Application of Bacillus biopreparations in reducing the contamination of antibiotic resistance genes in vegetables

Abstract

The present invention discloses the application of a Bacillus biological agent in reducing the contamination of antibiotic resistance genes in vegetables, belonging to the field of agricultural biotechnology. The present invention constructs a microbial agent containing one or more strains of Bacillus amyloliquefaciens B3, Bacillus subtilis B1, Bacillus atrophaeus B2, and Peribacillus frigoritolerans B4. By spraying the microbial agent constructed in the present invention on the leaf surface, the pressure of ARGs in hydroponic lettuce can be effectively alleviated, and the growth status and nutritional quality of hydroponic vegetables can be improved.

Description

Technical Field

[0001] The present invention relates to the application of Bacillus biological agents in reducing the pollution of antibiotic resistance genes in vegetables, and belongs to the field of agricultural biotechnology. Background Art

[0002] As an economic crop for daily human consumption, vegetables are an important environmental source of antibiotic resistance genes (ARGs) in the drug-resistant population and are closely related to human health. The remaining resistance genes and resistant bacteria in vegetables can be transmitted through the food chain, posing a potential threat to human health. A large amount of antibiotic residues caused by agricultural practices exert selective pressure on the bacterial community in the soil-vegetable system, promoting the occurrence and spread of horizontal gene transfer of ARGs, and enriching ARGs in vegetables. Consuming raw vegetables contaminated with ARGs may increase human drug resistance and exacerbate the risk of human death.

[0003] Bacillus belongs to probiotics, which are widely present in the soil-vegetable system and have strong survival ability and the function of maintaining environmental stability. When used as part of a biological agent, Bacillus can not only promote plant growth, but also reduce the level of ARGs in plants and soil by competitively excluding or directly inhibiting harmful microorganisms containing ARGs. By using the treatment method of Bacillus biological agents, the spread of antibiotic resistance can be slowed down and its threat to the environment and human health can be reduced. Summary of the Invention

[0004] The present invention provides a composition containing Bacillus, which contains Bacillus amyloliquefaciens; the Bacillus amyloliquefaciens (B3) is disclosed in the paper "Organic fertilizer and Bacillus amyloliquefaciens promote soil N availability via changing different mineralization–immobilization turnover rates in acidic soils".

[0005] In one embodiment, the composition further contains one or more strains of Bacillus subtilis B1, Bacillus atrophaeus B2, or Peribacillus frigoritolerans B4.

[0006] In one embodiment, the composition contains Bacillus subtilis (B1) and Bacillus amyloliquefaciens (B3).

[0007] In one embodiment, the composition contains Bacillus atrophaeus (B2) and Bacillus amyloliquefaciens (B3).

[0008] In one embodiment, the composition contains Bacillus amyloliquefaciens (B3) and Psychrobacter cryohalolentis (B4).

[0009] In one embodiment, the composition contains Bacillus subtilis (B1), Bacillus atrophaeus (B2), and Bacillus amyloliquefaciens (B3).

[0010] In one embodiment, the composition contains Bacillus atrophaeus (B2), Bacillus amyloliquefaciens (B3), and Psychrobacter cryohalolentis (B4).

[0011] In one embodiment, the composition contains Bacillus subtilis (B1), Bacillus atrophaeus (B2), Bacillus amyloliquefaciens (B3), and Psychrobacter cryohalolentis (B4).

[0012] In one embodiment, the bacterial concentration in the composition is ≥ 1×10 7 CFU / mL.

[0013] The present invention also provides a method for preparing the composition, which is to culture the composition in a medium for a period of time, collect the bacterial cells, and wash them to obtain a bacterial suspension with a specific concentration.

[0014] In one embodiment, the culturing is performed on a shaker at a constant temperature of 35 - 40°C and 220 rpm for 8 - 16 h.

[0015] In one embodiment, the washing is to wash the harvested bacteria twice in Phosphate Buffered Saline (PBS).

[0016] In one embodiment, the specific concentration refers to the OD of the bacterial suspension 600 being 0.08 - 0.12.

[0017] The present invention provides the use of Bacillus alone or in combination for reducing plant ARGs pollution and / or improving the nutritional quality of vegetables.

[0018] In one embodiment, the plants include, but are not limited to, edible fruits or vegetables.

[0019] In one embodiment, the vegetables include, but are not limited to, leafy vegetables.

[0020] In one embodiment, the vegetables include, but are not limited to, crown daisy, spinach, rape, lettuce, pea sprouts, Chinese cabbage, celery, and chicory.

[0021] In one embodiment, the application is to bring the Bacillus into contact with the plant.

[0022] In one embodiment, the application is to spray the bacterial suspension containing the Bacillus on the surface of the plant.

[0023] In one embodiment, the concentration of Bacillus in the bacterial suspension is ≥1×10 7 CFU / mL.

[0024] In one embodiment, the quantity ratio of Bacillus subtilis (B1) to Bacillus amyloliquefaciens (B3) in the bacterial suspension is 1:1.

[0025] In one embodiment, the quantity ratio of Bacillus atrophaeus (B2) to Bacillus amyloliquefaciens (B3) in the bacterial suspension is 1:1.

[0026] In one embodiment, the quantity ratio of Bacillus amyloliquefaciens (B3) to Bacillus frigoritolerans (B4) in the bacterial suspension is 1:1.

[0027] In one embodiment, the quantity ratio of Bacillus subtilis (B1), Bacillus atrophaeus (B2), and Bacillus amyloliquefaciens (B3) in the bacterial suspension is 1:1:1.

[0028] In one embodiment, the quantity ratio of Bacillus atrophaeus (B2), Bacillus amyloliquefaciens (B3), and Bacillus frigoritolerans (B4) in the bacterial suspension is 1:1:1.

[0029] In one embodiment, the quantity ratio of Bacillus subtilis (B1), Bacillus atrophaeus (B2), Bacillus amyloliquefaciens (B3), and Bacillus frigoritolerans (B4) in the bacterial suspension is 1:1:1:1.

[0030] In one embodiment, the improvement of the nutritional quality of vegetables includes, but is not limited to, increasing the vitamin content and / or protein content of vegetables; the vitamins include, but are not limited to, vitamin C.

[0031] The present invention also claims the application of the microbial preparation or the method in the cultivation of agricultural products.

[0032] Beneficial effects:

[0033] (1) The present invention constructs a microbial agent capable of reducing the abundance of antibiotic resistance genes in vegetables. Spraying the Bacillus agent on the leaf surface can effectively relieve the pressure of ARGs in hydroponic lettuce. Compared with the control group, the abundance of ARGs in hydroponic lettuce is reduced by 30% - 50%, and the abundance of class I integron intI1 is reduced by 6% - 30%.

[0034] (2) The microbial agent of the present invention can improve the growth status and nutritional quality of hydroponic vegetables while reducing the abundance of antibiotic resistance genes in hydroponic vegetables. Compared with the control group, growth indexes such as the plant height of hydroponic lettuce are improved, the vitamin C content is increased by 2% - 25%, and the protein content is increased by 1% - 10%.

[0035] (3) The present invention also studies the mechanism of action of the microbial agent in reducing the pollution of antibiotic resistance genes in vegetables. The results show that there is a negative correlation between the biofilm amount and ARGs in the mixed bacteria agent treatment group; in the single carbon source competition experiment, Bacillus shows a higher growth rate, and the growth rate of B23 treatment exceeds 1.4 times that of A1; the EPS extract of the experimental strains shows an antibacterial effect on antibiotic resistance bacteria (ARB), but there may be other antibacterial substances. Description of the Drawings

[0036] Figure 1 Total abundance of ARGs in different lettuce leaves;

[0037] Figure 2 Abundances of tetX(a), tetG(b), sul2(c), and sul3(d) in different lettuce leaves;

[0038] Figure 3 Abundances of qepA1(a), qnrS1(b), blaOXA-2(c), and floR(d) in different lettuce leaves;

[0039] Figure 4 Vitamin C content in different lettuces;

[0040] Figure 5 Growth curves of each Bacillus;

[0041] Figure 6 Biofilm amount of Bacillus;

[0042] Figure 7 Growth curves of Bacillus agents in malic acid (a), L-glutamic acid (b) and mixed drug-resistant strains in malic acid (c), L-glutamic acid (d);

[0043] Figure 8Growth rates of Bacillus and drug-resistant strains in malic acid (a) and L-glutamic acid (b);

[0044] Figure 9 It is the antagonistic effect between Bacillus and drug-resistant strains. Specific implementation manners

[0045] The present invention will be further described below in conjunction with specific embodiments.

[0046] The embodiments provided below are not intended to limit the scope covered by the present invention, nor are the described steps intended to limit their execution order. Obvious improvements made by those skilled in the art to the present invention in combination with the existing common general knowledge also fall within the protection scope required by the present invention.

[0047] (I) Technical terms

[0048] Antibiotic resistance pollution: The "antibiotic resistance pollution" mentioned in this application is a form of environmental pollution, mainly referring to the widespread presence and spread of antibiotic resistance genes (ARGs) and antibiotic resistance bacteria (ARB) in the environment (including but not limited to soil, water bodies, air). The spread of this pollution may occur through various channels such as medical wastewater, feces discharged from the livestock and poultry breeding industry, industrial emissions, and discharges from sewage treatment plants. During these processes, a large amount of antibiotics used are not completely absorbed or decomposed and directly or indirectly enter the environment and gradually accumulate.

[0049] Antibiotic resistance bacteria: The "antibiotic resistance bacteria" (abbreviated as ARB) mentioned in this application refer to bacteria that have developed resistance to one or more antibiotics through natural selection and gene transfer. These bacteria can survive and reproduce in an environment where antibiotics are present, thus greatly reducing the therapeutic effect of antibiotics.

[0050] Antibiotic resistance genes: The "antibiotic resistance genes" (abbreviated as ARGs) mentioned in this application refer to genes in the genomes of plants or microorganisms (mainly bacteria) that can encode proteins or enzymes resistant to antibiotics. In some embodiments of this application, the antibiotic resistance genes include but are not limited to sulfonamide antibiotic resistance genes (sul2, sul3), tetracycline antibiotic resistance genes (tetX, tetG), quinolone antibiotic resistance genes (qepA1, qnrS1), β-lactam antibiotic resistance genes (blaOXA-2), and florfenicol antibiotic resistance genes (floR).

[0051] (II) Culture media

[0052] LB liquid medium: 10.0 g of tryptone, 5.0 g of yeast extract, 10.0 g of sodium chloride, 1 L of deionized water.

[0053] M9 medium: 8.5 g of disodium hydrogen phosphate dihydrate, 3 g of potassium dihydrogen phosphate, 1 g of ammonium chloride, 0.5 g of sodium chloride, 1 L of deionized water, adjust the pH value to 7.0, and then perform autoclaving. The following components are separately filter-sterilized and then added to the sterilized medium: 1 mL of 0.1 mol / L calcium chloride dihydrate, 1 mL of 1 mol / L magnesium sulfate heptahydrate, 1 mL of 0.1 mol / L ferric chloride hexahydrate, and 10 mL of trace salt solution. The trace salt solution contains per liter: 170 mg of zinc chloride, 100 mg of manganese chloride tetrahydrate, 60 mg of chromium chloride hexahydrate, 60 mg of sodium molybdate dihydrate, 43 mg of copper chloride dihydrate.

[0054] (III) Biological materials

[0055] The strains involved in the specific implementation manners are all strains publicly disclosed in the prior art, and the specific information is as follows:

[0056] Bacillus strains: Bacillus subtilis (abbreviated as B1, published in the paper "Identification of ypqP as a new Bacillus subtilis biofilm determinant that mediates the protection of Staphylococcus aureus against antimicrobial agents in mixed-species communities"), Bacillus atrophaeus (B. atrophaeus 1942, abbreviated as B2, published in the paper "Bacillus atrophaeus: main characteristics and biotechnological applications - a review"), Bacillus amyloliquefaciens (abbreviated as B3, published in the paper "Organic fertilizer and Bacillus amyloliquefaciens promote soil N availability via changing different mineralization–immobilization turnover rates in acidic soils"), Peribacillus frigoritolerans (abbreviated as B4, published in the paper "Plant growth–promoting rhizobacteria: Peribacillus frigoritolerans 2RO30 and Pseudomonas sivasensis 2RO45 for their effect on canola growth under controlled as well as natural conditions").

[0057] Drug-resistant strains: Enterobacterales ludwigii (abbreviated as A1, published in Bloodstream infection due to Enterobacter ludwigii, correlating with massive aggregation on the surface of a central venous catheter), Enterobacterales kobei (abbreviated as A2, published in Enterobacter kobei sp. nov., a new species of the family Enterobacteriaceae resembling Enterobacter cloacae), Chryseobacterium planobacterium sp. (abbreviated as A3, published in Division of the genus Chryseobacterium: Observation of discontinuities in amino acid identity values, a possible consequence of major extinction events, guides transfer of nine species to the genus Epilithonimonas, eleven species to the genus Kaistella, and three species to the genus Halpernia gen. nov., with description of Kaistella daneshvariae sp. nov. and Epilithonimonas vandammei sp. nov. derived from clinical specimens), Chryseobacterium indologenes (abbreviated as A4, published in Chryseobacterium indologenes: an emerging infection in the USA), Priestia megaterium (abbreviated as A5, published in 《The “beauty in the beast”—the multiple uses of Priestia megaterium in biotechnology》).

[0058] Preparation method of Bacillus preparation in Example 1

[0059] Preparation of Bacillus preparation:

[0060] Bacillus subtilis (B1), Bacillus atrophaeus (B2), Bacillus amyloliquefaciens (B3), and Peribacillus frigoritolerans (B4) were respectively inoculated in LB liquid medium and cultured at 37 °C. The culture broth with an OD of 0.1 was collected. The harvested culture broth was washed twice in PBS and then resuspended in 0.9% sterile sodium chloride solution to make its absorbance (OD 600 ) be 0.1 for standby.

[0061] The prepared bacterial liquid was divided into 8 treatment groups, and bacterial agents with an OD 600 of 0.1 were respectively prepared:

[0062] B3 bacterial agent: The bacterial suspension of strain B3 in 0.9% sterile sodium chloride solution;

[0063] Equal-proportion mixed bacterial liquid groups of two strains: B13 (mixing strain B1 and strain B3 in equal volume ratio), B23 (mixing strain B2 and strain B3 in equal volume ratio), B34 (mixing strain B3 and strain B4 in equal volume ratio);

[0064] Equal-proportion mixed bacterial liquid groups of three strains: B123 (mixing strain B1, strain B2, and strain B3 in equal volume ratio), B134 (mixing strain B1, strain B3, and strain B4 in equal volume ratio), B234 (mixing strain B2, strain B3, and strain B4 in equal volume ratio);

[0065] Equal-proportion mixing of four strains: B1234 (mixing strain B1, strain B2, strain B3, and strain B4 in equal volume ratio).

[0066] Preparation of lettuce leaves under different groups in Example 2

[0067] Preparation of liquid organic fertilizer: Pig manure was soaked in water (m / v = 1 / 100) for three days, and stirred several times in the middle to increase the dissolution of the manure. Then it was filtered through five layers of gauze (with a pore size of 1 mm), and the pH was adjusted to 5.5 and then collected for standby.

[0068] Obtaining of lettuce contaminated with antibiotic resistance: First, soak lettuce seeds in a 5% hydrogen peroxide solution (w / w) for 30 minutes and wash them three times with deionized water. Then, place the seeds on moist filter paper for germination in the dark at 25°C. After 3 - 10 days of seed germination, select seedlings with similar growth and root length (about 1 cm) and sow them in a seedling tray filled with vermiculite. When the seedlings grow to the stage of having three true leaves and one heart leaf, select seedlings with consistent growth and transplant them into a hydroponic tank. In the hydroponic tank, first cultivate with 1 / 2 Hoagland nutrient solution, and then use full Hoagland nutrient solution until the lettuce has seven true leaves after survival and adaptation. Subsequently, switch to the prepared liquid organic fertilizer for continuous hydroponics for 15 days and then collect samples.

[0069] Treatment method for lettuce in the experimental group: The hydroponic solution used in the cultivation process of lettuce in the experimental group is the same as that of the lettuce contaminated with antibiotic resistance described above. The difference is that from the stage when the lettuce has grown to seven true leaves, use a spray gun to evenly spray the bacterial agents prepared in each group of Example 1 on the lettuce leaves, and the spraying dose is 2 mL. Replace the same liquid organic fertilizer as in the initial stage of cultivation every 6 days and continue to regularly spray the bacterial agents, repeating 2 times. The cultivation period is the same as that of the antibiotic resistance contamination group.

[0070] Treatment method for lettuce in the control group: The hydroponic solution used in the cultivation process of lettuce in the control group is the same as that for obtaining the lettuce contaminated with antibiotic resistance described above. The difference is that from the stage when the lettuce has grown to seven true leaves, use a spray gun to evenly spray sterile water on the lettuce leaves, replace the liquid organic fertilizer and spray sterile water every 6 days, repeating 2 times. The cultivation period is the same as that of the antibiotic resistance contamination group.

[0071] Comparison of ARGs abundances in lettuce leaves under different treatment conditions in Example 3

[0072] Collect lettuce leaves from the plants with uniform growth in each group of Example 2 and measure the abundances of ARGs in the lettuce leaves.

[0073] A total of 8 ARGs were measured in the lettuce leaves, namely sulfonamide ARGs (sul2, sul3), tetracycline ARGs (tetX, tetG), quinolone ARGs (qepA1, qnrS1), β - lactam ARGs (blaOXA - 2), and florfenicol ARGs (floR). The ARGs in all lettuce samples were characterized by quantitative PCR (qPCR) using a PCR instrument. The reaction system was 25 μL, including 1 μL of template DNA, 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, 12.5 μL of UltraSYBR fluorescent dye, and 10.5 μL of sterile water. qPCR was carried out under the following conditions: hold at 95°C for 10 seconds, then perform 39 cycles at 95°C for 15 seconds each and 1 minute at 60°C. The primer information is shown in Table 1.

[0074] Table 1 Fluorescent quantitative PCR primers

[0075]

[0076]

[0077] The gene copy number is calculated by the standard curve and Ct value. Calibration curves are established using different concentrations of plasmids carrying the target gene. The concentration and quality of the plasmids are determined by a NanoDrop spectrophotometer. A standard curve for qPCR is generated using 10-fold serial dilutions of the plasmid. The R 2 values of all standard curves for ARGs are higher than 0.99. Based on the standard curve, the copy number of the target ARGs is calculated by using the Ct value of the sample. The primers for the annotation curve are shown in Table 2:

[0078] Table 2 Standard curves for the absolute quantification of ARGs

[0079]

[0080] A total of 8 ARGs belonging to 4 categories were detected in lettuce leaves, among which the quinolone resistance gene (qnrS1) had the highest proportion, and the sulfonamide resistance genes (sul2, sul3) had the lowest proportion ( Figure 1 ). Previous studies have shown that tetracyclines, β-lactams, quinolones, macrolides, and sulfonamides are the main antibiotic resistance genotypes in pig farms. However, sulfonamide resistance genes were hardly detected, which may be due to the strict control of antibiotic use in the livestock and poultry breeding industry in China in recent years, resulting in a change in the choice of antibiotics in pig farms.

[0081] As Figure 2 shown, the tetracycline resistance gene tetX decreased significantly in all treatment groups (p<0.05). Except for the B13 treatment group, the mixed bacterial agent reduced the abundance of tetX in lettuce leaves more significantly than the B3 strain treatment group, and was reduced by more than 80% compared with the CK treatment group. Compared with the CK group, all treatment groups of tetG decreased, and the B23 treatment group decreased by 75%. Generally speaking, the treatment group containing the mixed bacterial agent with B3 strain had a further improved reduction effect compared with the B3 strain treatment group. In addition, the abundance of sulfonamide resistance genes (sul2, sul3) was below 4 copy g -1 , which may be related to the types of antibiotics used in the pig farm where the pig manure used in the experiment came from.

[0082] As Figure 3As shown, compared with the CK treatment group, the quinolone resistance gene qepA1 in the remaining treatment groups except the B23 and B234 treatment groups was significantly reduced (p<0.05), and the reduction range was approximately 35%-45%. Compared with the qnrS1 abundance of 9.31×10 3 copies / g in the CK treatment group, the qnrS1 abundances in the B3, B34, and B123 treatment groups were 4.9×10 3 , 5.1×10 3 , and 4.95.1×10 3 copies / g -1 , indicating that these three treatment groups had the best reduction effects. The B3 treatment group had the best effect on reducing the blaOXA-2 abundance, with a 50% reduction. And B13 had the best effect on reducing the floR abundance, with a 51% reduction.

[0083] Example 4 Comparison of the nutritional quality of lettuce leaves under different treatment conditions

[0084] Collect the lettuce leaves of each group in Example 2, measure the chlorophyll content (SPAD) value of lettuce using a chlorophyll content meter, measure the plant height using a ruler, measure the above-ground fresh weight and underground fresh weight using a balance, and measure the root surface area, root volume, and root tip number using a root scanner.

[0085] Vitamin C: The determination of vitamin C (Vc) was carried out by ultraviolet spectrophotometry. First, accurately weigh 0.50 g of the ground sample into a 2 mL centrifuge tube, add 1 mL of hydrochloric acid with a volume fraction of 0.01, and break it on a cell disruptor for 2 minutes. Then, centrifuge at 10000 rpm for 10 minutes using a high-speed refrigerated centrifuge, take 0.20 mL of the clarified liquid, add it to a 10 mL centrifuge tube containing 0.10 mL of hydrochloric acid with a volume fraction of 0.1%, and make up the volume to 10 mL with distilled water. Take a 5 mL centrifuge tube, add 2 mL of the volume-fixed sample solution, 1 mL of hydrochloric acid with a volume fraction of 0.1, and 0.3 mL of CuCl2 (Cu 2+ mass concentration of 100 μg / mL) solution, then place it in a constant temperature water bath at 70 °C, heat for 13 min, and cool it with ice water. Using distilled water as the blank, measure the optical density at 243.8 nm using an ultraviolet-visible spectrophotometer. The standard curve was measured in the same way, with the amount of Vc (μg) as the abscissa and the corresponding optical density as the ordinate, and the standard curve was plotted. Check the standard curve to calculate the Vc content in the sample.

[0086] The results showed that the overall growth of lettuce was good, and there were certain differences among different treatment groups. The growth indexes of lettuce are shown in Table 3 and Table 4. Compared with the CK treatment group, the plant height of the B34 treatment group increased by 1.97 cm. For the roots of lettuce, the underground fresh weight of the B34 and B134 treatment groups increased by more than 50%. The root length of the B13 treatment group reached 19.27 ± 1.93 cm, and the root surface area, root volume and root tip number of the 62.5% treatment group increased. Generally speaking, B34 had the best growth status among all treatment groups.

[0087] Table 3 Growth indexes of lettuce

[0088]

[0089]

[0090] Table 4 Growth indexes of lettuce roots

[0091]

[0092] Except for the B1234 treatment group, the Vc content in lettuce in the remaining treatment groups increased significantly (p < 0.05)( Figure 4 ). Among them, the Vc content of the B23 treatment group was as high as 25.54 mg / 100 g, which was 25% higher than that of the control group. In addition, the Vc content of the treatment groups in which the other three strains were mixed with strain B3 alone was higher than that of strain B3 alone, while the mixing of three strains and four strains did not have a better effect. Generally speaking, the lettuce nutritional indexes of the treatment groups in which strain B3 was mixed with the other three strains alone were higher than those of other treatment groups, and could effectively improve the nutritional quality of lettuce.

[0093] Example 5 Mechanism study on the reduction of ARGs by Bacillus preparations

[0094] Determination of growth curve:

[0095] Strains B1 - B4 were respectively inoculated into LB liquid medium and cultured in a constant temperature shaker at 37 °C and 220 rpm for 12 h. The vegetative cells of each strain were harvested from the overnight - cultured LB liquid medium by centrifugation at 5000 rpm for 15 minutes. The harvested cells were washed twice in PBS and then resuspended in PBS buffer to make their OD 600It is 0.05 for subsequent use. The prepared bacterial solutions were respectively formulated into treatment groups of B13 (equal - volume mixture of strain B1 and strain B3), B23 (equal - volume mixture of strain B2 and strain B3), B34 (equal - volume mixture of strain B3 and strain B4), B123 (equal - volume mixture of strain B1, strain B2 and strain B3), B134 (equal - volume mixture of strain B1, strain B3 and strain B4), B234 (equal - volume mixture of strain B2, strain B3 and strain B4) and B1234 (equal - volume mixture of strain B1, strain B2, strain B3 and strain B4). The initial OD 600 value was 0.05. They were cultured in LB medium at 37 °C for 12 h, and the OD 600 value in the above - mentioned treatment groups was measured every 2 h.

[0096] Drawing of the growth curve: The growth rate of the strain was determined by the OD600 value at the stationary phase. By comparing the growth curves ( Figure 5 ), it was found that the growth rate of B3 was 0.92 h -1 . Except for the B13 and B123 treatment groups, the growth rates of the remaining treatment groups were higher than that of the B3 treatment group. Among them, the growth rate of B134 was the largest, being 1.26 h -1 . Generally speaking, the growth rate of the mixed strains mainly depends on the growth of their single strains, and no mutual inhibition effect was found among the mixed strains. At the end of the strain culture, except for B2 and B13, the remaining treatment groups reached the stationary phase around 8 h.

[0097] Biofilm: The measurement of the biofilm was carried out using a 96 - well polystyrene microtiter plate (MTP). 230 μL of TSB medium and 20 μL of the bacterial suspension prepared for each strain according to 2.1.2 (resuspended in PBS, OD600 was 0.05) were added to the wells of a sterile 96 - well cell culture plate (16 treatment groups, 6 replicates for each group, and the blank control only contained TSB medium). The plate was incubated under static conditions at 30 °C for 48 h. The contents of the microtiter plate were poured out, and the wells were washed three times with 300 μL of PBS. The remaining attached bacteria were fixed with 250 μL of methanol per well. After 15 minutes, the microtiter plate was emptied and air - dried. Each well of the microtiter plate was stained with 250 μL of 1% crystal violet for Gram staining for 5 minutes. The excess stain was rinsed off by placing the microtiter plate under running tap water. After the microtiter plate was air - dried, 250 μL of 33% (v / v) glacial acetic acid was used to extract the dye bound to the adherent cells per well. It was left standing at 30 °C for 30 min, and then the OD570 was measured using a microplate reader.

[0098] The biofilm amount of the experimental strains was between 0 and 1, and there were certain differences among different groups ( Figure 6) Generally speaking, the biofilm amount in all treatment groups increased compared with that in the B3 treatment group. Among them, the biofilm amount in the B1234 mixed strain treatment group was the highest, reaching 0.9. The biofilm amounts in the remaining treatment groups ranged from 0.4 to 0.8. Overall, the three mixed strain treatment groups had higher biofilm amounts than the two mixed strain treatment groups. By comparing with Figure 6 There was no obvious relationship between the biofilm amount of the strains and their growth rate. Instead, the formation of biofilms was more affected by several parameters such as temperature, pH value, microbial surface structure and its charge, substrate surface roughness, bacterial metabolites, heavy metals, and water availability.

[0099] Comparing the relationship between the total ARGs abundance and biofilms in lettuce leaves ([[]] Figure 1 and Figure 6 ), a negative correlation between the two was found in the mixed inoculum treatment groups, but this pattern was not observed in the B3 strain single treatment group. Differently, the B23 treatment group had the highest biofilm amount and the lowest total ARGs abundance.

[0100] (1) Competitive effects of Bacillus on malic acid and L-glutamic acid:

[0101] According to et al.'s research (see "The distinct impact of multi-color LED light on nitrate, amino acid, soluble sugar and organic acid contents in red and green leaf lettuce cultivated in controlled environment"), lettuce leaves contain abundant substances such as sugars, amino acids, and organic acids that can serve as carbon sources for bacteria, such as glucose, fructose, malic acid, citric acid, L-glutamic acid, and L-aspartic acid. By cross-comparing the carbon sources available to pathogenic bacteria such as Bacillus and Escherichia coli, malic acid and L-glutamic acid were finally selected as carbon sources. Prepare the bacterial suspension of each strain according to 2.1.2 (resuspended in M9 medium with an OD600 of 0.05), add 180 μL to the wells of a sterile 96-well cell culture plate, and then add 20 μL of carbon source (0.1 mol / L) to each well (16 treatment groups, 6 replicates per group, and the blank control contains only M9 medium). Incubate on a shaker at 37 °C and 220 rpm / min, and use a microplate reader to measure the OD600 values at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h. The same operation was carried out for the 5 common pathogenic bacteria (A1 - A4). After the pretreatment, the growth curves were plotted, and the maximum growth rate of bacteria under a single carbon source condition was used to characterize the utilization intensity of the bacteria for this carbon source.

[0102] In the M9 medium with malic acid and L-glutamic acid as the sole carbon sources, both ARB and the experimental strains can grow. They enter the stationary phase at about 8 h, and the maximum growth amount is about 0.3. When malic acid is used as the carbon source, the growth of the two mixed treatment strain groups is delayed, and they enter the logarithmic growth phase later than the other treatment groups, with a large difference in the growth curve trend. When L-glutamic acid is used as the carbon source, the growth trends of the experimental strains are consistent, and there are differences in the maximum growth amounts of different treatment groups. ARB grows stably, and the growth ability of A5 is the lowest. ARB enters the stationary phase at 10 h, and the maximum growth amount of A1 is only 1 / 2 of that of A2.

[0103] By comparing the maximum growth rates of different strains in malic acid, it is found that the maximum growth rate of ARB is lower than that of the experimental strains, and B23 is 1.4 times that of A1. When L-glutamic acid is used as the carbon source, the proportion of experimental strains with a maximum growth rate exceeding that of ARB decreases, but the two mixed strain treatment groups are still more dominant, and B34 is 1.2 times that of A3. There are differences in the utilization efficiencies of different carbon sources by ARB and the experimental strains, which is due to the different carbon source metabolism and regulation mechanisms among different bacteria, and this also determines the ability of organisms to adapt to the environment. Compared with the intestinal habitat where Escherichia coli thrives, the natural habitat of Bacillus is in the soil, which makes it more dominant in competing for carbon sources on lettuce.

[0104] (2) Antagonistic effect between Bacillus and drug-resistant strains:

[0105] The antibacterial activity of Bacillus is related to exopolysaccharide (EPS). The extraction of EPS was carried out according to the research of Park et al. (in the reference "Cell-free supernatants of Bacillus subtilis and Bacillus polyfermenticus inhibit Listeria monocytogenes biofilm formation"). First, prepare the vegetative cells of each strain: inoculate the strains B1 - B4 into LB liquid medium and culture them in a shaker at a constant temperature of 37°C and 220 rpm for 12 h. Harvest the vegetative cells of each strain from the overnight-cultured LB liquid medium by centrifugation at 5000 rpm for 15 minutes. Secondly, prepare the EPS extract: after washing the vegetative cells by centrifugation with sterile PBS buffer three times, resuspend them in 2 mL of sterilized 0.05% NaCl solution and incubate in a water bath at 60°C for 30 min. Centrifuge at 4500 rpm for 5 min at room temperature to collect the supernatant. Filter the supernatant through a 0.22 μm filter membrane, and use glucose as a standard to determine the EPS content. Add 1 mL of the extract and 1 mL of phenol (5%) solution into a glass test tube, shake well, and then quickly add 5 mL of concentrated sulfuric acid. Incubate in a water bath at 40°C for 30 min. Measure the absorbance of the sample at a wavelength of 490 nm.

[0106] The Kirby-Bauer disk diffusion susceptibility test was used to detect the antibacterial activity of the Bacillus extract against five common pathogenic bacteria. Prepare the bacterial suspensions of the five pathogenic bacteria (resuspended in PBS, OD600 is 0.1), and use a spreading bead to spread 100 μL of each bacterial suspension on an agar (1.5%) LB plate in a sterile operating table. Use sterile forceps to place the drug sensitivity test paper on the inoculated plate within 15 minutes after inoculation. Place three drug sensitivity test papers on each plate, and add 5 μL of the EPS extract, with PBS as the blank control. After incubating the prepared plates statically at 30°C for 48 hours, observe the diameter of the inhibition zone to evaluate the antibacterial ability of Bacillus.

[0107] The EPS content of the four experimental strains is B3 > B2 > B4 > B1 (Table 5). Among them, the EPS content of strain B1 is much lower than that of the other strains, and the polysaccharide content of strain B3 is the highest. The high or low EPS content may be the reason why the bacterial agent B23 has a better effect on reducing the abundance of ARGs, while the bacterial agent B13 has a poor effect on reducing the absolute abundance of ARGs. However, in the Kirby-Bauer disk diffusion susceptibility test, the EPS extracts of the experimental strains failed to show antibacterial activity against ARB. Figure 9) It may be that due to the low content of EPS extracted, it cannot inhibit ARB. According to the latest research results, 47 antibacterial metabolites have been found in Bacillus, including polysaccharides, lipopeptides, enzymes (amylase, cellulase, chitinase, chitosanase, glucanase, and protease), etc. Therefore, there may be other substances that enable Bacillus to inhibit ARB.

[0108] Table 5 EPS content of Bacillus

[0109]

[0110] Application of Bacillus preparation in reducing plant antibiotic resistance pollution

[0111] This example is used to illustrate the application of Bacillus agent in reducing plant antibiotic resistance pollution; the plants include but are not limited to edible fruits or vegetables. Optionally, the vegetables include but are not limited to crown daisy, spinach, rape, lettuce, pea sprouts, Chinese cabbage, celery, and endive.

[0112] The specific steps are as follows:

[0113] (1) Prepare a Bacillus preparation with a bacterial concentration ≥ 1×10 7 CFU / mL according to the method of Example 1.

[0114] (2) Spray the prepared Bacillus preparation on the branches and leaves of the plants, or irrigate it at the roots of the plants for a period of time to obtain plants with reduced antibiotic resistance pollution.

[0115] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A composition, characterized in that, Composed of Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), B3, Bacillus subtilis ( Bacillus subtilis ), B1, Bacillus atrophaeus ( Bacillus atrophaeus ), B2 and Psychrobacillus cryophilus ( Peribacillus frigoritolerans ), B4.

2. The composition according to claim 1, wherein The bacterial concentration in the composition ≥ 1×10 7 CFU / mL.

3. The composition according to claim 1, wherein The cell concentrations of the microorganisms in the composition are of the same order of magnitude.

4. A method for preparing the composition according to claim 1, characterized in that, The microorganisms are cultured in a culture medium for a period of time, the cells are collected, and washed to prepare a bacterial suspension with a specific concentration.

5. The method according to claim 4, characterized in that The culturing is carried out at 35-40° C. for 8-16 hours.

6. The method according to claim 5, wherein The specific concentration refers to the OD of the bacterial suspension 600 being 0.08 - 0.

12.

7. Application of Bacillus amyloliquefaciens B3, Bacillus subtilis B1, Bacillus atrophaeus B2 and Bacillus psychrosporus B4 in reducing plant ARGs contamination and / or improving the nutritional quality of vegetables.

8. The application according to claim 7, wherein The plants include, but are not limited to, edible fruits or vegetables.

9. The application according to claim 8, wherein The use is to contact the bacillus with the plant.

10. The application according to claim 9, wherein The application is to spray the bacterial suspension containing the bacillus onto the surface of the plant.

11. The application according to claim 10, wherein The concentration of Bacillus in the bacterial suspension ≥ 1×10 7 CFU / mL.

12. The application according to any one of claims 7 to 11, characterized in that, The ARGs contamination includes, but is not limited to, sulfonamide antibiotic resistance gene contamination, tetracycline antibiotic resistance gene contamination, quinolone antibiotic resistance gene contamination, β-lactam antibiotic resistance gene contamination and florfenicol antibiotic resistance gene contamination.

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