Method for detecting mutual adhesion capacity between resistant live bacteria and application

Abstract

The invention discloses a method for detecting the mutual adhesion capacity between resistant live bacteria and application. The method comprises the following steps: providing a drug-resistant bacterium detection substrate with positive charges on the surface; the method comprises the following steps: culturing inherent bacteria of excrement by using ciprofloxacin hydrochloride and sulfametoxydiazine antibiotic liquid culture mediums respectively to obtain a first bacterial culture and a second bacterial culture; respectively obtaining a first bacterial culture solution and a second bacterial culture solution; respectively obtaining a first drug-resistant bacterium detection substrate and a second drug-resistant bacterium detection substrate; respectively transferring the first drug-resistant bacterium detection substrate and the second drug-resistant bacterium detection substrate to different sample tables of an atomic force microscope, and dropwise adding a sterile phosphate buffer salt solution to cover the sample tables; and respectively carrying out adhesive force test on the inherent bacteria of the excrement and the probe bacteria. According to the invention, the defect of non-activity detection between the bacterium probe and the resistant bacterium negative film can be effectively solved, and meanwhile, the real adhesion behavior between the bacteria in different environments can be really evaluated.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for detecting the mutual adhesion ability between resistant live bacteria and its application. Background Art

[0002] The use of antibiotics can lead to increased bacterial resistance to antibiotics. The reason for this increased resistance is that resistance genes are produced in bacteria. After bacteria come into contact with antibiotics, they trigger the production of resistance genes in their bodies. After accumulating a lot of resistance genes, the bacteria will become resistant bacteria. With the widespread use of antibiotics, the number of antibiotic-resistant bacteria driven by antibiotic resistance genes continues to increase, and the resistance genes are spread through horizontal or vertical migration. Specifically, as the number of resistant bacteria increases, they will transfer resistance genes to the outside through conjugation transfer. This transfer process requires bacteria to directly contact and adhere to each other. Transferring resistance genes to the outside through conjugation transfer means that some resistant bacteria will transfer resistance genes to sensitive bacteria. Resistant bacteria transfer resistance genes to sensitive bacteria, making sensitive bacteria resistant bacteria. Biofilm, as a protective layer formed on the surface of bacteria, can enhance the adhesion and drug resistance of bacteria because the conjugation transfer process depends on biofilm. When sensitive bacteria (without resistance genes, so they are sensitive to antibiotics and easily killed by antibiotics) turn into resistant bacteria, the bacteria's ability to adhere to host cells or biofilms increases, making it difficult for antibiotics to penetrate the bacteria and reach effective bactericidal concentrations, thereby increasing the bacteria's drug resistance. In addition, bacteria adhering to host cells may also enhance their resistance by changing their metabolic pathways or expressing specific resistance genes. This shows that biofilms are a double-edged sword and play an important role in the formation and development of resistant bacteria. The optical tweezers method, single-cell force spectroscopy technology and other methods that have been developed so far all test the bacterial adhesion force obtained when the bacteria are in an abnormal physiological metabolic state, and therefore cannot accurately realize the true adhesion behavior between resistant living bacteria while maintaining normal physiological metabolism. Summary of the invention

[0003] In order to solve the technical problems in the prior art, the present invention provides a detection method and application of the mutual adhesion ability between resistant live bacteria, which can accurately realize the real adhesion behavior between resistant live bacteria while maintaining normal physiological metabolism.

[0004] Specifically, the present invention includes the following contents.

[0005] The first aspect of the present invention provides a method for detecting the mutual adhesion ability between resistant live bacteria, comprising:

[0006] A method for detecting the mutual adhesion ability between resistant live bacteria, comprising:

[0007] Step S1, providing a drug-resistant bacteria detection substrate with a positive charge on the surface; Step S2, using a ciprofloxacin hydrochloride antibiotic liquid culture medium and a sulfamethoxazole antibiotic liquid culture medium to perform gradient concentration drug resistance stress culture on fecal intrinsic bacteria, to obtain a first bacterial culture and a second bacterial culture; Step S3, using a sterile phosphate buffered saline solution with a concentration of 0.1-1M to wash the first bacterial culture and the second bacterial culture for 2-5 times and then resuspend them to obtain a first bacterial culture solution and a second bacterial culture solution, and the concentrations of the first bacterial culture solution and the second bacterial culture solution are both adjusted to 10 7 -10 12 CFU / mL; Step S4, 5-10 μL of a concentration of 10 7 -10 12 The first bacterial culture solution and the second bacterial culture solution with a CFU / mL are uniformly and stably dripped onto the two modified drug-resistant bacteria detection substrates, and dried at room temperature to obtain the first drug-resistant bacteria detection substrate and the second drug-resistant bacteria detection substrate; step S5, respectively transferring the first drug-resistant bacteria detection substrate and the second drug-resistant bacteria detection substrate to different sample stages of an atomic force microscope, dripping sterile phosphate buffered saline solution on the first drug-resistant bacteria detection substrate and the second drug-resistant bacteria detection substrate to cover them, so that the fecal intrinsic bacteria in the first bacterial culture solution and the second bacterial culture solution maintain a normal physiological state; step S6, respectively performing adhesion tests on the fecal intrinsic bacteria on the first drug-resistant bacteria detection substrate and the second drug-resistant bacteria detection substrate and the probe bacteria on the spherical bacteria probe of the atomic force microscope, to obtain the first adhesion force curve and the second adhesion force curve between the fecal intrinsic bacteria on the first drug-resistant bacteria detection substrate and the second drug-resistant bacteria detection substrate and the probe bacteria.

[0008] Optionally, after step S6, the method further includes:

[0009] Step S7, using the underwater scanning tip probe of the atomic force microscope to respectively test the stability of the fecal intrinsic bacteria on the first drug-resistant bacteria detection substrate and the second drug-resistant bacteria detection substrate, and using a high-resolution laser confocal microscope to respectively detect the activity of the probe bacteria, the stability of the fecal intrinsic bacteria includes the stability of its position fixation and the stability of its morphology.

[0010] Optionally, in step S6, it further includes:

[0011] The adhesion force between the fecal inherent bacteria on the first drug-resistant bacteria detection substrate and the probe bacteria of the atomic force microscope is evaluated by the first adhesion force curve; the adhesion force between the fecal inherent bacteria on the second drug-resistant bacteria detection substrate and the probe bacteria of the atomic force microscope is evaluated by the second adhesion force curve.

[0012] Optionally, in step S6, a liquid phase test mode is selected by an atomic force microscope, and the fecal intrinsic bacteria fixed on the drug-resistant bacteria detection substrate are observed and located using the bright field module of the atomic force microscope, and the force curve array mode is used to collect the force spectra corresponding to the first adhesion force curve and the second adhesion force curve.

[0013] Optionally, in step S6, the probe bacteria is a Citrobacter freundii carrying a spontaneous green fluorescent protein sfgfp marker originating from the intestine, and the green fluorescence signal emitted by the super green fluorescent protein sfgfp carried by the probe bacteria is measured by using an EGFP module of a high-resolution laser confocal microscope, wherein the excitation light wavelength of the green fluorescence signal is 488nm.

[0014] Optionally, in step S1, providing a drug-resistant bacteria detection substrate having a surface carrying a positive charge comprises:

[0015] Materials including freshly dissociated mica sheets or glass slides are selected as drug-resistant bacteria detection substrates, and the surface of the drug-resistant bacteria detection substrate is modified by polyethyleneimine so that the surface of the drug-resistant bacteria detection substrate carries a positive charge.

[0016] Optionally, the mica sheet is muscovite, and the muscovite is used as the resistant bacteria detection substrate, and 30-100 μL of polyethyleneimine with a concentration of 0.1-4 mg / mL is used to coat the muscovite to modify the surface of the resistant bacteria detection substrate and thereby achieve the purpose of making the surface of the resistant bacteria detection substrate carry a positive charge.

[0017] Optionally, in step S2, the step of using a ciprofloxacin hydrochloride antibiotic liquid culture medium and a sulfamethoxazole antibiotic liquid culture medium to perform gradient concentration drug resistance stress culture on fecal intrinsic bacteria to obtain a first bacterial culture and a second bacterial culture comprises:

[0018] The single bacteria with the best growth advantage separated from the intestinal flora in the early stage were tested for MIC values ​​of ciprofloxacin hydrochloride and sulfamethoxazole, and ciprofloxacin hydrochloride and sulfamethoxazole were selected as antibiotic stress factors; the MIC value of the single bacteria with the best growth advantage was used as the exposure concentration, and the gradient concentrations of ciprofloxacin hydrochloride solution and sulfamethoxazole solution were prepared and exposed to the fecal intrinsic flora homologous to the intestinal flora for stress culture for 10-12 hours, thereby obtaining the first bacterial culture and the second bacterial culture.

[0019] Optionally, the method for determining the MIC value is selected as a microdilution method, and the total volume of the method for determining the MIC value is set to 180-220 μL; wherein the concentrations of ciprofloxacin hydrochloride and sulfonamide-methoxazole stress are set to three different antibiotic concentrations of 0.1×MIC, 1×MIC and 10×MIC, respectively; wherein the temperature of the drug resistance stress culture is 37°C±2°C, the time is 8-12h, and the condition is a 150rpm-200rpm constant temperature shaker.

[0020] The second aspect of the present invention provides an application of the method for detecting the mutual adhesion ability between resistant live bacteria according to any one of the above items in the field of antibiotic resistance transmission.

[0021] The technical effects of the present invention include but are not limited to:

[0022] The present invention can effectively and stably fix live bacteria (fecal intrinsic bacteria) with antibiotic resistance on the surface of the resistant substrate without shifting during the detection process, effectively solving the defect of inactive detection between the bacterial probe and the resistant bacteria film, and truly evaluating the actual adhesion behavior between bacteria in different environments.

[0023] Specifically, after the present invention modifies the muscovite base with polyethyleneimine to make it carry positive charge, the fecal bacteria in the substrate can be in a stable and evenly distributed state, because the bacteria are negatively charged, and if they are not modified, they cannot be fixed to the substrate due to charge repulsion. The liquid phase test mode is selected, and the bacteria fixed in the substrate are kept active by dripping sterile phosphate buffered saline (PBS solution); the adhesion force of the bacterial substrate is tested using a spherical bacterial probe and the force curve array mode (Forcevolume) is used to collect the force spectrum, which can evaluate the real adhesion behavior between the fecal bacteria on the first resistant bacteria detection substrate and the second resistant bacteria detection substrate under the stress of multiple pollution factors and the probe bacteria on the spherical bacteria probe of the atomic force microscope, respectively, which can be used to evaluate the influence of pollution factors on the formation of bacterial biofilms, the real adhesion behavior between bacteria, and the transfer of drug resistance.

[0024] Among them, the previous steps S1-S3 of the present invention are to simulate an antibiotic-contaminated environment to test whether the adhesion ability of highly resistant bacteria is stronger and whether it will increase with the increase of antibiotic concentration. Among them, the present invention needs to prove that liquid phase testing can maintain bacterial activity, and such test results are more consistent with the results in the actual environment. In fact, liquid phase testing can maintain bacterial activity, because the created liquid phase environment is an environment where bacteria can survive, and the fluorescent bacteria on the probe are also intestinal bacteria, which are homologous to the inherent bacteria in feces.

[0025] The result of the evaluation of the present invention is that the stress of antibiotics will increase the adhesion between bacteria, and the adhesion will increase with the increase of antibiotic concentration, that is, after the intestinal bacteria are affected by the ciprofloxacin hydrochloride antibiotic and the sulfamethoxazole antibiotic, the adhesion between bacteria will increase. Among them, the adhesion between bacteria described in the present invention refers to the direct adhesion between resistant bacteria and sensitive bacteria. By evaluating the size of this adhesion, it can be concluded that which sensitive bacteria is more likely to obtain resistance genes from resistant bacteria through conjugation transfer. Adhesion is used to evaluate the ability of bacteria in the environment to obtain resistance genes, that is, the ability to prevent non-resistant bacteria from obtaining resistance genes from resistant bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The effective adhesion force spectra between the fecal intrinsic bacteria on the first drug-resistant bacteria detection substrate and the second drug-resistant bacteria detection substrate and the probe bacteria respectively under the liquid phase test environment of the atomic force microscope provided in the embodiment of the present invention;

[0027] Figure 2 A schematic diagram of a first adhesion force curve and a second adhesion force curve provided in an embodiment of the present invention; wherein the first adhesion force curve refers to Figure 2 The curve of ciprofloxacin hydrochloride is marked on the left, and the second adhesion curve is Figure 2 The graph for sulfamethoxazole is labeled on the middle right;

[0028] Figure 3 A morphological image of fecal intrinsic bacteria fixed on a modified drug-resistant bacteria detection substrate in a liquid phase test environment of an atomic force microscope provided in an embodiment of the present invention;

[0029] Figure 4 This is a laser confocal fluorescence detection image of a spherical probe provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail. Unless otherwise specified, "%" is a percentage based on weight.

[0033] Those skilled in the art should understand that other steps or operations may be included before or after the steps of this embodiment, or between any steps, for example, to further optimize and / or improve the method described in the present invention.

[0034] Example 1

[0035] The method for detecting the mutual adhesion ability between resistant live bacteria in this embodiment is as follows:

[0036] The tropical clawed frog, which has a highly similar genome to that of humans, was selected as a model organism. After the tropical clawed frog was euthanized by freezing, fresh intestinal samples were obtained by dissecting it using sterile scissors and forceps.

[0037] The obtained intestinal sample was carefully transferred to a 2 mL sterile centrifuge tube, and was fully minced with sterile scissors. After 300 μL of sterile saline was added, the intestinal sample was ground into a homogenate using a pestle.

[0038] The intestinal homogenate obtained above was centrifuged at 2000 rpm for 5 min, and the upper bacterial layer was carefully pipetted into a new 2 mL centrifuge tube.

[0039] After gradient dilution of the above intestinal bacterial liquid, 100 μL was applied to LB solid culture medium and cultured at 37° C. for 24 h.

[0040] Single colonies with different colony morphology characteristics in the culture dish were selected for counting, and the colonies with the largest number in different morphologies were selected as the dominant bacteria representing the intestinal flora, and the single colony of the morphology was inoculated into LB medium for overnight culture.

[0041] The bacterial culture solution after overnight culture was transferred to new LB medium and re-cultured to the logarithmic growth phase.

[0042] Ciprofloxacin hydrochloride and sulfamethoxazole were selected as bacterial stress antibiotics, and the minimum inhibitory concentration (MIC) of the obtained intestinal dominant bacteria was determined by the microdilution method.

[0043] Add 100 μL of blank MH broth medium to each well of a 96-well plate, wherein the MH broth medium is a hydrolyzed casein broth medium, which is a culture medium specifically used for MIC experiments.

[0044] Add 100 μL of a sterile antibiotic solution with a concentration of 4096 μg / mL to the first well of the first three rows, and pipette thoroughly for at least three times to mix the drug (antibiotic solution) and MH broth thoroughly.

[0045] Pipette 100 μL of the liquid that has been pipetted evenly in the first well and pipet it evenly again in the next well. Repeat this process to the last well and then pipet 100 μL and discard it.

[0046] Wash and dilute to 10 9 100 μL of intestinal bacterial culture with a CFU / mL was added, and three replicates of the MIC value of an antibiotic against fecal intrinsic bacteria were formed; at this time, the drug concentrations in each well were from left to right: 1024 μg / mL, 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, and 0.5 μg / mL.

[0047] A negative control was made in the fourth row (only blank MH broth medium was added), and a positive control was made in the fifth row (only intestinal bacteria solution was added).

[0048] Cover the 96-well plate and incubate at 37°C for 18-24 hours, and observe the results.

[0049] After obtaining the MIC values ​​of ciprofloxacin hydrochloride and sulfamethoxazole against fecal intrinsic bacteria, three different antibiotic stress concentrations of 0.1×MIC, 1×MIC, and 10×MIC were set, respectively.

[0050] Select tropical clawed frog feces that are homologous to intestinal bacteria as the source of intestinal mixed bacteria. Pipette a certain amount of tropical clawed frog feces into a 50mL centrifuge tube and add an appropriate amount of glass beads. Vortex thoroughly and take the upper layer of liquid as the mixed bacterial inoculum source.

[0051] The intestinal mixed bacterial inoculum obtained above was inoculated into LB culture medium containing three different antibiotic stress solutions of 0.1×MIC, 1×MIC and 10×MIC respectively at a ratio of 1%.

[0052] In an ultra-clean workbench, the same volume of the above three mixed intestinal bacterial cultures obtained under the stress of ciprofloxacin hydrochloride and sulfamethoxazole was drawn into a 10 mL centrifuge tube, washed three times with a 0.01 M sterile PBS solution to remove the culture medium, and resuspended in a 0.01 M sterile PBS solution for use.

[0053] After dissociation and obtaining fresh muscovite, 50 μL of 1 mg / mL polyethyleneimine was added dropwise to coat the muscovite so that it carried a positive charge.

[0054] After the surface of the modified mica sheet is dried at room temperature, 5-10 μL of the mixed culture of intestinal bacteria is added dropwise onto the mica sheet and dried at room temperature.

[0055] When there is no obvious liquid on the mica sheet, it is transferred to the sample stage of the atomic force microscope, and an appropriate amount of 0.01 M sterile PBS solution is immediately added to keep the bacteria in a normal physiological state.

[0056] The spherical bacterial probe carrying the spontaneous green fluorescent protein sfGFP labeled Citrobacter freundii was selected for force spectroscopy curve testing.

[0057] Using an atomic force microscope in liquid phase mode, the bacteria immobilized on muscovite were located under bright field microscope observation.

[0058] In the liquid phase test mode, the spherical bacterial probe was used to test the adhesion of bacteria fixed on the mica substrate, and the force spectrum was collected using Forcevolume.

[0059] The force curve test results are shown in Figure 1 , where the abscissa represents distance and the ordinate represents force. The dotted line represents the process of spherical bacteria gradually approaching the bacterial substrate surface. The dotted line starts from point a. Before the spherical bacteria probe contacts the fecal bacteria substrate surface, the dotted line remains horizontal. At point b, the spherical bacteria probe contacts the fecal bacteria substrate surface, and the dotted line bends upward. It stops after reaching the set value at point c and maintains this state for 5s. The solid line represents the process of the spherical bacteria probe being gradually pulled away from the fecal bacteria substrate surface. The solid line starts from point d, and the cantilever straightens at point e. From point e to point f, the cantilever slowly pulls the bacteria in the spherical probe from the fecal bacteria substrate surface. By calculating the absolute value of the ordinate at point f, it can be obtained that the force curve from point f to point g becomes jagged. At point g, the spherical bacteria probe loses contact with the fecal bacteria substrate surface, and the force curve jumps from point g to point h. This proves that the bacterial force curve obtained in the liquid phase test mode is in line with expectations.

[0060] The results of the test on the adhesion between living bacteria under the stress of ciprofloxacin hydrochloride and sulfonamide at different concentrations are shown in Figure 2 , where the horizontal axis represents the antibiotic concentration and the vertical axis represents the adhesion. By analyzing the adhesion of bacteria (i.e., fecal bacteria) to the spherical probe after being stressed by different concentrations of ciprofloxacin hydrochloride and sulfonamide, it was found that the adhesion of fecal bacteria increased with the increase of ciprofloxacin hydrochloride and sulfonamide concentrations, indicating that as the drug resistance of bacteria increases, the mutual adhesion ability of bacteria is enhanced, which is in line with expectations. It shows that the method of the present invention can be used to evaluate the impact of pollution factors (antibiotics, heavy metals, disinfectants, and other environmental pollution factors) on the mutual adhesion of bacterial biofilms and the risk of drug resistance transmission between bacteria.

[0061] The spherical bacteria probe was replaced with an underwater scanning tip probe to test the stability of bacteria fixed on the substrate and obtain bacterial morphology images. The results are shown in Figure 3 It can be observed that after the bacteria are fixed on the modified substrate, the bacterial structure remains intact without damage. In the liquid phase test mode, the bacteria can be stably and firmly fixed on the substrate surface and do not move during the test.

[0062] After the test, the spherical bacterial probe was observed under a high-resolution laser confocal microscope to show that the green fluorescence of the bacteria was normal. Figure 4 , indicating that in the liquid phase testing mode, the bacteria can maintain an active state when the atomic force microscope is collecting bacterial morphology and force spectrum.

[0063] The modified bacterial substrate of the present invention can maintain a uniform and stable state of bacteria fixed on the substrate before and after atomic force microscopy testing; the resistant bacteria in the substrate can maintain normal physiological activity under the liquid phase testing mode, and can be used to evaluate the real adhesion behavior between bacteria under the influence of environmental pollution factors.

[0064] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims

1. A method for detecting the mutual adhesion ability between resistant live bacteria, characterized in that: include: Step S1, providing a drug-resistant bacteria detection substrate with a positively charged surface; Step S2, using a ciprofloxacin hydrochloride antibiotic liquid culture medium and a sulfamethoxazole antibiotic liquid culture medium to perform gradient concentration drug resistance stress culture on fecal intrinsic bacteria, to obtain a first bacterial culture and a second bacterial culture; Step S3, washing the first bacterial culture and the second bacterial culture 2-5 times with a sterile phosphate buffered saline solution with a concentration of 0.1-1 M, and then resuspending them to obtain a first bacterial culture solution and a second bacterial culture solution, and adjusting the concentrations of the first bacterial culture solution and the second bacterial culture solution to 10 7 -10 12 CFU / mL; Step S4, 5-10 μL of 10 7 -10 12 The first bacterial culture solution and the second bacterial culture solution with a concentration of CFU / mL are uniformly and stably added dropwise to the two modified drug-resistant bacteria detection substrates, and dried at room temperature to obtain a first drug-resistant bacteria detection substrate and a second drug-resistant bacteria detection substrate; Step S5, respectively transferring the first drug-resistant bacteria detection substrate and the second drug-resistant bacteria detection substrate to different sample stages of an atomic force microscope, dripping sterile phosphate buffered saline solution on the first drug-resistant bacteria detection substrate and the second drug-resistant bacteria detection substrate to cover them, so that the fecal intrinsic bacteria in the first bacterial culture solution and the second bacterial culture solution maintain a normal physiological state; Step S6, performing adhesion tests on the fecal intrinsic bacteria on the first drug-resistant bacteria detection substrate and the second drug-resistant bacteria detection substrate and the probe bacteria on the spherical bacteria probe of the atomic force microscope, respectively, to obtain a first adhesion curve and a second adhesion curve between the fecal intrinsic bacteria on the first drug-resistant bacteria detection substrate and the second drug-resistant bacteria detection substrate and the probe bacteria.

2. The method for detecting the mutual adhesion ability between resistant live bacteria according to claim 1, characterized in that: After step S6, the method further includes: Step S7, using the underwater scanning tip probe of the atomic force microscope to respectively test the stability of the fecal intrinsic bacteria on the first drug-resistant bacteria detection substrate and the second drug-resistant bacteria detection substrate, and using a high-resolution laser confocal microscope to respectively detect the activity of the probe bacteria, the stability of the fecal intrinsic bacteria includes the stability of its position fixation and the stability of its morphology.

3. The method for detecting the mutual adhesion ability between resistant live bacteria according to claim 1, characterized in that: In the step S6, it also includes: The adhesion between the fecal intrinsic bacteria of the first drug-resistant bacteria detection substrate and the probe bacteria of the atomic force microscope is evaluated by the first adhesion force curve; The adhesion force between the fecal intrinsic bacteria on the second drug-resistant bacteria detection substrate and the probe bacteria of the atomic force microscope is evaluated through the second adhesion force curve.

4. The method for detecting the mutual adhesion ability between resistant live bacteria according to claim 1, characterized in that: In step S6, the liquid phase test mode is selected by the atomic force microscope, the fecal intrinsic bacteria fixed on the drug-resistant bacteria detection substrate are observed and located using the bright field module of the atomic force microscope, and the force curve array mode is used to collect the force spectra corresponding to the first adhesion force curve and the second adhesion force curve.

5. The method for detecting the mutual adhesion ability between resistant live bacteria according to claim 1, characterized in that: In step S6, the probe bacteria are Citrobacter freundii carrying a spontaneous green fluorescent protein sfgfp marker originating from the intestine, and the green fluorescence signal emitted by the super green fluorescent protein sfgfp carried by the probe bacteria is measured by using the EGFP module of a high-resolution laser confocal microscope, wherein the excitation light wavelength of the green fluorescence signal is 488nm.

6. The method for detecting the mutual adhesion ability between resistant live bacteria according to claim 1, characterized in that: In step S1, providing a drug-resistant bacteria detection substrate having a positively charged surface includes: Materials including freshly dissociated mica sheets or glass slides are selected as drug-resistant bacteria detection substrates, and the surface of the drug-resistant bacteria detection substrate is modified by polyethyleneimine so that the surface of the drug-resistant bacteria detection substrate carries a positive charge.

7. The method for detecting the mutual adhesion ability between resistant live bacteria according to claim 6, characterized in that: The mica sheet is muscovite, which is used as the drug-resistant bacteria detection substrate, and 30-100 μL of polyethyleneimine with a concentration of 0.1-4 mg / mL is used to coat the muscovite to achieve the purpose of modifying the surface of the drug-resistant bacteria detection substrate and making the surface of the drug-resistant bacteria detection substrate carry a positive charge.

8. The method for detecting the mutual adhesion ability between resistant live bacteria according to claim 1, characterized in that: In step S2, the method uses a ciprofloxacin hydrochloride antibiotic liquid culture medium and a sulfamethoxazole antibiotic liquid culture medium to perform gradient concentration drug resistance stress culture on fecal intrinsic bacteria to obtain a first bacterial culture and a second bacterial culture, including: The single bacteria with the best growth advantage isolated from the intestinal flora in the early stage were tested for MIC values ​​of ciprofloxacin hydrochloride and sulfamethoxazole, and ciprofloxacin hydrochloride and sulfamethoxazole were selected as antibiotic stress factors; The MIC value of the single bacterium with the best growth advantage is used as the exposure concentration, and the first bacterial culture and the second bacterial culture are obtained by respectively preparing gradient concentrations of ciprofloxacin hydrochloride solution and sulfamethoxazole solution and exposing them to the fecal intrinsic flora homologous to the intestinal flora for stress culture for 10-12 hours.

9. The method for detecting the mutual adhesion ability between resistant live bacteria according to claim 8, characterized in that: The method for determining the MIC value is selected as the microdilution method, and the total volume of the method for determining the MIC value is set to 180-220 μL; wherein the concentrations of ciprofloxacin hydrochloride and sulfonamide-methoxazole stress are respectively set to three different antibiotic concentrations of 0.1×MIC, 1×MIC and 10×MIC; wherein the temperature of the drug resistance stress culture is 37°C±2°C, the time is 8-12h, and the condition is a 150rpm-200rpm constant temperature shaker.

10. Use of the method for detecting the mutual adhesion ability between resistant live bacteria according to any one of claims 1 to 9 in the field of antibiotic resistance transmission.

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