An antibacterial bacillus subtilis deserti strain and application thereof

Abstract

The present application relates to the technical field of microorganism, and particularly relates to a kind of antibacterial bacillus subtilis desert subsp strain and its application, the bacillus subtilis desert subsp D7-HSJ9 of the present application is obtained from mangrove rhizosphere soil by applicant, after detection, the strain has good salt tolerance, alkali tolerance, antibacterial characteristics, especially good antibacterial effect on streptococcus iniae, in addition, it also has good antibacterial effect on photobacterium damsel, aeromonas salmonicida and staphylococcus aureus and several other pathogenic bacteria, therefore, the strain can effectively replace antibiotic to improve the prevention and treatment of fish disease, provide good germplasm resource for preparing microecological preparation product in later period.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly relates to an antibacterial Bacillus subtilis desert subspecies strain and its application. Background Art

[0002] With the intensive development of aquaculture, the problem of aquatic diseases has become increasingly serious. Streptococcus iniae has the characteristics of being zoonotic, can cause human septicemia, cellulitis, and even lead to osteomyelitis, endocarditis and meningitis, and can also infect a variety of animals including fish. At present, there are reports on fish streptococcosis in many countries in the world, and the fish farming varieties affected are increasing day by day, such as tilapia, channel catfish, turbot, grouper, and pompano. Moreover, both seawater and freshwater fish can be infected by it. However, there is no effective commercial vaccine for the prevention and treatment of Streptococcus iniae, and for fish, the cost of the vaccine is relatively high and the promotion effect is not good. At present, the bacterial infections of fish are mostly treated with antibiotics. Although antibiotic treatment is effective, excessive use will produce a large number of drug-resistant bacteria in the environment, causing adverse effects on the environment. In order to find alternative treatment options, probiotics are usually used to replace antibiotics in the prior art. Probiotics have the characteristics of being non-toxic, harmless, residue-free, pollution-free, and capable of producing antibacterial substances, and are good alternative agents for disease prevention and treatment in aquaculture. However, the most important technical means of using probiotics is to continuously screen strains with antibacterial activity to provide sufficient germplasm resources for the later preparation of probiotic products. Summary of the Invention

[0003] In view of the above, it is necessary to screen strains with antibacterial activity against aquatic pathogenic bacteria to improve the prevention and treatment effect of fish diseases, reduce the harm of antibiotic resistance, and provide sufficient germplasm resources for the later preparation of probiotic products.

[0004] To achieve the above object, the present invention has screened a new strain: Bacillus inaquosorum strain D7-HSJ, its taxonomic name is: Bacillus inaquosorum D7-HSJ9, and its Chinese taxonomic name is: Bacillus subtilis desert subspecies D7-HSJ9, and the preservation number is CCTCC NO: M20241842; this strain is preserved in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, and the preservation date is August 26, 2024.

[0005] Furthermore, the 16sRNA sequence of the Bacillus inaquosorum strain D7-HSJ9 is as shown in SEQ ID NO:1.

[0006] The present invention also includes a detection gene for detecting the Bacillus inaquosorum strain D7-HSJ9, the nucleic acid sequence of which is shown in SEQ ID NO:2-SEQ ID NO:32.

[0007] The present invention also includes a bacterial agent comprising the Bacillus inaquosorum strain D7-HSJ9.

[0008] The present invention also includes the use of the Bacillus inaquosorum strain D7-HSJ9 or the bacterial agent in the preparation of antibacterial, acid-resistant and / or salt-resistant formulations.

[0009] Furthermore, the pathogens that the antibacterial agent can inhibit are: Photobacterium damselae subsp. piscicida, Aeromonassalmonicida, Staphylococcus aureus and / or Streptococcus iniae.

[0010] The present invention also includes a method for culturing the Bacillus inaquosorum strain D7-HSJ9, wherein the method comprises: inoculating the Bacillus inaquosorum strain D7-HSJ9 into a culture medium with a pH of 6-8, a salinity of 0‰-50‰ by mass, and a temperature of 26-35℃.

[0011] The present invention has the following beneficial effects:

[0012] The Bacillus subsp. desert subspecies D7-HSJ9 of this invention was obtained by the applicant from mangrove rhizosphere soil. Testing showed that this strain exhibits good salt and alkali tolerance, as well as antibacterial properties, particularly against Streptococcus dolphinii. Furthermore, it also shows good antibacterial activity against several aquatic bacteria, including *Bacillus flounder* subsp. *fish-killing*, *Aeromonas salmonii*, and *Staphylococcus aureus*. It also demonstrates high safety, indicating that this strain can effectively replace antibiotics to improve the prevention and treatment of fish diseases, providing high-quality germplasm resources for the later preparation of microecological preparations. Attached Figure Description

[0013] Figure 1 The image shows an electron microscope image of strain D7-HSJ9; the magnification of the electron microscope is 10K×.

[0014] Figure 2 This is a Gram staining microscopic image of strain D7-HSJ9.

[0015] Figure 3 This is a plate image of strain D7-HSJ9.

[0016] Figure 4 The image shows the hemolytic activity test results for strain D7-HSJ9.

[0017] Figure 5 This is a diagram showing the growth of strain D7-HSJ9 under different pH conditions.

[0018] Figure 6 This figure shows the growth of strain D7-HSJ9 under different salinity conditions.

[0019] Figure 7 This is a graph showing the growth of strain D7-HSJ9 at different temperatures.

[0020] Figure 8 The 16SRNA phylogenetic sequence is for strain D7-HSJ9.

[0021] Figure 9 This is a phylogenetic tree constructed based on the major housekeeping genes of Bacillus subtilis D7-HSJ9. The phylogenetic tree was constructed using the Neighbor-Joining (NJ) method, which selects 19 strains at the species level based on 31 housekeeping genes (dnaG, frr, infC, nusA, pgk, pyrG, rplA, rplB, rplC, rplD, rplE, rplF, rplK, rplL, rplM, rplN, rplP, rplS, rplT, rpmA, rpoB, rpsB, rpsC, rpsE, rpsI, rpsJ, rpsK, rpsM, rpsS, smpB, tsf). Detailed Implementation

[0022] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0023] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0024] Example 1

[0025] This embodiment describes a method for screening and identifying disease-resistant and antibacterial strains, as detailed below:

[0026] 1. Screening Method

[0027] ① Sampling: Rhizosphere soil of mangrove plants (Kandelia obovata)

[0028] ② Isolation and purification: The selective separation method is adopted. The isolation and purification method is as follows: Take the rhizosphere sample of mangrove plants, wash it 2 - 3 times with sterile normal saline, then add it to a conical flask containing glass beads and 45 mL of sterile water, shake it vigorously on a culture shaker for 30 min (37 °C), then keep it in a water bath at 80 °C for 30 min, 1000 r / min, 2 min. Dilute the suspension by 10 - fold gradient, take the original solution, 10 - fold, 100 - fold and 1000 - fold diluted solutions, coat them on LB solid plates, culture them at a constant temperature of 28 °C. After the colonies grow, pick the colonies and streak - purify them 2 - 3 times to obtain.

[0029] ③ Strain preservation: The strain preservation solution is 50% glycerol, and the ratio of the strain preservation solution to the bacterial solution is 1:1. Under sterile conditions, first use a pipette to aspirate 600 μL of 50% glycerol into a previously sterilized strain preservation tube, then aspirate 600 μL of the Bacillus fermentation broth, blow and suck to mix evenly, and store it at - 80 °C in the refrigerator.

[0030] The strain screened in this example is Bacillus inaquosorum strain D7 - HSJ9, and its taxonomic name is: Bacillus inaquosorum D7 - HSJ9, the Chinese taxonomic name is: Bacillus inaquosorum subsp. desert D7 - HSJ9, and the preservation number is CCTCC NO: M20241842; This strain is preserved in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, and the preservation date is August 26, 2024.

[0031] 4. Strain identification:

[0032] ① Morphological identification: The morphological identification is as Figure 1 、 Figure 2 and Figure 3 shown: Inoculate Bacillus inaquosorum subsp. desert D7 - HSJ9 on solid LB medium and culture it for 1 day. The colonies are light - yellow round semi - transparent colonies, with rough edges, dry surfaces, and a diameter of 2 - 5 mm; Bacillus inaquosorum subsp. desert D7 - HSJ9 is a Gram - positive bacterium, and the cells are rod - shaped, arranged continuously or scattered; Under an electron microscope, Bacillus inaquosorum subsp. desert D7 - HSJ9 presents a short - rod shape, and the length of a single cell is 0.7 - 0.8 × 2 - 3 μm, with uniform coloring.

[0033] ② Molecular identification of the strain: DNA was extracted from the strain using conventional methods. The 16SRNA gene was amplified using universal primers 27r-5'-AGAGTTTGATCCTGGCTCAG-3' and 1492f-5'-GGTTACCTTGTTACGACTT-3'. The amplified 16SRNA sequence is shown in SEQ ID NO:1. The species classification of strain D7-HSJ9 was analyzed, and the phylogenetic tree is shown below. Figure 8 The sequence comparison results are shown in Table 1. Figure 8 As shown in Table 1, strain D7-HSJ9 clusters with the genus Bacillus and belongs to the same branch as Bacillus subtilis.

[0034] Table 1. Results of rRNA nucleotide sequence alignment for strain D7-HSJ916S

[0035]

[0036]

[0037] To further classify the strains, we performed whole-genome sequencing on D7-HSJ9 and selected 19 strains most closely related at the species level based on 31 housekeeping genes (nucleic acid sequences such as SEQ ID NO:2-SEQ ID NO:32). A phylogenetic tree was constructed using the Neighbor-Joining (NJ) method with MEGA 6.0 software. The results are as follows: Figure 9 As shown, the housekeeping gene sequence of D7-HSJ9 shares 97.3% similarity with Bacillus inaquosorum, indicating that they belong to the same evolutionary branch. Therefore, we tentatively name this strain Bacillus inaquosorum D7-HSJ9.

[0038] Example 2

[0039] Safety tests were conducted on the screened Bacillus subsp. desert subspecies D7-HSJ9, as follows: D7-HSJ9 was added to blood agar plates and incubated at 28°C for 12 hours. Using strain HSJ8 (a laboratory-preserved strain) as a positive control, its hemolytic activity was tested. The results of the hemolysis test are as follows: Figure 4 As shown, strain D7-HSJ9 exhibits no hemolytic activity, indicating a certain level of safety. The hemolytic agar plates used were Columbia blood agar plates, purchased from Beijing Luqiao Technology Co., Ltd.

[0040] Example 3

[0041] Acid-base tolerance experiments were conducted on the screened Bacillus subsp. desert subspecies D7-HSJ9, as follows: Liquid LB medium was prepared, and the medium was adjusted to different pH values ​​using glacial acetic acid and NaOH, respectively. The medium was sterilized at 121℃ for 20 min. D7-HSJ9 bacterial suspension was inoculated at a ratio of 2% into the above-mentioned medium, with three replicates for each pH value. The culture was incubated at 28℃ with shaking, and the OD600 of the culture was measured to plot growth curves. Results are as follows: Figure 5 As shown in the figure, strain D7-HSJ9 grows fastest between pH 6 and 8, with no significant difference between the two.

[0042] Example 4

[0043] Salinity tolerance experiments were conducted on the screened Bacillus subsp. desert subspecies D7-HSJ9. The specific steps were as follows: LB liquid medium with different percentages of NaCl was prepared and sterilized at 121℃ for 20 min. Activated D7-HSJ9 bacterial suspension was added to the LB medium at 2% concentration. Three replicates were set up for each salinity level. The culture was incubated at 28℃ with shaking, and the OD600 of the culture was measured to plot growth curves. The LB medium composition was: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, pH 6.8–7.2, and 1 L pure water. The results are as follows: Figure 6 As shown in the figure, strain D7-HSJ9 has a strong tolerance to salinity, and its growth rate does not differ significantly within the salinity range of 0‰-50‰.

[0044] Example 5

[0045] The growth of Bacillus subtilis desert subspecies D7-HSJ9 was experimentally observed at different temperatures, as follows: Different temperature gradients were set, and D7-HSJ9 bacterial suspension was inoculated into LB medium at a ratio of 1:50. After 12 hours of incubation, the OD was measured. 600 The result is as follows Figure 7 As shown in the figure, the results indicate that strain D7-HSJ9 grows well at temperatures of 26-35℃. At 40℃, the growth of D7-HSJ9 is slower than at 30℃. At 20℃ and 50℃, the growth rate of D7-HSJ9 is inhibited.

[0046] Example 6

[0047] The selected Bacillus subtilis desert subspecies D7-HSJ9 was subjected to a screening experiment for resistance to aquatic pathogens, as detailed below:

[0048] Staphylococcus aureus, *Bacillus mermaidina*, *Streptococcus dolphinus*, and *Aeromonas salmonicida* were used as indicator bacteria. The Oxford cup perforation method was employed for antibacterial experiments.

[0049] 1. Strain activation: Activate Bacillus subtilis subsp. deserti D7-HSJ9 and indicator bacteria. Streptococcus iniae, Streptococcus agalactiae, Photobacterium damselae, Aeromonas salmonicida, and Staphylococcus aureus are inoculated into LB medium and cultured with shaking at 28°C and 180 r / min for 18 - 24 h to obtain activated bacterial solutions.

[0050] 2. Antibacterial experiment: Use the Oxford cup punching method to detect the antibacterial activity of the test strain D7-HSJ9. Place it in an incubator at 28°C for 12 - 16 h and observe the presence and size of the inhibition zones.

[0051] Antibacterial experiment by Oxford cup punching method: First, pour a layer of LB solid medium into a petri dish. After it solidifies, pipette 500 μl of the activated bacterial solution onto the plate, and then pour the non-solidified LB solid medium to mix the indicator bacterial solution. Use sterile forceps to pick up a sterilized Oxford cup and punch holes in the non-solidified medium. After the medium solidifies, use sterile forceps to pull out the Oxford cup, and use a sterile inoculation loop to pick out the agar blocks in the holes. Put 100 μl of the Bacillus subtilis subsp. deserti D7-HSJ9 bacterial solution into the Oxford cups, and use chloramphenicol (for Photobacterium damselae, Streptococcus iniae, Streptococcus agalactiae) at a concentration of 50 ppm, enrofloxacin (for Aeromonas salmonicida) at a concentration of 50 ppm, and penicillin (for Staphylococcus aureus) at a concentration of 50 ppm as positive controls, and sterile PBS as a negative control. Incubate the plates with samples added upright in an incubator at 28°C for 12 - 16 h and observe the size of the inhibition zones. The composition of the LB medium is: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 20 g of agar powder, 1 L of pure water, pH 6.8 - 7.2.

[0052] The obtained antibacterial results are shown in Table 2 below:

[0053] Table 2 Antibacterial activity of Bacillus subtilis subsp. deserti D7-HSJ9 bacterial solution

[0054]

[0055] Note: Size of inhibition zone (d): “+” represents: d < 10.0 mm; “++” represents: 10.0 mm < d < 20.0 mm; “+++” represents: d > 20.0 mm; “-” represents: no activity.

[0056] As can be seen from Table 2: Bacillus subtilis subsp. deserti D7-HSJ9 has a strong inhibitory and killing effect on Streptococcus iniae, a common pathogenic bacterium in dolphin farming. Secondly, it also has antibacterial activity against Staphylococcus aureus, Photobacterium damselae subsp. piscicida, and Aeromonas salmonicida. From the size of the inhibition zones, the antibacterial effects are in the order of: Streptococcus iniae > Staphylococcus aureus > Photobacterium damselae subsp. piscicida = Aeromonas salmonicida.

[0057] In summary, the Bacillus subtilis desert subspecies D7-HSJ9 of this invention was obtained by the applicant through screening. This strain originated from the rhizosphere soil of mangroves. After testing, this strain has good salt tolerance, alkali tolerance, and antibacterial properties, especially a good inhibitory effect on Streptococcus dolphinii. In addition, it has high safety, indicating that this strain can effectively replace antibiotics to improve the prevention and treatment of fish diseases and provide excellent germplasm resources for the later preparation of microecological preparation products.

[0058] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. Bacillus subtilis desert subspecies ( Bacillus inaquae The application of strain D7-HSJ9 in the preparation of antibacterial agents is characterized by, The desert subspecies of Bacillus subtilis ( Bacillus inaquae The preservation number of strain D7-HSJ9 is CCTCC NO: M20241842; The pathogen inhibited by the antibacterial agent is: Streptococcus dolphinus ( Streptococcus pneumoniae Staphylococcus aureus ( Staphylococcus aureus ), Mermaid bioluminescent bacteria fish-killing subspecies ( Photobacterium damselae subsp. piscicida and Aeromonas salmonida ( Aeromonas salmonicida ).

2. The application according to claim 1, characterized in that, The desert subspecies of Bacillus subtilis ( Bacillus waterless The 16sRNA sequence of strain D7-HSJ9 is shown in SEQ ID NO:

1.

3. The application according to claim 1, characterized in that, The desert subspecies of Bacillus subtilis ( Bacillus waterless The nucleic acid sequences of the detection gene of strain D7-HSJ9 are shown in SEQ ID NO:2-SEQ ID NO:

32.

4. Contains the Bacillus subtilis desert subspecies as described in claim 1 ( Bacillus inaquae Inoculum of strain D7-HSJ9.

5. The Bacillus subtilis desert subsp. as described in claim 1 ( Bacillus inaquae The use of strain D7-HSJ9 or the bacterial agent as described in claim 4 in the preparation of acid- and salt-resistant formulations.

6. Cultivation of Bacillus subtilis desert subsp. as described in claim 1 ( Bacillus inaquae The method for using strain D7-HSJ9 is characterized by, The method is as follows: Bacillus subtilis desert subsp. ( Bacillus inaquae Strains D7-HSJ9 were inoculated into a culture medium with a pH of 6-8, a salinity of 0-50‰, and a temperature of 26-35℃ and cultured.

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