A method for degrading free antibiotic resistance genes in water using nematodes

Abstract

The present invention discloses a method for degrading free antibiotic resistance genes (eARGs) in water bodies by using nematodes. The method adds nematodes widely present in the environment to water bodies containing eARGs, and utilizes the characteristics of nematodes secreting nucleases to degrade eARGs in water bodies. At 20°C, nematodes can degrade up to 5.26 orders of magnitude of eARGs within 84 hours, and reduce the conversion activity of eARGs by 170 times within 3 hours. The present invention has the advantages of simple operation, economic efficiency, green environmental protection, etc., and can achieve the purpose of curbing the pollution and spread of eARGs in actual sewage or surface water and other water bodies.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental protection, and in particular relates to a method for utilizing nematodes to degrade free antibiotic resistance genes in water. Background Art

[0002] The problem of antibiotic resistance seriously threatens global public health security and has become one of the most serious environmental health problems in the world. Antibiotic resistance genes can spread among bacteria, making the problem of antibiotic resistance even more serious. Transformation is an important way for antibiotic resistance genes to spread. It refers to the acquisition of resistance by competent bacteria that are widely present in the environment by directly absorbing free antibiotic resistance genes (eARGs). At present, the presence of eARGs has been detected in many aquatic environments, such as rivers, groundwater, tap water, and sewage treatment plants, suggesting that the transformation of eARGs occurs widely in aquatic environments. Therefore, it is urgent to develop a technology that can effectively degrade eARGs and block their spread in the environment to curb the development of resistance gene resistance problems.

[0003] At present, the common technologies for eliminating antibiotic resistance genes in water bodies include: traditional disinfection technology, new light disinfection technology, artificial wetlands, coagulation, filtration, etc. Traditional disinfection technologies, such as ultraviolet, chlorination, ozone, etc., are difficult to eliminate antibiotic resistance gene pollution problems, and may even release bacterial intracellular antibiotic resistance genes into the environment, increase the content of eARGs, and accelerate the spread of antibiotic resistance genes. New light disinfection methods include photocatalysis, photo-Fenton technology, etc., which have good removal effects on eARGs, but due to the addition of catalysts or oxidants, recovery and fixation problems are introduced, which increases the difficulty and cost of using new light elimination technologies. Artificial wetlands, coagulation, and membrane filtration technologies have poor removal effects on eARGs and may even bring the risk of secondary pollution. Therefore, it is necessary to develop a technology that is simple to operate, cost-effective, and green and environmentally friendly to degrade resistance genes.

[0004] Nematodes are the most numerous multicellular organisms in nature, accounting for 4 out of every 5 multicellular organisms. They are widely found in various natural or artificial aquatic environments, including freshwater, seawater, and artificial sewage treatment plants. In addition, nematodes can transform organic matter, enrich heavy metals, and participate in the chemical cycle of nutrients, and have great potential for application in the field of environmental remediation. Recent studies have shown that some parasitic nematodes, such as hookworms and Trichinella spiralis, can secrete DNase II to degrade extracellular traps produced by host immune cells, the main component of which is DNA. Interestingly, DNA fragments with unligatable ends (3' phosphate group and 5' hydroxyl group) produced by DNase II are predicted to prevent exogenous DNA from integrating into the host genome in lysosomes. There are a large number of free-living nematodes in the environment, and these nematodes may also have the ability to secrete DNase II to degrade eARGs in the water environment to curb the spread of eARGs. However, no studies have yet evaluated the ability of nematodes to mitigate eARG pollution in water bodies. The present invention finds that Caenorhabditis elegans can effectively degrade water eARGs and inhibit the transformation of eARGs, which will provide a simple, economical, green and efficient new technology for treating water eARGs pollution. Summary of the invention

[0005] The purpose of the present invention is to address the deficiencies of the prior art and provide a method for utilizing nematodes to degrade free antibiotic resistance genes in water.

[0006] The objective of the present invention is achieved through the following technical scheme: a method for utilizing nematodes to degrade free antibiotic resistance genes in water, wherein nematodes are added to water containing free antibiotic resistance genes, and the action of nucleases secreted by the nematodes is utilized to efficiently degrade the free antibiotic resistance genes in the water, thereby inhibiting the spread of the free antibiotic resistance genes.

[0007] Furthermore, the nematode is Caenorhabditis elegans.

[0008] Furthermore, the free antibiotic resistance gene is a tetracycline resistance gene or a kanamycin resistance gene.

[0009] Furthermore, the nematodes are added at a concentration of 1000 to 20000 nematodes / mL.

[0010] Furthermore, the optimal concentration of the nematodes added is 10,000 nematodes / mL.

[0011] The beneficial effects of the present invention are:

[0012] (1) This method can effectively degrade free antibiotic resistance genes in water, with a removal effect of up to 5.26 orders of magnitude;

[0013] (2) This method can effectively inactivate eARGs within a few hours, curb the spread of eARGs in the environment, and reduce the environmental risks of eARGs.

[0014] (3) This method utilizes nematodes that are widely present in the environment to solve the pollution problem of free antibiotic resistance genes in actual water bodies, which is in line with sustainable development and provides a simple, economical, efficient, and environmentally friendly technical method for the prevention and control of free antibiotic resistance gene pollution in water bodies and risk management. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a diagram showing the degradation effect of different numbers of Caenorhabditis elegans on the kan resistance gene in Example 1;

[0016] Figure 2 This is a diagram showing the degradation effect of the tetM resistance gene and the kan resistance gene in Caenorhabditis elegans in Example 1;

[0017] Figure 3 This is a diagram showing the effect of Caenorhabditis elegans on the conversion efficiency of eARGs in Example 1;

[0018] Figure 4 is the electrophoresis diagram of the degradation of eARGs by Caenorhabditis elegans in Example 1, wherein: Figure 4 (a) is the electrophoresis diagram of the group treated with Caenorhabditis elegans only. Figure 4 (b) is the electrophoresis diagram of the group treated with only the resistance plasmid. Figure 4 (c) is the electrophoresis diagram of the treatment group of Caenorhabditis elegans mixed with resistance plasmid;

[0019] Figure 5 This is a diagram showing the effect of degradation of the tetM resistance gene in Caenorhabditis elegans in Example 2, wherein: Figure 5 (a) is a graph showing the effect of different concentrations of natural organic matter in water on the degradation of the tetM resistance gene in Caenorhabditis elegans. Figure 5 (b) is a diagram showing the effect of different metal cations on the degradation of the tetM resistance gene in Caenorhabditis elegans. Figure 5 (c) is a diagram showing the effect of different temperatures on the degradation of the tetM resistance gene in Caenorhabditis elegans;

[0020] Figure 6 This is a diagram showing the degradation effect of Caenorhabditis elegans on the tetM resistance gene in the water of West Lake and the secondary sedimentation tank of Hongyu Wastewater Treatment Plant in Example 3. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0022] Example 1: Caenorhabditis elegans degrades eARGs

[0023] (1) Selection of the optimal number of Caenorhabditis elegans

[0024] In this example, 50 mmol / L hepes buffer (4-hydroxyethylpiperazineethanesulfonic acid) was used to simulate a neutral water environment. The pH of the hepes buffer was between 7.2 and 7.4 and was purchased from Meiyuan Biotechnology Co., Ltd. In this example, resistance plasmids carrying tetracycline antibiotic resistance genes (tetM resistance genes) and kanamycin resistance genes (kan resistance genes) were dissolved in the above buffer to simulate free antibiotic resistance genes (eARGs) in water to form a resistance plasmid solution.

[0025] The nematode used in this example is Caenorhabditis elegans, a common model animal (Caenorhabditis elegans, C.elegans). The culture of Caenorhabditis elegans includes the following three steps: Step 1: Prepare nematode growth medium (NGM medium). The preparation method of NGM medium is: weigh 2.5g peptone, 3.0g sodium chloride, and 20g agar powder in 975mL ultrapure water, and sterilize at high temperature (121°C, 30min), then add 1mL of 1.0mol / L sterile calcium chloride solution, 1mL of 1.0mol / L sterile magnesium sulfate solution, 1mL of 5mg / mL cholesterol (solvent is ethanol), and 25mL of 1mol / L potassium phosphate buffer (pH 6.0), shake well, and dispense into 9mm culture dishes, air dry and solidify; Step 2: Prepare food for Caenorhabditis elegans. Caenorhabditis elegans feeds on Escherichia coli OP50. Inoculate E. coli OP50 in LB culture medium (tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, high temperature sterilization), culture at 37°C overnight, take 100μL and spread it on NGM culture medium; Step 3: Inoculate Caenorhabditis elegans on NGM culture medium coated with E. coli OP50, the temperature is 20±2°C, the culture time is 1 week, and the NGM culture medium full of Caenorhabditis elegans can be harvested. Wash the NGM culture medium with M9 solution (5.8g / L disodium hydrogen phosphate, 3.0g / L potassium dihydrogen phosphate, 0.5g / L sodium chloride, 1.0g / L ammonium chloride, sterilized), transfer the obtained liquid to a small beaker, filter it with a 600-mesh sieve, and the filtrate is the Caenorhabditis elegans larvae solution. The Caenorhabditis elegans was washed and enriched by centrifugation and resuspension. The centrifugation conditions were as follows: resuspending the Caenorhabditis elegans 12 times with deionized water in a 1.5 mL centrifuge tube, with a centrifugal speed of 3000 rpm and a centrifugal time of 1 min.

[0026] Experimental group: C. elegans and the resistance plasmid solution were mixed in a 24-well plate to a concentration of 1 mg / L (the final concentration of the tetM resistance gene and the kan resistance gene was 10 8 copies / μL); then add 1000, 2000, 5000, 10000 or 20000 C. elegans / mL respectively. Control group: The number of C. elegans was 0 / mL, that is, no C. elegans resistance plasmid solution was added, and other conditions were consistent with the experimental group. Wrap the 24-well plate with tin foil and react in an environment of 20±2℃. Take samples every 12h. When sampling, place the 24-well plate on a vortex machine to mix, and then wait for the C. elegans in the well plate to naturally settle to the bottom of the plate, and take about 50μL of the supernatant into a centrifuge tube. The sample was stored in a -20℃ refrigerator, and only allowed to freeze and thaw once for subsequent analysis.

[0027] The content of tetM resistance gene or kan resistance gene was quantified by quantitative polymerase chain reaction (qPCR), and the logarithm of degradation rate was used to represent the degradation of tetM resistance gene or kan resistance gene by Caenorhabditis elegans, i.e., log10(Ct / C0), Ct represented the concentration of tetM resistance gene or kan resistance gene in the solution at time t (copies / μL), C0 represented the concentration of tetM resistance gene or kan resistance gene in the solution at time 0 (copies / μL), and Ct / C0 represented the degradation rate of tetM resistance gene or kan resistance gene. The primers used are shown in Table 1, wherein tetM-L is the long amplicon of tetM resistance gene, and tetM-S is the short amplicon of tetM resistance gene; kan-L is the long amplicon of kan resistance gene, and kan-S is the short amplicon of kan resistance gene.

[0028] Table 1 Amplicon information

[0029]

[0030] In order to determine the optimal addition concentration of Caenorhabditis elegans, the kan resistance gene was used as a representative and the kan-L primer was used to quantify the concentration change of the kan resistance gene in the sample. Figure 1 As shown, in the control group, the concentration of the kan resistance gene did not change within 84 hours, which indicates that the kan resistance gene was not degraded in the buffer solution. In the experimental group, Caenorhabditis elegans can effectively degrade the kan resistance gene within 84 hours. At 84 hours, the kan resistance gene corresponding to the concentrations of Caenorhabditis elegans of 1000, 2000, 5000, 10000, and 20000 / mL was reduced by 1.92, 2.67, 3.20, 5.10, and 5.20 orders of magnitude, respectively. Among them, when the concentration of Caenorhabditis elegans was 10000 / mL, the degradation effect of the kan resistance gene was close to that of 20000 / mL. Therefore, in this case, 10000 / mL of Caenorhabditis elegans was selected as the preferred material for subsequent experiments.

[0031] (2) Degradation effect of Caenorhabditis elegans on different eARGs

[0032] The experimental group was set up as follows: the dosage of Caenorhabditis elegans was 10,000 / mL, the concentration of the resistance plasmid solution was 1 mg / L (the concentration of the tetM resistance gene and the kan resistance gene was 10 8 The control group was set as follows: the dosage of Caenorhabditis elegans was 0, the concentration of the resistance plasmid solution was 1 mg / L (the concentration of the tetM resistance gene and the kan resistance gene was 10 8The other reaction conditions and experimental details were the same as those in the optimal Caenorhabditis elegans dosage experiment in Example 1. The degradation effect of Caenorhabditis elegans on the tetM resistance gene was tested using tetM-L and tetM-S primers, and the degradation effect of Caenorhabditis elegans on the kan resistance gene was tested using kan-L and kan-S primers.

[0033] The results are as follows Figure 2 As shown. The concentrations of long and short amplicons of tetM and kan resistance genes in the control group remained basically unchanged within 84 hours, indicating that the tetM resistance gene and kan resistance gene were not degraded in the buffer solution. In the experimental group, the long and short amplicons of tetM decreased by 5.26 and 4.95 orders of magnitude, respectively, and the long and short amplicons of kan decreased by 5.10 and 4.41 orders of magnitude, respectively. In the experiment, it was found that the degradation process can be divided into two stages, namely the rapid degradation stage and the slow degradation stage, in which the rapid degradation stage conforms to the first-order kinetics. For the long amplicons (tetM-L and kan-L), the rapid degradation stages occurred in the first 48 hours and the first 60 hours, respectively, with degradation rates of 0.10h and 0.20h, respectively. -1 and 0.083h -1 , followed by a slow degradation phase; for short amplicons (tetM-S and kan-S), the rapid degradation phase occurred 12 h after the start of the reaction, with degradation rates of 0.068 h -1 and 0.062h -1 . The order of degradation rate from large to small is tetM-L>kan-L>tetM-S>kan-S, among which there is no significant difference in degradation rate between tetM-S and kan-S. Amplicon length and degradation rate show a significant positive correlation (Spearman correlation coefficient r=0.705, significance test p<0.01), which means that the longer the amplicon length, the faster the degradation rate. It is worth noting that the fragment lengths of tetM-S and kan-S amplicons are similar, but the sequences are different, and there is no significant difference in their degradation rates, which suggests that the sequences of different eARGs do not affect the degradation rate of C. elegans, which means that C. elegans has no obvious preference for the sequence of eARGs for degradation and can degrade other types of eARGs.

[0034] (3) Caenorhabditis elegans reduces the conversion efficiency of eARGs

[0035] The experimental group was set up as follows: the dosage of Caenorhabditis elegans was 10,000 / mL, the concentration of the resistance plasmid solution was 1 mg / L (the concentration of the tetM resistance gene and the kan resistance gene was 10 8copies / μL), and sampling was performed every 1 h. Other reaction conditions and experimental details are the same as the optimal Caenorhabditis elegans dosage experiment in Example 1. The obtained samples were subjected to transformation experiments. The operating steps of the transformation experiment were carried out in accordance with the common production instructions, as follows: Competent Escherichia coli (Escherichia coli DH5α) were purchased from Shanghai Bioengineering Co., Ltd. Take out the competent Escherichia coli cells from the -80°C refrigerator, quickly insert them into the ice box, and wait for 30 minutes to dissolve them. Add 10 μL of sample to 100 μL of competent Escherichia coli, mix gently, and place on ice for 30 minutes. Then incubate in a 42°C water bath for 45 seconds, quickly put it back on ice and place it for about 2 minutes. Be careful not to shake the competent Escherichia coli cells. Then, 890 μL LB culture medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, high temperature sterilization) was added to the competent E. coli cells, with a total volume of 1 mL. After mixing evenly, the cells were shaken and revived at 180 rpm in a 37°C incubator for 1 hour. The revived E. coli cells were diluted step by step with physiological saline, and then 100 μL of E. coli cells were drawn and spread on a solid LB plate containing tetracycline and kanamycin for screening the successfully transformed E. coli. The preparation method of the plate was as follows: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, and 15 g agar powder were weighed in 1000 mL ultrapure water, and sterilized at high temperature (121°C, 30 min). When the culture medium was cooled to about 60°C, 200 μL of 50 mg / mL tetracycline and 400 μL of 50 mg / mL kanamycin were added, and the plates were divided into 9 mm culture dishes and air-dried to solidify. After culturing in a 37°C incubator for 36 hours, the grown E. coli were counted. The transformation efficiency was calculated by dividing the number of transformants (CFU) by the initial mass of the resistance plasmid (μg). Figure 3 As shown, in the presence of C. elegans, the transformation efficiency of the resistance gene decreased 170-fold within 3 h, and no transformants could be detected after 5 h, indicating that C. elegans greatly reduced the transformation risk of eARGs.

[0036] (4) Fragmented resistance plasmids in Caenorhabditis elegans

[0037] In order to characterize the phenomenon of Caenorhabditis elegans degradation of free antibiotic resistance genes (eARGs), agarose gel electrophoresis was used in this example to examine the degradation effect of Caenorhabditis elegans on resistance plasmids. The initial dosage of resistance plasmids carrying tetM and kan antibiotic resistance genes was increased to 10 mg / L, and the dosage of Caenorhabditis elegans remained at 10,000 / mL. The experiment set up three treatment groups. The first group was the Caenorhabditis elegans treatment group only: hepes buffer solution with only Caenorhabditis elegans present; the second group was the resistance plasmid treatment group only: hepes buffer solution with only resistance plasmid present; the third group was the treatment group in which Caenorhabditis elegans and resistance plasmids were mixed: hepes buffer solution mixed with Caenorhabditis elegans and resistance plasmids. The reaction conditions of agarose gel electrophoresis are: agarose concentration of 1%, electrophoresis voltage of 110-115V, and electrophoresis time of 40min. Figure 4 As shown in (a), no white bands were produced in the group treated with only Caenorhabditis elegans, indicating that the presence of Caenorhabditis elegans did not interfere with the detection of the resistance plasmid bands; Figure 4 As shown in (b), in the group treated with only the resistance plasmid, the resistance plasmid bands remained basically unchanged; Figure 4 As shown in (c), in the presence of C. elegans, the resistance plasmid band was degraded into smaller fragments over time, and at 72h, the fragment length did not exceed 250bp. This indicates that the fragmentation of the resistance plasmid is caused by C. elegans, that is, C. elegans can degrade the eARGs in the solution. In the experiment, we accidentally discovered that it was the protein secreted by C. elegans that caused the degradation of eARGs in the solution. This protein is NUC-1, which belongs to DNase II. Through bioinformatics analysis, it was found that the protein NUC-1 is ubiquitous and highly conserved in nematodes of the Strongylida, Rhabditida, and Trichinellida orders, which to some extent indicates that other free-living nematodes also have the ability to degrade free antibiotic resistance genes in water.

[0038] Example 2: Effects of different water quality parameters on eARGs degradation

[0039] This implementation case considers the effects of different concentrations of natural organic matter (NOM), metal cations or different temperatures on the degradation of eARGs. In this example, qPCR is used to detect the content of tetM resistance gene (tetM-L as primer) as an evaluation of the effect of nematode degradation on eARGs. The specific steps are as follows:

[0040] (1) Impact of NOM

[0041] Suwannee NOM was purchased from the International Humic Substances Society (IHSS). The preparation method of the resistance plasmid solution was as described in Example 1. The experimental group was set as follows: NOM was added to the resistance plasmid solution to make the final concentration of NOM 5, 10, and 20 mg / L, respectively, and the dosage of Caenorhabditis elegans was 10,000 / mL; the control group was set as 20 mg / L NOM mixed with the resistance plasmid solution, and no Caenorhabditis elegans was added. Other experimental conditions and experimental details were consistent with Example 1, such as Figure 5 As shown in (a), in the control group, the concentration of the tetM resistance gene did not change after 84 hours of reaction, which indicates that the presence of NOM will not affect the concentration change of the tetM resistance gene. In the experimental group, after 84 hours of reaction, Caenorhabditis elegans can degrade 5.26, 5.97, 6.14, and 6.22 orders of magnitude of tetM resistance genes when the concentration of NOM is 0, 5, 10, and 20 mg / L, respectively, which indicates that the addition of NOM is beneficial to the degradation of tetM resistance genes by Caenorhabditis elegans. At the same time, it can also be shown that the addition of NOM is beneficial to the degradation of eARGs by nematodes.

[0042] (2) Effect of metal cations

[0043] This case investigates an environmental concentration of 2.5mmol / L Ca 2+ and 2.0mmol / L Mg 2+ Their respective effects on the degradation of eARGs by nematodes.

[0044] The preparation method of the resistance plasmid solution is as described in Example 1. The experimental group is set as follows: 2.5mmol / L CaCl2 or 2.0mmol / L MgCl2 is added to the resistance plasmid solution, and the amount of Caenorhabditis elegans added is 10,000 / mL; the control group is set as follows: 2.5mmol / L CaCl2 or 2.0mmol / L MgCl2 is added to the resistance plasmid solution, and no Caenorhabditis elegans is added. The remaining experimental conditions and experimental details are consistent with Example 1. Figure 5 As shown in (b), in the absence of Caenorhabditis elegans, the concentration of the tetM resistance gene remained essentially unchanged, indicating that 2.5 mM Ca 2+ and 2.0 mM Mg 2+ The concentration of tetM resistance gene will not be affected. 2+ or 2.0 mM Mg 2+ In the presence of cations, Caenorhabditis elegans could degrade 5.02 and 5.04 orders of magnitude of tetM resistance genes within 84 h, and the degradation rates in the rapid degradation stage were 0.098 h -1and 0.11h -1 There is no significant difference between the degradation effect of the solution without metal ions and the environmental concentration of Ca 2+ and Mg 2+ It does not affect the degradation of tetM resistance gene in Caenorhabditis elegans. 2+ and Mg 2+ It does not affect the degradation of eARGs by nematodes.

[0045] (3) Effect of temperature

[0046] This case study investigates the effect of temperature on the degradation of eARGs by nematodes.

[0047] The preparation method of the resistance plasmid solution is as described in Example 1. The experimental group is set as follows: Caenorhabditis elegans is added to the resistance plasmid solution, the amount of Caenorhabditis elegans added is 10,000 / mL, and the reaction is placed in an environment of 1°C, 20°C, and 30°C respectively; the control group is set as follows: Caenorhabditis elegans is not added, and the resistance plasmid solution is directly placed in an environment of 1°C, 20°C, and 30°C. The other experimental conditions and experimental details are consistent with Example 1. Figure 5 As shown in (c), in the control group, the concentration of the tetM resistance gene did not change substantially, indicating that temperature did not affect the concentration change of the tetM resistance gene. In the experimental group, Caenorhabditis elegans could degrade 1.21 and 4.47 orders of magnitude at 1°C and 30°C, respectively, and the best degradation effect was 5.26 orders of magnitude at 20°C. At the same time, it can also be shown that at 20°C, Caenorhabditis elegans has the best degradation effect on eARGs.

[0048] In summary, NOM significantly promoted the degradation of eARGs in nematodes; metal ions (Mg 2+ and Ca 2+ ) has no significant effect on the degradation of eARGs by nematodes; temperature can affect the degradation of eARGs by nematodes, and the degradation of eARGs is best at a temperature of about 20°C in spring or autumn.

[0049] Example 3: Degradation effect of real water

[0050] The real water samples were collected from West Lake (Hangzhou, Zhejiang) and the secondary sedimentation tank of Hongyu Wastewater Treatment Plant (Zhuji, Zhejiang). The water samples were filtered through a 0.22 μm membrane, and the specific parameters are shown in Table 2.

[0051] Table 2 Water quality parameters of water samples

[0052]

[0053] Resistance plasmids were added to the filtered water from West Lake or the water from the secondary sedimentation tank of Hongyu Sewage Treatment Plant to prepare water from West Lake or the secondary sedimentation tank of Hongyu Sewage Treatment Plant containing tetM resistance gene and kan resistance gene. The experimental groups were set up as follows: Caenorhabditis elegans was added to the water from West Lake or the secondary sedimentation tank of Hongyu Sewage Treatment Plant containing tetM resistance gene and kan resistance gene, respectively. The amount of Caenorhabditis elegans added was 10,000 / mL, and the final concentration of tetM resistance gene and kan resistance gene was 10 8 The control group was set as: West Lake water or Hongyu sewage treatment plant secondary sedimentation tank water containing tetM resistance gene and kan resistance gene, without adding Caenorhabditis elegans, and the final concentration of tetM resistance gene and kan resistance gene was 10 8 The remaining experimental conditions and experimental details were consistent with those in Example 1. In this example, qPCR was used to detect the content of tetM (tetM-L was used as primer) to evaluate the degradation effect of Caenorhabditis elegans on eARGs in real water. Figure 6 As shown in the figure, in the control group without C. elegans, the concentration of tetM resistance gene in West Lake water or the water in the secondary sedimentation tank of Hongyu sewage treatment plant remained basically unchanged; in the experimental group, after 84 hours of reaction, the degradation effect of tetM resistance gene in West Lake water or the water in the secondary sedimentation tank of Hongyu sewage treatment plant was 4.78 and 3.23 orders of magnitude, respectively. This shows that C. elegans can effectively degrade tetM resistance gene in real water. At the same time, it can also be shown that nematodes can effectively degrade eARGs in real water.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for degrading free antibiotic resistance genes in water using nematodes, characterized in that: Nematodes are added to water bodies containing free antibiotic resistance genes, and the action of nucleases secreted by the nematodes is utilized to efficiently degrade the free antibiotic resistance genes in the water bodies and inhibit the spread of the free antibiotic resistance genes.

2. The method of using nematodes to degrade free antibiotic resistance genes in water according to claim 1, characterized in that: The nematode is Caenorhabditis elegans.

3. The method of using nematodes to degrade free antibiotic resistance genes in water according to claim 1, characterized in that: The free antibiotic resistance gene is a tetracycline resistance gene or a kanamycin resistance gene.

4. The method of using nematodes to degrade free antibiotic resistance genes in water according to claim 1, characterized in that: The nematode addition concentration is 1000 to 20000 nematodes / mL.

5. The method of using nematodes to degrade free antibiotic resistance genes in water according to claim 4, characterized in that: The optimal addition concentration of the nematodes is 10,000 nematodes / mL.

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