A Bacillus strain BAC4 that blocks the spread of ARGs by soil pathogens and its application

Abstract

The present invention discloses a strain of Bacillus BAC4 that blocks the transmission of ARGs by soil pathogens, which is classified and named as Bacillus pseudomycoides, and the strain deposit number is CGMCC NO.30925. Under the treatment of Bacillus BAC4 or its fermentation liquid, the frequency of cross-species transmission of ARGs by pathogens to recipient strains is significantly reduced. The present invention also discloses the application of the Bacillus BAC4 in blocking the transmission of ARGs by soil pathogens.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and relates to a strain of Bacillus pseudomycoides BAC4 for preventing the spread of antibiotic resistance genes (ARGs) of soil pathogenic bacteria and an application of the strain in preventing the spread of ARGs of soil pathogenic bacteria. Background Art

[0002] The soil biological complex pollution formed by the superposition and interaction of soil pathogens and antibiotic resistance genes (ARGs) seriously threatens the integrated health (One Health) of animals, humans, and the environment. During the outbreak of soil-borne diseases, pathogens spread high-risk ARGs across species in the soil-plant system and spread ARGs to the ecosystem through the food chain, causing serious ecological and health risks.

[0003] Commonly used measures to prevent the cross-species transmission of ARGs in the soil-plant system include physical, chemical, and biological measures. Physical and chemical measures include the use of soil conditioners, biomass, and nanomaterials. Soil conditioners can adjust the bioavailability of pollutants in the soil, optimize the physical and chemical properties of the soil, and regulate the soil microbial ecology, thereby indirectly inhibiting the spread of ARGs in microplastic-contaminated soil. Biological measures include adding plant residues [1] , bacteriophage [2] , soil animals [3] Compared with physical measures, biological measures have the advantages of sustainable effects, no need for repeated additions, and no damage to soil structure. However, the existing biological measures do not involve rhizosphere bacteria that can survive naturally in the soil. The present invention aims to screen rhizosphere bacteria from in situ soil and then apply them to prevent the cross-species transmission of ARGs, which has the advantages of strong survival and colonization ability. Summary of the invention

[0004] The purpose of the present invention is to provide a rhizosphere beneficial Bacillus BAC4 that can prevent soil pathogens from spreading ARGs. The inventors found that in indoor experiments, the effect of Bacillus BAC4 in preventing soil pathogens from spreading ARGs was as high as 87.37%, significantly reducing the maximum biomass of pathogens, significantly increasing the ability of recipient Escherichia coli to migrate, thereby reducing the frequency of recipient Escherichia coli accepting ARGs, and significantly increasing the production of active oxygen in donor pathogens and recipient Escherichia coli, causing the donor and recipient strains to produce oxidative stress reactions, thereby reducing the frequency of cross-species transmission, and significantly reducing the permeability of pathogenic bacteria membranes to reduce the ability of pathogens to communicate with the outside world. In a greenhouse potted plant experiment, Bacillus BAC4 was able to significantly reduce the frequency of Ralstonia solanacearum spreading ARGs to tomato stem microorganisms in an in situ soil system, reducing it by 54.38%. This shows that under the treatment of Bacillus BAC4 or its fermentation liquid, the frequency of cross-species transmission of ARGs by pathogens to recipient strains is significantly reduced, providing a basis for the development of rhizosphere beneficial bacteria that can effectively prevent pathogens from spreading ARGs.

[0005] The objective of the present invention is achieved through the following technical solutions:

[0006] A Bacillus strain BAC4 that blocks the spread of ARGs by soil pathogens was classified and named Bacillus pseudomycoides. It was deposited in the General Microbiology Center of China Culture Collection Administration on June 11, 2024. The address of the deposit unit is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the strain deposit number is CGMCCNO.30925.

[0007] Another object of the present invention is to provide a reagent for preventing and controlling the spread of antibiotic resistance genes (ARGs), wherein the reagent is a suspension of Bacillus BAC4 or a fermentation broth of Bacillus BAC4.

[0008] The concentration of Bacillus BAC4 in the Bacillus BAC4 bacterial suspension is 1×10 7 ~1×10 9 CFU / mL.

[0009] The Bacillus BAC4 bacterial suspension is prepared by the following method: Bacillus strain BAC4 is activated by TSA solid culture medium; a single colony of Bacillus BAC4 is picked and placed in TSB liquid culture medium, cultured at 28-32°C and 150-190rpm for 12-24h, centrifuged, the bacteria are collected, the bacteria are washed with sterile 1×PBS buffer to remove the culture medium, and the concentration of Bacillus BAC4 is adjusted to 1×10 7 ~1×10 9CFU / mL, and the obtained Bacillus BAC4 bacterial suspension. Wherein, the centrifugal speed is 6000 rpm, the centrifugal time is 5 min, and the centrifugal temperature is room temperature.

[0010] The fermentation broth of Bacillus subtilis BAC4 is prepared by the following method: Bacillus subtilis BAC4 is activated by using TSA solid culture medium; a single colony of Bacillus subtilis BAC4 is picked up and placed in TSB liquid culture medium, cultured at 28-32°C and 150-190rpm for 12-24h, and the concentration of Bacillus subtilis BAC4 is adjusted to 1×10 7 ~1×10 9 CFU / mL; inoculate Bacillus BAC4 into TSB liquid culture medium at a volume ratio of 0.5:100 to 2:100 for bacterial suspension and TSB liquid culture medium, culture for 12 to 24 hours at 28 to 32°C and 50 to 190 rpm, centrifuge the bacterial suspension, absorb the supernatant, filter with a filter membrane, and the filtrate is the fermentation liquid of Bacillus BAC4. The centrifugal speed is 6000 rpm, the centrifugal time is 5 minutes, and the filter membrane is a 0.22 μm filter membrane.

[0011] Another object of the present invention is to provide the application of the Bacillus BAC4 in preventing the spread of antibiotic resistance genes by soil pathogenic bacteria.

[0012] Another object of the present invention is to provide the use of the reagent for preventing and controlling the spread of antibiotic resistance genes in preventing and controlling the spread of antibiotic resistance genes by soil pathogens.

[0013] Another object of the present invention is to provide a method for preventing soil pathogens from spreading ARGs in in situ soil, comprising: 7 to 10 days after transplanting tomato seedlings with 3 to 5 true leaves, adding the agent into the rhizosphere soil by root irrigation.

[0014] When the reagent is a suspension of Bacillus subtilis BAC4, 10 mL of a concentration of 1×10 7 ~1×10 9 CFU / mL Bacillus BAC4 bacterial suspension.

[0015] The soil pathogenic bacteria is Ralstonia solanacearum.

[0016] Another object of the present invention is to provide the use of the Bacillus BAC4 or the reagent in preventing and controlling bacterial wilt or in preparing a reagent for preventing and controlling bacterial wilt. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the blocking effect of Bacillus fermentation broth on the transmission of ARGs from donor Rs1115-GFP to recipient DH5α.

[0018] Figure 2 The potted effect of Bacillus blocking donor Rs1115-GFP in transmitting ARGs to in situ soil microorganisms.

[0019] Figure 3 This is the effect of Bacillus fermentation broth on the maximum biomass of donor Rs1115-GFP and recipient DH5α.

[0020] Figure 4 This is the effect of Bacillus fermentation broth on the motility of donor Rs1115-GFP.

[0021] Figure 5 This is the effect of Bacillus fermentation broth on the motility of receptor DH5α.

[0022] Figure 6 This is the effect of Bacillus fermentation broth on the reactive oxygen production of donor Rs1115-GFP and acceptor DH5α.

[0023] Figure 7 This is the effect of Bacillus fermentation broth on the membrane permeability of donor Rs1115-GFP and acceptor DH5α.

[0024] Biodeposit Information:

[0025] The Bacillus strain BAC4, classified and named Bacillus pseudomycoides, was deposited in the General Microbiology Center of the China Culture Collection Administration on June 11, 2024. The address of the depository unit is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the culture collection number is CGMCC NO.30925. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described below in conjunction with specific implementation methods.

[0027] NB liquid culture medium: 10 g glucose, 5 g peptone, 3 g beef extract, 0.5 g yeast extract, 1000 mL deionized water, adjust pH to 7.2-7.4, and sterilize at 115°C for 30 min.

[0028] NA solid medium: NB liquid medium plus 2-3% (w / w) agar.

[0029] LB liquid culture medium: peptone 10 g, yeast extract 5 g, sodium chloride 10 g, deionized water 1000 mL, adjust pH to 7.2-7.4, and sterilize at 121°C for 20 min.

[0030] LB solid medium: LB liquid medium plus 2-3% (w / w) agar.

[0031] TSB liquid culture medium: 15 g peptone, 5 g soy peptone, 5 g sodium chloride, 1000 mL deionized water, adjust pH to 7.2-7.4, and sterilize at 121°C for 20 min.

[0032] TSA solid medium: TSB liquid medium plus 2-3% (w / w) agar.

[0033] Example 1

[0034] Isolation and identification of strain BAC4

[0035] Strain BAC4 was isolated from rhizosphere soil of tomato infected with bacterial wilt in Changsha, Hunan (112°58′E, 28°11′N).

[0036] Take 1 g of tomato rhizosphere soil and mix it with 9 mL of 1× PBS buffer in a 50 mL flask. Place the flask in a shaker at 30°C and 170 rpm for 30 min. Dilute the soil suspension to 10% with sterile water. -5 , 10 -6 , take 100 μL of diluted soil suspension and add it to a culture dish containing 1 / 10 times diluted TSA solid culture medium and spread it evenly; culture it in a 30℃ constant temperature incubator in the dark for 48 hours, then pick a single colony from the sample with a sterile toothpick, streak it on TSA solid culture medium, place it in a 30℃ constant temperature incubator in the dark for 48 hours, pick a pure single colony point, and inoculate it into a 96-well plate containing 100 μL TSB liquid culture medium, put the well plate in a 30℃, 170rpm shaker for incubation overnight, add 100 μL 30% glycerol and mix it thoroughly, and store it in a -80℃ refrigerator.

[0037] The strain BAC4 belongs to Bacillus pseudomycoides, and its main biological characteristics are Gram-positive G + , oxidase positive, catalase positive, indole negative, the bacteria are long rods, with spores, and can hydrolyze starch; the colonies are round, with neat and convex milky white edges.

[0038] The Bacillus strain BAC4, classified and named Bacillus pseudomycoides, was deposited in the General Microbiology Center of the China Culture Collection Administration on June 11, 2024. The address of the depository unit is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the culture collection number is CGMCC NO.30925.

[0039] Example 2

[0040] Study on the effect of strain BAC4 on the transmission of ARGs from donor Rs1115-GFP to recipient DH5α

[0041] Test antibiotics: Using chloroform as solvent, prepare nalidixic acid stock solution with a concentration of 15 mg / mL; using ethanol as solvent, prepare chloramphenicol stock solution with a concentration of 25 mg / mL; using ddH2O as solvent, prepare ampicillin stock solution with a concentration of 100 mg / mL and kanamycin stock solution with a concentration of 50 mg / mL; using DMSO as solvent, prepare tetracycline hydrochloride stock solution with a concentration of 10 mg / mL.

[0042] The pathogenic bacterial strain Rs1115-lacI-mCherry-GFP (abbreviated as Rs1115-GFP) is a two-color fluorescent solanacearum, classified and named Ralstonia solanacearum. For details, see the invention patent application filed by the applicant on March 30, 2022. The name of the invention is: A fluorescent labeling method for tracking the spread of resistance genes of soil-borne plant pathogens, patent number: ZL 2022103214030, publication number: CN 114807196 A. The strain has resistance to kanamycin, ampicillin, chloramphenicol and tetracycline antibiotics, and is used as a donor bacteria in the cross-species transmission experimental system.

[0043] The strain DH5α, classified as Escherichia coli, carries nalidixic acid resistance and serves as a high-risk recipient strain in the cross-species transmission experimental system.

[0044] The glycerol-preserved strain Rs1115-GFP was streaked on NA solid medium supplemented with 25 mg / L chloramphenicol, 100 mg / L ampicillin, 50 mg / L kanamycin, and 10 mg / L tetracycline hydrochloride, and cultured at 30°C for 2 days; a single colony on the plate was picked and transferred to NA liquid medium, and cultured overnight at 30°C and 170 rpm. Fresh bacterial liquid was taken and centrifuged at 6000 rpm for 5 min at room temperature to collect the cells, which were washed three times with sterile 1× PBS buffer, the culture medium was removed, and the concentration of the bacterial liquid was adjusted to 1×10 8 CFU / mL, and obtain a red and green dual-color fluorescent Ralstonia solanacearum suspension for standby use.

[0045] The glycerol-preserved strain DH5α was streaked and activated on solid LB medium supplemented with 15 mg / mL nalidixic acid and cultured at 37°C for 2 days; a single colony on the plate was picked and transferred to LB liquid medium and cultured at 37°C and 170 rpm overnight. Fresh bacterial liquid was centrifuged at 6000 rpm for 5 min at room temperature to collect the cells, which were washed three times with sterile 1× PBS buffer, the culture medium was removed, and the concentration of the bacterial liquid was adjusted to 1×108 CFU / mL, and obtain the E. coli suspension for later use.

[0046] The glycerol-preserved strain BAC4 was streaked on TSA solid medium for activation and cultured at 30°C for 2 days. A single colony was picked from the plate and transferred to TSB liquid medium, cultured in a shaker at 30°C and 170 rpm for 24 hours, and the bacterial solution concentration was adjusted to 1×10 8 CFU / mL, take 50μL of bacterial suspension and inoculate it into 5mL TSB liquid medium, and continue to shake and culture at 30℃ and 170rpm for 24h. Take the bacterial suspension in a sterile centrifuge tube, centrifuge it at 6000rpm for 5min, use a pipette to draw the supernatant into a new centrifuge tube, and then use a 5mL syringe to draw the supernatant, filter it with a 0.22μm filter membrane, and the filtrate is the Bacillus BAC4 fermentation broth, which is stored in a -80℃ refrigerator for later use.

[0047] A solid LB plate with 15 mg / L nalidixic acid added was prepared as a counting plate for all recipient Escherichia coli surviving in the cross-species transmission conjugation system, and Ralstonia solanacearum could not grow on this plate; a solid LB plate with 15 mg / L nalidixic acid, 25 mg / L chloramphenicol, 100 mg / L ampicillin, 50 mg / L kanamycin and 10 mg / L tetracycline hydrochloride added was prepared as a counting plate for recipient Escherichia coli that only accepts Ralstonia solanacearum ARGs in the cross-species transmission conjugation system, and Ralstonia solanacearum could not grow on this plate.

[0048] Cross-species transmission frequency = recipient E. coli that only accept R. solanacearum ARGs / all recipient E. coli

[0049] The experiment set up two treatments: 1) Bacillus fermentation broth group: add Bacillus BAC4 fermentation broth; 2) control group: replace Bacillus BAC4 fermentation broth with an equal volume of water. The cross-species transmission detection system was used for detection, as follows: 400 μL of the concentration was OD 600 =0.7 (i.e. 1×10 8 CFU / mL) of red-green fluorescent Ralstonia solanacearum suspension was centrifuged at 6000 rpm for 5 min, the precipitate was resuspended to 400 μL with 1× PBS, the culture medium was removed, the solution was washed twice, and finally resuspended to 200 μL with 1× PBS; 400 μL of the solution was taken as the OD 600 =0.7 (i.e. 1×10 8CFU / mL) of Escherichia coli suspension, centrifuge at 6000rpm for 5min, resuspend the precipitate in 1×PBS to 400μL, remove the culture medium, wash twice, and finally resuspend in 1×PBS to 200μL; mix the above donor and receptor suspensions, add 400μL of Bacillus BAC4 fermentation liquid, and vortex to mix. A circular filter paper with a diameter of 5mm is evenly attached to the NA plate, and then 5μL of the above mixed liquid is dripped on the filter paper, placed in a 30℃ incubator and cultured for 2h, and then the filter paper is transferred to 200μL of sterile water, vortexed, and the bacteria on the filter paper are washed to obtain a bacterial mixture, and the bacterial mixture is gradiently diluted to 10 with 1×PBS buffer. 0 , 10 -1 , 10 -2 , 10 -3 , 10 μL of the mixed solution of each gradient was taken and dropped onto the above two LB plates (i.e., solid LB medium with 15 mg / mL nalidixic acid added, and LB solid plate with 15 mg / L nalidixic acid, 25 mg / L chloramphenicol, 100 mg / L ampicillin, 50 mg / L kanamycin and 10 mg / L tetracycline hydrochloride added), and after the surface liquid was dried, it was transferred to a 37°C incubator and cultured for 12 h. The transmission frequency of ARGs from Ralstonia solanacearum to Escherichia coli was calculated after counting by the dilution spreading method.

[0050] The inhibitory effect of Bacillus on the transmission of ARGs from donor Rs1115-GFP to recipient DH5α is shown in Figure 1 and Table 1 , the results showed that compared with the control, Bacillus significantly reduced the cross-species transmission frequency of ARGs from donor Rs1115-GFP to recipient DH5α, which decreased by 87.37% (P<0.05).

[0051] Table 1. The inhibitory effect of Bacillus on the transmission of ARGs from donor Rs1115-GFP to recipient DH5α

[0052]

[0053] Note: Different letters in the same column in the table indicate significant differences at the 0.05 level, the same below.

[0054] Example 3

[0055] Potted effect of Bacillus sp. blocking donor Rs1115-GFP on transmitting ARGs to in situ soil microorganisms

[0056] Prepare Rs1115-GFP bacterial suspension (i.e., red and green dual-color fluorescent Ralstonia solanacearum suspension) and Bacillus BAC4 bacterial suspension with reference to Example 2, and finally adjust the concentration of Rs1115-GFP bacterial suspension to 1×10 9CFU / mL, the concentration of Bacillus BAC4 suspension is 1×10 9 CFU / mL, set aside.

[0057] Tomato variety: Red dwarf tomato.

[0058] After surface disinfection, tomato seeds were placed on a sterile plate lined with filter paper soaked in sterile deionized water and germinated at 30°C for 2 days. Seeds with uniform germination were taken and planted in a 50-hole seedling tray filled with sterilized substrate. After the tomato grew 3 true leaves, seedlings with uniform growth were transplanted into a 6-hole seedling tray.

[0059] The experiment set up two treatments: 1) Bacillus group: add Bacillus BAC4 suspension near the roots of tomatoes, 2) control group: replace Bacillus BAC4 suspension with an equal volume of water. 7 days after the tomato seedlings were transplanted, the Bacillus group: the Bacillus BAC4 suspension was evenly poured near the roots of tomatoes by root irrigation, and 10 mL of 1×10 9 CFU / mL of Bacillus BAC4 suspension, the final concentration in the soil is about 1×10 7 CFU / g, and the Bacillus BAC4 suspension was replaced by an equal volume of water as the control group; after 7 days of Bacillus colonization, the Rs1115-GFP suspension of Ralstonia solanacearum was evenly poured into the vicinity of the tomato roots by the root irrigation method, and 10 mL of 1×10 9 The final concentration of Ralstonia solanacearum Rs1115-GFP bacterial suspension in the soil was about 1×10 7 CFU / g; 3 days later, tomato stem microorganisms were collected. The method for collecting stem microorganisms is as follows: after removing tomato leaves and roots, 6g of tomato stem tissue was cut and ground, collected in a 50mL centrifuge tube, 20mL of 1×PBS buffer was added, the centrifuge tube was placed obliquely in a shaker, shaken at 30°C and 170rpm for 1h, filtered using 11cm medium-speed filter paper, the filtrate was taken, centrifuged at 6000rpm for 5min using a high-speed centrifuge, the supernatant was removed with a pipette, and the remaining 2mL of liquid was used as the collected microorganisms. The collected microorganisms were detected using a flow cytometer based on the presence or absence of green fluorescence, and the frequency of ARGs transmission from R. solanacearum Rs1115-GFP to tomato stem microorganisms was calculated according to the following formula.

[0060] Formula: Cross-species transmission frequency = number of microorganisms carrying green fluorescence / (number of microorganisms carrying green fluorescence + number of microorganisms not carrying green fluorescence)

[0061] The effect of Bacillus sp. on preventing Ralstonia solanacearum Rs1115-GFP from transmitting ARGs to tomato stem microorganisms is shown in Figure 2and Table 2. The results showed that Bacillus sp. BAC4 could significantly reduce the frequency of ARGs transmission from R. solanacearum to tomato stem microorganisms by 54.38% (P < 0.05).

[0062] Table 2. Frequency of ARGs transmitted by R. solanacearum Rs1115-GFP to tomato stem microorganisms

[0063]

[0064] Example 4

[0065] Indoor Effects of Bacillus on the Maximum Biomass of Donor Rs1115-GFP and Recipient DH5α

[0066] Referring to Example 2, prepare Rs1115-GFP bacterial suspension (ie, red-green dual-color fluorescent Ralstonia solanacearum suspension), DH5α bacterial suspension (ie, Escherichia coli suspension) and Bacillus BAC4 fermentation broth.

[0067] The maximum biomass of the donor pathogen Rs1115-GFP was determined. Two treatments were set up in the experiment: 1) Bacillus fermentation broth group: Bacillus BAC4 fermentation broth was added; 2) Control group: Bacillus BAC4 fermentation broth was replaced with an equal volume of water. Rs1115-GFP bacterial suspension and Bacillus BAC4 fermentation broth were added to NB liquid culture medium. The final concentration of Bacillus BAC4 fermentation broth was 2.5% (v / v), and the inoculation concentration of pathogenic bacteria Rs1115-GFP was 1% (final concentration of 1×10 6 CFU / mL), 30°C, 170 rpm shaking culture for 48 h, and OD was measured on a microplate reader 600 , investigate the growth of donor Ralstonia solanacearum Rs1115-GFP.

[0068] The maximum biomass of the recipient strain DH5α was determined. Two treatments were set up in the experiment: 1) Bacillus fermentation broth group: Bacillus BAC4 fermentation broth was added; 2) Control group: Bacillus BAC4 fermentation broth was replaced with an equal volume of water. The recipient strain DH5α bacterial suspension and Bacillus BAC4 fermentation broth were added to the LB liquid medium. The final concentration of Bacillus BAC4 fermentation broth was 2.5% (v / v), and the inoculation concentration of the recipient strain DH5α was 1% (final concentration of 1×10 6 CFU / mL), incubate at 37°C, 170 rpm, shake for 48 h, and measure OD on a microplate reader 600 , investigate the growth of the recipient Escherichia coli and Ralstonia solanacearum DH5α.

[0069] The effect of Bacillus on the maximum biomass of donor and recipient strains can be seen in Figure 3The results in Table 3 show that compared with the control group, Bacillus can significantly reduce the maximum biomass of Rs1115-GFP of the donor Ralstonia solanacearum (P<0.05), which decreased by 31.60%, thereby reducing the frequency of ARGs transmitted by the donor Ralstonia solanacearum. There was no significant effect on the recipient strain.

[0070] Table 3. Effect of Bacillus on the maximum biomass of donor Rs1115-GFP and recipient DH5α

[0071]

[0072] Example 5

[0073] In-house effects of Bacillus on the motility of donor Rs1115-GFP and recipient DH5α

[0074] Donor Ralstonia solanacearum Rs1115-GFP motility culture medium: swimming medium: 1 / 10 NB liquid culture medium, 0.3% agar; swarming medium: 1 / 10 NB liquid culture medium, 0.5% agar; gliding medium: 1 / 10 NB liquid culture medium, 0.7% agar.

[0075] Recipient Escherichia coli DH5α motility medium: swimming medium: 1 / 10 LB liquid medium, 0.3% agar; swarming medium: 1 / 10 LB liquid medium, 0.5% agar; motility medium: 1 / 10 LB liquid medium, 0.7% agar.

[0076] Referring to Example 2, a donor Rs1115-GFP bacterial suspension (i.e., a red-green fluorescent Ralstonia solanacearum suspension), a recipient DH5α bacterial suspension (i.e., an Escherichia coli suspension) and a Bacillus BAC4 fermentation broth were prepared.

[0077] Experiment on the swimming ability of Rs1115-GFP of Ralstonia solanacearum: Two treatments were set up: 1) Bacillus fermentation broth group: Bacillus BAC4 fermentation broth was added; 2) Control group: Bacillus BAC4 fermentation broth was replaced with an equal volume of water. Take 200 μL of 1×10 8 CFU / mL Rs1115-GFP bacterial suspension of Ralstonia solanacearum was centrifuged at 6000rpm for 5min, the precipitate was resuspended to 200μL with 1×PBS buffer, the culture medium was removed, washed twice, and finally resuspended to 100μL with 1×PBS buffer, 100μL of Bacillus BAC4 fermentation broth was added, and vortexed to mix. 2μL of the above mixed liquid was taken and spotted in the center of the Ralstonia solanacearum swimming culture medium, placed in an incubator, and cultured at 30℃ for 48h, and the swimming diameter of the donor Ralstonia solanacearum was measured by the cross method.

[0078] Determination of the swarming and motility abilities of donor Ralstonia solanacearum Rs1115-GFP: The method was the same as the experiment to determine the swimming ability of Ralstonia solanacearum Rs1115-GFP, except that the culture medium was replaced with Ralstonia solanacearum Rs1115-GFP swarming culture medium and motility culture medium, respectively.

[0079] The experiment of measuring the swimming ability of Escherichia coli DH5α was set up with two treatments: 1) Bacillus fermentation broth group: adding Bacillus BAC4 fermentation broth; 2) control group: replacing Bacillus fermentation broth with an equal volume of water. Take 200 μL of 1×10 8 CFU / mL of E. coli DH5α bacterial solution was centrifuged at 6000rpm for 5min, the precipitate was resuspended to 200μL with 1×PBS buffer, the culture medium was removed, washed twice, and finally resuspended to 100μL with 1×PBS buffer, 100μL of Bacillus BAC4 fermentation liquid was added, and vortexed to mix. 2μL of the above mixed liquid was taken and spotted at the center of the E. coli DH5α group swimming culture medium, placed in an incubator, and cultured at 37℃ for 48h. The swimming diameter of E. coli DH5α was measured by the cross method.

[0080] Determination of swarming and motility ability of Escherichia coli DH5α: The method is the same as the Escherichia coli DH5α swimming ability determination experiment, and the culture medium is replaced with Escherichia coli DH5α swarming culture medium and motility culture medium.

[0081] Results Figure 4 , Figure 5 As shown in Tables 4 and 5, compared with the control group, Bacillus significantly increased the escape ability of the recipient Escherichia coli by 8.028% (P<0.05), thereby reducing the frequency of the recipient strain accepting ARGs.

[0082] Table 4. Effect of Bacillus on the motility of donor Rs1115-GFP

[0083]

[0084] Table 5. Effects of Bacillus on the motility of receptor DH5α

[0085]

[0086] Example 6

[0087] Effects of Bacillus on the production of reactive oxygen species in donor Rs1115-GFP and acceptor DH5α

[0088] Referring to Example 2, prepare donor Rs1115-GFP bacterial suspension (i.e., red-green dual-color fluorescent Ralstonia solanacearum suspension), acceptor DH5α bacterial suspension (i.e., Escherichia coli suspension) and Bacillus BAC4 fermentation broth, and perform reactive oxygen detection on the donor and acceptor strains according to the instructions of the ROS detection kit (Biyuntian).

[0089] Donor Rs1115-GFP reactive oxygen species detection: Two treatments were set up: 1) Bacillus fermentation broth group: Bacillus BAC4 fermentation broth was added; 2) Control group: Bacillus BAC4 fermentation broth was replaced with an equal volume of water. Take 1 mL of a concentration of 1×10 8 CFU / mL of donor Rs1115-GFP bacterial suspension was added to a 2mL brown light-proof centrifuge tube, centrifuged to remove the culture medium and resuspended with 1×PBS buffer, centrifuged to remove 1×PBS buffer, added 1mL DCFH-DA probe (1000× diluted 1000 times), and cultured in an incubator at 37°C in the dark, inverted and mixed every 3-5min to allow the probe to fully contact the bacteria, and after 20min of culture, the mixed liquid was resuspended and washed 3 times with 1×PBS buffer to fully remove the DCFH-DA probe that did not enter the bacteria; 100μL of bacterial solution was dispensed into a light-proof centrifuge tube, centrifuged and resuspended with 1×PBS buffer to 50μL, and the bacterial solution was transferred to a black 96-well plate, and 50μL of Bacillus BAC4 fermentation liquid was added. Rosup (active oxygen positive control reagent) was added to the positive control group to fully stimulate the bacteria to produce ROS. After 30min, DCF (Ex / Em=488 / 525nm) was detected using a fluorescent microplate reader.

[0090] Two treatments were set up for the detection of active oxygen in the receptor DH5α: 1) Bacillus fermentation broth group: adding Bacillus BAC4 fermentation broth; 2) control group: replacing Bacillus BAC4 fermentation broth with an equal volume of water. The specific experimental method is the same as the donor Rs1115-GFP active oxygen detection method, except that the donor Rs1115-GFP bacterial suspension is replaced with the receptor DH5α bacterial suspension.

[0091] Results Figure 6 As shown in Table 6, compared with the control, the fermentation broth of Bacillus subtilis BAC4 could significantly increase the production of ROS in the donor Ralstonia solanacearum and the recipient Escherichia coli by 19.38 times and 7.995 times, respectively (P<0.05), causing oxidative stress in the donor and recipient strains, thereby reducing the frequency of cross-species transmission.

[0092] Table 6. Effects of Bacillus on the production of reactive oxygen species in donor Rs1115-GFP and acceptor DH5α

[0093]

[0094] Example 7

[0095] Effects of Bacillus on the membrane permeability of donor Rs1115-GFP and acceptor DH5α

[0096] Refer to Example 2 to prepare donor Rs1115-GFP bacterial suspension (i.e., red and green fluorescent Ralstonia solanacearum suspension), recipient DH5α bacterial suspension (i.e., Escherichia coli suspension) and Bacillus BAC4 fermentation broth, and perform membrane permeability detection on the donor and recipient strains according to the instructions of the Cell Activity and Cytotoxicity Detection Kit (Biyuntian).

[0097] Donor Rs1115-GFP membrane permeability test: Two treatments were set up: 1) Bacillus fermentation broth group: Bacillus BAC4 fermentation broth was added; 2) Control group: Bacillus BAC4 fermentation broth was replaced with an equal volume of water. Take 1 mL of a concentration of 1×10 8 CFU / mL of donor Rs1115-GFP bacterial suspension was added to a 2mL light-proof centrifuge tube, centrifuged to remove the culture medium, and resuspended to 500μL with 1×PBS buffer, and 500μL of Bacillus BAC4 fermentation liquid was added, vortexed, mixed, and the centrifuge tube was placed in a constant temperature incubator and cultured at 30°C for 2h; 100μL of the mixed liquid was taken to a brown light-proof centrifuge tube, centrifuged, and the mixed liquid was resuspended with 1×PBS buffer, washed 3 times, the residual fermentation liquid was removed, 100μL of PI detection working solution (1000× diluted 1000 times) was added, vortexed, and stood in the incubator at 37°C in the dark for 30min. The donor Rs1115-GFP killed by boiling water was used as a positive control to fully release reactive oxygen, and 6 replicates were set for each treatment. All liquids were transferred to a black 96-well plate, and red fluorescence (Ex / Em=535 / 617nm) was detected with a fluorescence microplate reader.

[0098] Membrane permeability test of receptor DH5α: Two treatments were set up: 1) Bacillus fermentation broth group: Bacillus BAC4 fermentation broth was added; 2) Control group: Bacillus BAC4 fermentation broth was replaced with an equal volume of water. The specific experimental method was the same as the membrane permeability test method of donor Rs1115-GFP, except that the donor Rs1115-GFP bacterial suspension was replaced with the receptor DH5α bacterial suspension.

[0099] Results Figure 7 As shown in Table 7, compared with the control, Bacillus BAC4 significantly reduced the membrane permeability of the donor Rs1115-GFP by 42.83% (P<0.05), reduced the ability of R. solanacearum to communicate with the outside world, and thus reduced the transmission frequency of ARGs.

[0100] Table 7. Effects of Bacillus on the membrane permeability of donor Rs1115-GFP and acceptor DH5α

[0101]

[0102] References:

[0103] [1]、Lu X M,Lu P Z.Distribution of antibiotic resistance genes in soilamended using Azolla imbricata and its driving mechanisms[J].Science of TheTotal Environment,2019,692:422-431.

[0104] [2]、Zhao Y,Ye M,Zhang X,et al.Comparing polyvalent bacteriophage andbacteriophage cocktails for controlling antibiotic-resistant bacteria insoil-plant system[J].Science of The Total Environment,2019,657:918-925.

[0105] [3]、Zhu D,Ding J,Yin Y,et al.Effects of earthworms on the microbiomesand antibiotic resistomes of detritus fauna and phyllospheres[J].Environmental Science&Technology,2020,54(10):6000-6008.

Claims

1. A strain of Bacillus BAC4, classified as Bacillus pseudomycoides ( Bacillus pseudomycoides ), was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 11, 2024, and the culture collection number is CGMCC NO.30925.

2. An agent for preventing and controlling the spread of antibiotic resistance genes, characterized in that: The reagent is a Bacillus BAC4 bacterial suspension or a fermentation liquid of Bacillus BAC4.

3. The agent for preventing and controlling the spread of antibiotic resistance genes according to claim 2, characterized in that: The concentration of Bacillus BAC4 in the Bacillus BAC4 bacterial suspension is 1×10 7 ~1×10 9 CFU / mL.

4. The agent for preventing and controlling the spread of antibiotic resistance genes according to claim 2 or 3, characterized in that: The Bacillus BAC4 bacterial suspension is prepared by the following method: picking a single colony of Bacillus BAC4 in TSB liquid culture medium, culturing at 28-32°C and 150-190 rpm for 12-24 hours, centrifuging, collecting the bacteria, washing the bacteria with sterile 1×PBS buffer to remove the culture medium, and then adjusting the concentration of Bacillus BAC4 to 1×10 7 ~1×10 9 CFU / mL, and obtain Bacillus BAC4 bacterial suspension.

5. The agent for preventing and controlling the spread of antibiotic resistance genes according to claim 2, characterized in that: The fermentation broth of Bacillus subtilis BAC4 is prepared by the following method: a single colony of Bacillus subtilis BAC4 is picked up and placed in TSB liquid culture medium, cultured at 28-32°C and 150-190 rpm for 12-24 h, and the concentration of Bacillus subtilis BAC4 is adjusted to 1×10 7 ~1×10 9 CFU / mL; inoculate Bacillus BAC4 into TSB liquid culture medium at a volume ratio of 0.5:100 to 2:100, and culture at 28-32°C and 150-190 rpm for 12-24 h; centrifuge the bacterial suspension, aspirate the supernatant, and filter it with a filter membrane. The filtrate is the fermentation broth of Bacillus BAC4.

6. The agent for preventing and controlling the spread of antibiotic resistance genes according to claim 2 or 5, characterized in that: The fermentation broth of Bacillus subtilis BAC4 is prepared by the following method: a single colony of Bacillus subtilis BAC4 is selected and placed in TSB liquid culture medium, cultured at 28-32°C and 150-190 rpm for 24 h, and the concentration of Bacillus subtilis BAC4 is adjusted to 1×10 7 ~1×10 9 CFU / mL; inoculate Bacillus BAC4 into TSB liquid culture medium at a volume ratio of 1:100, and culture at 28-32°C and 50-190 rpm for 24 h; centrifuge the bacterial suspension, aspirate the supernatant, and filter it with a filter membrane. The filtrate is the fermentation broth of Bacillus BAC4.

7. Use of the Bacillus BAC4 described in claim 1 or the reagent described in claim 2 in preventing soil pathogens from spreading antibiotic resistance genes; the soil pathogen is Ralstonia solanacearum.

8. A method for preventing soil pathogens from spreading antibiotic resistance genes, characterized in that: include: 7 to 10 days after the tomato seedlings with 3 to 5 true leaves are transplanted, the reagent according to claim 2 is added into the rhizosphere soil by root irrigation; the soil pathogen is Ralstonia solanacearum.

9. Use of the Bacillus BAC4 described in claim 1 or the reagent described in claim 2 in preventing and controlling bacterial wilt or in preparing a reagent for preventing and controlling bacterial wilt.

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