Phage vB PaeM phi 18 as well as preparation and application thereof

Abstract

The invention relates to the field of microorganisms, in particular to a bacteriophage vB PaeM phi 18 as well as a preparation and application thereof. The invention provides a bacteriophage vB PaeM phi 18, and the preservation number of the bacteriophage vB PaeM phi 18 is GDMCC No: 67429-B1. The bacteriophage provided by the invention has efficient splitting capacity on various pseudomonas aeruginosa environmental isolates including a model strain PAO1, has good biological stability, short incubation period and large splitting quantity, can effectively remove a biological membrane formed by pseudomonas aeruginosa, and has the advantages of high efficiency, high efficiency and high efficiency. And a new microbial resource and a technical basis are provided for developing a bacteriophage treatment preparation or a biological membrane scavenger aiming at the pseudomonas aeruginosa.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganisms, in particular to a bacteriophage vB PaeM Φ18 and a preparation and application thereof. BACKGROUND

[0002] Pseudomonas aeruginosa is a conditional pathogenic bacterium widely distributed in soil, water and other environments. The bacterium has strong environmental adaptability and metabolic diversity, and can survive and reproduce in various harsh environments. During the infection process, Pseudomonas aeruginosa secretes polymers such as exopolysaccharides, proteins and lipids through the regulation of the quorum sensing system, forming a complex biofilm structure. This biofilm structure not only provides a physical protective barrier for the bacteria, but also significantly enhances the tolerance of the bacteria to environmental stress (such as drought, ultraviolet light, and nutrient deficiency) and antibiotics and host immune systems through various mechanisms.

[0003] Phage therapy, as a new biological control method, uses bacteriophages to specifically infect and lyse host bacteria, has high specificity, and naturally disappears after completing lysis, without residual pollution to the environment. However, not all lytic bacteriophages have the ability to efficiently penetrate and clear biofilms.

[0004] Therefore, screening bacteriophages with both broad-spectrum lytic activity and high-efficiency anti-biofilm ability is still a technical difficulty and research focus in the field. SUMMARY

[0005] Therefore, the present application provides a bacteriophage vB PaeM Φ18 and a preparation and application thereof. The bacteriophage provided by the present application has high-efficiency lytic ability to various environmental isolates of Pseudomonas aeruginosa including the model strain PAO1, has good biological stability, a short latent period, a large amount of lysis, and can effectively clear the biofilm formed by Pseudomonas aeruginosa, thereby providing a new microbial resource and technical basis for developing a bacteriophage treatment preparation or biofilm clearing agent for Pseudomonas aeruginosa.

[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions.

[0007] The present application provides a bacteriophage vB PaeM Φ18, and the preservation number of the bacteriophage vB PaeM Φ18 is GDMCC No: 67429-B1.

[0008] The present application also provides a bacteriophage vB PaeM Φ18 preparation, which comprises any one of the following and an acceptable adjuvant:

[0009] (a) the bacteriophage vB PaeM Φ18 described above; and / or

[0010] (b) a metabolite, derivative, fermentation, culture, exosome, lysate or extract of the above-mentioned bacteriophage vB PaeM Φ18.

[0011] In some embodiments of the present application, in the above-mentioned bacteriophage vB PaeM Φ18 preparation, the titer of the bacteriophage vB PaeM Φ18 in the bacteriophage vB PaeM Φ18 preparation is not less than 10 9 PFU / mL.

[0012] The present application also provides a preparation method of the above-mentioned bacteriophage vB PaeM Φ18 preparation, comprising the following steps:

[0013] S1: preparing a host bacteria suspension;

[0014] S2: taking the host bacteria suspension, adding host bacteria PAO1, culturing, centrifuging, and obtaining the bacteriophage vB PaeM Φ18 preparation.

[0015] The present application also provides applications of the above-mentioned bacteriophage vB PaeM Φ18, the above-mentioned bacteriophage vB PaeM Φ18 preparation and / or the bacteriophage vB PaeM Φ18 preparation obtained by the above-mentioned preparation method in inhibiting and / or lysing Pseudomonas aeruginosa.

[0016] The present application also provides applications of the above-mentioned bacteriophage vB PaeM Φ18, the above-mentioned bacteriophage vB PaeM Φ18 preparation and / or the bacteriophage vB PaeM Φ18 preparation obtained by the above-mentioned preparation method in inhibiting the formation and / or regeneration of Pseudomonas aeruginosa biofilm.

[0017] The present application also provides applications of the above-mentioned bacteriophage vB PaeM Φ18, the above-mentioned bacteriophage vB PaeM Φ18 preparation and / or the bacteriophage vB PaeM Φ18 preparation obtained by the above-mentioned preparation method in preparing Pseudomonas aeruginosa inhibitors and / or products for inhibiting and / or lysing Pseudomonas aeruginosa.

[0018] The present application also provides applications of the above-mentioned bacteriophage vB PaeM Φ18, the above-mentioned bacteriophage vB PaeM Φ18 preparation and / or the bacteriophage vB PaeM Φ18 preparation obtained by the above-mentioned preparation method in preparing products for inhibiting the formation and / or regeneration of Pseudomonas aeruginosa biofilm.

[0019] In some embodiments of the present application, in the above-mentioned applications, the products comprise one or more of the following: drugs, disinfectants, antibacterial agents and antiseptics.

[0020] The present application also provides a method for inhibiting and / or lysing Pseudomonas aeruginosa, comprising administering to a subject any of the following:

[0021] (c) the above-mentioned bacteriophage vB PaeM Φ18; and / or

[0022] (d) the above-mentioned bacteriophage vB PaeM Φ18 preparation; and / or

[0023] (e) The bacteriophage vB PaeM Φ18 preparation obtained by the above preparation method.

[0024] The beneficial effects of this invention include:

[0025] (1) Broad lysis spectrum and strong host specificity: The bacteriophage vB PaeM Φ18 of this invention can effectively lyse all 24 strains of Pseudomonas aeruginosa isolated from the environment, with a lysis rate of 100%, while it has no lysis effect on Escherichia coli and Klebsiella pneumoniae.

[0026] (2) Strong proliferation capacity: short latency period (about 20 minutes), large burst size (about 100 PFU / cell), easy to industrialize and produce.

[0027] (3) Good stability: It is not sensitive to changes in temperature and pH, which makes it easy to store and transport the formulation.

[0028] (4) Wider temperature tolerance range: Under the same treatment conditions (60 min, 1 ... 9 At PFU / mL: vB PaeM Φ18 remains >10 at -20℃ 8 PFU / mL, still >10 at 65℃ 5 PFU / mL. This indicates that vB PaeM Φ18 has a wider temperature tolerance range, making it more suitable for practical applications and storage and transportation.

[0029] (5) It has significant anti-biofilm ability: This bacteriophage can not only effectively inhibit the formation of Pseudomonas aeruginosa biofilm, but also has a clear scavenging and degradation effect on the mature biofilm that has already formed. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0031] Figure 1 The morphology of plaques formed by bacteriophage vB PaeM Φ18 on host PAO1 is shown.

[0032] Figure 2 Transmission electron micrograph of bacteriophage vB PaeM Φ18;

[0033] Figure 3 Determination of the optimal multiple of infection (MOI) for phage vB PaeM Φ18;

[0034] Figure 4 The one-step growth curve of bacteriophage vB PaeM Φ18 is shown;

[0035] Figure 5 Stability curves of bacteriophage vB PaeM Φ18 at different temperatures are shown.

[0036] Figure 6 Stability curves of bacteriophage vB PaeM Φ18 at different pH values ​​are shown.

[0037] Figure 7 The effects of bacteriophage vB PaeM Φ18 and Φ10 on PAO1 biofilm removal are shown;

[0038] Figure 8 The effects of bacteriophage vB PaeM Φ18 and Φ10 on inhibiting PAO1 biofilm formation were demonstrated.

[0039] Biological Preservation Instructions

[0040] Biological material: Pseudomonas aeruginosa phage vB PaeM Φ18; Classification and nomenclature: Pseudomonas aeruginosa phage; Deposited on December 8, 2025 at Guangdong Provincial Center for Microbial Culture Collection; Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences Institute of Microbiology; Accession number: GDMCCNo: 67429-B1. Detailed Implementation

[0041] This invention discloses bacteriophage vB PaeM Φ18, its formulation, and its applications.

[0042] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0043] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0044] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0045] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0046] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0047] This invention obtains a highly active Pseudomonas aeruginosa lytic phage from environmental sewage samples, using the P. aeruginosa standard strain PAO1 as the host, through enrichment, separation and purification, tentatively named vB PaeM Φ18.

[0048] The specific technical content includes:

[0049] Isolation and identification of bacteriophages: Bacteriophages were isolated from wastewater samples using the double-layer agar plate method. The host bacterium was *Pseudomonas aeruginosa* type strain PAO1. After multiple purifications, single phage plaques with uniform morphology and size and clear, regular edges were obtained. Transmission electron microscopy images showed that the head diameter of the phage was approximately 84 nm and the tail length was approximately 155 nm.

[0050] Lysis spectrum determination: This bacteriophage produced clear and transparent plaques on all 24 strains of Pseudomonas aeruginosa isolated from environmental sewage, showing a broad lysis spectrum, while it had no lysis effect on common strains such as Klebsiella pneumoniae and Escherichia coli, indicating that it has high host specificity.

[0051] Biological characteristics study:

[0052] (1) Optimal multiplicity of infection: When bacteriophage vB PaeM Φ18 reacts with the corresponding host bacteria at a ratio of 0.0001, the titer of progeny bacteriophages produced is the highest, that is, the optimal multiplicity of infection for bacteriophage vB PaeM Φ18 is 0.0001.

[0053] (2) One-step growth curve: The results showed that the phage had a short latency period (20 minutes), a lysis period of 110 minutes, and a large burst size (100 PFU / cell).

[0054] (3) Stability test: Phage vB PaeM Φ18 showed stable activity and maintained a high titer in the pH range of 3 to 11, demonstrating good acid and alkali resistance; it could maintain a high titer for a long time under conditions of -20℃ to 65℃, and the PFU remained at 10. 5 above.

[0055] Assessment of biofilm removal / inhibition capabilities: Crystal violet staining confirmed that this phage could significantly inhibit the formation of PAO1 biofilm and had a significant removal and degradation effect on existing mature biofilms.

[0056] The culture media and reagents involved in this invention:

[0057] LB liquid medium: sodium chloride 10g / L, tryptone 10g / L, yeast extract 5g / L, prepared with pure water, pH 7.2, sterilized at 121℃ for 15min.

[0058] 0.6% LB semi-solid culture medium: sodium chloride 10g / L, tryptone 10g / L, yeast extract 5g / L, agar powder 6g / L, prepared with pure water, pH 7.2, sterilized at 121℃ for 15min.

[0059] 1.5% LB solid medium: sodium chloride 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, agar powder 15 g / L, prepared with pure water, pH 7.2, sterilized at 121℃ for 15 min.

[0060] The instruments and equipment used in this invention include: high temperature and high pressure sterilizer, analytical balance, ultra-clean workbench, 120 kV field emission transmission electron microscope, enzyme-linked immunosorbent assay (ELISA) reader, incubator, shaker, glow discharge instrument, etc.

[0061] In Examples 1 to 7 of this invention, all raw materials and reagents used can be purchased from the market.

[0062] The present invention will be further illustrated below with reference to the embodiments:

[0063] Example 1: Isolation and purification of bacteriophages

[0064] Environmental wastewater samples were collected and centrifuged at 10,000 rpm for 10 minutes at 4°C. The supernatant was filtered through 0.45 μm and 0.22 μm filter membranes. The filtrate was mixed with logarithmic-phase PAO1 bacterial culture and 5×LB medium and incubated at 37°C with shaking until the culture medium became clear, indicating that bacteriophages had lysed the host bacteria. The culture medium was centrifuged at 10,000 rpm for 10 minutes at 4°C, the supernatant was collected and filtered, and bacteriophages were isolated using the double-layer agar plate method. A single clear phage plaque was picked, and the above purification steps were repeated 5 times until the phage plaque morphology was uniform, obtaining a purified bacteriophage vB PaeM Φ18 suspension.

[0065] Example 2: Phage Morphological Observation

[0066] The purified phage suspension was dropped onto a copper grid, stained with 2% uranium acetate, and observed using a 120 kV transmission electron microscope. The results showed that the phage head was approximately 84 nm in diameter and the tail was approximately 155 nm long, with morphology consistent with the characteristics of the Myotail Phagesaceae family.

[0067] Example 3: Pyrolysis Spectroscopy Determination

[0068] All Pseudomonas aeruginosa standard strain PAO1 and 24 environmental isolates were revived and cultured to the logarithmic growth phase. Purified suspensions of vB PaeM Φ18 and Φ10 (phages of this strain were also isolated via PAO1 screening) were prepared, both with a titer of 10. 9 PFU / mL, take 100 μL of bacteria and mix it evenly with 7 mL of 0.6% semi-solid culture medium, quickly pour it onto the prepared 1.5% solid agar plate, wait for the 0.6% semi-solid to solidify, add 5 μL of isolated and purified bacteriophage to each host bacterial plate, let it stand for 10 min, place it upright in a 37℃ constant temperature incubator, and incubate overnight (12 h). The next day, observe whether there are phage plaques in each area.

[0069] Experimental results:

[0070] Table 1. Lysis profiles of phage vB PaeM Φ18 and Φ10 against 24 Pseudomonas aeruginosa environmental isolates and control strains.

[0071]

[0072] Note: The ability of bacteriophages to lyse strains is indicated by: "+" indicating that the bacteriophage can completely lyse the strain, forming clear plaques; "-" indicating that the bacteriophage cannot lyse the strain, and no plaques are visible to the naked eye.

[0073] Results analysis:

[0074] vB PaeM Φ18 was able to lyse all tested strains (100% lysis rate), while Φ10 could only lyse 88% of the strains, indicating that vB PaeM Φ18 has a broader host spectrum and stronger pan-host lysis ability.

[0075] Example 4: Determination of Optimal MOI and One-Step Growth Curve

[0076] The multiple of infection (MOI) is the initial ratio of the number of bacteriophages to the number of host bacteria when a bacteriophage infects a host bacteriophage. That is, MOI (PFU / CFU) = bacteriophage concentration (PFU / mL) / bacterial concentration (CFU / mL). Based on a MOI of 10...-6 10 -5 10 -4 10 -3 10 -2 10 -1 The phage / bacteria were mixed in a ratio of 1:10:100 and diluted to the corresponding values. 100 μL of each phage / bacteria mixture was added to 5 mL of LB medium and incubated at 37°C and 250 rpm for 3 h. The mixture was then centrifuged at 10000 x g for 10 min at 4°C. The supernatant was collected and filtered through a 0.22 μm filter. The phage titer was determined using the double-layer agar plate method. The ratio of phage concentration to bacterial concentration corresponding to the highest titer was the optimal MOI. Phage and host bacteria were mixed according to the optimal MOI, and samples were taken periodically to determine the titer and plot a growth curve. The results showed that the optimal MOI for vB PaeM Φ18 (0.0001) was significantly lower than that for Φ10 (0.1), indicating that highly efficient infection could be achieved at extremely low concentrations. Although the incubation period was slightly longer, its outbreak size was 5 times that of Φ10, indicating that a greater number of progeny phages were produced per infection cycle, resulting in stronger proliferation.

[0077] Experimental results:

[0078] Table 2

[0079]

[0080] Example 5 Stability Test

[0081] Temperature stability:

[0082] Add 500 μL of phage solution to a 1.5 mL EP tube, and then place it in a water bath at -20℃, 4℃, 37℃, 50℃, 65℃, 70℃, and 100℃ for 1 hour respectively. After standing, pour the solution into a double-layer plate to measure the titer. Figure 5 As shown, after treatment for 1 hour in a wide temperature range from -20℃ to 60℃, it still maintains extremely high activity, with titers all above 10. 8 The PFU / mL titer decreased slightly when the temperature rose to 65℃, but still remained above 10. 5 The level of PFU / mL indicates that the phage has excellent thermal stability, remaining partially viable even at temperatures up to 65°C.

[0083] pH stability:

[0084] The pH of LB medium was adjusted to 3, 5, 7, 9, and 11 using 1M HCl or 1M NaOH solution. 900 μL of LB was added to 1.5 mL centrifuge tubes for each tube, followed by 100 μL of phage solution. The tubes were incubated at 37°C for 30 min, and the phage titer was determined using the double-layer plate method. Figure 6As shown, the titer of this bacteriophage remained stable at 10 after treatment for 30 minutes within an extreme pH range of 3 to 11. 7 The concentration of PFU / mL is above 100%. This result demonstrates that the phage possesses both good acid and alkali resistance, is insensitive to changes in pH environment, and can maintain its infectivity under a wide range of pH conditions.

[0085] Example 6: Evaluation of Biofilm Scavenging Capacity

[0086] PAO1 was statically cultured in 24-well plates for 24 h to form a mature biofilm. Planktonic bacteria were discarded, and the plates were gently washed three times with PBS. 1 mL of phage suspension or 1 mL of LB medium (control) was added to each well, and the plates were incubated at 37°C for 12 h, after which the waste liquid was aspirated. 0.1% crystal violet was added for staining for 30 min, and unbound crystal violet solution was aspirated from the wells. The plates were washed three times with 500 μL PBS, and then dissolved in 500 μL of 30% acetic acid at room temperature for 3-5 min until the biofilm color was completely dissolved. The eluted dye was transferred to 96-well plates, and the absorbance was measured at 560 nm.

[0087] Table 3 Biofilm removal efficiency (OD) 560 (mean ± SD)

[0088]

[0089] In terms of removing established biofilms, vB PaeM Φ18 showed a significantly higher reduction rate than Φ10, demonstrating its comprehensive and efficient biofilm removal capabilities.

[0090] Example 7 Assessment of Biomembrane Inhibition Capacity

[0091] PAO1 and bacteriophage (MOI=10) were simultaneously added to 24-well plates and co-cultured at 37°C for 12 h. Waste liquid was discarded. 0.1% crystal violet was added for staining for 30 min. Unbound crystal violet solution was aspirated from the wells. The plates were washed three times with 500 μL PBS, and then dissolved in 500 μL 30% acetic acid at room temperature for 3–5 min until the biofilm color was completely dissolved. The eluted dye was transferred to 96-well plates, and absorbance was measured at 560 nm.

[0092] Table 4 Biomembrane inhibition effect (OD) 560 (mean ± SD)

[0093]

[0094] In terms of inhibiting biofilm formation, vB PaeM Φ18 showed a significantly higher reduction rate than Φ10, demonstrating its comprehensive and efficient ability to inhibit biofilm formation.

[0095] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Bacteriophage vB PaeM Φ18, characterized in that, The preservation number of the bacteriophage vB PaeM Φ18 is: GDMCCNo: 67429-B1.

2. A phage vB PaeM Φ18 formulation, characterized in that, Includes any of the following and acceptable adjuvants: (a) the phage vB PaeM Φ18 as described in claim 1; and / or (b) Metabolites, derivatives, fermentation broths, cultures, exosomes, lysates, or extracts of the bacteriophage vB PaeM Φ18 as described in claim 1.

3. The phage vB PaeM Φ18 formulation as described in claim 2, characterized in that, The titer of the bacteriophage vB PaeM Φ18 in the bacteriophage vB PaeM Φ18 formulation is not less than 10. 9 PFU / mL.

4. The method for preparing the phage vB PaeM Φ18 formulation as described in claim 2 or 3, characterized in that, Includes the following steps: S1: Prepare host bacterial suspension; S2: Take the host bacterial suspension, add the host bacterial PAO1, culture, centrifuge, and prepare the bacteriophage vB PaeM Φ18 preparation.

5. The use of the phage vB PaeM Φ18 as described in claim 1, the phage vB PaeM Φ18 preparation as described in claim 2 or 3, and / or the phage vB PaeM Φ18 preparation obtained by the preparation method as described in claim 4 in inhibiting and / or lysing Pseudomonas aeruginosa.

6. The use of the phage vB PaeM Φ18 as described in claim 1, the phage vB PaeM Φ18 preparation as described in claim 2 or 3, and / or the phage vB PaeM Φ18 preparation obtained by the preparation method as described in claim 4 in inhibiting the formation and / or regeneration of Pseudomonas aeruginosa biofilms.

7. The use of the phage vB PaeM Φ18 of claim 1, the phage vB PaeM Φ18 formulation of claim 2 or 3, and / or the phage vB PaeM Φ18 formulation obtained by the preparation method of claim 4 in the preparation of Pseudomonas aeruginosa inhibitors, and / or in the preparation of products that inhibit and / or lyse Pseudomonas aeruginosa.

8. The use of the phage vB PaeM Φ18 of claim 1, the phage vB PaeM Φ18 formulation of claim 2 or 3, and / or the phage vB PaeM Φ18 formulation obtained by the preparation method of claim 4 in the preparation of products that inhibit the formation and / or regeneration of Pseudomonas aeruginosa biofilms.

9. The application as described in claim 7 or 8, characterized in that, The products include one or more of the following: pharmaceuticals, disinfectants, antibacterial agents, and preservatives.

10. A method for inhibiting and / or lysing Pseudomonas aeruginosa, characterized in that, This includes administering any of the following to the test subjects: (c) the phage vB PaeM Φ18 as described in claim 1; and / or (d) the phage vB PaeM Φ18 formulation as described in claim 2 or 3; and / or (e) The phage vB PaeM Φ18 formulation obtained by the preparation method as described in claim 4.

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