A broad-spectrum Staphylococcus aureus phage SapYZUbeta and its applications

Abstract

This invention discloses a broad-spectrum Staphylococcus aureus bacteriophage, SapYZUbeta, and its applications. This bacteriophage has been deposited at the China Center for Type Culture Collection (CCTCC) on May 16, 2022, with accession number CCTCC NO: M 2022629. Identification revealed that the full-length gene of this bacteriophage is 17,790 bp, and it does not contain tRNAs or genes associated with lysogenicity, virulence, or antibiotic resistance. This genetic background confirms the safety of this bacteriophage for use against foodborne pathogens and for bacteriophage therapy. Furthermore, the bacteriophage exhibits a high titer of 1.9 × 10⁻⁶. 9 The phage, with a concentration of PFU / mL, exhibits stable activity within a pH range of 3–12 and a temperature range of -80–60°C, and can be applied to various matrices. The phage provided by this invention can lyse multidrug-resistant wild-type strains isolated from different regions, including MRSA and MSSA, and is characterized by a broad lysis spectrum and high lysis efficiency.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to a broad lysis spectrum Staphylococcus aureus bacteriophage SapYZUbeta and its applications. Background Technology

[0002] Staphylococcus aureus (SA) is a highly pathogenic bacterium that can cause a range of diseases in humans and livestock, from mild skin infections to life-threatening conditions such as pneumonia, toxic shock syndrome, and septicemia, seriously threatening the health and safety of the food industry and humans. It is highly resistant to environmental factors and has low nutritional requirements, thus it is widely distributed in nature, found in air, soil, water, and on food utensils. Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus aureus (MRSA) are frequently detected in clinical and livestock-related environments and food chains due to their phenotypic plasticity and adaptability, especially in milk samples. MRSA has a very balanced and mobile nutrient distribution, making it prone to rapid and difficult-to-control spread after contamination. However, ingestion of food contaminated with Staphylococcus aureus can lead to food poisoning, such as diarrhea and vomiting, and in severe cases, upper respiratory tract infections, purulent infections, and even death. Antibiotic therapy is generally used to prevent and treat Staphylococcus aureus, and this method is effective to some extent in the short term. Meanwhile, antibiotics are inexpensive and easy to administer, leading to over-reliance and excessive use, which in turn increases bacterial resistance. The most typical example is MRSA, known as a "superbug," causing enormous losses to humanity each year. In addition, physical methods for controlling Staphylococcus aureus have seen significant development and application in recent years, such as irradiation, pasteurization, and high hydrostatic pressure in food processing. However, some of these methods can cause adverse changes in the nutritional and sensory quality of food during the sterilization process. Furthermore, chemical methods for combating bacterial contamination have received considerable attention, but risks remain. For example, chemical disinfectants such as chlorine are not environmentally friendly, are harmful to public health, and may even form carcinogenic compounds under certain conditions. Therefore, there is an urgent need to develop novel biological antibacterial agents to reduce Staphylococcus aureus contamination.

[0003] Bacteriophages (or phages) are viruses that kill specific bacteria by binding to specific sites on the surface of bacterial cells and are widely distributed on Earth. Typically, bacteriophages replicate exponentially in the presence of host bacteria, eliminating target bacteria to achieve a therapeutic effect. To date, numerous research articles have been published on the use of bacteriophages in various matrices to treat different bacterial infections, and overall research results demonstrate their safety and efficacy. For example, in a 2020 human study, three bacteriophages, AB-SA01, used as adjunctive therapy, did not report any adverse reactions after 90 days of biochemical testing. As a therapeutic agent, bacteriophages have several advantages over antibiotics: targeted specificity that protects host bacteria, the ability to rapidly proliferate at the site of infection, harmlessness to humans, no development of resistance, and low production costs. However, highly efficient bacteriophages are needed for application or treatment, i.e., those screened based on their biological characteristics, thermal and pH stability, and lytic activity (in vitro, in vivo animal experiments, and food applications). Although bacteriophages are widely used and studied in the treatment of Staphylococcus aureus, finding candidates for Staphylococcus aureus bacteriophages is more challenging. Therefore, it remains necessary to isolate novel, specific bacteriophages with broad host range and strong lytic ability.

[0004] Common candidate bacteriophages for Staphylococcus aureus treatment are highly virulent phages belonging to Myoviridae (such as phages K and phiStau2A) and Podoviridae (such as phages CSA13 and phiSAP-2). Among them, Podoviridae phages, due to their smaller genomes, generally do not contain virulence genes, drug resistance genes, or lysogenic genes, and are considered potential biocontrol agents. Application studies of short-tailed phages are widely reported; their high specificity, safety, and strong lytic activity confirm their value as biocontrol agents. In 2019, Yoyeon Cha et al. isolated a highly lytic Podoviridae phage, CSA13, from pig farm wastewater. The results showed that this phage had a short latency period and a high burst rate, effectively inhibiting Staphylococcus aureus for up to 23 hours, confirming the inhibitory ability of phages against Staphylococcus aureus. A 2021 study demonstrated that high concentrations of the Podoviridae phage LSA2366, used alone or in combination with antibiotics, achieved the killing effect on over 90% of Staphylococcus aureus in milk. This suggests the potential for combined phage therapy with low-concentration antibiotics to treat mastitis in dairy cows. However, given the complex contamination of Staphylococcus aureus in food environments, isolating and screening for broad-spectrum phages from diverse sources remains essential. Summary of the Invention

[0005] Objective of the Invention: Addressing the problems of existing technologies, this invention provides a Staphylococcus aureus bacteriophage, SapYZUbeta. This bacteriophage has a broad host spectrum and exhibits strong lytic activity against multiple strains of Staphylococcus aureus from wild-type pigs, making it suitable for controlling Staphylococcus aureus contamination in food and production environments. Furthermore, it can withstand a wide temperature range (-80℃ to 70℃) and a wide pH range (2-13). This invention provides a bacteriophage with a broad host spectrum, which can prevent and reduce contamination by various Staphylococcus aureus strains. In addition, this bacteriophage can survive under high temperature and high acid / alkaline conditions, providing a guarantee for reducing the number of Staphylococcus aureus under extreme conditions.

[0006] The present invention also provides applications of the bacteriophage.

[0007] Technical solution: In order to achieve the above objectives, the broad host spectrum Staphylococcus aureus (SA) phage SapYZUbeta described in this invention has been deposited at the China Center for Type Culture Collection on May 16, 2022, with accession number CCTCC NO: M 2022629.

[0008] The bacteriophage of the present invention was isolated from sewage samples from agricultural markets in Yangzhou City, Jiangsu Province. It is a Staphylococcus aureus bacteriophage SapYZUbeta (Staphylococcusaureus phage SapYZUbeta) with highly efficient function of lysing multidrug-resistant wild Staphylococcus aureus.

[0009] The Staphylococcus aureus bacteriophage SapYZUbeta in this invention has the following biological characteristics:

[0010] (1) Morphological characteristics: When the bacteriophage SapYZUbeta uses Staphylococcus aureus ATCC 29213 as the host bacterium, it forms a plaque that is round, transparent, without a halo, and about 1-5 mm in diameter. According to the transmission electron microscope, the head of SapYZUbeta is symmetrical and has a short tail. Based on its morphological characteristics, the bacteriophage SapYZUbeta is classified into the family Brachypophagidae.

[0011] (2) Nucleic acid type: SapYZUbeta is a dsDNA phage.

[0012] (3) Genome characteristics: The SapYZUbeta genome is 17790bp in length, of which the total length of coding genes is 16815bp, with an average length of 764bp, accounting for 94.5% of the total length. The GC content is 29.2%, and it has 22 open reading frames (ORFs), including 10 hypothetical protein coding sequences and 12 sequences with known protein functions. It does not contain any known virulence genes.

[0013] (4) It has the function of strongly lysing Staphylococcus aureus from wild-type multidrug resistant wild pigs.

[0014] The application of the bacteriophage SapYZUbeta described in this invention in inhibiting Staphylococcus aureus.

[0015] The application of the bacteriophage SapYZUbeta in inhibiting Staphylococcus aureus in food, food production environment, or food production facilities.

[0016] The food products mentioned include grains, vegetables, dairy products, meat, eggs, and their derivatives.

[0017] The application of the bacteriophage SapYZUbeta in inhibiting a cocktail of Staphylococcus aureus (5 strains of MRSA mixed in a 1:1:1:1:1 ratio) in milk samples.

[0018] Preferably, the bacteriophage SapYZUbeta is used in the inhibition of Staphylococcus aureus under extreme temperature and / or extreme pH conditions.

[0019] The application of the bacteriophage SapYZUbeta described in this invention in the preparation of biological agents, antibacterial agents, or additives that inhibit Staphylococcus aureus.

[0020] The biological agent or antibacterial agent uses bacteriophage SapYZUbeta isolate or culture as the active ingredient or may also include excipients for preparing the agent.

[0021] The biological agent or antibacterial agent is used to remove Staphylococcus aureus and its contamination from food, production facilities, environment, and storage and transportation equipment.

[0022] Preferably, the biological agent or antibacterial agent is prepared as follows: SapYZUbeta phage is mixed with Staphylococcus aureus in logarithmic growth phase, placed at room temperature, and then added to LB liquid medium and cultured overnight with constant temperature shaking; the culture is transferred to a sterile centrifuge tube, centrifuged to collect the supernatant, filtered, and the phage proliferation solution is collected. PEG 8000 and NaCl are added, and the mixture is shaken until dissolved and incubated overnight; the supernatant is removed by centrifugation; SM buffer is added, and the reaction is carried out at room temperature; chloroform extraction is performed; the hydrophilic phase containing SapYZUbeta particles is recovered by centrifugation, and the obtained SapYZUbeta particles are mixed with SM buffer to prepare a stock solution of the phage biological agent or antibacterial agent.

[0023] The mother liquor of the bacteriophage SapYZUbeta biological agent or antibacterial agent is diluted with water and made into a spray solution or rinsing solution, which is used alone or in combination with other bactericides to spray or wash the production environment and production equipment to reduce the Staphylococcus aureus load and bacterial film formation in the food processing environment.

[0024] The biological agent or antibacterial agent is an additive in food raw material washing liquid, used to prevent the metabolism and reproduction of Staphylococcus aureus carried by food raw materials.

[0025] The present invention discloses a biological agent or bacteriostatic agent for inhibiting Staphylococcus aureus, which includes the bacteriophage SapYZUbeta or its culture as an active ingredient, alone or in combination to form a formulation.

[0026] This invention isolates a highly virulent Podoviridae bacteriophage from environmental wastewater, analyzes its physiological characteristics to determine its ability as a bioantibacterial agent, and preliminarily investigates its lytic activity against Staphylococcus aureus. The aim is to screen for highly efficient, broad-spectrum lytic Podoviridae bacteriophages as candidate bioantibacterial agents, providing a new strategy for effectively controlling Staphylococcus aureus growth and contamination. The bacteriophage provided by this invention can effectively control multiple strains of wild-swine-derived Staphylococcus aureus in milk samples, exhibiting high specificity, no residue, and safety compared to antibiotics or chemical preservatives. This invention can be used alone or in combination, providing a safe and non-toxic novel agent for controlling Staphylococcus aureus contamination.

[0027] This invention isolated a bacteriophage, SapYZUbeta, which differs significantly from the bacteriophage SapYZUalpha in the applicant's prior application (2022104112918). Firstly, this invention uses a short-tailed bacteriophage, while SapYZUalpha belongs to the myotailed bacteriophage family. Secondly, whole-genome sequencing shows that the complete genome length of this invention's bacteriophage is 17kb, while that of SapYZUalpha is 140kb, a significant difference that confirms they are not the same strain. Furthermore, this invention's bacteriophage exhibits good lysis ability against multiple strains of Staphylococcus aureus, with superior performance compared to the host bacterium ATCC 29213. For example, a doublet prepared using YZUsa12 as the host bacterium shows larger and clearer plaques.

[0028] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0029] 1. This invention isolates and obtains a bacteriophage, SapYZUbeta, which can efficiently lyse Staphylococcus aureus. It has unique morphological and genomic characteristics and is an effective bacteriophage for inhibiting Staphylococcus aureus. This bacteriophage can be used to prepare green and inexpensive Staphylococcus aureus inhibitors.

[0030] 2. The bacteriophage SapYZUbeta provided by this invention maintains good activity within a pH range of 3-12, and the phage titer does not change significantly, indicating that the phage can survive stably in various food matrices. Furthermore, phage SapYZUbeta can withstand a wide temperature range (-80℃-70℃), especially at low temperatures, where it maintains 100% activity. This allows the phage of this invention to effectively inhibit Staphylococcus aureus in foods stored at low temperatures and placed at high temperatures.

[0031] 3. The genome of the bacteriophage SapYZUbeta provided by this invention is only 17.8 kp, but its broad host spectrum confirms its uniqueness and shows potential application value. This bacteriophage can effectively infect and lyse all 53 Staphylococcus aureus strains isolated from human, pork, pig feces, pig snout, post-slaughter pork, and dust samples, and can also lyse Staphylococcus epidermidis ATCC 12228. However, it has no lytic ability against bacteria outside the Staphylococcus genus, such as Escherichia coli, Listeria monocytogenes, and 5 other bacteria, indicating that the bacteriophage SapYZUbeta has high specificity.

[0032] 4. The bacteriophage SapYZUbeta provided by this invention exhibits significant inhibitory effects on Staphylococcus aureus cocktail (a mixture of 5 MRSA strains in a 1:1:1:1:1 ratio) in LB broth, achieving a bactericidal rate as high as 99.99%. Furthermore, samples of milk contaminated with a simulated Staphylococcus aureus cocktail were used to evaluate the control effect of SapYZUbeta in food matrices. The results showed that the bacteriophage with an MOI of 100 showed good control effects against the Staphylococcus aureus cocktail in milk, with bactericidal rates of approximately 99.83% and 99.79% at 24 h and 48 h, respectively. In some studies, bacteriophages with an MOI of 10000 or higher have been selected to control Staphylococcus aureus in milk; for example, the Staphylococcus aureus bacteriophage LSA2308 only achieved a bactericidal rate of 97.79% (24 h) against a single strain of bacteria. Therefore, this invention can be used as a natural biological agent for the prevention and control of infections caused by Staphylococcus aureus, with better efficacy. Attached Figure Description

[0033] Figure 1 Plaque morphology of bacteriophage SapYZUbeta, the plaques shown were determined using Staphylococcus aureus ATCC29213 as the host;

[0034] Figure 2 Transmission electron micrograph of bacteriophage SapYZUbeta;

[0035] Figure 3pH stability of bacteriophage SapYZUbeta;

[0036] Figure 4 Thermodynamic stability of bacteriophage SapYZUbeta;

[0037] Figure 5 One-step growth curve of bacteriophage SapYZUbeta;

[0038] Figure 6 The whole genome sequence characteristics of bacteriophage SapYZUbeta;

[0039] Figure 7 Comparative structural analysis of bacteriophage SapYZUbeta and Staphylococcus aureus bacteriophage Sap-2;

[0040] Figure 8 Inhibitory effect of bacteriophage SapYZUbeta on wild-swine-derived Staphylococcus aureus cocktail (a mixture of 5 MRSA strains in a 1:1:1:1:1 ratio) in LB medium; where negative control (control group) represents the addition of 10 6 CFU / mL Staphylococcus aureus cocktail, the experimental groups were added with bacteriophage SapYZUbeta with MOI of 0.01, 1 and 100 respectively, and the OD value was measured during the isothermal incubation at 37℃.

[0041] Figure 9 The inhibitory effect of bacteriophage SapYZUbeta on the growth of a wild-type Staphylococcus aureus cocktail (a mixture of 5 MRSA strains in a 1:1:1:1:1 ratio) in a skim milk model (25°C). Figure A shows the change in the amount of the Staphylococcus aureus cocktail in milk; the negative control group represents the addition of 10... 6 CFU / mL Staphylococcus aureus cocktail was used in the experimental groups, with SapYZUbeta phage added at MOIs of 0.01, 1, and 100, respectively. The bacterial counts were varied during incubation at 37°C. Figure B shows the changes in phage counts at different MOIs in the milk.

[0042] Figure 10 The inhibitory effect of bacteriophage SapYZUbeta on the growth of a wild-type Staphylococcus aureus cocktail (a mixture of 5 MRSA strains in a 1:1:1:1:1 ratio) in a skim milk model (4°C). In the figure, A represents the change in the amount of Staphylococcus aureus cocktail in the milk, and negative (control group) represents the addition of 10... 6 CFU / mL Staphylococcus aureus cocktail was used in the experimental groups, with SapYZUbeta phage added at MOIs of 0.01, 1, and 100, respectively. The bacterial counts were varied during incubation at 37°C. Figure B shows the changes in phage counts at different MOIs in the milk. Detailed Implementation

[0043] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0044] Unless otherwise specified, all raw materials and reagents used in this invention are commercially available.

[0045] The bacteriophage host bacteria used in this invention, Staphylococcus aureus (strain number: ATCC 29213), was purchased from Beijing Biowell Biotechnology Co., Ltd.

[0046] The confirmatory strains in Table 2 of this invention, as well as the laboratory-isolated strains, were provided by Yangzhou University for isolation and screening or purchased commercially.

[0047] SM buffer (1L: NaCl 5.8g, MgSO4·7H2O 2.0g, 1M Tris-HCl (pH 7.4) 50mL)

[0048] Example 1

[0049] Phage isolation and purification preparation

[0050] bacteriophage isolation

[0051] The Staphylococcus aureus phage provided by this invention is isolated using ATCC 29213 as the host bacterium. 5000 mL of sewage sample was collected from a farmers' market in Yangzhou City, aliquoted into 50 mL centrifuge tubes, and centrifuged at 8,000 rpm for 10 min. The supernatant was filtered through 0.45 μm and 0.22 μm microporous filters to remove contaminating bacteria. 3 mL of the filtrate and 100 μL of the host bacterium cultured to the logarithmic growth phase were added to a test tube containing 3 mL of 2×LB culture medium and incubated overnight at 37°C and 125 rpm. The next day, the culture was centrifuged again and filtered through a membrane at 8,000 rpm for 10 min. The supernatant was filtered through 0.45 μm and 0.22 μm microporous filters. The resulting filtrate was the phage stock solution, stored at 4°C for later use.

[0052] The presence of bacteriophages was initially verified using a spotting method: 10 μL of phage stock solution was dropped onto the surface of a bilayer agar plate containing only host bacteria and incubated at 37°C for 12–18 h. The next day, if obvious plaques formed where the phage stock solution was added to the bilayer agar plate, the phage stock solution was serially diluted 10-fold with SM solution (10... -1 -10 -7 Furthermore, the double-layer plate method was adopted, and the culture was carried out overnight at 37°C.

[0053] Purification and preparation of bacteriophages

[0054] Pick a single, relatively independent, smooth-edged plaque from the cultured bilayer plate, inoculate it in 1 mL of SM buffer, mix well, and then perform serial dilutions (10⁻⁶). -1 -10 -7 Take an appropriate dilution (10). -6 Prepare a double-layer plate and incubate it upside down in a 37°C incubator. Repeat the above steps 5 times to purify the phage until phage plaques of approximately uniform size are obtained, which is the purified phage. Pick a single phage plaque from the purified double layer and place it in 1 mL of SM buffer, mix well, and incubate at 4°C for 24 h; collect the supernatant the next day and filter it through a 0.22 μm microporous membrane to obtain the phage purification solution. The titer of the phage purification solution is determined using a double-layer plate test. The specific procedure is as follows: perform a 10-fold serial dilution of the phage purification solution with SM buffer (10... -1 -10 -7 Prepare double-layer plates by mixing 100 μL of each dilution of phage with 100 μL of host bacteria in the logarithmic growth phase.

[0055] After solidification, the sample was inverted and incubated at 37°C for 12 hours. The resulting plaques were manually counted, and the titer was calculated. Results showed that each gradient of SapYZUbeta purification solution achieved a titer of approximately 10 for strain ATCC 29213. 9 PFU / mL or higher, and the phage plaques formed on the plate are bright, clear, and uniform in size (e.g., Figure 1 (As shown).

[0056] bacteriophage proliferation

[0057] Mix 100 μL of phage purification solution (prepared as above) with 100 μL of host bacteria in the logarithmic growth phase, incubate at room temperature for 5 min, then mix thoroughly with 3 mL of TSA semi-solid medium, pour onto LB agar plates, and incubate overnight at 37°C with shaking. The next day, remove the culture from the test tube, transfer it to a 10 mL sterile centrifuge tube, centrifuge at 8,000 rpm for 10 min, and filter the supernatant through a 0.22 μm sterile microporous membrane to obtain phage proliferation solution, which is stored at 4°C for later use.

[0058] Example 2

[0059] Characterization of bacteriophage SapYZUbeta

[0060] Phage morphological characteristics

[0061] The microscopic morphological characteristics of the bacteriophage SapYZUbeta isolated in Example 1 were observed using transmission electron microscopy. The purified bacteriophage solution obtained in Example 1 (10... 9 Concentrate (PFU / mL) to 10 10PFU / mL (concentrated phage suspension), take 20 μL of concentrated phage (10 10 A suspension of bacteriophage (pfu / mL) was dropped onto a 200-mesh carbon-coated copper grid. After adsorption for 15 minutes, the grid was removed and allowed to air dry for 2–3 minutes. The grid was then stained with 2% sodium phosphotungstenate solution (pH 7.6), blotted dry for 2 minutes, and allowed to air dry for 10 minutes. The phage was then observed using a transmission electron microscope (TEM, Hitachi H600A) at 100 kV, and clear images were selected for photographic analysis. The microscopic morphology of the purified bacteriophage SapYZUbeta is shown below. Figure 2 As shown, bacteriophage SapYZUbeta exhibits a symmetrical head with a diameter of approximately 36.96 nm and a short tail of approximately 12.53 nm. Based on its morphological characteristics, bacteriophage SapYZUbeta can be classified into the family Podoviridae.

[0062] Example 3

[0063] Effects of temperature and pH on bacteriophages

[0064] The valence processing and calculation methods are the same as in Example 1.

[0065] The valence is 10 8 1 mL of PFU / mL phage suspension was added to a test tube and incubated in a water bath at -80, -40, -20, 40, 50, 60, and 70°C for 1 h. Samples were taken every 20 min, and appropriate dilutions were performed. The phage titer was determined using the double-layer plate method. Each experiment was repeated three times. Phage thermostability (%) = Titer of surviving phage after heat treatment / Titer of phage before heat treatment × 100%.

[0066] The valence is 10 8 1 mL of PFU / mL phage suspension was added to a test tube, followed by 1 mL of SM buffer at pH values ​​of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0, respectively. The mixture was incubated at 37°C for 2 hours. The phage titer was then determined using the bilayer plate method after appropriate dilution. Each experiment was repeated three times. Phage pH stability (%) = (Title of surviving phage after pH treatment / Title of phage before pH treatment) × 100%.

[0067] Thermal stability results are as follows Figure 3As shown, the titer of bacteriophage SapYZUbeta did not change significantly after incubation for 1 hour in the range of -80℃ to -20℃, indicating that bacteriophage SapYZUbeta has good stability under low temperature conditions. When incubated at 40-60℃, the titer of bacteriophage decreased slightly, but the activity could still reach more than 80%. When incubated at 70℃ for 20 minutes, the activity of bacteriophage decreased to half of that under low temperature conditions. With the increase of time, the activity of bacteriophage gradually decreased until it was completely lost, indicating that bacteriophage SapYZUbeta has strong heat resistance.

[0068] Meanwhile, the pH stability results of bacteriophage SapYZUbeta are as follows: Figure 4 As shown, the results indicate that bacteriophage SapYZUbeta exhibits strong stability over a wide pH range of 3-12, demonstrating excellent acid and alkali tolerance. However, no live bacteriophages were detected when incubated at pH 2 and 13, as well as other extreme conditions.

[0069] Example 4

[0070] Determination of one-step growth curve

[0071] The purified phage fluid from Example 1 was mixed with Staphylococcus aureus ATCC 29213 (10) in the logarithmic phase. 8 The bacterial cell pellet (CFU / mL) was mixed at a ratio of 0.1 for a multiplicity of infection (MOI). After incubation at 37°C for 10 min, the mixture was centrifuged at 10000g for 30 s, the supernatant was discarded, and the pellet was resuspended in 10 mL of preheated LB medium. The pellet was then immediately placed in a shaker at 37°C (160 rpm). At different time points (0, 10, 20, 30, 40, 50, 60, 75, 90, 105, 120, 135, 150, 165, 180, 200, 220, and 240 min), 100 μL of culture medium was collected, and the phage titer was determined using the double-layer plate method. A one-step growth curve was plotted with sampling time on the x-axis and phage titer on the y-axis. Each experiment was performed in triplicate.

[0072] Implementation results are as follows Figure 5As shown, within 20 minutes of infection of the host bacteria, the titer of bacteriophage SapYZUbeta fluctuated slightly across three time periods without significant increases or decreases, indicating that the phage's latency period was approximately 20 minutes. From 20 minutes to 150 minutes, the phage titer continuously increased, indicating that the outbreak period of SapYZUbeta was approximately 130 minutes. After 150 minutes, the measured phage titer remained stable, indicating that the phage had entered its stationary phase. Calculations showed that the lysis rate of SapYZUbeta was approximately 152 PFU / CFU, demonstrating that the phage possesses strong lysis and replication capabilities, ensuring effective elimination of host bacteria within a short time.

[0073] Example 5

[0074] Whole genome characterization

[0075] The bacteriophage SapYZUbeta purification solution prepared in Example 1 above (10 9 Add DNase I to a final concentration of 5 μg / mL and RNase A to a final concentration of 1 μg / mL (PFU / mL), and incubate at 37°C for 1 h; add EDTA (pH 8.0) to a final concentration of 20 mmol / L; add proteinase K to a final concentration of 50 μg / mL and SDS to a final concentration of 0.5% (mg / mL), mix well, and incubate at 56°C for 1 h; add an equal volume of equilibrated phenol (pH 8.0) and shake to extract, centrifuge at 5000×g for 10 min, and collect the supernatant; extract with an equal volume of chloroform, centrifuge at 5000×g for 10 min, and collect the supernatant; add 1 / 10 volume of 3 mol / L NaAc (pH 5.2), then add two volumes of anhydrous ethanol to precipitate the nucleic acid, and incubate at -20°C overnight; centrifuge at 12000×g for 10 min at 4°C; wash the precipitate once with 70% ethanol and once with anhydrous ethanol, and air dry the precipitate for 10 min; use an appropriate amount of TE (pH 8.0) to extract the nucleic acid. 8.0) The phage DNA was quantified by suspension using a GeneQuant nucleic acid quantification instrument and stored at -20℃; the obtained phage DNA was extracted and sequenced using Illumina Hiseq.

[0076] Analysis of whole-genome sequencing results showed that ( Figure 6The SapYZUbeta phage genome consists of 17,790 bp of double-stranded DNA with an average GC content of 28.8%. GeneMarks gene prediction results (see Table 1 below) show that this phage has 22 open reading frames (ORFs) (16,806 bp), of which 12 ORFs encode functional proteins. Based on the protein sequence function prediction of the SapYZUbeta phage genome, it can be divided into three functional modules: the capsid morphogenesis module, the host cell lysis and tail morphogenesis module, and the DNA metabolism and assembly module. The capsid morphogenesis module contains 6 ORFs, 4 of which encode functional proteins: ORF3, ORF4, ORF5, and ORF6. A genome-wide comparison of SapYZUbeta phage with the highly homologous SAP-2 phage (92.6% homology) revealed… Figure 7 The capsid morphogenesis module of this phage shares 94.6% homology with that module of SAP-2. The host cell lysis and tail morphogenesis module contains five ORFs encoding two lysozymes (ORF7 and 11), one holin (ORF10), and two tail fibrins (ORF8 and 9). This region shares 92.1% nucleotide sequence identity with phage SAP-2. The two lysozymes and one holin encoded (see Table 2 below) show high sequence homology with Staphylococcus aureus phage LSA2366 (coverages of 100%, 100%, and 97%, and identity of 98.40%, 98.96%, and 100%, respectively). This phage exhibits a short latency period (10 min) and a high burst rate (258 PFU / mL), and can lyse 67% of drug-resistant Staphylococcus aureus (18 / 27). The DNA metabolism and assembly module consists of 11 ORFs, including those encoding DNA polymerase (ORF12), DNA packaging protein (ORF13), and DNA binding protein (ORF19), with a nucleotide sequence identity of 93.1% with this module of bacteriophage SAP-2. Furthermore, BLAST analysis showed that this bacteriophage is similar to the short-tailed bacteriophage CSA13 isolated from poultry samples (91.0% identity), indicating that bacteriophage SapYZUbeta belongs to a novel bacteriophage according to ICTV standards. In addition, no tRANA genes, lysogen genes, drug resistance genes, or virulence genes were found in the whole genome of bacteriophage SapYZUbeta, indicating that this bacteriophage can act as an inhibitory agent against Staphylococcus aureus.

[0077] Table 1. Complete genome annotation of SapYZUbeta

[0078]

[0079] Table 2. Gene sequences of ORF7, 10, and 11

[0080]

[0081]

[0082] The host range of bacteriophages was determined using the spot method. Sixty-two logarithmically cultured bacteria (100 μL) were evenly dispersed on LB agar plates. 10 μL of bacteriophage proliferation solution (10 μL) was then used. 8 PFU / mL was dropped onto the agar surface, with 10 μL of SM buffer as a control, and incubated overnight in an inverted 37°C incubator. The spots were categorized according to the transparency described in the literature (Li YK, Chen H, Shu M, et al. Isolation, characterization and application of an alkaline resistant viral bacteriophage JN01 against Escherichia coli O157:H7 in milk and beef[J]. LWT-Food Science and Technology. 2021, 144(2): 111266.). "+++" indicates transparent or semi-transparent spots; "++" indicates slightly turbid spots; "+" indicates turbid spots; and "-" indicates no spots. Each experiment was repeated three times.

[0083] The two genes encoding tail fibrin carried by bacteriophage SapYZUbeta share high amino acid sequence identity (83.6% and 97.6%) with bacteriophage CSA13. Studies have shown that bacteriophage CSA13 has a broad host spectrum, including MSSA, MRSA, and non-Staphylococcus aureus strains. Simultaneously, in the host spectrum determination results of this invention, bacteriophage SapYZUbeta also exhibits broad host characteristics; it can effectively infect and lyse all 53 Staphylococcus aureus strains isolated from human, pork, pig feces, pig snouts, pig carcasses, and dust samples. However, this bacteriophage cannot infect bacteria outside the Staphylococcus genus, such as Salmonella and Escherichia coli, indicating that bacteriophage SapYZUbeta has high specificity and particularity towards its host bacteria. It is worth noting that all the Staphylococcus aureus isolates tested came from different regions (Guangzhou, Xiamen, and Yangzhou), with a large geographical range. Furthermore, the phage was also able to lyse Staphylococcus epidermidis ATCC 12228, which is not Staphylococcus aureus. This indicates that the lysis spectrum of phage SapYZUbeta is extensive, as detailed in Table 3 below.

[0084] Table 3. Host spectrum of SapYZUbeta

[0085]

[0086]

[0087]

[0088] “+++” indicates that the plaque is clear and translucent.

[0089] "++" indicates that the plaque is slightly turbid.

[0090] “+” indicates plaque turbidity, and “-” indicates no plaque.

[0091] ATCC, American Type Culture Collection.

[0092] Example 6

[0093] Antibacterial effect of bacteriophage SapYZUbeta in LB broth

[0094] Five strains of Staphylococcus aureus from wild-caught pigs (laboratory isolates: YZUsa1, YZUsa4, YZUsa12, and YZUsa14; standard strain: MRSA JCSC 4744) were cultured to the logarithmic growth phase, centrifuged at 9000g for 2 min, washed with PBS (pH 7.4) to remove the culture medium, and the concentration of each bacterial strain was adjusted to 10. 6 CFU / mL was mixed at a volume ratio of 1:1:1:1:1 (Staphylococcus aureus cocktail). Then, 100 μL of the Staphylococcus aureus cocktail was mixed with bacteriophages at different MOI ratios (0.01, 1, and 100) in equal volumes (mixtures). 2 μL of the mixture was placed in a 96-well plate containing 198 μL of TSB medium and incubated at 37 °C for 24 h. The OD value was measured at OD590 nm every 3 h. The experiment was repeated 5 times.

[0095] Implementation results are as follows Figure 8As shown, the number of bacteria in the control group (without phage) did not change significantly within 9 hours, but bacterial growth began after 9 hours, and the number continued to increase. When different concentrations of phage SapYZUbeta were added, the OD value of bacteria in the broth was controlled at around 0.05 within 15 hours, similar to the blank group, indicating that the phage had a very significant control effect on the five-strain bacterial cocktail. After co-incubation with the phage for 24 hours, it was observed that when the MOI was 0.01, the control of bacteria decreased slightly, but its OD value was still far below that of the control (1 / 3). In addition, when the broth was treated with phages at MOIs of 1 and 100, it continuously and significantly inhibited the Staphylococcus aureus cocktail throughout the test period, with an inhibition rate as high as 99.99%.

[0096] Example 7

[0097] Antibacterial effect of bacteriophage SapYZUbeta against Staphylococcus aureus cocktail in milk

[0098] Five porcine Staphylococcus aureus strains (YZUsa1, YZUsa4, YZUsa12, YZUsa14 and MRSAJCSC 4744) from the host spectrum were cultured to the logarithmic growth phase, and then the concentration of each strain was adjusted to 10. 6 CFU / mL, mixed at a volume ratio of 1:1:1:1:1 (Staphylococcus aureus cocktail), set aside. Purchase ultra-high temperature pasteurized skim milk from the supermarket, artificially inoculate 100uL of Staphylococcus aureus cocktail into 10mL of milk, and add to a final concentration of 10... 4 10 6 and 10 8 PFU / mL phage suspension (MOIs of 0.01, 1, and 100), while the control group received only 100 μL of a mixed bacterial suspension of Staphylococcus aureus (S. aureus) and its cocktail. Both groups were incubated at 25°C for 48 h, with 1 mL samples taken at 0, 3, 6, 9, 12, 24, and 48 h for processing. Alternatively, both groups were incubated at 4°C for 7 days, with 1 mL samples taken every other day for processing. Finally, the bacteria and phages in the samples were separated. Specifically, the samples were centrifuged at 6000g for 10 min to separate the bacteria and phages. The precipitated bacterial cells were resuspended in 1 mL of PBS, and this centrifugation and resuspension process was repeated three times. The collected bacterial cells were plated onto mannitol agar, and viable counts were performed. The supernatants from the three separation processes were collected, filtered through a 0.22 μm filter, and then used to count the phages using the double-layer plate method. Each group of experiments was repeated three times, with three parallel trials.

[0099] Implementation results are as follows Figure 9 and Figure 10To verify the bactericidal effect of bacteriophage SapYZUbeta in a food matrix, milk samples were simulated at 25°C and 4°C. At both temperatures, SapYZUbeta effectively controlled the amount of bacteria in the milk mixture. In milk at 25°C (… Figure 9 A) In the control group, the bacterial count continued to increase to approximately 9 Log10 CFU / mL over time. When treated with phages at MOIs of 1 and 0.01, there was no significant inhibitory effect on the bacterial cocktail within 12 hours, but both reduced the number of the five bacterial strains to varying degrees after 12 hours (1.4 Log10 CFU / mL and 0.5 Log10 CFU / mL, respectively). At an MOI of 100, phage SapYZUbeta co-grows with the bacteria before 6 hours; however, after 6 hours, this phage showed better control over the total number of the five bacterial strains in the milk, reducing the overall bacterial count by 2.8 Log10 CFU / mL and 2.7 Log10 CFU / mL at 24 and 48 hours, respectively, with sterilization rates of approximately 99.83% and 99.78%.

[0100] In addition, milk samples artificially inoculated with Staphylococcus aureus cocktail at 4°C using the above method were incubated for 7 days. Figure 10 A) The growth of the five Staphylococcus aureus strains was relatively slow, increasing only from approximately 4 Log10 CFU / mL to 4.4 Log10 CFU / mL. In contrast, the phage treatment groups with the three MOIs all significantly reduced the total bacterial count. When MOIs were added at 0.01 and 1, the inhibition rates against the Staphylococcus aureus cocktail reached approximately 60% (day 7) and 50% (day 2), respectively. Furthermore, in the phage treatment group with an MOI of 100, the best bacterial control effect in milk was observed only on day 2, with a sterilization rate of approximately 62%. However, during continuous culture, its bacterial control effect was lower than that of the phage treatment groups with MOIs of 0.01 and 1. Research indicates that the application of phages is closely related to temperature, as phage growth primarily relies on the growth of the host to reproduce. Bacteria typically grow faster within their optimal temperature range, indirectly promoting phage replication. Low temperatures, on the other hand, indirectly lead to a significant reduction or cessation of phage replication. Therefore, the overall results show that the bacteriophage SapYZUbeta was more effective at controlling a mixture of five wild-swine-derived Staphylococcus aureus strains in milk at room temperature than at low temperature. Furthermore, since 4°C is the optimal storage temperature for both bacteria and bacteriophages, meaning that the numbers of bacteriophages and bacteria do not increase or decrease significantly at this temperature, bacterial growth is slower, and bacteriophage reproduction depends on the host bacteria; therefore, the effect was not as good as at room temperature.

[0101] In addition to studying the changes in bacterial colony counts in skim milk, the changes in the number of bacteriophages in the system were also measured. At 25°C ( Figure 9 B) When the bacteriophage SapYZUbeta with an MOI of 100 controlled multiple bacterial strains, the number of bacteriophages in the milk system steadily increased to 7.0 log10 PFU / mL within the first 24 hours, and then dropped sharply to the initial value at 24 hours, indicating that the bacteriophage reached an extreme value at 24 hours, i.e., it had the best effect on controlling the number of mixed bacteria. At MOIs of 1 and 0.01, the number of bacteriophages was relatively stable within 6 hours, gradually increasing from 6 hours, eventually reaching a maximum and stabilizing. On the other hand, under conditions of 4℃ ( Figure 10 B) The number of phages of the three MOIs in the liquid did not change significantly, and this temperature may be a good temperature for short-term storage of phages.

Claims

1. A broad-spectrum Staphylococcus aureus (Staphylococcus aureus) SA ） Bacteriophage SapYZUbeta has been deposited at the China Center for Type Culture Collection on May 16, 2022, with accession number CCTCC NO: M2022629.

2. The use of the bacteriophage SapYZUbeta according to claim 1 in inhibiting Staphylococcus aureus.

3. The application according to claim 2, characterized in that, The application of the bacteriophage SapYZUbeta in inhibiting Staphylococcus aureus in food, food production environment, or food production facilities.

4. The use of the bacteriophage SapYZUbeta according to claim 1 in the preparation of an antibacterial agent for inhibiting Staphylococcus aureus.

5. The application of the bacteriophage SapYZUbeta according to claim 1 in the preparation of an additive to inhibit Staphylococcus aureus, wherein the additive is a food raw material washing liquid additive used to prevent Staphylococcus aureus carried by food raw materials from metabolizing and multiplying.

6. The application according to claim 4, characterized in that, The antibacterial agent uses bacteriophage SapYZUbeta isolates or cultures as its active ingredient.

7. The application according to claim 6, characterized in that, The antibacterial agent also includes excipients for preparing the formulation.

8. The application according to claim 4, characterized in that, The antibacterial agent is used to remove Staphylococcus aureus contamination from food and production facilities, the environment, and storage and transportation equipment.

9. The application according to claim 4, characterized in that, The antibacterial agent is diluted with water to make a spray or rinsing solution, which can be used alone or in combination with other bactericides to spray or wash the production environment and equipment to reduce the Staphylococcus aureus load in the food processing environment.

Images