Staphylococcus aureus bacteriophage vB_SauM_GZMU_SAY13 and application thereof

Abstract

This invention discloses a lytic Staphylococcus aureus bacteriophage vB_SauM_GZMU_SAY13 and its applications, relating to the fields of microbial biotechnology and anti-infection technology. The preservation number of this Staphylococcus aureus bacteriophage vB_SauM_GZMU_SAY13 is GDMCC No: 67758-B1. This bacteriophage can effectively lyse Staphylococcus aureus, and its titer is stable in an environment with a pH of 4-10 and a temperature of 4-50℃, with a maximum titer of up to 10. 10 PFU / mL or higher. This invention provides important technical support for the development of novel antibacterial drugs and treatment regimens against Staphylococcus aureus, demonstrating broad application prospects and potential value.

Description

Technical Field

[0001] This invention relates to the fields of microbial biotechnology and anti-infection technology, and in particular to a lytic Staphylococcus aureus bacteriophage vB_SauM_GZMU_SAY13 and its applications. Background Technology

[0002] Staphylococcus aureus is a common Gram-positive pathogen in clinical practice, widely colonizing human skin, mucous membranes, and nasal cavity. It can cause mild community-acquired skin and soft tissue infections and folliculitis, as well as serious infections such as pneumonia, sepsis, and endocarditis in immunocompromised individuals, making it a significant source of both hospital-acquired and community-acquired infections. In recent years, due to antibiotic overuse, the problem of Staphylococcus aureus resistance has become increasingly serious. Methicillin-resistant Staphylococcus aureus (MRSA), in particular, is completely resistant to β-lactam antibiotics and can spread rapidly through contact in hospitals, nursing homes, and other settings, posing a significant obstacle to clinical anti-infective treatment.

[0003] Bacteriophages, as a class of viruses that specifically infect and lyse bacteria, possess unique advantages such as strong host specificity, rapid proliferation rate, high lysis efficiency, and low susceptibility to inducing drug resistance, making them a core candidate for novel anti-infective strategies to replace traditional antibiotics. Among them, Staphylococcus aureus phages, as a class of viruses that specifically infect Staphylococcus aureus, have shown significant advantages in controlling pathogenic Staphylococcus aureus infections and have important research and application value. With the increasingly serious problem of antibiotic resistance, phage therapy, as a natural and specific antibacterial strategy, is gradually becoming a new treatment option against drug-resistant bacterial infections. Therefore, developing novel Staphylococcus aureus phages with broad-spectrum lytic activity and high stability is of significant clinical and public health importance for overcoming the treatment bottlenecks of drug-resistant strains such as MRSA, filling the technological gap in phage therapy for Gram-positive drug-resistant bacteria, and solving the dilemma of clinical anti-infective treatment. Summary of the Invention

[0004] The purpose of this invention is to provide a lytic Staphylococcus aureus bacteriophage vB_SauM_GZMU_SAY13 and its applications to solve the problems existing in the prior art. This bacteriophage can lyse Staphylococcus aureus, especially methicillin-resistant Staphylococcus aureus, providing technical support for the development of novel antibacterial drugs and antibacterial strategies to inhibit Staphylococcus aureus, and has good application prospects.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a Staphylococcus aureus bacteriophage ( Staphylococcus aureusThe accession number of the Staphylococcus aureus phage vB_SauM_GZMU_SAY13 is GDMCCNo: 67758-B1.

[0006] This invention provides the use of the above-mentioned Staphylococcus aureus bacteriophage vB_SauM_GZMU_SAY13 in the preparation of medicaments for the prevention and / or treatment of Staphylococcus aureus infections.

[0007] This invention provides the application of the above-mentioned Staphylococcus aureus bacteriophage vB_SauM_GZMU_SAY13 in the preparation of Staphylococcus aureus bactericides.

[0008] Optionally, the Staphylococcus aureus includes methicillin-resistant Staphylococcus aureus (MRSA).

[0009] This invention provides a medicament for the prevention and / or treatment of Staphylococcus aureus infection, the active ingredient of which includes the Staphylococcus aureus bacteriophage vB_SauM_GZMU_SAY13 described above.

[0010] Optionally, the drug may also include pharmaceutically acceptable excipients.

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

[0012] Optionally, the drug may also include other active ingredients that have antibacterial activity against Staphylococcus aureus.

[0013] This invention provides a Staphylococcus aureus bactericide, the active ingredient of which includes the above-mentioned Staphylococcus aureus bacteriophage vB_SauM_GZMU_SAY13.

[0014] Optionally, the formulation of the Staphylococcus aureus bactericide is a spray, powder, gel, granule, or lyophilized agent.

[0015] The present invention discloses the following technical effects: This invention discovers a novel lytic Staphylococcus aureus bacteriophage ( Staphylococcus aureus The phage (vB_SauM_GZMU_SAY13) exhibits typical lytic phage characteristics. This phage shows stable titers within a pH range of 4-10 and a temperature range of 4-50℃, with a maximum titer reaching 1.7 × 10⁻⁶. 10A concentration of PFU / mL or higher can effectively lyse Staphylococcus aureus. This invention provides important technical support for the development of novel antibacterial drugs and treatment regimens against Staphylococcus aureus, demonstrating broad application prospects and potential value. 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 Electron micrograph of bacteriophage particles; Figure 3 A phylogenetic analysis diagram constructed based on distance analysis using whole-genome sequences; Figure 4 The graph shows the results of the thermal stability test. Figure 5 The result of pH stability measurement is shown in the figure. Figure 6 The graph shows the results of the optimal multiplicity of infection determination; Figure 7 The figure shows the results of the pyrolysis kinetics determination. Figure 8 This is a one-step growth curve of bacteriophage; Figure 9 The figure shows the results of the bacteriophage inhibition of biofilm assay. 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] Example 1: Isolation, identification, and preservation of bacteriophages 1. Isolation and purification of bacteriophages Water samples were collected from river water and sewage in Xiufa Village, Xinzhao Town, Panyu District, Guangzhou City, Guangdong Province, China. The water samples were first centrifuged (5000 rpm for 10 min) to remove most of the impurities. Then, the liquid samples were filtered using a 0.22 µm filter membrane for sterile filtration to remove bacteria and other macromolecular substances.

[0024] Staphylococcus aureus strains were selected as host bacteria for phage isolation. The treated samples and host bacteria were inoculated together into LB broth and cultured at 37°C with shaking until the OD of the bacterial culture reached a certain value. 600 = 0.6 to ensure normal growth of the host bacteria. The filtered liquid sample was mixed with the host bacteria, added to LB liquid medium, and co-cultured overnight in a 37°C shaker. The culture medium was centrifuged to remove bacterial residue, and the supernatant (i.e., the liquid containing bacteriophages) was collected and filtered through a 0.22 µm sterile filter membrane to remove residual bacteria, obtaining the enriched solution.

[0025] A spot test was used to detect the enrichment solution and determine the presence of the target phage in the sample. The phage sample to be tested was spotted onto a host bacterial culture plate and incubated overnight at 37°C. The appearance of transparent lysis spots was observed; these spots represent the lysis regions formed after phage infection of the bacteria. The phage was purified using a double-layer agar plate method until the phage spots on the plate were uniform in size. After multiple purifications, a purified phage suspension was obtained, ultimately yielding a lytic Staphylococcus aureus phage strain. Staphylococcus aureus Phage) vB_SauM_GZMU_SAY13 (hereinafter referred to as phage SAY13), phage SAY13 morphology as follows Figure 1 As shown.

[0026] 2. Phage titer detection The titer of phage SAY13 was determined and calculated using the double-layer agar plate method. First, the bottom layer of agar was poured into culture plates and allowed to solidify and cool completely before use. The purified phage suspension was serially diluted, and the diluted solution was mixed with the logarithmic-phase host bacterial suspension. After incubation at 37°C for 15 min, it was mixed thoroughly with 0.7% semi-solid LB agar medium (LB agar concentration of 7 g / L) and spread on top of the bottom layer plate. The mixture was then incubated overnight at 37°C. If the phage successfully infected bacteria, the bacteria would lyse, forming clear lysis plaques (phage plaques). Plates with 30-300 plaques per field of view were selected for plaque counting. The titer (PFU / mL) was calculated as: number of plaques × 10 × dilution factor. The results showed that the highest titer of this phage could reach 1.7 × 10⁻⁶. 10 PFU / mL or higher.

[0027] 3. Phage identification (1) Observation using transmission electron microscopy (TEM) To observe the morphological characteristics of bacteriophage SAY13, the sample was treated with phosphotungstic acid negative staining. First, the activated bacteriophage SAY13 suspension was dropped onto a 400-mesh carbon copper grid and allowed to stand for 2 minutes to allow the bacteriophage to fully adhere to the grid surface. Next, excess liquid around the grid was gently blotted away with filter paper, and a 1% phosphotungstic acid solution was added to the grid for negative staining for 1 minute. Excess phosphotungstic acid solution was again blotted away with filter paper, and the grid was allowed to air dry at room temperature. The morphology was observed using a Hitachi transmission electron microscope with an accelerating voltage of 80 kV, and the structural dimensions of the bacteriophage were measured using ImageJ software. Figure 2 As shown in the figure. The results show that the bacteriophage is a myotail bacteriophage with a contractile sheath structure at the tail, an icosahedral head with a head diameter of about 96 nm, a tail length of about 189 nm, and a tail tube length of about 23 nm.

[0028] (2) Phage whole genome analysis Single-virus genome sequencing of the bacteriophage was performed using the Illumina sequencing platform. After obtaining the sequencing data, quality control was performed, including assessing data quality and removing low-quality data to ensure the reliability of subsequent analyses. After removing host contamination, the genome was assembled using Megahit software, and the assembly performance was evaluated using Checkv.

[0029] Phylogenetic tree constructed using VIPTree default parameters ( Figure 3 The results showed that Staphylococcus aureus phage ( Staphylococcus aureus phage)vB_SauM_GZMU_SAY13 clusters with known members of the Kayvirus genus in the same evolutionary cluster and forms an independent branch within the cluster, suggesting that it is a new species of the Kayvirus genus.

[0030] To clarify the relationship between genome similarity and evolution, the distance between viral genomes (VIRIDIC) was used to analyze Staphylococcus aureus phages (…). Staphylococcus aureus A genome-wide nucleotide similarity analysis of phage vB_SauM_GZMU_SAY13 with 30 known Staphylococcus phages showing the highest similarity was less than 90%; among them, the similarity with... Staphylococcus Phage phiSA12 (NC_023573.1), Staphylococcus Phage K (NC_005880.2) Staphylococcus Phage J-Sa36 (MK417516.1), Staphylococcus The VIRIDIC Identity values ​​of phage vB_SauH_IME522 (MN304941.1) were 86.98%, 84.96%, 84.95%, and 84.57%, respectively (Table 1). Based on the International Committee on Taxonomy of Viruses (ICTV) standard for defining a new species as having a genome-wide nucleotide identity greater than 70% and less than 95%, Staphylococcus aureus phage (…) can be identified. Staphylococcus aureus phage)vB_SauM_GZMU_SAY13 is a novel bacteriophage of the Kayvirus genus.

[0031] Further, multiple tools such as BLAST (Identity, Cover, Identity×Cover), Matcher, and ClustalW were used to analyze Staphylococcus aureus phage ( Staphylococcus aureusThe genomes of the phage vB_SauM_GZMU_SAY13 were compared and verified with the four Kayvirus phages mentioned above, and the results are shown in Table 1. The results showed significant differences in the consistency patterns across multiple tools. The BLAST Identity×Cover values ​​ranged from 87.88% to 89.44%; the Matcher Identity values ​​showed significant divergence, with an Identity value of 79.80% with vB_SauH_IME522, but only 51.60%-51.70% with the other three phages; the ClustalW Identity value showed even more pronounced differences, with an Identity value of 80.81% with vB_SauH_IME522, but only 16.65%-16.67% with the other three phages. This discrepancy further corroborates the presence of Staphylococcus aureus phage (vB_SauM_GZMU_SAY13). Staphylococcus aureus The independence of the classification status of phage)vB_SauM_GZMU_SAY13.

[0032] In summary, Staphylococcus aureus bacteriophage ( Staphylococcus aureus Although phage)vB_SauM_GZMU_SAY13 possesses typical characteristics of the Kayvirus genus, it has a unique genetic background. Based on phylogenetic analysis, genome similarity assessment, and multi-tool whole-genome alignment results, it can be confirmed that it is a new species member of the Kayvirus genus.

[0033] Table 1. Comparison of the whole genomes of Staphylococcus aureus phage SAY13 and other members of the Kayvirus genus using different tools. 4. Preservation of bacteriophages Staphylococcus aureus phage ( Staphylococcus aureus phage)vB_SauM_GZMU_SAY13 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 28, 2026. Its taxonomic name is Staphylococcus aureus Phage, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 67758-B1.

[0034] Example 2: Host profile determination of Staphylococcus aureus bacteriophage vB_SauM_GZMU_SAY13 A dot-matrix assay was used to detect different host bacteria (Table 2) to determine the host range of Staphylococcus aureus phage vB_SauM_GZMU_SAY13. 100 μL of host bacteria in the logarithmic phase was mixed thoroughly with 0.7% semi-solid LB agar and spread onto a culture dish to prepare a plate containing the bacteria. 10 µL of Staphylococcus aureus phage vB_SauM_GZMU_SAY13 suspension (titer 10) was added... 10 A drop of PFU / mL was added to the surface of the plate and incubated overnight at 37°C. The appearance of plaques indicated that the phage could infect the corresponding host bacteria. The results are shown in Table 2. The lysis rate of Staphylococcus aureus phage vB_SauM_GZMU_SAY13 was 66% (23 / 35), which contained 3 methicillin-resistant Staphylococcus aureus strains.

[0035] Table 2. Host spectrum determination of 35 Staphylococcus aureus strains by Staphylococcus aureus bacteriophage vB_SauM_GZMU_SAY13 Note: "-" indicates no cleavage; "+" indicates cleavage.

[0036] Example 3: Biological characteristics of Staphylococcus aureus bacteriophage vB_SauM_GZMU_SAY13 1. Thermal stability Take 100 μL of Staphylococcus aureus phage vB_SauM_GZMU_SAY13 suspension (titer 10). 10 PFU / mL was added to 900 μL of sterile LB broth medium. Different temperature conditions were set (4℃, 37℃, 50℃, 60℃, with the corresponding LB broth medium pre-cooled or preheated). After the mixture was incubated at the set temperature for 1 hour, the titer of Staphylococcus aureus phage vB_SauM_GZMU_SAY13 was detected using the double-layer agar plate method. The results are as follows: Figure 4 As shown in the figure. The results show that Staphylococcus aureus phage vB_SauM_GZMU_SAY13 can maintain a relatively high titer in the range of 4℃-50℃, and Staphylococcus aureus phage vB_SauM_GZMU_SAY13 is inactivated when the temperature reaches 60℃ or above.

[0037] 2. pH stability Add hydrochloric acid or sodium hydroxide solution to sterile LB liquid medium to adjust the pH to the range of 1-13, then filter sterilize using a 0.22 μm microporous membrane. Take 100 μL of Staphylococcus aureus bacteriophage vB_SauM_GZMU_SAY13 suspension (titer 10). 10PFU / mL was added to 900 μL of LB liquid medium at different pH values ​​and incubated at 37°C for 1 hour. The phage titer was determined using the double-layer agar plate method. The results are as follows: Figure 5 As shown in the figure. The results show that Staphylococcus aureus bacteriophage vB_SauM_GZMU_SAY13 can maintain a high titer within a pH range of 4-10.

[0038] 3. Optimal Multiplicity of Infection To determine the optimal multiplicity of infection for this bacteriophage, host bacterial culture in the logarithmic growth phase was diluted to 1 × 10⁻⁶. 7 The cells were inoculated with CFU / mL and then infected with Staphylococcus aureus phage suspensions of different titers (vB_SauM_GZMU_SAY13). The phage titers at different multiples of infection (MOI) were determined using the double-layer agar plate method. The experimental results are shown below. Figure 6 As shown in the figure. The results indicate that the optimal multiplicity of infection for bacteriophage vB_SauM_GZMU_SAY13 is 10.

[0039] 4. Fracturing kinetics The logarithmic-phase host bacteria and Staphylococcus aureus phage vB_SauM_GZMU_SAY13 suspension were mixed at a 1:1 volume ratio to prepare seven different multiplicity of infection (MOI) mixtures (1000, 100, 10, 1, 0.1, 0.01, and 0.001), which were then added to 96-well plates. The logarithmic-phase host bacteria suspension served as a control. The 96-well plates were incubated at 37°C and 220 rpm on a shaker, with samples taken every hour to measure the OD. 600 The value was determined, and the experiment was repeated three times. The results are as follows: Figure 7 As shown, the absorbance of the pure bacterial culture in the control group increased rapidly. After 4 hours of culture, the host bacteria entered the logarithmic phase, at which point the OD of the Staphylococcus aureus phage vB_SauM_GZMU_SAY13 group increased significantly. 600 The values ​​showed statistical differences. All phage groups could inhibit the growth of Staphylococcus aureus for 9 h, and the inhibitory ability was relatively strong; however, after 9 h of culture, Staphylococcus aureus in the low infection complex group developed phage resistance and the strain began to grow slowly, while the high infection complex group could still inhibit the growth of Staphylococcus aureus for 12 h, indicating that the phage had a better inhibitory effect on the growth of Staphylococcus aureus.

[0040] 5. One-step growth curve According to the optimal multiplicity of infection ratio, the suspension of Staphylococcus aureus phage vB_SauM_GZMU_SAY13 was mixed with the logarithmic-phase bacterial culture and co-cultured on a shaker at 37°C for 15 minutes. Afterwards, the mixture was centrifuged (10 minutes), the supernatant was discarded, the precipitate was resuspended in LB liquid medium and washed, and then incubated on a shaker at 37°C and 220 r / min. Samples were taken every 20 minutes to determine the phage titer. The results are as follows: Figure 8 As shown in the figure. The results show that the incubation period of Staphylococcus aureus phage vB_SauM_GZMU_SAY13 is about 20 minutes, the outbreak phase occurs between 20 and 120 minutes, and the plateau phase occurs at about 180 minutes of culture.

[0041] 6. Staphylococcus aureus bacteriophage vB_SauM_GZMU_SAY13 inhibits biofilm formation. Crystal violet staining was used to investigate biofilm inhibition. Log-phase host bacterial suspension and Staphylococcus aureus bacteriophage vB_SauM_GZMU_SAY13 suspension were mixed at a 1:1 volume ratio and added to 96-well plates. MOI values ​​were set at 100, 10, and 1. An equal volume of log-phase host bacterial suspension 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 once with 200 μL PBS to remove airborne bacteria. After washing and air drying, 200 μL of 1% crystal violet solution was added for staining for 30 minutes. The staining solution was discarded, and the plates were washed three times. 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 in the figure. The results showed that the OD values ​​of all Staphylococcus aureus phage vB_SauM_GZMU_SAY13 groups were statistically significantly different from those of the positive control group (PC), indicating that Staphylococcus aureus phage vB_SauM_GZMU_SAY13 can effectively inhibit the formation of host biofilms, ultimately achieving the prevention and / or treatment of Staphylococcus aureus infection.

[0042] 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 Staphylococcus aureus bacteriophage ( Staphylococcus aureus phage)vB_SauM_GZMU_SAY13, characterized in that, The accession number of the Staphylococcus aureus bacteriophage vB_SauM_GZMU_SAY13 is GDMCCNo: 67758-B1.

2. The use of the Staphylococcus aureus bacteriophage vB_SauM_GZMU_SAY13 according to claim 1 in the preparation of medicaments for the prevention and / or treatment of Staphylococcus aureus infection.

3. The application of the Staphylococcus aureus bacteriophage vB_SauM_GZMU_SAY13 according to claim 1 in the preparation of Staphylococcus aureus bactericide.

4. The application according to claim 2 or 3, characterized in that, The Staphylococcus aureus mentioned includes methicillin-resistant Staphylococcus aureus.

5. A drug for the prevention and / or treatment of Staphylococcus aureus infection, characterized in that, The active ingredient includes the Staphylococcus aureus bacteriophage vB_SauM_GZMU_SAY13 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 Staphylococcus aureus.

9. A Staphylococcus aureus bactericide, characterized in that, The active ingredient includes the Staphylococcus aureus bacteriophage vB_SauM_GZMU_SAY13 as described in claim 1.

10. The Staphylococcus aureus bactericide according to claim 9, characterized in that, The Staphylococcus aureus bactericide is available in the form of spray, powder, gel, granules, or lyophilized agent.

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