CN119351582A

CN119351582A - Detection method of the existence form and absolute content of antibiotic resistance genes and its application

Info

Publication number: CN119351582A
Application number: CN202411193136.9A
Authority: CN
Inventors: 张卓然; 刘翔; 张宁; 李涵
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2024-08-28
Filing date: 2024-08-28
Publication date: 2025-01-24

Abstract

The present application provides a method for detecting the existence form and absolute content of antibiotic resistance genes and its application. The aforementioned 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 and dead bacteria in the bacterial suspension; filtering the bacterial suspension using a predetermined filter membrane to extract nucleic acids in 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 and 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. 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, avoids underestimation of ARGs in the sample, makes up for the deficiency of the existing technology in being unable to accurately detect the form in which ARGs in the water body enter the porous medium to participate in physical processes such as adsorption, convection-diffusion, and provides technical support for the risk assessment of ecological water replenishment aquifers for the management and control of recycled water.

Description

Method for detecting existence form and absolute content of antibiotic resistance gene and application thereof

Technical Field

The application relates to the technical field of migration mechanisms of novel pollutants in the environment, in particular to a detection method for existence form and absolute content of antibiotic resistance genes and application thereof.

Background

Antibiotic resistance problems are the first among six new environmental problems worldwide. The excessive use of antibiotics for human or veterinary use makes the antibiotics not completely degraded in vivo, and the body or byproducts thereof are discharged into the environment through feces or urine, which causes drug resistance pressure to the environment and stimulates the spread of drug resistance in soil and groundwater. Researchers have conducted extensive research on the distribution characteristics and propagation laws of antibiotic resistance genes (antibiotics RESISTANCE GENES, ARGs) in typical pollution sources and various receiving environments.

The transmission of ARGs, whether the source of the contamination or the receiving environment, is accompanied by a transition in intracellular morphology (iARGs) and extracellular morphology (eARGs) and a change in quantity. Although eARGs is 2-3 orders of magnitude less detected than iARGs, it penetrates more easily into the formation and drives the propagation of ARGs in the environment by Horizontal Gene Transfer (HGT) into environmental bacteria. Currently, related techniques can quantify iARGs and eARGs in a body of water, respectively, but the migration rules of iARGs and eARGs in a body of water during infiltration replenishment of aquifers are different. eARGs can be regarded as a solute, and iARGs exists in the form of a biological colloid and follows different physical laws. The content of iARGs and eARGs in the water body can change along with the growth, division, decay and rupture of cells, and the content of ARGs in the water body and the form of the content in the water body enter a porous medium to participate in physical processes such as adsorption, convection and dispersion cannot be distinguished.

Therefore, the existing detection method of ARGs in the water body still needs to be improved. By improving the detection method ARGs, the risk evaluation of the antibiotic resistance in the reclaimed water ecological moisturizing aquifer can be assisted.

Disclosure of Invention

The present application aims to solve at least one of the technical problems existing in the prior art. Therefore, an object of the present application is to provide a method for accurately detecting the existence of the morphology and the absolute content of ARGs in the water.

Specifically, the technical scheme of the application is as follows:

In a first aspect, the present application provides a method for detecting the presence of the antibiotic resistance gene ARGs in a morphology and absolute content. According to the embodiment of the application, the method comprises the steps of preprocessing a sample to be detected to obtain bacterial suspension, carrying out dyeing identification treatment on thalli in the bacterial suspension to determine the proportion of live bacteria and dead bacteria in the bacterial suspension, carrying out filtering treatment on the bacterial suspension by adopting a preset filtering membrane to respectively extract nucleic acids in the filtering membrane and filtrate, respectively determining ARGs copy numbers in the filtering membrane and filtrate based on the nucleic acids, and determining the copy numbers of live bacteria, dead bacteria and free ARGs copy numbers in the sample to be detected based on the proportion of live bacteria and dead bacteria and ARGs copy numbers in the filtering membrane and filtrate.

In some examples of the application, the method can effectively detect the existence form of ARGs in the sample to be detected and the absolute content under different existence forms, avoid underestimation of ARGs in the sample, and make up for the defect that the prior art can not accurately detect the content of ARGs in the water body and the form of the content enter a porous medium to participate in physical processes such as adsorption, convection-dispersion and the like.

In some examples of the present application, the method for detecting the presence and absolute content of the antibiotic resistance gene may further include at least one of the following additional technical features:

In some examples of the application, the foregoing dye identification process is performed by a fluorescent dye. Wherein the fluorescent dye is selected from SYTO9 dye and PI dye.

The aforementioned SYTO9 dye is a green fluorescent dye, commonly used for staining bacterial nucleic acids. It can penetrate cell membrane and combine with DNA and RNA in cell after entering cell, so that green fluorescence is emitted. In some examples of the application, SYTO9 is used to stain live and dead bacteria in bacterial suspensions.

The PI dye is a red fluorescent dye, and is commonly used for nucleic acid staining. Unlike SYTO9, PI cannot penetrate the intact cell membrane, so it stains only those cells whose cell membrane has been damaged, such as apoptotic or dead cells. In some examples of the application, the dead bacteria in the bacterial suspension are stained with PI dye.

The method for performing dyeing identification treatment on the bacterial suspension by using SYTO9 dye and PI dye comprises the steps of 1) mixing the SYTO9 dye, the PI dye and the bacterial suspension, 2) incubating the mixed treatment product for 13-18 min in a dark place for fluorescence detection, and 3) determining the ratio of live bacteria to dead bacteria in the bacterial suspension based on the fluorescence ratio.

In the mixed treatment system of the above step 1), the SYTO9 dye concentration is selected from 5. Mu.M to 6. Mu.M, and the PI dye concentration is selected from 30. Mu.M to 31. Mu.M. The inventor optimizes the dye concentration to ensure the best dyeing effect and the accuracy of subsequent flow type experiments.

The light-shielding incubation time of the mixed treatment product in the step 2) is preferably 15min. For ensuring adequate dyeing.

In some examples of the application, the predetermined filtration membrane employed in the present method is selected from 0.22 μm nitrocellulose filtration membranes. The filter membrane is capable of effectively separating intracellular antibiotic resistance gene ARGs from episomal antibiotic resistance gene ARGs. Wherein ARGs copies of the filter membrane include live and dead bacterial ARGs copies, and ARGs copies of the filtrate include free AR Gs copies. Based on the total ARGs copies in the filtrate and the proportional relationship between the live bacteria and the dead bacteria, the ARGs copies of the live bacteria and the dead bacteria can be determined.

In some examples of the application, the sample to be tested is selected from environmental water samples. Wherein the environmental water sample comprises at least one of river, sewage treatment plant inlet and outlet water, livestock wastewater and industrial wastewater.

In other examples of the present application, the sample to be tested may be selected from soil or fecal samples.

In some examples of the application, the aforementioned antibiotic resistance gene is selected from sul1, sul2, tetW, tetO, ermB, m ecA, aadA, vanA, vanB, strA, strB, vanC, or mcr-1. In a specific example of the present application, the inventor takes sul1 as an example to perform copy number detection, and the detection principle of the method is equally applicable to copy number detection of different existing forms of other types of resistance genes, and is limited for reasons of space and will not be described herein.

In a second aspect, the present application provides a method for simulating attenuation of an antibiotic resistance gene ARGs. According to an embodiment of the application, the method comprises the steps of determining copy numbers of each existing form of the antibiotic resistance gene ARGs of a sample to be tested at a plurality of times based on the method in the first aspect, and simulating ARGs changes in the sample to be tested based on ARGs copy numbers of the antibiotic resistance genes in different forms at different times.

In some examples of the application, the method can be used for long-term monitoring of the number change of the antibiotic resistance genes ARGs in the sample to be tested, and provides guidance for drug resistance risk evaluation of the effluent ecological water-replenishing river and lake of the sewage treatment plant.

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

In some examples of the application, the foregoing simulation is performed using first order reaction kinetics.

In a third aspect, the application provides an application of the detection method of the existence form and absolute content of the antibiotic resistance gene in ecological water-replenishing drug resistance risk evaluation.

In some examples of the application, the existence form of ARGs and the absolute content under different existence forms are determined based on the method, so that the drug resistance risk of ecological water supplement can be effectively evaluated, for example, the stable existence time of ARGs in a water body is short, the reaction rate constant of an attenuation process is large, and the drug resistance risk to underground water and soil is smaller in the ecological water supplement process.

It should be noted that the features and advantages described herein for the screening method are applicable to other aspects, and are not described herein.

Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application.

Drawings

The foregoing and/or additional aspects and advantages of the application will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1 is a schematic diagram of the results of a bacterial staining control group provided by the embodiment of the application, wherein (a) is SYTO9 single-stained live bacteria, (b) is PI single-stained dead bacteria, (c) is mixed-stained bacteria (live bacteria: dead bacteria=1:1), and (d) is negative control;

FIG. 2 is a schematic view of the flow assay for detecting bacterial cell definition provided by the embodiments of the present application;

FIG. 3 is a schematic diagram of a control group of bacteria detected by flow analysis, wherein (a) is SYTO9 single-contaminated live bacteria, (b) is PI single-contaminated dead bacteria, (c) is mixed-contaminated bacteria (live bacteria: dead bacteria=1:1), and (d) is a negative control;

FIG. 4 is a schematic diagram of a scale calculation result of viable bacteria in a water body according to an embodiment of the present application;

FIG. 5 is a schematic diagram showing the existence form and the absolute content change result of sul1 in still water within 30 days according to the embodiment of the present application.

Detailed Description

The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. Further, it is understood that various changes and modifications may be made by those skilled in the art, and that such equivalents fall within the scope of the application as defined by the appended claims.

In the present disclosure, the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present application, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise.

The technology of the present application will be illustrated by the following specific examples. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1 detection of copy number of antibiotic resistance Gene sul 1 in environmental Water

In this example, the absolute content of the sul1 gene was measured in different forms.

1. Cultivation of resistant bacteria

1.1 Strains from the subject group cultured sulfonamide resistant bacteria were screened from underground water samples, identified as pseudomonas (Stutzerimonas stuzer.) by strain identification, stored at-20 ℃ using 30% glycerol stock.

1.2 The strains were inoculated into LB liquid medium containing 1mg/L Sulfadimidine (SMZ) for activation.

After 1.3, the bacterial suspension cultured overnight with OD 600=1.18 was inoculated at a ratio of 2% into 200mL of LB broth containing 1mg/L SMZ to obtain bacterial suspension required for experiments, which was used for control experiments of water preparation rich in ARGs and staining at presence detection.

2. Preparation of water containing ARGs environmental bodies

2.1 Filtration of 100mL of undisturbed groundwater with a 0.22 μm nitrocellulose filter;

2.2 to 5mL of cells obtained by centrifugation at 10000 Xg for 1-3min, OD ₆₀₀ =1.0 was added to prepare a still aqueous solution containing intracellular ARGs.

3. Preparation of bacterial suspension

3.1, Sampling 1mL of the prepared water body containing ARGs environments every week, continuously sampling for 5 weeks, and centrifuging at 10000 x g for 1-3min to obtain thalli in the water body;

3.2, preparing pure resistance bacterial suspension as a control group sample according to the above-mentioned steps of the culture of the resistance bacteria before each sampling preparation test ARGs copies and existence state, sampling 4mL, centrifuging 10000 Xg for 1-3min to obtain bacterial suspension;

3.3 the supernatant was aspirated off and the pellet was resuspended with 1mL of 0.85% sodium chloride solution.

4. Staining thalli in the bacterial suspension, and determining the proportion of live bacteria to dead bacteria in the bacterial suspension

4.1 For two samples of SYTO9 single-stained live bacteria and PI single-stained dead bacteria in the control group, 332. Mu.L each of either live bacteria or dead bacteria was taken, 1. Mu.L of 3.34mM SYTO9 or 20mM PI was added;

4.2 for the mixed samples in the control, 165. Mu.L of live bacteria and 165. Mu.L of dead bacteria were added, the samples resuspended in 2mL centrifuge tubes, and 3. Mu.L of dye pre-1:1 mixed with 3.34mM SYTO9 and 20mM PI were added to 2mL centrifuge tubes;

4.3 for the experimental group, 332. Mu.L of dye pre-1:1 mixed with 3.34mM SYTO9 and 20mM PI was directly taken in a 2mL centrifuge tube and added;

4.4, the dyeing time is 15min under the condition of light-shielding at room temperature;

4.5 samples stained were centrifuged at 10000 Xg for 1-3min and the supernatant was removed to preserve the stained bacteria. Bacteria of the control group and the experimental group were resuspended in 333. Mu.L of physiological saline;

4.6 determining the ratio of live bacteria to dead bacteria in the bacterial suspension by a flow analyzer, and adopting confocal microscopy for verification, wherein the method comprises the following specific steps:

1) Bacterial staining state observation is carried out by using an Andor turntable confocal microscope (Dragonfly), the SYTO9 and PI respectively adopt 520/40nm and 620/40nm emission light reception, and the experimental result is shown in figure 1;

2) Quantitatively measuring the quantity of dead bacteria and living bacteria in the solution by using a Berle ZE5 flow cytometer, and calculating the proportion of the dead bacteria and the living bacteria;

3) SYTO9 and PI are excited by excitation light of 488 and 561nm, respectively, and SYT O9 is hardly excited at 561nm, so that the interference of SYTO9 can be reduced. Therefore, two monochromatic control tones are not compensated for in the experiment. The selected thalli are smaller, and a 405nm laser is additionally selected for identifying the circling thalli cells;

4) A negative control which was not stained was loaded, a scattergram (fig. 2) was created with fsc405_10-a-log as the abscissa and SSC488_10-a-log as the ordinate, and the voltage of the two channels was adjusted so that the cell signals were in the center of the scattergram, and the cell was discriminated from the background and circled as shown in fig. 2. The parameters of each channel during the experiment are set forth in table 1.

5) Data the gate-demarcation analysis was performed using flowjo_v10 software to calculate the ratio of live and dead bacteria, as shown in fig. 3, which is a gate schematic diagram of the control group in the experiment. A scatter plot was created with SYTO9 on the abscissa and PI on the ordinate, and the area was delimited to 4 quadrants according to negative control and double-stained (L: d=1:1) samples. The Q1 area is positive to PI and represents the number of dead cells, the Q2 area is double positive to PI and SYTO9, the PI only dyes dead cells, and the dyed cells are dead cells with broken cell membranes due to competitive adsorption with SYTO9, the Q3 area is positive to SYTO9 and represents the number of living cells, and the Q4 area is negative and represents undyed cells.

TABLE 1 flow cytometric basic parameter settings

6) Before beginning the sample measurement, preparing a solution with known live bacteria ratio and a control group, and comparing the number of Q3 zone events with the sum of the numbers of Q1 and Q2 eventsLinear fitting was performed with the known viable bacteria ratio P in the actual control group samples (fig. 4), yielding formula (1), R ² =0.97.

5. Filtering the bacterial suspension, and respectively extracting nucleic acid from the filter membrane and the filtrate;

5.1 disrupting the microorganisms on the filter membrane using a kit (DNA easy Power Soil Pro Kit, qiagen) to extract intracellular ARGs, steps referred to the kit instructions;

5.2, extracting and enriching extracellular DNA in filtrate by using magnetic beads, wherein the steps are as follows:

a. To 200. Mu.L of the sample filtrate, 400. Mu.L of isopropanol and 300. Mu.L of Cl buffer (proteinase K was dissolved in 30mM Tris-Cl, tiangen Biotech, china) were added and mixed well to remove impurities (e.g., proteins).

B. Then, 6.5. Mu.L of a magnetic bead stock solution (30 g/L) (1 μm nucleic acid extraction of silica hydroxyl magnetic beads, zhongkelei (Beijing)), vortex for 3-4min, mix solution adsorbs nucleic acid carried by the magnetic beads with a magnet, and discard supernatant without magnetic beads.

C. the beads were washed with 0.5mL CW1 buffer (7 mol/L guanidine hydrochloride in 50% isopropanol) and the supernatant removed again by magnet adsorption.

D. the beads were washed twice with 0.5ml CW2 buffer (75% ethanol) and then left at room temperature for 5min to evaporate the remaining ethanol.

E. mu.L of eluent (10 mM Tris-HCl, pre-heated at pH 8.5,55 ℃) was added to the beads and incubated for 5min. During incubation, vortex for 20s per minute.

F. finally, the mixture was placed on a magnetic rack and the supernatant was collected for subsequent DNA analysis.

Determining ARGs copies of the filter and filtrate, respectively, based on the nucleic acids;

1) After the pretreatment is completed, the absolute content of ARGs in the sample is determined by using a fluorescent quantitative PCR instrument.

2) And (3) calculating according to a formula (2) to obtain the absolute copy number of the target gene in the sample.

Absolute copy number (copies/mL) = [ machine-readable value (copies/μl) ×total amount of sample DNA (μl) ]/total volume of extracted sample (m L), equation (2).

Based on the calculation of the steps, the copy number of the sul1 gene in the active bacteria is 3.75, the copy number of the sul1 gene in the dead bacteria is 6.9 and the copy number of the free sul1 gene is 3.85 in 50mL of environmental water.

Example 2 simulation of attenuation curve of antibiotic resistance Gene sul 1 in environmental Water

This example is based on the procedure of example 1, and shows the absolute content changes in the number of copies of live bacterial intracellular sul1, dead bacterial intracellular sul1, extracellular sul1 in still water over 30 days (1 d,8d,19d,25d,28 d) are collected continuously as shown in FIG. 5. Extracellular sul1 content increases in still water with lysis of dead bacteria within three weeks, peaking at 21 d. Meanwhile, the number of living bacteria in still water is increased and then reduced, so that the increase of sul1 in living bacteria cells is brought, and the number of intracellular ARGs carried by dead bacteria is continuously reduced, so that the bacteria decays exponentially and sharply in 20-25 d. The absolute change of total sul1 content in still water without antibiotics added at room temperature was simulated with time using an exponential function, and the simulation was performed using first-order reflection kinetics (formula 3). The results showed that the first order reaction kinetics was met.

C=c ₀*e^-0.1t, equation (3);

Wherein, C is the total absolute content (copies/mL) of sul1 in still water, C ₀ is the initial absolute content (copies/mL) of sul1 in still water, and R ² is 0.59.

In the description of the present specification, reference to the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the spirit and scope of the invention as defined by the appended claims and their equivalents.

Claims

1. A method for detecting the presence and absolute content of antibiotic resistance genes ARGs, characterized in that it includes:

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 is 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 staining and identification treatment to determine the ratio of live bacteria to dead bacteria in the bacterial suspension, comprising:

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, comprising:

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 is 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.