阅读说明：本技术 一种抑制细菌在金属诱导下抗生素抗性基因表达的试剂及其制备方法与应用 (Reagent for inhibiting expression of antibiotic resistance gene of bacteria under metal induction, preparation method and application thereof ) 是由 吴平霄 李义豪 王慧敏 牛文超 王锐 朱能武 党志 于 2020-05-29 设计创作，主要内容包括：本发明公开了一种抑制细菌在金属诱导下抗生素抗性基因表达的试剂及其制备方法与应用。该方法包括：将蒙脱石加入水中,离心取沉淀,将沉淀重新加入水中,重复离心直到上清液澄清,烘干,过筛,加入溶剂中,混匀,得到所述抑制细菌在金属诱导下抗生素抗性基因表达的试剂。该试剂通过利用Mt调节Cd<Sup>2+</Sup>诱导下细菌抗生素抗性基因的表达模式和代谢,抵抗低剂量的抗生素应激,从而抑制<I>E. coli</I>抗生素抗性基因的表达。最小抑菌浓度和荧光定量PCR的结果也表明Mt能减弱Cd<Sup>2+</Sup>诱导下细菌抗生素抗性的表达。因此,Mt可作为环境材料缓解重金属胁迫下细菌抗生素抗性基因的表达,对控制因此造成的ARGs污染具有重要意义。(The invention discloses a reagent for inhibiting the expression of antibiotic resistance genes of bacteria under the induction of metal, a preparation method and application thereof. The method comprises the following steps: adding montmorillonite into water, centrifuging to obtain precipitate, and collecting the precipitateAnd adding the precipitate into water again, repeatedly centrifuging until the supernatant is clear, drying, sieving, adding the supernatant into a solvent, and uniformly mixing to obtain the reagent for inhibiting the expression of the antibiotic resistance gene of the bacteria under the induction of metal. The reagent regulates Cd by utilizing Mt 2+ Inducing the expression pattern and metabolism of bacterial antibiotic resistance genes against low-dose antibiotic stress, thereby inhibiting E. coli Expression of antibiotic resistance genes. The results of minimum inhibitory concentration and fluorescent quantitative PCR also show that Mt can weaken Cd 2+ The expression of bacterial antibiotic resistance is induced. Therefore, Mt can be used as an environmental material to relieve the expression of bacterial antibiotic resistance genes under heavy metal stress, and has important significance for controlling ARGs pollution caused by the expression.)

1. An agent for inhibiting the expression of an antibiotic resistance gene in a bacterium induced by a metal, comprising montmorillonite.

2. The agent for inhibiting the expression of an antibiotic resistance gene in a bacterium induced by a metal according to claim 1, wherein the concentration of montmorillonite is 8 to 16gL^-1。

3. The agent for inhibiting the expression of an antibiotic resistance gene in a bacterium induced by a metal according to claim 1, wherein the pH of the agent is 6.0 to 8.0.

4. The agent for inhibiting the expression of an antibiotic resistance gene in a bacterium induced by a metal according to claim 1, wherein the metal is Cd²⁺Ion induced environment.

5. The agent for inhibiting the expression of an antibiotic resistance gene in a bacterium induced by a metal according to claim 4, wherein the Cd is²⁺The concentration of the ions is 16-128 mug ∙ mL^-1。

6. A method for preparing an agent for inhibiting the expression of an antibiotic resistance gene in a bacterium induced by a metal according to any one of claims 1 to 5, comprising the steps of:

(1) adding montmorillonite into water, centrifuging, removing supernatant, collecting precipitate, adding the precipitate into water again, repeating the centrifuging step until the supernatant is clear, oven drying, sieving, and sterilizing at high temperature to obtain pretreated montmorillonite;

(2) and (2) adding the pretreated montmorillonite obtained in the step (1) into a solvent, and uniformly mixing to obtain the reagent for inhibiting the expression of the antibiotic resistance gene of the bacteria under the induction of metal.

7. The method for preparing the agent for inhibiting the expression of the antibiotic resistance gene of bacteria induced by metal according to claim 6, wherein the rotation speed of the centrifugation in the step (1) is 8000-10000 rpm; the drying temperature in the step (1) is 55-65 ℃; the size of the sieve holes of the sieve in the step (1) is 200 meshes.

8. The agent for inhibiting the expression of antibiotic resistance genes in bacteria induced by metals as claimed in claim 6, wherein the temperature of the high temperature sterilization treatment is 100-120 ℃, and the time of the high temperature sterilization treatment is 30-60 min.

9. The method for preparing an agent for inhibiting the expression of an antibiotic resistance gene in bacteria induced by a metal according to claim 6, wherein the solvent in the step (2) is water.

10. Use of an agent for inhibiting the expression of an antibiotic resistance gene in a bacterium according to any one of claims 1 to 5 under metal induction for the preparation of an antibacterial agent.

Technical Field

The invention belongs to the fields of mineralogy and environmental microbiology, and particularly relates to a reagent for inhibiting the expression of antibiotic resistance genes of bacteria under the induction of metal, and a preparation method and application thereof.

Background

Antibiotic resistance is one of the most serious threats to public health. Overuse of antibiotics induces the production of bacterial Antibiotic Resistance Genes (ARGs), which become a key cause of the spread of antibiotic resistance. The ARGs are novel environmental pollutants and have great harm to environmental media, particularly soil environment.

A large number of researches show that the ARGs are induced by not only antibiotics, but also non-antibiotic substances such as heavy metals and organic matters. There is a close relationship between heavy metals and antibiotic resistance, which can enhance antibiotic resistance through a co-selection mechanism. In a heavy metal pollution area, heavy metal can induce bacteria to generate heavy metal resistance and can also induce bacteria to generate multiple antibiotic resistance, and the existence level of the ARGs is in positive correlation with the heavy metal pollution level. More noteworthy is that the content of heavy metals in the environmental medium is much higher than that of antibiotics and that heavy metals are not degraded compared to antibiotics, which will put longer-term stress on the environment. Therefore, the research on reducing the diffusion of the ARGs under the induction of the heavy metals has important significance on reducing the environmental health risks.

Clay minerals are the most common substances in the terrestrial environment and have complex interfacial reactions with microorganisms and heavy metals in soil. On the one hand, microbial activity can promote dissolution, precipitation and transformation of minerals, thereby accelerating biogeochemical cycles and influencing the evolution of landforms and the earth ecosystem. On the other hand, the clay mineral can regulate the growth and metabolism of microorganisms, and has great influence on the distribution, activity, diversity, gene expression and transformation of the microorganisms under external pressure. However, few researchers have deeply explored from a molecular biology perspective how clay minerals regulate the antibiotic resistance gene generation process of microorganisms under heavy metal stress. The Mt has excellent biocompatibility, and can enable bacteria to have the potential of regulating gene expression mode and growth metabolic activity under heavy metal stress, so that heavy metal resistance is enhanced to relieve the environmental stress of heavy metal pollution and the environmental stress and ecological risk brought by the generation of antibiotic resistance genes under the induction of heavy metals. Therefore, the research on the antibiotic resistance expression mechanism of Mt to bacteria under the metal stress can further provide a new idea and a new technology for promoting heavy metal pollution treatment and relieving the expression of ARGs.

Disclosure of Invention

In order to overcome the problem of pollution of antibiotic resistance genes, the invention provides a reagent for inhibiting the expression of antibiotic resistance genes of bacteria under the induction of metal, and a preparation method and application thereof.

The purpose of the invention is realized by at least one of the following technical solutions.

The invention provides a reagent for inhibiting the expression of antibiotic resistance genes of bacteria under the induction of metal, which comprises montmorillonite.

Further, the montmorillonite (Mt) needs to be sterilized at high temperature before use, and the temperature is 100-120 ℃.

Further, the concentration of Mt is 10-20gL^-1。

Further, the pH of the reagent is 6.0-8.0.

Further, the metal is induced to Cd²⁺Ion induced environment.

Further, the Cd²⁺The concentration of the ions is 16-128 mug.mL^-1。

The invention provides a method for preparing an agent for inhibiting the expression of an antibiotic resistance gene of bacteria under the induction of metal, which comprises the following steps:

(1) adding montmorillonite into water, centrifuging, removing supernatant, collecting precipitate, adding the precipitate into water again, repeating the centrifuging step until the supernatant is clear, oven drying, sieving, and sterilizing at high temperature to obtain pretreated montmorillonite;

(2) and (2) adding the pretreated montmorillonite obtained in the step (1) into a solvent, and uniformly mixing to obtain the reagent for inhibiting the expression of the antibiotic resistance gene of the bacteria under the induction of metal.

Further, the rotation speed of the centrifugal treatment in the step (1) is 8000-; the drying temperature in the step (1) is 55-65 ℃.

Further, the size of the sieve holes of the sieve in the step (1) is 200 meshes.

Further, the temperature of the high-temperature sterilization treatment in the step (1) is 100-120 ℃, and the time of the high-temperature sterilization treatment is 30-60 min.

Further, the solvent in the step (2) is deionized water.

The invention provides an application of a reagent for inhibiting the expression of a bacterial antibiotic resistance gene in the preparation of an antibacterial agent.

The invention utilizes the influence of Mt on the growth, the propagation and the metabolism of microorganisms, regulates the gene expression mode of bacteria, resists metal-induced antibiotic stress under low dose, further reduces the possibility of resisting the expression of the ARGs, and has important significance for controlling the ARGs pollution caused by the ARGs.

The effect of the agent for inhibiting the expression of the bacterial antibiotic resistance gene provided by the invention can be verified through the following experiments. The validation experiment included the following steps.

1) Mt pretreatment:

washing Mt raw soil with deionized water, centrifuging at 8000rpm to remove soluble impurities on the surface, and treating for multiple times until the deionized water is clear to obtain a precipitate which is pure Mt solid. Then the sample is put into an oven at 60 ℃ for blast drying, sieved by a 200-mesh sieve and subjected to high-temperature sterilization treatment (100 ℃ and 120 ℃) for subsequent analysis.

2) And (3) culturing bacteria:

the bacterium was Escherichia coli ATCC25922(E.coli) wild type, cultured in MHB medium at 37 ℃ and pH 7.0 with shaking at 200 rpm.

3) Gradient concentration induction of heavy metals on bacteria in the presence or absence of Mt

The heavy metal is cadmium (Cd)²⁺) Starting from the sub-inhibitory concentration of bacteria, Cd is utilized²⁺Coli induces antibiotic resistance in antibiotic-susceptible bacteria e. Coli to 30mL of Cd at a concentration of 16. mu.g.mL in the presence and absence of Mt at a ratio of 1:100, respectively^-1、32μg·mL^-1、64μg·mL^-1And 128. mu.g.mL^-1Was cultured for three generations in fresh NB medium at 37 ℃ and pH 7.0 at 200rpm for 24 hours each. Then transferred to the medium under the same conditions for 3 passages, each for 24 h.

4) Analysis of the Minimum Inhibitory Concentration (MIC) of bacteria for heavy metals and antibiotics

Experiments the MIC of heavy metals was determined using the broth microdilution method. Diluting the tested bacteria with LB culture medium and dispersing to Cd²⁺The concentration was 16. mu.g/mL^-1、32μg·mL^-1、48μg·mL^-1、64μg·mL^-1、80μg·mL^-1、98μg·mL^-1、112μg·mL^-1、128μg·mL^-1And ensuring that the number of bacteria per well is 10⁴And (4) CFU. Subsequently, the 96-well plate was placed in a constant temperature incubator (37 ℃) and incubated for 24 hours, and then the results were checked. Determination of MIC valueThe concentration at which no bacterial growth began to occur in the first column was determined.

In addition, the MIC of antibiotics was also determined by broth microdilution. The bacteria to be tested (after Cd or Cd-Mt induction) were diluted with MHB medium and dispersed to contain 0.5. mu.g.mL^-1、1μg·mL^-1、2μg·mL^-1、4μg·mL^-1、8μg·mL^-1、16μg·mL^-1、32μg·mL^-1、64μg·mL^-1、128μg·mL^-196-well plates with various antibiotic concentrations and with a bacterial count of 10 per well⁴And (4) CFU. Subsequently, the 96-well plate was placed in a constant temperature incubator (37 ℃) and incubated for 24 hours, and then the results were checked. The determination of the MIC value is the concentration at which no bacterial growth begins to occur in the first line.

5) Cd induced changes in the MIC of bacteria compared to the presence and absence of Mt.

And (3) acquiring the change of the Cd-induced bacteria ARGs under the conditions that the Mt exists and the Mt does not exist by utilizing real-time fluorescent quantitative PCR. Expression levels of antibiotic-associated genes such as penicillin (e.g., mrdA, mrcA, dacB/D), tetracycline (e.g., accC), erythromycin (e.g., suhB), and chloramphenicol (e.g., tktA). And the expression conditions of genes related to multidrug resistance/multiple antibiotic resistance/antibiotic response (such as mdtQ, marB/R, arnA/B/C/D) and a large number of toxin efflux related genes (such as emrA, mprA, ydhC, marA) of the bacteria under Cd stress.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the reagent provided by the invention resists low-dose antibiotic stress by using Mt to regulate the expression mode and metabolism of bacteria under the induction of heavy metals, so that the expression of gene mutation is inhibited, and the expression of antibiotic resistance genes is reduced; the preparation method provided by the invention has the advantages of simple steps, no need of special equipment, easily obtained raw materials, low investment and the like.

Drawings

FIG. 1 is a flow chart of the preparation of a reagent for inhibiting the expression of antibiotic resistance genes induced by bacterial metals.

Detailed Description

The following examples are presented to further illustrate the practice of the invention, but the practice and protection of the invention is not limited thereto. It is noted that the processes described below, if not specifically described in detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.