Bacillus megaterium strain si-jd-01 against drug-resistant bacteria and application thereof

Abstract

The application discloses a bacillus megaterium strain SI-JD-01 resistant to drug-resistant bacteria and an application thereof, and belongs to the technical field of microorganisms. The bacillus megaterium has been preserved in the China Center for Type Culture Collection on March 9, 2023, and the preservation name is bacillus megaterium SI-JD-01, and the preservation number is CCTCC NO: M 2023278. The beneficial effect is that the strain with antagonistic activity to drug-resistant bacteria and non-drug-resistant bacteria which are easy to cause infectious diseases is found in the environment air, specifically a bacillus megaterium strain SI-JD-01 isolated from the environment air, and the genetic background is rare. The strain secretes broad-spectrum bacteriostatic substances, and has broad-spectrum antibacterial activity to common drug-resistant bacteria and non-drug-resistant bacteria which are easy to cause infectious diseases, which lays a foundation for mining probiotic preparations for maintaining the stability of the microbial community of the body.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a Bacillus megaterium strain SI-JD-01 resistant to drug-resistant bacteria and its applications. Background Technology

[0002] The microbiome (such as gut microbiota, lung microbiota, and skin microbiota) has established a close relationship with its host over a long period of evolution, playing important physiological functions, including biological barrier function, immune function, metabolic function, and nutritional function, and influencing the body's growth, development, and aging. The number of symbiotic microorganisms in the human body is 10 times the number of human cells, and the number of genes is approximately 150 times that of humans. They are mainly distributed in the digestive tract, respiratory tract, and skin. Under normal circumstances, the microbiome and host are in balance, maintaining homeostasis. When this balance is disrupted, harmful microorganisms can easily induce disease, leading to or accelerating the occurrence and progression of diseases such as tumors, inflammation, or obesity.

[0003] Ecological imbalance includes an imbalance in the microbiota of the skin or organ tissues (including gut microbiota, lung microbiota, urogenital microbiota, and skin microbiota). In the microbiota of healthy tissues, non-pathogenic bacteria can secrete inhibitory substances or occupy their ecological niches, thereby directly inhibiting or indirectly killing pathogenic bacteria. Once the balance is broken, pathogenic bacteria will occupy the ecological niches and proliferate abnormally, leading to or accelerating the occurrence of inflammatory and / or metabolic diseases.

[0004] Antibiotics are commonly used to treat microbial infections, but their use can easily lead to dysbiosis of the body's microbiome and can also cause bacterial resistance.

[0005] Chinese patent application CN116004435A discloses a strain of Bacillus megaterium and its applications. The strain is named Bacillus megaterium, strain number Tu27, and accession number CCTCCNO: M20221115. This strain exhibits inhibitory effects on the growth of rice pathogenic fungi in confrontation culture; this strain can synthesize silver nanoparticles with antibacterial and bactericidal activities. The silver nanoparticles synthesized by this strain have a particle size of 15±6 nm and show significant inhibitory effects on the growth of rice blast fungus. However, this strain does not possess broad-spectrum antibacterial activity. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to provide a strain of Bacillus megaterium with broad-spectrum antibacterial activity.

[0007] The present invention solves the above-mentioned technical problems through the following technical means:

[0008] The first aspect of this invention provides a Bacillus megaterium strain SI-JD-01 resistant to drug-resistant bacteria. This Bacillus megaterium was deposited on March 9, 2023, at the China Center for Type Culture Collection (CCTCC, address: No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China), with the deposit name Bacillus megaterium SI-JD-01 and the accession number CCTCC NO: M 2023278.

[0009] Beneficial effects: This Bacillus megaterium strain SI-JD-01 has good antagonistic activity against a variety of drug-resistant and non-drug-resistant microorganisms.

[0010] Preferably, the 16S rRNA sequence of the strain is shown in SEQ ID NO.1.

[0011] A second aspect of the present invention proposes the application of the above-mentioned Bacillus megaterium strain SI-JD-01 in antibacterial applications.

[0012] Preferably, the antibacterial agent is effective against both drug-resistant and non-drug-resistant microorganisms.

[0013] Preferably, the drug-resistant microorganisms include one or more of the following: ESBLs (Escherichia coli), CRE (Escherichia coli), MRSA, MRCNS (Staphylococcus aureus), VRE (Enterococcus faecalis), PDR-AB, and PDR-PA.

[0014] Preferably, the non-drug-resistant microorganisms include: G + cocci, G - bacilli, G + One or more of the following bacteria.

[0015] Preferably, the G + Cocci include one or more of the genera Staphylococcus, Enterococcus, and Streptococcus.

[0016] Preferably, the Staphylococcus genus includes one or more of Staphylococcus aureus, Staphylococcus hominis, and Staphylococcus saprophyticus.

[0017] Preferably, the Enterococcus genus includes one or more of Enterococcus avium, Enterococcus faecalis, and Enterococcus faecium.

[0018] Preferably, the Streptococcus genus includes Streptococcus tenuissus, etc.

[0019] Preferably, the G - Bacilli include Escherichia coli, etc.

[0020] Preferably, the G + Bacilli include: Corynebacterium, etc.

[0021] Preferably, the Corynebacterium genus includes Corynebacterium striatum, etc.

[0022] A third aspect of the present invention provides an antibacterial agent, the raw materials of which include one or more of the above-mentioned strain SI-JD-01, fermentation supernatant of strain SI-JD-01, and lysate of strain SI-JD-01.

[0023] The advantages of this invention are:

[0024] This invention relates to a strain of *Bacillus megaterium*, strain SI-JD-01, isolated from ambient air, which exhibits antagonistic activity against various common drug-resistant and non-drug-resistant bacteria that easily cause infectious diseases. This strain secretes broad-spectrum antibacterial substances and possesses broad-spectrum antibacterial activity against common drug-resistant and non-drug-resistant bacteria that easily cause infectious diseases. This lays the foundation for developing probiotic preparations that maintain the homeostasis of the body's microbiome. Attached Figure Description

[0025] Figure 1 This is a colony morphology diagram of strain SI-JD-01 on a blood agar plate.

[0026] Figure 2 The image shows the morphology of strain SI-JD-01 under a microscope (1000×) after Gram staining.

[0027] Figure 3 MALDI-TOF MS identification pattern of strain SI-JD-01;

[0028] Figure 4 A phylogenetic tree of strain SI-JD-01 constructed based on the 16S rRNA gene;

[0029] Figure 5 The diagram shows the antagonistic effect of strain SI-JD-01 on Bacillus subtilis.

[0030] Figure 6 The antagonistic effect of different treatments on MRSA after dialysis and freeze-drying of fermentation supernatant of strain SI-JD-01 is shown in the figure.

[0031] Figure 7 The strain SI-JD-01 is used to treat common clinical G... - Graph showing the antagonistic effect of CRE-Escherichia coli on bacilli;

[0032] Figure 8 The strain SI-JD-01 is used to treat common clinical G... + Antagonistic effects of cocci, MRSA, Staphylococcus aureus, and VRE-Enterococcus faecium;

[0033] Figure 9The diagram shows the antagonistic effect of strain SI-JD-01 against Enterococcus avium, Enterococcus faecalis, and Streptococcus tenuissus.

[0034] Figure 10 The diagram shows the antagonistic effect of strain SI-JD-01 against *Staphylococcus saprophyticus*.

[0035] Figure 11 This is a diagram showing the antagonistic effect of strain SI-JD-01 on Corynebacterium striatum. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Example 1:

[0038] (1) Isolation and identification of the strain: This strain was discovered on May 16, 2022, in Laboratory 306, South Building, Comprehensive Experimental Building, Hefei Institutes of Physical Science, Chinese Academy of Sciences. It was collected from indoor ambient air. Through systematic screening of strains from ambient air, a strain capable of inhibiting the growth of Staphylococcus aureus was found. The strain was purified by streaking on blood agar plates. Using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI Biotyper 3.1 software, the strain was preliminarily identified as Bacillus megaterium, with a score of 2.451. The 16S rRNA gene was amplified using the bacterial universal primer (47F / 1492R). The gene sequence obtained after PCR product analysis (GenBank Accession: OR064032) was compared and analyzed in NCBI and MEGA was used. The phylogenetic tree of this strain was constructed using software 11.0 to clarify its taxonomic position.

[0039] (2) Colony morphology and cell structure of the strain: On blood agar plates, the colonies of this strain are light yellow, opaque, smooth, moist, and round with no hemolytic zone. Under an optical microscope, the cells of this strain are spherical or cylindrical, with spores and flagella, and are Gram-positive.

[0040] (3) Using the Oxford cup method—plate inhibition test (Oxford cup diameter 6 mm), *Bacillus megaterium* SI-JD-01 was found to have antagonistic activity against Gram-positive cocci (*Staphylococcus aureus*, *Staphylococcus hominis*, *Enterococcus avium*, *Enterococcus faecium*, *Staphylococcus saprophyticus*, *Enterococcus faecalis*, and *Streptococcus tenuis*), Gram-negative bacilli (*Escherichia coli*), and Gram-positive bacilli (*Corynebacterium striatum*). This indicates that strain SI-JD-01 has broad-spectrum antibacterial activity. Notably, *Bacillus megaterium* SI-JD-01 also exhibits antagonistic activity against other non-*Bacillus megaterium* strains, suggesting that its secreted active substance is likely a bacteriocinogen.

[0041] (4) The antagonistic effect of dialysis and freeze-drying of the fermentation supernatant of Bacillus megaterium SI-JD-01 on MRSA was investigated, as well as the antagonistic effect of freeze-drying of the precipitate after dialysis of the fermentation supernatant through a 3 kDa dialysis membrane (dialysis for 24 h, dialysis solution was ultrapure water). Further observation was conducted on the antagonistic effect of freeze-drying the precipitate after dialysis through a 3 kDa dialysis membrane, dissolving it in ultrapure water (total protein concentration of 100 μg / mL), and then subjecting it to UV irradiation for 30 min, boiling for 30 min, and protease treatment (final concentration of 10 μM proteinasek) for 30 min on MRSA. This indicates that the effector component secreted by Bacillus megaterium SI-JD-01 is below 3 kDa, and is heat-resistant, protease-resistant, and UV-resistant, exhibiting good stability.

[0042] (5) The Bacillus megaterium SI-JD-01 described in this invention was deposited at the China Center for Type Culture Collection (Wuhan University) on March 9, 2023, with the deposit name Bacillus megaterium SI-JD-01 and the accession number CCTCC No: M 2023278.

[0043] Example 2:

[0044] Antagonistic activity of Bacillus megaterium SI-JD-01 against MRSA and non-MRSA strains

[0045] 1. Activation of the strain

[0046] Bacillus megaterium SI-JD-01, 15 MRSA strains, and 15 non-MRSA strains, stored at -80℃, were streaked onto sheep blood agar plates and incubated at 37℃ for 16–20 hours. The incubation was repeated twice.

[0047] 2. Preparation of target bacteria culture

[0048] The 15 activated MRSA strains and 15 non-MRSA strains were each prepared into bacterial suspensions with a turbidity of 0.5 McFarland using sterile physiological saline. Then, 100 μL of the 0.5 McFarland turbidity bacterial suspension was added to a 1.5 mL EP tube containing 900 μL of sterile physiological saline, mixed thoroughly, and a 10-fold dilution of the target bacterial suspension was prepared for later use.

[0049] 3. Preparation of Bacillus megaterium SI-JD-01 bacterial culture

[0050] Prepare a bacterial suspension of the activated Bacillus megaterium SI-JD-01 to 1.0 McFarland turbidity using sterile physiological saline. Set aside for later use.

[0051] 4. Oxford cup plate antibacterial test

[0052] Inside the biosafety cabinet:

[0053] (1) Using a sterile cotton swab, dip it into the target bacterial solution diluted 10 times above, rotate and squeeze out the excess bacterial solution on the inner wall of the tube, spread it evenly on the surface of the MH plate 3 times, rotate the plate 60 degrees each time, and finally spread it around the edge of the agar in the plate.

[0054] (2) After inoculating the target bacterial solution, MH plates are dried at room temperature for 3-5 minutes.

[0055] (3) Using sterile forceps, pick up the sterilized Oxford cup (6.0 mm aperture) and gently place it on the inner surface of the above MH plate. Press lightly to make the bottom of the Oxford cup fit tightly against the agar surface to prevent leakage.

[0056] (4) Use a pipette to add 30 μL of Bacillus megaterium SI-JD-01 suspension to the Oxford cup and let it stand open for 30 minutes.

[0057] (5) After the bacterial suspension in the Oxford cup has been completely absorbed by the agar, use sterile tweezers to gently remove the Oxford cup.

[0058] (6) Place the MH plate in an incubator and incubate at 30°C for 16-18 hours.

[0059] (7) The inhibitory activity against the target bacteria was evaluated by observing whether there was an inhibition zone around the Bacillus megaterium SI-JD-01 colony and measuring the diameter of the inhibition zone.

[0060] Table 1: Evaluation of the antagonistic activity of Bacillus megaterium SI-JD-01 against MRSA and non-MRSA strains

[0061] Target bacteria categories Diameter of the inhibition zone (mm) Inhibition rate (%) MRSA 11.5±1.26 100 Non-MRSA 10.9±1.05 100

[0062] Figure 1The image shows the colony morphology of strain SI-JD-01 on blood agar plates. As can be seen from the image, the colonies are light yellow, opaque, rough, moist, 0.5-3 mm in diameter, and round and raised. There is no hemolytic zone around the colonies.

[0063] Figure 2 The image shows the morphology of strain SI-JD-01 under a microscope (1000×) after Gram staining. As can be seen from the image, the bacteria turn purple after Gram staining, indicating that Bacillus megaterium is a Gram-positive bacterium.

[0064] Figure 3 MALDI-TOF MS identification pattern of strain SI-JD-01.

[0065] Figure 4 A phylogenetic tree for strain SI-JD-01 constructed based on the 16S rRNA gene.

[0066] Figure 5 The figure shows the antagonistic effect of strain SI-JD-01 on Bacillus. It can be seen from the figure that strain SI-JD-01 has a significant killing effect on Bacillus. The diameter of the inhibition zone was measured to be about 10 mm.

[0067] Figure 6 The graph shows the antagonistic effect of different treatments on MRSA after dialysis and freeze-drying of the fermentation supernatant of strain SI-JD-01 using the Oxford cup method. As can be seen from the graph, the supernatant of strain SI-JD-01 fermentation, after being dialyzed through a 3kDa dialysis membrane and then freeze-dried (with the total protein concentration of the solution adjusted to 100 μg / mL), exhibits a significant killing effect on MRSA after UV irradiation, boiling, or protease treatment. The control group used an equal volume of LB broth medium, and the total volume of the solution added to the Oxford cup was 100 μL.

[0068] Figure 7 The figure shows the antagonistic effect of strain SI-JD-01 against common clinical Gram-negative bacilli, CRE-Escherichia coli. As can be seen from the figure, strain SI-JD-01 has a significant killing effect on CRE-Escherichia coli. The diameter of the inhibition zone was measured to be approximately 8 mm.

[0069] Figure 8 This is a graph showing the antagonistic effects of strain SI-JD-01 against common clinical Gram-positive cocci, MRSA, Staphylococcus aureus, and VRE-Enterococcus faecalis. The graph illustrates the antagonistic effects of strain SI-JD-01 against common clinical Gram-positive cocci, MRSA, Staphylococcus aureus, and VRE-Enterococcus faecium. + Cocci have a significant killing effect; the diameter of the inhibition zone was measured to be approximately 10 mm.

[0070] Figure 9The figure shows the antagonistic effect of strain SI-JD-01 against Enterococcus spp., Enterococcus avium, Enterococcus faecalis and Streptococcus tenuis. It can be seen from the figure that strain SI-JD-01 has a significant killing effect on Enterococcus avium, Enterococcus faecalis and Streptococcus tenuis. The diameter of the inhibition zone was measured to be about 10 mm.

[0071] Figure 10 The figure shows the antagonistic effect of strain SI-JD-01 against Staphylococcus spp. and Staphylococcus saprophyticus. It can be seen from the figure that strain SI-JD-01 has a significant killing effect on Staphylococcus saprophyticus. The diameter of the inhibition zone was measured to be about 10 mm.

[0072] Figure 11 The image shows the antagonistic effect of strain SI-JD-01 on Corynebacterium striatum. As can be seen from the image, strain SI-JD-01 has a significant killing effect on Corynebacterium striatum. The diameter of the inhibition zone was measured to be approximately 10 mm.

[0073] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Bacillus megaterium resistant to drug-resistant bacteria ( Bacillus megaterium SI-JD-01, characterized in that, This strain was deposited at the China Center for Type Culture Collection on March 9, 2023, with accession number CCTCC NO: M 2023278.

2. The Bacillus megaterium resistant to drug-resistant bacteria as described in claim 1 ( Bacillus megaterium SI-JD-01, characterized in that, The 16S rRNA sequence of the strain is shown in SEQ ID NO.

1.

3. The Bacillus megaterium resistant to drug-resistant bacteria as described in claim 1 ( Bacillus megaterium Application of SI-JD-01 in the preparation of antibacterial agents.

4. The application according to claim 3, characterized in that, The antibacterial agents are effective against both drug-resistant and non-drug-resistant microorganisms.

5. The application according to claim 4, characterized in that, The drug-resistant microorganisms include one or more of the following: ESBLs (Escherichia coli), CRE (Escherichia coli), MRSA, MRCNS (Staphylococcus aureus), VRE (Enterococcus faecium), PDR-AB, and PDR-PA.

6. The application according to claim 4, characterized in that, The non-drug-resistant microorganisms include G. + cocci, G - bacilli and G + One or more of the following bacteria.

7. The application according to claim 6, characterized in that, The G + Cocci include one or more of the genera Staphylococcus, Enterococcus, and Streptococcus; the G - Bacilli include Escherichia coli; the G... + Bacilli include the genus Corynebacterium.

8. The application according to claim 7, characterized in that, The *Staphylococcus* genus includes one or more of *Staphylococcus aureus*, *Staphylococcus hominis*, and *Staphylococcus saprophyticus*; the *Enterococcus* genus includes one or more of *Enterococcus avium*, *Enterococcus faecium*, and *Enterococcus faecalis*; and the *Streptococcus* genus includes *Streptococcus tenacis*.

9. The application according to claim 7, characterized in that, The Corynebacterium genus includes Corynebacterium striatum.

10. An antibacterial agent, characterized in that, Its raw materials include Bacillus megaterium as described in claim 1 ( Bacillus megaterium SI-JD-01.

Images