Pseudomonas aeruginosa phage cocktail and application thereof
By developing a phage cocktail containing Pseudomonas aeruginosa phages PB17, PB57 and PB67, the drug resistance of drug-resistant Pseudomonas aeruginosa to traditional antibiotic therapy was solved, and the efficient and specific bactericidal effect was achieved, which significantly broadened the bactericidal spectrum.
Patent Information
- Application Number
- CN202411836261.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-13
- Publication Date
- 2025-05-09
AI Technical Summary
The prior art is difficult to effectively deal with the threat of drug-resistant Pseudomonas aeruginosa, and traditional antibiotic therapy has limited effects.
A Pseudomonas aeruginosa phage cocktail was developed, containing three different phages, PB17, PB57 and PB67. The combination of these phages was significantly improved in bactericidal efficiency of Pseudomonas aeruginosa.
The bactericidal spectrum of this phage cocktail on Pseudomonas aeruginosa was increased to 99.53%, significantly broadening the bactericidal range and being highly specific, avoiding the impact on other bacteria.
Smart Images
Figure CN119955740A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of bioengineering, and in particular to a Pseudomonas aeruginosa phage cocktail and an application thereof. Background Art
[0002] Pseudomonas aeruginosa is an important zoonotic pathogen that can cause acute or chronic infections in immunocompromised individuals with chronic obstructive pulmonary disease, cystic fibrosis, cancer, trauma, burns, sepsis, and ventilator-associated pneumonia. Due to the extensive use of antibiotics, P. aeruginosa has gradually become more resistant, especially to β-lactams, fluoroquinolones, and aminoglycosides. In 2024, the World Health Organization (WHO) changed carbapenem-resistant Pseudomonas aeruginosa (CRPA) from a critical priority to a high priority, indicating its serious threat to public health. To address this threat, there is an urgent need to develop new antimicrobial strategies against drug-resistant P. aeruginosa.
[0003] Phage therapy is one of the methods to replace antibiotics and solve the problem of antibiotic resistance. Phages are natural obligate parasitic viruses of bacteria that can infect and kill bacteria that are sensitive and resistant to antibiotics. Compared with traditional antibiotic therapy, phage therapy has the advantages of host specificity, high safety, and no residual effect. Virulent phages will lyse host bacteria after infection, and will be released into the outside world with the death of host bacteria, continuing to infect susceptible bacteria in the surrounding environment. Domestic and foreign studies have shown that phages have great potential in preventing and controlling bacterial infections. Phage therapy can be used as a single phage or a mixture of phages. Compared with a single phage, phage cocktails have more advantages, such as resisting bacterial mutations, broadening the host spectrum, and inhibiting the formation of biofilms. Therefore, the development of Pseudomonas aeruginosa phage cocktails is of great significance for the treatment of drug-resistant Pseudomonas aeruginosa. Summary of the invention
[0004] The purpose of the present invention is to solve the shortcomings of the prior art, and to propose a Pseudomonas aeruginosa phage cocktail and its application. The phage cocktail is composed of three different Pseudomonas aeruginosa phages, including Pseudomonas aeruginosa phage PB17 (deposit number CCTCC M 20242120), Pseudomonas aeruginosa phage PB57 (deposit number CCTCC M 20242119) and Pseudomonas aeruginosa phage PB67 (deposit number CCTCC M 20242121). The Pseudomonas aeruginosa phage cocktail has strong bactericidal activity. Compared with a single phage, the bactericidal spectrum of the phage cocktail against Pseudomonas aeruginosa is increased to 99.53% (210 / 211).
[0005] In order to achieve the above object, the present invention adopts the following technical solutions: A Pseudomonas aeruginosa phage cocktail, comprising three phages PB17, PB57 and PB67 isolated from nature and capable of specifically killing Pseudomonas aeruginosa, which were deposited in the China Center for Type Culture Collection on September 29, 2024, with the deposit numbers being CCTCC M 20242120, CCTCC M 20242119 and CCTCC M20242121, respectively.
[0006] Use of the above-mentioned Pseudomonas aeruginosa phage cocktail as an effective ingredient in the preparation of a drug for preventing and / or treating infectious diseases caused by Pseudomonas aeruginosa.
[0007] A phage cocktail preparation for killing Pseudomonas aeruginosa, comprising the above phage cocktail as an active ingredient.
[0008] A composition for preventing or treating infectious diseases caused by Pseudomonas aeruginosa, comprising the above-mentioned phage cocktail as an effective ingredient.
[0009] Preferably, the composition comprises one of a liquid preparation, a lyophilized preparation, and an oral solid preparation.
[0010] The above-mentioned Pseudomonas aeruginosa phage cocktail is used as an effective ingredient or directly used as a spray to kill Pseudomonas aeruginosa in a space environment; the space environment includes an animal breeding environment and a medical environment.
[0011] Preferably, the breeding environment includes breeding equipment such as the ground, walls, feces, bedding, feed, water and feed troughs.
[0012] Preferably, the medical environment is such as a ward, a treatment room, etc.
[0013] The above-mentioned Pseudomonas aeruginosa phage cocktail is used as an effective ingredient in the preparation of a drug for killing Pseudomonas aeruginosa on the surface of livestock and poultry and in the body of livestock and poultry.
[0014] The beneficial effects of the present invention are: 1. Highly efficient bactericidal activity: The phage cocktail exhibits a strong bactericidal effect against Pseudomonas aeruginosa, significantly broadening the bactericidal spectrum against Pseudomonas aeruginosa.
[0015] 2. Strong specificity: Phage cocktail is highly specific to Pseudomonas aeruginosa. While killing Pseudomonas aeruginosa, it will not affect other bacteria, thus avoiding the side effects that may be caused by broad-spectrum antibacterial preparations.
[0016] 3. Compound compatibility: It can be used in combination with other substances to further enhance application flexibility and effect.
[0017] 4. Safe and non-toxic: As a safe and non-toxic phage disinfection product, it can be widely used in the treatment of Pseudomonas aeruginosa infections in vivo and in vitro, pathogen control in aquaculture, and disinfection and purification of Pseudomonas aeruginosa in the environment. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 Plaques of phages PB17, PB57, and PB67; Figure 2 The morphological observations of bacteriophages PB17, PB57, and PB67 were performed; Figure 3 The temperature stability of phages PB17, PB57, and PB67; Figure 4 is the pH stability of bacteriophages PB17, PB57, and PB67; Figure 5 is the host spectrum of phages PB17, PB57, and PB67; Figure 6 It is the antibacterial activity of bacteriophages PB17, PB57 and PB67 and their mixture. DETAILED DESCRIPTION
[0019] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.
[0020] A Pseudomonas aeruginosa phage cocktail, comprising three phages PB17, PB57 and PB67 isolated from nature and capable of specifically killing Pseudomonas aeruginosa, which were deposited in the China Center for Type Culture Collection on September 29, 2024, with the deposit numbers CCTCC M 20242120, CCTCC M 20242119 and CCTCC M20242121, respectively; address: Wuhan University, Wuhan, China.
[0021] Example 1: Phage Isolation and Preparation The sewage samples in the present invention were collected from Changchun Park in Changchun, and the host bacteria were Pseudomonas aeruginosa BPA17, BPA57 and BPA67. The sewage was collected, filtered with gauze, the supernatant was taken, and the treated sewage was used instead of ddH2O to prepare LB medium (100 mL). 1 mL of overnight cultured host bacteria was added to the culture medium, and cultured at 37°C for 10-12 h. 1 mL of the culture was taken, centrifuged at 12,000 r / min for 5 min, the supernatant was filtered with a 0.22 μm filter and stored, and the obtained filtrate was used for plaque testing to check whether it included bacteriophages that could lyse the host bacteria.
[0022] The plaque test is as follows: Pseudomonas aeruginosa BPA17, BPA57 and BPA67 are inoculated in 5 mL LB liquid medium at a ratio of 1%, and cultured at 37°C for 12 h. Take 100 μL of the above-prepared bacterial solution and drop it in the center of the plate, and spread the bacterial solution evenly with a coating rod. After it dries, take 10 μL of the above filtrate and drop it in the center of the plate. After natural drying, invert it in a 37°C incubator for 10 h, and then observe whether there are plaques in the area where the filtrate is added. If a clear plaque is produced in the area where the filtrate is added, it can be judged that the filtrate contains bacteriophages that can kill the host bacteria.
[0023] Take the above filtrate and dilute it with sterile PBS. Take 100 μL of the diluted filtrate and 100 μL of the overnight cultured bacterial solution and mix them thoroughly. Incubate at room temperature for about 5 min, then add them to 7 mL of LB semi-solid medium at 45°C. After mixing, quickly pour it on 1.5% LB agar medium to make a double-layer plate. After it solidifies, place it in a 37°C incubator and incubate it for 16-20 h to observe the growth of plaques.
[0024] Example 2: Phage amplification and purification On the double-layer plate where plaques are formed, pick a single plaque with a larger diameter, relatively round and transparent, and inoculate it into 5 mL LB liquid culture medium. At the same time, add 100 μL of phage host bacterial liquid, mix well, and culture at 37°C until the liquid gradually becomes clear. Centrifuge at 12,000 r / min and 4°C for 10 min, and take the supernatant. Repeat the double-layer plate experiment, and pick a single plaque for purification 4-5 times.
[0025] Take 1 mL of freshly cultured host bacteria, add 300 μL of phage lysis solution (with a single phage culture and host bacteria in a ratio of 1:1, 1:10, and 1:100, respectively), and incubate at 37°C for 20 min to allow the phage particles to adsorb to the host bacteria; add 800 mL of LB liquid culture medium, shake and culture at 37°C for 6-8 h, centrifuge at 12,000 r / min, 4°C for 10 min, and take the supernatant, which is the phage lysis solution.
[0026] Phage purification: RNase A and DNaseⅠ were added to the phage lysate to a final concentration of 1 μg / mL, and the mixture was placed at room temperature for 30 min. NaCl was added to a final concentration of 1 mol / L, and the mixture was mixed and placed on ice for 1~2 h. The supernatant was collected after centrifugation at 8,000 r / min for 15-20 min in a 4 ℃ centrifuge. 10 g PEG8000 was added to each 100 ml mixed solution, and the mixture was stirred gently to fully dissolve. The mixture was placed on ice for 12 h to allow the phage to form a precipitate under the action of PEG8000. The mixture was then centrifuged at 12,000 r / min at 4 ℃ for 20 min. 2 mL SM solution was added to wash the precipitate thoroughly, and the mixture was placed at room temperature for 1 h. An equal volume of chloroform was added for extraction, and the mixture was gently shaken for 30 s. The organic phase and the hydrophilic phase were separated by centrifugation at 5,000 r / min at 4 ℃ for 10 min, and the hydrophilic phase containing the phage particles was recovered to obtain the purified phage.
[0027] The double-layer plate method was used to detect the phage titer: the purified phage solution was diluted 10 times in a gradient, 100 μL of the corresponding phage dilution solution was taken and mixed with 100 μL of the host bacterial solution, and then spread on a double-layer agar plate. The plate was cultured at 37°C for about 8 hours, and the plaques were counted on each agar plate. The plate with about 100-300 plaques was selected, and the phage titer was obtained by calculating the initial phage concentration based on the dilution multiple. The purified phage was as follows Figure 1 shown.
[0028] The purified phages PB17, PB57 and PB67 are deposited in the China Center for Type Culture Collection, the address of the depository is Wuhan University, Wuhan, China, with the deposit numbers being CCTCC M 20242120, CCTCC M 20242119 and CCTCC M 20242121, respectively, and the deposit date is September 29, 2024.
[0029] Example 3: Transmission electron microscopy observation of bacteriophages PB17, PB57 and PB67 The purified phage in Example 2 was observed under an electron microscope. The specific operation steps were as follows: 10 μL of the sample was dropped on the copper mesh, and the sample was allowed to settle for 15 min. The excess liquid was removed with filter paper, and the sample was stained with 2% phosphotungstic acid (PTA) for 1-2 min. After drying, the sample was observed under a transmission electron microscope (Hitachi H-7650). The results are shown in FIG. Figure 2As shown in the figure, the heads of PB17, PB57 and PB67 are all icosahedral. According to the "Taxonomy of Viruses - The Eighth Report of the International Committee on Taxonomy of Viruses" published by the International Committee on Taxonomy of Viruses (ICTV) in 2005, PB17, PB57 and PB67 belong to the order Caudovirales.
[0030] Example 4: Temperature stability determination of bacteriophages PB17, PB57 and PB67 The titers of phages PB17, PB57, and PB67 were adjusted to 107 PFU / mL and incubated in water baths at 4°C, 25°C, 37°C, 50°C, and 60°C, respectively. Samples were taken every 20 min until 80 min had passed. The titers of phages in different samples were determined using the double-layer plate method. Figure 3 As shown, within 80 minutes, the titer of phage PB17 remained basically stable when the temperature was below 50°C, but starting from 60°C, the titer of phage PB17 gradually decreased or even disappeared as the reaction time increased; the titer of phage PB57 and phage PB67 remained basically stable when the temperature was below 60°C, but starting from 70°C, the titer of phage PB57 and phage PB67 gradually decreased as the reaction time increased, indicating that the three phages have strong temperature tolerance.
[0031] Example 5: pH stability determination of bacteriophages PB17, PB57 and PB67 Phages PB17, PB57, and PB67 were mixed with SM buffers of different pH values (pH 2-13), and then incubated at 37°C for 1 h. The phage titers in different samples were then determined. Figure 4 As shown, the bactericidal activity of phage PB17, phage PB57 and phage PB67 remained basically stable in environments of pH = 3, 4, 5, 6, 7, 8, 9, 10 and 11, while in an acidic environment with a pH lower than 3 or an alkaline environment with a pH higher than 11, the phage activity was basically lost, indicating that the three phages have strong pH tolerance.
[0032] Example 6: Host spectrum determination of bacteriophages PB17, PB57 and PB67 The titers of phages PB17, PB57 and PB67 obtained in Example 2 were all adjusted to 108 PFU / mL for standby use. 211 strains of Pseudomonas aeruginosa to be tested were selected in the experiment, and the host spectrum of phages PB17, PB57 and PB67 was analyzed. The specific operation was as follows: Plaque test: Take 100 μL of the overnight culture of the strain to be tested, drop it in the center of the 1.5% LB medium plate, and use a spreading stick to spread them into a uniform bacterial lawn. Take 10 μL of bacteriophage PB17, PB57 and PB67 and drop them on the surface of the bacterial lawn. After the droplet dries, invert it in a 37℃ incubator and culture it for 12~16 hours. Observe the results. If plaques are produced, record it as "+", otherwise it is "-". The results are as follows Figure 5 As shown, phage PB17 can produce plaques on the plates of 186 tested Pseudomonas aeruginosa, with a lysis rate of 88.15% (186 / 211); PB57 can produce plaques on the plates of 183 tested Pseudomonas aeruginosa, with a lysis rate of 86.73% (183 / 211); PB67 can produce plaques on the plates of 160 tested Pseudomonas aeruginosa, with a lysis rate of 75.83% (160 / 211). According to the principle of host spectrum complementarity, the bacteriophage cocktail made of three phages will have a wider range of bactericidal effects, and can produce plaques on the plates of 210 tested Pseudomonas aeruginosa, with a lysis rate of up to 99.53% (210 / 211).
[0033] Example 7: Determination of antibacterial activity of phages PB17, PB57 and PB67 and their mixture Pseudomonas aeruginosa BPA17, BPA57 and BPA67 were cultured in LB liquid medium until the logarithmic growth phase. The three phages and their phage cocktails were mixed with Pseudomonas aeruginosa X-34 at an MOI of 0.01, and then cultured in ordinary LB medium, and samples were taken every 2 hours until 12 hours. The results are shown in Figure 6 As shown in the figure, when phages and phage cocktails were co-cultured with bacteria for 12 h, the number of resistant bacteria in the phage cocktail group did not increase significantly compared with the single phage group. Therefore, the phage cocktail group has a better antibacterial effect than the single phage group.
[0034] The deposit information is as follows: bacteriophage PB17, bacteriophage PB57, and bacteriophage PB67 all belong to Pseudomonas aeruginosa phage.
[0035] Among them, phage PB17 and phage PB57 belong to the Podoviridae family, and phage PB67 belongs to the Myoviridae family.
[0036] The full name of the institution that deposited the three phages: China Center for Type Culture Collection The address of the three phage strains deposited is: Wuhan University, Wuhan, China. The date of deposit is: September 29, 2024 The deposit numbers of the three phage strains are: phage PB17 (deposit number is CCTCC M 20242119); phage PB57 (deposit number is CCTCC M 20242120); phage PB67 (deposit number is CCTCC M 20242121).
[0037] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.
Claims
1. A Pseudomonas aeruginosa phage cocktail, characterized in that: The phage cocktail includes three phages PB17, PB57 and PB67 isolated from nature and capable of specifically killing Pseudomonas aeruginosa. They were deposited in the China Center for Type Culture Collection on September 29, 2024, with the deposit numbers being CCTCC M 20242120, CCTCC M 20242119 and CCTCC M 20242121, respectively.
2. Use of the Pseudomonas aeruginosa phage cocktail as claimed in claim 1 as an effective ingredient in the preparation of a drug for preventing and / or treating infectious diseases caused by Pseudomonas aeruginosa.
3. A phage cocktail preparation for killing Pseudomonas aeruginosa, comprising the phage cocktail according to claim 1 as an active ingredient.
4. A composition for preventing or treating infectious diseases caused by Pseudomonas aeruginosa, comprising the phage cocktail according to claim 1 as an active ingredient.
5. A composition for preventing or treating infectious diseases caused by Pseudomonas aeruginosa according to claim 4, characterized in that: The composition includes one of a liquid preparation, a freeze-dried preparation, and an oral solid preparation.
6. Use of the Pseudomonas aeruginosa phage cocktail as claimed in claim 1 as an active ingredient or directly used as a spray to kill Pseudomonas aeruginosa in a space environment; the space environment includes an animal breeding environment and a medical environment.
7. Use of the Pseudomonas aeruginosa phage cocktail as claimed in claim 1 as an effective ingredient in the preparation of a product for killing Pseudomonas aeruginosa on the surface of livestock and poultry and in the body of livestock and poultry.