Antibacterial bacillus subtilis subsp. Deserticola strain and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to an antibacterial bacillus subtilis subsp. Deserticola strain and application thereof.Bacillus subtilis subsp. Deserticola D7-HSJ9 is obtained from mangrove forest rhizosphere soil by the applicant, and it is detected that the strain has good salt resistance, alkali resistance and antibacterial characteristics, and the bacillus subtilis subsp. Deserticola D7-HSJ9 can be used as a bacillus subtilis subsp. Deserticola strain. According to the present invention, the bacterial strain has characteristics of good antibacterial effect, particularly good antibacterial effect on streptococcus iniae, and good antibacterial effect on a plurality of pathogenic bacteria such as photobacterium dama subspecies, aeromonas salmonicida, staphylococcus aureus and the like, such that the bacterial strain can effectively replace the antibiotic so as to improve the prevention and control of fish diseases, and a good germplasm resource is provided for later preparation of microecological preparation products.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to an antibacterial Bacillus subtilis subspecies deserticola strain and application thereof. Background Art

[0002] With the intensive development of aquaculture, the problem of aquatic diseases is becoming increasingly serious. Streptococcus dolphinus has zoonotic characteristics and can cause sepsis, cellulitis, and even osteomyelitis, endocarditis and meningitis in humans. It can also infect a variety of animals including fish. At present, there are reports of fish streptococcal disease in many countries in the world, and the number of fish species endangered is also increasing, such as tilapia, channel catfish, turbot, grouper, ovate pomfret, etc., and both marine and freshwater fish can be infected by it. However, there is no effective commercial vaccine for the prevention and treatment of Streptococcus dolphinus, and for fish, the cost of the vaccine is high and the promotion effect is not good. At present, bacterial infections in 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, microecological preparations are usually used to replace antibiotics in the existing technology. Microecological preparations are non-toxic, harmless, residue-free, pollution-free, and can produce antibacterial substances. They are a good alternative agent for disease prevention and control in aquaculture; however, the most important technical means of using microecological preparations is to continuously screen strains with antibacterial activity to provide sufficient germplasm resources for the later preparation of microecological preparation products. Summary of the invention

[0003] In view of the above, it is necessary to improve the prevention and treatment of fish diseases, reduce the hazards of antibiotic resistance, screen strains with antibacterial activity against aquatic pathogens, and provide sufficient germplasm resources for the subsequent preparation of microecological preparations.

[0004] To achieve the above purpose, the present invention screened out a new strain: Bacillus inaquosorum strain D7-HSJ9, whose classification name is: Bacillus inaquosorum D7-HSJ9, and its Chinese classification name is: Bacillus subtilis desert subspecies D7-HSJ9, and the preservation number is CCTCC NO: M20241842; the strain is deposited 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 shown in SEQ ID NO:1.

[0006] The present invention also includes a detection gene for detecting the Bacillus inaquosorum strain D7-HSJ9, and the nucleic acid sequence of the detection gene 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 related preparations.

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

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

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

[0012] The desert Bacillus subtilis subspecies D7-HSJ9 of the present invention is obtained by screening by the applicant from the rhizosphere soil of mangroves. After testing, the strain has good salt resistance, alkali resistance and antibacterial properties, especially has a good antibacterial effect on dolphin Streptococcus. In addition, it can also have a good antibacterial effect on several aquatic bacteria such as mermaid photobacterium piscicidial subspecies, salmonicidal Aeromonas and Staphylococcus aureus. At the same time, it also has high safety, indicating that the strain can effectively replace antibiotics to improve the prevention and treatment of fish diseases, and provide high-quality germplasm resources for the later preparation of microecological preparation products. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0016] Figure 4 This is the hemolytic test result of strain D7-HSJ9.

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

[0018] Figure 6 This is a graph showing 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 This is the 16sRNA evolutionary tree sequence of strain D7-HSJ9.

[0021] Fig. 9 This is an evolutionary tree constructed based on the main housekeeping genes of Bacillus subtilis D7-HSJ9. The figure is 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). The phylogenetic tree was constructed using the NJ (Neighbor-Joining) method using MEGA6.0 software. DETAILED DESCRIPTION

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

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

[0024] Example 1

[0025] This embodiment is a method for screening and identifying disease-resistant and bacteriostatic strains, which is as follows:

[0026] 1. Screening method

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

[0028] ②Separation and purification: A selective separation method was used, and the separation and purification method was as follows: Take the rhizosphere sample of mangrove plants, wash it with sterile physiological saline for 2-3 times, add it to a conical flask containing glass beads and 45mL sterile water, shake it vigorously on a culture shaker for 30min (37℃), and then keep it in a water bath at 80℃ for 30min, 1000r / min, 2min. Dilute the suspension 10 times, take the original solution, 10 times, 100 times and 1000 times dilutions, spread them on LB solid plates, and culture them at 28℃. After the colonies grow, pick the colonies and streak them for purification 2-3 times.

[0029] ③ Preservation of strains: The strain preservation solution is 50% glycerol, and the ratio of strain preservation solution to bacterial solution is 1:1. Under sterile conditions, first use a pipette to draw 600μL of 50% glycerol into a pre-sterilized strain preservation tube, then draw 600μL of Bacillus fermentation solution, pipette and mix well, and store in a refrigerator at -80℃.

[0030] The strain screened in this example is Bacillus inaquosorum strain D7-HSJ9, which is classified and named as Bacillus inaquosorum D7-HSJ9, and its Chinese classification name is Bacillus subtilis desert subspecies D7-HSJ9, and its preservation number is CCTCC NO: M20241842; the strain is deposited 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 Morphological identification such as Figure 1 , Figure 2 and Figure 3 As shown: Bacillus subtilis desert subsp. D7-HSJ9 was inoculated on solid LB medium and cultured for 1 day. The colonies were light yellow, round, translucent colonies with rough edges, dry surface, and a diameter of 2-5 mm. Bacillus subtilis desert subsp. D7-HSJ9 was a Gram-positive bacterium with rod-shaped cells arranged continuously or scattered. Under an electron microscope, Bacillus subtilis desert subsp. D7-HSJ9 appeared to be short rod-shaped, with a single cell length of 0.7-0.8×2-3 um and uniform coloring.

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

[0034] Table 1 Comparison results of rRNA nucleotide sequences of strain D7-HSJ916S

[0035]

[0036]

[0037] For further classification, we sequenced the whole genome of D7-HSJ9 and selected the 19 strains that were closest to it at the species level based on 31 housekeeping genes (nucleotide sequences such as SEQ ID NO: 2-SEQ ID NO: 32). We used MEGA6.0 software to select the NJ (Neighbor-Joining) method to construct a phylogenetic tree. The results are shown in the figure. Fig. 9 As shown: the housekeeping gene sequence of D7-HSJ9 is 97.3% similar to that of Bacillus inaquosorum, and they belong to the same branch in evolution. Therefore, we tentatively identified this strain as Bacillus inaquosorum D7-HSJ9.

[0038] Example 2

[0039] The safety test of the selected Bacillus subtilis desert subspecies D7-HSJ9 was carried out as follows: D7-HSJ9 was added to the blood agar plate and cultured at 28°C for 12 hours. The strain HSJ8 was used as a positive control (the strain HSJ8 was a laboratory preserved strain) to detect its hemolytic activity. Figure 4 As shown, strain D7-HSJ9 has no hemolytic activity, indicating that it has certain safety. The hemolytic plate used is Columbia blood agar plate, purchased from Beijing Luqiao Technology Co., Ltd.

[0040] Example 3

[0041] The selected Bacillus subtilis desert subspecies D7-HSJ9 was subjected to an acid-base tolerance experiment as follows: liquid LB medium was prepared, the medium was adjusted to different pH values ​​with glacial acetic acid and NaOH, sterilized at 121°C for 20 min, D7-HSJ9 bacterial solution was inoculated into the above conditioned medium at a ratio of 2%, 3 groups were paralleled for each pH value, shaken and cultured at 28°C, OD600 of the culture solution was measured, and a growth curve was drawn. The results are shown in Figure 5 As shown in the figure, strain D7-HSJ9 grew fastest between pH 6 and 8, and there was no significant difference.

[0042] Example 4

[0043] The selected Bacillus subtilis desert subspecies D7-HSJ9 was subjected to a salinity tolerance experiment, as follows: LB liquid culture medium with different percentages of NaCl concentration was prepared, sterilized at 121°C for 20 minutes, and the activated D7-HSJ9 bacterial solution was added to the LB culture medium at 2%; three parallels were set for each salinity, and shaken and cultured at 28°C; the OD600 of the cultured bacterial solution was measured, and a growth curve was drawn. The LB culture medium components are: 10g tryptone, 5g yeast extract, 10g sodium chloride, pH 6.8-7.2, and 1L pure water. The results are as follows: Figure 6 As shown in the figure, the strain D7-HSJ9 has a strong tolerance to salinity, and there is no significant difference in growth rate within the salinity range of 0‰-50‰.

[0044] Example 5

[0045] The growth of the selected Bacillus subtilis desert subspecies D7-HSJ9 was experimentally observed at different temperatures. The details were as follows: different temperature gradients were set, and the D7-HSJ9 bacterial solution was inoculated into the LB medium at a ratio of 1:50. After culturing for 12 hours, the OD 600 The results are as follows Figure 7 As shown in the figure, the results show that strain D7-HSJ9 grows better at 26-35°C. The growth of D7-HSJ9 at 40°C is slower than that at 30°C. At 20°C and 50°C, the growth rate of D7-HSJ9 is inhibited.

[0046] Example 6

[0047] The selected Bacillus subtilis desert subspecies D7-HSJ9 was subjected to an anti-aquatic pathogen screening experiment, as follows:

[0048] Staphylococcus aureus, Photobacterium mermani, Streptococcus dolphinii, Streptococcus agalactiae, Aeromonas salmonicida were used as indicator bacteria. The antibacterial experiment was carried out by the Oxford cup punch method.

[0049] 1. Strain activation: Activate Bacillus subtilis subsp. deserti D7-HSJ9 and the 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 the activated bacterial liquid.

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

[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 liquid onto the plate, and then pour the non-solidified LB solid medium to mix the indicator bacterial liquid. 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 block in the hole. Put 100 μl of the Bacillus subtilis subsp. deserti D7-HSJ9 bacterial liquid into the Oxford cup, 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 respectively, and use sterile PBS as a negative control. Incubate the plate upright in an incubator at 28°C for 12 - 16 h and observe the size of the inhibition zone. 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 liquid

[0054]

[0055] Note: Size of the 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 properties against Staphylococcus aureus, Photobacterium damselae subsp. piscicida, and Aeromonas salmonicida. From the size of the inhibition zone, 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 the present invention was obtained by the applicant's own screening. The strain is derived from the rhizosphere soil of mangroves. After testing, the strain has good salt resistance, alkali resistance, and antibacterial properties, especially has a good antibacterial effect on Streptococcus dolphinus. In addition, it has high safety, indicating that the 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 above-described embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. Bacillus inaquosorum strain D7-HSJ9, whose deposit number is CCTCCNO: M20241842.

2. The 16sRNA sequence of the Bacillus inaquosorum strain D7-HSJ9 as claimed in claim 1 is shown in SEQ ID NO:

1.

3. Detection of the detection gene of Bacillus inaquosorum strain D7-HSJ9 as claimed in claim 1, characterized in that: The nucleic acid sequences of the detection genes are shown in SEQ ID NO: 2-SEQ ID NO:

32.

4. A bacterial agent comprising the Bacillus inaquosorum strain D7-HSJ9 according to any one of claims 1 to 2.

5. Use of the Bacillus inaquosorum strain D7-HSJ9 according to any one of claims 1 to 2 or the bacterial agent according to claim 3 in the preparation of antibacterial, acid-resistant and / or salt-resistant preparations.

6. The use according to claim 5, characterized in that The pathogenic bacteria that can be inhibited by the antibacterial agent are: Streptococcus iniae, Staphylococcus aureus, Photobacterium damselae subsp. piscicida and / or Aeromonas salmonicida.

7. A method for culturing the Bacillus inaquosorum strain D7-HSJ9 according to any one of claims 1 to 2, characterized in that: The method comprises the following steps: inoculating Bacillus subtilis desert subspecies (Bacillus inaquosorum) strain D7-HSJ9 into a culture medium with a pH value of 6-8, a salinity mass percentage of 0-50‰ and a temperature of 26-35°C for cultivation.

Images