Detection method for existence form and absolute content of antibiotic resistance gene and application thereof

Abstract

The invention provides a method for detecting the existence form and absolute content of an antibiotic resistance gene and application of the method. The method comprises the following steps: pretreating a to-be-detected sample to obtain a bacterial suspension; carrying out dyeing identification treatment on thalli in the bacterial suspension, and determining the ratio of viable bacteria to dead bacteria in the bacterial suspension; carrying out filtering treatment on the bacterial suspension by adopting a preset filtering membrane, and respectively extracting nucleic acid in the filtering membrane and filtrate; based on the nucleic acid, respectively determining the copy number of ARGs in the filter membrane and the filtrate; based on the ratio of the viable bacteria to the dead bacteria and the copy number of the ARGs in the filter membrane and the filtrate, the copy number of the viable bacteria, the dead bacteria and the free ARGs in the to-be-detected sample is determined. According to the method, the existence forms of the ARGs in the to-be-detected sample and the absolute content of the ARGs in different existence forms can be effectively detected, underestimation of the ARGs in the sample is avoided, and the defect that in the prior art, which forms of the ARGs in a water body enter a porous medium to participate in physical processes of adsorption, convection-dispersion and the like cannot be accurately detected is overcome; and a technical support is provided for managing and controlling the risk evaluation of the reclaimed water ecological water supplementing aquifer.

Description

Technical Field

[0001] The present application relates to the technical field of migration mechanism of new pollutants in the environment. Specifically, the present application relates to a method for detecting the existence form and absolute content of antibiotic resistance genes and its application. Background Art

[0002] Antibiotic resistance ranks first among the six major emerging environmental problems in the world. Excessive use of antibiotics for human or animal use means that antibiotics cannot be completely degraded in the body, and the antibiotics themselves or their byproducts are discharged into the environment through feces or urine, causing resistance pressure on the environment and stimulating the spread of resistance in soil and groundwater. Researchers have conducted extensive research on the distribution characteristics and propagation patterns of antibiotic resistance genes (ARGs) in typical pollution sources and various receiving environments.

[0003] Whether it is the pollution source or the receiving environment, the spread of ARGs involves the transformation of intracellular forms (iARGs) and extracellular forms (eARGs) as well as changes in quantity. Although the detection amount of eARGs is 2-3 orders of magnitude smaller than that of iARGs, it is easier for them to penetrate the strata and transfer to environmental bacteria through horizontal gene transfer (HGT), thereby driving the spread of ARGs in the environment. At present, there are related technologies that can quantify iARGs and eARGs in water bodies respectively, but the migration patterns of iARGs and eARGs in water bodies are different during the infiltration and recharge of aquifers. eARGs can be regarded as solutes, and iARGs exist in the form of biological colloids, following different physical laws. The content of iARGs and eARGs in water bodies will change with the growth, division, decay and rupture of cells. It is impossible to judge the content of ARGs in water bodies and the form in which they enter porous media to participate in physical processes such as adsorption and convection-diffusion.

[0004] Therefore, the existing detection methods of ARGs in water bodies still need to be improved. By improving the detection methods of ARGs, it can assist in the risk assessment of antibiotic resistance in reclaimed water ecological recharge aquifers. Summary of the invention

[0005] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, one purpose of the present application is to propose a method that can accurately detect the presence form and absolute content of ARGs in water.

[0006] Specifically, the technical solution of this application is as follows:

[0007] In the first aspect, the present application proposes a method for detecting the presence form and absolute content of antibiotic resistance gene ARGs. According to an embodiment of the present application, the method includes: pre-treating the sample to be tested to obtain a bacterial suspension; staining and identifying the bacteria in the bacterial suspension to determine the ratio of live bacteria to dead bacteria in the bacterial suspension; filtering the bacterial suspension using a predetermined filter membrane to extract nucleic acids from the filter membrane and the filtrate respectively; based on the nucleic acid, determining the number of ARGs copies in the filter membrane and the filtrate respectively; based on the ratio of live bacteria to dead bacteria and the number of ARGs copies in the filter membrane and the filtrate, determining the number of live bacteria, dead bacteria and free ARGs copies in the sample to be tested.

[0008] In some examples of the present application, the aforementioned method can effectively detect the existence form of ARGs in the sample to be tested and the absolute content in different existence forms, avoiding the underestimation of ARGs in the sample, and making up for the deficiency of the existing technology that cannot accurately detect the content of ARGs in water bodies and the form in which they enter the porous medium to participate in physical processes such as adsorption, convection-diffusion, etc.

[0009] In some examples of the present application, the aforementioned method for detecting the presence form and absolute content of antibiotic resistance genes may also include at least one of the following additional technical features:

[0010] In some examples of the present application, the aforementioned staining identification process is performed by a fluorescent stain, wherein the fluorescent stain is selected from SYTO9 dye and PI dye.

[0011] The aforementioned SYTO9 dye is a green fluorescent dye, which is commonly used to dye bacterial nucleic acids. It can penetrate the cell membrane, enter the cell and bind to the DNA and RNA in the cell, thereby emitting green fluorescence. In some examples of the present application, SYTO9 is used to dye live bacteria and dead bacteria in a bacterial suspension.

[0012] The aforementioned PI dye is a red fluorescent dye commonly used for nucleic acid staining. Unlike SYTO9, PI cannot penetrate intact cell membranes, so it will only stain cells whose cell membranes are damaged, such as apoptotic or dead cells. In some examples of the present application, PI dye is used to stain dead bacteria in bacterial suspensions.

[0013] The steps of staining and identifying the bacterial suspension using SYTO9 dye and PI dye include: 1) mixing SYTO9 dye, PI dye and the aforementioned bacterial suspension; 2) incubating the mixed product in the dark for 13 minutes to 18 minutes, and performing fluorescence detection; 3) determining the ratio of live bacteria to dead bacteria in the aforementioned bacterial suspension based on the fluorescence ratio.

[0014] In the mixed treatment system of step 1), the concentration of SYTO9 dye is selected from 5 μM-6 μM; the concentration of PI dye is selected from 30 μM-31 μM. The inventors optimized the dye concentration to ensure the best staining effect and the accuracy of subsequent flow cytometry experiments.

[0015] The mixed product in step 2) is preferably incubated in the dark for 15 minutes to ensure adequate staining.

[0016] In some examples of the present application, the predetermined filter membrane used in the present method is selected from a 0.22 μm nitrocellulose filter membrane. The filter membrane can effectively separate intracellular antibiotic resistance gene ARGs and free antibiotic resistance gene ARGs. The number of ARGs copies in the filter membrane includes live bacteria and dead bacteria ARGs copies, and the number of ARGs copies in the aforementioned filtrate includes free ARGs copies. Based on the total ARGs copies in the filtrate and the above-mentioned ratio of live bacteria to dead bacteria, the ARGs copies of live bacteria and dead bacteria can be determined.

[0017] In some examples of the present application, the aforementioned sample to be tested is selected from environmental water samples, wherein the environmental water sample includes at least one of a river, inlet and outlet water of a sewage treatment plant, livestock and poultry wastewater, and industrial wastewater.

[0018] In other examples of the present application, the aforementioned sample to be tested may also be selected from soil or feces samples, etc.

[0019] In some examples of the present application, the aforementioned antibiotic resistance gene is selected from sul1, sul2, tetW, tetO, ermB, mecA, aadA, vanA, vanB, strA, strB, vanC or mcr-1. In a specific example of the present application, the inventors use sul1 as an example to detect the copy number. The detection principle of this method is also applicable to the copy number detection of other types of resistance genes in different forms. Due to space limitations, it will not be repeated here.

[0020] In the second aspect, the present application proposes a method for simulating the attenuation of antibiotic resistance genes ARGs. According to an embodiment of the present application, the method comprises: based on the method described in the first aspect, determining the copy number of each existing form of antibiotic resistance genes ARGs in the sample to be tested at multiple times; based on the copy number of antibiotic resistance genes ARGs in different forms at different times, simulating the change of ARGs in the sample to be tested.

[0021] In some examples of the present application, the aforementioned method can be used for long-term monitoring of changes in the number of antibiotic resistance genes ARGs in the test samples, providing guidance for the risk assessment of drug resistance in rivers and lakes where ecological water is replenished by sewage treatment plant effluent.

[0022] In some examples of the present application, the aforementioned antibiotic resistance gene ARGs attenuation simulation method may also include at least one of the following additional technical features:

[0023] In some examples of the present application, the aforementioned simulations were performed using first-order reaction kinetics.

[0024] On the third aspect, the present application proposes the application of a method for detecting the presence form and absolute content of the aforementioned antibiotic resistance genes in the risk assessment of ecological water replenishment resistance.

[0025] In some examples of the present application, the existence forms of ARGs and the absolute contents in different existence forms are determined based on the aforementioned method, so that the resistance risk of ecological water replenishment can be effectively evaluated. For example, the ARGs in the water body have a short stable existence time and a large reaction rate constant in the attenuation process. During the ecological water replenishment process, the resistance risk to groundwater and soil is lower.

[0026] It should be noted that the features and advantages described in this article for the screening method are applicable to other aspects and will not be repeated here.

[0027] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0029] Figure 1 Schematic diagram of the results of the bacterial staining control group provided in the examples of the present application; wherein (a) is live bacteria stained with SYTO9 alone; (b) is dead bacteria stained with PI alone; (c) is mixed bacteria (live bacteria: dead bacteria = 1:1); (d) is a negative control;

[0030] Figure 2 A schematic diagram of the delineation of bacterial cells by flow cytometry detection provided in the embodiment of the present application;

[0031] Figure 3 Schematic diagram of the control group of bacteria detected by flow cytometry provided in the embodiment of the present application; wherein (a) is live bacteria stained with SYTO9 alone; (b) is dead bacteria stained with PI alone; (c) is mixed bacteria (live bacteria: dead bacteria = 1:1); (d) is a negative control;

[0032] Figure 4 A schematic diagram of the calculation result of the live bacteria ratio in water provided in the embodiment of the present application;

[0033] Figure 5This is a schematic diagram of the existence form and absolute content change results of sul1 in static water within 30 days provided in the examples of the present application. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0035] In the present application, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present application, "plurality" means at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0036] The technology of this application is described below through specific examples. If no specific technology or conditions are specified in the examples, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0037] Example 1: Detection of the copy number of the antibiotic resistance gene sul 1 in environmental water

[0038] This example takes the sulfonamide-resistant bacteria resistance gene sul1 as an example to detect its absolute content in different existence forms.

[0039] 1. Cultivation of resistant bacteria

[0040] 1.1 The strain was from sulfonamide-resistant bacteria screened and cultured from groundwater samples by the research group. The strain was identified as Pseudomonas stuzeri. It was stored in 30% glycerol tubes and preserved at -20°C.

[0041] 1.2 The strain was inoculated into LB liquid culture medium containing 1 mg / L sulfadimethazine (SMZ) for activation.

[0042] After 1.3, the bacterial suspension with OD600=1.18 of overnight culture was inoculated into 200mL LB culture medium containing 1mg / L SMZ at a ratio of 2% to obtain the bacterial suspension required for the experiment, which was used for the preparation of ARGs-rich water and the control group experiment of staining during the presence status detection.

[0043] 2. Preparation of ARGs-containing Environmental Water

[0044] 2.1 Filter 100 mL of original groundwater using a 0.22 μm nitrocellulose filter membrane;

[0045] 2.2 Add 5 mL OD 600 =1.0 The bacterial cells obtained by centrifugation at 10000*g for 1-3 min were used to prepare a static aqueous solution containing intracellular ARGs.

[0046] 3. Preparation of Bacterial Suspension

[0047] 3.1 Take 1 mL of the prepared ARGs-containing environmental water sample every week for 5 consecutive weeks, and centrifuge at 10,000*g for 1-3 minutes to obtain the bacteria in the environmental water;

[0048] 3.2 At the same time, before each sampling to test the copy number and existence status of ARGs, prepare a pure resistant bacterial suspension as a control sample according to the above resistant bacteria culture steps, sample 4 mL, and centrifuge at 10000*g for 1-3 minutes to obtain a bacterial suspension;

[0049] 3.3 Aspirate the supernatant and resuspend the pellet with 1 mL of 0.85% sodium chloride solution.

[0050] 4. Stain the bacteria in the bacterial suspension to determine the ratio of live and dead bacteria in the bacterial suspension

[0051] 4.1 For the two samples of SYTO9 single staining of live bacteria and PI single staining of dead bacteria in the control group, take 332 μL of live bacteria or dead bacteria and add 1 μL of 3.34 mM SYTO9 or 20 mM PI;

[0052] 4.2 For the mixed samples in the control group, add 165 μL of live bacteria and 165 μL of dead bacteria, resuspend the sample in a 2 mL centrifuge tube, and add 3 μL of a dye pre-mixed with 3.34 mM SYTO9 and 20 mM PI in a ratio of 1:1 to the 2 mL centrifuge tube;

[0053] 4.3 For the experimental group, directly take 332 μL into a 2 mL centrifuge tube and add 1 μL of a dye pre-mixed with 3.34 mM SYTO9 and 20 mM PI in a ratio of 1:1;

[0054] 4.4 Staining time conditions: incubate at room temperature in the dark for 15 min;

[0055] 4.5 Centrifuge the stained samples at 10000*g for 1-3 minutes, remove the supernatant and preserve the stained bacteria. Resuspend the bacteria in the control group and the experimental group in 333μL of normal saline;

[0056] 4.6 Determine the ratio of live and dead bacteria in the bacterial suspension by flow cytometry and verify it by confocal microscopy. The specific steps are as follows:

[0057] 1) Use an Andor spinning disk confocal microscope (Dragonfly) to observe the bacterial staining status. SYTO9 and PI use 520 / 40nm and 620 / 40nm emission and reception, respectively. The experimental results are as follows: Figure 1 As shown;

[0058] 2) Use Bio-Rad ZE5 flow cytometer to quantify the number of dead and live bacteria in the solution and calculate the ratio between the two;

[0059] 3) SYTO9 and PI were excited with 488 and 561 nm excitation light respectively. SYTO9 was hardly excited at 561 nm, which could reduce the interference of SYTO9. Therefore, there was no need to adjust the compensation for the two single-color controls in the experiment. The selected bacteria were small, so a 405 nm laser was selected to identify and delineate bacterial cells;

[0060] 4) Load the unstained negative control and create a scatter plot with FSC405_10-A-log as the abscissa and SSC488_10-A-log as the ordinate ( Figure 2 ), adjust the voltage of the two channels so that the bacterial signal is in the center of the scatter plot, distinguish the bacterial cells from the background and perform gating. Figure 2 The parameter settings of each channel during the experiment are shown in Table 1.

[0061] 5) The data were analyzed by gate demarcation using FlowJo_V10 software, and the ratio of live and dead bacteria was calculated. Figure 3 This is a schematic diagram of the gate for the control group in the experiment. A scatter plot with SYTO9 as the horizontal axis and PI as the vertical axis was established, and the area was divided into 4 quadrants according to the negative control and double-stained (L:D=1:1) samples. Among them, the Q1 area is PI positive, representing the number of dead cells; the Q2 area is the PI and SYTO9 double positive area. Since PI only stains dead cells, there is competitive adsorption with SYTO9, so the stained cells are still dead cells with ruptured cell membranes; the Q3 area is SYTO9 positive, representing the number of living cells; the Q4 area is negative, representing unstained cells.

[0062] Table 1 Basic parameter settings for flow cytometry analysis

[0063]

[0064] 6) Before starting the sample test, prepare a gradient solution with a known percentage of live bacteria and a control group, and calculate the ratio of the number of events in the Q3 zone to the sum of the number of events in Q1 and Q2. A linear fit is performed with the known proportion of live bacteria P in the actual control group sample ( Figure 4 ), we get formula (1), R 2 =0.97.

[0065]

[0066] 5. Filter the bacterial suspension and extract the nucleic acid in the filter membrane and the filtrate respectively;

[0067] 5.1 Use a kit (DNA easy Power Soil Pro Kit, Qiagen) to rupture the microorganisms on the filter membrane and extract intracellular ARGs. The steps can be referred to the kit instructions;

[0068] 5.2 Use magnetic beads to extract the extracellular DNA in the enriched filtrate, the steps are as follows:

[0069] a. Add 400 μL isopropanol and 300 μL Cl buffer (proteinase K dissolved in 30 mM Tris-Cl at 20 g / L, Tiangen Biotech, China) to 200 μL sample filtrate and mix thoroughly to remove impurities (such as proteins).

[0070] b. Then add 6.5 μL magnetic bead stock solution (30 g / L) (1 μm nucleic acid extraction silicon hydroxy magnetic beads, Zhongke Leiming (Beijing)), vortex for 3-4 minutes. The mixed solution is adsorbed with a magnet to absorb the nucleic acid carried by the magnetic beads, and the supernatant without magnetic beads is discarded.

[0071] c. The magnetic beads were washed with 0.5 mL CW1 buffer (7 mol / L guanidine hydrochloride dissolved in 50% isopropanol) and the supernatant was removed by magnet adsorption again.

[0072] d. Wash the beads twice with 0.5 mL CW2 buffer (75% ethanol) and then place at room temperature for 5 min to evaporate the remaining ethanol.

[0073] e. Add 30 μL of elution buffer (10 mM Tris-HCl, pH 8.5, preheated at 55°C) to the magnetic beads and incubate for 5 min. During the incubation period, vortex for 20 s per minute.

[0074] Finally, the mixture was placed on a magnetic rack and the supernatant was collected for subsequent DNA analysis.

[0075] Based on the nucleic acid, determining the copy number of ARGs in the filter membrane and the filtrate, respectively;

[0076] 1) After pretreatment, the absolute content of ARGs in the samples was determined using a fluorescence quantitative PCR instrument.

[0077] 2) Calculate the absolute copy number of the target gene in the sample according to formula (2).

[0078] Absolute copy number (copies / mL) = [machine read value (copies / μL) × total sample DNA volume (μL)] / total volume of extracted sample (mL), formula (2).

[0079] Based on the above steps, it can be calculated that in 50 mL of environmental water, the copy number of the sul1 gene in active bacteria is 3.75; the copy number of the sul1 gene in dead bacteria is 6.9; and the copy number of the free sul1 gene is 3.85.

[0080] Example 2: Simulation of the decay curve of the antibiotic resistance gene sul 1 in environmental water

[0081] This example is based on the method of Example 1. The absolute content changes of the copies of intracellular sul1 of live bacteria, intracellular sul1 of dead bacteria, and extracellular sul1 in static water were continuously collected for 30 days (1d, 8d, 19d, 25d, 28d). Figure 5 As shown. In still water, the extracellular sul1 content increased within three weeks with the lysis of dead bacteria, reaching a peak at 21 days. At the same time, the number of live bacteria in still water first increased and then decreased, resulting in an increase in intracellular sul1 in live bacteria. As the number of intracellular ARGs carried by dead bacteria continued to decrease, it decayed exponentially rapidly at 20 to 25 days. An exponential function was used to simulate the change of the absolute content of total sul1 in still water without adding antibiotics at room temperature, and first-order reaction kinetics was used for simulation (Formula 3). The results show that it conforms to first-order reaction kinetics.

[0082] C=C0*e -0.1t , formula (3);

[0083] Where C is the total absolute content of sul1 in still water (copies / mL), C0 is the initial absolute content of sul1 in still water (copies / mL), R 2 It is 0.59.

[0084] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0085] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A method for detecting the presence and absolute content of antibiotic resistance gene ARGs, characterized in that: include: Pre-treat the sample to be tested to obtain a bacterial suspension; Performing staining and identification treatment on the bacteria in the bacterial suspension to determine the ratio of live bacteria to dead bacteria in the bacterial suspension; The bacterial suspension is filtered using a predetermined filter membrane to extract nucleic acids from the filter membrane and the filtrate respectively; Based on the nucleic acid, determining the copy number of ARGs in the filter membrane and the filtrate, respectively; Based on the ratio of live bacteria to dead bacteria and the number of ARGs copies in the filter membrane and the filtrate, the number of live bacteria, dead bacteria and free ARGs copies in the sample to be tested is determined.

2. The method according to claim 1, characterized in that The staining identification process is performed by a fluorescent stain, and the fluorescent stain is selected from SYTO9 dye and PI dye.

3. The method according to claim 2, characterized in that The bacteria in the bacterial suspension are subjected to dyeing and identification treatment to determine the ratio of live bacteria to dead bacteria in the bacterial suspension, including: 1) mixing SYTO9 dye, PI dye and the bacterial suspension; 2) Incubate the mixed product in the dark for 13-18 minutes and perform fluorescence detection; 3) determining the ratio of live bacteria to dead bacteria in the bacterial suspension based on the fluorescence ratio; Optionally, in the mixed treatment system, the SYTO9 dye concentration is selected from 5 μM-6 μM; the PI dye concentration is selected from 30 μM-31 μM.

4. The method according to claim 1, characterized in that The predetermined filter membrane is selected from a 0.22 μm nitrocellulose filter membrane.

5. The method according to claim 4, characterized in that The ARGs copy number in the filter membrane includes live bacteria and dead bacteria ARGs copies, and the ARGs copy number in the filtrate includes free ARGs copies.

6. The method according to any one of claims 1 to 5, characterized in that: The sample to be tested is selected from environmental water samples; Optionally, the environmental water sample includes at least one of a river, inlet and outlet water of a sewage treatment plant, livestock and poultry wastewater, and industrial wastewater.

7. The method according to any one of claims 1 to 5, characterized in that: The antibiotic resistance gene is selected from sul1, sul2, tetW, tetO, ermB, mecA, aadA, vanA, vanB, strA, strB, vanC or mcr-1.

8. A method for simulating the attenuation of antibiotic resistance genes ARGs, characterized in that: include: Based on the method described in any one of claims 1 to 7, determining the copy number of each existing form of ARGs in the sample to be tested at multiple times; Based on the ARGs copy numbers at different times and in different forms, the changes of ARGs in the samples to be tested are simulated.

9. The method according to claim 8, characterized in that The simulation was performed based on first-order reaction kinetics.

10. Application of the method according to any one of claims 1 to 6 in risk assessment of ecological water replenishment drug resistance.

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