A bacteriophage and uses thereof

Abstract

The application belongs to the technical field of bacteriophages, and provides a bacteriophage, named Microbacterium oxidans bacteriophage MO526, which is preserved in the China General Microbiological Culture Collection Center on May 8, 2023, and the preservation number is CGMCC NO.45566. The application also provides application of the bacteriophage in preparation of a medicine for treating diseases caused by Microbacterium oxidans. The application also provides application of the bacteriophage in preparation of a preparation for killing Microbacterium oxidans. The application also provides an environmental disinfectant, wherein the effective component of the environmental disinfectant comprises the bacteriophage. The bacteriophage MO526 provided by the application can be obtained from nature, has excellent killing effect on Microbacterium oxidans, can be used as an effective component in corresponding medicines or disinfectants, is easy to be industrially produced, can reduce cost, and has the advantages of green environmental protection.

Description

Technical Field

[0001] This invention belongs to the field of bacteriophage technology, and particularly relates to a bacteriophage and its application. Background Technology

[0002] Bacteriophages are viruses that can kill bacteria in their target host. They can inject genetic material into bacteria and replicate within them. Compared with antibiotics, bacteriophages have many advantages, such as not disrupting the host's normal microbial flora, having strong host specificity, replicating only at the site of infection and having no serious side effects. Moreover, they can be used in synergistic therapy with antibiotics to achieve better therapeutic effects, making them an ideal antibiotic alternative.

[0003] Microbacterium oxysporum is a common environmental contaminant, belonging to the Gram-positive bacillus family. This strain is round with a smooth, short rod-like shape. Microbacterium oxysporum can be left in central venous catheters in patients, causing varying degrees of systemic infection symptoms. It is also a major cause of microbial corrosion of metal pipes, forming biofilms on the inner walls and interacting with the metal to accelerate corrosion. Summary of the Invention

[0004] The purpose of this invention is to provide a bacteriophage that addresses the problems mentioned in the background section.

[0005] The present invention is implemented as follows: a bacteriophage named Microbacillus oxytocinoides phage MO526 was deposited on May 8, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.45566.

[0006] Another objective of this invention is to provide the application of the above-mentioned bacteriophage in the preparation of a drug for treating diseases caused by Microbacterium oxysporum.

[0007] Another objective of this invention is to provide an application of the above-mentioned bacteriophage in the preparation of a formulation for eliminating oxidizing microbes.

[0008] Another objective of this invention is to provide an environmental disinfectant, wherein the effective component of the environmental disinfectant includes the aforementioned bacteriophage.

[0009] Preferably, the titer of the bacteriophage is at least 1 × 10⁻⁶. 8 pfu / mL.

[0010] Preferably, the environmental disinfectant also includes excipients.

[0011] Preferably, the environmental disinfectant is a liquid preparation.

[0012] The bacteriophage MO526 provided in this invention can be obtained from nature and has excellent disinfection effect on oxidizing microbes. It can be used as an effective ingredient in corresponding drugs or disinfectants, and is easy to carry out industrial production. It can not only reduce costs, but also has the advantages of being green and environmentally friendly. Attached Figure Description

[0013] Figure 1 Image of phage MO526 provided in Embodiment 1 of the present invention;

[0014] Figure 2 This is a transmission electron microscope image of bacteriophage MO526 provided in Example 1 of the present invention;

[0015] Figure 3 Another transmission electron microscope image of phage MO526 provided in Embodiment 1 of the present invention;

[0016] Figure 4 This refers to the surgical instrument with a positive surface for *Microbacterium oxidans* provided in Embodiment 2 of the present invention;

[0017] Figure 5 Colonies were collected and cultured from the surgical instruments after disinfection in the experimental group provided in Example 2 of the present invention.

[0018] Figure 6 Colonies were collected and cultured from surgical instruments after disinfection, which served as the control group in Example 2 of this invention. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0020] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0021] Example 1

[0022] Isolation and identification of bacteriophage MO526:

[0023] The host bacteria MO isolated from clinical samples were inoculated on solid Columbia agar plates using the three-zone streak method and cultured overnight at 37°C. After 48 hours, single colonies on the solid plates were observed, picked and inoculated into test tubes containing 5 mL of BHI liquid medium, and cultured at 37°C in a shaker until the bacterial solution became turbid (approximately 24 hours).

[0024] Wastewater from the northern wastewater treatment plant in Changchun City was selected for separation. 50 mL of wastewater sample was centrifuged (4℃, 10000 r / min, 10 min) to separate bacteria and other impurities. The supernatant was filtered through a 0.22 μm microporous membrane for sterilization. The filtrate was collected in a sterile Erlenmeyer flask. 100 mL of liquid BHI medium and 5 mL of host bacterial culture were added to the wastewater sample. After mixing, the mixture was incubated overnight at 37℃. The next day, the sample was filtered for sterilization. 0.3 mL of the filtrate was placed in a glass test tube, and 0.3 mL of host bacterial culture was added. The mixture was mixed and placed at room temperature for 15 min to allow the bacteriophages to fully adsorb onto the host bacteria. Then, 3 mL of semi-solid medium was added. The mixture was shaken with a micro-shaker and immediately poured onto a solid LB agar plate to prepare a double-layer agar plate. The plate was incubated at 37℃. Plaques were observed the next day. If plaques were observed, it indicated that the bacteriophages had been successfully screened.

[0025] The largest single plaque with smooth edges was selected from the obtained plaques and cultured in 3 mL of MO bacterial solution (37℃, 200 r / min) for 6 h. After centrifugation (10000 r / min, 30 min), the supernatant was filtered through a 0.22 μm filter membrane for sterilization. The plating method was repeated 5 times to obtain purified phage.

[0026] Observe the bacteriophage, such as Figure 1 As shown, it can form clear, empty plaques with clear and regular edges in agar medium, with a diameter of about 1.5 mm, which is typical of lytic bacteriophages;

[0027] Using the phosphotungstic acid negative staining method, a drop of purified high-titer phage solution was placed on a copper grid and absorbed for 10 minutes. The residual suspension was then aspirated, and the grid was stained with 2% phosphotungstic acid (PTA, 2% w / v) for 2 minutes. The copper grid was then dried using an infrared lamp, and the results were observed under a transmission electron microscope. Figure 2 and 3 As shown, transmission electron microscopy revealed that this bacteriophage belongs to the order Caudovirales and the family Longtailed Phages.

[0028] It was sent to the China General Microbiological Culture Collection Center for preservation, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and classified as Microbacterium oxysporum MO526, with the accession number CGMCCNO.45566.

[0029] Example 2

[0030] Experiment on the elimination of oxidizing microbes by bacteriophage MO526:

[0031] Experimental subjects: Numerous oxidizing bacteria were found on the surface of surgical forceps that were not promptly sterilized after use. Four identical surgical instruments with positive oxidizing bacteria surfaces were collected, and samples were taken from four locations prone to oxidizing bacteria deposition before sterilization (e.g., ...). Figure 4 (As shown), place in a petri dish;

[0032] Experimental group: Bacteriophage disinfectant, prepared as follows: Bacteriophage fluid and host bacteria were mixed 1:1 in BHI broth medium and incubated at 37℃ and 150 rpm for 12–24 h until the liquid was clear and bacterial fragments were visible. The clear liquid was filtered to remove bacterial fragments and residues. The bacteriophage fluid was then diluted 100 times to obtain a titer of 1×10⁻⁶. 8 The pfu / mL phage disinfectant solution, sealed and stored at 2-8℃, is ready for use.

[0033] Control group: 75% alcohol;

[0034] Experimental procedure: Two surgical instruments were disinfected using the experimental group, while the other two surgical instruments were disinfected using the control group. After 30 minutes of disinfection, samples were taken from the same location on the surgical instruments in both the experimental and control groups for culture.

[0035] The sampling petri dishes before and after disinfection were placed in a 37°C incubator and incubated for 36–48 hours. The experimental group's disinfected petri dishes were as follows: Figure 5 As shown, the sterilized petri dishes of the control group are as follows: Figure 6 As shown, the bacterial cultures were counted, and the results (average values) are shown in Table 1:

[0036] Table 1

[0037] Disinfection area Number of tests bacterial extinction rate in the control group Bacterial mortality rate in the experimental group 1 10 80％ 100％ 2 10 70％ 100％ 3 10 70％ 100％ 4 10 90％ 90％ total 40 77.5％ 97.5％

[0038] according to Figure 5 , Figure 6 As shown in Table 1, after disinfection with 75% alcohol, the bacterial elimination rate on the surface of surgical instruments was 77.5%, while after disinfection with phage MO526 disinfectant, the bacterial elimination rate on the surface of surgical instruments reached 97.5%. It can be seen that the disinfection effect of phage MO526 is significantly better than that of conventionally used 75% alcohol, and it is also safer to use. Phage MO526 has excellent disinfection effect against microorganisms and can be promoted and applied as a new type of biological disinfectant.

[0039] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A bacteriophage, characterized in that, The bacteriophage was named Microbacterium oxysporum phage MO526, and it was deposited on May 8, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.45566.

2. The use of the bacteriophage as described in claim 1 in the preparation of a medicament for treating diseases caused by Microbacterium oxysporum.

3. The use of the bacteriophage as described in claim 1 in the preparation of an agent for disinfecting oxidizing microbes.

4. An environmental disinfectant, characterized in that, The active ingredient of the environmental disinfectant includes the bacteriophage as described in claim 1.

5. The environmental disinfectant according to claim 4, characterized in that, The titer of the bacteriophage is at least 1 × 10⁻⁶. 8 pfu / mL.

6. The environmental disinfectant according to claim 4, characterized in that, The environmental disinfectant also includes excipients.

7. The environmental disinfectant according to claim 4, characterized in that, The environmental disinfectant is a liquid preparation.

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