Screening method of high-risk drug-resistant bacteria in biogas slurry application soil

Abstract

The invention belongs to the field of biology, and particularly relates to a method for screening high-risk drug-resistant bacteria in biogas slurry application soil. The method starts with antibiotic selection, sorts and selects important antibiotic types which are currently and clinically used massively and mainly depend on in future clinics, and directly analyzes whether biogas slurry application soil is enriched with drug-resistant bacteria corresponding to the important clinical antibiotics or not. Meanwhile, aiming at the obtained drug-resistant bacteria, combined with a drug sensitive test, multiple drug resistance expressed by the drug-resistant bacteria is detected, so that a set of convenient and rapid method for screening the high-risk drug-resistant bacteria in the biogas slurry application soil is established. In the prior art, the high-risk multi-drug resistance threat of the strain is often ignored, and the multi-drug resistance of the strain is creatively identified by using a drug sensitive test, so that a drug-resistant bacterium risk assessment system is perfected.

Description

Technical Field

[0001] The invention belongs to the biological field, and in particular relates to a method for screening high-risk drug-resistant bacteria in soil where biogas slurry is applied. Background Art

[0003] The risks of bacterial resistance to humans are mainly reflected in: 1. Whether it is enriched in human-related environments; 2. Whether the resistant bacteria are highly pathogenic; 3. The proliferation and spread of resistant bacteria. Although there have been many studies on bacterial resistance in soils with biogas slurry application, most of them focus on the analysis of resistance to veterinary antibiotics, ignoring the risk of clinical drug resistance that may arise in soils with biogas slurry application. Clinically used antibiotics undoubtedly have a higher protective value. Isolating and identifying clinical antibiotic-resistant bacteria and analyzing whether they are multidrug-resistant can predict the pollution circulation process of resistant bacteria, which is of great significance for further inhibiting the spread of resistant bacteria.

[0004] Current research mainly uses the dilution plate method to isolate drug-resistant bacteria in soils with biogas slurry application. The types of drug-resistant bacteria are mainly traditional antibiotic-resistant bacteria or veterinary antibiotic-resistant bacteria. The types of antibiotics selected are less directly related to important clinical antibiotics for humans, and it is impossible to focus on typical high-risk drug-resistant bacteria in soils with biogas slurry application. At the same time, few patents combine the dilution plate method with drug sensitivity testing, so it is impossible to analyze the multi-drug resistance and potential risks of drug-resistant bacteria in soils with biogas slurry application.

[0005] The existing technical methods are mainly as follows:

[0006] 1. A method for screening and identifying antibiotic-resistant bacteria, which is a method for efficient batch screening and identification of antibiotic-resistant bacteria: take a soil sample and mix it with a phosphate buffer, and make soil suspensions of different gradients after dilution. Add antibiotic solution (the target antibiotics are erythromycin, streptomycin, and polymyxin B sulfate veterinary drugs) to LB culture medium, pour the plate and spread the soil suspension on the plate, culture and observe to determine the optimal dilution gradient. Spread the plate in batches at the optimal gradient, pick the colonies with a sterilized pipette tip after culture, shake and culture, and use glycerol for preservation. The strain is identified using high-throughput sequencing.

[0007] 2. Wang Y, Sutton NB, Zheng YH, et al. Effect of Wheat Crops on the Persistence and Attenuation of Antibiotic Resistance Genes in Soil after Swine Wastewater Application[J]. Journal of Hazardous Materials, 2024: 133759, informed the following method: different types of soil samples and biogas slurry were collected (different soil types are divided into: clay, sandy soil, loam), and a certain amount of biogas slurry was applied to the soil samples. After DNA was extracted, high-throughput sequencing was used to analyze the changes in drug resistance in different experimental groups. The experimental groups were divided into soil with vegetation and bare soil and different soil types. The target antibiotics were mostly veterinary tetracyclines, sulfonamides, etc.

[0008] 3. Pu ChengJun PCJ, Liu Hang LH, Ding GuoChun DGC, et al. Impact of direct application of biogas slurry and residue in fields: in situ analysis of antibiotic resistance genes from pig manure to fields[J]. 2018. The following method was reported: soil samples that had been applied with biogas slurry for 10 years and soils of similar soil types that had not been applied with biogas slurry were collected as controls. The soil samples were dissolved in physiological saline to prepare a soil suspension, which was then gradiently diluted and spread on a selective culture medium (the target antibiotics were sulfamethoxypyridine (SUL); tetracycline (TET), norfloxacin (NOR), erythromycin (ERY), vancomycin (VAN); veterinary drugs such as ceftriaxone (CTX) and streptomycin (STR)). The number of colonies was counted after inverted culture, and the comparison was used to determine whether the application of biogas slurry induced the enrichment of drug-resistant bacteria.

[0009] The above prior art has the following disadvantages:

[0010] 1. The antibiotics selected are mostly traditional antibiotics and veterinary antibiotics, ignoring the resistance of resistant bacteria in the soil where biogas slurry is applied to important clinical antibiotics.

[0011] 2. It is impossible to identify the multi-drug resistance of strains, and the analysis of bacterial resistance risk is incomplete. Summary of the invention

[0012] The technical problem to be solved by the present invention is to provide a method for screening high-risk drug-resistant bacteria in soil where biogas slurry is applied.

[0013] Note: Both the selection of clinically important antibiotics and multidrug resistance are manifestations of drug resistance. Therefore, high-risk resistant bacteria include resistant bacteria that are resistant to important antibiotics (including multidrug resistance).

[0014] In order to solve the above technical problems, the present invention provides a method for screening high-risk drug-resistant bacteria in soil where biogas slurry is applied, comprising the following steps:

[0015] 1) Set the antibiotics to be tested:

[0016] These include cephalosporins, quinolones, carbapenems, and glycopeptide antibiotics;

[0017] 2) Screening of drug-resistant bacteria includes the following steps:

[0018] 2.1) Prepare soil suspension with gradient concentration;

[0019] The soil includes a soil sample to be tested that has been subjected to long-term biogas slurry application and a control soil sample with a similar geographical location and soil properties to the soil sample to be tested;

[0020] 2.2) Adding antibiotics and cycloheximide of set concentrations to the R2A nutrient medium to prepare a selective medium;

[0021] Note: Add antibiotics at a set concentration to the R2A nutrient medium. The set concentration is the maximum amount that the bacteria can withstand as set by the Clinical and Laboratory Standards Institute (CLSI) of the United States, to ensure that the cultured bacteria have strong drug resistance.

[0022] 2.3) Evenly spread the soil suspension on the selective culture medium with the gradient concentration corresponding to the antibiotic, and invert the culture medium (which can be cultured at a constant temperature of 7°C) to determine the best dilution gradient of the antibiotic; the maximum gradient when the number of colonies in the plate is no more than 200 is determined as the best dilution gradient;

[0023] 2.4) After culturing the soil samples to be tested with different dilution gradients of biogas slurry for a long time on the selective medium corresponding to the antibiotic for 2 days, the soil suspension with the best dilution gradient was selected, and the plates were spread in batches on the selective medium corresponding to the antibiotic, inverted for cultivation, and independent colonies with better growth were picked;

[0024] Similarly, after the soil samples to be tested with long-term biogas slurry application at different dilution gradients were cultured on the selective medium corresponding to the antibiotic for 2 days, the soil suspension at the best dilution gradient was selected, and the plates were spread in batches on the selective medium corresponding to the antibiotic and cultured in an inverted manner;

[0025] The optimal gradient culture medium corresponding to the same antibiotic is selected, and the number of colonies grown on the plates of the soil sample applied with biogas slurry and the control soil sample is counted to achieve screening of drug-resistant bacteria (i.e., the enrichment of culturable related antibiotic-resistant bacteria is obtained).

[0026] As an improvement of the method for screening high-risk drug-resistant bacteria in soil after biogas slurry application of the present invention, the method further includes the following step 3):

[0027] For the acquired drug-resistant bacteria, drug sensitivity tests are performed to identify strains with multidrug resistance.

[0028] As a further improvement of the method for screening high-risk drug-resistant bacteria in soil after biogas slurry application of the present invention:

[0029] The cephalosporin antibiotics include cefuroxime and cefoperazone;

[0030] The quinolone antibiotics include moxifloxacin and levofloxacin;

[0031] The carbapenem antibiotics include imipenem and meropenem;

[0032] The glycopeptide antibiotics include vancomycin (vancomycin hydrochloride) and polymyxin B.

[0033] As a further improvement of the method for screening high-risk drug-resistant bacteria in soil after biogas slurry application of the present invention:

[0034] The step 2.1) is:

[0035] Soil samples were mixed with tryptone soy broth (TSB) at a ratio of 1 g soil sample to 9 ml TSB to activate the bacterial flora in the soil sample. After shaking, the supernatant was aspirated with a pipette and graded diluted with sterile deionized water to prepare soil suspensions of different gradients (gradient dilution is convenient for counting);

[0036] Soil suspensions of different gradients, specifically:

[0037] 9 ml of TSB solution was mixed with 1 g of soil sample to prepare ×10 times TSB donor, 1 ml of ×10 times TSB donor was mixed with 9 ml of deionized water to prepare ×100 times TSB donor; 1 ml of ×100 times TSB donor was mixed with 9 ml of deionized water to prepare ×1k times TSB donor; and so on, ×100, ×1k, ×1w, ×10w, ×100w TSB donors were prepared; as soil suspensions of different gradients;

[0038] When the soil sample is a soil sample to which biogas slurry has been applied for a long time, the obtained soil suspensions are soil suspensions of different gradients corresponding to the soil sample to which biogas slurry has been applied for a long time;

[0039] When the soil sample is a control soil sample, the obtained soil suspensions are of different gradients corresponding to the control soil sample.

[0040] Note: “×100” represents 1% of the above-set concentration, and ×1k represents 1‰ of the above-set concentration; and the rest can be deduced by analogy.

[0041] As a further improvement of the method for screening high-risk drug-resistant bacteria in soil after biogas slurry application of the present invention:

[0042] The set concentration of antibiotics is: Imipenem: 16ug·ml -1 , Meropenem: 16ug·ml -1 , Levofloxacin: 8ug·ml -1 , Moxifloxacin: 8ug ml -1 , Cefoperazone: 64ug·ml -1 , Cefuroxime acid: 32ug·ml -1 , Vancomycin (vancomycin hydrochloride): 32ug·ml -1 , Polymyxin B: 4ug·ml -1 ;

[0043] The set concentration of cycloheximide is: 100ug ml -1 .

[0044] As a further improvement of the method for screening high-risk drug-resistant bacteria in soil after biogas slurry application of the present invention:

[0045] The specific identification of multi-drug resistance of strains by drug sensitivity test is as follows:

[0046] Pick out the independent colonies with better growth in the biogas slurry application plate (culture in the optimal gradient for two days, and pick out the colonies with better growth after culture), mix with TSB liquid culture medium, and spread on MH(A) culture medium (MH(A) culture medium is a culture medium commonly used for drug sensitivity test), paste different types of drug sensitivity paper slips on it, invert and culture, observe and measure the diameter of the inhibition zone produced therein, and compare with the CLSI drug sensitivity standard to obtain the drug resistance of the bacteria to different antibiotics (in addition to the test bacteria, daily quality control is also required, that is, the scientific nature of the drug sensitivity test results of the day are tested using standard strains).

[0047] The present invention starts with the selection of antibiotics, sorts out and selects the important antibiotics currently used in large quantities in clinical practice and mainly relied on in the future, and directly analyzes whether the resistant bacteria corresponding to the above important clinical antibiotics are enriched in the soil where biogas slurry is applied. At the same time, for the obtained resistant bacteria, combined with drug sensitivity tests, the multi-drug resistance expressed by them is detected, so as to try to establish a convenient and fast method for screening high-risk resistant bacteria in soil where biogas slurry is applied.

[0048] The beneficial effects of the present invention are mainly reflected in:

[0049] 1. We selected types of antibiotics that are widely used in clinical practice or will become relied upon in the future by humans, and monitored clinical antibiotic-resistant bacteria enriched in soil after biogas slurry application.

[0050] The present invention starts with the selection of antibiotics and studies the types of antibiotics that are currently widely used in clinical practice or will become relied upon in the future. The corresponding drug-resistant bacteria undoubtedly pose a greater risk to human society. At the same time, it warns related research fields not to ignore the clinical drug resistance risks that may arise from the application of biogas slurry to soil.

[0051] 2. An innovative drug sensitivity test was added to detect the multi-drug resistance expressed by the extracted strains.

[0052] The existing technology often ignores the high-risk multi-drug resistance threat of strains. The present invention innovatively uses drug sensitivity tests to identify the multi-drug resistance of strains, thereby improving the risk assessment system for drug-resistant bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0054] Figure 1 This is the intensity chart of clinical antibiotic use in China from 2001 to 2015;

[0055] Figure 2 The trend of the intensity of clinical antibiotic use in China from 2001 to 2015. (A) The total change rate of the intensity of clinical antibiotic use in China from 2001 to 2015; (B) The heat map of the annual change of the intensity of clinical antibiotic use in China from 2001 to 2015. DETAILED DESCRIPTION

[0056] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0057] The culture medium involved in the present invention is as follows:

[0058] Tryptone soy broth (TSB) (per 30 g): tryptone 17 g, soy peptone 3 g, sodium chloride 5 g, potassium dihydrogen phosphate 2.5 g, glucose 2.5 g;

[0059] R2A nutrient medium (per 15.2 g): tryptone 0.25 g, acid hydrolyzed casein 0.5 g, soluble starch 0.5 g, magnesium sulfate 0.1 g, agar 12.0 g, peptone 0.25 g, yeast extract powder 0.5 g, glucose 0.5 g, dipotassium hydrogen phosphate 0.3 g, sodium pyruvate 0.3 g;

[0060] MH(A) medium (per 36.5 g): starch 1.5 g, agar 12.5 g, beef extract powder 5 g, casein hydrolyzate 17.5 g.

[0061] 1. Choice of clinical antibiotics:

[0062] We used the data website (https: / / resistancemap.onehealthtrust.org / AntibioticUse.php, with existing literature citations as evidence) to retrieve the usage intensity of various types of clinical antibiotics in China from 2000 to 2015. Combined with the data from relevant literature research, we analyzed and confirmed that there are currently large-scale clinical uses and a significant upward trend, and these antibiotics may become the types of antibiotics that will be relied upon in clinical practice in the future.

[0063] Supplementary data:

[0064] Figure 1 This reflects the clinical use intensity of various antibiotics in China from 2001 to 2015. Figure 1 It can be seen that the five most commonly used antibiotics in China from 2001 to 2015 are Cephalosporins, Broadspectrum, Tetracyclines, Fluoroquinolones, and Macrolides. Among the five antibiotics mentioned above, tetracyclines are widely used in livestock and poultry farming. [1] , and there are currently a lot of studies on tetracycline resistance in livestock and poultry farming. At the same time, the clinical application of macrolide antibiotics has been hindered in recent years. For example, the new type of pneumonia Mycoplasma is highly resistant to it, and even the resistance rate in China has reached more than 90%. Studies have also shown that there are many types of adverse reactions to macrolide antibiotics. [2-4] Penicillin antibiotics are the earliest discovered type of antibiotics and are indispensable in clinical applications. However, in the long-term application process, the resistance rate and adverse reaction rate of penicillin antibiotics are also increasing. It has been reported that the number of adverse reactions caused by penicillin in a certain hospital from 2006 to 2020 ranked second, accounting for 21.9%. [5] At the same time, in recent years, penicillin antibiotics have been used more frequently in combination, such as acamycin combined with piperacillin to treat severe pneumonia in the elderly. [6]Etc. The resistance rates of the three antibiotics Broad specturm (broad-spectrum penicillins), Macrolides (macrolides), and Tetracclines (tetracyclines) are constantly increasing, and there are many studies of the same type, so they will not be the focus of this study. In comparison, cephalosporin antibiotics are the most used anti-infective drugs. From 2012 to 2018, a hospital in Beijing found that cephalosporins had the highest sales share among different types of antibiotics, accounting for more than half of the total sales. Similarly, hospitals in Lanzhou and Jiangxi also found in a 2022 survey that the most frequently used drug in recent years was cefuroxime, and the drug with the highest amount of money was cefoperazone. [7-10] .

[0065] Figure 2 It reflects the changing trend of the intensity of clinical antibiotic use in China from 2001 to 2015. It can be seen from the figure that carbaenems (carbapenem antibiotics) and glycopeptides (glycopeptide antibiotics) have increased significantly, while tetracyclines and quinolone antibiotics have increased slightly, macrolide antibiotics have remained basically unchanged, and the remaining antibiotics have all shown a downward trend. The intensity of use of quinolone antibiotics has remained high in recent years. A hospital surveyed in 2018 and found that quinolone antibiotics were the most used antibiotic category in the selection of antibiotics in the gastroenterology department. There were also reports in 2023 that the intensity of use of levofloxacin in a hospital from January 2021 to March 2022 ranked among the top ten in all departments. [11,12] A hospital in Shanghai also reported in 2023 that moxifloxacin was the main temporary antibacterial drug purchased in the past year.

[13] With the continuous improvement of bacterial resistance, carbapenem antibiotics have been widely used in recent years due to their unique advantages in dealing with serious infections and complicated infections. A hospital in Jiangsu reported in 2021 that the clinical use of carbapenem antibiotics DDDs, DUI, total consumption amount, and annual total use showed an upward trend in the four years from 2015 to 2018

[14] . A hospital in Shanxi also reported that clinical carbapenem antibiotics were mainly imipenem and meropenem, and the frequency of use was also increasing year by year.

[15] Glycopeptide antibiotics are a new type of antibacterial drug that has emerged in recent years. Vancomycin was once used as the last line of defense against Gram-positive bacterial infections.

[16] However, in 2019, it was reported that among the 187 traditional antibacterial projects in clinical development, only about 10% targeted Gram-positive bacteria.

[17] . In recent years, due to the continuous iteration of multidrug-resistant bacteria, the clinical application of glycopeptide antibiotics has received attention. In 2023, there were reports showing that the intensity of vancomycin use was at a high level. In the same year, a hospital in Shanghai also issued a document stating that the highest number of applications and costs for temporary procurement of antibacterial drugs between July 2021 and June 2022 were for injection of polymyxin B sulfate.[11,13] .

[0066] In summary, the present invention finally selected cephalosporins (cefuroxime, cefoperazone), quinolones (moxifloxacin, levofloxacin), carbapenems (imipenem, meropenem), and glycopeptides (vancomycin (i.e., vancomycin hydrochloride), polymyxin B) antibiotics as research objects.

[0067] 2. Screening of drug-resistant bacteria:

[0068] Note: The present invention is all an aerobic environment.

[0069] 2.1) Collect soil samples with long-term biogas slurry application and control soil samples with similar geographical locations and soil properties. That is, the control soil samples are soil samples with similar soil properties to the biogas slurry application soil samples, but without artificial biogas slurry application.

[0070] Take a soil sample and mix it with tryptone soy broth (TSB) medium in the ratio of 1g soil sample to 9ml TSB medium (to activate the bacterial flora in the soil sample). After shaking, use a pipette to aspirate the supernatant and use sterile deionized water to perform gradient dilution to prepare soil suspensions of different gradients (gradient dilution is convenient for counting).

[0071] therefore:

[0072] When the soil sample is a soil sample to which biogas slurry has been applied for a long time, the obtained soil suspensions are soil suspensions of different gradients corresponding to the soil sample to which biogas slurry has been applied for a long time;

[0073] When the soil sample is a control soil sample, the obtained soil suspensions are of different gradients corresponding to the control soil sample.

[0074] 2.2) Add any antibiotic of the concentration described in Table 1 and cycloheximide to the R2A nutrient medium to prepare a selective medium corresponding to the antibiotic.

[0075] Note: The set concentrations of different antibiotics are the maximum amounts that the strains can withstand as set by the Clinical and Laboratory Standards Institute (CLSI) of the United States, to ensure that the cultured strains have extremely strong drug resistance, as shown in Table 1 below.

[0076] Table 1

[0077] Drug Name Target concentration / ug·ml-1 Imipenem 16 Meropenem 16 Levofloxacin 8 Moxifloxacin 8 Cefoperazone 64 Cefuroxime Acid 32 Vancomycin Hydrochloride 32 Polymyxin B 4 Cycloheximide 100

[0078] The gradient of soil suspension was set to ×100, ×1k, ×1w, ×10w, and ×100w;

[0079] illustrate:

[0080] ×100 represents 1% of the above-set concentration, ×1k represents 1‰ of the above-set concentration, and the rest can be deduced by analogy.

[0081] The dilution gradient is only for soil suspension; the concentration of antibiotics directly determines the strength of the selective culture medium, so it is fixed to the concentration shown in Table 1 (i.e., the maximum drug resistance concentration of each CLSI strain).

[0082] 2.3) Spread the soil suspensions with different dilution gradients evenly on the selective culture medium with the corresponding concentration of the antibiotic, invert and culture (7°C constant temperature culture) and observe and determine the best soil suspension dilution gradient for each antibiotic (when the number of colonies in the plate is no more than 200, it is determined to be the best soil suspension dilution gradient corresponding to the antibiotic).

[0083] 2.4) The soil suspension with the best dilution gradient is applied to the antibiotic-related selective culture medium in batches and plated and inverted for culture. The number of colonies grown on the plates of the biogas slurry-applied soil sample and the control soil sample is counted and compared to obtain the enrichment of culturable related antibiotic-resistant bacteria.

[0084] 3. Drug sensitivity test to identify multi-drug resistance of strains:

[0085] Pick out the independent colonies with better growth in the biogas slurry application plate (culture in the optimal gradient for two days, and pick out the colonies with better growth after culture), mix with TSB liquid culture medium, and spread on MH(A) culture medium (MH(A) culture medium is a culture medium commonly used for drug sensitivity test), paste different types of drug sensitivity paper slips on it, invert and culture, observe and measure the diameter of the inhibition zone produced therein, and compare with the CLSI drug sensitivity standard to obtain the drug resistance of the bacteria to different antibiotics (in addition to the test bacteria, daily quality control is also required, that is, the scientific nature of the drug sensitivity test results of the day are tested using standard strains).

[0086] Experiment 1

[0087] 1. Select the antibiotics shown in Table 1;

[0088] 2. The soil samples for long-term biogas slurry application were selected from vegetable garden soil in Jinhua, Zhejiang Province, where biogas slurry had been applied for 18 years. The control soil samples were selected from soil with similar geographical location and soil properties.

[0089] According to the above "II. Screening of drug-resistant bacteria", the specific process is as follows:

[0090] 1) Preparation of TSB donor

[0091] 1. Weigh 7.5 g of TSB solid powder, dissolve it in 250 ml of deionized water and place it in a 500 ml conical flask to obtain TSB solution.

[0092] 2. Place the TSB solution and pipette tips in a high temperature and high pressure sterilizer at 121℃ for 15 minutes.

[0093] 3. Weigh 1g of soil sample and set aside.

[0094] 4. Wait for the TSB solution to cool down before operating on a sterile operating table.

[0095] 5. Use a pipette to draw up 9 ml of TSB solution and mix it with 1 g of soil sample to make a ×10 TSB donor, and place it in a conical flask.

[0096] Seal the conical flask with gauze and place it on a shaker at 28°C and 50 rpm for 12 h.

[0097] 2) Serial dilution of TSB donor

[0098] 1. Use a measuring cylinder to measure 250 ml of deionized water into a 500 ml conical flask.

[0099] 2. Sterilize deionized water, pipette tips, and 10ml centrifuge tubes in a high-temperature and high-pressure sterilizer at 121℃ for 15 minutes.

[0100] 3. Use a pipette to draw 1 ml of ×10 TSB donor and mix it with 9 ml of deionized water to prepare ×100 TSB donor.

[0101] 4. Use a pipette to draw 1 ml of ×100 TSB donor and mix it with 9 ml of deionized water to prepare ×1k TSB donor.

[0102] 5. Similarly, prepare TSB donors of ×100, ×1k, ×1w, ×10w, and ×100w; use this as soil suspension with gradient concentration.

[0103] 6. Store the donor in a 4°C refrigerator.

[0104] 3) Screening test

[0105] 1. According to the instructions provided by Solebow, weigh 3.8 g of R2A culture medium powder, dissolve it in 250 ml of deionized water and place it in a 500 ml conical flask to obtain R2A culture medium.

[0106] 2. Seal the R2A culture medium, pipette tip, and glass applicator and place them in a high-temperature and high-pressure sterilizer at 121℃ for 15 minutes.

[0107] 3. Adjust the water bath to 55°C. After sterilization, place the R2A medium in a water bath to cool down.

[0108] 4. Operate on a sterile operating table after cooling.

[0109] 5. Use a pipette to add cycloheximide of a specified concentration (as described in Table 1 above) and any of the antibiotics in Table 1 to the R2A medium, and shake it slightly to obtain a selective medium corresponding to the antibiotic; three parallel selective mediums corresponding to each antibiotic are set up;

[0110] Note: Cycloheximide is added to inhibit fungal growth. Cycloheximide should be added to the culture medium each time and an antibiotic should be selected for addition;

[0111] 6. Quickly pour the selective culture medium of corresponding concentration into the blank culture dish and open the lid of the culture dish to release water vapor.

[0112] 7. After the culture medium solidifies, close the lid, mark the name and turn it upside down.

[0113] 8. Use a pipette to draw 100ul of TSB donor dilution and inject it on the surface of the selective culture medium as the experimental group;

[0114] 9. Place the applicator under the flame of an alcohol lamp for 3 seconds and then cool for 10 seconds.

[0115] 10. Use a spreader to evenly spread the TSB donor dilution on the solid culture medium and then invert it.

[0116] 11. Place the culture medium in a 37°C incubator for 2 days and count.

[0117] The final results are shown in Table 2:

[0118] Table 2

[0119]

[0120] Note: The above values ​​represent the number of resistant bacteria that can be cultured in the plate multiplied by the corresponding dilution factor and then multiplied by 10 (i.e. the number of resistant colonies contained in 1g of soil).

[0121] In the preliminary experiment, the present invention found that the morphology of the colonies grown on the plate was similar, so a colony with better growth among the same type of colonies was selected for drug sensitivity test; if there were two obvious types of colonies on the plate, the colony with better growth among each type would be selected for subsequent tests.

[0122] 2. The obtained "independent colonies with better growth in the biogas slurry application plate" were subjected to the above "3. Drug sensitivity test to identify multi-drug resistance of strains". The specific process is as follows:

[0123] 1) Isolation of drug-resistant bacteria

[0124] 1. Weigh 7.5 g of TSB solid powder, dissolve it in 250 ml of deionized water and place it in a 500 ml conical flask to obtain TSB culture medium.

[0125] 2. Seal the conical flask containing TSB medium, together with a 10ml centrifuge tube and a 5ml pipette tip with gauze and kraft paper, sterilize it in a high-temperature and high-pressure sterilizer at 121℃ for 15 minutes, and cool it for later use.

[0126] 3. In a sterile operating table, pipette 10 ml of cooled TSB culture medium into a centrifuge tube for later use.

[0127] 4. Use an alcohol lamp to burn the inoculation loop until it turns slightly red, then cool it down, and repeat this process three times.

[0128] 5. Select relatively independent and full colonies in the screening experiment, pick them up with an inoculation loop, immerse them in TSB culture medium and wait for them to fuse to obtain the test bacterial solution, label them and store them in a refrigerator at 2-8℃.

[0129] 2) Paper diffusion method drug sensitivity test

[0130] 1. Weigh 9.125 g of MH(A) solid powder, dissolve it in 250 ml of deionized water and place it in a 500 ml conical flask to obtain MH(A) culture medium.

[0131] 2. Place the conical flask containing MH(A) medium, together with a 1 ml pipette tip and a coating rod, seal it with gauze and kraft paper in a high-temperature and high-pressure sterilizer at 121°C for 15 minutes, and then place it in a 55°C water bath to cool down.

[0132] 3. Spread the drug-sensitive bacterial solution evenly on the MH(A) solid culture medium.

[0133] 4. Evenly stick the drug-sensitive paper strips on the MH(A) medium coated with the bacterial solution (no more than 4 strips per plate, and the center distance between the strips should be no less than 24 mm), and then culture in a 37°C incubator for 16-18 hours.

[0134] Note: Daily drug sensitivity tests must be supported by the drug sensitivity of the quality control strains of the day.

[0135] 5. Record the diameter of the inhibition zone and compare it with the inhibition zone standard (Table 3) to determine the drug sensitivity.

[0136] Table 3. Determination of drug sensitivity results

[0137]

[0138]

[0139] The final results are shown in Table 4 below:

[0140] Table 4

[0141]

[0142]

[0143] illustrate:

[0144] The naming rule is JH (Jinhua place name)-1 (sample number)-1 (simple number of colonies picked out from plates with different antibiotics). Generally, 1-2 more representative strains are picked from the plates corresponding to each antibiotic.

[0145] Verification 1. Experiment 1 The drug sensitivity test was quality controlled according to the conventional methods of the industry. The specific methods are as follows:

[0146] 1. Weigh 9.125 g of MH(A) solid powder, dissolve it in 250 ml of pure water and place it in a 500 ml conical flask to obtain MH(A) culture medium.

[0147] 2. Place the conical flask containing MH(A) medium, together with a 1 ml pipette tip and a coating rod, seal it with gauze and kraft paper in a high-temperature and high-pressure sterilizer at 121°C for 15 minutes, and then place it in a 55°C water bath to cool down.

[0148] 3. Pick ATCC25922 and ATCC25923 standard strains and spread them evenly on MH(A) solid culture medium.

[0149] 4. Evenly stick the drug-sensitive paper strips on the MH(A) medium coated with the bacterial solution (no more than 4 strips per plate, and the center distance between the strips should be no less than 24 mm), and then culture in a 37°C incubator for 16-18 hours.

[0150] 5. Record the diameter of the inhibition zone and compare it with the QC standard range (Table 5) to judge whether the drug sensitivity test environment is qualified (the failure rate does not exceed 10%).

[0151] Table 5. QC range of disk diffusion method

[0152]

[0153]

[0154] The results are shown in Table 6 below:

[0155] Table 6

[0156]

[0157]

[0158] Note: —— represents not tested, √ represents data within the range of the above table "QC Standard for Paper Diffusion Method", and × represents data not within the range

[0159] A pass rate of over 90% proves that the drug sensitivity test environment is qualified and the drug sensitivity data is valid.

[0160] Experiment 2: Change the soil sample in Experiment 1 to paddy field soil that has been treated with biogas slurry for 5 years; the rest is the same as Experiment 1.

[0161] The results are shown in Table 7 below:

[0162] Table 7

[0163]

[0164]

[0165] Experiment 3: Change the soil sample in Experiment 1 to: soil from vegetable fields that have been treated with biogas slurry for 10 years; the rest is the same as Experiment 1.

[0166] The results are shown in Table 8 below:

[0167] Table 8

[0168]

[0169] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

[0170] The references involved in the present invention are as follows:

[0171] [1]TANG

[0172] [2] Tan Jianqing. The occurrence and guidance of adverse reactions of macrolide antibiotics[J]. Northern Pharmacy, 2022, 19(03): 100-2.

[0173] [3]GUO DX,HU WJ,WEI R,et al.Epidemiology and mechanism of drugresistance of Mycoplasma pneumoniae in Beijing,China:A multicenter study[J].Bosnian journal of basic medical sciences,2019,19(3):288.

[0174] [4]GUO Z, LIU L, GONG J, et al. Molecular features and antimicrobialsusceptibility of Mycoplasma pneumoniae isolates from paediatric inpatients in Weihai, China: characteristics of M. pneumoniae in Weihai[J]. Journal of Global Antimicrobial Resistance, 2022, 28: 180-4.

[0175] [5] Yang Wenfen, Wang Chunsheng. Analysis of adverse reactions to antimicrobial drugs in Hangu Hospital of Traditional Chinese Medicine, Binhai New Area, Tianjin from 2006 to 2020[J]. Modern Drugs and Clinics, 2021, 36(08): 1728-32.

[0176] [6] Yu Jihong, Yan Zhongqiang, Zhao Xiumei, et al. Clinical efficacy of amikacin combined with piperacillin-tazobactam sodium in the treatment of severe pneumonia in the elderly[J]. Chinese Journal of Hospital Infection, 2020, 30(18): 2759-63.

[0177] [7] Kong Yuwei. Analysis of trends and characteristics of antibiotic and anti-tuberculosis drug use in hospitals in Beijing from 2012 to 2018[D]; Bengbu Medical College, 2020.

[0178] [8] Wang Min, Wang Hui. Investigation on the use of antibiotics in our hospital in the past three years and analysis of rational drug use[J]. Gansu Science and Technology, 2022.

[0179] [9] Wang Yusheng. Development of cephalosporin drugs and process development of cefuroxime[J]. Journal of Applied Technology, 2023, 23(03): 255-64.

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[10] Zhang Chuanlin, Xu Xiaoting, Liu Xiaolian. Analysis of the use of antibiotics in hospitalized patients in a certain hospital from 2021 to 2022[J]. Anti-Infective Pharmacy, 2023, 20(04): 394-7+402.

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[11] Yang Yanlin, Wang Chao, Liu Jie, et al. Investigation and analysis of antibiotic use in primary hospitals[J]. Journal of Traditional Chinese Medicine Management, 2023, 31(04): 99-102.

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[12] Zhu Xiaoli, Xue Yun. Analysis of antibiotic use in a ward of the gastroenterology department of our hospital from January to December 2018[J]. Chinese Journal of Drug Abuse Prevention and Treatment, 2020, 26(2):76-7.

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[13] Qu Haijing, Gao Yue. Analysis of temporary procurement and application of antimicrobial drugs in hospitals[J]. Chinese Journal of Pharmaceutical Science, 2023, 13(10): 171-4.

[0184]

[14] Zhang Yi, Cheng Yunjie. Analysis of clinical use of carbapenem antibiotics in a certain hospital from 2015 to 2018[J]. Anti-Infective Pharmacy, 2021, 18(02): 174-8.

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[15] Lü Jian, Lü Lei. Analysis of the use of carbapenem antibiotics in our hospital from 2017 to 2018[J]. Medical Theory and Practice, 2020, 33(15): 2548-9.

[0186]

[16] MERKEL AB,TEMPLE GK,BURKART MD,et al.Purification,crystallization and preliminary structural studies of dTDP-4-keto-6-deoxy-glucose-5-epimerase(EvaD)from Amycolatopsis orientalis,the fourth enzyme in the dTDP-L-epivancosamine biosynthetic pathway[J].Acta CrystallographicaSection D:Biological Crystallography,2002,58(7):1226-8.

[0187]

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Claims

1. A method for screening high-risk drug-resistant bacteria in soil after biogas slurry application, characterized in that The following steps are involved: 1) Set the antibiotics to be tested: These include cephalosporins, quinolones, carbapenems, and glycopeptide antibiotics; 2) Screening of drug-resistant bacteria includes the following steps: 2.1) Prepare soil suspension with gradient concentration; The soil includes a soil sample to be tested that has been long-term treated with biogas slurry and a control soil sample with similar geographical location and soil properties to the soil sample to be tested; 2.2) Add antibiotics and cycloheximide of set concentrations to the R2A nutrient medium to prepare a selective medium; 2.3) Evenly spread the soil suspension on the selective culture medium with the corresponding gradient concentration of the antibiotic, invert the culture medium, and determine the best dilution gradient of the antibiotic; the maximum gradient when the number of colonies in the plate is no more than 200 is determined as the best dilution gradient; 2.4) After culturing the soil samples to be tested with different dilution gradients of biogas slurry for a long time on the selective medium corresponding to the antibiotic for 2 days, the soil suspension with the best dilution gradient was selected, and the plates were spread in batches on the selective medium corresponding to the antibiotic, inverted for cultivation, and independent colonies with better growth were picked; Similarly, after the soil samples to be tested with long-term biogas slurry application at different dilution gradients were cultured on the selective medium corresponding to the antibiotic for 2 days, the soil suspension at the best dilution gradient was selected, and the plates were spread in batches on the selective medium corresponding to the antibiotic and cultured in an inverted manner; The optimal gradient culture medium corresponding to the same antibiotic was selected, and the number of colonies grown on the plates of the soil samples treated with biogas slurry and the control soil samples was counted to screen for drug-resistant bacteria.

2. The method for screening high-risk drug-resistant bacteria in soil after biogas slurry application according to claim 1, characterized in that It also includes the following step 3): For the acquired drug-resistant bacteria, drug sensitivity tests are performed to identify strains with multidrug resistance.

3. The method for screening high-risk drug-resistant bacteria in soil after biogas slurry application according to claim 1 or 2, characterized in that: The cephalosporin antibiotics include cefuroxime and cefoperazone; The quinolone antibiotics include moxifloxacin and levofloxacin; The carbapenem antibiotics include imipenem and meropenem; The glycopeptide antibiotics include vancomycin and polymyxin B.

4. The method for screening high-risk drug-resistant bacteria in soil after biogas slurry application according to claim 3, characterized in that: The step 2.1) is: Soil samples were mixed with tryptone soy broth (TSB) at a ratio of 1 g soil sample to 9 ml TSB to activate the bacterial flora in the soil samples. After shaking, the supernatant was aspirated with a pipette and graded diluted with sterile deionized water to prepare soil suspensions of different gradients. Soil suspensions of different gradients, specifically: 9 ml TSB solution was mixed with 1 g soil sample to prepare ×10 times TSB donor, 1 ml ×10 times TSB donor was mixed with 9 ml deionized water to prepare ×100 times TSB donor; 1 ml ×100 times TSB donor was mixed with 9 ml deionized water to prepare ×1k times TSB donor; and so on, ×100, ×1k, ×1w, ×10w, ×100w TSB donors were prepared; as soil suspensions of different gradients; When the soil sample is a soil sample to which biogas slurry has been applied for a long time, the obtained soil suspensions are soil suspensions of different gradients corresponding to the soil sample to which biogas slurry has been applied for a long time; When the soil sample is a control soil sample, the obtained soil suspensions are of different gradients corresponding to the control soil sample.

5. The method for screening high-risk drug-resistant bacteria in soil subjected to biogas slurry application according to claim 4, characterized in that: The set concentration of antibiotics is: Imipenem: 16ug·ml -1 , Meropenem: 16ug·ml -1 , Levofloxacin: 8ug·ml -1 , Moxifloxacin: 8ug ml -1 , Cefoperazone: 64ug·ml -1 , Cefuroxime acid: 32ug·ml -1 , Vancomycin: 32ug·ml -1 , Polymyxin B: 4ug·ml -1 ; The set concentration of cycloheximide is: 100ug ml -1 .

6. The method for screening high-risk drug-resistant bacteria in soil subjected to biogas slurry application according to any one of claims 2 to 5, characterized in that: The specific identification of multi-drug resistance of strains by drug sensitivity test is as follows: Independent colonies with better growth in the biogas slurry application plate were picked up, mixed with TSB liquid culture medium, and then spread on MH(A) culture medium. Different types of drug-susceptibility paper slips were pasted on the medium. After inverted culture, the diameter of the inhibition zone produced was observed and measured, and compared with the CLSI drug sensitivity standard, the drug resistance of the bacteria to different antibiotics was obtained.

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