Bacillus velezensis nxpm130 for inhibiting erysipelothrix rhusiopathiae and application thereof

Abstract

This invention discloses a strain of *Bacillus belyssima* NXPM130 that inhibits *Erysipelothrix rhusiopathiae* and its applications, belonging to the field of microbial technology. The *Bacillus belyssima* NXPM130 has the accession number CGMCC No. 24334. This strain was obtained through targeted screening and identification of *Erysipelothrix rhusiopathiae* as a specific target from the intestinal contents of healthy piglets. In vitro antibacterial tests and in vivo models in piglets validated that this strain not only significantly reduces the *Erysipelothrix rhusiopathiae* load in the intestines and feces but also improves intestinal barrier function and promotes growth performance. This invention fills the technological gap in specific probiotic spores targeting *Erysipelothrix rhusiopathiae*, providing a safe, efficient, and drug-resistant new biocontrol solution for pig farming.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Bacillus vesiculosus NXPM130 that inhibits Erysipelothrix rhusiopathiae and its applications. Background Technology

[0002] Swine erysipelas ( Erysipelothrix rhusiopathiae Erysipelothrix rhusiopathiae is an important zoonotic pathogen widely found in pig populations worldwide. It primarily causes swine erysipelas, with acute infection manifesting as high fever, cyanosis, and hemorrhagic erythema, accompanied by septicemia, and a mortality rate exceeding 50%. Chronic infection leads to arthritis, endocarditis, and skin necrosis, severely impacting growth performance and carcass quality in pigs. Epidemiological surveys show that, in addition to typical clinical symptoms, the colonization and increased abundance of Erysipelothrix rhusiopathiae in the intestines are closely related to host metabolic disorders. Recent studies have confirmed a significant correlation between abnormally increased Erysipelothrix rhusiopathiae abundance and the occurrence of obesity, colorectal cancer, and inflammatory bowel disease (IBD).

[0003] In pig farming practices, the persistent presence of Erysipelas posed a serious threat to the industry. Currently, prevention and control of this disease mainly rely on vaccination and antibiotic treatment. However, existing technologies have significant shortcomings: (1) Limitations of vaccines: There are many serotypes of Erysipelas (28 serotypes have been identified so far), and the cross-protection between different serotypes is weak. Moreover, the protective effect of current vaccines against heterologous serotypes is unstable. In addition, vaccination often causes stress response and the duration of immunity is short, requiring frequent booster immunizations, which increases breeding costs and management difficulty.

[0004] (2) Risk of drug resistance: Although antibiotics can control the epidemic, long-term abuse has led to the emergence of drug-resistant strains, which not only weakens the treatment effect, but also brings drug residues and food safety risks.

[0005] (3) Intestinal microecological imbalance: The use of antibiotics often disrupts the intestinal microecological balance, which in turn creates conditions for secondary infection of pathogens.

[0006] To address the aforementioned issues, the use of probiotics for biocontrol has become an important strategy for replacing antibiotics and improving gut health. Bacillus (…) Bacillus Bacillus spp. (Bacillus subtilis) is a promising candidate for feed probiotics due to its ability to form heat- and dry-resistant spores, as well as its unique advantages such as resistance to stomach acid and bile salts, and ease of granulation and storage. Through competitive exclusion, the production of antibacterial substances (such as bacteriocins and organic acids), and modulation of host immunity, Bacillus can effectively inhibit the colonization of intestinal pathogens.

[0007] However, although various Bacillus strains with broad-spectrum antibacterial activity have been disclosed in existing technologies, highly effective inhibitory strains specifically targeting swine erysipelas remain a gap. Existing technologies mostly focus on the inhibition of Escherichia coli or Salmonella, lacking targeted screening and functional verification for erysipelas as a specific target. Summary of the Invention

[0008] The purpose of this invention is to provide a strain of Bacillus beleilli NXPM130 that inhibits Erysipelothrix rhusiopathiae and its application, in order to solve the problems existing in the prior art. This strain can not only significantly reduce the load of Erysipelothrix rhusiopathiae in the intestine and feces, but also improve intestinal barrier function and promote growth performance, filling the technical gap of specific probiotic Bacillus beleilli erysipelothrix rhusiopathiae, and providing a safe, efficient and drug-free new biological control solution for pig farming.

[0009] To achieve the above objectives, the present invention provides the following solution: This invention provides a Bacillus belesiensis ( Bacillus velezensis The Bacillus belyssus NXPM130 has the accession number CGMCC No. 24334.

[0010] The present invention also provides a bacterial agent comprising the aforementioned Bacillus belyssus NXPM130.

[0011] The present invention also provides a composition comprising the aforementioned Bacillus belye NXPM130 or the bacterial agent of claim 2, and acceptable excipients or carriers.

[0012] Preferably, the composition is a feed additive or veterinary drug.

[0013] This invention also provides the aforementioned Bacillus vesiculosus NXPM130 or the aforementioned bacterial agent or the aforementioned composition in the preparation of a product for inhibiting Erysipelothrix rhusiopathiae (… Erysipelothrix rhusiopathiae Applications in products.

[0014] Preferably, the inhibition of Erysipelothrix includes inhibiting the growth of Erysipelothrix and / or reducing the abundance of Erysipelothrix in the intestine.

[0015] The present invention also provides the use of the described Bacillus belyssus NXPM130 or the described bacterial agent or the described composition in the preparation of products for improving animal growth performance.

[0016] Preferably, the improvement in animal growth performance includes increasing average daily feed intake, increasing average daily weight gain, increasing final body weight, reducing feed conversion ratio, and / or increasing apparent digestibility of energy or crude protein.

[0017] Preferably, the animal is a pig.

[0018] The present invention also provides the use of the described Bacillus belyssus NXPM130 or the described bacterial agent or the described composition in the preparation of products for the prevention or treatment of diseases caused by Erysipelothrix rhusiopathiae.

[0019] Preferably, the diseases caused by Erysipelothrix include swine erysipelas, intestinal damage, and / or growth retardation.

[0020] The present invention discloses the following technical effects: This invention has identified a strain, NXPM130, with strong antagonistic activity against *Erysipelothrix rhusiopathiae* from the intestinal contents of healthy piglets. This strain was identified as *Bacillus belyssus*. In vitro antibacterial tests and in vivo models in piglets validated that this strain not only significantly reduces the *Erysipelothrix rhusiopathiae* load in the intestines and feces but also improves intestinal barrier function and promotes growth performance. The *Bacillus belyssus* NXPM130 provided by this invention originates from the native flora of the pig intestines, exhibits high safety and non-pathogenicity, and possesses excellent high-temperature granulation resistance due to its spore formation. This invention fills the technological gap in specific probiotic spore-forming bacteria targeting *Erysipelothrix rhusiopathiae*, providing an effective solution for preventing and alleviating intestinal damage and growth retardation caused by *Erysipelothrix rhusiopathiae*. It can replace or partially replace antibiotics, offering a safe, efficient, and drug-free biocontrol solution for pig farming, with significant economic benefits and application prospects. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the 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.

[0022] Figure 1 The flowchart and initial screening results of probiotics for inhibiting Erysipelas are shown in Figure A. A flowchart shows the screening process for Erysipelas and its antagonistic probiotics. Figure B shows the size of the inhibition zone after treating Erysipelas with cell-free supernatant (CFS) of different strains. Figure 2 A represents the replication validation results of strain 130; A shows the statistical results of the inhibition zone size of the control group (LB) and different candidate strains; B shows the inhibition zone size of the control group (LB) and different candidate strains on GAM solid medium. Figure 3 Phylogenetic analysis of Bacillus belyssus NXPM130; Figure 4 To detect the in vivo inhibition of Erysipelothrix erysipelas abundance by Bacillus belysin NXPM130; Figure 5The effect of Bacillus vesiculosus NXPM130 on a growth-retarded piglet model is shown in Figure 1. A represents initial body weight (IBW), B represents final body weight (FBW), C represents average daily feed intake (ADFI), D represents average daily weight gain (ADG), E represents feed conversion ratio (F:G), and F represents apparent digestibility. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] like Figure 1As shown in Figure A, this invention selectively screened a strain NXPM130 from the intestinal contents of healthy piglets, which exhibits strong antagonistic activity against Erysipelothrix rhusiopathiae. In vitro and in vivo experiments verified that strain NXPM130 can significantly inhibit Erysipelothrix rhusiopathiae and effectively improve the growth performance of piglets, providing an effective solution for preventing and alleviating intestinal damage and growth retardation caused by Erysipelothrix rhusiopathiae. The following examples further illustrate this approach.

[0029] Example 1: Bacillus belye ( Bacillus velezensis Separation and Identification of NXPM130 1. Establish a Bacillus strain resource bank 1.1 Strain Isolation Samples were collected from fresh jejunal contents and feces of healthy, fast-growing (high-weight) piglets. 1 g of sample was added to 9 mL of sterile physiological saline, vortexed, and heated in an 80°C water bath for 20 minutes to kill non-spore-forming bacteria. After cooling, 100 μL of the suspension was spread onto LB agar plates and incubated at 37°C for 24 hours. Typical colonies were picked and streaked three times for purification.

[0030] 1.2 DNA sequencing Genomic DNA was extracted from purified colonies, and 16S rDNA was amplified using universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2). The PCR products were verified by agarose gel electrophoresis and then sequenced.

[0031] 1.3 Identification and Classification of Microbial Species Sequence alignment was performed in the NCBI database using BLASTN software, and the bacterial species were determined based on a similarity of ≥99%. A total of 150 Bacillus strains were isolated from the intestinal contents of piglets, and a Bacillus strain resource bank was established.

[0032] 2. Initial screening 2.1 Preparation of Cell-Free Supernatant (CFS) After activation, each candidate Bacillus strain was inoculated into 1 mL of LB liquid medium and pre-cultured at 37°C and 900 rpm for 12 hours. The culture was then transferred 1:100 to 20 mL of LB medium and incubated at 37°C and 200 rpm for 24 hours. The culture was centrifuged at 10,000 × g for 10 minutes at 4°C, and the supernatant was filtered through a 0.22 μm filter membrane and aliquoted for storage at -80°C.

[0033] 2.2 Initial screening for antibacterial activity Using the common pathogen *Erysipelothrix rhusiopathiae* as an indicator bacterium, a liquid mixed medium of BHI medium and 30% fetal bovine serum was used. After activation and culture for 24 hours in an incubator at 37°C and 5% CO2, 100 μL of *Erysipelothrix rhusiopathiae* culture was diluted 100-fold with 10 mL of sterile water. Before the solidified GAM medium was heated to high temperature, the diluted culture was added to the medium at a ratio of 1:20, mixed thoroughly, and quickly poured into petri dishes. The dishes were then cooled and solidified in a laminar flow hood. One end of a hollow iron rod was sterilized by flaming over an alcohol lamp. After cooling, the rod was used to poke holes in the solidified GAM plates. The holes were marked on the bottom of the petri dishes. The supernatant of the centrifuged culture was filtered through a 0.22 μm sterile filter and 35 μL was transferred to the holes. Three biological replicates were performed for each candidate bacterium, with LB medium as a control. After adding the culture, the plates were placed in a laminar flow hood and the holes were blown dry until no liquid remained. The plates were then inverted and incubated in a 37°C, 5% CO2 incubator for 24 hours. The inhibitory effect of a bacterial species on Erysipelothrix rhusiopathiae was determined by the presence and size of the inhibition zone. Results were as follows: Figure 1 As shown in Figure B, strains numbered 56, 73, 74, 82, and 130 all exhibited significantly higher antibacterial effects than the LB control. P Since the value was <0.05, the above strains were selected as candidate strains for further screening.

[0034] 2.3 Antibacterial rescreening The strains exhibiting the ability to inhibit the growth of Erysipelas were reactivated and passaged for 10 generations. The above antibacterial experimental steps were then repeated, and the size of the inhibition zone for each candidate strain was further measured. Results are as follows: Figure 2 As shown, the inhibition diameter of strain 130 can reach 26 mm, and the antibacterial effect is the most significant, indicating that strain 130 can significantly inhibit the growth of swine erysipelas. Strain 130 was identified as an excellent strain for subsequent molecular identification and animal experimental verification.

[0035] 3. Molecular identification of strain 130 a. 16S rDNA sequencing: Genomic DNA of strain 130 was extracted, amplified and sequenced using primers 27F / 1492R.

[0036] The amplified sequence (SEQ ID NO.3) is as follows:

[0037] b. Phylogenetic analysis: Sequencing results were BLAST-aligned against the NCBI database, and a phylogenetic tree was constructed using the Neighbor-Joining method with MEGA 7.0 software. The results are as follows: Figure 3 As shown, strain 130 and Bacillus velezensis GCF 001461825.1 clustered together, with a 16S rRNA gene sequence similarity of 97%, confirming strain 130 as Bacillus belesii ( ). Bacillus velezensis It was named NXPM130.

[0038] The above-mentioned Bacillus belye ( Bacillus velezensis NXPM130 was deposited on January 17, 2022, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 24334.

[0039] Example 2: In vivo validation of Bacillus belyssus NXPM130 in a growth-retarded piglet model 1. Animal grouping Twenty-four piglets of the same age were selected and divided into three groups (n=8) according to their body weight: a positive control group (high-weight group, HW group, fed with basal diet), a low-weight group (LW group, basal diet), and an LW+NXPM130 group (basal diet + 10 live bacteria count). 9 CFU / kg Bacillus belyssus NXPM130). Feeding was conducted for 21 days, and body weight, feed intake, and health status were recorded. Fecal samples were collected three days before the end of the experiment for digestibility testing. After the experiment, intestinal and ileal digesta samples were collected from slaughtered animals and sequenced using 16S rDNA for gut microbiota analysis. One-way ANOVA and graphing were then performed using GraphPad Prism 10.1.2 software.

[0040] 2. Results and Analysis The results are as follows Figure 4 As shown, compared to the LW group, the addition of Bacillus belye NXPM130 to the feed significantly reduced the incidence of Erysipelothrix spp. in the intestines of stunted piglets. Erysipelothrix The relative abundance of ).

[0041] The results are as follows Figure 5 As shown: Compared with the LW group, the LW+NXPM130 group of piglets showed significantly increased average daily feed intake, average daily weight gain, and final body weight. P <0.05, the material weight ratio decreased significantly ( P<0.05%. Compared to the HW group, the average daily weight gain in the LW+NXPM130 group significantly increased and approached the average daily weight gain of the HW group ( P >0.05). Furthermore, compared to the HW and LW groups, the LW+NXPM130 group showed a significant increase in both energy and apparent crude protein digestibility ( P <0.05). The above results indicate that strain 130 can effectively improve the growth performance of low-weight piglets caused by growth retardation.

[0042] Example 3: Industrial Application To address the lack of effective control measures for bacterial diseases such as swine erysipelas in the feed industry, this invention provides a non-antibiotic biocontrol solution. This strain can effectively reduce the colonization of *Erysipelothrix rhusiopathiae* in the intestines, thus reducing the need for clinical medication. The *Bacillus belyssus* NXPM130 obtained through screening exhibits good in vitro and in vivo inhibitory activity against *Erysipelothrix rhusiopathiae* and can be prepared as a feed additive or drug for animal health management, improving growth performance, and preventing intestinal damage, showing significant application potential.

[0043] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of Bacillus velezensis (NXPM130) inhibiting Dermatophilus conglaeratus, characterized in that, Bacillus velezensis The Bacillus belyssus NXPM130 has the accession number CGMCC No. 24334. ​ 2. A microbial agent, characterized in that, It includes Bacillus niger NXPM130 as described in claim 1.

3. A composition, characterized in that, It comprises Bacillus niger NXPM130 as described in claim 1 or the inoculum as described in claim 2, and acceptable excipients or carriers.

4. The composition according to claim 3, characterized in that, The composition is a feed additive or veterinary drug.

5. The Bacillus vesiculosus NXPM130 as described in claim 1, or the bacterial agent as described in claim 2, or the composition as described in claim 3 or 4, in the preparation of a product for inhibiting Erysipelothrix rhusiopathiae (… Erysipelothrix rhusiopathiae Applications in products.

6. The application as described in claim 5, characterized in that, The inhibition of Erysipelothrix includes inhibiting the growth of Erysipelothrix and / or reducing the abundance of Erysipelothrix in the intestine.

7. The use of Bacillus belyssus NXPM130 as described in claim 1, or the bacterial agent as described in claim 2, or the composition as described in claim 3 or 4 in the preparation of products for improving animal growth performance.

8. The application as described in claim 7, characterized in that, The animal in question is a pig.

9. The use of Bacillus vesiculosus NXPM130 as described in claim 1, or the bacterial agent as described in claim 2, or the composition as described in claim 3 or 4, in the preparation of products for the prevention or treatment of diseases caused by Erysipelothrix rhusiopathiae.

10. The application as described in claim 9, characterized in that, The diseases caused by Erysipelothrix include swine erysipelas, intestinal damage, and / or growth retardation.

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