Bacillus siamensis strain for reducing antibiotic resistance and bacterial inoculant and use thereof

Abstract

The application belongs to the field of biological environmental protection technology, and particularly relates to a kind of antibiotic resistance reduction bacillus siamensis and microbial agent and application thereof.The preservation number of the antibiotic resistance reduction bacillus siamensis is CCTCC NO: M 20232193.The bacillus siamensis provided by the application can effectively reduce the antibiotic resistance of soil, can significantly reduce the abundance of antibiotic resistance bacteria and antibiotic resistance genes in soil, the average reduction rate of different antibiotic resistance bacteria and antibiotic resistance genes reaches 38.92% and 46.32%, can greatly reduce the proliferation and diffusion of ARGs and ARB in soil, significantly reduces the enrichment level, provides a new solution for efficient, pollution-free elimination of soil antibiotic resistance, has the potential to be applied to promote sustainable agriculture, animal husbandry development and improve soil health;In addition, the strain has strong biofilm formation ability, and is expected to be used as a biocontrol agent.

Description

Technical Field

[0001] This invention relates to the field of bio-environmental protection technology, and in particular to a Bacillus sicca for reducing antibiotic resistance and its inoculant and application. Background Technology

[0002] In large-scale and intensive livestock and poultry farming, antibiotics are widely used as feed additives to prevent and treat infections, promote animal growth, and improve feeding efficiency. However, approximately 60-90% of antibiotics cannot be completely absorbed by animals and are excreted in feces as parent compounds or secondary metabolites. This results in a high diversity and abundance of antibiotic resistance genes in feces, making it a significant source of antibiotics in the environment. The widespread accumulation of antibiotics in the environment will stress and induce the evolution of antibiotic resistance in environmental microorganisms, leading to the generation of antibiotic resistance genes (ARGs) in organisms. Simultaneously, microorganisms can induce the emergence of new antibiotic resistance bacteria (ARBs) through gene mutations or horizontal gene transfer within the same or different bacterial populations via mobile gene elements such as plasmids, integrons, and transposons. A growing body of research demonstrates that ARGs and ARBs are mobile and persistent, and their proliferation and spread in the natural environment are more difficult to manage than antibiotic residues, posing a serious threat to human health, plant and animal growth, and the ecological environment. Therefore, antibiotic resistance in the environment is an increasingly serious global public health problem, and its potential harm cannot be ignored. Antibiotic-resistant bacteria and antibiotic resistance genes have been identified as novel pollutants. How to efficiently remove ARGs and ARBs from the environment has become a key focus and challenge of current research.

[0003] Currently, research on ARGs removal technologies is still in its early stages. Reported ARGs removal technologies mainly include ultraviolet and chlorination disinfection, coagulation and sedimentation using aluminum and iron salts, and combined coagulation, sedimentation, and disinfection treatment. However, these technologies all require the addition of numerous water treatment units and equipment, as well as the addition of large amounts of chemical agents, significantly increasing treatment costs, land occupation, and operational management difficulties, thus exhibiting considerable limitations. Microorganisms are the hosts of ARGs, and their abundance is strongly influenced by the microbial community. Furthermore, similar to plant pathogens, soil ARBs are affected by direct antagonism, indirect nutrient competition, or niche occupation by other microorganisms. Therefore, the enrichment of ARGs and ARBs in the environment can be reduced by the addition of exogenous antagonistic microbial agents. Thus, the introduction of exogenous agents may provide an effective means of controlling soil antibiotic resistance.

[0004] Soil possesses high bacterial community diversity and complexity. Fecal antibiotics and antibiotic resistance genes (ARGs) can be transferred into the soil through the application of organic fertilizers, altering the soil microbial community structure and antibiotic resistance profile. Furthermore, the high abundance and diversity of mobile genetic elements can facilitate the transfer of ARGs between exogenous and native soil microorganisms. These factors inevitably make soil an important reservoir for ARGs and ARBs. Screening for soil-borne beneficial microorganisms with ARG and ARB reduction capabilities holds great potential for mitigating the potential ecological risks of antibiotic resistance and offers broad prospects for developing healthy agricultural and livestock systems. However, currently, there are few reports on microorganisms with antibiotic resistance reduction capabilities. Summary of the Invention

[0005] To address the lack of microorganisms with antibiotic resistance reduction capabilities in existing technologies, this invention provides a Bacillus sicca for reducing antibiotic resistance, a bacterial agent containing the Bacillus sicca, and the application of the aforementioned Bacillus sicca and bacterial agent in reducing antibiotic resistance. The aim is to solve the problems in the prior art or at least alleviate some of the problems in the prior art.

[0006] This invention is specifically achieved through the following technical solutions:

[0007] The first aspect of the present invention provides a Bacillus siamensis strain that reduces antibiotic resistance, with accession number CCTCC NO: M 20232193. This strain was deposited on November 10, 2023, at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province (Wuhan University), and the culture name is Bacillus siamensis zz07.

[0008] This invention isolated a strain from soil that had been treated with chicken manure and fermented soldier fly larvae fertilizer for a long period of time. This strain antagonized multiple soil antibiotic-resistant bacteria (ARBs) and reduced multiple antibiotic resistance genes (ARGs). It was identified as a Bacillus sicca strain. The strain was then subjected to a 4 × 10⁻⁶ ppm fermentation process. 8Inoculated into the soil at a rate of CFU / kg, and after 20 days of treatment, soil samples were collected to determine the colony count of resistant bacteria. The results showed that this strain reduced the number of six ARBs (antibiotics) in the soil by 73.02%, 26.56%, 100.00%, 12.53%, 78.86%, and 51.76%, respectively, against vancomycin, penicillin, oxytetracycline, sulfadiazine, thiamphenicol, and streptomycin-resistant bacteria. Simultaneously, the abundance of ARGs was measured, revealing that this strain reduced the number of 13 ARGs in the soil by 81.95%, 70%, and 80%, respectively, against strB, sul2, floR, qacB, tetX, blaTEM, tetB, mdtB, mdtC, mdtH, emrR, fox5, and bmr genes. The percentages of antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARBs) in the present invention were 0.75%, 24.78%, 30.20%, 98.11%, 73.65%, 73.95%, 46.34%, 38.64%, 69.89%, 31.66%, 27.80%, and 98.33%, respectively. This demonstrates that the *Bacillus sicca* zz07 strain of the present invention can effectively reduce the abundance of antibiotic resistance genes and antibiotic-resistant bacteria, with average reduction rates of 46.32% and 38.92%, respectively. This significantly reduces the proliferation and spread of ARGs and ARBs in the soil, lowering their accumulation levels in the environment. It is an excellent microbial strain for alleviating antibiotic resistance pollution and provides a new solution for eliminating soil antibiotic resistance using biological methods, possessing broad market prospects and significant ecological importance. Furthermore, the Siamese Bacillus zz07 of the present invention has a strong biofilm-forming ability. The biofilm can stabilize the environment around the strain, improve the availability of nutrients and oxygen, and help it quickly seize the ecological niche in the soil. It plays a competitive role in nutrition and ecological niche against antibiotic-resistant bacteria or pathogenic bacteria, thereby limiting the growth and reproduction of the aforementioned microorganisms. It is expected to be used as a biocontrol agent in the field of plant disease control and has a wide range of application prospects.

[0009] A second aspect of the present invention provides a bacterial agent for reducing antibiotic resistance, including Bacillus sicca as described above.

[0010] The advantages of the antibiotic resistance-reducing bacterial agent compared to the prior art are the same as those of the antibiotic resistance-reducing Bacillus sicca compared to the prior art, as described above, and will not be repeated here.

[0011] Furthermore, this also includes acceptable functional adjuvants. Acceptable functional adjuvants are components that do not interfere with the efficacy of the active ingredient, namely the biological activity of *Bacillus sicca*, and do not have significant toxicity or pathological effects on the body (including humans, animals, or plants) at the concentrations in which they are applied. These include any one or a combination of at least two of solvents, dispersants, stabilizers, wetting agents, emulsifiers, synergists, thickeners, defoamers, suspending agents, antioxidants, lyophilizers, preservatives, and pH adjusters. The use of the above components in microbial preparations is well known in the art. For example, solvents may include water and / or organic reagents, as well as mixtures of these solvents; antioxidants may include one or more of benzoic acid, ascorbic acid, 2,6-di-tert-butyl-4-methylphenol, sodium sulfite, or sodium bisulfite; pH adjusters may include one or more of sodium carbonate, sodium bicarbonate, phosphoric acid, dipotassium hydrogen phosphate, sodium hydroxide, and ammonia. The biocide of the present invention can be prepared into wettable powder, oil, aqueous solution, suspension or lyophilized agent by means of the aforementioned acceptable functional additives.

[0012] Furthermore, the bacterial agent is an aqueous solution, and the concentration of the Bacillus sicca is 1×10⁻⁶. 6 -1×10 10 CFU / mL.

[0013] A third aspect of the invention provides the use of the antibiotic-resistant Bacillus sicca described above or the antibiotic-resistant bacterial agent described above in the reduction of antibiotic resistance.

[0014] The advantages of the Bacillus sicca or bacterial agent for reducing antibiotic resistance compared to existing technologies are the same as those of the Bacillus sicca for reducing antibiotic resistance compared to existing technologies, and will not be repeated here.

[0015] Furthermore, the reduction of antibiotic resistance includes reducing antibiotic-resistant bacteria and / or reducing antibiotic resistance genes.

[0016] Furthermore, the antibiotic-resistant bacteria include at least one of vancomycin, penicillin, oxytetracycline, sulfadiazine, thiamphenicol, and streptomycin-resistant bacteria.

[0017] Furthermore, the antibiotic resistance gene includes at least one of the following: aminoglycosides, quinolones, tetracyclines, β-lactams, macrolides, lincosamides, streptozotocins, sulfonamides, and multidrug-resistant genes.

[0018] Specifically, aminoglycoside resistance genes include mdtB, mdtC, and strB; quinolone resistance genes include mdtH, qacB, and emrR; tetracycline resistance genes include tetB and tetX; β-lactam resistance genes include fox5 and blaTEM; macrolide, lincosamide, and streptozotocin resistance genes include floR; sulfonamide resistance genes include sul2; and multidrug resistance genes include bmr.

[0019] Furthermore, the application method includes: applying the Bacillus sicca at a concentration of 1×10⁻⁶. 7 -1×10 10 The dosage of CFU / kg soil is applied to the soil.

[0020] The advantages and positive effects of this invention are as follows:

[0021] 1. The *Bacillus sicca* provided by this invention can effectively reduce soil antibiotic resistance, mainly by effectively reducing the abundance of antibiotic-resistant bacteria and antibiotic resistance genes in the soil. The average reduction rates for six ARBs (vancomycin, penicillin, oxytetracycline, sulfadiazine, thiamphenicol, and streptomycin-resistant bacteria) and 13 ARGs (strB, sul2, floR, qacB, tetX, blaTEM, tetB, mdtB, mdtC, mdtH, emrR, fox5, and bmr genes) are 38.92% and 46.32%, respectively. This can significantly reduce the proliferation and spread of ARGs and ARBs in the soil and significantly reduce their enrichment level in the environment. It provides a new solution for the efficient and pollution-free elimination of soil antibiotic resistance and has the potential to be applied to promote sustainable agriculture and animal husbandry development and improve soil health.

[0022] 2. The Bacillus sicca provided by this invention has a strong biofilm-forming ability, which can quickly seize ecological niches and compete with antibiotic-resistant bacteria or pathogenic bacteria for nutrients and ecological niches, thereby limiting the growth and reproduction of the aforementioned microorganisms. It is expected to be used as a biocontrol agent in the fields of preventing and controlling plant diseases and promoting the growth of plants and animals. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a phylogenetic tree diagram of Bacillus zz07 from Example 1 of the present invention;

[0025] Figure 2 This is a colony morphology diagram of Bacillus sicca zz07 on LB solid culture medium in Example 1 of the present invention;

[0026] Figure 3 The colony counts of different antibiotic-resistant bacteria in the soil 20 days after inoculation with Bacillus sicca zz07 of Example 2 of the present invention;

[0027] Figure 4 A heatmap showing the abundance of different antibiotic resistance genes in the soil 20 days after inoculation with Bacillus sicca zz07 of Example 2 of the present invention;

[0028] Figure 5 This is a graph showing the biofilm formation ability test results of Bacillus sicca zz07 in Example 3 of the present invention;

[0029] Figure 6 The relative abundance of the key biofilm synthesis gene epsA in the soil 20 days after inoculation with Bacillus sicca zz07 of Example 3 of the present invention. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0031] Based on the information contained herein, various changes to the precise description of the invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.

[0032] To better understand the invention and not to limit its scope, all figures and other numerical values ​​used in this invention to indicate amounts, percentages, etc., should in all cases be understood to be modified by the word "about". Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods. In this invention, "about" means within 10%, preferably within 5%, of a given value or range. Furthermore, it should be noted that the terms "comprising," "including," "containing," "having," etc., are non-limiting in meaning, allowing for the addition of other steps and other components that do not affect the result.

[0033] Unless otherwise specified, all embodiments of the present invention are performed under normal temperature conditions. Normal temperature refers to the natural room temperature conditions in all four seasons, without additional cooling or heating treatment. Generally, the normal temperature is controlled between 10-30°C, preferably between 15-25°C.

[0034] In the following examples, the test soil was taken from the vegetable greenhouse of the Vegetable Research Institute of Wuhan Academy of Agricultural Sciences, Huangpi District, Wuhan City, Hubei Province. This soil had been treated with soldier fly larvae bio-organic fertilizer for a long period at a rate of 5000 kg / ha. The soldier fly larvae bio-organic fertilizer was produced as follows: 300,000 eight-day-old soldier fly larvae were inoculated into 300 kg of fresh chicken manure, and biotransformation was carried out for 10 days in a greenhouse at 28°C and 70% relative humidity. The fresh chicken manure was taken from the chicken farm of Wuhan Chaotuo Ecological Agriculture Co., Ltd., and the moisture content was adjusted to 70% with bran.

[0035] In the following examples, the culture media used included: 1) liquid LB medium: 10 g / L peptone, 5 g / L yeast extract and 10 g / L sodium chloride, pH 7.0; 2) solid LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride and 15 g / L agar, pH 7.0; all culture media were sterilized at 121°C for 20 min before use.

[0036] In the following examples, Bacillus subtilis 168 was obtained from the collection of the School of Life Sciences, Hubei University.

[0037] Unless otherwise specified, the experimental methods described in the following examples are generally performed according to the conditions recommended by the manufacturer or conventional experimental methods in the art.

[0038] Example 1: Isolation and Identification of Bacillus siamensis zz07

[0039] Soil from vegetable greenhouses at the Wuhan Academy of Agricultural Sciences' Vegetable Research Institute, which had been using soldier fly larvae bio-organic fertilizer for a long time, was used as the test soil. 10g of soil was added to an Erlenmeyer flask containing 90mL of sterile water, and the mixture was shaken at 180rpm for 30min to prepare a soil suspension. The suspension was then heated in a 90℃ water bath for 20min. Since Bacillus can form heat-resistant spores, it could still survive after heat treatment. After heat treatment, the suspension was serially diluted, and the diluted solutions were spread onto LB agar plates and incubated at 37℃ for 48h. Based on morphology and colony characteristics (rough, opaque colony surface, dirty white or slightly yellow), suspected Bacillus colonies were preliminarily identified. These candidate Bacillus colonies were then spread onto LB agar plates for purification.

[0040] Purified Bacillus subtilis was inoculated into LB liquid medium, and bacterial DNA was extracted and amplified by PCR. The 16S rRNA gene was amplified using universal bacterial primers 27F (DNA sequence 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (DNA sequence 5'-GGTTACCTTGTTACGACTT-3'). The PCR reaction program was as follows: 94℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 50 s, for 25 cycles, followed by a final extension at 72℃ for 10 min. The purified PCR products were electrophoresed on a 1% agarose gel, and the electrophoretic bands were recovered and sequenced.

[0041] The sequenced sequences were aligned using the NCBI online BlastN tool, and phylogenetic analysis was performed using MEGA 5.0 software. The phylogenetic tree is shown below. Figure 1 As shown, the strain of this invention belongs to Bacillus siamensis in phylogenetic taxonomy and is named Bacillus siamensis zz07. Its colony morphology is as follows. Figure 2 As shown.

[0042] The 16S rRNA gene sequence of Bacillus siamensis zz07 is shown below:

[0043]

[0044] The aforementioned Bacillus siamensis zz07 was deposited on November 10, 2023, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province (Wuhan University), with accession number CCTCC NO: M20232193, and the culture was classified as Bacillus siamensis.

[0045] Example 2: Study on the reduction of soil antibiotic resistance by Bacillus siamensis zz07

[0046] Tomato seeds were grown in moist vermiculite for 20 days and then transplanted into potting soil. *Bacillus sicca* zz07 was inoculated into 200 mL of LB broth and cultured at 37°C and 170 rpm for 2 days. The bacterial culture was then centrifuged, washed, and resuspended in sterile PBS solution to obtain a density of 102. 7 Inoculation solution at CFU / mL. Inoculate 20 mL of the bacterial suspension per pot surface, with an addition rate of 4 × 10⁻⁶ bacteria per kg of soil. 8 CFU was used in triplicate for each treatment. A blank control group was used, which was not inoculated but had the same amount of PBS solution added. A control group was used, which was inoculated with the same amount of Bacillus subtilis 168. Watering was carried out every two days during the inoculation period, and the rest of the time the conditions were left natural.

[0047] Soil samples were collected 20 days after inoculation with Bacillus zz07 strain of Siamese bacteria. The soil samples were diluted and spread on LB solid medium containing seven antibiotics (streptomycin, thiamphenicol, vancomycin, penicillin, ciprofloxacin, oxytetracycline and sulfadiazine, all at a concentration of 50 mg / L). The samples were incubated at 37°C for 24 h, and the number of antibiotic-resistant bacterial colonies on the solid medium was counted. For colony counting on solid culture media, select dilutions with colony counts between 30 and 300, multiplied by the dilution factor. If the average colony count of all dilutions is >300, multiply the average colony count of the highest dilution by the dilution factor. For example, if the colony counts at 1000x and 10000x dilutions both exceed 300, multiply the dilution factor by 10000. If the average colony count of all dilutions is <30, multiply the average colony count of the lowest dilution by the dilution factor. For example, if the colony counts at 1000x and 10000x dilutions both are below 30, multiply the dilution factor by 1000. The reduction rate of antibiotic-resistant bacteria is calculated using the following formula:

[0048] Antibiotic-resistant bacteria reduction rate = (A 空白对照 -A 实验组 ) / A 对照 ×100%; where A空白对照 A represents the number of antibiotic-resistant bacteria in the soil of the PBS-treated group. 实验组 This indicates the number of antibiotic-resistant bacteria in the soil of the inoculated strain treatment group.

[0049] The effects of Bacillus sicca zz07 on the growth of antibiotic-resistant bacteria, such as Figure 3 As shown, the results indicate that the addition of Bacillus sicca strain zz07 was more effective than Bacillus subtilis strain 168 in reducing various antibiotic-resistant bacteria in the soil. The reduction rates of vancomycin, penicillin, oxytetracycline, sulfadiazine, thiamphenicol, and streptomycin-resistant bacteria in the soil were 73.02%, 26.56%, 100.00%, 12.53%, 78.86%, and 51.76%, respectively. The average reduction rate of the six antibiotic-resistant bacteria reached 38.92%, which was calculated by (number of all resistant bacteria in the PBS group - number of all resistant bacteria in the experimental group) / number of all resistant bacteria in the PBS group.

[0050] Soil samples were collected 20 days after inoculation with Bacillus sicca strain zz07. DNA was extracted from the potted soil using the SoilDNA Kit (Omega Bio-Tek, USA, D5625-01) according to the manufacturer's instructions. The concentration and quality of the extracted DNA were determined using a Nanodrop 2000 spectrophotometer (Thermo Scientific, USA). DNA samples were stored at -80°C for subsequent quantitative PCR (qPCR) detection. A total of 15 ARGs were measured, including 3 aminoglycosides (mdtB, mdtC, strB), 3 quinolones (mdtH, qacB, emrR), 2 tetracyclines (tetB, tetX), 2 β-lactams (fox5, blaTEM), 2 MLSBs (floR, lnuC), 1 sulfonamide (sul2), 1 vancomycin (vanG), and 1 multidrug-resistant strain (bmr). qPCR detection was performed using the QuantStudio 5 system from Applied Biosystems, USA, with SYBR Green I fluorescent dye. Primer sequences for each ARG detection are shown in Table 1. The 16S rRNA gene was used as an internal reference gene. It should be noted that macrolides, lincosamides, and streptogramin B are three types of antimicrobial drugs with different structures but the same or overlapping target sites. Microorganisms can exhibit cross-resistance to these three classes of antimicrobial drugs simultaneously, a phenomenon known as macrolide-lincosamide-streptogramin B (MLSB) resistance.

[0051] Table 1 Primer sequences for quantitative real-time PCR detection of antibiotic resistance genes

[0052]

[0053]

[0054] The qPCR reaction system (per 10 μL) included: 5 μL SYBR green Master Mix, 0.2 μL forward primer, 0.2 μL reverse primer, 1 μL DNA template, and 3.6 μL ddH2O. The reaction program was as follows: 95℃ pre-denaturation for 2 min, 95℃ denaturation for 15 s, 53℃ annealing for 30 s, and 72℃ extension for 15 s, for 39 amplification cycles. Melting curve analysis was performed, with subsequent reactions at 65℃ for 5 s and 95℃ for 5 s. The reduction rate of antibiotic resistance genes was calculated using the following formula:

[0055] Antibiotic resistance gene reduction rate = (B 空白对照 -B 实验组 ) / B 对照 ×100%; where B 空白对照 Indicates the abundance of soil antibiotic resistance genes in the PBS-treated group, B 实验组 This indicates the abundance of soil antibiotic resistance genes in the inoculated strain treatment group.

[0056] The effect of Bacillus sicca zz07 on the abundance of different antibiotic resistance genes, such as Figure 4 As shown in Table 2, the results indicate that the addition of Bacillus sicca zz07 was more effective than Bacillus subtilis 168 in reducing multiple antibiotic resistance genes in the soil. The reduction rates of strB, sul2, floR, qacB, tetX, blaTEM, tetB, mdtB, mdtC, mdtH, emrR, fox5, and bmr genes in the soil were 81.95%, 70.75%, 24.78%, 30.20%, 98.11%, 73.65%, 73.95%, 46.34%, 38.64%, 69.89%, 31.66%, 27.80%, and 98.33%, respectively. The average reduction rate of the 13 ARGs reached 46.32%, which was calculated by (total abundance of ARGs in the PBS group - total abundance of ARGs in the experimental group) / total abundance of ARGs in the PBS group.

[0057] Table 2. Gene abundance of antibiotic resistance genes

[0058]

[0059]

[0060] Analysis of the Biofilm Formation Ability of Bacillus siamensis zz07 in Example 3

[0061] Bacillus siamensis zz07 was cultured in LB liquid medium for 24 h. 100 μL of LB liquid medium was added to each well of a 96-well polystyrene microplate and 10 μL of the bacterial fermentation broth was inoculated, and then incubated statically at 37 °C for 48 h. Then, the biofilm formation ability of the strain was measured according to the following steps: 1) The culture broth was aspirated, and 200 μL of sterile PBS buffer was added to each well to wash the wells 3 times; 2) 100 μL of methanol was added to each well to fix for 15 min, then the methanol in the culture wells was aspirated and air-dried naturally; 3) 100 μL of 1% crystal violet solution was added to each well and stained at room temperature for 5 min; 4) After aspirating the crystal violet staining solution in the culture wells, the excess dye was rinsed off with running water; 5) The microplate was inverted on filter paper to remove the residual water, and dried in an oven at 37 °C or air-dried at room temperature; 6) After complete drying, 100 μL of 33% glacial acetic acid solution was added to each well and allowed to act in an incubator at 37 °C for 30 min to dissolve the crystal violet; 7) The OD value of the solution in the culture wells was measured with a microplate reader at 590 nm; Each strain was replicated in 3 wells for each experiment, and the experimental values were averaged 3 times (D value). The culture broth without inoculated bacteria was used as a negative control, and twice the negative value was used as the cut-off value (Dc).

[0062] Result determination: Strong biofilm-forming strain (D > 2×Dc); Weak biofilm-forming strain (Dc < D < 2×Dc); No biofilm-forming strain (D < Dc). As Figure 5 shown, the D value of Bacillus siamensis zz07 strain was 0.42 ± 0.08, and the experimental Dc was 0.08 ± 0.01, indicating that it has strong biofilm formation ability. There are various main biological macromolecules in the biofilm, such as proteins, polysaccharides (such as exopolysaccharides (EPS)), DNA, RNA, peptidoglycan, lipids, and phospholipids, etc., which can wrap the bacterial community to form a bacterial aggregate film; Therefore, the presence of the biofilm can create a stable environment for the life activities of cells as a barrier, improve the availability of surrounding nutrients and oxygen, facilitate its rapid occupation of ecological niches in the soil, and play a role in competing for nutrients and ecological niches against pathogenic bacteria, etc. That is, the strain of the present invention has strong application potential, and can not only be used in the field of eliminating antibiotic resistance, but also has applications in the fields of biological control of plant diseases, promotion of the growth of animals and plants, etc.

[0063] In view of the role of biofilm formation in the reduction of antibiotic-resistant bacteria, a qPCR detection method was used to detect the relative abundance of the key biofilm polysaccharide biosynthesis gene (epsA). The amplification system and procedure were the same as above, and the primer sequences for detection are shown below:

[0064] epsA-F: 5'-TCGAATCTCAGTGACATCCA-3' (see SEQ ID NO. 34);

[0065] epsA-R: 5'-AGATAGGTGCAATTCCGC-3' (see SEQ ID NO. 35).

[0066] like Figure 6 As shown, inoculating soil with the fermentation broth of this strain can significantly increase the relative abundance of the epsA gene in the soil, which is conducive to the rapid formation of biofilm by soil microorganisms, thereby inhibiting the growth and reproduction of antibiotic-resistant or pathogenic bacteria.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of Bacillus sicca that reduces antibiotic resistance ( Bacillus siamensis ), characterized in that, The accession number is CCTCC NO: M 20232193.

2. A bacterial agent for reducing antibiotic resistance, characterized in that, Including the antibiotic-resistant Bacillus sicca as described in claim 1.

3. The antibiotic resistance-reducing bacterial agent according to claim 2, characterized in that, The concentration of the *Bacillus sicca* was 1 × 10⁻⁶. 6 -1×10 10 CFU / mL.

4. The application of the antibiotic-resistant Bacillus sicca as described in claim 1 or the antibiotic-resistant bacterial agent as described in any one of claims 2-3 in the reduction of antibiotic resistance, characterized in that, The reduction of antibiotic resistance refers to the reduction of antibiotic-resistant bacteria or the reduction of antibiotic resistance genes. The antibiotic-resistant bacteria are at least one of vancomycin, penicillin, oxytetracycline, sulfadiazine, thiamphenicol, and streptomycin-resistant bacteria. The antibiotic resistance gene is at least one of mdtB, mdtC, strB, mdtH, qacB, emrR, tetX, tetB, fox5, blaTEM, floR, sul2, and bmr genes.

5. The application of the antibiotic-resistant Bacillus sicca or antibiotic-resistant bacterial agent according to claim 4 in the reduction of antibiotic resistance, characterized in that, The application includes: dispensing the Bacillus sicca at a concentration of 1×10⁻⁶. 7 -1×10 10 A dose of CFU / kg was applied to the soil.

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