Pseudomonas aeruginosa single-stranded RNA bacteriophage and application thereof

Abstract

The application discloses a Pseudomonas aeruginosa single-stranded RNA bacteriophage and application thereof, and belongs to the technical field of bioengineering, wherein the Pseudomonas aeruginosa single-stranded RNA bacteriophage is named Pseudomonas aeruginosa phage Ppa61, is preserved in the China Center for Type Culture Collection, and has a preservation number of CCTCC No: M 20231334. The bacteriophage has strong bactericidal activity and a wide host spectrum on Pseudomonas aeruginosa, is specific to Pseudomonas aeruginosa, cannot kill other bacteria, and can be used alone or in combination with other substances, thereby providing a safe and non-toxic bacteriophage killing product for in-vivo and in-vitro Pseudomonas aeruginosa infection treatment, Pseudomonas aeruginosa disinfection and purification in aquaculture and other environments.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a single-stranded RNA bacteriophage of Pseudomonas aeruginosa and its applications. Background Technology

[0002] Pseudomonas aeruginosa is an opportunistic pathogen that can cause a variety of infections, including ventilator-associated pneumonia, burn and wound infections, sepsis, and chronic lung infections in patients with cystic fibrosis.

[0003] Antibiotics are the first-line treatment for Pseudomonas aeruginosa infections. However, due to the high intrinsic and acquired resistance of Pseudomonas aeruginosa to many antibiotics, as well as antibiotic overuse, multidrug-resistant Pseudomonas aeruginosa have emerged. The World Health Organization (WHO) has listed carbapenem-resistant Pseudomonas aeruginosa as a key pathogen requiring new treatment strategies. To address this threat, there is an urgent need to develop new antimicrobial strategies against drug-resistant Pseudomonas aeruginosa.

[0004] Among numerous novel antibacterial drugs, bacteriophages hold promise as an alternative to antibiotics. Bacteriophages are viruses that use bacteria as hosts and are widely distributed in nature. Virulent bacteriophages can infect and lyse host bacteria, and are released into the environment after the host bacteria die, continuing to infect susceptible bacteria in the surrounding environment. Compared with antibiotics and chemical antibacterial drugs, bacteriophages have advantages such as host specificity, high safety, and no residual effects. Furthermore, they can rapidly kill specific pathogens without affecting the body's normal flora, and also have good killing effects on drug-resistant strains. Therefore, developing novel bacteriophages with preventive and therapeutic effects against multidrug-resistant Pseudomonas aeruginosa is of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide a single-stranded RNA phage of Pseudomonas aeruginosa to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] A single-stranded RNA phage of Pseudomonas aeruginosa, named Pseudomonas aeruginosa phage Ppa61, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number M20231334.

[0008] Another objective of this invention is to provide the use of the above-mentioned Pseudomonas aeruginosa single-stranded RNA phage as an active ingredient in the preparation of a drug for the prevention and / or treatment of infectious diseases caused by Pseudomonas aeruginosa.

[0009] Another objective of this invention is to provide the application of the above-mentioned Pseudomonas aeruginosa single-stranded RNA phage as an active ingredient in the preparation of compositions for killing Pseudomonas aeruginosa on and / or inside livestock and poultry.

[0010] Another objective of this invention is to provide an application of the above-mentioned Pseudomonas aeruginosa single-stranded RNA phage as an active ingredient in killing Pseudomonas aeruginosa in the space environment.

[0011] Preferably, the spatial environment includes at least an animal breeding environment and / or a medical environment.

[0012] Preferably, the Pseudomonas aeruginosa single-stranded RNA phage is used as a component of the spraying agent.

[0013] Another object of the present invention is to provide a composition for specifically killing Pseudomonas aeruginosa, comprising the above-mentioned Pseudomonas aeruginosa single-stranded RNA bacteriophage as an active ingredient.

[0014] This invention provides a novel single-stranded RNA bacteriophage for Pseudomonas aeruginosa. This bacteriophage exhibits strong bactericidal activity and a broad host spectrum against Pseudomonas aeruginosa, while also being specific to Pseudomonas aeruginosa without harming other bacteria. It can be used alone or in combination with other substances, providing a safe and non-toxic bacteriophage disinfection product for the treatment of Pseudomonas aeruginosa infections in vivo and in vitro, as well as for the disinfection and purification of Pseudomonas aeruginosa in aquaculture and other environments. Attached Figure Description

[0015] Figure 1 This is a plaque image of Pseudomonas aeruginosa phage Ppa61 provided in an embodiment of the present invention;

[0016] Figure 2 This is a morphological observation image of Pseudomonas aeruginosa bacteriophage Ppa61 provided in an embodiment of the present invention;

[0017] Figure 3 The MOI determination results of Pseudomonas aeruginosa bacteriophage Ppa61 provided in an embodiment of the present invention are shown in the figure.

[0018] Figure 4 The figure shows the one-step growth curve determination results of Pseudomonas aeruginosa phage Ppa61 provided in the embodiments of the present invention. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] In one embodiment of the present invention, a single-stranded RNA phage of *Pseudomonas aeruginosa* is provided, named *Pseudomonas aeruginosa phage* Ppa61, which was deposited at the China Center for Type Culture Collection (CCTCC) on July 19, 2023, with accession number CCTCC No: M 20231334. This phage exhibits strong bactericidal activity against *Pseudomonas aeruginosa* and a broad host spectrum.

[0021] Bacteriophages are bacterial-specific viruses capable of infecting specific bacteria and inhibiting their growth. They are viruses that contain single-stranded or double-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) as their genetic material. The bacteriophage provided in this embodiment of the invention is a novel single-stranded RNA bacteriophage isolated from sewage. This bacteriophage is icosahedral and can form clear plaques on LB agar medium.

[0022] In another embodiment of the present invention, the above-mentioned Pseudomonas aeruginosa single-stranded RNA phage is also provided as an active ingredient in the preparation of a drug for the prevention and / or treatment of infectious diseases caused by Pseudomonas aeruginosa.

[0023] In another embodiment of the present invention, the above-mentioned Pseudomonas aeruginosa single-stranded RNA phage is also provided as an active ingredient in the preparation of a composition for killing Pseudomonas aeruginosa on and / or inside livestock and poultry.

[0024] In another embodiment of the present invention, the above-mentioned Pseudomonas aeruginosa single-stranded RNA phage is also provided as an active ingredient for killing Pseudomonas aeruginosa in the space environment.

[0025] In a preferred embodiment of the present invention, the spatial environment includes at least an animal husbandry environment and / or a medical environment. Specifically, the animal husbandry environment includes, but is not limited to, the ground, walls, feces, bedding, feed, water, and feeding troughs and other animal husbandry equipment; the medical environment includes, but is not limited to, wards and treatment rooms.

[0026] In a preferred embodiment of the present invention, the Pseudomonas aeruginosa single-stranded RNA phage is used as a component of the spraying agent.

[0027] Another object of the present invention is to provide a composition for specifically killing Pseudomonas aeruginosa, comprising the above-mentioned Pseudomonas aeruginosa single-stranded RNA bacteriophage as an active ingredient.

[0028] In the above applications, Pseudomonas aeruginosa phage Ppa61 can be used alone or in combination with other substances to provide a safe and non-toxic phage disinfection product for the treatment of Pseudomonas aeruginosa infections in vivo and in vitro, as well as for the disinfection and purification of Pseudomonas aeruginosa in aquaculture and other environments.

[0029] The following embodiments are examples of practical applications of the technical solution of the present invention, but are not limited thereto.

[0030] Example 1: This example provides a method for isolating single-stranded RNA bacteriophages from Pseudomonas aeruginosa, specifically including the following steps:

[0031] S1. The wastewater sample used in this embodiment of the invention was collected from Changchun Park in Changchun City. The host bacterium was Pseudomonas aeruginosa BPA61, which was donated by the Changchun Special Products Research Institute of the Chinese Academy of Agricultural Sciences. Wastewater was collected, filtered with gauze, and the supernatant was taken. LB medium (100 mL) was prepared using treated wastewater instead of ddH2O. 1 mL of overnight cultured host bacteria BPA61 was added to the medium and cultured at 37℃ for 10-12 h. 1 mL of the culture was taken, centrifuged at 12000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm filter and stored. The obtained filtrate was used for plaque testing to check whether it contained bacteriophages capable of lysing Pseudomonas aeruginosa BPA61.

[0032] S2. Plaque test: Inoculate 2% of *Pseudomonas aeruginosa* BPA61 into 5 mL of LB broth and incubate at 37°C with shaking for 12 h. Place 100 μL of the prepared bacterial culture solution in the center of a plate and spread it evenly using a spreader. After drying, place 10 μL of the filtrate in the center of the plate. After air drying, invert the plate and incubate at 37°C for 10 h. Then observe whether plaques form in the area where the filtrate was added. If a transparent area appears in the area where the filtrate was added, it can be determined that the filtrate contains bacteriophages capable of killing *Pseudomonas aeruginosa* BPA61.

[0033] S3. Take the above-obtained filtrate and serially dilute it with sterile PBS. Take 100 μL of the diluted filtrate and mix it thoroughly with 100 μL of host bacteria cultured overnight. Incubate at room temperature for about 5 min. Then add it to 7 mL of LB semi-solid medium at 45℃. After mixing, quickly pour it onto 1.5% LB agar medium to make a double-layer plate. After solidification, place it in a 37℃ incubator for 16-20 h and observe the growth of phage plaques.

[0034] Example 2: This example provides a method for amplifying and purifying single-stranded RNA phages of Pseudomonas aeruginosa, specifically including the following steps:

[0035] S1. On the double-layer plate where plaques have formed, use a sterile pipette tip to pick up a single plaque that is relatively large in diameter, round, and clear. Inoculate it into 5 mL of LB liquid medium, add 200 μL of phage host culture, mix well, and incubate at 37°C until the liquid gradually becomes clear. Centrifuge at 12000 r / min, 4°C for 10 min, and collect the supernatant. Repeat the double-layer plate experiment, picking up single plaques 4-5 times in this way for purification.

[0036] S2. Take 1 mL of freshly cultured host bacteria, add 300 μL of phage lysis buffer (at a ratio of 1:1, 1:10, and 1:100 for single phage culture and host bacteria, respectively), and incubate at 37°C for 20 min to allow phage particles to adsorb onto the host bacteria; add 800 mL of LB liquid medium, incubate at 37°C with shaking for 6-8 h, centrifuge at 12000 rpm at 4°C for 10 min, and collect the supernatant, which is the phage lysis buffer.

[0037] S3, PEG purification: Add RNase A and DNase I to the phage lysis buffer to a final concentration of 1 μg / mL and incubate at room temperature for 30 min; add NaCl to a final concentration of 1 mol / L, mix thoroughly, and incubate on ice for 1-2 h; centrifuge at 8000 r / min for 15-20 min at 4 °C and collect the supernatant; add 10 g of PEG8000 to every 100 mL of the mixed solution, stir gently to dissolve completely, and incubate on ice overnight to allow the phage to precipitate under the action of PEG8000; the next day, centrifuge at 12000 r / min for 10-20 min at 4 °C, recover the precipitated phage particles, add 2 mL of SM solution, wash the precipitate thoroughly, and incubate at room temperature for 1 h; add an equal volume of chloroform for extraction, and gently shake for 30 s; centrifuge at 5000 r / min for 10 min at 4 °C to separate the organic and hydrophilic phases, recover the hydrophilic phase containing phage particles, and obtain purified phage.

[0038] S4. Phage titer was determined using the double-layer agar plate method: The purified phage solution was serially diluted 10-fold. 100 μL of each of the corresponding phage dilutions was thoroughly mixed with 100 μL of the host bacterial solution. The mixture was then plated onto double-layer agar plates and incubated at 37°C for approximately 8 hours. Plaques were counted on each agar plate. Plates showing approximately 100-200 plaques were selected. The initial phage concentration calculated based on the dilution factor is the phage titer. The purified phage is shown below. Figure 1As shown. The purified phage described above is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC No. M 20231334, named Pseudomonas aeruginosa phage (Pseudomonas aeruginosa phag e) Ppa61, and deposited on July 19, 2023.

[0039] Example 3: The transmission electron microscopy observation of phage Ppa61 obtained in Example 2 above is as follows:

[0040] The bacteriophages purified in Example 2 were observed under an electron microscope. The specific procedure was as follows: 10 μL of sample was added to a copper grid and allowed to precipitate for 15 min. Excess liquid was absorbed with filter paper, and the sample was stained with 2% phosphotungstic acid (PTA) for 1-2 min. After drying, the sample was observed using a transmission electron microscope (Hitachi H-7650). The observation results are as follows: Figure 2 As shown, the head is icosahedral. According to the International Committee on Taxonomy of Viruses (ICTV) 2005 report, "Taxonomy of Viruses—Eighth Report of the International Committee on Taxonomy of Viruses," the aforementioned bacteriophage Ppa61 belongs to the family Le viviridae.

[0041] Example 4: The optimal MOI determination experiment for phage Ppa61 obtained in Example 2 above is as follows:

[0042] Adjust the bacterial culture that has reached the logarithmic growth phase to a concentration of 10. 7 CFU / mL, then phage Ppa61 was mixed with bacteria at phage / bacteria ratios of 0.001, 0.01, 0.1, 1, and 10, and transferred to LB broth. The mixture was incubated at 37°C with shaking for 8 hours. The culture was centrifuged at 10000g for 15 minutes at 4°C. The supernatant was filtered through a disposable 0.22μm filter to obtain the phage propagation solution. The titer of the propagation solution was determined using the double-layer plate method. The phage / bacteria ratio with the highest titer was the optimal MOI. The results are as follows: Figure 3 As shown.

[0043] Example 5: The one-step growth curve determination experiment of phage Ppa61 obtained in Example 2 above is as follows:

[0044] The host bacteria cultured to the logarithmic growth phase were mixed with bacteriophage Ppa61 at the optimal MOI ratio and incubated at 37°C for 10 min. After incubation, the mixture was centrifuged at 4°C (10000g, 10 min). The precipitate was resuspended in 10 mL of fresh LB liquid medium and incubated with shaking at 37°C. Samples were taken at 0 min, 5 min, 10 min, 15 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, and 120 min to determine the titer of bacteriophage Ppa61, thus plotting a one-step growth curve of bacteriophage Ppa61 infection of bacteria. The results are as follows: Figure 4 As shown.

[0045] Example 6: The host spectrum analysis experiment of phage Ppa61 obtained in Example 2 above is as follows:

[0046] The titer of phage Ppa61 obtained in Example 2 was adjusted to 10. 8 Pfu / mL was prepared for use. Fifty test strains were selected for the experiment, and the host spectrum of phage Ppa61 was analyzed. The specific procedures are as follows:

[0047] Plaque assay: 100 μL of overnight culture of each test strain was dropped into the center of a 1.5% LB agar plate and spread evenly using a spreader. 10 μL of bacteriophage Ppa61 was dropped onto the surface of the bacterial motif. After the droplet dried, the plate was inverted and incubated at 37℃ for 12-16 h. The results were observed; if plaques appeared, it was marked as "+", otherwise as "-". The results are shown in Table 1.

[0048] Table 1

[0049]

[0050] In Table 1, the superscript numbers indicate the following: 1 represents Pseudomonas aeruginosa; 2 represents Acinetobacter baumannii; 3 represents Klebsiella pneumoniae; and 4 represents Salmonella.

[0051] Table 1 shows that 50 test strains were selected to determine the host spectrum of bacteriophage Ppa61, including 16 strains of *Pseudomonas aeruginosa*, 10 strains of *Klebsiella pneumoniae*, 13 strains of *Acinetobacter baumannii*, and 11 strains of *Salmonella*. The results showed that in the plaque assay, bacteriophage Ppa61 only produced plaques on the plates of 11 tested *Pseudomonas aeruginosa* strains, with a lysis rate of 68.75% (11 / 16). This indicates that the bacteriophage has a broad lysis spectrum against *Pseudomonas aeruginosa*.

[0052] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. A single-stranded RNA phage of *Pseudomonas aeruginosa*, characterized in that, It was named Pseudomonas aeruginosa phage Ppa61 and is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC No: M 20231334.

2. The use of the Pseudomonas aeruginosa single-stranded RNA phage as described in claim 1 as an active ingredient in the preparation of a medicament for the prevention and / or treatment of infectious diseases caused by Pseudomonas aeruginosa.

3. The use of the Pseudomonas aeruginosa single-stranded RNA phage as described in claim 1 as an active ingredient in the preparation of compositions for killing Pseudomonas aeruginosa on and / or inside livestock and poultry.

4. The application of the Pseudomonas aeruginosa single-stranded RNA phage as described in claim 1 as an active ingredient in killing Pseudomonas aeruginosa in the space environment.

5. The application according to claim 4, characterized in that, The spatial environment includes at least an animal breeding environment and / or a medical environment.

6. The application according to claim 4, characterized in that, The Pseudomonas aeruginosa single-stranded RNA phage is used as a component of the spraying agent.

7. A composition for specifically killing Pseudomonas aeruginosa, characterized in that, It contains the Pseudomonas aeruginosa single-stranded RNA phage as described in claim 1 as an active ingredient.

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