Experimental method for antibiotic resistance gene transmission and transfer research

Abstract

The invention discloses an experimental method for studying propagation and transfer of antibiotic resistance genes. The experimental method comprises the following steps: preparing an experimental material and a porous medium material, designing a soil column simulation system, operating the soil column simulation system, changing and measuring ARGs in effluent, and calculating an experimental result. The fitting value and the measured value of the effluent show that the ARGs may enter underground water through a soil layer to cause environmental health risks in the surface infiltration process after the reclaimed water is supplemented, theoretical support is provided for the environmental risk assessment and pollution control of the ARGs, and in the actual site application process, the one-dimensional model of pollutants can be utilized to improve the environmental risk assessment and pollution control of the ARGs. And predicting the migration concentration of the ARGs according to actual parameters such as an actual site retention factor, a dispersion coefficient, an actual water flow velocity, medium density and the like, and evaluating the environmental risk.

Description

Technical Field

[0001] The present invention is applicable to the study of the spread and pollution of antibiotic resistance genes in the environment, and particularly relates to the spread and transfer of ARGs in various media involved in the process of reclaimed water as an important groundwater recharge, and is specifically an experimental method for studying the spread and transfer of antibiotic resistance genes. Background Art

[0002] In 2006, Pruden et al. first proposed that antibiotic resistance genes (ARGs) be regarded as a new type of environmental pollutant. Unlike other pollutants, ARGs are hereditary and can be transmitted between parents and offspring through vertical transfer, or horizontally transferred to bacteria that originally did not have ARGs through mobile genetic elements. Therefore, it will cause long-term and irreversible impacts on the environment. If humans and animals are infected with pathogenic bacteria carrying ARGs, the effect of antibiotics during treatment will be greatly reduced, increasing the difficulty of cure. Studies have shown that about 75%-95% of antibiotics used in medical treatment or animal breeding are excreted in the form of original drugs or metabolites. Due to the frequent use and discharge of antibiotics, a certain level of accumulation is formed in the environment, and a selection pressure for antibiotic resistance is formed, which in turn promotes the spread and transformation of ARGs. Usually in environmental media, strains rely on horizontal gene transfer (HGT) to acquire antibiotic resistance. Since HGT can be transmitted between strains of the same or different species, it greatly accelerates the emergence of resistance genes.

[0003] ARGs exist in the environment in two forms: free (DNA fragments) and intracellular ARGs carried by bacteria. Researchers have used model strains or plasmids to study the changes of ARGs in different forms in a single environment, but have not considered the migration changes of ARGs between different media. In particular, the mutual influence of ARGs in the migration process between solid and liquid phases during the recharge of rivers, lakes and groundwater with recycled water. In fact, the spread of ARGs in the environment in multiphase environmental media mainly includes horizontal gene transfer (HGT) within microbial communities, the growth and reproduction of individuals, and the physical effect of ARGs carriers in the migration of water in the medium. However, there are few studies on the migration and transformation of ARGs in different forms in multiphase media, and the migration model has not yet been established. Summary of the invention

[0004] The object of the present invention is to provide an experimental method for studying the transmission and transfer of antibiotic resistance genes to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] An experimental method for studying the transmission and transfer of antibiotic resistance genes comprises the following steps:

[0007] (1) Experimental materials:

[0008] ① Intracellular ARGs: Pseudomonas putida KT2400 is the donor bacteria, and Escherichia coli ATCC25922 is the recipient bacteria;

[0009] ② Extracellular ARGs: The constructed sul1 plasmid was used as the target ARGs;

[0010] (2) Preparation of porous media materials

[0011] Gravel, quartz sand, undisturbed soil, and fine sand are used as porous media fillers;

[0012] The porous media were sterilized by high-pressure steam to exclude the influence of DNA-degrading enzymes and biological inhibitors in the microbial community;

[0013] Soil medium was inoculated with 5 mL OD 600 = 1.0 Escherichia coli ATCC25922 obtained by centrifugation at 10000*g for 1-3 min;

[0014] (3) The soil column is made of plexiglass, with an inner diameter of 6 cm and a height of 30 cm;

[0015] The bottom of the simulation column is filled with 2 cm gravel as a drag layer, and the top is filled with 2 cm fine sand as a water distribution layer to achieve uniform water distribution. The middle is a filler layer, and the filler layer uses the collected original soil with a filling thickness of 16 cm. A water outlet is set at the bottom of the soil column to collect the total water after passing through the soil column, which is used to measure the changes in ARGs in the total water outlet.

[0016] When filling the soil column, tamp every 3-5cm of soil added;

[0017] (4) Operation of soil column simulation system

[0018] The soil column simulation system is mainly divided into three stages:

[0019] In the first stage, bromide ions were used as tracer elements to draw Br penetration curves. 40 mg / L NaBr solution was continuously injected into two soil columns for 5 days. Samples were taken at different times and the concentration of Br was measured using an anion chromatograph to determine the simulated column diffusion coefficient.

[0020] The second stage is the cleaning stage, using soil background solution for continuous infiltration for 7 days. The soil background solution is 0.01 mol / LC aCl2 and 200mg / LNaN3 mixed solution. The main purpose of this stage is to wash out the original soluble impurities in the soil column, inhibit the growth of bacteria, balance the soil ion concentration level and obtain stable effluent water quality, prepare for subsequent tests and ensure the stability of the system effluent;

[0021] The third stage is the ARGs propagation and transfer experimental stage;

[0022] Four soil column experiments were carried out at the same time, and the absolute abundance of the target gene in the influent was designed to be 2×10 5 copies / μL, 2.9×10 7 cells / mL bacterial solution. During the infiltration period, the water head of the soil column was kept at 3 cm. Samples were taken from the water outlet of the soil column on the 6th, 8th, 10th, 15th and 20th day after infiltration to determine the absolute abundance of the target gene or the number of bacteria in the water outlet.

[0023] (5) Changes and determination of ARGs in effluent

[0024] By comparing the differences in the number of target genes or bacteria in the soil column effluent, the migration process of antibiotic resistance genes in the soil column simulation system was studied;

[0025] Confocal microscopy was used to analyze the gene transfer of intracellular ARGs; qPCR was used to detect the migration process of extracellular ARGs;

[0026] ① Measure the number of bacteria in the cell using confocal microscopy and flow cytometry

[0027] The fluorescence expression of the effluent samples of soil column 1 was detected by Olympus laser confocal microscope FV3000. The recipient bacteria ATCC25932 was used as a negative control without fluorescence, and the donor bacteria KT2440 was used as a red fluorescence positive control. The fluorescence excitation spectrum was selected to be 488nm and 561nm respectively. 100μl of the effluent samples after pretreatment at different times were taken and placed in a confocal dish, and the same zoom value was selected to detect the fluorescence expression.

[0028] The stained bacteria were analyzed using a Bio-Rad ZE5 flow cytometer, and three lasers, 488nm and 561nm, were selected for excitation. Before the flow analysis, the test samples were washed three times by centrifugation with phosphate buffered saline (PBS), and finally resuspended with PBS. The 561nm and 488nm lasers were selected, and positive and negative controls were set up respectively. The number of green fluorescent and non-fluorescent bacteria in the samples was analyzed, and the gene horizontal transfer rate T / R was calculated accordingly.

[0029] The number of green bacteria in the water from soil column 1 is N T1

[0030]

[0031] Where, f: bonding transfer rate;

[0032] N T : The number of green fluorescent conjugated cells measured by flow cytometry, unit:

[0033] N o : Total number of sorted cells, in units;

[0034] N D : The number of red fluorescent donor bacterial cells measured by flow cytometry, unit:

[0035] ②Only measure the copy number

[0036] After pretreatment, the absolute content of ARGs in the sample was measured using a fluorescent quantitative PCR instrument to calculate the absolute copy number q1 of the target gene in the sample, and the long-term existence of the plasmid in static water was used as the control q0, and q was the adsorption of the column medium to the plasmid q = q0-q1;

[0037] The changes in the number of bacteria caused by adsorption during the washing of the column with bacterial liquid were studied. The biodegradation and other complex processes were not considered. The long-term culture of bacteria in static water was used as a control. Finally, the content of KTT240 in the water discharged from the column was determined by flow cytometry.

[0038] The number of control bacteria in still water is c0, the number of bacteria in the column outlet water is c1, c is the adsorption amount of bacteria by the column medium c = c0-c1;

[0039] (6) Experimental results and calculations

[0040] ①Simulation of joint transfer of soil column 1

[0041] The abundance of KT2440 was detected in soil column 1 on the 6th day, and the abundance of KT2440 and zygotes in the effluent gradually increased with the increase of soil column operation time, reaching the maximum value of 4.09×10 6 cells / μL, an increase of 28% compared with the initial bacterial count, and the abundance value decreased to 3.11×10 cells / μL after 20 days. 6 cells / μL, and the abundance of KT2440 in the effluent of the soil column at 20 days reached 2.90×10 6 cells / μL, which was basically the same as the abundance of KT2440 in the control, indicating that KT2440 was adsorbed in the soil column medium during its migration, and the exogenous bacteria were constantly multiplying while adsorbing to the medium;

[0042] ②Simulating the migration of extracellular ARGs in soil column 2

[0043] A small amount of plasmid concentration was detected in the effluent of extracellular ARGs in soil column 2 on the second day. The absolute abundance of sul1 in the effluent gradually increased with time and reached 1.13×10 4 The absolute abundance of sul1 in the control group decreased with time, and after 20 days, it dropped to 1.59×10 4 copies / μL, indicating that in the absence of any medium under natural conditions, the plasmid solution has a self-attenuation process, and when the plasmid is injected into the soil column medium, the attenuation process is alleviated due to the adsorption protection of the soil particles in the medium;

[0044] ③ Migration pattern of ARGs colloids in porous media

[0045] In order to compare the migration and transformation of ARGs of different forms in the experimental column, the physicochemical changes and biological reproduction of pollutants in the process were not considered. The convection-diffusion equilibrium model of CXTFIT software was used to simulate the temporal changes of KT2440 and sul1 in the effluent of the soil column. The one-dimensional model of the pollutants was described as follows:

[0046]

[0047]

[0048] In the formula, R d : retention factor;

[0049] D: diffusion coefficient, unit: m 2 / d;

[0050] u: actual flow velocity of water, unit: m / d;

[0051] ρ b : Medium density, unit: g / cm 3 ;

[0052] η e : porosity;

[0053] K d : Linear adsorption constant, cm 3 / g;

[0054] When the solution condition is set to t = 0, C(x, t) = 0; when x = 0, C(x, t) = C0; when x tends to infinity, C(x, t) = 0, the analytical solution is as follows:

[0055]

[0056] Where D L : Hydrodynamic dispersion coefficient, unit: m2 / d;

[0057] u: actual flow velocity of water, unit: m / d;

[0058] C: pollutant concentration, copies / μL or cells / μL;

[0059] In order to compare the migration and transformation of ARGs with different forms in the experimental column, the convection-diffusion equilibrium model of CXTFIT software was used to simulate the temporal changes of KT2440 and sul1 in the effluent water of the soil column without considering the physicochemical changes and biological reproduction of the pollutants in the process, and the fitting results were good.

[0060] As a further solution of the present invention: Pseudomonas putida KT2400: carries a wide-host plasmid RP4 marked with green fluorescent protein, and the chromosome of the bacterium is marked with red fluorescent protein and lacIq gene, which can express red fluorescence but inhibit the expression of plasmid green fluorescent protein. If conjugation transfer occurs, the plasmid RP4 will be transferred to the recipient bacteria to make the recipient bacteria emit green fluorescence, and the antibiotic-sensitive Escherichia coli ATCC25922 is used as the plasmid receptor.

[0061] As a further solution of the present invention: in order to remove the influence of impurities on the surface of gravel, quartz sand and fine sand media on the experimental results, the surface of the media is cleaned, and the treatment method is as follows:

[0062] 1) Soak in 0.1 mol nitric acid for 3 hours;

[0063] 2) Rinse with deionized water until the conductivity of the rinse water is low;

[0064] 3) Soak in 0.1 mol NaOH solution for 3 h;

[0065] 4) Then rinse with deionized water until the ionic strength IS of the rinse solution is less than 0.01 mM;

[0066] 5) Place the cleaned porous medium in an autoclave and sterilize at 121°C for 20 minutes;

[0067] 6) Finally, dry at 105°C and seal in a beaker for later use.

[0068] Compared with the prior art, the present invention has the following beneficial effects:

[0069] The indoor simulated soil column test of the present invention is an effective method for studying the migration and transformation laws of pollutants in soil. Compared with the batch test method, the soil column test can be closer to the actual conditions by simulating the on-site conditions. The fitting value and the measured value of the effluent of the present invention both indicate that ARGs may enter the groundwater through the soil layer during the surface infiltration process after the reclaimed water replenishment and cause environmental health risks, providing theoretical support for the environmental risk assessment and pollution control of ARGs. In the actual site application process, the one-dimensional model of the pollutant can be used to predict the migration concentration of ARGs according to the actual parameters such as the actual site retention factor, diffusion coefficient, actual water flow velocity, medium density, etc., to assess the environmental risk. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 Schematic diagram of the experimental approach used to study the spread of antibiotic resistance genes.

[0071] Figure 2 This is a picture showing the difference in the surface of quartz sand before and after treatment and cleaning using a high-performance field emission scanning electron microscope.

[0072] Figure 3 Design diagram of the soil column simulation system.

[0073] Figure 4 This is the abundance change diagram of KT2440 in the water outlet of soil column 1.

[0074] Figure 5 This is a graph showing the abundance changes of plasmid sul1 in the water effluent from soil column 2.

[0075] Figure 6 This is the one-dimensional model fitting result diagram of KT2440 in the water outlet of soil column 1.

[0076] Figure 7 This is the one-dimensional model fitting result of sul1 in the water outlet of soil column 2. DETAILED DESCRIPTION

[0077] Various exemplary embodiments, features and aspects of the present application will be described in detail below with reference to the accompanying drawings. The same reference numerals in the accompanying drawings represent elements with the same or similar functions. Although various aspects of the embodiments are shown in the accompanying drawings, the drawings are not necessarily drawn to scale unless otherwise specified.

[0078] The word “exemplary” is used exclusively herein to mean “serving as an example, example, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

[0079] In addition, in order to better illustrate the present application, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In some examples, methods, means, and elements well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present application.

[0080] In order to understand the migration and transformation of ARGs during surface water infiltration after reclaimed water replenishment in rivers, to reasonably predict the distribution and propagation patterns of ARGs in recharge infiltration, and to predict the risk of ARGs gene transfer, this study conducted a 30-day simulated soil column experiment in the laboratory.

[0081] The present invention aims to study the propagation of ARGs of different forms in a multiphase environmental medium, and in particular to study a propagation and transfer model of ARGs in a river-soil-groundwater system.

[0082] Indoor simulated soil column test is an effective method to study the migration and transformation laws of pollutants in soil. Compared with batch test methods, column test can be closer to actual conditions by simulating field conditions.

[0083] An experimental method for studying the transmission and transfer of antibiotic resistance genes comprises the following steps:

[0084] (1) Experimental materials:

[0085] ① Intracellular ARGs: Pseudomonas putida KT2400 is the donor bacteria, and Escherichia coli ATCC25922 is the recipient bacteria;

[0086] See also Figure 1 Pseudomonas putida KT2400: carries a wide-host plasmid RP4 marked with green fluorescent protein, and the chromosome of the bacterium is marked with red fluorescent protein and lacIq gene, which can express red fluorescence but inhibit the expression of plasmid green fluorescent protein. If conjugation transfer occurs, plasmid RP4 will be transferred to the recipient bacteria, making the recipient bacteria emit green fluorescence. The antibiotic-sensitive Escherichia coli ATCC25922 is used as the plasmid receptor.

[0087] ② Extracellular ARGs: The constructed sul1 plasmid was used as the target ARGs;

[0088] (2) Preparation of porous media materials

[0089] Gravel, quartz sand, undisturbed soil, and fine sand are used as porous media fillers;

[0090] The porous media were sterilized by high-pressure steam to exclude the influence of DNA-degrading enzymes and biological inhibitors in the microbial community;

[0091] Soil medium was inoculated with 5 mL OD600 = 1.0 Escherichia coli ATCC25922 obtained by centrifugation at 10000*g for 1-3 min;

[0092] In order to remove the influence of impurities on the surface of gravel, quartz sand and fine sand media on the experimental results, the surface of the media is cleaned. The treatment method is as follows:

[0093] 1) Soak in 0.1 mol nitric acid for 3 hours;

[0094] 2) Rinse with deionized water until the conductivity of the rinse water is low;

[0095] 3) Soak in 0.1 mol NaOH solution for 3 h;

[0096] 4) Then rinse with deionized water until the ionic strength IS of the rinse solution is less than 0.01 mM;

[0097] 5) Place the cleaned porous medium in an autoclave and sterilize at 121°C for 20 minutes;

[0098] 6) Finally, dry at 105°C and seal in a beaker for later use.

[0099] See also Figure 2 Using a high-performance field emission scanning electron microscope Zeiss Merlin to observe the difference in the surface of quartz sand before and after cleaning, it can be seen that the impurities on the surface of quartz sand are significantly reduced after cleaning.

[0100] See also Figure 3 ,(3) The soil column material is plexiglass, with an inner diameter of 6 cm and a height of 30 cm;

[0101] The bottom of the simulation column is filled with 2 cm gravel as a drag layer, and the top is filled with 2 cm fine sand as a water distribution layer to achieve uniform water distribution. The middle is a filler layer, and the filler layer uses the collected original soil with a filling thickness of 16 cm. A water outlet is set at the bottom of the soil column to collect the total water after passing through the soil column, which is used to measure the changes in ARGs in the total water outlet.

[0102] When filling the soil column, tamp every 3-5cm of soil added;

[0103] (4) Operation of soil column simulation system

[0104] The soil column simulation system is mainly divided into three stages of operation. The water inflow and operation time of each stage are as follows:

[0105]

[0106] Table: Soil column operation mode

[0107] In the first stage, bromide ions were used as tracer elements to draw Br penetration curves. 40 mg / L NaBr solution was continuously injected into two soil columns for 5 days. Samples were taken at different times and the concentration of Br was measured using an anion chromatograph to determine the simulated column diffusion coefficient.

[0108] The second stage is the cleaning stage, using soil background solution for continuous infiltration for 7 days. The soil background solution is 0.01 mol / LC a Cl2 and 200mg / LNaN3 mixed solution. The main purpose of this stage is to wash out the original soluble impurities in the soil column, inhibit the growth of bacteria, balance the soil ion concentration level and obtain stable effluent water quality, prepare for subsequent tests and ensure the stability of the system effluent;

[0109] The third stage is the ARGs propagation and transfer experimental stage;

[0110] Four soil column experiments were carried out at the same time, and the absolute abundance of the target gene in the influent was designed to be 2×10 5 copies / μL, 2.9×10 7 cells / mL bacterial solution. During the infiltration period, the water head of the soil column was kept at 3 cm. Samples were taken from the water outlet of the soil column on the 6th, 8th, 10th, 15th and 20th day after infiltration to determine the absolute abundance of the target gene or the number of bacteria in the water outlet.

[0111]

[0112] (5) Changes and determination of ARGs in effluent

[0113] By comparing the differences in the number of target genes or bacteria in the soil column effluent, the migration process of antibiotic resistance genes in the soil column simulation system was studied;

[0114] Confocal microscopy was used to analyze the gene transfer of intracellular ARGs; qPCR was used to detect the migration process of extracellular ARGs;

[0115] ① Measure the number of bacteria in the cell using confocal microscopy and flow cytometry

[0116] The fluorescence expression of the effluent samples of soil column 1 was detected by Olympus laser confocal microscope FV3000. The recipient bacteria ATCC25932 was used as a negative control without fluorescence, and the donor bacteria KT2440 was used as a red fluorescence positive control. The fluorescence excitation spectrum was selected to be 488nm and 561nm respectively. 100μl of the effluent samples after pretreatment at different times were taken and placed in a confocal dish, and the same zoom value was selected to detect the fluorescence expression.

[0117] The stained bacteria were analyzed using a Bio-Rad ZE5 flow cytometer, and three lasers, 488nm and 561nm, were selected for excitation. Before the flow analysis, the test samples were washed three times by centrifugation with phosphate buffered saline (PBS), and finally resuspended with PBS. The 561nm and 488nm lasers were selected, and positive and negative controls were set up respectively. The number of green fluorescent and non-fluorescent bacteria in the samples was analyzed, and the gene horizontal transfer rate T / R was calculated accordingly.

[0118] The number of green bacteria in the water from soil column 1 is N T1

[0119]

[0120] Where, f: bonding transfer rate;

[0121] N T : The number of green fluorescent conjugated cells measured by flow cytometry, unit:

[0122] N o : Total number of sorted cells, in units;

[0123] N D : The number of red fluorescent donor bacterial cells measured by flow cytometry, unit:

[0124] ②Only measure the copy number

[0125] After pretreatment, the absolute content of ARGs in the sample was measured using a fluorescent quantitative PCR instrument to calculate the absolute copy number q1 of the target gene in the sample, and the long-term existence of the plasmid in static water was used as the control q0, and q was the adsorption of the column medium to the plasmid q = q0-q1;

[0126] The changes in the number of bacteria caused by adsorption during the washing of the column with bacterial liquid were studied. The biodegradation and other complex processes were not considered. The long-term culture of bacteria in static water was used as a control. Finally, the content of KTT240 in the water discharged from the column was determined by flow cytometry.

[0127] The number of control bacteria in still water is c0, the number of bacteria in the column outlet water is c1, c is the adsorption amount of bacteria by the column medium c = c0-c1;

[0128] (6) Experimental results and calculations

[0129] ①Simulation of joint transfer of soil column 1

[0130] See also Figure 4 The figure shows the changes in the abundance of KT2440 in the effluent of soil column 1 over time, the changes in the green fluorescent conjugates produced by the horizontal transfer of KT2440 in the effluent, and the changes in the abundance of KT2440 in the control sample A over time.

[0131] It can be seen that the abundance of KT2440 was detected in soil column 1 on the 6th day, and the abundance of KT2440 and zygotes in the effluent gradually increased with the increase of soil column operation time, reaching the maximum value of 4.09×10 6 cells / μL, an increase of 28% compared with the initial bacterial count, and the abundance value decreased to 3.11×10 cells / μL after 20 days. 6 cells / μL, and the abundance of KT2440 in the effluent of the soil column at 20 days reached 2.90×10 6 cells / μL, which was basically the same as the abundance of KT2440 in the control, indicating that KT2440 was adsorbed in the soil column medium during its migration, and the exogenous bacteria were constantly multiplying while adsorbing to the medium;

[0132] ②Simulating the migration of extracellular ARGs in soil column 2

[0133] See also Figure 5 In the effluent of soil column 2, a small amount of plasmid concentration was detected on the second day. The absolute abundance of sul1 in the effluent gradually increased with time and reached 1.13×10 4 The absolute abundance of sul1 in the control group decreased with time, and after 20 days, it dropped to 1.59×10 4 copies / μL, indicating that in the absence of any medium under natural conditions, the plasmid solution has a self-attenuation process, and when the plasmid is injected into the soil column medium, the attenuation process is alleviated due to the adsorption protection of the soil particles in the medium;

[0134] ③ Migration pattern of ARGs colloids in porous media

[0135] In order to compare the migration and transformation of ARGs of different forms in the experimental column, the physicochemical changes and biological reproduction of pollutants in the process were not considered. The convection-diffusion equilibrium model of CXTFIT software was used to simulate the temporal changes of KT2440 and sul1 in the effluent of the soil column. The one-dimensional model of the pollutants was described as follows:

[0136]

[0137]

[0138] In the formula, R d : retention factor;

[0139] D: diffusion coefficient, unit: m 2 / d;

[0140] u: actual flow velocity of water, unit: m / d;

[0141] ρ b : Medium density, unit: g / cm 3 ;

[0142] η e : porosity;

[0143] K d : Linear adsorption constant, cm 3 / g;

[0144] When the solution condition is set to t = 0, C(x, t) = 0; when x = 0, C(x, t) = C0; when x tends to infinity, C(x, t) = 0, the analytical solution is as follows:

[0145]

[0146] Where D L : Hydrodynamic dispersion coefficient, unit: m 2 / d;

[0147] u: actual flow velocity of water, unit: m / d;

[0148] C: pollutant concentration, copies / μL or cells / μL;

[0149] See also Figure 6-7 In order to compare the migration and transformation of ARGs with different forms in the experimental column, the convection-diffusion equilibrium model of CXTFIT software was used to simulate the temporal variation of KT2440 and sul1 in the effluent of the soil column without considering the physicochemical changes and biological reproduction of the pollutants in the process, and the fitting results were good.

[0150] It should be specifically stated that the indoor simulated soil column test of the present invention is an effective method for studying the migration and transformation laws of pollutants in soil. Compared with the batch test method, the soil column test can be closer to the actual conditions by simulating the on-site conditions. The fitting value and the measured value of the effluent of the present invention both indicate that ARGs may enter the groundwater through the soil layer during the surface infiltration process after reclaimed water replenishment and cause environmental health risks, providing theoretical support for the environmental risk assessment and pollution control of ARGs. In the actual site application process, the one-dimensional model of the pollutant can be used to predict the migration concentration of ARGs according to actual parameters such as the actual site retention factor, diffusion coefficient, actual water flow velocity, medium density, etc., to assess the environmental risk.

[0151] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0152] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. An experimental method for studying the transmission and transfer of antibiotic resistance genes, characterized in that: The following steps are involved: (1) Experimental materials: ① Intracellular ARGs: Pseudomonas putida KT2400 is the donor bacteria, and Escherichia coli ATCC25922 is the recipient bacteria; ② Extracellular ARGs: The constructed sul1 plasmid was used as the target ARGs; (2) Preparation of porous media materials Gravel, quartz sand, undisturbed soil, and fine sand are used as porous media fillers; The porous media were sterilized by high-pressure steam to exclude the influence of DNA-degrading enzymes and biological inhibitors in the microbial community; Soil medium was inoculated with 5 mL OD 600 = 1.0 Escherichia coli ATCC25922 obtained by centrifugation at 10000*g for 1-3 min; (3) The soil column is made of plexiglass, with an inner diameter of 6 cm and a height of 30 cm; The bottom of the simulation column is filled with 2 cm gravel as a drag layer, and the top is filled with 2 cm fine sand as a water distribution layer to achieve uniform water distribution. The middle is a filler layer, and the filler layer uses the collected original soil with a filling thickness of 16 cm. A water outlet is set at the bottom of the soil column to collect the total water after passing through the soil column, which is used to measure the changes in ARGs in the total water outlet. When filling the soil column, tamp every 3-5cm of soil added; (4) Operation of soil column simulation system The soil column simulation system is mainly divided into three stages: In the first stage, bromide ions were used as tracer elements to draw Br penetration curves. 40 mg / L NaBr solution was continuously injected into two soil columns for 5 days. Samples were taken at different times and the concentration of Br was measured using an anion chromatograph to determine the simulated column diffusion coefficient. The second stage is the cleaning stage, using soil background solution for continuous infiltration for 7 days. The soil background solution is 0.01 mol / LC a Cl2 and 200mg / LNaN3 mixed solution. The main purpose of this stage is to wash out the original soluble impurities in the soil column, inhibit the growth of bacteria, balance the soil ion concentration level and obtain stable effluent water quality, prepare for subsequent tests and ensure the stability of the system effluent; The third stage is the ARGs propagation and transfer experimental stage; Four soil column experiments were carried out at the same time, and the absolute abundance of the target gene in the influent was designed to be 2×10 5 copies / μL, 2.9×10 7 cells / mL bacterial solution. During the infiltration period, the water head of the soil column was kept at 3 cm. Samples were taken from the water outlet of the soil column on the 6th, 8th, 10th, 15th and 20th day after infiltration to determine the absolute abundance of the target gene or the number of bacteria in the water outlet. (5) Changes and determination of ARGs in effluent By comparing the differences in the number of target genes or bacteria in the soil column effluent, the migration process of antibiotic resistance genes in the soil column simulation system was studied; Confocal microscopy was used to analyze the gene transfer of intracellular ARGs; qPCR was used to detect the migration process of extracellular ARGs; ① Measure the number of bacteria in the cell using confocal microscopy and flow cytometry The fluorescence expression of the effluent samples of soil column 1 was detected by Olympus laser confocal microscope FV3000. The recipient bacteria ATCC25932 was used as a negative control without fluorescence, and the donor bacteria KT2440 was used as a red fluorescence positive control. The fluorescence excitation spectrum was selected to be 488nm and 561nm respectively. 100μl of the effluent samples after pretreatment at different times were taken and placed in a confocal dish, and the same zoom value was selected to detect the fluorescence expression. The stained bacteria were analyzed using a Bio-Rad ZE5 flow cytometer, and three lasers, 488nm and 561nm, were selected for excitation. Before the flow analysis, the test samples were washed three times by centrifugation with phosphate buffered saline (PBS), and finally resuspended with PBS. The 561nm and 488nm lasers were selected, and positive and negative controls were set up respectively. The number of green fluorescent and non-fluorescent bacteria in the samples was analyzed, and the gene horizontal transfer rate T / R was calculated accordingly. The number of green bacteria in the water from soil column 1 is N T1 Where, f: bonding transfer rate; N T : The number of green fluorescent conjugated cells measured by flow cytometry, unit: N o : Total number of sorted cells, in units; N D : The number of red fluorescent donor bacterial cells measured by flow cytometry, unit: ②Only measure the copy number After pretreatment, the absolute content of ARGs in the sample was measured using a fluorescent quantitative PCR instrument to calculate the absolute copy number q1 of the target gene in the sample, and the long-term existence of the plasmid in static water was used as the control q0, and q was the adsorption of the column medium to the plasmid q = q0-q1; The changes in the number of bacteria caused by adsorption during the washing of the column with bacterial liquid were studied. The biodegradation and other complex processes were not considered. The long-term culture of bacteria in static water was used as a control. Finally, the content of KTT240 in the water discharged from the column was determined by flow cytometry. The number of control bacteria in still water is c0, the number of bacteria in the column outlet water is c1, c is the adsorption amount of bacteria by the column medium c = c0-c1; (6) Experimental results and calculations ①Simulation of joint transfer of soil column 1 The abundance of KT2440 was detected in soil column 1 on the 6th day, and the abundance of KT2440 and zygotes in the effluent gradually increased with the increase of soil column operation time, reaching the maximum value of 4.09×10 6 cells / μL, an increase of 28% compared with the initial bacterial count, and the abundance value decreased to 3.11×10 cells / μL after 20 days. 6 cells / μL, and the abundance of KT2440 in the effluent of the soil column at 20 days reached 2.90×10 6 cells / μL, which was basically the same as the abundance of KT2440 in the control, indicating that KT2440 was adsorbed in the soil column medium during its migration, and the exogenous bacteria were constantly multiplying while adsorbing to the medium; ②Simulation of the migration of extracellular ARGs in soil column 2 A small amount of plasmid concentration was detected in the effluent of extracellular ARGs in soil column 2 on the second day. The absolute abundance of sul1 in the effluent gradually increased with time and reached 1.13×10 4 The absolute abundance of sul1 in the control group decreased with time, and after 20 days, it dropped to 1.59×10 4 copies / μL, indicating that in the absence of any medium under natural conditions, the plasmid solution has a self-attenuation process, and when the plasmid is injected into the soil column medium, the attenuation process is alleviated due to the adsorption protection of the soil particles in the medium; ③ Migration pattern of ARGs colloids in porous media In order to compare the migration and transformation of ARGs of different forms in the experimental column, the physicochemical changes and biological reproduction of pollutants in the process were not considered. The convection-diffusion equilibrium model of CXTFIT software was used to simulate the temporal changes of KT2440 and sul1 in the effluent of the soil column. The one-dimensional model of the pollutants was described as follows: In the formula, R d : retention factor; D: diffusion coefficient, unit: m 2 / d; u: actual flow velocity of water, unit: m / d; ρ b : Medium density, unit: g / cm 3 ; η e : porosity; K d : Linear adsorption constant, cm 3 / g; When the solution condition is set to t = 0, C(x, t) = 0; when x = 0, C(x, t) = C0; when x tends to infinity, C(x, t) = 0, the analytical solution is as follows: Where D L : Hydrodynamic dispersion coefficient, unit: m 2 / d; u: actual flow velocity of water, unit: m / d; C: pollutant concentration, copies / μL or cells / μL; In order to compare the migration and transformation of ARGs with different forms in the experimental column, the convection-diffusion equilibrium model of CXTFIT software was used to simulate the temporal changes of KT2440 and sul1 in the effluent water of the soil column without considering the physicochemical changes and biological reproduction of the pollutants in the process, and the fitting results were good.

2. The experimental method for studying the transmission and transfer of antibiotic resistance genes according to claim 1, characterized in that: Pseudomonas putida KT2400: carries a broad-host plasmid RP4 marked with green fluorescent protein, and the chromosome of the bacterium is marked with red fluorescent protein and lacIq gene. It can express red fluorescence but inhibit the expression of plasmid green fluorescent protein. If conjugation transfer occurs, plasmid RP4 will be transferred to the recipient bacteria, making the recipient bacteria emit green fluorescence. The antibiotic-sensitive Escherichia coli ATCC25922 is used as the plasmid receptor.

3. The experimental method for studying the spread and transfer of antibiotic resistance genes according to claim 1, characterized in that: In order to remove the influence of impurities on the surface of gravel, quartz sand and fine sand media on the experimental results, the surface of the media is cleaned. The treatment method is as follows: 1) Soak in 0.1 mol nitric acid for 3 hours; 2) Rinse with deionized water until the conductivity of the rinse water is low; 3) Soak in 0.1 mol NaOH solution for 3 h; 4) Then rinse with deionized water until the ionic strength IS of the rinse solution is less than 0.01 mM; 5) Place the cleaned porous medium in an autoclave and sterilize at 121°C for 20 minutes; 6) Finally, dry at 105°C and seal in a beaker for later use.

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