Method for determining and evaluating influence of phycosphere microenvironment on conjugational transfer capability of ARGs

Abstract

The invention discloses a method for determining and evaluating the influence of an algal microenvironment on the conjugational transfer capability of ARGs. Comprising the following steps: preparing donor bacteria, recipient bacteria and an algae solution, evaluating the influence of the density of microalgae in the interalgal microenvironment on the conjugational metastasis of ARGs, evaluating the influence of the number of binding-state bacteria in the interalgal microenvironment on the conjugational metastasis of ARGs, evaluating the influence of the EPS content in the interalgal microenvironment on the conjugational metastasis of ARGs, and evaluating the influence of the EPS component in the interalgal microenvironment on the conjugational metastasis of ARGs. According to the method, macroscopic and microscopic processes of microalgae in a water body are considered, the influence on the conjugational transfer capability of the antibiotic resistance genes of the drug-resistant bacteria in the interalgal microenvironment can be systematically and comprehensively evaluated, and the determination method can provide powerful theoretical basis and support for behavior research and use management of the antibiotic resistance genes in the water environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of water treatment, and particularly relates to a method for measuring the influence of the phycosphere microenvironment on the conjugative transfer ability of ARGs and evaluating it. Background Art

[0002] Antibiotics Resistance Genes (ARGs) can be widely spread in the environment through conjugative transfer, transposition, transformation, etc. with the help of mobile elements such as plasmids, transposons, and integrons, resulting in the continuous enhancement of microbial drug resistance.

[0003] Urban landscape water bodies are an important "sink" for ARGs, gathering ARGs from many sources such as reclaimed water recharge, direct sewage discharge, sewage overflow during the rainy season, and urban non-point source pollution. In some lakes mainly replenished with reclaimed water, the Antibiotic Resistance Indexes (ARI) of antibiotics are as high as 14 - 16. Urban landscape water bodies are often located in densely populated areas and shoulder the functions of urban landscapes and residents' hydrophilic activities. Hydrophilic activities are important exposure routes for pollutants. Therefore, the risk of the spread and diffusion of ARGs in urban landscape water bodies cannot be ignored. In addition, urban landscape water bodies are prone to eutrophication, leading to a large growth of microalgae. The phycosphere microenvironment formed by microalgae will enrich a large number of bacteria in the water body, including harmful bacteria such as antibiotic-resistant bacteria and pathogenic bacteria. The aggregation of a large number of bacteria in a small area creates favorable conditions for the exchange of genetic material between bacteria.

[0004] Previous studies on the biological safety risks of water bodies in the prior art mainly focused on algal blooms and pathogen infections, and rarely paid attention to the transmission process and mechanism of ARGs in the phycosphere microenvironment of water bodies. Therefore, studying the transmission characteristics of ARGs in the phycosphere microenvironment of water bodies has important theoretical and practical significance for understanding the environmental fate behavior of ARGs, accurately assessing the biological safety risks of water bodies, and formulating effective prevention and control measures. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for measuring the influence of the phycosphere microenvironment on the conjugative transfer ability of ARGs and evaluating it, which can systematically and comprehensively evaluate the influence of the phycosphere microenvironment on the conjugative transfer ability of antibiotic resistance genes in drug-resistant bacteria, taking into account both the macroscopic and microscopic processes of microalgae in the water body, and providing strong theoretical basis and support for the behavior research and use management of antibiotic resistance genes in the water environment.

[0006] The purpose of the present invention is achieved as follows, including: (1) Respectively take recipient bacteria and donor bacteria and incubate them in the phycosphere microenvironment with different algal densities; (2)Determine the number of conjugants and recipient bacteria containing conjugation transfer treatment, and evaluate the effect of microalgae density in the phycosphere microenvironment on the conjugation transfer frequency of ARGs; (3)Determine the number of bound bacteria and the number of free bacteria in the phycosphere microenvironment, and evaluate the effect of the number of bound bacteria in the phycosphere microenvironment on the conjugation transfer of ARGs; (4)Determine the total organic carbon of EPS (extracellular secretions) in the phycosphere microenvironment to quantify the EPS content, and evaluate the effect of EPS content in the phycosphere microenvironment on the conjugation transfer of ARGs; (5)Determine the polysaccharide and protein contents in the EPS of the phycosphere microenvironment, and evaluate the effect of EPS components in the phycosphere microenvironment on the conjugation transfer of ARGs.

[0007] The determination of algal density can be carried out by methods such as ultraviolet absorbance method, cell counter, hemocytometer counting method and flow cytometer. The ultraviolet absorbance method requires establishing a standard curve of the absorbance of the algal solution at 680 nm and the algal density. However, due to the influence of algal cell growth metabolites and possible cell damage during the decline period, it may affect its accuracy. The cell counter cannot distinguish intact cells from damaged cells. The hemocytometer has a large workload and many error factors. Therefore, it is recommended to use a flow cytometer to determine the algal density.

[0008] Preferably, (1) the algal species in the phycosphere microenvironment is Chlorella ( Chlorella.sp ), the donor bacterium is Escherichia coli Eoli .DH5α, and the recipient bacterium is Escherichia coli Eoli .HB101.

[0009] Preferably, inoculate the donor bacterium E.coli DH5α in an LB medium containing 100 μg / mL Amp (ampicillin), 50 μg / mL Kan (kanamycin) and 10 μg / mL Tet (tetracycline), and inoculate the recipient bacterium E.coli HB101 in an LB medium containing 30 μg / mL Str, and synchronously place them in a shaker at 37 °C and 120 rpm / min for overnight shaking culture; centrifuge the overnight-cultured donor and recipient bacteria in a 50 mL centrifuge tube at 8000 rpm for 5 min, then wash the bacterial solution 3 times with PBS buffer solution, and finally resuspend the bacterial solution with PBS buffer solution; transfer the algal cells cultured to the stationary phase to a 50 mL sterilized centrifuge tube, centrifuge at 10000 rpm for 10 min, discard the supernatant, add 30 mL sterilized PBS buffer solution, gently suspend the cells for washing, and finally suspend the cells in PBS buffer solution; count the algal cells using a microscope counting method based on a hemocytometer; take 10 mL of the donor bacterial solution and 10 mL of the recipient bacterial solution into a 50 mL sterilized glass tube respectively, add algal cells at different concentrations, and control the final concentration of algal cells to be 1×102 cells / mL, 1×10 3 cells / mL, 1×10 4 cells / mL, 1×10 5 cells / mL, 1×10 6 cells / mL, 1×10 7 cells / mL. Three replicates were set for each treatment; meanwhile, a control group with the same bacteria but without algae was set; fully mixed to establish a microcosm system for conjugative transfer in the phycosphere microenvironment; the conjugative transfer system was placed in a constant temperature shaker at 37 °C for 6 h, and the shaker speed was 120 rpm; the recipient bacteria and / or donor bacteria used for conjugation were in the logarithmic growth phase.

[0010] Preferably, in (2), for the method of measuring the number of conjugants and recipient bacteria in the conjugative transfer treatment: Take the bacterial liquid plate containing the conjugants of the conjugative transfer treatment for counting, and spread it on an LB solid medium containing 100 mg / L of Amp, 10 mg / L of Tet, 50 mg / L of Kan, and 30 mg / L of Str (streptomycin) to screen for transfer conjugants, culture at 37 °C for 24 h, and count the conjugant colonies by the plate counting method; at the same time, use a selective LB medium containing 30 mg / L of Str to plate count the recipient bacteria; three replicates were set for each treatment; The formula for calculating the conjugative transfer frequency is as follows: Conjugative transfer frequency (ƒ) = N T / N S In the formula: N T : The number of transfer conjugants, CFU / mL; N S : The number of recipient bacteria, CFU / mL.

[0011] Preferably, in (3), when measuring the number of bound bacteria and the number of free bacteria in the phycosphere microenvironment, the filtration method is used to separate the phycosphere microenvironment-bound bacteria and the water body-free bacteria. Specifically, first filter 20 mL of the mixed system through a 5-μm pore size metal filter mesh, and collect the filtrate; the entire filtration process uses a vacuum pump to ensure a faster filtration speed, and stop filtering when the filtration speed of the 5-μm filter mesh decreases; put the filter mesh into 100 mL of phosphate buffer solution (PBS, pH = 7.2 ± 0.2), fully dissolve it, the 5-μm filter membrane is the bound bacteria in the phycosphere microenvironment, and the filtrate is the water body-free bacteria.

[0012] Preferably, in (4), the specific method for extracting EPS in the phycosphere microenvironment is: Extraction method of soluble extracellular polymeric substances (S-EPS): First, perform centrifugation (centrifuge at 8000 rpm / min for 10 min at 4°C). After collecting the supernatant, then filter the supernatant using a 0.45-μm PTFE membrane. The obtained filtrate is the S-EPS component; collect and subpackage the filtrate and store it in a -80°C refrigerator for later use; Extraction method of loosely bound extracellular polymeric substances (LB-EPS): Resuspend the lower-layer residue from which S-EPS has been extracted in a sterilized conical flask with 0.05% NaCl solution; after centrifugation (centrifuge at 10000 rpm / min for 10 min at 4°C), obtain the supernatant, filter it using a 0.45-μm PTFE membrane to obtain the LB-EPS component; subpackage the filtrate and store it in a -80°C refrigerator.

[0013] Preferably, in (5), the phenol-sulfuric acid method is used to determine the polysaccharide content in EPS in the phycosphere microenvironment. Specifically: Mix 1 mL of the sample solution with 2 mL of 6% phenol, add 5 mL of 98% sulfuric acid, boil for 20 min, and after cooling to room temperature, use a microplate reader to measure the OD value at 490 nm; the BCA method is used to determine the protein content in EPS in the phycosphere microenvironment. Specifically: Mix 20 μL of the sample solution with 200 μL of BCA working solution (enhanced BCA protein detection kit), incubate at 37°C for 30 min, and use a microplate reader to measure the OD value at 562 nm.

[0014] Advantages of the present invention: 1. The present invention establishes the relationship between the algal density and the conjugation transfer frequency of ARGs in the phycosphere microenvironment, which will supplement the understanding of the spread of cyanobacteria and ARGs in water and provide theoretical guidance for evaluating the conjugation transfer risk of antibiotic resistance genes in urban landscape water bodies and formulating effective prevention and control measures; 2. The present invention provides strong evidence for clarifying that the phycosphere microenvironment accelerates the spread risk of antibiotic resistance genes by increasing the contact between bacteria through aggregating bacteria by evaluating the influence of bound bacteria in the phycosphere microenvironment on the conjugation transfer frequency of antibiotic resistance genes; 3. The present invention further clarifies the mechanism for promoting the spread of antibiotic resistance genes through conjugation transfer in the phycosphere microenvironment by measuring the EPS content and EPS components in the phycosphere microenvironment, and provides theoretical guidance for formulating prevention and control strategies for the spread risk of ARGs in urban landscape water bodies. Description of the drawings

[0015] Figure 1 For the conjugation transfer frequency of ARGs in the phycosphere microenvironment with different algal densities in Example 1; Figure 2 For the numbers of bound bacteria and free bacteria in the phycosphere microenvironment with different algal densities in Example 1; Figure 3EPS content in the phycosphere microenvironment with different algal densities in Example 1; Figure 4 Protein (A) and polysaccharide (B) contents in EPS in the phycosphere microenvironment with different algal densities in Example 1; Detailed implementation method

[0016] The present invention will be further described below in conjunction with examples and drawings, but the present invention is not limited in any way. Any transformation or replacement based on the teachings of the present invention falls within the protection scope of the present invention.

[0017] Example 1 Method for evaluating the influence of the phycosphere microenvironment on the conjugative transfer ability of ARGs. The Chlorella used in this example was Chlorella sp purchased from the Freshwater Algae Culture Collection of the Chinese Academy of Sciences. The donor bacterium E.coli DH5α and the recipient bacterium E. coli HB101 were both sourced from commercial channels. The method includes: (1) Preparation of donor bacteria, recipient bacteria, and algal solution: Inoculate the donor bacterium E.coli DH5α in LB medium containing Amp (100 μg / mL), Kan (50 μg / mL), and Tet (10 μg / mL), and inoculate the recipient bacterium E.coli HB101 in LB medium containing Str (30 μg / mL). Synchronously place them in a shaker at 37 °C and 120 rpm / min for overnight shaking culture. After overnight culture, centrifuge the donor bacteria and recipient bacteria in a 50 mL centrifuge tube at 8000 rpm for 5 min, then wash the bacterial solution 3 times with PBS buffer solution, and finally resuspend the bacterial solution with PBS buffer solution. Measure the OD 600 value of the bacterial solution, that is, the optical density of the strain, and adjust the suspension concentrations of the donor bacteria and recipient bacteria to OD 600 = 0.5 for subsequent experiments; Chlorella is cultured using BG-11 culture medium. The algal species is cultured in a light constant temperature shaking incubator. The culture conditions are pH value 7.1 ± 1 °C, light intensity 3000 Lux, light-dark ratio 12 h:12 h, rotation speed 120 rpm, and randomly change the placement position every day. Transfer the algal cells cultured to the stationary phase to a 50 mL sterilized centrifuge tube, centrifuge at 10000 rpm for 10 min, discard the supernatant, add 30 mL sterilized PBS buffer solution, gently suspend the cells for washing, and finally suspend the cells in PBS buffer solution. Count the algal cells using a microscope counting method based on a hemocytometer; Establishment of the conjugation transfer system of ARGs in the phycosphere microenvironment: Take 10 mL of donor bacterial solution and 10 mL of recipient bacterial solution into a 50 mL sterilized glass tube, add algal cells at different concentrations, and control the final concentration of algal cells to be 1×10 2 cells / mL, 1×10 3 cells / mL, 1×10 4 cells / mL, 1×10 5 cells / mL, 1×10 6 cells / mL, 1×10 7 cells / mL. Set three replicates for each treatment; at the same time, set a control group with the same bacteria but without algae; mix well to establish a conjugation transfer microcosm system in the phycosphere microenvironment; place the conjugation transfer system in a constant temperature shaker at 37°C (rotation speed is 120 rpm) for 6 h; the relationship between the conjugation transfer frequency of antibiotic resistance genes and algal density in the phycosphere microenvironment; the influence of bound bacteria in the phycosphere microenvironment on the conjugation transfer frequency of antibiotic resistance genes; the influence of extracellular polymeric substances (EPS) in the phycosphere microenvironment on the conjugation transfer frequency of antibiotic resistance genes; the influence of the main components of EPS in the phycosphere microenvironment on the conjugation transfer frequency of antibiotic resistance genes; (2) Influence of microalgal density on the conjugation transfer of ARGs in the phycosphere microenvironment: Measure the conjugation transfer frequency of ARGs in the phycosphere microenvironment with different algal densities; Method for measuring the number of conjugants and recipient bacteria: Take the bacterial liquid plate containing the conjugants of the conjugation transfer treatment, coat it on the LB solid medium containing 100 mg / L Amp, 10 mg / L Tet, 50 mg / L Kan, and 30 mg / L Str at the same time to screen for transfer conjugants, culture at 37°C for 24 h, and count the conjugant colonies by the plate counting method; at the same time, use the selective LB medium containing 30 mg / L Str to plate count the recipient bacteria; set three replicates for each treatment; The formula for calculating the conjugation transfer frequency is as follows: Conjugation transfer frequency (ƒ) = N T / N S Where: N T : The number of transfer conjugants, CFU / mL; N S : The number of recipient bacteria, CFU / mL; The results are as Figure 1 shown. In the phycosphere microenvironment with different Chlorella densities, it all shows promotion of bacterial conjugation transfer; among them, the microalgal density is 10 2 cell / mL, 10 3 cell / mL, 10 4 cell / mL, 10 5The bacterial conjugation transfer frequencies in the microalgae-bacteria conjugation system were 7.6*10 -5 ,7.6*10 -5 ,7.7*10 -5 ,7.7*10 -5 , while the microalgae density is 10 6 cell / mL, 10 7 The bacterial conjugation transfer frequencies in the microalgae-bacteria conjugation system were 8.2*10 -5 , 11.4*10 -5 Compared with the control group (algae density of 0 cell / mL), the algae density in the microalgae-bacteria conjugation system was 10 6 cell / mL and 10 7 The bacterial conjugation transfer frequency of 10 cell / mL increased significantly, increasing by about 1.2 times and 1.5 times, respectively; in contrast, 2 cell / mL, 10 3 cell / mL, 10 4 cell / mL and 10 5 cell / mL bacterial conjugative transfer frequency increased only about 1.0-1.1-fold; (3) Effect of the number of conjugated bacteria in the algal microenvironment on the conjugative transfer of ARGs: The filtration method was used to separate the bacteria bound to the algal microenvironment and the free bacteria in the water. Specifically, 20 mL of the mixed system was first filtered with a 5 μm pore size metal filter and the filtrate was collected. A vacuum pump was used to ensure a faster filtration speed during the entire filtration process. When the filtration speed of the 5 μm filter decreased, the filtration was stopped. The filter was placed in 100 mL of phosphate buffer and fully dissolved. The 5 μm filter membrane contained the bound bacteria in the algal microenvironment, and the filtrate contained the free bacteria in the water. The detection of the number of bound and free bacteria in the algal microenvironment in the microalgae-bacteria conjugation system with different Chlorella densities found that Figure 2 As shown in Figure 2, the number of bound bacteria in the algae microenvironment increases with the increase of microalgae density in the microalgae-bacteria conjugation system; 2 cell / mL, 10 3 cell / mL, 10 4 cell / mL, 10 5 cell / mL, 10 6 The number of bacteria bound to the algae microenvironment in the microalgae-bacteria conjugation system was 2.5*10 6 CFU / mL, 3*10 6 CFU / mL, 5*10 6 CFU / mL, 6*106 CFU / mL, 9*10 6 CFU / mL. When the microalgae density in the microalgae-bacteria conjugation system is 10 7 cell / mL, the number of bound bacteria in the phycosphere microenvironment reaches 37*10 6 CFU / mL, which is approximately 18.5 times higher than that of the control group (microalgae density of 0 cell / mL); in contrast, at other microalgae densities in the microalgae-bacteria conjugation system, the number of bound bacteria in the phycosphere microenvironment only increases by about 2 to 8 times compared to the control group; when the microalgae density is 10 2 cell / mL, 10 3 cell / mL, 10 4 cell / mL, 10 5 cell / mL, 10 6 cell / mL in the microalgae-bacteria conjugation system, the number of free bacteria is 53*10 6 CFU / mL, 52*10 6 CFU / mL, 51*10 6 CFU / mL, 49*10 6 CFU / mL, 47*10 6 CFU / mL, which is approximately 21 times, 17 times, 10 times, 8 times, 4 times, and 3 times that of the number of bound bacteria in the phycosphere microenvironment; when the microalgae density in the microalgae-bacteria conjugation system reaches 10 7 cell / mL, the number of free bacteria is 21*10 6 CFU / mL, which is significantly reduced by approximately 50% compared to the control group, and the number of bound bacteria in its phycosphere microenvironment is approximately twice that of the number of free bacteria; (4) Influence of EPS content in the phycosphere microenvironment on the conjugative transfer of ARGs: Extraction method of soluble extracellular polymeric substances (S-EPS): First, perform centrifugation (centrifuge at 8000 rpm / min for 10 min at 4°C). After collecting the supernatant, then filter the supernatant using a 0.45 μm PTFE membrane. The obtained filtrate is the S-EPS fraction; collect and aliquot the filtrate and store it in a -80°C refrigerator for later use; Extraction method of loosely bound extracellular polymeric substances (LB-EPS): Resuspend the remaining lower layer from which S-EPS has been extracted in a sterilized conical flask with 0.05% NaCl solution; after centrifugation (centrifuge at 10000 rpm / min for 10 min at 4°C), obtain the supernatant and filter it using a 0.45 μm PTFE membrane to obtain the LB-EPS fraction; aliquot the filtrate and store it in a -80°C refrigerator; EPS can be divided into soluble EPS (S-EPS), loosely bound EPS (LB-EPS), and tightly bound EPS (TB-EPS); among them, EPS (S-EPS) and loosely bound EPS (LB-EPS) were studied, and they had higher contents and greater effects on bacterial conjugation transfer; the total organic carbon contents in different types of EPS in the microalgae-bacteria conjugation system with different Chlorella densities were evaluated; as Figure 3 shown, the total amount of EPS in the Chlorella-bacteria conjugation system also increased with the increase in microalgae density. Among them, the total TOC of EPS in the microalgae-bacteria conjugation systems with microalgae densities of 10 2 cell / mL, 10 3 cell / mL, 10 4 cell / mL, 10 5 cell / mL, and 10 6 cell / mL were 20 mg / L, 21 mg / L, 22 mg / L, 22 mg / L, and 23 mg / L respectively. When the microalgae density in the microalgae-bacteria conjugation system was 10 7 cell / mL, the total TOC of EPS in the phycosphere microenvironment reached 27 mg / L, which was significantly increased by about two times compared with the control group (microalgae density of 0 cell / mL); in contrast, at other microalgae densities in the microalgae-bacteria conjugation system, the total TOC of EPS in the phycosphere microenvironment was only increased by about 1.1 to 1.3 times compared with the control group; (5) Effect of EPS components in the phycosphere microenvironment on the conjugation transfer of ARGs: The polysaccharide content in EPS was determined by the sulfuric acid-phenol method. Specifically: 1 mL of the sample solution was mixed with 2 mL of 6% phenol, 5 mL of 98% sulfuric acid was added, boiled for 20 min, and after cooling to room temperature, the OD value at 490 nm was measured using a microplate reader; the protein content in EPS in the phycosphere microenvironment was determined by the BCA method. Specifically: 20 μL of the sample solution was mixed with 200 μL of the BCA working solution, incubated at 37 °C for 30 min, and the OD value at 562 nm was measured using a microplate reader; The protein and polysaccharide contents of different types of EPS in the bacteria-microalgae conjugation systems with different Chlorella densities were analyzed; as Figure 4 shown in A, the total protein amount in the bacteria-microalgae conjugation systems with different Chlorella densities changed little compared with the control group (microalgae density of 0 cells / mL), and the protein contents of different types of EPS in the bacteria-microalgae conjugation systems with different Chlorella densities changed little compared with the control group (algae density of 0 cells / mL); at the same time, the polysaccharide contents of different types of EPS in the microalgae-bacteria conjugation systems with different Chlorella densities were evaluated, as Figure 4As shown in Figure B, the total polysaccharide content in EPS increases with the increase of Chlorella density in the microalgae-bacteria conjugation system. The total polysaccharide contents of EPS in the microalgae-bacteria conjugation systems with Scenedesmus density of 10 2 cell / mL, 10 3 cell / mL, 10 4 cell / mL, 10 5 cell / mL, 10 6 cell / mL are 0.21 mg / L, 0.22 mg / L, 0.23 mg / L, 0.23 mg / L, and 0.24 mg / L respectively. When the Scenedesmus density in the microalgae-bacteria conjugation system is 10 7 cell / mL, the total polysaccharide content of EPS in the phycosphere microenvironment reaches 0.3 mg / L, which is about 1.5 times significantly higher than that of the control group (microalgae density of 0 cell / mL).

Claims

1. A method for evaluating the effect of algae microenvironment on the conjugation and transfer ability of ARGs, characterized in that include: (1) Take the recipient bacteria and donor bacteria separately and incubate them in algal microenvironments containing different algae densities; (2) Determine the number of conjugants and recipient bacteria in the conjugative transfer treatment and evaluate the effect of microalgae density in the algal microenvironment on the frequency of ARGs conjugative transfer; (3) Determine the number of bound bacteria and the number of free bacteria in the algal microenvironment, and evaluate the effect of the number of bound bacteria in the algal microenvironment on the conjugation and transfer of ARGs; (4) Determine the total organic carbon of EPS in the algae microenvironment to quantify the EPS content and evaluate the effect of EPS content in the algae microenvironment on the conjugation and transfer of ARGs; (5) Determine the content of polysaccharides and proteins in the EPS of the algae microenvironment and evaluate the effect of EPS components in the algae microenvironment on the conjugation and transfer of ARGs.

2. The method for evaluating the effect of algae microenvironment on the conjugation and transfer ability of ARGs according to claim 1, characterized in that (1) The algae species in the algae microenvironment is Chlorella vulgaris, and the donor bacteria is Escherichia coli Eoli .DH5α, the recipient bacterium is Escherichia coli Eoli .HB101.

3. The method for evaluating the effect of algae microenvironment on the conjugation and transfer ability of ARGs according to claim 2, characterized in that The donor bacteria E. coli DH5α was inoculated in LB medium containing 100 μg / mL Amp, 50 μg / mL Kan and 10 μg / mL Tet. E. coli HB101 was inoculated in LB medium containing 30 μg / mL Str, and simultaneously placed in a shaker at 37°C and 120 rpm / min for overnight shaking culture; the donor bacteria and the recipient bacteria after overnight culture were centrifuged at 8000 rpm for 5 min in a 50 mL centrifuge tube, and then the bacterial solution was washed 3 times with PBS buffer solution, and finally the bacterial solution was resuspended with PBS buffer solution; the algae cells cultured to the stable period were transferred to a 50 mL sterile centrifuge tube, centrifuged at 10000 rpm for 10 min, and then the supernatant was discarded, 30 mL sterile PBS buffer was added, the bacteria were gently suspended for washing, and finally the bacteria were suspended in PBS buffer; The algae cells were counted using a microscope counting method based on a hemocytometer. 10 mL of the donor bacterial solution and 10 mL of the recipient bacterial solution were taken into 50 mL sterile glass tubes, and different concentrations of algae cells were added to control the final concentration of algae cells to be 1×10 2 cells / mL、1×10 3 cells / mL、1×10 4 cells / mL、1×10 5 cells / mL、1×10 6 cells / mL、1×10 7 cells / mL, with three replicates for each treatment; a control group with the same bacteria but without algae was set up at the same time; the mixture was fully mixed to establish an algal microenvironment conjugation transfer microcosm system; the conjugation transfer system was set up in a 37°C constant temperature shaker for 6 hours with a shaker speed of 120 rpm; the recipient bacteria and / or donor bacteria used for conjugation were in the logarithmic growth phase.

4. The method for evaluating the effect of algae microenvironment on the conjugation and transfer ability of ARGs according to claim 1, characterized in that Method for determining the number of conjugants and recipient bacteria in the conjugation transfer treatment in (2): Take a plate containing the bacterial solution of the conjugants in the conjugation transfer treatment and count it, spread it on an LB solid culture medium containing 100 mg / L Amp, 10 mg / L Tet, 50 mg / L Kan and 30 mg / L Str to select the transferred conjugants, culture at 37°C for 24 hours, and count the conjugant colonies using the plate counting method; at the same time, use a selective LB culture medium containing 30 mg / L Str to count the recipient bacteria on the plate; set up three replicates for each treatment; The formula for calculating the junction transfer frequency is as follows: Engagement transfer frequency (ƒ) = N T / N S Where: N T : The number of transferred conjugates, CFU / mL; N S : Number of recipient bacteria, CFU / mL.

5. The method for evaluating the effect of algae microenvironment on the conjugation and transfer ability of ARGs according to claim 1, characterized in that When determining the number of bound bacteria and the number of free bacteria in the algal microenvironment in (3), the filtration method was used to separate the bound bacteria in the algal microenvironment and the free bacteria in the water. Specifically, 20 mL of the mixed system was first filtered with a 5 μm pore size metal filter and the filtrate was collected. A vacuum pump was used to ensure a fast filtration speed during the entire filtration process. When the filtration speed of the 5 μm filter decreased, the filtration was stopped. The filter was placed in 100 mL of phosphate buffer and fully dissolved. The 5 μm filter membrane contained bound bacteria in the algal microenvironment, and the filtrate contained free bacteria in the water.

6. The method for evaluating the effect of algae microenvironment on the conjugation and transfer ability of ARGs according to claim 1, characterized in that The specific EPS extracted from the algae microenvironment in (4) is: The S-EPS extraction method is as follows: firstly, centrifuge and collect the supernatant, then filter the supernatant using a 0.45 μm PTFE membrane, and the obtained filtrate is the S-EPS component; collect and subpackage the filtrate and store it in a -80°C refrigerator for later use; Extraction method of LB-EPS: resuspend the lower layer residue from which S-EPS is extracted in a sterile conical flask with 0.05% NaCl solution; obtain the supernatant after centrifugation, filter it using a 0.45 μm PTFE membrane to obtain the LB-EPS component; divide the filtrate into portions and store it in a -80°C refrigerator.

7. The method for evaluating the effect of algae microenvironment on the conjugation and transfer ability of ARGs according to claim 1, characterized in that In (5), the polysaccharide content in EPS in the algal microenvironment was determined by the sulfuric acid-phenol method. Specifically, 1 mL of sample solution was mixed with 2 mL of 6% phenol, 5 mL of 98% sulfuric acid was added, and the mixture was boiled for 20 min. After cooling to room temperature, the OD value at 490 nm was measured using an enzyme-labeled instrument. The protein content in EPS in the algal microenvironment was determined by the BCA method. Specifically, 20 μL of sample solution was mixed with 200 μL of BCA working solution, incubated at 37°C for 30 min, and the OD value at 562 nm was measured using an enzyme-labeled instrument.

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