Bacillus cereus phage and application thereof

Abstract

The invention provides a bacillus cereus bacteriophage and application thereof, and belongs to the technical field of bacteriophages. The preservation number of the bacillus cereus phage provided by the invention is CCTCC (China Center For Type Culture Collection) NO: M 20251545. The bacteriophage provided by the invention can specifically split bacillus cereus and remarkably reduce the colonization amount of the bacillus cereus in the respiratory tract, so that lung inflammatory response caused by swine influenza virus infection is effectively relieved, lung tissue injury is reduced, infection symptoms are relieved, and remarkable interference on normal florae of a host is avoided. According to the technical scheme, a new direction is provided for development and utilization of the bacteriophage in the antiviral field, and compared with traditional antibiotics, the bacteriophage serving as a natural antibacterial agent has the advantages of precise targeting, self-replication, low toxicity and the like and has a wide application prospect in the aspect of prevention and treatment of influenza virus infection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bacteriophages, and particularly relates to a Bacillus cereus bacteriophage and application thereof. BACKGROUND

[0002] Influenza virus (IAV) belongs to the Orthomyxoviridae family and is a single-stranded negative-strand RNA virus that mainly infects humans or mammals. IAV has high genetic variability and constantly evolves through antigenic drift and antigenic shift, which limits the protective effect of existing vaccines. Influenza A and B viruses are the main causes of seasonal influenza, and severe symptoms can cause lung damage or death. About 250,000 to 500,000 people die from influenza every year, causing a huge burden on public health. Although vaccination is the main means of preventing and controlling IAV, the rapid mutation of the virus and the mismatch between the vaccine strain and the epidemic strain make the prevention and control effect unstable. In addition, the problem of drug resistance caused by antibiotic abuse also increases the difficulty of treating secondary bacterial infections.

[0003] Bacillus cereus (B.C) is a gram-positive facultative anaerobic bacterium widely distributed in the environment, belonging to the Bacillus genus, which can form heat-resistant and dry-resistant spores. It is often spread through contaminated food, medical devices or the environment, causing gastrointestinal and extraintestinal infections in humans and animals. Bacteriophages (phages) are viruses that can specifically infect and lyse bacteria. Unlike broad-spectrum antibiotics, phages have high host specificity and can accurately target specific pathogenic bacteria without affecting the normal flora of the host. In recent years, phages have received widespread attention in the prevention and control of bacterial infections due to their strong specificity and good bactericidal effect. However, there is relatively little research on the use of phages in the prevention and control of influenza virus infection. Existing research on Bacillus cereus phages has mainly focused on the control of Bacillus cereus contamination in the food industry, and there is no relevant report on its application in animal disease prevention and control, especially in relation to influenza virus infection. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a Bacillus cereus bacteriophage that can be used to prevent and / or treat influenza virus infection.

[0005] To achieve the above-mentioned purpose of the application, the present application provides the following technical solutions.

[0006] The present application provides a Bacillus cereus bacteriophage, which is Bacillus cereus bacteriophage P315, has a preservation number of CCTCC NO:M 20251545, and was preserved in the China Center for Type Culture Collection on July 7, 2025.

[0007] The application also provides application of the above-mentioned bacteriophage of Bacillus cereus in preparation of a product for preventing and treating influenza virus infection.

[0008] The application also provides application of the above-mentioned bacteriophage of Bacillus cereus in preparation of a product for preventing and treating lung inflammation caused by influenza virus infection.

[0009] The application also provides application of the above-mentioned bacteriophage of Bacillus cereus in preparation of a product for preventing and treating lung tissue damage caused by influenza virus infection.

[0010] The application also provides application of the above-mentioned bacteriophage of Bacillus cereus in preparation of a product for preventing and treating lung tissue damage caused by influenza virus infection.

[0011] The application also provides application of the above-mentioned bacteriophage of Bacillus cereus in preparation of a product for preventing and treating lung tissue damage caused by influenza virus infection.

[0012] Preferably, the influenza virus comprises an H1N1 subtype.

[0013] Preferably, the product comprises a medicine.

[0014] The application also provides a composition for preventing and treating influenza virus infection, wherein the composition comprises the above-mentioned bacteriophage of Bacillus cereus.

[0015] Preferably, the preparation type of the composition comprises a nasal preparation and an inhalation preparation.

[0016] The application has the following beneficial effects:

[0017] The bacteriophage provided by the application can specifically lyse Bacillus cereus, significantly reduce the colonization amount of Bacillus cereus in the respiratory tract, and effectively reduce lung inflammation caused by influenza virus infection, reduce lung tissue damage, and relieve infection symptoms, and will not cause significant interference to the normal flora of the host.

[0018] The technical scheme of the application provides a new direction for the development and utilization of bacteriophages in the field of antiviral agents. As a natural antibacterial agent, the bacteriophage has the advantages of precise targeting, self-replication, low toxicity, etc. compared with traditional antibiotics, and has a broad application prospect in the prevention and treatment of influenza virus infection.

[0019] The technical scheme of the application makes up for the technical defects of the prior art in preventing influenza virus infection, such as poor vaccine effect caused by virus variation, drug resistance, etc., and provides a new and effective solution for preventing influenza virus infection.

[0020] Depositary

[0021] The classification name of the bacteriophage is Bacillus cereus bacteriophage P315, the preservation unit is China Center for Type Culture Collection, the address is China, Wuhan, Wuhan University, the preservation number is CCTCC NO: M 20251545, and the preservation date is July 7, 2025. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is a plaque map of the bacteriophage.

[0023] Figure 2 It is an electron microscope map of the bacteriophage P315.

[0024] Figure 3 It is a titer detection result of the bacteriophage P315, wherein 1-7 respectively represent gradient dilution multiples 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 and 10 -7 .

[0025] Figure 4 It is a mouse weight change curve, * indicates P<0.05, and ** indicates P<0.01.

[0026] Figure 5 It is a mouse symptom severity, wherein A is a fur wrinkle condition, B is an appetite decrease condition, * indicates P<0.05, and ** indicates P<0.01.

[0027] Figure 6 It is a mouse lung tissue pathology map (the scale is 200 μm), A is a CON group, B is an H1N1 group, C is a B.C group, and D is a B.C+H1N1+P315 group.

[0028] Figure 7 It is the immune cell number of each group of mice, A is a lung T cell, B is a lung B cell, C is a lung macrophage, and D is a bronchoalveolar lavage fluid neutrophil.

[0029] Figure 8 It is the lung immune cell percentage of mice, A is a lung T cell, B is a lung B cell, C is a lung macrophage, and D is a bronchoalveolar lavage fluid neutrophil, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and **** indicates P<0.0001.

[0030] Figure 9 It is a mouse lung flora composition map, A is a door level analysis, and B is an attribute level analysis.

[0031] Figure 10 The percentage of B. cereus in the lung flora, and ** indicates P<0.01. DETAILED DESCRIPTION

[0032] The present application provides a B. cereus bacteriophage, which is B. cereus bacteriophage P315, the preservation number is CCTCC NO: M 20251545, and the preservation date is July 7, 2025 in the China Center for Type Culture Collection.

[0033] The B. cereus bacteriophage P315 of the present application belongs to the Caudovirales family of the Siphoviridae family, the head diameter is 60.72±1.08 nm, the tail length is 160.51±1.21 nm, and the titer can reach 10 8 PFU / mL or so.

[0034] The present application also provides the application of the above-mentioned B. cereus bacteriophage in the preparation of products for preventing and treating influenza virus infection, lung inflammation caused by influenza virus infection or lung tissue damage caused by influenza virus infection.

[0035] In the present application, the influenza virus preferably includes the H1N1 subtype. In the present application, the prevention and treatment refer to prevention and / or treatment. The bacteriophage of the present application specifically lyses B. cereus, reduces its colonization in the respiratory tract, and thus prevents influenza virus infection. In the present application, the preventive effect is reflected in reducing the lung inflammatory response caused by influenza virus infection, alleviating lung tissue damage and relieving infection symptoms. In the present application, the product preferably includes a drug. The core advantage of the bacteriophage used in the present application for targeted treatment of influenza virus lies in its high specificity, efficient penetration, immune synergy and clinical application flexibility. By precisely lysing B. cereus without destroying the symbiotic flora, the bacteriophage breaks through the biofilm barrier to achieve deep sterilization, restores macrophage function and reduces inflammation to synergize against viruses; its non-residual toxicity, multi-pathway drug delivery characteristics and low-cost advantage provide a safer and sustainable solution for influenza virus and bacterial co-infection than traditional antibiotics, especially for the prevention and control of drug-resistant strains. Mechanism verification based on a mouse model shows that the bacteriophage therapy of the present application plays a role through “flora-immune-metabolism” multidimensional regulation. Future development of pig-specific bacteriophage combinations can further promote industrial application. The object of the application is preferably a mammal, and the mammal is preferably a mouse or a pig.

[0036] The present application also provides the application of a B. cereus inactivator in the preparation of products for preventing and treating influenza virus infection.

[0037] The present application first proposes that Bacillus cereus can significantly increase the viral load after influenza virus infection. In the experimental mouse model, Bacillus cereus can be planted in the lung by nasal inoculation, destroy the function of alveolar barrier, and aggravate the inflammatory response (such as the increase of IL-6 and TNF-α levels). When co-infected with influenza virus, Bacillus cereus can promote virus replication by inhibiting the type I interferon signaling pathway, leading to aggravated lung tissue damage (such as bleeding and neutrophil infiltration). Under the conditions of animal immunosuppression (such as co-infection of circovirus) or antibiotic abuse, Bacillus cereus may become an opportunistic pathogen, and the hemolysin and phospholipase secreted by Bacillus cereus may synergize with porcine respiratory tract intrinsic pathogens (such as Haemophilus parasuis) to aggravate pneumonia lesions. In the present application, the Bacillus cereus inactivator preferably includes the above-mentioned Bacillus cereus bacteriophage.

[0038] The present application also provides the use of the above-mentioned Bacillus cereus bacteriophage in the preparation of a Bacillus cereus lysis product.

[0039] Bacillus cereus is a common foodborne pathogen (can produce emetic toxin and diarrheal toxin), and the bacteriophage provided by the present application can be used to inhibit the propagation of Bacillus cereus in food. Bacillus cereus can cause opportunistic infections (such as septicemia and meningitis) in immunocompromised people, and when the bacteria develop resistance to antibiotics, the Bacillus cereus bacteriophage provided by the present application can be used as an alternative. Some Bacillus cereus strains can act as plant pathogens (such as causing crop wilt), and the bacteriophage of the present application can be used for field spraying to inhibit the propagation of the pathogen and reduce the use of pesticides. Adding the bacteriophage of the present application to feed or drinking water can control the overproliferation of Bacillus cereus in the intestinal tract of livestock and poultry, reduce the risk of diarrhea and other diseases, and replace antibiotics for growth promotion.

[0040] The present application also provides a composition for preventing and treating influenza virus infection, which comprises the above-mentioned Bacillus cereus bacteriophage.

[0041] In the present application, the formulation type of the composition preferably includes nasal and inhalation formulations, which preferably include nasal sprays, aerosol inhalation formulations or nose drops. The composition of the present application preferably further includes pharmaceutical excipients. The present application is not particularly limited to the specific types of pharmaceutical excipients. In a mouse model, the composition (bacteriophage preparation) provided by the present application significantly reduces the viral load after C. difficile infection (P<0.05), improves the lung inflammatory response (P<0.01), and regulates the level of immune cells (macrophages increase and neutrophils decrease) by reducing the colonization of C. difficile. Experimental data show that the composition (preparation) of the present application can be safely delivered to the respiratory tract without significant disturbance to the host flora. Based on the verification results of the mouse experiment, the present application further proposes to apply the bacteriophage preparation to C. difficile infection prevention, to enhance the host's resistance to C. difficile by targeted elimination of C. difficile, and to reduce the economic losses caused by C. difficile in the livestock industry. The present application provides an innovative solution for the precise prevention and control of respiratory infections in livestock.

[0042] The technical solutions provided by the present application will be described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0043] In the following examples, unless otherwise specified, the methods are conventional methods.

[0044] In the following examples, unless otherwise specified, the materials, reagents, etc. used can be obtained from commercial channels.

[0045] Example 1

[0046] Isolation and purification of bacteriophages

[0047] 200 mL of water sample was collected from a lake near Qiqihar University, 1.2 g of CaCl2 solid was added and dissolved thoroughly, and after standing for 30 minutes, the supernatant was obtained by centrifugation at 4000 rpm for 15 minutes. 15 mL of sewage supernatant, 1.5 mL of C. difficile HBL-AI bacterial solution cultured to the logarithmic growth phase, and 15 mL of LB medium were placed in a 50 mL conical flask, and cultured at 37°C, 160 rpm overnight. The culture suspension was centrifuged at 4000 rpm for 10 minutes, and the supernatant was filtered with a 0.45 μm filter. 100 μL of the filtrate was added to a semi-solid culture medium plate containing C. difficile HBL-AI, and after incubation at 37°C overnight, round transparent plaques without halos were observed, as shown in Figure 1

[0048] ​A single phage plaque was selected, and 5 mL of SM buffer was added. The plaque was incubated at 37°C with shaking for 5 hours, followed by centrifugation at 8000 rpm and 4°C for 8 minutes. The supernatant was filtered through a 0.22 μm filter. The filtrate was serially diluted, and the semi-solid plate addition process was repeated 5-7 times to obtain purified phage P315. Electron microscopy observation showed (see...) Figure 2 Phage P315 has a head diameter of 60.72±1.08 nm and a tail length of 160.51±1.21 nm, belonging to the order Tailed Phages and the family Longtail Phagesidae.

[0049] The purified phage was serially diluted (10⁻⁶ ppm). -1 10 -2 10 -3 10 -4 10 -5 10 -6 and 10 -7 Afterwards, it was inoculated into Bacillus cereus HBL-AI culture medium in the logarithmic growth phase, and amplified by shaking culture at 37℃. Its titer was determined using the double-layer plate method, and the results are as follows: Figure 3 As shown, the titer of bacteriophage P315 is 1×10⁻⁶. 8 PFU / mL.

[0050] Example 2

[0051] The impact of phage-modified lung microbiota on IAV infection

[0052] Twenty-four 4-week-old male BALB / c rats were placed in a standard animal housing environment for acclimatization (temperature 20–25°C, humidity 50%–60%, 12-hour light / dark cycle) for 7 days. They were then randomly divided into four groups (n=6 per group): blank control group (CON group), IAV model group (H1N1 group), Bacillus cereus HBL-AI-IAV group (B.C+H1N1 group, also labeled as BC group), and Bacillus cereus HBL-AI-IAV-phage P315 group (B.C+H1N1+P315 group, also labeled as DA group or P group).

[0053] Use 10 6 A colonization model was established in 5-week-old BALB / c mice by intranasal drops of CFU-Bacillus cereus HBL-AI solution (bacterial solution prepared with sterile PBS). The IAV model group used PBS instead of the bacterial solution. After 5 days, 100 μL of PBS was added to the solution. 6 TCID 50 / 0.1mL dose nasal drops for H1N1 virus infection, the bacteriophage obtained in Example 1 was used at 1×10 8Pfu / kg dose was administered once daily via intranasal route during the establishment of the B. cereus HBL-AI colonization H1N1 infection model (1d-5d), and the B. cereus HBL-AI-IAV group was replaced with TM buffer. During this period, the body weight, fur wrinkle, and appetite decrease of each group of mice were recorded. The mice were euthanized on the 7th day after infection, and the bronchoalveolar lavage fluid was collected for microbiome analysis, and the lung tissue was collected for metabolomics analysis, HE staining, and immune cell detection.

[0054] The results show that the body weight loss of the B.C group (also referred to as the B.C+H1N1 group) was significantly higher than that of the single SIV infected mice (referred to as the H1N1 group) (see Figure 4 ), and the fur wrinkle and appetite decrease were more severe (see Figure 5 ), indicating that the lung pathogenic load was significantly increased. It was observed through HE staining (see Figure 6 ) that a large number of inflammatory cells gathered in the alveolar region of the B.C group mice, further proving that B. cereus HBL-AI significantly aggravated the IAV infection symptoms. It can be seen that B. cereus HBL-AI may exacerbate the IAV infection driven lung pathological damage by promoting the overactivation of the immune system.

[0055] In view of the core role of immune cells in IAV infection, the changes in the number of different types of immune cells in the lung tissue were further detected by flow cytometry, and the results are shown in Figure 7 and Figure 8 It can be seen that the phage modified lung flora can alleviate the aggravation of IAV infection caused by B. cereus HBL-AI colonization and relieve lung pathological changes.

[0056] Through microbiome analysis of the bronchoalveolar lavage fluid, it was confirmed that the phage P315 can effectively target and remove B. cereus HBL-AI in the lung, and has no effect on other genera (see Figure 9 and Figure 10 ). As can be seen from Figure 8 , B. cereus HBL-AI colonization (B.C group) caused a significant decrease in the number of macrophages in the lungs of mice, and phage treatment (P group) can restore the number of macrophages by removing B. cereus HBL-AI, and further restore its immune function to resist the deterioration of IAV infection.

[0057] In combination with the above results, it can be seen that the increase in the colonization amount of B. cereus HBL-AI may weaken the lung immune defense by directly or indirectly reducing the number of macrophages, thereby exacerbating the symptoms of IAV infection. The phage P315 modified lung flora of mice can target to reduce the content of B. cereus HBL-AI in the lung flora of mice, restore the immune function mediated by lung macrophages, and reverse the deterioration process of IAV infection mediated by B. cereus HBL-AI.

[0058] The above merely preferred embodiments of the present application, it should be noted that for those of ordinary skill in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered within the scope of the present application.

Claims

1. A Bacillus cereus bacteriophage, characterized in that, The Bacillus cereus phage mentioned is Bacillus cereus phage P315, with accession number CCTCC NO:M 20251545, deposited at the China Center for Type Culture Collection on July 7, 2025.

2. The use of the Bacillus cereus phage according to claim 1 in the preparation of products for preventing and treating influenza virus infection.

3. The use of the Bacillus cereus phage according to claim 1 in the preparation of products for preventing and treating lung inflammation caused by influenza virus infection.

4. The use of the Bacillus cereus phage according to claim 1 in the preparation of products for preventing and treating lung tissue damage caused by influenza virus infection.

5. Application of Bacillus cereus inactivating agent in the preparation of products for the prevention and treatment of influenza virus infection.

6. The use of the Bacillus cereus phage according to claim 1 in the preparation of lysed Bacillus cereus products.

7. The application according to any one of claims 2 to 5, characterized in that, The influenza virus includes the H1N1 subtype.

8. The application according to claims 2 to 6, characterized in that, The products include pharmaceuticals.

9. A composition for preventing and treating swine influenza virus infection, characterized in that, The composition comprises the Bacillus cereus phage as described in claim 1.

10. The composition according to claim 9, characterized in that, The formulations of the composition include nasal and inhaled formulations.

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