Staphylococcus aureus bacteriophage with resistance to gastric juice treatment and persistent bacteriostatic effect and application thereof

Abstract

The application belongs to the technical field of microorganisms, and specifically discloses a staphylococcus aureus bacteriophage resistant to gastric fluid treatment and having a persistent bacteriostatic effect and application thereof. The bacteriophage was preserved in the China General Microbiological Culture Collection Center on May 28, 2026, and the preservation number is CGMCC No. 47052. The bacteriophage remains high stability in the pH 5-11 range, retains about 10 6 PFU / mL activity after being treated in simulated gastric fluid containing pepsin for 120 min, and the activity does not decrease by more than 2 orders of magnitude, and the bacteriophage exhibits good thermal stability under the condition of 30-50 DEG C. At the same time, the bacteriophage has a sustained inhibition effect of about 42.6 h on staphylococcus aureus. The bacteriophage of the application can maintain a high survival rate under the conditions of acid digestion environment and temperature stress, and maintain a long-term bacteriostatic effect after action, thereby improving the stability of the bacteriophage in oral and complex environment applications. The bacteriophage of the application can be used for preparing a preparation for resisting staphylococcus aureus infection.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Staphylococcus aureus bacteriophage that is resistant to gastric juice treatment and has a long-lasting antibacterial effect and its application. Background Technology

[0002] Staphylococcus aureus is a common zoonotic foodborne pathogen with high pathogenicity. It is widely distributed in nature and can cause various diseases in humans and animals, including localized purulent infections, pneumonia, osteomyelitis, and septicemia. This bacterium has low nutritional requirements and strong resistance to environmental factors. It is widely present in air, soil, water bodies, and livestock farming environments, with particularly prominent contamination issues in dairy products, raw meat, and frozen foods. It has been reported that food poisoning incidents caused by Staphylococcus aureus account for more than 30% of bacterial food poisoning incidents worldwide, and it is also relatively common among bacterial foodborne illnesses in my country.

[0003] Currently, the prevention and treatment of Staphylococcus aureus infection mainly relies on antibiotics. However, with the long-term, large-scale, and irrational use of antibiotics in clinical and aquaculture fields, drug-resistant strains of Staphylococcus aureus are constantly emerging, significantly limiting the therapeutic effect of traditional antibiotics and necessitating the development of new antibacterial strategies. Bacteriophages, as a class of viruses capable of specifically infecting bacteria, possess characteristics such as strong host specificity, minimal impact on the host microecology, and self-replication, and are considered a potential alternative strategy for combating drug-resistant bacterial infections. However, in practical applications, existing bacteriophages still have certain limitations: on the one hand, most natural bacteriophages are prone to structural damage or loss of activity in low pH and protease-containing environments, especially exhibiting poor stability in highly acidic environments such as simulated gastric juice and enzymatic environments, limiting their application potential in complex physiological or physiological-like environments; on the other hand, existing bacteriophages often suffer from limited duration of action and insufficient antibacterial persistence during in vitro antibacterial processes, making it difficult to achieve long-term stable inhibition of bacteria.

[0004] Therefore, how to obtain a bacteriophage that can maintain high stability in the gastric fluid environment and achieve long-term sustained inhibition of Staphylococcus aureus remains a technical problem that urgently needs to be solved in this field.

[0005] The information disclosed in this background section is only intended to enhance the understanding of the background technology of this application and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] To address the problems of existing bacteriophages being easily inactivated in low pH and pepsin-containing environments, and having a limited duration of inhibitory effect on Staphylococcus aureus, this invention provides a Staphylococcus aureus bacteriophage that is resistant to gastric juice treatment and has a long-lasting antibacterial effect, as well as its applications.

[0007] In a first aspect, this invention provides a Staphylococcus aureus bacteriophage strain resistant to gastric juice treatment and possessing long-lasting antibacterial activity, named RDP-SR-25010, which was deposited on May 28, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 47052, and classified as Staphylococcus aureus bacteriophage. Staphylococcus aureus bacteriophage.

[0008] Preferably, after treatment with simulated gastric juice containing NaCl and pepsin at pH 2.0 for 120 min, the titer of the bacteriophage decreases by no more than two orders of magnitude, and the titer is not lower than 10. 6 PFU / mL.

[0009] Preferably, the bacteriophage exhibits a sustained inhibitory effect on Staphylococcus aureus for up to 42.6 hours.

[0010] Preferably, the titer of the bacteriophage does not decrease by an order of magnitude after treatment at pH 5-11 for 1 h, and the titer decreases by no more than two orders of magnitude after treatment at strong acid conditions (pH 2-3) or strong alkaline conditions (pH 12) for 1 h.

[0011] Preferably, the titer of the bacteriophage decreases by no more than two orders of magnitude after treatment at 30–50°C for 6 hours, and it retains its infectious activity after treatment at 70°C for 2 hours.

[0012] Secondly, the present invention provides the use of the above-mentioned Staphylococcus aureus phage in the preparation of formulations for inhibiting or inactivating Staphylococcus aureus.

[0013] Preferably, the formulation is an oral formulation, based on the fact that the bacteriophage retains approximately 10% of its contents after treatment with simulated gastric juice at pH 2.0 and containing pepsin for 120 min. 6 The physiological characteristics of PFU / mL activity make it suitable for oral delivery systems.

[0014] Preferably, the preparation is a topical antibacterial preparation or a wound treatment preparation, based on the physiological characteristic that the bacteriophage maintains stable activity in the pH range of 5 to 11.

[0015] Preferably, the formulation is an environmental treatment or disinfection formulation, based on the physiological characteristic that the bacteriophage retains high activity after being treated at 30–50°C for 6 hours.

[0016] Preferably, the formulation is used to prepare a sustained-release antimicrobial agent based on the physiological characteristic that the bacteriophage has a sustained inhibitory effect on Staphylococcus aureus for about 42.6 h.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) According to existing technology, bacteriophages are generally considered to be prone to structural damage or significant reduction in activity in simulated gastric juice environments with low pH and containing proteases, making it difficult to maintain infectious activity in such complex environments. The bacteriophage provided by this invention can still maintain high activity under simulated gastric juice (pH 2.0, containing NaCl and pepsin) treatment conditions, and the titer decreases by no more than two orders of magnitude after 120 min, indicating that it has excellent gastric juice environment stability, which is beneficial to its survival and application in complex acidic and enzyme-containing environments; (2) The bacteriophage of the present invention has a long-term sustained inhibitory effect on Staphylococcus aureus in the in vitro culture system, with an inhibition time of up to 42.6 h, which is significantly better than the short-term antibacterial characteristics of conventional bacteriophages, indicating that it has good sustained antibacterial ability; (3) The bacteriophages of the present invention maintain good activity stability after treatment in different pH environments (pH 2.0~12.0) and certain temperature conditions (30~50℃). After treatment with pH 2.0~13.0, the titer decreases by no more than 3 orders of magnitude, and after treatment at 30~50℃ for 6 h, the titer decreases by no more than 3 orders of magnitude, which provides a basic guarantee for its stability in complex storage, transportation and application environments. Attached Figure Description

[0018] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the specific embodiments will be briefly introduced below. Obviously, the following description is only a part of the embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The plaque is from RDP-SR-25010.

[0020] Figure 2 Transmission electron microscope image of RDP-SR-25010.

[0021] Figure 3 The results show the acid and base tolerance of RDP-SR-25010.

[0022] Figure 4 The results are from the temperature stability test of RDP-SR-25010.

[0023] Figure 5 Results of the antibacterial duration of RDP-SR-25010. Detailed Implementation

[0024] This invention presents a Staphylococcus aureus bacteriophage that is resistant to gastric juice treatment and has a long-lasting antibacterial effect, and its application. To facilitate understanding of this invention by those skilled in the art, the specific embodiments of this invention are described below with reference to the accompanying drawings.

[0025] In this invention, unless otherwise specified, the equipment and raw materials used are commercially available or commonly used in the art. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0026] Example 1: Isolation and purification of Staphylococcus aureus bacteriophage RDP-SR-25010 1.1 Experimental Methods (1) Isolation of bacteriophages Fecal samples were collected from the farm, and an appropriate amount of SM buffer was added. The samples were centrifuged at 12000 rpm for 10 minutes. The supernatant was filtered through a 0.22 μm filter membrane to obtain the phage stock solution. 200 μL of the filtrate was mixed with 200 μL of Staphylococcus aureus bacterial culture (concentration approximately 10). 8 The phage (CFU / mL) was placed in 5 mL of LB broth and cultured at 37°C and 160 rpm for 6 h with shaking to allow the phage to fully interact with the host bacteria.

[0027] After the culture was completed, the mixture was filtered through a 0.22 μm filter membrane to remove bacteria, and a preliminary phage lysate was obtained.

[0028] The above lysis buffer was serially diluted 10-fold, and 100 μL of each of the different dilutions was taken. -1 10 -2 10 -3 10 -4 The sample was mixed with 100 μL of host bacteria, then 5 mL of LB semi-solid medium was added. After mixing well, the mixture was quickly poured onto the surface of an LB solid plate to form a two-layer plate system. After the plates solidified, they were incubated upside down in a 37°C incubator overnight. Plaque formation was observed the next day.

[0029] (2) Purification of bacteriophages Select a single, clear phage plaque and inoculate it into 5 mL of LB broth containing 100 μL of Staphylococcus aureus. Incubate at 37°C and 160 rpm for 6 h with shaking for amplification. Filter the amplified solution again through a 0.22 μm filter to obtain the phage filtrate. Observe the morphology of the plaques using the double-layer agar plate method. Repeat the above purification process 3–5 times until phage plaques of uniform shape and size are obtained; in this example, the process was repeated 3 times.

[0030] 1.2 Experimental Results and Analysis Observed using the double-layer plate method, such as Figure 1 As shown, the phage formed transparent plaques with a diameter of about 0.5 mm on LB plates, with clear edges and no obvious halo, indicating that it has strong lytic activity and good phage population uniformity.

[0031] Example 2: Morphological observation of RDP-SR-25010 2.1 Experimental Methods Place the copper mesh on clean filter paper and use a pipette to draw 20 μL of phage suspension (titer 10). 10 PFU / mL was added to a copper grid and allowed to stand at room temperature for 15 min to allow the phages to fully adsorb onto the grid surface. Then, excess liquid was removed by siphoning through the edge with filter paper. After air drying, 1 drop of 2% phosphotungstic acid staining agent (PTA, pH 6.8) was added to cover the sample area and stained at room temperature for 10 min. After staining, excess staining solution was absorbed with filter paper, and after air drying, the phage morphology and structure were observed under a transmission electron microscope.

[0032] 2.2 Experimental Results and Analysis like Figure 2 As shown, RDP-SR-25010 exhibits a typical long-tailed bacteriophage morphology, consisting of a head and a tail. The head has a polyhedral symmetry structure with a diameter of approximately 75 nm; the tail is slender, with a length of approximately 270 nm. According to the classification criteria of the International Committee on Taxonomy of Viruses (ICTV), this bacteriophage belongs to the typical tailed bacteriophage family, and its morphological characteristics are consistent with those of long-tailed virus phages.

[0033] Example 3 Whole-genome sequencing analysis of RDP-SR-25010 3.1 Experimental Methods After amplification and culture of the purified phage fluid, the phage particles were enriched by precipitation using a mixture of 10% PEG8000 and 0.5 M NaCl at 4°C overnight. The next day, the precipitate was collected by centrifugation at 12000 rpm for 30 min, resuspended in SM buffer, and phage genomic DNA was extracted using the standard λ phage DNA extraction method. After passing quality testing, the extracted DNA samples were sent to a sequencing institution (BGI Genomics) for whole-genome sequencing.

[0034] 3.2 Experimental Results and Analysis The whole genome size of phage RDP-SR-25010 is 50478 bp. No known virulence genes, drug resistance genes, or lysogen-associated integrase genes were detected in the phage genome.

[0035] Example 4: Acid and base tolerance test of RDP-SR-25010 4.1 Experimental Methods Take the purified phage stock solution (potency approximately 10). 9 Phage titers (PFU / mL) were mixed in equal volumes with PBS buffers of different pH gradients (pH 2.0, 3.0, 4.0, 5.0, 7.0, 9.0, 10.0, 11.0, 12.0, 13.0). The mixtures were then incubated in a 37°C water bath for 1 h to fully expose the phages to different acidic and alkaline environments. Immediately after treatment, the reaction was terminated by an ice bath, and the samples were treated with a 10-fold serial dilution method. The phage titers (PFU / mL) of each treatment group were then determined using the double-layer plate method, with three parallel samples in each group, and the average value was taken as the final result.

[0036] 4.2 Experimental Results and Analysis like Figure 3 As shown, the potency of RDP-SR-25010 is basically stable in the pH range of 5 to 11, with a potency of approximately 10. 9 The PFU / mL concentration did not show a significant order-of-magnitude decrease; the potency decrease after treatment under strong acidic conditions (pH 2-3) and strong alkaline conditions (pH 12) was no more than two orders of magnitude, and even after treatment at pH 13, the potency decrease was no more than three orders of magnitude. Overall, it exhibits good survival ability over a wide pH range, and retains some activity even under extreme acidic and alkaline conditions, indicating that it possesses strong environmental adaptability and structural stability.

[0037] It should be noted that the acid-base stability test results mainly reflect the tolerance of bacteriophages in a single chemical environment, but cannot directly predict their stability in complex physiological environments containing proteases, thus providing a comparative basis for subsequent experiments simulating gastric juice environment tolerance.

[0038] Example 5: Gastric juice tolerance test of RDP-SR-25010 5.1 Experimental Methods To evaluate the stability of the bacteriophage of this invention in oral-related physiological environments, simulated gastric juice (pH 2.0, containing pepsin) was prepared according to literature and conventional methods, specifically as follows: Based on NaCl solution, pepsin was added to a final concentration of 0.32% (w / v), and the pH was adjusted to 2.0 with HCl to obtain simulated gastric juice containing pepsin. A pH 2.0 control solution without pepsin was also prepared.

[0039] The phage stock solution was mixed with simulated gastric fluid at a volume ratio of 1:9 (i.e., 100 μL of phage solution was mixed with 900 μL of simulated gastric fluid) and incubated in a 37°C water bath. Samples were taken at 0, 15, 30, 60, 90, and 120 min, with 20 μL of sample taken each time. Immediately afterward, 180 μL of pre-cooled SM buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM MgSO4·7H2O, 0.01% gelatin) was added to terminate pepsin activity and neutralize the acidic environment. Subsequently, the samples were serially diluted 10-fold using SM buffer, and the phage titer (PFU / mL) at each time point was determined using the double-layer plate method. Three replicates were set up for each group, and the average value was taken.

[0040] Two control groups were set up: (1) a control group with pH 2.0 but without pepsin; and (2) a positive control group with pH 7.2 SM buffer (without pepsin). The operation procedure was the same as that of the experiment with simulated gastric juice.

[0041] 5.2 Experimental Results and Analysis As shown in Table 1, in a simulated gastric fluid environment containing pepsin, the titer of RDP-SR-25010 gradually decreased over time, but the overall decrease was limited. After 120 min of treatment, the titer decreased by no more than two orders of magnitude, and the phage titer remained at approximately 10. 6 PFU / mL level. Meanwhile, control experiments showed that in a pH 2.0 environment without pepsin, the titer change trend was basically consistent with that of the pepsin-containing group, with no significant additional decrease observed.

[0042] The results show that the bacteriophage of the present invention can not only tolerate strong acid environment, but also has a strong resistance to structural damage caused by pepsin. It can still maintain a high survival rate under the dual action of "low pH + protease", indicating that the bacteriophage has excellent characteristics for development into oral formulation.

[0043] It should be noted that, in the prior art, it is generally believed that bacteriophages are prone to rapid inactivation in a simulated gastric fluid environment where strong acid and proteases coexist. However, the bacteriophage of the present invention still shows significant survival ability in this complex environment, which is a stability performance that exceeds conventional expectations.

[0044] Table 1. Gastric juice tolerance results of RDP-SR-25010

[0045] Example 6 Temperature stability test of RDP-SR-25010 6.1 Experimental Methods Take the purified phage stock solution (potency approximately 10). 9 Phage titers (PFU / mL) were dispensed into sterile centrifuge tubes and treated at different temperatures (30℃, 40℃, 50℃, 60℃, and 70℃) for 1 h, 2 h, 4 h, and 6 h, respectively. After treatment, the centrifuge tubes were removed and cooled immediately, and the samples were treated with a 10-fold serial dilution method. Subsequently, the phage titers (PFU / mL) of each treatment group were determined using the double-layer plate method, with 3 parallel samples set up for each group.

[0046] 6.2 Experimental Results and Analysis like Figure 4 As shown, after treatment at 30℃~40℃ for 6 h, the titer of RDP-SR-25010 remained relatively stable without a significant decrease of orders of magnitude. After treatment at 50℃ for 6 h, the phage titer decreased by about two orders of magnitude. After treatment at 60℃ for 6 h, the phage titer decreased by about four orders of magnitude, but phage particles with infectious activity could still be detected. After treatment at 70℃ for 2 h, the phage still retained some activity, but gradually became inactive after 4 h.

[0047] The above results indicate that the bacteriophage of the present invention has good thermal stability and can maintain high activity under normal ambient temperature and moderate heat stress conditions. This is beneficial for maintaining its biological activity stability during transportation, storage and practical application, and provides a basic support for its industrial application.

[0048] Example 7: Durable antibacterial experiment of RDP-SR-25010 7.1 Experimental Methods Under aseptic conditions, add 20 mL of LB liquid culture medium to a 50 mL Erlenmeyer flask, and add 10 mL of LB liquid culture medium at a 2% inoculum volume. 9 CFU / mL Staphylococcus aureus culture was thoroughly mixed and then dispensed into sterile stoppered colorimetric tubes.

[0049] The experimental groups are as follows: Group 1 is the Staphylococcus aureus culture control group; Group 2 is the phage treatment group (adding 10 at a 2% inoculum rate). 9 PFU / mL RDP-SR-25010 phage stock solution); Group 3# blank culture medium control group.

[0050] All samples were placed in a microbial growth instrument for culture and monitoring. The culture conditions were constant temperature of 37℃ for 84 h. The OD600 value was measured every 10 min to reflect the dynamic changes in bacterial growth.

[0051] 7.2 Experimental Results and Analysis like Figure 5 As shown, in the control group (1#), Staphylococcus aureus showed a normal exponential growth trend, with OD600 continuously increasing; the phage-treated group (2#) showed a significant inhibitory effect from the initial stage, with OD600 remaining at a very low level for approximately 42.6 h (2560 min), and no obvious bacterial proliferation trend was observed. With prolonged culture time, the phage-treated group remained significantly lower than the control group, indicating that the phage has a sustained inhibitory effect on Staphylococcus aureus, rather than a short-term transient bactericidal effect.

[0052] The above results show that the bacteriophage of the present invention can maintain its inhibitory effect on the host bacteria for a long time after action, and has good sustained antibacterial ability, providing functional support for its application in complex environments.

[0053] In summary, phage RDP-SR-25010 maintains high stability over a wide pH range (pH 5–11), retaining approximately 10% of its original pH after treatment with simulated gastric fluid containing pepsin for 120 min. 6 It exhibits PFU / mL activity (decreased by no more than two orders of magnitude) and good thermal stability at 30–50°C. Furthermore, it demonstrates sustained inhibition against Staphylococcus aureus for approximately 42.6 h, indicating good environmental adaptability and sustained antibacterial activity.

[0054] The embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A Staphylococcus aureus bacteriophage resistant to gastric juice treatment and possessing long-lasting antibacterial activity, characterized in that: The bacteriophage was named RDP-SR-25010 and was deposited on May 28, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 47052.

2. The bacteriophage according to claim 1, characterized in that: After treatment with simulated gastric juice containing NaCl and pepsin at pH 2.0 for 120 min, the titer of the bacteriophage decreased by no more than two orders of magnitude, and the titer was not lower than 10. 6 PFU / mL.

3. The bacteriophage according to claim 1, characterized in that: The bacteriophage exhibited a sustained inhibitory effect on Staphylococcus aureus for 42.6 hours.

4. The bacteriophage according to claim 1, characterized in that: The phages showed no order-of-magnitude decrease in titer after treatment at pH 5–11 for 1 h, and the titer decrease after treatment at strongly acidic conditions (pH 2–3) or strongly alkaline conditions (pH 12) for 1 h did not exceed two orders of magnitude.

5. The bacteriophage according to claim 1, characterized in that: The phages showed a titer decrease of no more than two orders of magnitude after treatment at 30–50°C for 6 hours, and maintained their infectious activity after treatment at 70°C for 2 hours.

6. The use of a Staphylococcus aureus phage as described in any one of claims 1 to 5 in the preparation of an agent for inhibiting or inactivating Staphylococcus aureus.

7. The application according to claim 6, characterized in that: The preparation is an oral preparation.

8. The application according to claim 6, characterized in that: The preparation is a topical antibacterial preparation or a wound treatment preparation.

9. The application according to claim 6, characterized in that: The preparation is an environmental treatment or disinfection preparation.

10. The application according to claim 6, characterized in that: The formulation is used to prepare a sustained-release antibacterial agent.

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