Bacillus subtilis kc66 and medical use for preventing and treating leptospirosis

Abstract

The application discloses bacillus subtilis KC66, and also provides medical use of the strain in preventing and treating leptospirosis, and expands the medical range of the bacillus subtilis KC66. The application provides a new idea for preventing leptospirosis, can effectively reduce the abuse of antibiotics, and is helpful for the healthy development of the animal husbandry.

Description

Technical Field

[0001] This invention discloses a strain of Bacillus subtilis KC66, and also provides the medical use of this strain in the prevention and treatment of leptospirosis, belonging to the field of pharmaceutical application. It also relates to the design of pharmaceutical preparations of Bacillus subtilis KC66 and their uses. In particular, it relates to the pharmaceutical use of Bacillus subtilis KC66 in the prevention of leptospirosis. Background Technology

[0002] Leptospirosis is a zoonotic disease that occurs worldwide. Infection is linked to occupational or daily contact with leptospires and is prevalent in flood-prone areas, particularly in developing countries and tropical regions. Both humans and animals are susceptible, and the clinical manifestations are diverse, making diagnosis challenging. The leptospirosis pathogen persists within the host, further spreading and contaminating the environment, infecting other humans and animals, posing a serious threat. Currently, prevention and treatment methods for leptospirosis include vaccination and antibiotics.

[0003] Bacillus subtilis is a Gram-positive bacterium that produces endospores and possesses strong resistance to adverse conditions, high temperatures, high pressure, and easy storage. Belonging to the genus Bacillus, its proliferation in the intestines consumes oxygen, inhibits the growth of harmful bacteria, promotes the growth of beneficial bacteria, and produces various metabolites such as enzymes and bacteriocins. In animal husbandry, the application of Bacillus can improve animal growth performance, improve intestinal flora structure, enhance immunity, and purify the farming environment. However, there are few reports on its use in the prevention and treatment of leptospirosis. The application of Bacillus subtilis can reduce the overuse of antibiotics and effectively control leptospirosis outbreaks. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Bacillus subtilis KC66, and also to disclose the medical use of this strain in the prevention and treatment of leptospirosis.

[0005] The Bacillus subtilis strain KC66 disclosed in this invention was deposited at the China General Microbiological Culture Collection Center of the China Committee for the Preservation of Type Microorganisms on September 16, 2022, and named: Bacillus subtilis. Bacillus subtilis The model strain was KC66, with the accession number CGMCC NO.25731.

[0006] The use of the *Bacillus jilinensis* KC66 described in this invention in the preparation of drugs for the prevention and treatment of leptospirosis.

[0007] The positive effects of this invention are: it provides a novel *Bacillus subtilis* strain, KC66, and also offers its medicinal applications in the prevention and treatment of leptospirosis, expanding the medicinal scope of *Bacillus subtilis* KC66. This new approach to preventing leptospirosis can effectively reduce the overuse of antibiotics and greatly contribute to the healthy development of animal husbandry. Attached Figure Description

[0008] Figure 1 This study investigated the resistance of golden hamsters to leptospirosis infection after oral administration of Bacillus subtilis KC66 according to the present invention (survival rate of golden hamsters in the control and experimental groups).

[0009] Figure 2 The present invention demonstrates the scavenging effect of Bacillus subtilis KC66 on Leptospira in vivo (Leptospira load in the kidneys, livers, and lungs of golden hamsters in the control and experimental groups).

[0010] Figure 3 This invention demonstrates the protective effect of Bacillus subtilis KC66 on the major organs of golden hamsters (pathological sections of kidney, liver, and lung from golden hamsters in the control and experimental groups; a, b, and c are the liver, kidney, and lung from the control group; d, e, and f are the liver, kidney, and lung from the experimental group). Implementation

[0011] The present invention is further illustrated by the following embodiments, which are not intended to limit the invention in any way. Any modifications or alterations made to the present invention that are easily implemented by those skilled in the art without departing from the technical solutions of the present invention shall fall within the scope of the claims of the present invention. Example 1

[0012] The isolation and culture of Bacillus subtilis KC66 were performed using the following culture medium:

[0013] Tryptone-Soy Broth (TSB) liquid culture medium: tryptone 17.0 g / L, soybean peptone 3.0 g / L, sodium chloride 5.0 g / L, dipotassium hydrogen phosphate 2.5 g / L, glucose 2.5 g / L, pH 7.3 ± 0.2;

[0014] Bacillus solid culture medium: glucose 10.0 g / L, calcium phosphate 5.0 g / L, ammonium sulfate 0.5 g / L, potassium chloride 0.2 g / L, magnesium sulfate heptahydrate 0.1 g / L, manganese sulfate 0.0001 g / L, ferrous sulfate 0.0001 g / L, yeast extract 0.5 g / L, agar 20.0 g / L, pH 7.0 ± 0.2;

[0015] Bacillus subtilis was streaked onto Bacillus solid medium and cultured at 37°C for 48 hours to obtain single colonies. Single colonies were picked and inoculated into TSB liquid medium and shaken in a shaker at 37°C for 5 hours to obtain bacterial suspension. Glycerol (20-30% of total volume) was added to the bacterial suspension and stored at -80°C.

[0016] The specific method for preparing Bacillus subtilis suspension is as follows:

[0017] 1. Sample collection

[0018] Milk samples were collected from a healthy dairy cow at a dairy farm in Jilin City. The samples were placed in centrifuge tubes containing 20-30% glycerol by total volume, stored in an insulated box with ice packs, and brought back to the laboratory and quickly placed in a -80℃ freezer for separation and screening.

[0019] 2. Isolation and culture of bacterial strains

[0020] 1) Dilution and plating: Take about 0.5g of the contents stored in 20-30% glycerol and add it to a 10mL centrifuge tube containing 4.5mL of physiological saline under sterile conditions to obtain a 10-1 dilution. Repeat the above dilution steps to obtain 10-2, 10-3, 10-4, 10-5, 10-6, 10-7, and 10-8 dilutions in sequence.

[0021] 2) Streaking culture: Use an inoculation loop to take the four dilutions of 10⁻¹, 10⁻², 10⁻³, and 10⁻⁴ from step (1) and streak them on Bacillus solid medium. Incubate at 37°C for 48 hours to obtain streak plates.

[0022] 3) Spreading culture: Take 50 μL of the four dilutions of 10⁻⁵, 10⁻⁶, 10⁻⁷ and 10⁻⁸ from step (1) onto Bacillus solid medium, spread evenly with a spreader, and incubate at 37°C for 48 h to obtain diluted spread plates.

[0023] 4) Bacterial culture: Pick single colonies from the streak plate in step (2) and the diluted spread plate in step (3) and inoculate them into TSB liquid medium. Shake in a shaker at 37°C for 5 hours. When the colony count is 1×109 CFU / mL, add 20-30% of the total volume of glycerol and store at -80°C.

[0024] 3. Identification of fungal strains

[0025] 1) The bacterial culture from step 2 (4) was sent to Jilin Kumei Biotechnology Co., Ltd. for gene sequencing; the assembled sequence was uploaded to NCBI's BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST) for species confirmation; the comparison results showed that it was Bacillus subtilis.

[0026] 2) Extract the bacterial DNA from step 2 (4) as a template for conventional PCR amplification;

[0027] 3) The reaction program for the conventional PCR amplification is as follows: pre-denaturation at 94℃ for 5 min, followed by the following cycles: denaturation at 94℃ for 30 s; annealing at 55℃ for 30 s; extension at 72℃ for 30 s; run 35 cycles each, followed by a final extension at 72℃ for 5 min.

[0028] 4) The reaction system for conventional PCR amplification is 25 μL, and the reaction system is as follows: ddH2O, 8.5 μL; forward primer and reverse primer, 1 μL each; bacterial template, 2 μL; Taq DNA polymerase, 12.5 μL;

[0029] 5) The primers designed for Bacillus subtilis are as follows:

[0030] The forward primer is: 5'-TCAAGACTTGGGAATACGA-3'.

[0031] The reverse primer is: 5'-GCAAAGCGACTGAAGAACA-3';

[0032] 6) PCR products are subjected to nucleic acid electrophoresis (e.g.) Figure 1 After analysis and confirmation, the sample was sent to Jilin Kumei Biotechnology Co., Ltd. for gene sequencing. The assembled sequence was uploaded to NCBI's BLAST for species confirmation. After comparison, it was confirmed to be Bacillus subtilis, and its 16S rDNA amplification sequence is shown in SEQ ID NO.1.

[0033] 4. Preservation of microbial strains

[0034] The strain was deposited on September 16, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. It was named Bacillus subtilis KC66, classified as Bacillus subtilis, and its accession number is CGMCC NO.25731.

[0035] Experimental Example 1

[0036] The experimental steps for investigating the role of Bacillus subtilis KC66 in preventing Leptospirosis infection are as follows:

[0037] 1. Preparation of Bacillus subtilis KC66 gavage solution

[0038] Bacillus subtilis KC66 was streaked onto Bacillus solid medium and cultured at 37°C for 48 h to obtain single colonies. Single colonies from the solid medium were picked and inoculated into TSB liquid medium and shaken at 37°C for 5 h to obtain a bacterial suspension. Glycerol (20-30% of total volume) was added to the suspension and stored at -80°C. Before the experiment, the Bacillus subtilis KC66 suspension was centrifuged, the supernatant was discarded, and the suspension was washed and resuspended with an equal volume of sterile physiological saline to obtain a Bacillus subtilis KC66 gavage solution (1×10⁻⁶). 9 CFU / mL);

[0039] 2. Strains and experimental animals

[0040] Leptospira strain 56601 was cultured and preserved in the laboratory. Leptospira were grown in EMJH liquid medium at 29°C. The virulence of Leptospira was maintained by infection with golden hamsters. Leptospira were passaged less than three times. Leptospira concentrations were determined using a Petrov-Hausser counting chamber and dark-field microscopy prior to infection. Twenty four-week-old female golden hamsters, randomly divided into two groups, were provided by Liaoning Changsheng Biotechnology Co., Ltd. All hamsters were housed in a clean environment with a light / dark cycle of 12h / 12h, during which they had free access to food.

[0041] 3. Experimental Grouping

[0042] Twenty four-week-old female golden hamsters were randomly divided into two groups of 10 each: a control group and an experimental group.

[0043] 4. Experiment Content

[0044] (1) Control group: administered physiological saline by gavage daily;

[0045] (2) Experimental group: Bacillus subtilis KC66 gavage solution was administered to golden hamsters at a dose of 100ul / hamster every day;

[0046] (3) One day later, the experimental group and the control group were given an intraperitoneal injection of 1×10 7 Hooked spirochete;

[0047] (4) Record the survival status of the two groups of golden hamsters every day;

[0048] (5) The experimental period is 21 days;

[0049] 5. Experimental Results

[0050] like Figure 1 As shown, the survival rate of the experimental group treated with Bacillus subtilis KC66 via gavage was 50%, while all golden hamsters in the control group died. This indicates that Bacillus subtilis KC66 can effectively prevent leptospirosis infection.

[0051] Experimental Example 2

[0052] The efficacy of Bacillus KC66 in eliminating Leptospira in golden hamsters was demonstrated through the following steps:

[0053] 1. The preparation of Bacillus subtilis KC66 gavage solution was the same as in Experiment 1;

[0054] 2. The experimental animals were the same as in Experiment 1;

[0055] 3. The experimental groups are the same as in Experiment Example 1;

[0056] 4. Experiment Content

[0057] (1) Control group: administered physiological saline by gavage daily;

[0058] (2) Experimental group: Bacillus subtilis KC66 gavage solution was administered to golden hamsters at a dose of 100ul / hamster every day;

[0059] (3) One day later, the experimental group and the control group were given an intraperitoneal injection of 1×10 7 Hooked spirochete;

[0060] (4) The experimental period is 21 days;

[0061] (5) Samples were taken from golden hamsters in the control group during their near-death period. Samples were taken from golden hamsters in the experimental group on day 22. Liver, kidney and lung samples were collected from each group of golden hamsters. Leptospira DNA was extracted from the tissues and then quantitative real-time PCR was performed to analyze the leptospira load in each organ of the golden hamsters.

[0062] 5. Experimental Results

[0063] like Figure 2 As shown, compared with the control group that was only given saline by gavage, the leptospirosis load in the liver, kidneys and lungs of golden hamsters given Bacillus subtilis KC66 by gavage was significantly reduced.

[0064] Experimental Example 3

[0065] The protective effect of Bacillus KC66 on the major organs of golden hamsters is demonstrated through the following steps:

[0066] 1. The preparation of Bacillus subtilis KC66 gavage solution was the same as in Experiment 1;

[0067] 2. The experimental animals were the same as in Experiment 1;

[0068] 3. The experimental groups are the same as in Experiment Example 1;

[0069] 4. Experiment Content

[0070] (1) Control group: administered physiological saline by gavage daily;

[0071] (2) Experimental group: Bacillus subtilis KC66 gavage solution was administered to golden hamsters at a dose of 100ul / hamster every day;

[0072] (3) One day later, the experimental group and the control group were given an intraperitoneal injection of 1×10 7 Hooked spirochete;

[0073] (4) The experimental period is 21 days;

[0074] (5) Samples were taken from golden hamsters in the control group during their near-death period, and samples were taken from golden hamsters in the experimental group on day 22. The livers, kidneys, and lungs of the collected golden hamsters from each group were made into pathological sections and then stained with hematoxylin and eosin (HE). The samples were observed under a 100x optical microscope.

[0075] 5. Experimental Results

[0076] like Figure 3 As shown, the liver, kidneys, and lungs of the control group golden hamsters showed varying degrees of hemorrhage and inflammatory lesions, while the pathological changes in the liver, kidneys, and lungs of the experimental group golden hamsters were not particularly obvious, with basically no hemorrhage points and inflammatory lesions.

[0077] See the comparison of the survival rates of golden hamsters. Figure 1 Leptospira load in the kidneys, livers, and lungs of the two groups of golden hamsters is shown in the figure. Figure 2 ; Pathological sections of the kidneys, livers, and lungs of the two groups of golden hamsters are shown in Figure 3 As shown in the figure, Bacillus subtilis KC66 can improve the survival rate of Leptospira infection; reduce the Leptospira load in golden hamsters after Leptospira infection; and protect the major organs of golden hamsters after Leptospira infection.

Claims

1. The use of Bacillus subtilis KC66 in the preparation of drugs for the prevention of leptospirosis, characterized in that: The Bacillus subtilis KC66 described herein was deposited at the China General Microbiological Culture Collection Center on September 16, 2022, and named Bacillus subtilis. Bacillus subtilis The model strain was KC66, with the accession number CGMCC NO.25731.

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