene transmission inhibitor, a preparation method and an application thereof. Background Art

[0002] The long-term overuse and misuse of various antibiotics to prevent pathogens and promote livestock growth has accelerated the emergence and spread of drug-resistant bacteria and antibiotic resistance genes, posing a serious threat to global health and the economy. Numerous studies have found that non-pathogenic bacteria in the environment can horizontally transmit antibiotic resistance genes to clinical pathogens via mobile genetic elements such as plasmids and transposons, leading to the emergence of super-resistant bacteria and exacerbating human health risks.

[0003] Soil is the material foundation for human survival and development, and the contamination and spread of antibiotic resistance genes in soils has attracted considerable attention. Currently, much research is focused on the mechanisms of the spread of antibiotic resistance genes in soils. Research into controlling these genes primarily focuses on source control and in-situ remediation. Source control primarily involves controlling the presence of antibiotic resistance genes in organic fertilizers introduced into the soil to reduce their entry into the soil.

[0004] Chinese Patent Application No. CN202210547091.5 is a method for reducing the abundance of antibiotic resistance genes and mobile gene elements in pig manure compost through aerobic composting; Application No. CN202210056661.0 is a method for introducing ozone into the compost during the cooling period of aerobic composting of organic solid waste to enhance the removal of antibiotic resistance genes. However, these methods only minimize the antibiotic resistance genes contained in organic fertilizers before they enter the soil, but do not effectively control the antibiotic resistance genes already present in the soil. In situ remediation methods, on the other hand, primarily involve introducing exogenous additives into soil contaminated with antibiotic resistance genes to control the spread and diffusion of antibiotic resistance genes. Application number: 201610693735.6 A method for preparing quorum sensing inhibitors by mixing crude natural plant extracts with chemical reagents to reduce soil antibiotic resistance gene contamination. This method introduces pyrrolidone and 2,5-diketopiperazine into the soil. These substances are stable at room temperature and pressure, have certain contact toxicity, etc., and may have potential impacts on the soil; Application number: CN202010506308.9 A technology for killing antibiotic-resistant bacteria and inactivating antibiotic resistance genes with loaded nano-zinc oxide has been developed, but these bactericidal materials are not selective. While removing pathogenic microorganisms carrying antibiotic resistance genes, they will also kill beneficial microorganisms, posing a potential hazard of destroying the original ecological balance in the soil.

[0005] Existing technologies offer limited strategies for controlling soil antibiotic resistance gene contamination. Adding soil amendments is the primary method used to control these genes, but the types of soil amendments currently available are limited, and some have negative impacts on the soil itself. Therefore, there is an urgent need to develop environmentally friendly methods for inhibiting soil antibiotic resistance genes. Summary of the Invention

[0006] In response to the problems of the existing technology, the present application provides a method for preparing plant essential oils and artificial root secretions as soil conditioners and their application in alleviating or inhibiting the spread of antibiotic resistance genes in soil. The method relies on the bactericidal properties of plant essential oils and their ability to inhibit pathogenic bacteria, as well as the root secretions to improve the effectiveness of soil nutrients and promote the growth performance of microorganisms. That is, by inhibiting harmful host microorganisms, the proliferation and spread of antibiotic resistance genes in the soil are reduced, while the growth of beneficial microorganisms is promoted. The method is an efficient, broad-spectrum, environmentally friendly and low-cost technology for inhibiting the spread of antibiotic resistance genes in soil.

[0007] The purpose of the present invention is to provide a soil antibiotic resistance gene spread inhibitor.

[0008] Another object of the present invention is to provide a method for preparing a soil antibiotic resistance gene spread inhibitor.

[0009] Another object of the present invention is to provide an application of a soil antibiotic resistance gene transmission inhibitor in alleviating or inhibiting the spread of soil antibiotic resistance genes.

[0010] Another object of the present invention is to provide an application method of a soil antibiotic resistance gene spread inhibitor.

[0011] The inhibitor of the present invention is composed of the following components in parts by weight: 100 parts of plant residues, 1-5 parts of artificial root secretions, and 5-20 parts of plant essential oils.

[0012] Preferably, the inhibitor of the present invention is composed of the following components in parts by weight: 100 parts of plant residues, 5 parts of artificial root exudates, and 15 parts of plant essential oils.

[0013] The plant essential oil of the present invention is prepared by the following steps:

[0014] (1) Wash lavender, oregano, and pine oil branches and leaves with water, air-dry, and grind them separately in a grinder, pass through a 20-35 mesh nylon sieve to obtain fine powder, seal, and store at room temperature for later use;

[0015] (2) taking 10-50 parts of lavender, 10-50 parts of oregano, and 20-60 parts of pine oil powder obtained in step (1), mixing them in a round-bottom flask, adding sodium chloride solution at a concentration of 1.5%, adding distilled water at a liquid-to-material ratio of 6-10:1, and soaking for 1.5-2.5 hours; transferring the material and distilled water together to a distillation apparatus for atmospheric distillation for 2-4 hours, controlling the temperature of the heating mantle to 200-240° C., until no oil is distilled out, collecting the distillate and the remaining powdered plant branch and leaf residue;

[0016] (3) The effluent collected in step (2) is transferred to a separatory funnel and allowed to stand for 1-2 hours to allow the stratification to proceed naturally; the upper layer of oily extract is collected to obtain the plant essential oil.

[0017] Preferably, the plant essential oil of the present invention is prepared by the following steps:

[0018] (1) Wash lavender, oregano, and pine oil branches and leaves with water, air-dry, and grind them separately in a grinder, pass through a 20-mesh nylon sieve to obtain fine powder, and seal and store at room temperature for later use;

[0019] (2) 30 parts of lavender, 30 parts of oregano, and 40 parts of pine oil powder obtained in step (1) were mixed and placed in a round-bottom flask, sodium chloride solution was added at a concentration of 1.5%, and distilled water was added at a liquid-to-material ratio of 6:1, and the mixture was soaked for 2 hours; the material and distilled water were transferred to a distillation apparatus and distilled at normal pressure for 3 hours, with the heating mantle temperature controlled at 220°C, until no oil was distilled out, and the distillate and the remaining powdered plant branch and leaf residue were collected;

[0020] (3) The effluent collected in step (2) was transferred to a separatory funnel and allowed to stand for 1 hour to allow the stratification to proceed naturally; the upper layer of oily extract was collected to obtain the plant essential oil.

[0021] The plant residues described in the present invention are powdered plant branch and leaf residues left after plant essential oil extraction.

[0022] The artificial root exudate of the present invention is made of six components: D-fructose, D-glucose monohydrate, sucrose, succinic acid, L-malic acid, and urea;

[0023] The preparation method comprises the following steps: weighing appropriate amounts of D-fructose, D-glucose monohydrate, sucrose, succinic acid, L-malic acid and urea, preparing D-fructose to a concentration of 50-70 mmol / L, D-glucose monohydrate to a concentration of 30-50 mmol / L, sucrose to a concentration of 40-60 mmol / L, succinic acid to a concentration of 30-50 mmol / L, L-malic acid to a concentration of 30-50 mmol / L and urea to a concentration of 35-40 mmol / L, mixing the mixtures in equal volumes, and stirring them thoroughly with a glass rod to obtain artificial root exudates.

[0024] Preferably, the preparation method of the artificial root exudate of the present invention is: weigh appropriate amounts of D-fructose, D-glucose monohydrate, sucrose, succinic acid, L-malic acid, and urea, prepare D-fructose to a concentration of 60 mmol / L, D-glucose monohydrate to a concentration of 40 mmol / L, sucrose to a concentration of 50 mmol / L, succinic acid to a concentration of 40 mmol / L, L-malic acid to a concentration of 37.5 mmol / L, mix them in equal volumes, and stir them thoroughly with a glass rod to obtain artificial root exudate.

[0025] The method for preparing the soil antibiotic resistance gene transmission inhibitor of the present invention comprises the following steps:

[0026] (1) Wash lavender, oregano, and pine oil branches and leaves with water, air-dry, and grind them separately in a grinder, pass through a 20-35 mesh nylon sieve to obtain fine powder, seal, and store at room temperature for later use;

[0027] (2) taking 10-50 parts of lavender, 10-50 parts of oregano, and 20-60 parts of pine oil powder obtained in step (1), mixing them in a round-bottom flask, adding sodium chloride solution at a concentration of 1.5%, adding distilled water at a liquid-to-material ratio of 6-10:1, and soaking for 1.5-2.5 hours; transferring the material and distilled water together to a distillation apparatus for atmospheric distillation for 2-4 hours, controlling the temperature of the heating mantle to 200-240° C., until no oil is distilled out, collecting the distillate and the remaining powdered plant branch and leaf residue;

[0028] (3) transferring the effluent collected in step (2) into a separatory funnel and allowing it to stand for 1-2 hours to allow it to separate naturally; collecting the upper oily extract to obtain plant essential oil; and air-drying the powdered plant branch and leaf residue collected in step (2) to obtain plant residue;

[0029] (4) Weigh appropriate amounts of D-fructose, D-glucose monohydrate, sucrose, succinic acid, L-malic acid, and urea, prepare D-fructose to a concentration of 50-70 mmol / L, D-glucose monohydrate to a concentration of 30-50 mmol / L, sucrose to a concentration of 40-60 mmol / L, succinic acid to a concentration of 30-50 mmol / L, L-malic acid to a concentration of 30-50 mmol / L, and urea to a concentration of 35-40 mmol / L, mix them in equal volumes, and stir them thoroughly with a glass rod to obtain artificial root exudates;

[0030] (5) Evenly mixing the plant residue obtained in step (3), the artificial root secretions obtained in step (4), and the plant essential oil obtained in step (3) in a mass ratio of 100:1-5:5-20 to obtain the product.

[0031] Preferably, the method for preparing the soil antibiotic resistance gene transmission inhibitor of the present invention comprises the following steps:

[0032] (1) Wash lavender, oregano, and pine oil branches and leaves with water, air-dry, and grind them separately in a grinder, pass through a 20-mesh nylon sieve to obtain fine powder, and seal and store at room temperature for later use;

[0033] (2) 30 parts of lavender, 30 parts of oregano, and 40 parts of pine oil powder obtained in step (1) were mixed and placed in a round-bottom flask, sodium chloride solution was added at a concentration of 1.5%, and distilled water was added at a liquid-to-material ratio of 6:1, and the mixture was soaked for 2 hours; the material and distilled water were transferred to a distillation apparatus and distilled at normal pressure for 3 hours, with the heating mantle temperature controlled at 220°C, until no oil was distilled out, and the distillate and the remaining powdered plant branch and leaf residue were collected;

[0034] (3) transferring the effluent collected in step (2) into a separatory funnel and allowing it to stand for 1 hour to allow it to separate naturally; collecting the upper oily extract to obtain plant essential oil; and air-drying the powdered plant branch and leaf residue collected in step (2) to obtain plant residue;

[0035] (4) Weigh appropriate amounts of D-fructose, D-glucose monohydrate, sucrose, succinic acid, L-malic acid, and urea. Prepare a mixture of D-fructose at a concentration of 60 mmol / L, D-glucose monohydrate at a concentration of 40 mmol / L, sucrose at a concentration of 50 mmol / L, succinic acid at a concentration of 40 mmol / L, L-malic acid at a concentration of 40 mmol / L, and urea at a concentration of 37.5 mmol / L. Mix the mixture in equal volumes and stir thoroughly with a glass rod to obtain artificial root exudates.

[0036] (5) Evenly mixing the plant residue obtained in step (3), the artificial root secretions obtained in step (4), and the plant essential oil obtained in step (3) in a mass ratio of 100:5:15 to obtain the product.

[0037] The present invention relates to an application of the soil antibiotic resistance gene transmission inhibitor in alleviating or inhibiting the spread of soil antibiotic resistance genes. The application method comprises directly applying the antibiotic resistance gene inhibitor at an addition amount of 2 g / kg of soil dry weight to soil contaminated with antibiotic resistance genes and mixing the inhibitor evenly. The antibiotic resistance genes comprise at least one of glycopeptides, sulfonamides, quinolones, aminoglycosides, β-lactams, macrolides, multiple drug resistance genes, and tetracycline resistance genes, and also comprise mobile genetic elements.

[0038] The present invention measures the abundance of antibiotic resistance genes on the 28th day after adding the above-mentioned antibiotic resistance gene inhibitor; the calculation formula for the relative abundance of antibiotic resistance genes is:

[0039]

[0040] Where CT represents the number of cycles that each gene's fluorescence signal reaches the set threshold.

[0041] The weight parts can be μg, mg, g, kg and other weight units known in the medical field.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1. The present invention is verified by experiments. The abundance of antibiotic resistance genes is measured on the 28th day after adding the antibiotic resistance gene inhibitor. The results are as follows: (1) The antibiotic resistance gene inhibitor prepared by adding air-dried plant residues, artificial root secretions and plant essential oils in a mass ratio of 100:1:20 can reduce the abundance of antibiotic resistance genes; (2) When the mass ratio is 100:5:15, it is more suitable to reduce the abundance of antibiotic resistance genes, and the effect is more significant; (3) When the mass ratio is 100:10:5, the abundance of antibiotic resistance genes is increased; therefore, the present invention preferably uses a "mass ratio" of "100:5:15", that is, the antibiotic resistance gene inhibitor prepared by adding air-dried plant residues, artificial root secretions and plant essential oils in a mass ratio of 100:5:15 can most significantly reduce the abundance of multiple antibiotic resistance genes.

[0044] 2. The antibiotic resistance gene inhibitor of the present invention is effective. The present invention uses a microcosm test, through DNA extraction and gene determination, to obtain an antibiotic resistance gene inhibitor. After 28 days of application, the test results are as follows:

[0045] (1) The removal efficiency of the sulfonamide antibiotic resistance gene (sul1) was 91.4%;

[0046] (2) The removal efficiency of aminoglycoside antibiotic resistance gene (aadA1) was 76.7%; (3) The removal efficiency of aminoglycoside antibiotic resistance gene (ANT(4')-Ia, apmA, APH(6)-Ia) was 81.3%;

[0047] (4) The removal rate of β-lactam antibiotic resistance genes (OXA-10, CTX-M beta-lac, ACT beta-lac, TEMbeta-lac) was 66.7%;

[0048] (5) The removal rate of quinolone antibiotic resistance genes (QnrB4, QepA_1_2) was 90.9%;

[0049] (6) The removal rate of glycopeptide antibiotic resistance genes (vanHB, vanYD) was 100%;

[0050] (7) The removal rate of macrolide antibiotic resistance genes (mphA, ErmB, ErmE, ErmG, Erm(35), EreA) was 90.3%;

[0051] (8) The removal rate of multidrug antibiotic resistance genes (ceoA, mdtA, mdtG) was 43.3%;

[0052] (9) The removal rate of tetracycline antibiotic resistance genes (tetD, tetPB, tetR, tetM, tetA(P)) was 16.7%;

[0053] (10) The total removal rate of mobile genetic elements (Integrase, MGE, Plasmid, Plasmid-inc) was 92.8%.

[0054] 3. The artificial root secretions prepared by the present invention mainly contain C and N nutrients, which not only do not produce adverse effects, but also provide carbon and nitrogen sources for the growth of soil microorganisms, which is conducive to promoting the growth of beneficial microorganisms.

[0055] 4. The antibiotic resistance gene inhibitor of the present invention has a simple preparation method, is easy to store and transport, and is simple to operate. It is a green and environmentally friendly material.

[0056] 5. The antibiotic resistance gene inhibitor obtained by the present invention has good application in inhibiting the spread of antibiotic resistance genes in soil, can significantly reduce the abundance of multiple antibiotic resistance genes, and provides an ideal strategy for blocking the spread of antibiotic resistance genes in soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Flow chart of the preparation process of antibiotic resistance gene inhibitors

[0058] Figure 2 Abundance of sulfonamide antibiotic resistance gene (sul1) 28 days after application of antibiotic resistance gene inhibitor

[0059] Figure 3 Abundance of aminoglycoside antibiotic resistance gene (aadA1) 28 days after application of antibiotic resistance gene inhibitor

[0060] Figure 4Figure 2. Relative abundance of seven antibiotic resistance genes 28 days after application of antibiotic resistance gene inhibitors. (Aminoglycoside antibiotic resistance genes (ANT(4')-Ia, apmA, APH(6)-Ia), β-lactam antibiotic resistance genes (OXA-10, CTX-M beta-lac, ACT beta-lac, TEM beta-lac), quinolone antibiotic resistance genes (QnrB4, QepA_1_2), glycopeptide antibiotic resistance genes (vanHB, vanYD), macrolide antibiotic resistance genes (mphA, ErmB, ErmE, ErmG, Erm(35), EreA), multidrug antibiotic resistance genes (ceoA, mdtA, mdtG), tetracycline antibiotic resistance genes (tetD, tetPB, tetR, tetM, tetA(P)))

[0061] Figure 5 Relative abundance of mobile genetic elements 28 days after application of an antibiotic resistance gene inhibitor. DETAILED DESCRIPTION

[0062] The specific technical effects of the present application will be described in detail below through embodiments and in conjunction with the accompanying drawings, so that those skilled in the art can better understand the essence of the present application. It is worth noting that the described embodiments are only some embodiments of the present application, not all embodiments. All other embodiments obtained by those of ordinary skill in the art without making creative efforts are within the scope of protection of this application.

[0063] Example 1 Preparation of Antibiotic Resistance Gene Inhibitors

[0064] (1) Wash lavender, oregano, and pine oil branches and leaves with water, air-dry, grind the three raw materials in a grinder, pass through a 20-mesh nylon sieve, seal, and store at room temperature for later use;

[0065] (2) 30 g of lavender, 30 g of oregano, and 40 g of pine oil powder obtained in step (1) were mixed and placed in a round-bottom flask, sodium chloride solution was added at a concentration of 1.5%, and distilled water was added at a liquid-to-material ratio of 6:1, and the mixture was soaked for 2 h; the material and distilled water were transferred to a distillation apparatus and distilled at atmospheric pressure for 3 h, with the heating mantle temperature controlled at 220° C., until no oil was distilled out, and the distillate and plant residue were collected;

[0066] (3) transferring the effluent collected in step (2) into a separatory funnel and allowing it to stand for 1 hour to allow it to separate naturally; collecting the upper oily extract to obtain the plant essential oil; and air-drying the plant residue collected in step (2) for later use;

[0067] (4) Weigh appropriate amounts of D-fructose, D-glucose monohydrate, sucrose, succinic acid, L-malic acid, and urea. Prepare a mixture of D-fructose at a concentration of 60 mmol / L, D-glucose monohydrate at a concentration of 40 mmol / L, sucrose at a concentration of 50 mmol / L, succinic acid at a concentration of 40 mmol / L, L-malic acid at a concentration of 40 mmol / L, and urea at a concentration of 37.5 mmol / L. Mix the mixture in equal volumes and stir thoroughly with a glass rod to obtain artificial root exudates.

[0068] (5) Evenly mixing the plant residue obtained in step (3), the artificial root secretions obtained in step (4), and the plant essential oil obtained in step (3) in a mass ratio of 100:5:15 to obtain the product.

[0069] Example 2 Preparation of Antibiotic Resistance Gene Inhibitors

[0070] (1) Same as Example 1;

[0071] (2) Same as Example 1;

[0072] (3) Same as Example 1;

[0073] (4) Same as Example 1;

[0074] (5) Evenly mixing the plant residue obtained in step (3), the artificial root secretions obtained in step (4), and the plant essential oil obtained in step (3) in a mass ratio of 100:1:20 to obtain the product.

[0075] Example 3 Preparation of Antibiotic Resistance Gene Inhibitors

[0076] (1) Same as Example 1;

[0077] (2) Same as Example 1;

[0078] (3) Same as Example 1;

[0079] (4) Same as Example 1;

[0080] (5) Evenly mixing the plant residue obtained in step (3), the artificial root secretions obtained in step (4), and the plant essential oil obtained in step (3) in a mass ratio of 100:5:5 to obtain the product.

[0081] Example 4 Preparation of Antibiotic Resistance Gene Inhibitors

[0082] (1) Same as Example 1;

[0083] (2) Same as Example 1;

[0084] (3) Same as Example 1;

[0085] (4) Same as Example 1;

[0086] (5) Evenly mixing the plant residue obtained in step (3), the artificial root secretions obtained in step (4), and the plant essential oil obtained in step (3) in a mass ratio of 100:3:5 to obtain the product.

[0087] Example 5 Preparation of Antibiotic Resistance Gene Inhibitors

[0088] (1) Wash lavender, oregano, and pine oil branches and leaves with water, air-dry, grind the three raw materials in a grinder, pass through a 20-mesh nylon sieve, seal, and store at room temperature for later use;

[0089] (2) 30 g of lavender, 30 g of oregano, and 40 g of pine oil powder obtained in step (1) were mixed and placed in a round-bottom flask, sodium chloride solution was added at a concentration of 1.5%, and distilled water was added at a liquid-to-material ratio of 6:1, and the mixture was soaked for 2 h; the material and distilled water were transferred to a distillation apparatus and distilled at atmospheric pressure for 3 h, with the heating mantle temperature controlled at 220° C., until no oil was distilled out, and the distillate and plant residue were collected;

[0090] (3) transferring the effluent collected in step (2) into a separatory funnel and allowing it to stand for 1 hour to allow it to separate naturally; collecting the upper oily extract to obtain the plant essential oil; and air-drying the plant residue collected in step (2) for later use;

[0091] (4) Weigh appropriate amounts of D-fructose, D-glucose monohydrate, sucrose, succinic acid, L-malic acid, and urea. Prepare a D-fructose concentration of 50 mmol / L, a D-glucose monohydrate concentration of 30 mmol / L, a sucrose concentration of 40 mmol / L, a succinic acid concentration of 30 mmol / L, a L-malic acid concentration of 30 mmol / L, and a urea concentration of 35 mmol / L. Mix equal volumes and stir thoroughly with a glass rod to obtain artificial root exudates.

[0092] (5) Evenly mixing the plant residue obtained in step (3), the artificial root secretions obtained in step (4), and the plant essential oil obtained in step (3) in a mass ratio of 100:1:20 to obtain the product.

[0093] Example 6 Preparation of Antibiotic Resistance Gene Inhibitors

[0094] (1) Wash lavender, oregano, and pine oil branches and leaves with water, air-dry, grind the three raw materials in a grinder, pass through a 20-mesh nylon sieve, seal, and store at room temperature for later use;

[0095] (2) 50 g of lavender, 50 g of oregano, and 60 g of pine oil powder obtained in step (1) were mixed and placed in a round-bottom flask, sodium chloride solution was added at a concentration of 1.5%, and distilled water was added at a liquid-to-material ratio of 10:1, and the mixture was soaked for 2.5 h; the material and distilled water were transferred to a distillation apparatus and distilled at atmospheric pressure for 4 h, with the heating mantle temperature controlled at 240 ° C. until no oil was distilled out, and the distillate and plant residue were collected;

[0096] (3) transferring the effluent collected in step (2) into a separatory funnel and allowing it to stand for 2 hours to allow it to separate naturally; collecting the upper oily extract to obtain the plant essential oil; and air-drying the plant residue collected in step (2) for later use;

[0097] (4) Weigh appropriate amounts of D-fructose, D-glucose monohydrate, sucrose, succinic acid, L-malic acid, and urea. Prepare a mixture of D-fructose at a concentration of 70 mmol / L, D-glucose monohydrate at a concentration of 50 mmol / L, sucrose at a concentration of 60 mmol / L, succinic acid at a concentration of 50 mmol / L, L-malic acid at a concentration of 50 mmol / L, and urea at a concentration of 40 mmol / L. Mix the mixture in equal volumes and stir thoroughly with a glass rod to obtain artificial root exudates.

[0098] (5) Evenly mixing the plant residue obtained in step (3), the artificial root secretions obtained in step (4), and the plant essential oil obtained in step (3) in a mass ratio of 100:5:5 to obtain the product.

[0099] Example 7 Preparation of Antibiotic Resistance Gene Inhibitors

[0100] (1) Wash lavender, oregano, and pine oil branches and leaves with water, air-dry, grind the three raw materials in a grinder, pass through a 20-mesh nylon sieve, seal, and store at room temperature for later use;

[0101] (2) 10 g of lavender, 10 g of oregano, and 20 g of pine oil powder obtained in step (1) were mixed and placed in a round-bottom flask, sodium chloride solution was added at a concentration of 1.5%, and distilled water was added at a liquid-to-material ratio of 6:1, and the mixture was soaked for 1.5 h; the material and distilled water were transferred to a distillation apparatus and distilled at atmospheric pressure for 2 h, with the heating mantle temperature controlled at 200 ° C. until no oil was distilled out, and the distillate and plant residue were collected;

[0102] (3) transferring the effluent collected in step (2) into a separatory funnel and allowing it to stand for 1 hour to allow it to separate naturally; collecting the upper oily extract to obtain the plant essential oil; and air-drying the plant residue collected in step (2) for later use;

[0103] (4) Weigh appropriate amounts of D-fructose, D-glucose monohydrate, sucrose, succinic acid, L-malic acid, and urea. Prepare a D-fructose concentration of 50 mmol / L, a D-glucose monohydrate concentration of 30 mmol / L, a sucrose concentration of 40 mmol / L, a succinic acid concentration of 30 mmol / L, a L-malic acid concentration of 30 mmol / L, and a urea concentration of 35 mmol / L. Mix equal volumes and stir thoroughly with a glass rod to obtain artificial root exudates.

[0104] (5) Evenly mixing the plant residue obtained in step (3), the artificial root secretions obtained in step (4), and the plant essential oil obtained in step (3) in a mass ratio of 100:1:5 to obtain the product.

[0105] Example 8 Preparation of Antibiotic Resistance Gene Inhibitors

[0106] (1) Wash lavender, oregano, and pine oil branches and leaves with water, air-dry, grind the three raw materials in a grinder, pass through a 20-mesh nylon sieve, seal, and store at room temperature for later use;

[0107] (2) 20 g of lavender, 20 g of oregano, and 50 g of pine oil powder obtained in step (1) were mixed and placed in a round-bottom flask, sodium chloride solution was added at a concentration of 1.5%, and distilled water was added at a liquid-to-material ratio of 6:1, and the mixture was soaked for 2 h; the material and distilled water were transferred to a distillation apparatus and distilled at atmospheric pressure for 3 h, with the heating mantle temperature controlled at 220° C., until no oil was distilled out, and the distillate and plant residue were collected;

[0108] (3) transferring the effluent collected in step (2) into a separatory funnel and allowing it to stand for 1 hour to allow it to separate naturally; collecting the upper oily extract to obtain the plant essential oil; and air-drying the plant residue collected in step (2) for later use;

[0109] (4) Weigh appropriate amounts of D-fructose, D-glucose monohydrate, sucrose, succinic acid, L-malic acid, and urea. Prepare a mixture of D-fructose at a concentration of 60 mmol / L, D-glucose monohydrate at a concentration of 40 mmol / L, sucrose at a concentration of 50 mmol / L, succinic acid at a concentration of 40 mmol / L, L-malic acid at a concentration of 40 mmol / L, and urea at a concentration of 37.5 mmol / L. Mix the mixture in equal volumes and stir thoroughly with a glass rod to obtain artificial root exudates.

[0110] (5) Evenly mixing the plant residue obtained in step (3), the artificial root secretions obtained in step (4), and the plant essential oil obtained in step (3) in a mass ratio of 100:3:10 to obtain the product.

[0111] Example 9 Preparation of Antibiotic Resistance Gene Inhibitors

[0112] (1) Wash lavender, oregano, and pine oil branches and leaves with water, air-dry, grind the three raw materials in a grinder, pass through a 20-mesh nylon sieve, seal, and store at room temperature for later use;

[0113] (2) 30 g of lavender, 30 g of oregano, and 40 g of pine oil powder obtained in step (1) were mixed and placed in a round-bottom flask, sodium chloride solution was added at a concentration of 1.5%, and distilled water was added at a liquid-to-material ratio of 8:1, and the mixture was soaked for 2 h; the material and distilled water were transferred to a distillation apparatus and distilled at atmospheric pressure for 3 h, with the heating mantle temperature controlled at 220° C., until no oil was distilled out, and the distillate and plant residue were collected;

[0114] (3) transferring the effluent collected in step (2) into a separatory funnel and allowing it to stand for 1 hour to allow it to separate naturally; collecting the upper oily extract to obtain the plant essential oil; and air-drying the plant residue collected in step (2) for later use;

[0115] (4) Weigh appropriate amounts of D-fructose, D-glucose monohydrate, sucrose, succinic acid, L-malic acid, and urea. Prepare a mixture of D-fructose at a concentration of 60 mmol / L, D-glucose monohydrate at a concentration of 40 mmol / L, sucrose at a concentration of 50 mmol / L, succinic acid at a concentration of 40 mmol / L, L-malic acid at a concentration of 40 mmol / L, and urea at a concentration of 37.5 mmol / L. Mix the mixture in equal volumes and stir thoroughly with a glass rod to obtain artificial root exudates.

[0116] (5) Evenly mixing the plant residue obtained in step (3), the artificial root secretions obtained in step (4), and the plant essential oil obtained in step (3) in a mass ratio of 100:3:5 to obtain the product.

[0117] Example 10 Preparation of Antibiotic Resistance Gene Inhibitors

[0118] (1) Wash lavender, oregano, and pine oil branches and leaves with water, air-dry, grind the three raw materials in a grinder, pass through a 20-mesh nylon sieve, seal, and store at room temperature for later use;

[0119] (2) 40 g of lavender, 40 g of oregano, and 50 g of pine oil powder obtained in step (1) were mixed and placed in a round-bottom flask, sodium chloride solution was added at a concentration of 1.5%, and distilled water was added at a liquid-to-material ratio of 6:1, and the mixture was soaked for 2 h; the material and distilled water were transferred to a distillation apparatus and distilled at atmospheric pressure for 3 h, with the heating mantle temperature controlled at 220° C., until no oil was distilled out, and the distillate and plant residue were collected;

[0120] (3) transferring the effluent collected in step (2) into a separatory funnel and allowing it to stand for 1 hour to allow it to separate naturally; collecting the upper oily extract to obtain the plant essential oil; and air-drying the plant residue collected in step (2) for later use;

[0121] (4) Weigh appropriate amounts of D-fructose, D-glucose monohydrate, sucrose, succinic acid, L-malic acid, and urea. Prepare a mixture of D-fructose at a concentration of 60 mmol / L, D-glucose monohydrate at a concentration of 40 mmol / L, sucrose at a concentration of 50 mmol / L, succinic acid at a concentration of 40 mmol / L, L-malic acid at a concentration of 40 mmol / L, and urea at a concentration of 37.5 mmol / L. Mix the mixture in equal volumes and stir thoroughly with a glass rod to obtain artificial root exudates.

[0122] (5) Evenly mixing the plant residue obtained in step (3), the artificial root secretions obtained in step (4), and the plant essential oil obtained in step (3) in a mass ratio of 100:4:15 to obtain the product.

[0123] Example 11 Application of Antibiotic Resistance Gene Inhibitors

[0124] The antibiotic resistance gene inhibitor used was obtained from Example 2

[0125] Soil collection

[0126] The soil used in the experiment was collected in March 2022 from cultivated land in Guiyang, Guizhou Province, China. This soil was contaminated with a sulfonamide antibiotic resistance gene (sul1). Surface soil (0-15 cm) was collected using a five-point sampling method, avoiding plant litter and rocks on the soil surface. The soil was then transported to the laboratory in a refrigerated sampling box, air-dried, and passed through a 2 mm sieve to obtain the sample.

[0127] Microcosm Experiment

[0128] 14 g (dry weight) of the aforementioned soil sample was weighed into a 50 ml centrifuge tube. 2 g / kg of the antibiotic resistance gene inhibitor was added to the sample soil and mixed thoroughly. Soil without the antibiotic resistance gene inhibitor served as a control. Each treatment was repeated three times. The soil moisture content was adjusted to approximately 60% of its maximum field water holding capacity and incubated at 25°C in a constant-temperature incubator for 28 days. The resulting soil samples were then stored at -80°C. Sterile water was used to maintain a constant soil moisture content throughout the incubation process.

[0129] DNA extraction and sul1 gene detection

[0130] DNA was extracted using the Omega Soil DNA Miniprep Kit (refer to the kit instructions for specific steps). Real-time fluorescence quantitative PCR was used to quantify the sul1 gene.

[0131] The effects of antibiotic resistance gene inhibitors Figure 2 As shown in Figure 2, the absolute abundance of the sulfonamide resistance gene (sul1) in the original soil was 5.22×10 6±9.88×10 5 copies / g soil dry weight, and its absolute abundance after 28 days without treatment was 2.94×10 7 ±1.29×10 7 copies / g soil dry weight, and its absolute abundance after treatment with antibiotic resistance gene inhibitors was 2.53×10 6 ±1.78×10 5 copies / g soil dry weight, and the removal rate of sulfonamide resistance gene (sul1) was 91.4%.

[0132] Example 12 Application of Antibiotic Resistance Gene Inhibitors

[0133] The antibiotic resistance gene inhibitor used was obtained from Example 1

[0134] Soil collection

[0135] The soil used in the experiment was collected in March 2022 from cultivated land in Guiyang, Guizhou Province, China. This soil was contaminated with an aminoglycoside antibiotic resistance gene (aadA1). Surface soil (0-15 cm) was collected using a five-point sampling method, avoiding plant litter and rocks on the soil surface. The soil was then transported to the laboratory in a refrigerated sampling box, air-dried, and passed through a 2 mm sieve to obtain the sample.

[0136] Microcosm Experiment

[0137] 14 g (dry weight) of the aforementioned soil sample was weighed into a 50 ml centrifuge tube. 2 g / kg of the antibiotic resistance gene inhibitor was added to the sample soil and mixed thoroughly. Soil without the antibiotic resistance gene inhibitor served as a control. Each treatment was repeated three times. The soil moisture content was adjusted to approximately 60% of its maximum field water holding capacity and incubated at 25°C in a constant-temperature incubator for 28 days. The resulting soil samples were then stored at -80°C. Sterile water was used to maintain a constant soil moisture content throughout the incubation process.

[0138] DNA extraction and determination of aadA1 gene

[0139] DNA was extracted using the Omega Soil DNA Miniprep Kit (refer to the kit instructions for specific steps). Fluorescence quantification of the aadA1 gene was performed using a real-time fluorescence quantitative PCR instrument.

[0140] The effects of antibiotic resistance gene inhibitors Figure 3 As shown in Figure 2, the absolute abundance of aminoglycoside antibiotic resistance gene (aadA1) in the original soil was 7.68×10 4 ±2.78×10 4copies / g soil dry weight, and its absolute abundance was 1.31×10 5 ±5.93×10 4 copies / g soil dry weight, and its absolute abundance was 3.05×10 4 ±7.37×10 2 copies / g soil dry weight, and the removal rate of aminoglycoside antibiotic resistance gene (aadA1) was 76.7%.

[0141] Example 13 Application of Antibiotic Resistance Gene Inhibitors

[0142] The antibiotic resistance gene inhibitor used was obtained from Example 1

[0143] Soil collection

[0144] The soil used in the experiment was collected in March 2022 from farmland in Guiyang, Guizhou Province, China, where organic fertilizers had been applied for many years. Surface soil (0-15 cm) was collected using a five-point sampling method, avoiding plant litter and rocks on the soil surface. The soil was then transported to the laboratory in a refrigerated sampling box, air-dried, and passed through a 2 mm sieve to obtain the sample.

[0145] Microcosm Experiment

[0146] 14 g (dry weight) of the aforementioned soil sample was weighed into a 50 ml centrifuge tube. 2 g / kg of the antibiotic resistance gene inhibitor was added to the sample soil and mixed thoroughly. Soil without the antibiotic resistance gene inhibitor served as a control. Each treatment was repeated three times. The soil moisture content was adjusted to approximately 60% of its maximum field water holding capacity and incubated at 25°C in a constant-temperature incubator for 28 days. The resulting soil samples were then stored at -80°C. Sterile water was used to maintain a constant soil moisture content throughout the incubation process.

[0147] DNA extraction and detection of multiple antibiotic resistance genes

[0148] DNA was extracted using the Omega soil DNA miniprep kit; the specific steps are described in the kit instructions. High-throughput quantitative PCR was used for relative quantification of resistance genes to glycopeptides (vanHB, vanYD), quinolones (QnrB4, QepA_1_2), aminoglycosides (ANT(4')-Ia, apmA, APH(6)-Ia), β-lactams (OXA-10, CTX-M beta-lac, ACT beta-lac, TEMbeta-lac), macrolides (mphA, ErmB, ErmE, ErmG, Erm(35), EreA), multidrug resistance (ceoA, mdtA, mdtG), and tetracycline resistance (tetD, tetPB, tetR, tetM, tetA(P)).

[0149] The effects of antibiotic resistance gene inhibitors Figure 4 As shown. After 28 days of treatment with antibiotic resistance gene inhibitors, the removal rate of aminoglycoside antibiotic resistance genes (ANT(4')-Ia, apmA, APH(6)-Ia) in the soil was 81.3%, and the removal rate of β-lactam antibiotic resistance genes (OXA-10, CTX-M beta-lac, ACT beta-lac, TEM beta-lac) was removed by 66.7%, quinolone antibiotic resistance genes (QnrB4, QepA_1_2) by 90.9%, glycopeptide antibiotic resistance genes (vanHB, vanYD) by 100%, macrolide antibiotic resistance genes (mphA, ErmB, ErmE, ErmG, Erm(35), EreA) by 90.3%, multidrug antibiotic resistance genes (ceoA, mdtA, mdtG) by 43.3%, and tetracycline antibiotic resistance genes (tetD, tetPB, tetR, tetM, tetA(P)) by 16.7%.

[0150] Effects of antibiotic resistance gene inhibitors on the removal of mobile genetic elements in soil Figure 5 As shown in Figure 2, 28 days after the application of antibiotic resistance gene inhibitors, the total removal rate of the relative abundance of mobile genetic elements (Integrase, MGE, Plasmid, Plasmid-inc) was 92.8%.

[0151] In order to further verify the feasibility of the present invention, the inventors conducted a series of experiments, the steps of which are as follows:

[0152] 1. Extraction of plant essential oils

[0153] Essential oils have been widely used in flavoring additives, pharmaceutical preparations, cosmetics, pesticides, antioxidants, anti-inflammatory agents, anti-allergic agents, and anti-cancer agents. Notably, the significant antimicrobial properties of essential oils are attributed to their primary components, monoterpenes and sesquiterpenes, as well as their oxidized derivatives, and are widely used in agriculture and the environment as environmentally functional materials. Essential oils can alter cell membrane interface properties, disrupt cell membrane integrity, damage DNA structure, or inhibit gene expression. Based on this hypothesis, essential oils may effectively inhibit the spread of ARGs in soil by altering cell membrane interface properties, inhibiting bacterial activity, and reducing the frequency of conjugation and transformation of drug-resistant bacteria, thereby reducing horizontal gene transfer.

[0154] The raw materials selected for essential oil extraction in the present invention are lavender, oregano, and Chinese pine branches and leaves. Before preparation, the three plant branches and leaves are first washed with water and air-dried. After air-drying, the three raw materials are respectively pulverized in a grinder, passed through a 20-mesh nylon sieve, and stored in sealed bags for drying. During extraction, 3 parts of lavender, 3 parts of oregano, and 4 parts of pine oil are mixed and placed in a round-bottom flask. Sodium chloride solution is added at a concentration of 1.5%, and distilled water is added at a liquid-to-solid ratio of 6:1. The mixture is soaked for 2 hours. The material and distilled water are then transferred to a distillation apparatus and distilled at atmospheric pressure for 3 hours. The heating mantle temperature is controlled at 220°C until no oil is distilled out. The effluent in the collection flask is then transferred to a separatory funnel and allowed to stand for 1 hour to allow natural stratification. The upper layer of oily extract is collected to obtain the essential oil. The plant residue is air-dried for later use.

[0155] 2. Preparation of Artificial Root Exudates

[0156] Root exudates have the ability to increase soil nutrients and promote the growth of microorganisms. The preparation method of the artificial root exudates prepared by the present invention is as follows:

[0157] D-fructose was prepared to a concentration of 60 mmol / L, D-glucose monohydrate to a concentration of 40 mmol / L, sucrose to a concentration of 50 mmol / L, succinic acid to a concentration of 40 mmol / L, L-malic acid to a concentration of 40 mmol / L, and urea to a concentration of 37.5 mmol / L. The prepared reagents were mixed in equal volumes according to Table 1, and stirred thoroughly with a glass rod to obtain artificial root exudates, which were then stored in the dark for later use.

[0158] The reagents used in the artificial root secretions only contain C and N elements. The concentrations of the reagents are shown in Table 1. The reagents are mixed in equal volumes and stirred evenly with a glass rod.

[0159] Table 1 Preparation of artificial root exudates

[0160]

[0161] 3. Preparation of Antibiotic Resistance Gene Inhibitors

[0162] Preparation method 1

[0163] 100 parts of the air-dried plant residue obtained under item "1", 1 part of the artificial root secretion obtained under item "2" and 20 parts of the plant essential oil obtained under item "1" are mixed and manually stirred to fully mix, thereby obtaining an antibiotic resistance gene inhibitor.

[0164] Preparation method 2

[0165] 100 parts of the air-dried plant residue obtained under item "1", 5 parts of the artificial root secretions obtained under item "2" and 15 parts of the plant essential oil obtained under item "1" are mixed and manually stirred to fully mix, thereby obtaining an antibiotic resistance gene inhibitor.

[0166] Preparation method 3

[0167] 100 parts of the air-dried plant residue obtained under item "1", 10 parts of the artificial root secretions obtained under item "2" and 5 parts of the plant essential oil obtained under item "1" are mixed and manually stirred to fully mix, thereby obtaining an antibiotic resistance gene inhibitor.

[0168] IV. Application of Antibiotic Resistance Gene Inhibitors

[0169] The present invention verifies and evaluates the application effect of the antibiotic resistance gene inhibitor in inhibiting the spread of soil antibiotic resistance genes through laboratory experiments, and measures the abundance of antibiotic resistance genes on the 28th day after adding the antibiotic resistance gene inhibitor.

[0170] The abundance of antibiotic resistance genes refers to the number of copies of various antibiotic resistance genes in the genome. The larger the number, the higher the abundance. It includes absolute abundance and relative abundance, where relative abundance is used to describe the percentage of a single gene to the number of bacterial 16S ribosomal RNA genes.

[0171] The formula for calculating the relative abundance of antibiotic resistance genes is:

[0172]

[0173] Where CT represents the number of cycles that each gene's fluorescence signal reaches the set threshold.

[0174] The specific steps are as follows:

[0175] The antibiotic resistance gene inhibitor used in Experimental Example 1 was obtained from Preparation Method 1

[0176] Soil collection

[0177] The soil used in the experiment was collected in March 2022 from cultivated land in Guiyang, Guizhou Province, China. This soil was contaminated with a sulfonamide antibiotic resistance gene (sul1). Surface soil (0-15 cm) was collected using a five-point sampling method, avoiding plant litter and rocks on the soil surface. The soil was then transported to the laboratory in a refrigerated sampling box, air-dried, and passed through a 2 mm sieve to obtain the sample.

[0178] Microcosm Experiment

[0179] 14 g (dry weight) of the aforementioned soil sample was weighed into a 50 ml centrifuge tube. 2 g / kg of the antibiotic resistance gene inhibitor was added to the sample soil and mixed thoroughly. Soil without the antibiotic resistance gene inhibitor served as a control. Each treatment was repeated three times. The soil moisture content was adjusted to approximately 60% of its maximum field water holding capacity and incubated at 25°C in a constant-temperature incubator for 28 days. The resulting soil samples were then stored at -80°C. Sterile water was used to maintain a constant soil moisture content throughout the incubation process.

[0180] DNA extraction and sul1 gene detection

[0181] DNA was extracted using the Omega Soil DNA Miniprep Kit (refer to the kit instructions for specific steps). Real-time fluorescence quantitative PCR was used to quantify the sul1 gene.

[0182] The effects of antibiotic resistance gene inhibitors Figure 2 As shown in Figure 2, the absolute abundance of the sulfonamide resistance gene (sul1) in the original soil was 5.22×10 6 ±9.88×10 5 copies / g soil dry weight, and its absolute abundance after 28 days without treatment was 2.94×10 7 ±1.29×10 7 copies / g soil dry weight, and its absolute abundance after treatment with antibiotic resistance gene inhibitors was 2.53×10 6 ±1.78×10 5 copies / g soil dry weight, and the removal rate of sulfonamide resistance gene (sul1) was 91.4%.

[0183] Experimental Example 2: The antibiotic resistance gene inhibitor is obtained by Preparation Method 2

[0184] Soil collection

[0185] The soil used in the experiment was collected in March 2022 from cultivated land in Guiyang, Guizhou Province, China. This soil was contaminated with an aminoglycoside antibiotic resistance gene (aadA1). Surface soil (0-15 cm) was collected using a five-point sampling method, avoiding plant litter and rocks on the soil surface. The soil was then transported to the laboratory in a refrigerated sampling box, air-dried, and passed through a 2 mm sieve to obtain the sample.

[0186] Microcosm Experiment

[0187] 14 g (dry weight) of the aforementioned soil sample was weighed into a 50 ml centrifuge tube. 2 g / kg of the antibiotic resistance gene inhibitor was added to the sample soil and mixed thoroughly. Soil without the antibiotic resistance gene inhibitor served as a control. Each treatment was repeated three times. The soil moisture content was adjusted to approximately 60% of its maximum field water holding capacity and incubated at 25°C in a constant-temperature incubator for 28 days. The resulting soil samples were then stored at -80°C. Sterile water was used to maintain a constant soil moisture content throughout the incubation process.

[0188] DNA extraction and determination of aadA1 gene

[0189] DNA was extracted using the Omega Soil DNA Miniprep Kit (refer to the kit instructions for specific steps). Fluorescence quantification of the aadA1 gene was performed using a real-time fluorescence quantitative PCR instrument.

[0190] The effects of antibiotic resistance gene inhibitors Figure 3 As shown in Figure 2, the absolute abundance of aminoglycoside antibiotic resistance gene (aadA1) in the original soil was 7.68×10 4 ±2.78×10 4 copies / g soil dry weight, and its absolute abundance was 1.31×10 5 ±5.93×10 4 copies / g soil dry weight, and its absolute abundance was 3.05×10 4 ±7.37×10 2 copies / g soil dry weight, and the removal rate of aminoglycoside antibiotic resistance gene (aadA1) was 76.7%.

[0191] Experimental Example 3: The antibiotic resistance gene inhibitor is obtained by Preparation Method 2

[0192] Soil collection

[0193] The soil used in the experiment was collected in March 2022 from farmland in Guiyang, Guizhou Province, China, where organic fertilizers had been applied for many years. Surface soil (0-15 cm) was collected using a five-point sampling method, avoiding plant litter and rocks on the soil surface. The soil was then transported to the laboratory in a refrigerated sampling box, air-dried, and passed through a 2 mm sieve to obtain the sample.

[0194] Microcosm Experiment

[0195] 14 g (dry weight) of the aforementioned soil sample was weighed into a 50 ml centrifuge tube. 2 g / kg of the antibiotic resistance gene inhibitor was added to the sample soil and mixed thoroughly. Soil without the antibiotic resistance gene inhibitor served as a control. Each treatment was repeated three times. The soil moisture content was adjusted to approximately 60% of its maximum field water holding capacity and incubated at 25°C in a constant-temperature incubator for 28 days. The resulting soil samples were then stored at -80°C. Sterile water was used to maintain a constant soil moisture content throughout the incubation process.

[0196] DNA extraction and detection of multiple antibiotic resistance genes

[0197] DNA was extracted using the Omega soil DNA miniprep kit; the specific steps are described in the kit instructions. High-throughput quantitative PCR was used for relative quantification of resistance genes to glycopeptides (vanHB, vanYD), quinolones (QnrB4, QepA_1_2), aminoglycosides (ANT(4')-Ia, apmA, APH(6)-Ia), β-lactams (OXA-10, CTX-M beta-lac, ACT beta-lac, TEMbeta-lac), macrolides (mphA, ErmB, ErmE, ErmG, Erm(35), EreA), multidrug resistance (ceoA, mdtA, mdtG), and tetracycline resistance (tetD, tetPB, tetR, tetM, tetA(P)).

[0198] The effects of antibiotic resistance gene inhibitors Figure 4As shown. After 28 days of treatment with antibiotic resistance gene inhibitors, the removal rate of aminoglycoside antibiotic resistance genes (ANT(4')-Ia, apmA, APH(6)-Ia) in the soil was 81.3%, and the removal rate of β-lactam antibiotic resistance genes (OXA-10, CTX-M beta-lac, ACT beta-lac, TEM beta-lac) was removed by 66.7%, quinolone antibiotic resistance genes (QnrB4, QepA_1_2) by 90.9%, glycopeptide antibiotic resistance genes (vanHB, vanYD) by 100%, macrolide antibiotic resistance genes (mphA, ErmB, ErmE, ErmG, Erm(35), EreA) by 90.3%, multidrug antibiotic resistance genes (ceoA, mdtA, mdtG) by 43.3%, and tetracycline antibiotic resistance genes (tetD, tetPB, tetR, tetM, tetA(P)) by 16.7%.

[0199] Effects of antibiotic resistance gene inhibitors on the removal of mobile genetic elements in soil Figure 5 As shown in Figure 2, 28 days after the application of antibiotic resistance gene inhibitors, the total removal rate of the relative abundance of mobile genetic elements (Integrase, MGE, Plasmid, Plasmid-inc) was 92.8%.

[0200] Experimental Example 4: The antibiotic resistance gene inhibitor obtained by Preparation Method 3

[0201] Soil collection

[0202] The soil used in the experiment was collected in March 2022 from farmland in Guiyang, Guizhou Province, China, where organic fertilizers had been applied for many years. Surface soil (0-15 cm) was collected using a five-point sampling method, avoiding plant litter and rocks on the soil surface. The soil was then transported to the laboratory in a refrigerated sampling box, air-dried, and passed through a 2 mm sieve to obtain the sample.

[0203] Microcosm Experiment

[0204] 14 g (dry weight) of the aforementioned soil sample was weighed into a 50 ml centrifuge tube. 2 g / kg of the antibiotic resistance gene inhibitor was added to the sample soil and mixed thoroughly. Soil without the antibiotic resistance gene inhibitor served as a control. Each treatment was repeated three times. The soil moisture content was adjusted to approximately 60% of its maximum field water holding capacity and incubated at 25°C in a constant-temperature incubator for 28 days. The resulting soil samples were then stored at -80°C. Sterile water was used to maintain a constant soil moisture content throughout the incubation process.

[0205] DNA extraction and tetM gene detection

[0206] DNA was extracted using the Omega Soil DNA Miniprep Kit (refer to the kit instructions for specific steps). Real-time fluorescence quantitative PCR was used to quantify the tetM gene.

[0207] The absolute abundance of tetracycline resistance genes (tetM) in the original soil was 7.73×10 6 ±1.72×10 6 copies / g soil dry weight, and its absolute abundance was 8.38×10 6 ±1.56×10 6 copies / g soil dry weight, and its absolute abundance after treatment with antibiotic resistance gene inhibitors was 2.43×10 8 ±1.74×10 7 co pies / g soil dry weight, and the absolute abundance of tetracycline antibiotic resistance genes (tetM) increased 28-fold.

[0208] In summary, the antibiotic resistance gene inhibitor prepared by the mass ratio of air-dried plant residues to artificial root secretions and plant essential oils of 100:1:20 can reduce the abundance of antibiotic resistance genes; when the mass ratio is 100:5:15, it is suitable for reducing the abundance of antibiotic resistance genes, and the effect is more significant; when the mass ratio is 100:10:5, the abundance of antibiotic resistance genes is increased; that is, the antibiotic resistance gene inhibitor prepared within the range of 100:(1-5):(5-20) can significantly reduce the abundance of multiple antibiotic resistance genes, but the antibiotic resistance gene inhibitor prepared at a mass ratio of 100:5:15 can most significantly reduce the abundance of multiple antibiotic resistance genes, so the preferred "mass ratio" of the present invention is "100:5:15".

[0209] The inhibitor prepared by the present invention effectively reduces the spread of antibiotic resistance genes in soil, providing an ideal strategy for preventing and controlling the spread of antibiotic resistance genes in soil.

[0210] The embodiments and test examples described above merely represent several aspects of the present application. While their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present application, and these variations and improvements are all within the scope of protection of the present application.

Claims

1. A soil antibiotic resistance gene transmission inhibitor, characterized in that The inhibitor is composed of the following components in parts by weight: 100 parts of plant residues, 1-5 parts of artificial root secretions, and 5-20 parts of plant essential oils; The plant essential oil is prepared by the following steps: (1) Wash lavender, oregano, and pine oil branches and leaves with water, air-dry, and grind them separately in a grinder, pass through a 20-35 mesh nylon sieve to obtain fine powder, and seal and store at room temperature for later use; (2) taking 10-50 parts of lavender, 10-50 parts of oregano, and 20-60 parts of pine oil powder obtained in step (1), mixing them in a round-bottom flask, adding sodium chloride solution at a concentration of 1.5%, adding distilled water at a liquid-to-material ratio of 6-10:1, and soaking for 1.5-2.5 hours; transferring the material and distilled water together to a distillation apparatus for atmospheric distillation for 2-4 hours, controlling the temperature of the heating mantle to 200-240° C., until no oil is distilled out, collecting the distillate and the remaining powdered plant branch and leaf residue; (3) transferring the effluent collected in step (2) into a separatory funnel and allowing it to stand for 1-2 hours to allow it to naturally separate into layers; collecting the upper oily extract to obtain the plant essential oil; The plant residue is the powdered plant branch and leaf residue left after the plant essential oil extraction. The artificial root exudate is prepared from six components: D-fructose, D-glucose monohydrate, sucrose, succinic acid, L-malic acid, and urea. The preparation method comprises the following steps: weighing appropriate amounts of D-fructose, D-glucose monohydrate, sucrose, succinic acid, L-malic acid, and urea; preparing the components to have a concentration of 50-70 mmol / L of D-fructose, 30-50 mmol / L of D-glucose monohydrate, 40-60 mmol / L of sucrose, 30-50 mmol / L of succinic acid, 30-50 mmol / L of L-malic acid, and 35-40 mmol / L of urea; mixing the components in equal volumes; and stirring the components thoroughly with a glass rod to obtain the artificial root exudate.

2. The soil antibiotic resistance gene spread inhibitor according to claim 1, characterized in that The inhibitor is composed of the following components in parts by weight: 100 parts of plant residues, 5 parts of artificial root secretions, and 15 parts of plant essential oils.

3. The soil antibiotic resistance gene spread inhibitor according to claim 1, characterized in that The plant essential oil is prepared by the following steps: (1) Wash lavender, oregano, and pine oil branches and leaves with water, air-dry, and grind them separately in a grinder, pass through a 20-mesh nylon sieve to obtain fine powder, and seal and store at room temperature for later use; (2) 30 parts of lavender, 30 parts of oregano, and 40 parts of pine oil powder obtained in step (1) were mixed and placed in a round-bottom flask, sodium chloride solution was added at a concentration of 1.5%, and distilled water was added at a liquid-to-material ratio of 6:1, and the mixture was soaked for 2 hours; the material and distilled water were transferred to a distillation apparatus and distilled at normal pressure for 3 hours, with the heating mantle temperature controlled at 220°C, until no oil was distilled out, and the distillate and the remaining powdered plant branch and leaf residue were collected; (3) The effluent collected in step (2) was transferred to a separatory funnel and allowed to stand for 1 hour to allow the stratification to proceed naturally; the upper layer of oily extract was collected to obtain the plant essential oil.

4. The soil antibiotic resistance gene spread inhibitor according to claim 1, characterized in that The preparation method of the artificial root exudate comprises the following steps: weighing appropriate amounts of D-fructose, D-glucose monohydrate, sucrose, succinic acid, L-malic acid, and urea, preparing the D-fructose to a concentration of 60 mmol / L, the D-glucose monohydrate to a concentration of 40 mmol / L, the sucrose to a concentration of 50 mmol / L, the succinic acid to a concentration of 40 mmol / L, the L-malic acid to a concentration of 40 mmol / L, and the urea to a concentration of 37.5 mmol / L, mixing the mixture in equal volumes, and stirring the mixture thoroughly with a glass rod to obtain the artificial root exudate.

5. A method for preparing the soil antibiotic resistance gene transmission inhibitor according to claim 1, characterized in that: The method comprises the following steps: (1) Wash lavender, oregano, and pine oil branches and leaves with water, air-dry, and grind them separately in a grinder, pass through a 20-35 mesh nylon sieve to obtain fine powder, and seal and store at room temperature for later use; (2) taking 10-50 parts of lavender, 10-50 parts of oregano, and 20-60 parts of pine oil powder obtained in step (1), mixing them in a round-bottom flask, adding sodium chloride solution at a concentration of 1.5%, adding distilled water at a liquid-to-material ratio of 6-10:1, and soaking for 1.5-2.5 hours; transferring the material and distilled water together to a distillation apparatus for atmospheric distillation for 2-4 hours, controlling the temperature of the heating mantle to 200-240° C., until no oil is distilled out, collecting the distillate and the remaining powdered plant branch and leaf residue; (3) transferring the effluent collected in step (2) into a separatory funnel and allowing it to stand for 1-2 hours to allow it to separate naturally; collecting the upper oily extract to obtain plant essential oil; and air-drying the powdered plant branch and leaf residue collected in step (2) to obtain plant residue; (4) Weigh appropriate amounts of D-fructose, D-glucose monohydrate, sucrose, succinic acid, L-malic acid, and urea, prepare D-fructose to a concentration of 50-70 mmol / L, D-glucose monohydrate to a concentration of 30-50 mmol / L, sucrose to a concentration of 40-60 mmol / L, succinic acid to a concentration of 30-50 mmol / L, L-malic acid to a concentration of 30-50 mmol / L, and urea to a concentration of 35-40 mmol / L, mix them in equal volumes, and stir them thoroughly with a glass rod to obtain artificial root exudates; (5) Evenly mixing the plant residue obtained in step (3), the artificial root secretions obtained in step (4), and the plant essential oil obtained in step (3) in a mass ratio of 100:1-5:5-20 to obtain the product.

6. The soil antibiotic resistance gene spread inhibitor according to claim 5, characterized in that The method comprises the following steps: (1) Wash lavender, oregano, and pine oil branches and leaves with water, air-dry, and grind them separately in a grinder, pass through a 20-mesh nylon sieve to obtain fine powder, and seal and store at room temperature for later use; (2) 30 parts of lavender, 30 parts of oregano, and 40 parts of pine oil powder obtained in step (1) were mixed and placed in a round-bottom flask, sodium chloride solution was added at a concentration of 1.5%, and distilled water was added at a liquid-to-material ratio of 6:1, and the mixture was soaked for 2 hours; the material and distilled water were transferred to a distillation apparatus and distilled at normal pressure for 3 hours, with the heating mantle temperature controlled at 220°C, until no oil was distilled out, and the distillate and the remaining powdered plant branch and leaf residue were collected; (3) transferring the effluent collected in step (2) into a separatory funnel and allowing it to stand for 1 hour to allow it to separate naturally; collecting the upper oily extract to obtain plant essential oil; and air-drying the powdered plant branch and leaf residue collected in step (2) to obtain plant residue; (4) Weigh appropriate amounts of D-fructose, D-glucose monohydrate, sucrose, succinic acid, L-malic acid, and urea. Prepare a mixture of D-fructose at a concentration of 60 mmol / L, D-glucose monohydrate at a concentration of 40 mmol / L, sucrose at a concentration of 50 mmol / L, succinic acid at a concentration of 40 mmol / L, L-malic acid at a concentration of 40 mmol / L, and urea at a concentration of 37.5 mmol / L. Mix the mixture in equal volumes and stir thoroughly with a glass rod to obtain artificial root exudates. (5) Evenly mixing the plant residue obtained in step (3), the artificial root secretions obtained in step (4), and the plant essential oil obtained in step (3) in a mass ratio of 100:5:15 to obtain the product.

7. A use of the soil antibiotic resistance gene transmission inhibitor as claimed in claim 1 in alleviating or inhibiting the spread of soil antibiotic resistance genes; the application method is to directly apply the antibiotic resistance gene inhibitor at an addition amount of 2 g / kg soil dry weight to soil contaminated with antibiotic resistance genes and mix it evenly; the antibiotic resistance gene comprises at least one of glycopeptides, sulfonamides, quinolones, aminoglycosides, β-lactams, macrolides, multiple drug and tetracycline resistance genes, or comprises mobile genetic elements.

Images