A halophilic Bacillus DY299 strain with biofilm inhibition ability and its application in the field of anti-MRSA microbial control agents

Abstract

The present invention relates to the technical field of microbial bacillus, and specifically provides a halophilic bacillus DY299 with the ability to inhibit biofilm and its application in the field of anti-MRSA microbial control agents, which is classified and named Bacillus halotolerans and has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20242371. The halophilic bacillus DY299 of the present invention has excellent ability to inhibit Staphylococcus aureus biofilm; the halophilic bacillus DY299 of the present invention has broad application prospects and huge economic value in the fields of microorganisms, biomedicine, etc.

Description

Technical Field

[0001] The invention relates to the technical field of microbial bacillus, in particular to a halophilic bacillus DY299 with the ability to inhibit biofilm and application thereof in the field of anti-MRSA microbial control agents. Background Art

[0002] Staphylococcus aureus is the most common pathogen causing atopic dermatitis and other skin infections. Methicillin-resistant Staphylococcus aureus (MRSA) is the most common drug-resistant strain. Since its discovery, the infection has spread almost all over the world and has become a major healthcare problem worldwide. Biofilm is a complex structure composed of microbial cells and their secretions when bacteria attach to the surface of an object. This not only increases its resistance to antibiotics, but also may cause the disease to become chronic and increase the difficulty of treatment. First of all, due to the low immunity and susceptible skin of the human body, Staphylococcus aureus colonizes on the skin surface, secretes virulence factors such as immunomodulatory proteins, toxins and superantigens, activates the immune system, and produces an inflammatory response. With the formation of biofilm, Staphylococcus aureus is 10-1000 times more resistant to antimicrobial drugs than planktonic bacteria, and induces phagocytic dysfunction of macrophages.

[0003] Bacillus halotolerans is a Gram-positive non-pathogenic bacterium that survives in a high-salt environment to maintain its cell structure and function. Under such extreme conditions, salt-tolerant microorganisms have formed a unique metabolic mechanism, special group characteristics, and new specific metabolites. Therefore, it is regarded by scientists as a new source of natural products with great application potential. Summary of the invention

[0004] The purpose of the present invention is to provide a halophilic Bacillus DY299 with the ability to inhibit biofilm and its application in the field of anti-MRSA microbial control agents to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A strain of Bacillus halotolerans DY299 (Bacillus halotoleransDY299) with the ability to inhibit biofilm, hereinafter referred to as Bacillus halotolerans DY299, is classified and named Bacillus halotolerans. The strain was deposited in the China Center for Type Culture Collection on October 29, 2024, with the deposit address being Wuhan University, Wuhan, and the deposit number being CCTCC NO: M 20242371.

[0007] Furthermore, the halophilic Bacillus DY299 was cultured in RM liquid culture medium at a culture temperature of 37° C., a culture pH of 7, and a growth salt concentration of 6%.

[0008] The halophilic bacillus DY299 is used as an anti-MRSA microbial control agent.

[0009] Furthermore, the application of halophilic Bacillus DY299 in the field of biomedicine adopts the following method:

[0010] Halophilic Bacillus DY299 was cultured in RM broth at 37°C with shaking, and then centrifuged at 12000 rpm / min for 2 min. The collected supernatant was filtered through a 0.22 µm water filter membrane to obtain the filtered supernatant (DY299-CFS).

[0011] Furthermore, after co-culturing Staphylococcus aureus with DY299-CFS for 24 h, crystal violet staining showed that DY299-CFS had the ability to inhibit the biofilm of Staphylococcus aureus.

[0012] Furthermore, the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA).

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The halophilic Bacillus DY299 of the present invention has an excellent ability to inhibit the formation of MRSA biofilm. After the halophilic Bacillus DY299 is co-cultured with MRSA for 24 hours, the amount of MRSA biofilm formation is effectively inhibited. In the field of biomedicine, the halophilic Bacillus DY299 is worthy of further research as an anti-MRSA microbial control agent, and is expected to provide a solution strategy and research direction for the prevention and control of multidrug-resistant bacterial infections. It has broad application prospects and huge economic value in the field of biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the colony morphology of halophilic Bacillus DY299;

[0016] Figure 2 is a microscope image of halophilic Bacillus DY299;

[0017] Figure 3 This is the phylogenetic tree of the 16S rDNA sequence of Halobacillus sp. DY299;

[0018] Figure 4 The pH curve of Halobacillus subtilis DY299

[0019] Figure 5 It is the temperature curve of halophilic Bacillus DY299;

[0020] Figure 6 It is the salt concentration curve of halophilic Bacillus DY299;

[0021] Figure 7 is the amount of biofilm formed by Bacillus halophilus DY299 after treatment with different concentrations of DY299-CFS (**p<0.01, ***p<0.001, control group);

[0022] Figure 8 is the biofilm inhibition rate of halophilic Bacillus DY299 after treatment with different concentrations of DY299-CFS (**p<0.01, ***p<0.001, control group);

[0023] Fig. 9 The live bacteria counts in the biofilm of Halobacillus halophilus DY299 after being treated with different concentrations of DY299-CFS;

[0024] Fig.10 This is the effect of halophilic Bacillus DY299 on the MRSA biofilm structure. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] See also Figures 1 to 10 The present invention provides a strain of Bacillus halotolerans DY299 with the ability to inhibit biofilm, which is derived from the natural Arshan water of Jibuhulangtu Sumu, Hulunbuir, Inner Mongolia Autonomous Region, and is preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: M 20242371.

[0027] Example 1: Molecular Biological Identification of Halophilic Bacillus DY299

[0028] The halophilic Bacillus DY299 strain was revived and amplified using the P1 / P2 bacterial universal 16SrRNA primers, where:

[0029] The sequence of the P1 primer was 5′-ACTCCTACGGGAGGCAGCAG-3′ (SEQ ID NO: 1);

[0030] The sequence of the P2 primer was 5′-GGACTACHVGGGTWTCTAAT-3′ (SEQ ID NO: 2);

[0031] The colony morphology of halophilic Bacillus DY299 is shown in the figure Figure 1 As shown, the microscope image of halophilic Bacillus DY299 is as follows Figure 2 As shown;

[0032] The amplified product was detected by 0.1% agarose gel electrophoresis and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. After comparing the sequencing results with the sequences in the database, it was found that the homology with Bacillus was high and it belonged to a type of Bacillus halotolerans. The phylogenetic tree is shown in the figure below. Figure 3 As shown,

[0033] The DNA sequence of Halobacillus sp. DY299 (SEQ ID NO: 3) is described as follows:

[0034] GCGTTCCAGTCCATGGACGAGTCTGACGGAGCACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGC GGTTCCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCAA.

[0035] Example 2: Growth characteristics experiment of halophilic Bacillus DY299

[0036] (1) Growth pH experiment of Halophilic Bacillus DY299:

[0037] Halophilic Bacillus DY299 was inoculated into RM liquid culture medium with pH values ​​of 4, 5, 6, 7, 8, 9, 10, 11 and 12, respectively, and cultured at 37°C for 24 hours. The absorbance value (OD600) of the 24-hour culture medium was measured. The absorbance icon is as follows: Figure 4 As shown, the growth state of the bacteria was judged according to the absorbance value; the results showed that the halophilic Bacillus DY299 grew at pH 5, 6, 7, 8, 9, and 10, and the optimal growth pH was 7. The determination results are shown in Table 1 below:

[0038] Table 1: Growth pH experimental table of Halophilic Bacillus DY299

[0039]

[0040] (2) Growth temperature experiment of halophilic Bacillus DY299:

[0041] Halophilic Bacillus DY299 was inoculated into RM liquid culture medium, and cultured at 4℃, 10℃, 15℃, 20℃, 25℃, 30℃, 37℃, and 45℃ for 24h. The absorbance value (OD600) of the 24h culture solution was measured. The absorbance icon is as follows: Figure 5 As shown, the growth state of the bacteria was judged according to the absorbance value. The results showed that the halophilic Bacillus DY299 grew at 10°C, 15°C, 20°C, 25°C, 30°C, 37°C, and 45°C, and 37°C was the optimal growth temperature. The determination structure is shown in Table 2 below:

[0042] Table 2: Growth temperature experimental table of Halophilic Bacillus DY299

[0043]

[0044] (3) Growth salt concentration experiment of halophilic Bacillus DY299:

[0045] Halophilic Bacillus DY299 was inoculated into RM liquid culture medium with salt concentrations of 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20, respectively, and cultured at 37°C for 24 hours with shaking. The absorbance value (OD600) of the 24-hour culture medium was measured. The absorbance icon is as follows: Figure 6 As shown, the growth state of the bacteria was judged according to the absorbance value. The results showed that the growth salt concentration of halophilic Bacillus DY299 was between 2% and 10%, and the optimal growth salt concentration was 4% to 6%. The determination structure is shown in Table 3 below:

[0046] Table 3: Experimental table of growth salt concentration of halophilic Bacillus DY299

[0047]

[0048] Example 3: Inhibition experiment of halophilic Bacillus DY299 on MRSA biofilm

[0049] After halophilic Bacillus DY299 was cultured in RM broth at 37°C with shaking, the supernatant of halophilic Bacillus DY299 was obtained by centrifugation at 12000 rpm / min for 2 min. The collected supernatant of halophilic Bacillus DY299 was filtered through a 0.22 µm water filter membrane to obtain the filtered supernatant (DY299-CFS); DY299-CFS was divided and stored in a -20°C refrigerator; 50 ml of 24-CFS was freeze-dried by a vacuum freeze dryer, and the obtained powder was re-dissolved with sterile water to a final volume of 5 mL to obtain a 5-fold concentrated supernatant (CFS-Con50µL);

[0050] (1) Quantitative biofilm staining with crystal violet:

[0051] 100 µL of MRSA bacterial solution (1×106 CFU / mL) was added to a 96-well plate. Except for the control well, 50 µL of DY299-CFS cultured at different times was added to each well and supplemented to 200 µL with LB medium. Different concentrations of DY299-CFS (CFS-Con50 µL, 50 µL, 25 µL, 12.5 µL, 6.25 µL, 3.125 µL) were added to each well and supplemented to 200 µL with LB medium. After culturing for 24 h, crystal violet staining was used to quantify biofilm. The experimental results of biofilm formation are shown in Figure 2. Figure 7 The results of the biofilm inhibition experiment are shown in Figure 8 As shown; From the results, DY299-CFS has the ability to inhibit the formation of MRSA biofilm;

[0052] (2) Scanning electron microscopy observation of biofilm:

[0053] The slides were placed in a 24-well plate, and the bacterial culture and drug treatment were the same as crystal violet staining. After 24 hours of culture, the bacterial solution was aspirated, and the floating bacteria were gently washed off with sterile saline, and 2.5% glutaraldehyde solution was added for 4 hours. Rinse twice with PBS buffer, dry, and gold-plated. The samples were observed using a SU-8220 field emission scanning electron microscope. The experimental results are shown in the figure. Fig. 9 As shown, from the results, DY299-CFS can effectively destroy the bacterial structure of MRSA, has a damaging effect on MRSA, and can cause the leakage of cell contents;

[0054] (3) Quantification of live bacteria in biofilm:

[0055] Bacterial culture and drug treatment were the same as crystal violet staining. After 24 h of culture, the culture medium was aspirated and washed twice with PBS. The attached biofilm was knocked down with PBS to collect the biofilm. The collected biofilm was dispersed with an ultrasonic instrument for 10 min. After the suspension was diluted 105 continuously, 100 μL was evenly spread on the LB agar plate and incubated upside down at 37 ° C for 24 h, and then the CFU was calculated. The experimental results are shown in Fig.10 As shown in the results, DY299-CFS can kill live bacteria in MRSA biofilm.

[0056] Through the above three embodiments, it can be seen that halophilic Bacillus DY299 has the following characteristics:

[0057] Growth characteristics: The culture temperature of Halophilic Bacillus DY299 is 10-45°C, and the optimal culture temperature is 37°C. The culture pH is 5-10, and the optimal culture pH is 7. The growth salt concentration is 2%-10%, and the optimal salt concentration is 4%-6%;

[0058] Biofilm inhibition properties: Halophilic Bacillus DY299 can inhibit the formation of MRSA biofilm, effectively destroy the bacterial structure of MRSA, cause the leakage of cell contents, and kill live bacteria in MRSA biofilm.

[0059] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A halophilic Bacillus DY299 having the ability to inhibit biofilm, characterized in that: Its classification name is Bacillus halotolerans and it has been deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M20242371.

2. Use of the halophilic Bacillus DY299 having the ability to inhibit biofilm as claimed in claim 1 in the preparation of an anti-MRSA microbial control agent.

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