Bacillus siamensis and application thereof

Abstract

The invention relates to the technical field of microbial control, and provides bacillus siamensis and application thereof, the bacillus siamensis has been preserved in China General Microbiological Culture Collection Center (CGMCC) on June 17, 2025, and the preservation number is CGMCC No.34929. The bacillus siamensis has been applied to the field of microbial control. According to the technical scheme, the problem that no bacillus siamensis is applied to prevention and treatment of fish nocardia disease in the prior art is solved. Research finds that the bacillus siamensis provided by the invention has a very good effect in the aspect of biological prevention and control of fish nocardia disease, and a new strain resource is provided for biological prevention and control of fish nocardia disease, so that the bacillus siamensis has a good practical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial control, and in particular to a siam bacillus and application thereof. Background Art

[0002] With the expansion of high-density, intensive aquaculture models, aquaculture diseases are becoming increasingly common, with mortality rates reaching 100% in severe cases, resulting in significant economic losses. For example, fish nocardiosis, while a chronic disease with subtle early symptoms, develops multiple nodules in the fish's internal organs in the later stages, making it difficult to treat. For a long time, bacterial diseases in aquaculture have mostly been treated with chemical methods. While effective, these methods can easily lead to antibiotic residues, bacterial resistance, and damage the aquaculture environment. Therefore, green and environmentally friendly methods are urgently needed for the prevention and control of aquaculture diseases.

[0003] Biological control is a green and safe prevention and control method that primarily utilizes biocontrol bacteria and the antibacterial substances they produce, while also inhibiting harmful bacteria through competition for nutrients or growth space. Furthermore, some biocontrol bacteria can, to a certain extent, promote the growth of farmed animals and improve feed conversion efficiency, meeting the needs of sustainable aquaculture. Currently, a wide range of biocontrol bacteria are used for fish disease control, including Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus velezensis. Bacillus siamensis is a new species of Bacillus first reported in 2010, isolated from pickled crab in Thailand. However, there is no published data demonstrating its antagonistic effect against Nocardia spp. in yellowtail.

[0004] Therefore, the present invention screened out siam bacillus with a strong antagonistic effect on Nocardia spp. from the intestine of snakehead fish, and the bacterium is not pathogenic to snakehead fish, has good biosafety, and has the ability to prevent and control fish nocardiosis. It has broad application prospects in preventing and controlling fish nocardiosis in aquaculture. Summary of the Invention

[0005] The present invention provides a strain of Siamese Bacillus and application thereof, which solves the problem that Siamese Bacillus has not yet been applied to the prevention and treatment of fish nocardiosis in the prior art.

[0006] The technical solution of the present invention is as follows: a strain of Bacillus siamensis, which has been deposited in the General Microbiology Center of the China Culture Collection Administration on June 17, 2025, with a deposit number of CGMCC No.34929.

[0007] The present invention also provides a microbial agent, which contains the Bacillus siamese according to claim 1.

[0008] As a further technical solution, the microbial agent includes a solid-state microbial agent and a liquid-state microbial agent.

[0009] The application also provides an application of the Paenibacillus thailandicus in feed.

[0010] The application also provides an application of the microbial agent in feed.

[0011] The application also provides a disinfectant containing the Paenibacillus thailandicus.

[0012] As a further technical solution, the disinfectant further includes an auxiliary agent.

[0013] The application also provides a disinfectant containing the microbial agent.

[0014] The working principle and beneficial effects of the application are as follows:

[0015] 1. The Paenibacillus thailandicus provided by the application has a strong antagonistic effect on Nocardia seriolae, can effectively prevent or treat diseases caused by Nocardia seriolae infection in aquaculture, and greatly reduces the incidence probability of Nocardia seriolae disease; at the same time, the microbial agent containing the bacteria can be used for disinfection of breeding environment, breeding supplies and breeding feed, and reduces the morbidity and mortality of aquatic animals caused by Nocardia seriolae from different sources.

[0016] 2. The Paenibacillus thailandicus provided by the application is screened from the intestinal tract of healthy Ophicephalus argus, and as probiotics, the bacteria can effectively solve the infection caused by Nocardia seriolae and the water problem caused by the massive proliferation of Nocardia seriolae, and avoid the problems of antibiotic residues and drug resistance of Nocardia seriolae caused by the use of antibiotics. In addition, the aquatic product, disinfectant or detection reagent prepared from the bacteria also has the advantages of safe use and green environmental protection. BRIEF DESCRIPTION OF DRAWINGS

[0017] The application will be further described in detail below in combination with the drawings and specific embodiments.

[0018] Figure 1 It is a preliminary screening and gram staining microscopic examination diagram of the Paenibacillus thailandicus BA-3.

[0019] Figure 2 It is a 16S r RNA electrophoresis diagram and NCBI comparison result of the Paenibacillus thailandicus BA-3.

[0020] Figure 3 It is an inhibition effect of the Paenibacillus thailandicus BA-3 on Nocardia seriolae in vitro.

[0021] Figure 4The growth curve of the bacillus siamensis BA-3 in 24 hours;

[0022] Figure 5 The hemolysis determination and biofilm characteristic analysis of the bacillus siamensis BA-3;

[0023] Figure 6 The growth of the bacillus siamensis BA-3 after high-temperature treatment;

[0024] Figure 7 The influence of the mixed feed containing the bacillus siamensis BA-3 on the serum enzyme activity of the snakehead;

[0025] Figure 8 The influence of the mixed feed containing the bacillus siamensis BA-3 on the pathological changes of the spleen tissue of the infected snakehead. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor also fall within the protection scope of the present application.

[0027] The present application provides a bacillus siamensis, named as BA-3. The bacillus siamensis is isolated from the intestinal tract of healthy snakehead, and has been preserved in the China General Microbiological Culture Collection Center on June 17, 2025, with the preservation number of CGMCC No.34929.

[0028] The isolated bacillus siamensis BA-3 has white round colony color, dry and wrinkled surface, and presents rod-shaped bacteria under a microscope, and is positive in gram staining. After continuous shaking for about 2 hours at 37 DEG C in a shaking table, it all enters the logarithmic growth phase, and the growth cycle is stable. It is identified as beta hemolysis, and the colony surface is smooth and does not produce biofilm on the 8% Congo red LB agar plate. After being incubated at 80 DEG C, 90 DEG C and 100 DEG C for 10 minutes respectively, it can still continue to grow, and has good high-temperature tolerance. The BA-3 is resistant to streptomycin, polysporin and neomycin, and moderately sensitive to rifampicin, clindamycin, kanamycin and tetracycline.

[0029] The present application provides a method for isolating, purifying and identifying the bacillus siamensis, and the specific method is as follows:

[0030] The healthy experimental fish, Erpetoichthys calabaricus, was fished out and placed in a bucket of water mixed with eugenol (1:10000) for 2-3 minutes of anesthesia. After anesthesia, the fish was placed on a dissection plate and the surface of the fish was wiped with a 75% alcohol cotton ball. The fish was cut open with sterile scissors and the intestinal tract was carefully dissected. The obtained fish intestinal tract sample was taken out and cut longitudinally. The residual feces was washed off with sterile water without over-washing. The entire intestinal tract was cut into three sections, i.e., the front, middle and rear intestines, and placed into a glass homogenizer. 5 ml of sterile water was added for grinding. After sufficient homogenization, the homogenate was diluted to different dilution ratios. 10-4, 10-5, 10-6 and 10-7 dilutions of the bacterial solution were taken and placed in a 80°C water bath for 15 min. After cooling to room temperature, 100 μL of each of the 10-4, 10-5, 10-6 and 10-7 dilutions of the bacterial solution were spread on LB plates. The two plates were blown dry in a clean bench and incubated at 30°C for 24 h. The colony morphological characteristics were observed and recorded. Single colonies were inoculated in MRS liquid medium and LB liquid medium for expansion. Through multiple streaking purification, purified single colonies were obtained and the isolated bacteria morphology was observed by Gram staining. The results are shown in Figure 1 .

[0031] To accurately identify the isolated strains, bacterial DNA was extracted using a bacterial DNA extraction kit from Tiangeng Biology and the 16S RNA gene was amplified. The primers used included 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR reaction system was 20 systems: 10 μL of 2×Mix ExTap, 1 μL of 27F and 1492R each, 1 μL of bacterial DNA, and 7 μL of ddH2O. The reaction conditions were 95°C for 5 min, 95°C for 30 s, 55°C for 30 s, 72°C for 90 s, 30 cycles, and 72°C for 10 min. The amplification quality was detected by 1% agarose gel electrophoresis. The successfully amplified PCR products were sent to Shanghai Shenguo Biological Company for nucleic acid sequencing. In order to perform homologous comparison analysis, the BLAST tool from NCBI (https: / / www.ncbi.nlm.nih.gov / ) was used to compare the obtained sequences. It was found that BA-3 had high homology with Bacillus siamensis. The results are shown in Figure 2 .

[0032] The samples were subjected to biochemical identification using a biochemical identification kit. The results are shown in the table below.

[0033]

[0034]

[0035] The application adopts the paper sheet method to verify the antagonistic effect of Bacillus siamensis on Nocardia seriolae, and the specific process is as follows:

[0036] The paper sheet method is adopted, the blank drug sensitivity paper sheet is purchased and sterilized and dried, and then used. 100 muL of Nocardia seriolae ground by a pipette gun is added to a flat plate and uniformly coated to prepare a bacterial plate. The previously treated blank drug sensitivity paper sheet is pasted in the middle of the above-mentioned bacterial plate, 10 muL of the bacterial suspension to be tested is absorbed on the filter paper sheet, and the diffusion is carried out at room temperature for 10 min, and then placed in a 28 DEG C incubator for culture for 48 hours, and the obvious inhibition ring on the plate is observed, and the results are shown in Figure 3 .

[0037] The application also provides a biological characteristic determination of Bacillus siamensis, and the specific content is as follows:

[0038] (1) Determination of growth curve: 1 mL of fresh antagonistic bacteria liquid is inoculated in 100 mL of LB liquid medium, and the growth is monitored by using an enzyme label instrument to measure OD600 every two hours at 37 DEG C constant temperature culture of shaking table 150 rpm, and the growth is monitored for 24 h, and the results are shown in Figure 4 .

[0039] (2) Hemolytic determination and biofilm determination: the single colony of the antagonistic bacteria is inoculated in a sheep blood plate in the super-clean bench, and the hemolytic ring with clear boundary and complete transparency around the colony is observed after 24 h of incubation in a 37 DEG C constant temperature incubator, and then the beta-hemolysis is determined. In the super-clean bench, the isolate is streaked on the LB agar plate (containing 0.8% Congo red), and incubated at 37 DEG C for 48 h. The smooth red colonies on the plate indicate that the strain does not produce biofilm, and the results are shown in Figure 5 .

[0040] (3) Determination of high temperature tolerance of antagonistic bacteria strain

[0041] Fresh overnight culture liquid is taken, centrifuged at 10000xg, 4 DEG C for 5 min, and the supernatant is discarded and the bacteria are collected. 1 mL of ddH2O is used for resuspension, and the resuspension liquid is treated at high temperature (80 DEG C, 90 DEG C, 100 DEG C) for 10 min, and the control group is treated at room temperature, and the treatment time is recorded as 0 min; after high temperature treatment, 25 mL of LB liquid medium is added, and the culture is carried out at 37 DEG C shaking table (150 rpm) for 12 h, and it is found that the Bacillus siamensis starts to enter the logarithmic growth phase after about 2 h of shaking, and the results are shown in Figure 6 .

[0042] (5) Antibiotic sensitivity test: 100 μL of antagonistic bacteria solution was spread on LB plate, antibiotic paper was placed on the plate with sterile forceps, and the plate was incubated at 37℃ for 24h. The diameter of the inhibition zone was measured and recorded. It was found that BA-3 was resistant to streptomycin, polymyxin and neomycin, moderately sensitive to rifampicin, clindamycin, kanamycin and tetracycline, and the results are shown in Table 2.

[0043] antibiotic Bacillus siamensis BA-3 rifampicin R tetracycline R gentamicin S Streptomyces R chloramphenicol S florfenicol S cefladine S amikacin S oxacillin S doxycycline S ofloxacin S piperacillin R furazolidone S clindamycin S polymyxin B R vancomycin S ciprofloxacin S cefazolin S cefalexin S erythromycin I kanamycin S cefuroxime S neomycin I ampicillin R

[0044] Note: S represents high sensitivity (D inhibition zone > 20mm); R represents resistance (D inhibition zone < 10mm); I represents moderate sensitivity (D inhibition zone 10-20mm)

[0045] The application also provides the immune protection effect of the antagonistic bacteria Bacillus siamensis on the snakehead against Nocardia seriolae, and specifically as follows:

[0046] (I) Experimental method

[0047] (1) Preparation of mixed feed: ① Activate the bacteria solution in advance; ② Take the activated bacteria solution and inoculate it at a rate of 1% for overnight culture (12h; 37℃ / 150rpm); ③ Take the bacteria solution and count it by dilution coating method; ④ Centrifuge the remaining bacteria solution at 10000xg for 5min to collect the bacteria, discard the culture medium and adjust the concentration to 1x10 8 cfu / mL with PBS; ⑤ Spray the probiotics evenly on the feed with a watering can, place it in a 16℃ air-conditioned room to dry, and then feed it the next day. The excess is stored in a 4℃ refrigerator (to ensure fresh feed).

[0048] (2) The purchased snakeheads were temporarily raised for one week and then divided into four groups. Groups A and B were the prevention groups, i.e., they were first fed with the treated feed and then attacked by the virus. Groups C and D were the treatment groups, i.e., they were first attacked by the virus and then fed with the treated feed for treatment. The control groups A and C were fed with normal feed, and the experimental groups B and D were fed with mixed feed containing Bacillus siamensis (80mL of antagonistic bacteria with a concentration of 1x10 8 cfu / mL was added to 1000g of feed), and the feeding amount was 3% of the average weight of the experimental fish, which was fed twice a day (9:00 and 15:00). The water was changed twice a week, and the amount of water changed was 2 / 3.

[0049] (3) The experimental fish for attack were injected with 100μL of Nocardia seriolae with a concentration of 1x10 7 cfu / mL diluted with sterile PBS in the abdominal cavity. The control group was injected with an equal amount of sterile PBS. At 0d and 14d after infection, 3 fish from the infection group and the control group were randomly collected for dissection, and the kidney, liver and spleen were taken and quickly frozen in liquid nitrogen and then stored at -80℃. When the fish died, they were dissected in time and the pathological tissues were collected.

Claims

1. A strain of Bacillus siamensis, characterized in that: The Siamese Bacillus was deposited in the General Microbiology Center of the China Culture Collection Administration on June 17, 2025, with the deposit number CGMCC No.34929.

2. A microbial agent containing the siam bacillus according to claim 1.

3. The microbial agent according to claim 2, characterized in that The microbial agent includes a solid microbial agent and a liquid microbial agent.

4. Use of the siam bacillus according to claim 1 in feed.

5. Use of the microbial agent according to claim 2 or 3 in feed.

6. A disinfectant containing the siam bacillus according to claim 1.

7. The disinfectant according to claim 6, characterized in that The disinfectant also includes an auxiliary agent.

8. A disinfectant containing the microbial agent according to claim 2 or 3.

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