Enterococcus faecalis bacteriophage vBEfaPFA3, bacteriophage composition and application thereof

Abstract

The invention discloses an enterococcus faecalis bacteriophage vBEfaPFA3, a bacteriophage composition and application of the enterococcus faecalis bacteriophage vBEfaPFA3, the enterococcus faecalis bacteriophage vBEfaPFA3 has a specific lysis effect on enterococcus faecalis and also has a cross-species lysis effect on salmonella, the acid-base adaptability of the bacteriophage is high, and the enterococcus faecalis bacteriophage vBEfaPFA3 can be used as an active component to be prepared into the bacteriophage composition or a bacteriophage pharmaceutical preparation. The enterococcus faecalis bacteriophage and the bacteriophage composition are applied to preparation of drugs or bacteriostatic agents for preventing and treating enterococcus faecalis and salmonella infection, are safe to use, have no side effects, are easy to industrially produce, and are wide in lysis spectrum and wide in application range based on the cross-species lysis performance of the enterococcus faecalis bacteriophage and the bacteriophage composition.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Enterococcus faecalis bacteriophage vB_EfaP_FA3, a bacteriophage composition, and its applications. Background Technology

[0002] Enterococcus faecalis is an aerobic or facultative anaerobic Gram-positive coccus, widely distributed in nature and part of the normal gut microbiota of humans and animals. Although a commensal bacterium, Enterococcus faecalis is also a common opportunistic pathogen. It frequently translocates across the mucosal barrier, causing a range of inflammatory responses such as urinary tract infections, endocarditis, and oral infections. Due to the inherent resistance of many Enterococcus faecalis to antibiotics such as penicillin, ampicillin, and piperacillin, coupled with the non-standard use of antibiotics in medical interventions, the clinical manifestations of drug resistance in Enterococcus faecalis are highly complex, making it a significant cause of nosocomial infections. Therefore, there is an urgent need to find and develop new antimicrobial agents to increase treatment options for Enterococcus faecalis infections.

[0003] Bacteriophages are bacterial viruses capable of specifically infecting and killing their host bacteria. Their numbers in nature are enormous, forming the largest known group of viruses. Almost every type of bacteria has multiple corresponding bacteriophages. Bacteriophages are classified into virulent and temperate phages. Virulent phages can rapidly invade host bacteria, quickly replicate, and lyse them. Using bacteriophage lysis to treat pathogenic infections is called "phage therapy." Naturally virulent bacteriophages are suitable candidates for phage therapy. As a novel biocontrol method, phage therapy utilizes the host specificity of bacteriophages to infect bacteria, causing them to lyse or reducing their pathogenicity, thereby treating bacterial infections in humans and animals. During treatment and prevention, bacteriophages target only their corresponding host bacteria, without disrupting the intestinal flora balance of the animal and without side effects. Some studies suggest that bacteriophages may be a suitable alternative drug for treating Enterococcus faecalis infections.

[0004] However, there are currently no safe and efficient phage products with high lysis performance specifically targeting Enterococcus faecalis in clinical practice. Therefore, the development of an effective and safe phage preparation has great potential for treating infections caused by Enterococcus faecalis.

[0005] Therefore, the existing technology needs further improvement. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a self-selected Enterococcus faecalis phage vB_EfaP_FA3 and its phage composition. This phage exhibits specific lytic effects against Enterococcus faecalis and also demonstrates cross-species lytic effects against Salmonella. The phage is highly adaptable to acid and alkali conditions, making it suitable for various harsh environments and applicable to a wide range of conditions.

[0007] To address the above problems, this application provides the following technical solution: Firstly, we provide a *Enterococcus faecalis* bacteriophage vB_EfaP_FA3, with accession number CGMCCNO.45792.

[0008] The Enterococcus faecalis phage vB_EfaP_FA3 was isolated from nearshore seabed sediments. Its Latin name is Enterobacter phage. The phage was deposited on December 1, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.45792. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] The head of Enterococcus faecalis phage vB_EfaP_FA3 is approximately 55–65 nm long and 60–70 nm wide, while the tail is approximately 115–125 nm long. According to the classification criteria of the International Committee on Taxonomy of Viruses (ICTV), Ninth Report, its morphology conforms to the characteristics of the Myotail Phage family, hence the name vB_EfaP_FA3. On solid culture media, it forms translucent plaques that are uniform in size and shape, with clear and regular edges, and a plaque diameter of approximately 1.5 mm.

[0010] This bacteriophage exhibits strong acid-base stability and good tolerance to acidic and alkaline environments, making it suitable for use in various extreme conditions. Experiments have shown that this bacteriophage maintains relatively stable activity within a pH range of 2–13.

[0011] In a second aspect, the present invention provides a phage composition comprising the aforementioned Enterococcus faecalis phage.

[0012] Enterococcus faecalis phage vB_EfaP_FA2 can be combined with other Enterococcus faecalis phages or other Salmonella phages to extend the bactericidal and bacteriostatic effects of the phage composition and broaden its antibacterial spectrum.

[0013] Preferably, the phage composition further includes one or both of vB_EfaP_FA1 with accession number CGMCC NO.45790 and vB_EfaP_FA3 with accession number CGMCC NO. 45791.

[0014] Phage compositions can be prepared by combining vB_EfaP_FA1 and vB_EfaP_FA3, or vB_EfaP_FA3 and vB_EfaP_FA2, or vB_EfaP_FA1, vB_EfaP_FA2 and vB_EfaP_FA3, thereby expanding the lysis spectrum of the phage composition.

[0015] Among them, the aforementioned Enterococcus faecalis phage vB_EfaP_FA1 was isolated from nearshore seabed sediments. Its Latin name is Enterobacter phage. This phage was deposited on December 1, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.45790. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0016] The head of Enterococcus faecalis phage vB_EfaP_FA1 is approximately 65–75 nm long and 55–65 nm wide, while the tail is approximately 85–95 nm long. According to the classification criteria of the International Committee on Taxonomy of Viruses (ICTV), its morphology conforms to the characteristics of the Myotail Phage family, hence the name vB_EfaP_FA1. On solid culture media, it forms translucent plaques with uniform shape and size, clear and regular edges, and a plaque diameter of approximately 0.8 mm.

[0017] Enterococcus faecalis phage vB_EfaP_FA2 was isolated from nearshore seabed sediments. Its Latin name is Enterobacter phage. This phage was deposited on December 1, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.45791. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0018] The head of Enterococcus faecalis phage vB_EfaP_FA2 is approximately 60–70 nm long and 60–70 nm wide, while the tail is approximately 120–130 nm long. According to the classification criteria of the International Committee on Taxonomy of Viruses (ICTV), Ninth Report, its morphology conforms to the characteristics of the Myotail Phage family, and it is named vB_EfaP_FA2. On solid culture media, it forms translucent plaques that are uniform in size and shape, with clear and regular edges, and a plaque diameter of approximately 2.0 mm.

[0019] Thirdly, the present invention also provides the use of the above-mentioned Enterococcus faecalis phage or the aforementioned phage composition in the inhibition of Enterococcus faecalis and / or Salmonella.

[0020] The aforementioned Enterococcus faecalis phage vB_EfaP_FA2 exhibits relatively specific lysis against Enterococcus faecalis. It also shows a broad lysis range against Salmonella, capable of lysing various serotypes of Salmonella, including Salmonella Typhimurium, Salmonella Dublin, and Salmonella Enteritidis.

[0021] Based on the aforementioned lysis properties of this bacteriophage, it can be applied to the inhibition of specific host bacteria Enterococcus faecalis and the broad-spectrum inhibition of Salmonella.

[0022] Fourthly, the present invention also provides the use of the above-described Enterococcus faecalis phage or the above-described phage composition in the preparation of medicaments for the prevention and treatment of diseases caused by Enterococcus faecalis and / or Salmonella infection. The term "prevention" herein refers to all actions that inhibit or delay the disease by administering the phage. The term "treatment" herein refers to all actions that improve or alleviate the disease by administering the phage.

[0023] Based on the aforementioned lytic properties of the phage, the aforementioned Enterococcus faecalis phage or the aforementioned phage composition can be used in the preparation of medicaments for the prevention and treatment of diseases caused by Enterococcus faecalis and / or Salmonella infections. These diseases include various illnesses caused by Enterococcus faecalis or Salmonella infections, including: urinary tract infections, wound infections, bacteremia, or endocarditis caused by Enterococcus faecalis infection, and sepsis and enteritis caused by Salmonella infection.

[0024] Fourthly, the present invention also provides a phage drug formulation, the active ingredient of which includes the aforementioned Enterococcus faecalis phage or the aforementioned phage composition.

[0025] Optionally, the phage drug formulation further comprises a pharmaceutically acceptable carrier in the form of a solution, powder, gel, granule, or lyophilized product. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to the organism and does not eliminate the biological activity and properties of the administered active ingredient. For the pharmaceutical composition to be formulated into a liquid formulation, the pharmaceutically acceptable carrier must be suitable for sterility and biocompatibility. Examples include saline, sterile water, Ringer's solution, buffered physiological saline, albumin infusion, glucose solution, maltodextrin solution, glycerol, and ethanol. They can be used alone or in any combination thereof. Other conventional additives, such as antioxidants, buffers, and antibacterial agents, may be added if desired. The compositions of the present invention can also be formulated into injections (e.g., aqueous solutions, suspensions, and emulsions), or pills, capsules, granules, or tablets, when also combined with diluents, dispersants, surfactants, binders, and / or lubricants.

[0026] The present invention has the following beneficial effects: 1. This invention provides a self-selected Enterococcus faecalis phage vB_EfaP_FA2, and also provides a phage composition composed of the phage, vB_EfaP_FA1, and vB_EfaP_FA3. The phage and the phage composition have cross-species lysis performance, not only having a specific lysis effect on Enterococcus faecalis, but also having a broad lysis spectrum and high lysis rate against clinically obtained Salmonella, and can lyse multiple serotypes such as Salmonella typhimurium, Salmonella Dublin, and Salmonella enteritidis.

[0027] 2. This bacteriophage can be used to prepare drugs for the prevention and treatment of Enterococcus faecalis and Salmonella infections. It provides a rapid, efficient, and safe solution to these infections while avoiding the problem of antibiotic resistance, thus effectively improving treatment efficiency. The bacteriophage is obtained from nature and is easy to produce industrially. Drugs prepared from this bacteriophage not only reduce costs but also have the advantages of being environmentally friendly.

[0028] 3. Due to its cross-species lytic activity and broad lytic spectrum, this phage can be combined with other Enterococcus faecalis or Salmonella phages to form phage compositions, further expanding its lytic spectrum and improving its efficacy. Furthermore, by further preparing it into different dosage forms such as powders, aqueous solutions, or lyophilized formulations, the product can be further improved and its application facilitated. Attached Figure Description

[0029] Figure 1 Image of a plaque from bacteriophage vB_EfaP_FA1; Figure 2 Electron micrograph of bacteriophage vB_EfaP_FA1; Figure 3 This is a one-step growth curve of bacteriophage vB_EfaP_FA1; Figure 4 Results of thermal stability of bacteriophage vB_EfaP_FA1; Figure 5 The pH stability results for bacteriophage vB_EfaP_FA1; Figure 6 The in vitro antibacterial effect of bacteriophage vB_EfaP_FA1 on Enterococcus faecalis F563 is shown; where 2216E represents the liquid culture medium component, F563 represents the Enterococcus faecalis host bacterium, and A1 represents bacteriophage vB_EfaP_FA1. Figure 7 Image of a plaque from bacteriophage vB_EfaP_FA2; Figure 8 Electron micrograph of bacteriophage vB_EfaP_FA2; Figure 9This is a one-step growth curve of bacteriophage vB_EfaP_FA2; Figure 10 Results for the thermal stability of bacteriophage vB_EfaP_FA2; Figure 11 The pH stability results for bacteriophage vB_EfaP_FA2; Figure 12 The in vitro antibacterial effect of bacteriophage vB_EfaP_FA2 on Enterococcus faecalis F563 is shown; where 2216E represents the culture medium component, F563 represents the host bacteria of Enterococcus faecalis, and A2 represents bacteriophage vB_EfaP_FA2. Figure 13 Image of a plaque from bacteriophage vB_EfaP_FA3; Figure 14 Electron micrograph of bacteriophage vB_EfaP_FA3; Figure 15 This is a one-step growth curve of bacteriophage vB_EfaP_FA3; Figure 16 Results for the thermal stability of bacteriophage vB_EfaP_FA3; Figure 17 The pH stability results for bacteriophage vB_EfaP_FA3; Figure 18 The in vitro antibacterial effect of bacteriophage vB_EfaP_FA3 on Enterococcus faecalis F563 is shown; where 2216E represents the culture medium component, F563 represents the Enterococcus faecalis host bacterium, and A3 represents bacteriophage vB_EfaP_FA3. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0031] Example 1: Isolation and Purification of Bacteriophages 1.1 Isolation and culture of host bacteria In this example, the host bacteriophage of Enterococcus faecalis was a clinical strain of Enterococcus faecalis, which was isolated from the blood of a patient in the ICU of a tertiary hospital in Ningbo, Zhejiang Province.

[0032] Host bacteria F563 was inoculated into 2216E solid medium and cultured overnight. Then, a single colony was picked and inoculated into 2216E liquid medium and cultured at 37°C and 180 rpm for 24 hours as the host bacteria culture for later use.

[0033] 1.2 Isolation, Culture and Purification of Bacteriophages Add an appropriate amount of nearshore seabed sediment to a 1000ml Erlenmeyer flask containing Enterococcus faecalis bacterial solution and mix well. Add an appropriate amount of 2216E liquid culture medium and incubate at 37℃ and 180rpm for 3-5 days with shaking. Centrifuge the mixture at 5000rpm for 15min, collect the supernatant and filter it through a 0.22μm bacterial filter. Store at 4℃ in the dark.

[0034] The obtained filtrate was subjected to 10... -2 10 -4 10 -6 Serial dilutions were performed, with 1 ml of the diluted solution mixed with 1 ml of logarithmically grown bacterial suspension. Then, 5 ml of 0.6% agar (0.6 g agar per 100 mL of 2216E liquid medium; equilibrate to 52°C in a water bath before use) was added. The mixture was poured onto a pre-prepared bottom solid agar plate (1.5% 2216E solid medium). After the top agar solidified, the plate was sealed with Parafilm and incubated upright in a 37°C biochemical incubator overnight. Observation was conducted for plaque formation. If plaques appeared, they were removed using a smooth pipette tip and placed in 1 ml of SM buffer (diluted to 1X with ultrapure water and autoclaved). The plate was vortexed for 1 minute to obtain a plaque suspension. The suspension was then stored in a 4°C refrigerator overnight, protected from light. Dilute the plaque suspension obtained in the previous step by 10%. -2 10 -4 10 -6 Repeat the double-layer plate method to purify the phage, about 3-5 times, until the phage plaques on the plates are all the same size and shape.

[0035] The results are as follows Figure 1 , Figure 7 and Figure 13 As shown, three bacteriophage strains were isolated. These bacteriophages exhibited consistent plaque morphology, size, and clarity, and demonstrated typical lytic activity. These three bacteriophages were subsequently named vB_EfaP_FA1, vB_EfaP_FA2, and vB_EfaP_FA3, respectively.

[0036] Example 2: Identification of bacteriophages 2.1 Observation and identification of bacteriophage morphology (1) Experimental method: Add 9 mL of cesium chloride solution (67 g cesium chloride + 82 mL sterile SM buffer, final density 1.5 g / mL) and 3 mL of phage resuspension to a centrifuge tube. Centrifuge at 33600 rpm, 4℃, for 8 h. After centrifugation, aspirate the supernatant and store at 4ºC protected from light. Add the phage sample solution obtained after cesium chloride density gradient centrifugation to a 30 kDa ultrafiltration tube, add SM buffer, and remove cesium chloride by multiple centrifugations (5000 g, 4℃, 5 min) to obtain pure phage sample solution, which is then stored at 4ºC protected from light. Two μL of phage suspension, purified by cesium chloride density gradient centrifugation and ultrafiltration, was added to the center of a 200-mesh copper mesh and allowed to dry thoroughly in air. After drying, the sample was stained with 2% uranium acetate for 3 minutes and then observed under a Hitachi H-7650 transmission electron microscope with an accelerating voltage of 80 kV. Images were acquired using a Gatan Inc. system. The head width, tail length, and width of the acquired virus images were analyzed to determine their taxonomic position.

[0037] (2) Experimental results: like Figure 2 As shown in the electron micrograph, the head of Enterococcus faecalis phage vB_EfaP_FA1 is about 65-75 nm long, about 55-65 nm wide, and about 85-95 nm long. According to the classification criteria of the International Committee on Taxonomy of Viruses (ICTV), its morphology conforms to the characteristics of Myotail Phages, and it is named vB_EfaP_FA1.

[0038] like Figure 8 As shown in the electron microscope image, the head of Enterococcus faecalis phage vB_EfaP_FA2 is about 60-70 nm long and 60-70 nm wide, and the tail is about 120-130 nm long. According to the classification criteria of the Ninth Report of the International Committee on Taxonomy of Viruses (ICTV), its morphology conforms to the characteristics of Myotail Phages, and it is named vB_EfaP_FA2.

[0039] like Figure 14 As shown in the electron microscope images, the head of Enterococcus faecalis phage vB_EfaP_FA3 is about 55-65 nm long, about 60-70 nm wide, and about 115-125 nm long. According to the classification criteria of the Ninth Report of the International Committee on Taxonomy of Viruses (ICTV), its morphology conforms to the characteristics of Myotail Phages, and it is named vB_EfaP_FA3.

[0040] 2.2 Whole genome analysis of bacteriophages (1) Experimental method: After extracting the genomes of bacteriophages vB_EfaP_FA1, vB_EfaP_FA2 and vB_EfaP_FA3, they were sent for sequencing. After sequencing, sequence analysis was performed.

[0041] (2) Experimental results and analysis The genomes of bacteriophages vB_EfaP_FA1, vB_EfaP_FA2, and vB_EfaP_FA3 were analyzed using tRNAscan-SE software and found to contain no tRNA genes. mmseq2 analysis showed that the genomes contained no drug resistance genes or virulence genes. PHASTEST analysis showed that the genomes contained no lysogenic genes.

[0042] In the genome of bacteriophage vB_EfaP_FA1: the sequence of the tail fiber protein gene, which is related to phage host recognition, is shown in SEQ ID NO:1 of the sequence listing; the sequence of the highly conserved terminal enzyme large subunit protein gene is shown in SEQ ID NO:2 of the sequence listing. Sequence similarity analysis of the phage genome was performed using the online BLAST tool (http: / / blast.ncbi.nlm.nih.gov / ). The bacterium with the highest homology was Proteus mirabilis strain PM-OXA-09, with a homology of only 86%. These results indicate that bacteriophage vB_EfaP_FA1 is a novel Enterococcus faecalis phage, and its phylogenetic relationship with existing closely related bacteriophages is relatively distant.

[0043] In the genome of bacteriophage vB_EfaP_FA2: the sequence of the tail fiber protein gene, which is related to phage host recognition, is shown in Sequence 3 of the sequence listing; the sequence of the highly conserved terminal enzyme large subunit protein gene is shown in Sequence 4 of the sequence listing. Sequence similarity analysis of the phage genome was performed using the online BLAST tool (http: / / blast.ncbi.nlm.nih.gov / ). The bacterium with the highest homology was Proteus mirabilis strain XH1569, with a homology of only 87%. These results indicate that bacteriophage vB_EfaP_FA2 is a novel Enterococcus faecalis phage, and its phylogenetic relationship with existing closely related bacteriophages is relatively distant.

[0044] In the genome of bacteriophage vB_EfaP_FA3: the sequence of the tail fiber protein gene, which is related to phage host recognition, is shown in Sequence 5 of the sequence listing; the sequence of the highly conserved terminal enzyme large subunit protein gene is shown in Sequence 6 of the sequence listing. Sequence similarity analysis of the phage genome was performed using the online BLAST tool (http: / / blast.ncbi.nlm.nih.gov / ). The bacterium with the highest homology was Proteus mirabilis strain XH1569, with a homology of only 87%. These results indicate that bacteriophage vB_EfaP_FA3 is a novel Enterococcus faecalis phage, and its phylogenetic relationship with existing closely related bacteriophages is relatively distant.

[0045] Table 1 Gene Sequence Information Table

[0046] Example 3: Determination of the biological characteristics of bacteriophages 2.1 Determination of the optimal multiple of infection (MOI) of bacteriophages (1) Experimental method: Enterococcus faecalis bacteriophages vB_EfaP_FA1, vB_EfaP_FA2, and vB_EfaP_FA3, along with the host bacterium Enterococcus faecalis F563, were cultured using standard methods for expansion. When the Enterococcus faecalis reached the logarithmic growth phase, the corresponding OD value was measured. 600 Enterococcus faecalis in its logarithmic growth phase was serially diluted, and 10 μl was spread onto solid plates for each gradient. The plates were then incubated overnight at 37°C. The experiment was repeated three times. The result was: when OD... 600 When the concentration of host bacteria is approximately 0.2, the concentration is approximately 1 × 10⁻⁶. 8 CFU / ml. After serial dilution of the phage suspension, the initial titer of the phage suspension was determined using the bilayer plate method and adjusted to a uniform titer. Phage and host bacteria were mixed at ratios of 0.001, 0.01, 0.1, and 1 in sterile Erlenmeyer flasks containing 2216E liquid medium to form a 10 mL reaction system. The mixture was incubated at 37°C and 180 rpm for 24 h in a shaker. 1 mL of the solution was transferred to an Eppendorf tube and centrifuged at 5000 rpm for 15 min at room temperature. The supernatant was filtered through a disposable 0.22 μm filter membrane, and the titer of the filtrate was determined. The experiment was repeated three times. The number of phage plaques at different multiplicity of infection (MOI) was determined using the bilayer plate method, and the phage titer was calculated. The MOI of the phage was determined by the highest MOI.

[0047] Table 2 Results of Optimal Multiple of Infection (MOI) determination for bacteriophage vB_EfaP_FA1

[0048] Table 3. Results of Optimal Multiple of Infection (MOI) determination for bacteriophage vB_EfaP_FA2

[0049] Table 4. Results of Optimal Multiple of Infection (MOI) determination for bacteriophage vB_EfaP_FA3

[0050] (2) Experimental results: The results from the three tables above show that the optimal multiplicity of infection for bacteriophage vB_EfaP_FA1 is 0.01. Under these conditions, the titers of progeny bacteriophages produced by phage infection of the host bacteria are 4.8 × 10⁻⁶. 8 The PFU / ml titer was the highest among the four multiples of infection of the same phage.

[0051] The optimal multiplicity of infection (MNI) for bacteriophage vB_EfaP_FA2 is 0.1. Under this condition, the titer of progeny bacteriophages produced by phage infection of the host bacteriophage is 3.16 × 10⁻⁶. 8 PFU / ml.

[0052] The optimal multiplicity of infection (MNI) for bacteriophage vB_EfaP_FA3 is 0.01. Under this condition, the titer of progeny bacteriophages produced by phage infection of the host bacteriophage is 4.5 × 10⁻⁶. 8 PFU / ml.

[0053] 2.2 Determination of the one-step growth curve of bacteriophage (1) Experimental method: Phage suspensions were added to the cultures of the three Enterococcus faecalis strains at optimal multiplicity of infection and in the exponential growth phase, and the cultures were incubated on a shaker (37°C, 180 rpm) for 5 hours to allow the phages to fully adsorb onto the bacterial surface. Then, the cultures were centrifuged. Collect bacterial cells (10000 g, 4℃, 5 min), add 2216E liquid medium, rinse the precipitate, centrifuge to collect bacterial cells (10000 g, 4℃, 5 min), repeat rinsing twice to remove free bacteriophages that failed to adsorb onto the bacterial cell surface, and finally transfer the bacterial cell precipitate to 300 mL of 2216E liquid medium and incubate in a shaker (37℃, 180 rpm).

[0054] Samples were taken every 10 minutes during the initial 1 hour, and then every 30 minutes thereafter. 1.5 ml of bacterial culture medium was centrifuged (10000 g, 4℃, 5 min), and immediately filtered through a disposable 0.22 μm filter membrane to collect the supernatant. The filtrate was temporarily stored in a medical refrigerator protected from light. After 390 min, all filtrates were serially diluted, and the phage titer was determined using the double-layer plate method. A one-step growth curve was plotted with infection time on the x-axis and phage titer on the y-axis to determine the phage latency, outbreak period, and outbreak quantity.

[0055] like Figure 3 As shown, the lysis cycle of bacteriophage vB_EfaP_FA1 is approximately 220 min. After infecting the host bacteria, the titer of the bacteriophage is basically stable within 50 min, indicating that the phage latency period is approximately 50 min. Within 50-270 min after infecting the host bacteria, the number of bacteriophages increases sharply, indicating that the lysis period of the bacteriophage is approximately 220 min, and the burst quantity of bacteriophage vB_EfaP_FA1 is approximately 220 PFU / cell.

[0056] like Figure 9 As shown, the lysis cycle of bacteriophage vB_EfaP_FA2 is approximately 260 min. After infecting the host bacteria, the titer of the bacteriophage is basically stable within 40 min, indicating that the phage latency period is approximately 40 min. Within 40-300 min after infecting the host bacteria, the number of bacteriophages increases sharply, indicating that the lysis period of the bacteriophage is approximately 260 min. The burst quantity of bacteriophage vB_EfaP_FA2 is approximately 180 PFU / cell.

[0057] like Figure 15 As shown, the lysis cycle of bacteriophage vB_EfaP_FA3 is approximately 300 min. After infecting the host bacteria, the titer of the bacteriophage is basically stable within 30 min, indicating that the phage latency period is approximately 30 min. Within 30-300 min after infecting the host bacteria, the number of bacteriophages increases sharply, indicating that the lysis period of the bacteriophage is approximately 270 min, and the burst quantity of bacteriophage vB_EfaP_FA3 is approximately 200 PFU / cell.

[0058] 2.3 Thermal stability test of bacteriophage vB_EfaP_FA1 3.25×10 6 Lysis buffer of phage vB_EfaP_FA1 at PFU / ml was incubated in a water bath at 40℃, 50℃, 60℃, and 70℃ for 30 min and 60 min respectively, with two replicates at each temperature. The titer of each phage was determined using the double-layer plate method.

[0059] 2.78×10 6Lysis buffer of phage vB_EfaP_FA2 at PFU / ml was incubated in a water bath at 40℃, 50℃, 60℃, and 70℃ for 30 min and 60 min, respectively, with two replicates at each temperature. The titer of each phage was determined using the double-layer plate method.

[0060] 4.77×10 6 Lysis buffer of phage vB_EfaP_FA3 at PFU / ml was incubated in a water bath at 40℃, 50℃, 60℃, and 70℃ for 30 min and 60 min respectively, with two replicates at each temperature. The titer of each phage was determined using the double-layer plate method.

[0061] Experimental results are as follows Figure 4 As shown, bacteriophage vB_EfaP_FA1 maintained its original activity after being exposed to 40℃-50℃ for 60 min; the plaque formation rate after exposure to 60℃ for 60 min was only half that after exposure to 40℃ for 60 min; and it essentially lost its activity after exposure to 70℃ for 60 min. The experimental results indicate that bacteriophage vB_EfaP_FA1 can tolerate high temperatures below 50℃.

[0062] Experimental results are as follows Figure 10 As shown, bacteriophage vB_EfaP_FA2 maintained its original activity after being exposed to 40℃-50℃ for 60 min; the plaque formation rate decreased significantly after exposure to 60℃ for 60 min; and it essentially lost its activity after exposure to 70℃ for 60 min. These experimental results indicate that bacteriophage vB_EfaP_FA2 can tolerate high temperatures below 50℃.

[0063] Experimental results are as follows Figure 16 As shown, bacteriophage vB_EfaP_FA3 maintained its original activity after being exposed to 40℃-50℃ for 60 min; however, the plaque formation rate decreased significantly after exposure to 60℃ for 60 min. These experimental results indicate that bacteriophage vB_EfaP_FA3 can tolerate temperatures below 50℃.

[0064] 2.4 pH stability experiment of bacteriophage vB_EfaP_FA1 Add 4.5 ml of culture medium with different pH values ​​(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) to three sterile test tubes. Place each tube in a water bath at 37°C. After the temperature stabilizes, add 500 μl of phage lysis buffer to each tube, mix well, and incubate at 37°C for 1 h, 2 h, and 3 h. After the water bath, add an appropriate amount of HCl or NaOH to the mixture to adjust the pH to approximately 7. Determine the phage titer using the double-layer plate method.

[0065] like Figure 5As shown, within the pH range of 2–13, the titer of bacteriophage vB_EfaP_FA1 remained almost unchanged, maintaining a value of 10. 5 When the concentration of PFU / ml is above a certain level, the titer of the bacteriophage vB_EfaP_FA1 only decreases slightly after being exposed to pH 2, pH 3, pH 10, pH 11, pH 12 and pH 13 for 3 hours. This indicates that the bacteriophage vB_EfaP_FA1 can tolerate certain acidity and alkalinity and has good tolerance to acidic and alkaline environments.

[0066] like Figure 11 As shown, within the pH range of 2-13, the titer of bacteriophage vB_EfaP_FA2 remained almost unchanged, still maintaining 10. 3 -10 4 The titer of PFU / ml decreased only slightly after 3 hours of exposure to pH 2, indicating that phage vB_EfaP_FA2 can tolerate certain acidity and alkalinity and has good tolerance to acidic and alkaline environments.

[0067] like Figure 17 As shown, within the pH range of 2-13, the titer of bacteriophage vB_EfaP_FA3 remained almost unchanged, still maintaining a value of 10. 5 The PFU / ml indicates that phage vB_EfaP_FA3 can tolerate certain acidity and alkalinity, and has good tolerance to acidic and alkaline environments.

[0068] Example 4: In vitro lysis experiment of bacteriophage vB_EfaP_FA1 Enterococcus faecalis F563 and bacteriophages vB_EfaP_FA1 / vB_EfaP_FA2 / vB_EfaP_FA3 were added in equal proportions to 96-well plates according to the optimal multiplicity of infection. The control group was prepared with the same volume of 2216E liquid medium as the bacteriophage solution. Three parallel experiments were performed for each group. The bacterial culture and bacteriophages were mixed and incubated at 37°C and 180 rpm using a Multiskan FC microplate reader with continuous shaking. OD was measured every 10 minutes. 600 Until the OD value results are basically stable.

[0069] The results are as follows Figure 6 , Figure 12 and Figure 18 As shown in the results, vB_EfaP_FA1, vB_EfaP_FA2, and vB_EfaP_FA3 have a good lysis effect on Enterococcus faecalis F563. At the optimal multiplicity of infection, the absorbance half an hour after infection is significantly lower than that of the positive control, and no rebound phenomenon occurs within 8 hours.

[0070] Example 5: Determination of phage lysis profile 1. Experimental Methods The lysis spectrum of bacteriophages was determined using the double-layer plate method. The steps are as follows: The host bacterial suspension was obtained according to the method in Example 1. In this experiment, 75 human host bacteria were used to determine the lysis spectrum of bacteriophages vB_EfaP_FA1, vB_EfaP_FA2 and vB_EfaP_FA3 respectively.

[0071] The 75 host bacteria included 11 clinical Enterococcus faecalis strains, multiple clinical Salmonella strains, and 3 other clinical Vibrio vulnificus strains. These host bacteria were derived from tissues or blood of patients from different departments of a hospital in Ningbo. The aforementioned clinical Salmonella strains included 14 strains of Salmonella enteritidis (…). Salmonella enteritidis ), 29 strains of Salmonella typhimurium ( Salmonella typhimurium ), 1 strain of Salmonella Birkenhead ( Salmonella birkenhead ), 2 strains of Salmonella Chester Salmonella chester ), 2 strains of Salmonella Delphi ( Salmonella derby ), 1 strain of Salmonella Dublin Salmonella dublin ), 3 strains of Salmonella Gold Coast ( Salmonella Gold Coast ), 4 strains of Salmonella Riessen Salmonella rissen ), 1 strain of Litchfield Salmonella ( Salmonella litchfield ), 3 strains of Salmonella London Salmonella London ), 1 strain of Salmonella infantis ( Salmonella infantis See Table 3 below for details.

[0072] After incubating 100 μl of each phage with the above-mentioned clinical strains at 37°C for 24 h, the phages were added to the upper agar to prepare a double-layer plate. After the agar solidified, the plate was placed in a constant temperature incubator at 37°C and cultured upright for 24 h to observe the lysis results.

[0073] The pyrolysis results in Table 3 show that: (1) Bacteriophage vB_EfaP_FA3 can lyse strain F563 of 11 Enterococcus faecalis strains, showing high lysis specificity for Enterococcus faecalis. Enterococcus faecalis strain F563 was deposited on November 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 32693, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0074] The bacteriophage vB_EfaP_FA3 exhibits broad-spectrum antibacterial activity against multiple subspecies of Salmonella. It can lyse 8 out of 14 Salmonella enteritidis strains with a lysis rate of 57.1%; lyse 25 out of 29 Salmonella typhimurium strains with a lysis rate of 58.1%; and lyse 28 out of 61 Salmonella strains. Furthermore, bacteriophage vB_EfaP_FA3 does not lyse Vibrio vulnificus, demonstrating high lysis specificity.

[0075] (2) Bacteriophage vB_EfaP_FA1 also lysed strain F563 of 11 Enterococcus faecalis strains, showing high lysis specificity for Enterococcus faecalis. This bacteriophage could lyse 12 of 14 Salmonella enteritidis strains, with a lysis rate of 85.7%; it could lyse 23 of 29 Salmonella typhimurium strains, with a lysis rate of 79.3%, and the total lysis rate for 61 Salmonella strains reached 68.8%.

[0076] (3) Bacteriophage vB_EfaP_FA2 also lysed strain F563 of 11 Enterococcus faecalis strains, showing high lysis specificity against Enterococcus faecalis. Bacteriophage vB_EfaP_FA2 has broad-spectrum antibacterial activity against multiple subspecies of Salmonella, and can lyse 9 of 14 Salmonella enteritidis strains with a lysis rate of 64.2%; can lyse 15 of 29 Salmonella typhimurium strains with a lysis rate of 51.7%; and can lyse 35 of 61 Salmonella strains with a total lysis rate of 57.3%.

[0077] Due to the differences in the lysis spectra of the three bacteriophages and their high complementarity, the lysis performance of the phage composition composed of these three bacteriophages is better. In particular, the lysis spectrum of the phage composition composed of these three bacteriophages is greatly improved, and it can lyse 14 out of 14 Salmonella enteritidis strains with a lysis rate of 100%; it can lyse all 29 Salmonella typhimurium strains with a lysis rate of 100% for Salmonella typhimurium and also achieve a lysis rate of 100% for 61 Salmonella strains.

[0078] Therefore, bacteriophage vB_EfaP_FA3 is a novel transspecies lytic bacteriophage that can be used alone or in combination with vB_EfaP_FA1 and / or vB_EfaP_FA2 to inhibit Enterococcus faecalis and various Salmonella strains and to prevent and treat diseases caused by them, showing promising application prospects.

[0079] Table 3. Lysis of three bacteriophage strains on different clinical bacterial strains.

[0080] Note: "+" indicates that it can be split; "-" indicates that it cannot be split.

[0081] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A strain of Enterococcus faecalis bacteriophage vB_EfaP_FA3, characterized in that, Its accession number is CGMCC NO. 45792.

2. A bacteriophage composition, characterized in that, Includes Enterococcus faecalis phage vB_EfaP_FA3 as described in claim 1.

3. The phage composition according to claim 1, characterized in that, Also includes: One or both of vB_EfaP_FA1 with accession number CGMCC NO. 45790 and vB_EfaP_FA2 with accession number CGMCC NO. 45791.

4. The use of the Enterococcus faecalis phage according to claim 1 or the phage composition according to claim 2 or 3 in the inhibition of Enterococcus faecalis and / or Salmonella.

5. The use of the Enterococcus faecalis phage according to claim 1 or the phage composition according to claim 2 or 3 in the preparation of medicaments for the prevention and treatment of diseases caused by Enterococcus faecalis and / or Salmonella infection.

6. A bacteriophage drug formulation, characterized in that, Its active ingredients include the Enterococcus faecalis phage as described in claim 1 or the phage composition as described in claim 2 or 3.

7. The phage drug formulation according to claim 6, characterized in that, The phage drug formulation also includes a pharmaceutically acceptable carrier, and its dosage form is a solution, powder, gel, granule or lyophilized form.

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