Pseudomonas aeruginosa phage vB_PaeS_GZMU_S12 and application thereof

Abstract

The application discloses a pseudomonas aeruginosa bacteriophage vB_PaeS_GZMU_S12 and application thereof, and relates to the technical field of biotechnology.The pseudomonas aeruginosa bacteriophage vB_PaeS_GZMU_S12 is preserved in the Guangdong Microbial Culture Collection Center on April 14, 2026, the address of the preservation center is No. 59 Building, 5th Floor, Guangzhou Xianlie Middle Road 100, and the preservation number is GDMCC No: 68086-B1.The bacteriophage has typical characteristics of a lytic bacteriophage, the titer is stable in an environment with a pH of 4-12 and a temperature of 4-50 DEG C, the highest titer can be 10 9 PFU / mL or more, can lyse the pseudomonas aeruginosa, and has a highest genomic similarity of 47.2% with a known bacteriophage, and belongs to a new genus in the bacteriophage.The application provides technical support for developing a new type of antibacterial drug and an antibacterial scheme for inhibiting the pseudomonas aeruginosa, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a strain of Pseudomonas aeruginosa bacteriophage vB_PaeS_GZMU_S12 and its applications. Background Technology

[0002] Pseudomonas aeruginosa ( Pseudomonas aeruginosa Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a Gram-negative pathogen that poses a significant challenge to clinical treatment due to its resistance to multiple antimicrobial agents. The increasing prevalence of CRPA has become a global concern, with multidrug-resistant strains accounting for 15%-30% of clinical isolates in many regions. This bacterium can form biofilms on the surfaces of medical devices such as catheters and endotracheal tubes, further complicating treatment. Its antibiotic resistance to biofilm-related infections is 2-3 orders of magnitude higher than that of planktonic bacteria. Therefore, addressing the drug resistance problem of Pseudomonas aeruginosa and developing new alternative treatments has become an urgent problem to be solved.

[0003] With antibiotic research resources dwindling, phage therapy has once again emerged as an effective strategy to combat multidrug-resistant pathogens. Lytic phages are viruses that can specifically infect and lyse bacteria without integrating into the host genome, possessing unique advantages: high host specificity, the ability to self-reproduce at the infection site, and the evolutionary capacity to overcome bacterial resistance. While phage research targeting *Pseudomonas aeruginosa* has made some progress, it still faces a series of challenges, including scarce phage resources, difficulties in screening and identification, and challenges in clinical translation. Therefore, developing a novel *Pseudomonas aeruginosa* phage with strong stability and significant efficacy is of great importance in filling this technological gap and solving the treatment dilemma of drug-resistant bacteria. Summary of the Invention

[0004] The purpose of this invention is to provide a *Pseudomonas aeruginosa* bacteriophage vB_PaeS_GZMU_S12 and its applications, thereby addressing the problems existing in the prior art. This bacteriophage is capable of lysing *Pseudomonas aeruginosa* and represents a new genus within the family Longtail Phagesaceae. It provides technical support for the development of novel antibacterial drugs and antibacterial strategies to inhibit *Pseudomonas aeruginosa*, and has promising application prospects.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a strain of Pseudomonas aeruginosa bacteriophage ( PseudomonasThe Pseudomonas aeruginosa phage vB_PaeS_GZMU_S12 was deposited on April 14, 2026, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 68086-B1.

[0006] The present invention also provides the use of the above-mentioned Pseudomonas aeruginosa phage vB_PaeS_GZMU_S12 in the preparation of medicaments for the prevention and / or treatment of Pseudomonas aeruginosa infections.

[0007] The present invention also provides the application of the above-mentioned Pseudomonas aeruginosa bacteriophage vB_PaeS_GZMU_S12 in the preparation of Pseudomonas aeruginosa bactericides.

[0008] Furthermore, the *Pseudomonas aeruginosa* is a multidrug-resistant *Pseudomonas aeruginosa*.

[0009] The present invention also provides a drug for preventing and / or treating Pseudomonas aeruginosa infection, the active ingredient of which includes the above-mentioned Pseudomonas aeruginosa bacteriophage vB_PaeS_GZMU_S12.

[0010] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0011] Furthermore, the dosage form of the drug is an injection, powder, gel, granule, or lyophilized preparation.

[0012] Furthermore, the drug also includes other active ingredients that have antibacterial effects against Pseudomonas aeruginosa.

[0013] The present invention also provides a bactericide for Pseudomonas aeruginosa, the active ingredient of which includes the above-mentioned Pseudomonas aeruginosa bacteriophage vB_PaeS_GZMU_S12.

[0014] Furthermore, the formulation of the Pseudomonas aeruginosa bactericide is a spray, powder, gel, granule, or lyophilized agent.

[0015] The present invention discloses the following technical effects: This invention discovers a novel *Pseudomonas aeruginosa* bacteriophage, vB_PaeS_GZMU_S12, which exhibits typical lytic phage characteristics. This phage is stable in titer at pH 4-12 and temperatures 4-50°C, with a maximum titer reaching 10. 9 The concentration of PFU / mL or higher is sufficient to lyse *Pseudomonas aeruginosa*, and the highest genomic similarity with known bacteriophages is 47.2%, classifying it as a novel genus of bacteriophages. This invention provides technical support for the development of novel antibacterial drugs and antibacterial strategies to inhibit *Pseudomonas aeruginosa*, and has promising application prospects. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a diagram of bacteriophage morphology. Figure 2 This is a phylogenetic analysis diagram based on distance-based whole-genome sequences; Figure 3 Electron micrograph of bacteriophage particles; Figure 4 The result of pH stability measurement is shown in the figure. Figure 5 The graph shows the results of the thermal stability test. Figure 6 The figure shows the results of the pyrolysis kinetics determination. Figure 7 The graph shows the antibacterial effect test results under different MOIs after 8 hours of treatment; Figure 8 The graph shows the antibacterial effect test results under different MOIs after 12 hours of treatment; Figure 9 This is a graph showing the detection results of the inhibitory effect of bacteriophages on biofilms. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Biological Preservation:

[0024] The Pseudomonas aeruginosa phage of the present invention ( Pseudomonas Phage vB_PaeS_GZMU_S12 was deposited on April 14, 2026 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 68086-B1.

[0025] Example 1: Isolation and Identification of Bacteriophages 1. Isolation and purification of bacteriophages Water samples were collected from Guangzhou, Guangdong Province, China. After centrifugation, larger impurities and most bacteria were removed using a 0.22 μm microporous membrane to obtain the filtrate. *Pseudomonas aeruginosa* (…) Pseudomonas aeruginosa The host bacteria were inoculated into LB liquid medium and cultured at 37°C with shaking until the bacterial culture reached OD500. 600 =0.6. Take 10 mL of filtrate and 1 mL of host bacterial culture, add 5 mL of LB nutrient broth (3 times concentration), and co-culture overnight in a shaker at 37℃. After centrifugation, take the supernatant and filter it through a 0.22 μm microporous membrane to obtain the first enrichment solution. Repeat the above operation to obtain the second enrichment solution. The presence of phage plaques is detected by a dot test. Purify the phage using the double-layer agar plate method until the phage plaques on the plate are uniform in size. After several rounds of purification, a phage strain is obtained and named *Pseudomonas aeruginosa* phage (…). Pseudomonas Phage) vB_PaeS_GZMU_S12 (hereinafter referred to as PS12), the phage plaques are round, transparent, and have clear boundaries, such as Figure 1 As shown.

[0026] 2. Phage titer determination Phage titer was determined using the double-layer plate method. Phage solutions were serially diluted. 100 μL of the diluted phage solution was mixed 1:1 with logarithmic-phase host bacteria and incubated at 37°C for 15 min. Then, it was mixed with 0.7% semi-solid LB agar and evenly spread on top of 1.5% solid LB agar pre-spread on culture dishes. The dishes were then incubated overnight at 37°C. Several translucent phage plaques were visible on the plates. Plates with 30-300 plaques per field of view were selected for plaque counting. Titer (PFU / mL) = number of plaques × 10 × dilution factor. Results showed that the titer of phage PS12 could reach up to 10. 9 PFU / mL or higher.

[0027] 3. Phage whole genome analysis Single-virus genome sequencing of bacteriophage PS12 was performed using the Illumina sequencing platform. After obtaining the sequencing data, SOAPnuke was used for data quality control, i.e., low-quality data was evaluated and removed to ensure the reliability of subsequent analysis results. After removing host contamination, high-quality reads were assembled using MEGAHIT. Virulence factors and antibiotic resistance genes in the bacteriophage genome were compared with the VFDB database (http: / / www.mgc.ac.cn / VFs / ) and the CARD database (https: / / card.mcmaster.ca / ). The results showed that the bacteriophage PS12 genome size was 49500 bp, the GC content was 59.3%, and the genome contained no virulence genes or antibiotic resistance genes, indicating the safety of this bacteriophage at the gene level.

[0028] Using MEGA 11.0.13 software, a phylogenetic tree was constructed using the distance method after 1000 bootstrap iterations. The results are as follows. Figure 2As shown. According to the standards of the International Committee on Taxonomy of Viruses (ICTV), when the genomic sequence similarity of two bacteriophages is less than 95%, they should be classified as different species. In the NCBI database, the similarity of bacteriophage PS12 with other bacteriophages was compared and analyzed using the Local Base Alignment Search (BLAST) tool. The highest genomic similarity observed in bacteriophage PS12 (Pseudomonas phage Haboob; GenBank number: OR683404.1) was 47.2%. Referring to the classification guidelines of the Bacteria and Archaeaviruses Subcommittee (BAVS) of the ICTV, viral populations with nucleotide sequence similarity exceeding 70% can be classified into the same genus. New species are considered to have a nucleotide level difference of more than 5% from existing species, i.e., a similarity of no more than 95% (see the literature "Turner D, Kropinski AM, Adriaenssens E M. A roadmap for genome-based phage taxonomy[J]. Viruses, 2021, 13(3): 506."). The highest alignment similarity of phage PS12 (47.2%) was far below the ICTV-recommended genus threshold of 70%, indicating that phage PS12 represents a new genus.

[0029] Table 1. Comparison of bacteriophage similarities

[0030] Example 2: Phage Host Profile Determination The host range of bacteriophage PS12 was determined by detecting different strains using a spot test. 100 μL of logarithmic-phase host bacteria was mixed with 0.7% semi-solid LB agar and spread onto a culture dish to prepare a bacterial plate. 10 µL of bacteriophage solution was dropped onto the surface of the plate and incubated overnight at 37°C. The appearance of phage plaques indicated that the bacteriophage had infected the corresponding host bacteria. The results (Table 2) showed that the lysis rate of bacteriophage PS12 was 55.6% (30 / 54).

[0031] The 54 host bacteria selected in this experiment included clinical isolates with various drug resistance phenotypes. Antimicrobial susceptibility testing identified 13 of them as multidrug-resistant (MDR) bacteria, meaning they exhibited resistance or intermediate susceptibility to three or more classes of antibiotics, including carbapenems, quinolones, and cephalosporins. Among the 30 strains lysed by PS12, 7 were MDR-resistant (e.g., B-PA-37, Pa-1, Pa-22).

[0032] Table 2. Host spectrum determination of bacteriophages against 54 strains of Pseudomonas aeruginosa

[0033] Example 3: Biological Characteristics Analysis of Bacteriophages 1. Morphological observation of bacteriophages Observation was performed using phosphotungstic acid negative staining. Activated bacteriophage PS12 was attached to a 400-mesh carbon copper grid and allowed to adsorb. After 2 minutes, residual liquid around the copper grid was blotted away with filter paper. 1% phosphotungstic acid was then applied to the copper grid for staining for 1 minute. The staining solution was blotted away with filter paper, and the grid was washed twice with deionized water and allowed to dry. Observation was performed using a Hitachi electron microscope with an accelerating voltage of 80 kV. The structural dimensions of the bacteriophages were measured using Image J. Figure 3 As shown, morphological examination by transmission electron microscopy (TEM) revealed that bacteriophage PS12 has an icosahedral head and a long, non-retractable tail, consistent with the morphology of long-tailed bacteriophages. Precise measurements showed that the head diameter was approximately 660 Å and the tail length was approximately 1760 Å.

[0034] 2. pH stability To evaluate the effect of different pH values ​​on phage titers, hydrochloric acid or sodium hydroxide solutions were pre-added to sterile LB liquid medium to adjust the pH to a range of 1-14, and the medium was then filtered through a 0.22 μm microporous membrane for sterilization. 100 μL of phage solution was added to 900 μL of LB liquid medium at different pH values ​​and incubated at 37°C for 1 hour. Titer determination was performed using the double-layer agar plate method. The experimental results are shown below. Figure 4 As shown.

[0035] The results showed that bacteriophage PS12 maintained a relatively stable titer in environments with pH values ​​ranging from 4 to 12, and the titer could reach 10 in all of these environments. 9 A concentration of PFU / mL or higher indicates that it has good stability and activity within this pH range.

[0036] 3. Thermal stability To evaluate the effect of different temperatures on phage titer, 100 μL of phage solution was added to 900 μL of sterile LB broth pretreated at different temperatures, and incubated for 1 hour at 4℃, 37℃, 50℃, 60℃, and 70℃. Titer was determined using the double-layer agar plate method. The experimental results are shown below. Figure 5 As shown.

[0037] The results showed that phage PS12 exhibited high thermal stability, maintaining a titer of approximately 10 after incubation at 4-50°C for 1 hour. 9 PFU / mL. However, its survival rate decreased significantly at 60°C and was almost completely lost at 70°C.

[0038] 4. In vitro antibacterial activity The efficiency of phage infection was evaluated by adjusting the MOI to 1000, 100, 10, 1, 0.1, 0.01, and 0.0001, respectively. The OD was compared... 600 To assess the inhibitory effect on bacterial growth, the experiment was repeated three times over 12 hours. A 1:1 mixture of logarithmic-phase host bacteria and phage fluid was added to 96-well plates, with an equal volume of logarithmic-phase bacterial culture serving as a control. The plates were incubated at 37°C with shaking at 220 rpm. Results are as follows: Figures 6-8 As shown, within the first 5 hours of co-culture, the higher concentration group (MOI ≥ 1) rapidly and effectively inhibited bacterial growth; around 8 hours, the high MOI group exhibited a stronger antibacterial effect. Continued observation up to 12 hours revealed that, except for the extremely low concentration group (MOI = 0.0001), which showed no significant antibacterial effect, all other treatment groups exhibited varying degrees of growth inhibition. In particular, the treatment groups with MOI = 100 and MOI = 1000 showed significantly higher OD values. 600 It remained at a low level and was significantly lower than other groups.

[0039] 5. Inhibition of biofilms Crystal violet staining was used to assess biofilm inhibition. Log-phase host bacteria and phage fluid were mixed 1:1 and added to 96-well plates with MOIs of 100, 10, 1, 0.1, and 0.01. An equal volume of log-phase bacterial culture served as the positive control (PC), and an equal volume of LB medium served as the blank control (NC). The plates were incubated at 37°C for 24 hours to allow biofilm formation, with three replicates per well. After incubation, the culture medium was discarded, and the plates were washed twice with 200 μL PBS to remove airborne bacteria. The plates were fixed with methanol solution for 15 minutes, discarded, and air-dried. Then, 100 μL of 1% crystal violet solution was added for staining for 30 minutes, discarded, and the plates were washed twice. After air-drying, anhydrous ethanol was added for destaining for 5 minutes. The eluent was transferred to a new sterile 96-well plate, and the OD value at 595 nm was measured using a multi-mode microplate reader. The results are shown below. Figure 9 As shown.

[0040] The results showed that the OD values ​​of all phage groups and the PC group were statistically significant. The biofilm inhibition rates of MOIs of 100, 10, 1, 0.1 and 0.01 were 48.63%, 51.86%, 30.47%, 34.63% and 47.32%, respectively, indicating that phage PS12 effectively inhibited the formation of host biofilm.

[0041] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of Pseudomonas aeruginosa bacteriophage ( Pseudomonas phage)vB_PaeS_GZMU_S12, characterized in that, The Pseudomonas aeruginosa bacteriophage vB_PaeS_GZMU_S12 was deposited on April 14, 2026 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 68086-B1.

2. The use of the Pseudomonas aeruginosa bacteriophage vB_PaeS_GZMU_S12 as described in claim 1 in the preparation of a medicament for the prevention and / or treatment of Pseudomonas aeruginosa infection.

3. The application of the Pseudomonas aeruginosa bacteriophage vB_PaeS_GZMU_S12 as described in claim 1 in the preparation of a Pseudomonas aeruginosa bactericide.

4. The application according to claim 2 or 3, characterized in that, The *Pseudomonas aeruginosa* strain mentioned is a multidrug-resistant *Pseudomonas aeruginosa* strain.

5. A drug for the prevention and / or treatment of Pseudomonas aeruginosa infection, characterized in that, The active ingredient includes the Pseudomonas aeruginosa phage vB_PaeS_GZMU_S12 as described in claim 1.

6. The drug according to claim 5, characterized in that, The drug also includes pharmaceutically acceptable excipients.

7. The drug according to claim 6, characterized in that, The dosage form of the drug is injection, powder, gel, granule or lyophilized.

8. The drug according to claim 5, characterized in that, The drug also includes other active ingredients that have antibacterial effects against Pseudomonas aeruginosa.

9. A fungicide for Pseudomonas aeruginosa, characterized in that, The active ingredient includes the Pseudomonas aeruginosa phage vB_PaeS_GZMU_S12 as described in claim 1.

10. The Pseudomonas aeruginosa bactericide according to claim 9, characterized in that, The formulation of the Pseudomonas aeruginosa bactericide is a spray, powder, gel, granule or lyophilized agent.

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