Enterococcus faecalis phage vBEfaS-1017 and application thereof

Abstract

The invention provides enterococcus faecalis bacteriophage vBEfaS-1017 and application thereof, the enterococcus faecalis bacteriophage vBEfaS-1017 is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO.46182, and the preservation time is September 6, 2024. The bacteriophage vBEfaS-1017 is high in specificity, short in incubation period and ultrahigh in stability, and has activity under the conditions of extreme pH, high temperature and ultraviolet radiation; by evaluating the in-vitro bactericidal efficacy of the bacteriophage vBEfaS-1017, it is determined that the bacteriophage vBEfaS-1017 can effectively remove a biological membrane formed by host bacteria Ef 10-17, the removal rate is as high as 90%, and the bacteriophage vBEfaS-1017 is expected to become a scavenger for solving the biological membrane. Further experiments prove that the bacteriophage vBEfaS-1017 has in-vivo and in-vitro safety. Through combined use of the bacteriophage and different antibiotics, it is found that combined use of the bacteriophage vBEfaS-1017 and the antibiotic levofloxacin can effectively treat bacteremia of mice, the treatment time after infection can be prolonged by using the bacteriophage in advance, and the bacteriophage has a good application prospect in the aspect of preventing and treating enterococcus faecalis.

Description

Technical Field

[0001] This invention belongs to the field of bacteriophage technology, and specifically relates to an Enterococcus faecalis bacteriophage vB_EfaS-1017 and its applications. Background Technology

[0002] Enterococcus faecalis, a common opportunistic pathogen, poses a significant threat to human health. Antibiotics have played a crucial role in the fight against bacteria, but their overuse and the transfer of antibiotic resistance genes within Enterococcus faecalis have made its control difficult. Bacteriophages, widely distributed in nature, can effectively kill pathogens, and their diversity lays the foundation for their development for biocontrol. Therefore, bacteriophages have returned to the forefront of research as a tool to alleviate the worsening global health crisis. Research on bacteriophages is increasing, and they are being applied in various fields. However, to date, there are relatively few cases of using Enterococcus faecalis bacteriophages for treatment.

[0003] Therefore, how to obtain Enterococcus faecalis bacteriophages with good stability and effective biofilm removal is a problem that needs to be solved. Summary of the Invention

[0004] In view of the problems existing in the prior art, the purpose of this invention is to provide a falciparum phage vB_EfaS-1017 and its application.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] The first aspect of this invention provides a *Enterococcus faecalis* bacteriophage, named vB_EfaS-1017, screened from untreated medical wastewater, and deposited at the China General Microbiological Culture Collection Center (CGMCC); address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China; classification name: *Caudovirales* Siphoviridae; accession number: CGMCC NO. 46182; deposit date: September 6, 2024. This bacteriophage exhibits strong lytic activity against *Enterococcus faecalis*.

[0007] Furthermore, the bacteriophage vB_EfaS-1017 exhibits good tolerance under conditions of 4℃ to 80℃ and pH values ​​of 4 to 13.

[0008] Furthermore, the phage vB_EfaS-1017 titer was 2.0 × 10⁻⁶ under the condition of MOI = 0.001. 10 pfu / mL, after 5 min of incubation, it enters the lysis phase and the burst reaches 97 pfu / cell.

[0009] Furthermore, the bacteriophage vB_EfaS-1017 belongs to the class Caudoviricetes, which are tailed bacteriophages.

[0010] A second aspect of the invention provides the use of the Enterococcus faecalis phage vB_EfaS-1017, having any of the following uses:

[0011] (a) Use in the inhibition of Enterococcus faecalis in non-disease treatment;

[0012] (b) Use in the preparation of formulations that broadly inhibit Enterococcus faecalis;

[0013] (c) Use in the preparation of products for the prevention or treatment of diseases caused by Enterococcus faecalis;

[0014] (d) Use in the preparation of products that inhibit / eliminate biofilms produced by Enterococcus faecalis.

[0015] Furthermore, this bacteriophage was used to inhibit Enterococcus faecalis Ef 10-17.

[0016] Furthermore, the diseases include urinary tract infections, abdominal infections, pelvic infections, soft tissue infections, endocarditis infections, and bacteremia caused by Enterococcus faecalis.

[0017] Furthermore, in the preparation of products for preventing biofilm formation by Enterococcus faecalis, the products include antibacterial agents, drugs, disinfectants, or cleaning agents.

[0018] A third aspect of the present invention provides a bactericidal composition for preventing and controlling Enterococcus faecalis, wherein the composition uses the above-mentioned Enterococcus faecalis bacteriophage vB_EfaS-1017 as an active ingredient, and a carrier or excipient may or may not be added to the composition.

[0019] Furthermore, the bactericidal composition is used in combination with an antibiotic when used to prevent or treat diseases or contamination caused by Enterococcus faecalis. Preferably, the antibiotic is levofloxacin.

[0020] The advantages of this invention compared to the prior art are as follows:

[0021] 1. This invention isolates a long-tailed bacteriophage vB_EfaS-1017 from medical wastewater using Enterococcus faecalis as the host bacterium. It has high specificity, short latency period, and extremely strong stability, exhibiting activity under extreme pH and high temperature conditions, and has good application prospects.

[0022] 2. By evaluating the in vitro bactericidal efficacy of bacteriophage vB_EfaS-1017, vB_EfaS-1017 can effectively remove biofilms formed by host bacterium Ef 10-17, with a clearance rate as high as 90%, showing promise as a biofilm removal agent. Further experiments confirmed that bacteriophage vB_EfaS-1017 has in vivo and in vitro safety. Furthermore, by combining the bacteriophage with different antibiotics, it was found that the combined use of the bacteriophage vB_EfaS-1017 described in this application and the antibiotic levofloxacin can effectively treat bacteremia in mice. Preventive use of the bacteriophage can prolong the treatment time after infection, showing good application prospects in the prevention and control of Enterococcus faecalis. Attached Figure Description

[0023] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0024] Figure 1 The morphology of bacteriophage vB_EfaS-1017 is shown; (A) is a photograph of the phage plaque, and (B) is a transmission electron microscope image of the phage.

[0025] Figure 2 This shows the optimal multiplicity of infection for bacteriophage vB_EfaS-1017;

[0026] Figure 3 The one-step growth curve of bacteriophage vB_EfaS-1017;

[0027] Figure 4 The stability of bacteriophage vB_EfaS-1017 is shown; where (A) pH stability of bacteriophage, (B) temperature stability of bacteriophage, and (C) UV stability of bacteriophage are shown.

[0028] Figure 5 The in vitro bactericidal kinetics of bacteriophage vB_EfaS-1017 are shown;

[0029] Figure 6 The results of the biofilm clearance and inhibition assays are shown below; (A) the clearance of biofilm by different titers of bacteriophages was determined by the crystal violet method; (B) the clearance efficiency of bacteriophages on biofilms was determined by the colony counting method; (C) the inhibition of biofilms by different titers of bacteriophages; and (D) a comparison of biofilm clearance by bacteriophages and EDTA. Statistical analysis was performed using t-tests (*, p < 0.5; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

[0030] Figure 7 To image biofilms for scanning electron microscopy; (A) biofilms formed by Ef 10-17, (B) biofilms treated with bacteriophage, (C) biofilms treated with a mixture of bacteriophage and EDTA, and (D) biofilms treated with EDTA alone.

[0031] Figure 8 The combined bactericidal effect of bacteriophage vB_EfaS-1017 with different types of antibiotics is shown;

[0032] Figure 9 The results of in vitro safety assays for bacteriophage vB_EfaS-1017 are shown; (A) LO2 cell viability, (B) erythrocyte hemolytic activity.

[0033] Figure 10 The results of the in vivo safety assessment of the bacteriophage are shown; (A) scores of mice in each group after injection, and (B) daily changes in mouse body weight.

[0034] Figure 11 Survival curves for a mouse bacteremia model; where 2-1 represents injection of sterile PBS, and 2-2 represents injection of 8×10⁻⁶ PBS. 10 The host bacterial culture volume is CFU / mL, and 2-3 indicates an injection of 4×10⁻⁶. 10 Host bacterial volume, 2-4 indicates an injection of 2×10 10 The host bacterial culture volume is represented by CFU / mL, and 2-5 indicates an injection of 1×10⁻⁵. 10 The host bacterial culture volume is represented by CFU / mL, and 2-6 indicates an injection of 1×10⁻⁶. 9 CFU / mL of host bacterial culture, 2-7 indicates an injection of 1×10 8 CFU / mL host bacterial culture volume;

[0035] Figure 12 Survival curves for mice after infection and treatment;

[0036] Figure 13 The mouse status score and mouse weight are displayed;

[0037] Figure 12 , 13 In the diagram, 3-1 indicates injection of sterile PBS, 3-2 indicates injection of host bacterial solution for infection, 3-3 indicates post-infection treatment with bacteriophage alone, 3-4 indicates post-infection treatment with levofloxacin, and 3-5 indicates post-infection treatment with a combination of bacteriophage and antibiotics.

[0038] Figure 14 The results show the bacterial and plaque counts in mouse organs; (A) the number of colonies in the liver, spleen and blood of each group of mice, and (B) the number of plaques in the liver, spleen and blood of each group of mice.

[0039] Figure 15 The images show the pathological sections of the liver, kidney, and spleen of mice in each group.

[0040] Figure 16The results of ELISA are shown; (A) changes in TNF-α levels in the blood of mice in groups 3-1 to 3-5, (B) determination of IL-1β levels in the blood of mice in each group, and (C) measurement of IL-6 levels in the blood of mice in each group.

[0041] Figure 17 The results of routine blood tests in mice are shown; (A) the number of white blood cells in each group of mice, and (B) the number of neutrophils in the blood of mice.

[0042] Figure 18 Survival curves for mice after infection and treatment are shown below; 4-1 is the group injected with sterile PBS, 4-2 is the Ef10-17 infection group, 4-3 is the group injected with phage 12 hours in advance and then not treated, 4-4 is the group injected with phage 24 hours in advance and then not treated, 4-5 is the group injected with phage 12 hours in advance and then treated with combined therapy 1 hour later, 4-6 is the group injected with phage 24 hours in advance and then treated with combined therapy 1 hour later, 4-7 is the group treated with combined therapy immediately after infection, 4-8 is the group treated with combined therapy 6 hours after infection, and 4-9 is the group injected with phage 24 hours in advance and then treated with combined therapy 6 hours later.

[0043] Figure 19 The mouse condition scores and mouse weights are shown; (A) changes in scores of mice in groups 4-1 to 4-9 over one week, and (B) changes in weight of mice in each group over one week.

[0044] Figure 20 The in vivo metabolism of bacteriophage vB_EfaS-1017 is shown; among which, the plaque count in mouse liver, spleen and blood is shown.

[0045] Figure 21 The table shows the number of immune cells in mice; (A) the white blood cell count was determined by taking mouse blood samples at different times, and (B) the neutrophil count was determined by taking mouse blood samples at different times.

[0046] Figure 22 The table shows the number of immune cells in mouse blood; (A) the percentage of CD3 cells in mouse blood after blood was taken at different times, and (B) the NK cell content in mouse blood. Detailed Implementation

[0047] The strains used in the following examples were all clinically collected strains from Nanjing Public Health Medical Center.

[0048] Example 1: Isolation and purification of bacteriophage vB_EfaS-1017

[0049] In this embodiment, Enterococcus faecalis 10-17 (hereinafter referred to as Ef 10-17) was used as the host bacterium to screen and isolate a strain of Enterococcus faecalis bacteriophage from medical wastewater. The specific steps include:

[0050] (1) Take 200 mL of untreated sewage from the sewage pool of Nanjing Public Health Medical Center, set a pre-cooled high-speed refrigerated centrifuge at 12000 rpm for 15 min, discard the sediment and collect the supernatant, and filter it with a 0.22 μm filter membrane to remove large particles and bacteria from the supernatant.

[0051] (2) Add 20 mL of filtered wastewater, 1 mL of logarithmic bacterial culture and 10 mL of 3×BHI to a centrifuge tube, and add CaCl2 to a final concentration of 10 nM. After 1 h, transfer the centrifuge tube to a shaker and incubate for 24 h. Take 1 mL of the overnight culture liquid, 12000 rpm, 10 min, and filter using a 0.22 μm filter membrane.

[0052] (3) Mix 0.5 mL of Enterococcus faecalis suspension with 0.1 mL of the above liquid, add 6 mL of semi-solid BHI medium cooled to about 55°C, and quickly pour the mixture onto a solid BHI plate. Set up a double-layer plate with only 0.5 mL of bacterial suspension as a control. After the plate solidifies, place it in a constant temperature incubator at 37°C overnight. The next day, the plate containing the sewage bacterial suspension can be observed to become transparent. Record the experimental results, add 5 mL of 1×SM Buffer solution to the plate, and after shaking for 2 hours, aspirate the liquid, stir at 12000 rpm for 5 minutes, discard the precipitate, and filter the supernatant again using a 0.22 μm filter membrane.

[0053] (4) The filtered liquid was serially diluted and measured using the double-layer plate method. 0.1 mL of the appropriately diluted serially filtered liquid was mixed with 0.5 mL of the host bacterial suspension, and 6 mL of BHI medium with an agar content of 0.8% was added. The mixture was poured onto a solid BHI plate, and a plate without dilution was set up as a control. The plate was placed in a constant temperature incubator at 37°C overnight.

[0054] (5) Remove a single transparent plaque from the petri dish and place it into 5 mL of 1×SM Buffer solution. Shake to mix well. Repeat step (4) until the plaques obtained in the entire petri dish are of uniform size.

[0055] After bacteria are lysed by bacteriophages, tiny, transparent, circular plaques are observed on the agar plate, such as... Figure 1 In case A, a single plaque was removed, diluted after shaking, and passaged. After being mixed with Ef 10-17 again, a translucent plaque of the same size could still be observed. Therefore, it was determined that this lytic phage strain was highly efficient and stable in infecting Ef 10-17.

[0056] Transmission electron microscopy (TEM) helps us observe the morphology of bacteriophages more directly. When we use cesium chloride density gradient centrifugation to ultracentrifuge bacteriophages, most bacterial fragments are found in a density layer below 1.3, while tailed bacteriophages aggregate in the middle density layer (1.4-1.7), appearing as a milky white density band. Gently aspirating this middle milky white density band and determining the titer using the double-layer plate method, we found that the titer of the bacteriophage layer collected directly after ultracentrifugation was 10. 9 PFU / mL, the collected phage layer fluid was placed in a dialysis bag and dialyzed overnight. This dialysis step displaced the phages from the cesium chloride buffer into 1×SM buffer. The phages were then observed using a transmission electron microscope. Figure 1 As can be seen from electron microscopy, the bacteriophage possesses a polyhedral capsid, belonging to the typical class Caudoviricetes. Based on its electron microscopic morphology, the bacteriophage is named vB_EfaS-1017.

[0057] Example 2: Study on the biological characteristics of bacteriophage vB_EfaS-1017

[0058] 1. Phage lysis profile determination

[0059] The host range of bacteriophage vB_EfaS-1017, isolated and purified in the example, was determined using the droplet method. As shown in Table 1, the bacteriophage could lyse the three tested Enterococcus faecalis strains, but did not lyse other strains. This indicates that bacteriophage vB_EfaS-1017 has high specificity and a narrow host range.

[0060] Table 1. Host range of bacteriophages

[0061]

[0062] Note: "+" indicates that the cytokine can lyse and form plaques, and "-" indicates that the cytokine cannot lyse and no plaques will form.

[0063] 2. Determination of the optimal multiple of infection (MOU) of bacteriophages

[0064] according to Figure 2 It can be seen that at MOI = 0.001, the phage titer reaches 2.0 × 10⁻⁶. 10 PFU / mL, the value when the titer is higher than other MOI values, therefore at 1×10 8 When the host bacterial concentration of Ef 10⁻¹⁷ is CFU / mL, use 1×10⁻¹⁷. 5 The optimal MOI can be achieved with a phage concentration of PFU / mL, which also indicates that phage vB_EfaS-1017 has strong lytic ability.

[0065] 3. Determination of one-step growth curve of bacteriophage

[0066] This embodiment further adds bacteriophage and host bacteria at the optimal MOI, takes samples at specific time intervals, and measures the bacteriophage titer to determine the state of the bacteriophage in the latent, lytic, and stationary phases. Figure 3 As shown, this bacteriophage has a short latency period; within 5 minutes, it rapidly enters the lysis phase, and the number of bacteriophages increases dramatically. Based on the formula for calculating the burst rate, the burst rate is approximately 97 PFU / cell. The assessment of the latency period and burst rate demonstrates the significant application potential of bacteriophage vB_EfaS-1017.

[0067] 4. Phage stability assay

[0068] The acid-base stability of bacteriophage vB_EfaS-1017 was determined, such as... Figure 4 As shown in Figure A, the phage fluid exhibits the highest stability at pH 8, which is the optimal pH for its existence. The phage remains active at pH 4-13, indicating that it has a wide pH tolerance range. However, the phage survival rate is only 40% at pH 13 and approximately 75% at pH 4. Therefore, it can be inferred that this phage prefers a slightly acidic environment. Figure 4 Figure B shows the results of a temperature sensitivity experiment on the bacteriophage. It reveals that at 50℃, the phage survival rate is 90%, and when the temperature increases to 80℃, approximately 50% of the phage remains. These data indicate that the bacteriophage possesses strong temperature tolerance. Under ultraviolet light irradiation, such as... Figure 4 As shown in Figure C, the amount of bacteriophage decreased sharply after 20 minutes, and after 40 minutes, bacteriophage could not be detected by the double-layer plate method. Therefore, if bacteriophage is accidentally spilled during the experiment and causes contamination, ultraviolet irradiation can be used to reduce the risk.

[0069] 5. Phage whole genome characterization

[0070] The complete genome of bacteriophage vB_EfaS-1017 is 40766 bp, with a GC content of 34.82%. Using the extracted phage genome as a template, PCR amplification was performed, followed by 5 μL of electrophoresis. The results showed bands with the same number of nucleotides as expected from the primer design. Sequencing by the company and alignment with the genome sequence showed high similarity, indicating that the genome is a circular DNA sequence. The number of ORFs predicted for the vB_EfaS-1017 genome was 65, with the longest being 4371 bp and the shortest 105 bp. Among these, 23 ORFs encode proteins whose functions can be identified through searching. These proteins can be mainly divided into three categories: genes encoding tail fimbriae related to host recognition, genes related to DNA binding and cleavage, and genes related to phage lysins. No virulence genes are present. The location of these genes can be accurately identified using the circular image presented by CGView.

[0071] 6. Phage phylogenetic analysis

[0072] Using BLASTN nucleic acid alignment in NCBI, it was found that the whole genome sequence of bacteriophage vB_EfaS-1017 had 91.19% identity with vB_EfaS-SRH2, with a coverage rate of 79%, making it the bacteriophage with the highest similarity. The second most similar was vB_EfaS-IME196, with 88.79% identity and 79% coverage, respectively.

[0073] The terminal large subunit participates in genome packaging. While this sequence exhibits high variability in double-stranded DNA bacteriophages, it is strictly conserved functionally. The evolution of capsid proteins in tailed bacteriophages enables them to withstand environmental stresses, and the stable, conserved capsid proteins are suitable for constructing phylogenetic trees. Therefore, the nucleic acid sequence encoding the terminal large subunit and the amino acid sequence of the capsid protein in the genome were selected for constructing a phylogenetic tree. These data indicate that bacteriophage vB_EfaS-1017 belongs to the class Caudoviricetes.

[0074] Example 3: In vitro bactericidal activity of bacteriophage vB_EfaS-1017

[0075] 1. Bacteriophage bactericidal activity experiment

[0076] Colonies of Ef 10-17 were inoculated into 50 mL of liquid BHI and cultured at 37℃ and 300 rpm for 4 h with shaking. The OD of the bacterial culture was then measured. 600When the value is around 0.5, the bacterial culture is centrifuged at 5000 rpm for 10 min. The supernatant is discarded, and the precipitate is washed twice with an equal volume of PBS and resuspended. Bacteriophage vB_EfaS-1017 is added at MOI = 0.001, and the culture is carried out at 37℃ and 300 rpm in a shaker. Samples are taken at the initial time point, 30 min, 1 h, 2 h, 4 h, and 8 h, and the colony count is recorded. The experiment is repeated three times.

[0077] The bacterial pellet from the logarithmic phase was resuspended in sterile PBS, and samples were taken at different time points with an initial bacterial count of 100%. After dilution, the pellets were spread onto BHI solid medium, and bacterial colony counts were determined. Figure 5 It can be seen that within 30 minutes, the bacterial colony count decreases by more than 90%, i.e., by 1 Log. As time gradually increases, the lysis efficiency no longer increases, indicating that bacteriophage vB_EfaS-1017 has a strong in vitro bactericidal ability. If bacteriophage is added to the bacterial culture in the Erlenmeyer flask during the logarithmic phase, and another flask of host bacteria is inoculated under the same conditions and cultured for another 8 hours, the clarity of the culture medium is observed again. It can be found that the flask with bacteriophage vB_EfaS-1017 added is clearer than the flask with the bacterial culture.

[0078] 2. Bacteriophage clearance and inhibition experiments on biofilms

[0079] (1) Culture of Enterococcus faecalis Ef 10-17 to form a biofilm:

[0080] Select a 96-well plate made of polystyrene and add OD in columns A1-H 11. 600 0.2 mL of bacterial suspension (0.5 g / mL) was added to 8 wells (A12-H12) and BHI liquid medium supplemented with 1% glucose was added as a negative control. The plate was then capped and incubated at 37°C for 24 hours. After 24 hours, a thick membrane was observed at the bottom of the 96-well plate, except for the control group. The bacterial suspension was then discarded, and the biofilm formation was measured using crystal violet staining: The 96-well plate was gently washed three times with sterile water, inverted and dried at 37°C for 10 minutes. 0.2 mL of 0.1% crystal violet solution was added to each well, and the plate was incubated at 37°C for 30 minutes. The plate was then washed three times with sterile water, dried again for 10 minutes, and 0.2 mL of anhydrous ethanol was added. After 30 minutes, the plate was placed in a microplate reader to measure the OD. 570 The experiment was repeated three times.

[0081] (2) The biofilm formed was removed using bacteriophage vB_EfaS-1017:

[0082] Pre-inoculated bacterial culture in the logarithmic growth phase was transferred at 0.2 mL per well to columns 1-10 of a 96-well polystyrene plate. Wells A11-H 11 were used as positive controls, and wells A12-H ​​12 as negative controls. The plate was incubated at 37°C for 24 hours to create a biofilm. The bacterial culture was then gently aspirated along the plate wall, avoiding contact with the bottom of the plate. Serially diluted phage solution was prepared and added to each well at different dilutions according to the number of columns. The plates were incubated at 37°C for 4 hours. Three wells from each column were randomly selected, and the biofilm in the wells was repeatedly aspirated and pipetted using 0.2 mL of PBS. The liquid was then transferred to sterile centrifuge tubes and serially diluted. The remaining bacterial count was determined by colony counting, and the OD was measured using crystal violet staining. 570 Verify the cleaning effect.

[0083] (3) Bacteriophages inhibit biofilm formation

[0084] Take 0.1 mL of bacterial culture in the logarithmic phase and 0.1 mL of culture from 10... 8 Initially, 0.1 mL of phage solution diluted 10-fold was added to each well of a 96-well plate and incubated at 37°C for 24 hours. Wells containing only bacterial suspension and wells containing only culture medium were set up as controls. OD was measured using a microplate reader with crystal violet staining. 570 The experiment was repeated three times.

[0085] (4) Comparison of biofilm clearance effects of bacteriophage vB_EfaS-1017 and EDTA

[0086] Biofilms were cultured in 96-well plates using the method described above. After biofilm formation, 0.1 mL each of high-titer bacteriophage, a mixture of bacteriophage and 5 mM EDTA, and 5 mM EDTA were added to the 96-well plates. The plates were incubated at 37°C for 24 hours. The wells were then cut and placed into tubes containing 2.5% glutaraldehyde solution, and the tubes were incubated overnight at 4°C. The results were observed using a scanning electron microscope and verified again using crystal violet staining.

[0087] Experimental results:

[0088] OD after crystal violet staining 570 Around 1.5, and the colony count is 10. 9 CFU / mL, while the initial bacterial culture volume was 10. 7 CFU / mL indicates good biofilm formation. Figure 6 As shown in A and B, when using the crystal violet method to determine the biofilm clearance effect of different titers of bacteriophage, the bacteriophage effectively cleared existing biofilms compared to the control group, and there was no difference in biofilm clearance between different titers of bacteriophage. After continuously blowing and aspirating the biofilm, the bacterial colony count was measured, indicating that the bacteriophage achieved a biofilm clearance efficiency of 90%. Figure 6 As shown in Figure C, different titers of bacteriophages have a good inhibitory effect on biofilms and can inhibit biofilm formation. Figure 6 In the study, it was observed that when comparing the biofilm removal agents EDTA and bacteriophages, bacteriophages were more effective at removing biofilms than EDTA. Furthermore, the combined use of both was less effective than the use of bacteriophages alone. Similar results were observed in... Figure 7 As observed, bacterial colonies aggregate to form dense biofilms. After using bacteriophages to remove the biofilms, the bacterial count is significantly reduced. Therefore, bacteriophages are an effective way to solve biofilm pollution.

[0089] Example 4: Study on the bactericidal effect of bacteriophage vB_EfaS-1017 combined with antibiotics

[0090] The minimum inhibitory concentration (MIC) of antibiotics used clinically to treat Enterococcus faecalis was determined (the MIC of different antibiotics against host bacteria Ef 10-17 was determined by broth dilution method).

[0091] Prepare the bacterial culture in the logarithmic growth phase in advance, and measure the OD of the bacterial culture. 600 Adjust to OD 600 =0.02, and phage solution diluted serially to the appropriate concentration, and antibiotic solution with a 2×MIC value were prepared. First, the antibiotic was serially diluted, then the pre-diluted phage solution was added to each well, and finally the bacterial culture was added. Antibiotic validation, phage inhibition at different dilutions, pure bacteria, and blank culture medium were set up as positive and negative controls, respectively, with replicate wells. The samples were placed in a microplate reader and the OD was measured at 37℃. 600 Set a 24-hour continuous measurement program. Repeat the experiment three times for each antibiotic. After the measurement is completed, export, organize, and plot the data.

[0092] As shown in Table 2, Ef 10-17 is a vancomycin-resistant Enterococcus faecalis. Based on the measured MIC, antibiotics with different mechanisms of action were selected to investigate the combined effects. Figure 8 It can be seen that bacteriophage alone inhibits bacterial growth within 10 hours, followed by the growth of phage-resistant bacteria, after which the bacteriophage can no longer inhibit bacterial growth. However, the use of drugs such as levofloxacin (Lev), ciprofloxacin, linezolid, and daptomycin effectively prolongs the inhibition time of drug-resistant bacteria. In particular, levofloxacin, when used in combination with bacteriophage at a concentration of 1 / 2 × MIC, can inhibit bacterial growth for up to 24 hours. The figure also shows that penicillin and vancomycin exhibit antagonistic effects when combined with bacteriophage, weakening the antibacterial effect of bacteriophage used alone.

[0093] Table 2. MICs of different antibiotics against host bacteria

[0094]

[0095] The above results demonstrate that bacteriophage vB_EfaS-1017 can effectively remove biofilms formed by host bacterium Ef 10-17, achieving a removal rate of 90% or higher. Bacteriophage vB_EfaS-1017 exhibits a good synergistic effect with levofloxacin; simultaneous use with levofloxacin at 1 / 2×MIC can effectively and stably inhibit bacteria for 24 hours or more.

[0096] Example 5: Study on the therapeutic effect of bacteriophage vB_EfaS-1017 on bacteremia

[0097] Bacteremia often occurs secondary to intra-abdominal infections, urinary tract infections, etc., and severe bacteremia can occur in immunocompromised patients. In the search for prevention and treatment of bacteremia, phage therapy has been put into practice. Compared with antibiotics, phage therapy has advantages such as host specificity and efficient clearance of biofilms.

[0098] However, phage therapy also presents challenges, including issues with phage injection concentration, route of administration, and frequency of dosing. Although oral phage administration is generally considered safe, phage translocation may inhibit the production of immune factors, thereby downregulating the host's immune response and affecting the therapeutic effect. Studies have also shown that after phage injection into mice, the mice's innate immune response clears the phage. The inflammatory response induced by phage varies depending on the site of infection, and different individuals and phages also produce different effects. Therefore, the immune response triggered by phages after entering the body still requires further investigation.

[0099] In this embodiment, a fecal enterococcal bacteremia model was established by intraperitoneal injection. In Example 4, antibiotics with synergistic effects were selected for treatment using a combination of antibiotics and bacteriophages, and the treatment effect was observed.

[0100] Experimental animals: 7-8 week old female mice purchased from Spiford (Suzhou) Biotechnology Co., Ltd.

[0101] 1. Establishment of a mouse bacteremia model

[0102] Seven-week-old Kunming mice were divided into seven groups of six for a transitional period of one week. The mice were grouped according to the concentration of bacteria injected intraperitoneally, as shown in Table 3.

[0103] Table 3 Grouping of mouse bacteremia models

[0104]

[0105] After challenging mice with the bacteria via intraperitoneal injection, their body weight was measured daily, their condition was observed regularly, and they were scored according to a scoring system for one week. The bacterial concentration that ultimately led to the complete death of the mice was taken as the treatment dose.

[0106] 2. Evaluation of the efficacy of phage therapy

[0107] Seven-week-old Kunming mice were separated and transitioned to a different cage for one week. Mice were then challenged with bacteria according to the bacteremia-inducing doses determined in Table 3. With the challenge time point designated as 0, the concentration of the antibiotic levofloxacin was 30 mg / kg, and the injection rate of bacteriophage vB_EfaS-1017 was 1×10⁻⁶ mg / kg. 9 PFU / mL.

[0108] To address the question of whether phage therapy alone is highly effective in current phage therapy, we compared the therapeutic effects of using antibiotics or phages alone with those of using levofloxacin in combination with phages. Specific groupings are shown in Table 4.

[0109] Table 4 Grouping of mouse bacteremia models

[0110]

[0111] For one week, the growth status of each group of mice was observed, and the mice were scored and weighed. Nine hours after intraperitoneal challenge, intact spleen, kidneys, and liver were harvested for pathological sections to observe the degree of organ damage. Spleen, liver, and blood were ground, serially diluted, and plated. Colonies grown on the plates from the combined treatment group's liver were used to determine phage susceptibility, exploring whether the mortality was due to the development of phage-resistant bacteria, and to observe the presence of phages through double-layer plate counting. Blood samples obtained from the orbital fossa were analyzed using ELISA kits to detect the levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) to assess changes in the immune response after phage treatment. The number of white blood cells and neutrophils in the blood was also measured using routine blood tests to observe fluctuations in the mouse's immune system.

[0112] The impact of the timing of phage injection on the effectiveness of treatment after infection is controversial. Therefore, mice were grouped according to the injection time after infection, as shown in Table 5.

[0113] Table 5 Grouping of mouse bacteremia models treated with phage injection at different time points

[0114]

[0115] 3. Metabolism of bacteriophages in mice after intraperitoneal injection

[0116] To investigate whether preventive injection of bacteriophage vB_EfaS-1017 affects the innate immune cells of mice and whether the bacteriophage works with the host's immune system to fight infection, Kunming mice of about 7 weeks old were transitionally fed in four groups of 6 mice each, and bacteriophage fluid and host bacterial fluid in the logarithmic growth phase were prepared in advance.

[0117] Starting at 9:00 AM on the day of the experiment, 0.2 mL of phage stock solution was injected intraperitoneally into each mouse. Samples were collected by dissection at intervals of 1 hour, 6 hours, 12 hours, and 24 hours. Fresh liver and spleen organs were placed in 1.5 mL Eppendorf tubes pre-loaded with magnetic beads and immediately ground in a cryo-mortar. Blood was collected via the orbital sac and placed in anticoagulant tubes containing EDTA salt. A small portion was immediately sent to the Department of Laboratory Medicine at the Tangshan Branch of Nanjing Second Hospital for complete blood count (CBC) analysis, which can be used to analyze changes in white blood cells and neutrophils in mice after phage injection. A portion of the diluted sample, along with the ground organs, was serially diluted. 0.1 mL and 0.5 mL of the host bacteria were mixed using appropriate titers of different diluents, and the remaining amount of phage in the organs after a certain time was determined using the double-layer plate method. The blood samples obtained in the experiment were analyzed using flow cytometry. Specifically, mouse peripheral blood mononuclear cells (PBMCs) were obtained using Ficoll density gradient centrifugation. FITC-anti-CD3 and APC-anti-DR were mixed in a 1:1 ratio, and 1.5 μL was collected. The mixture was incubated at 4°C for 30 min. After centrifugation at 300 × g for 5 min, the supernatant was discarded, and the cells were resuspended in 250 μL of sterile PBS solution (pH 7.4). Flow cytometry analysis was then performed.

[0118] Experimental results:

[0119] 1. In vitro safety assessment of bacteriophages

[0120] By enriching phage lysate on plates, a concentration of 1×10⁻⁶ can be achieved. 12 The PFU / mL phage solution indicates that this method can improve phage titer. By placing the bacterial solution in a shaker and culturing for the same amount of time, the colony count of the host bacterium Ef 10⁻¹⁷ can be stably maintained at 1 × 10⁻¹⁷. 9 CFU / mL.

[0121] Hemolytic activity assays are the most common preliminary rapid tests used for toxicity assessment, such as... Figure 9 The results from the middle A study showed that the phage titer was as high as 10. 11 At PFU / mL, vB_EfaS-1017 did not show a significant inhibitory effect on LO2 cells. Figure 9 In the treatment group B, Triton X-100, the hemolysis rate of cells was 100%, but when using 1.5 × 10⁻⁶ cells... 9 When phage concentrations of PFU / mL are applied, hemolysis of red blood cells is not induced. These data indicate that the phage exhibits good in vitro safety and can be further investigated for therapeutic purposes.

[0122] 2. In vivo safety assessment of bacteriophages

[0123] Bacteriophages were injected into mice via intraperitoneal and tail vein injections. Scores were continuously recorded and monitored at set times. The scores showed that the mice remained healthy throughout, exhibiting no adverse reactions. All mice, including the control group, scored 0. Figure 10 In case A, by weighing the mice daily, it was found that the mice experienced a temporary decrease in body weight after injection. Figure 10 In case B, it is speculated that the phage triggered a series of reactions in the mice, leading to decreased appetite and weight loss. However, no abnormalities were observed in the mice's feces, and there was no difference compared to the control group. This confirms that the phage injection is safe. Therefore, whether injected intraperitoneally or via the tail vein, there is no difference in the mice's health. The next experiment will use intraperitoneal injection to investigate whether the mice's innate immunity fluctuates after phage injection.

[0124] 3. Establishment of a mouse bacteremia model

[0125] Different bacterial suspension concentrations were set, and mice were challenged with the bacteria via intraperitoneal injection. The mice's condition was observed and scored at regular intervals, and survival curves were constructed. Figure 11 It can be seen that intraperitoneal injection of 1×10 10 CFU / mL is the complete lethal dose for mice. Furthermore, the mortality rate increased with increasing bacterial concentration; mice died more rapidly 6 hours after injection, with groups 2-2 receiving 8×10⁻⁶ CFU / mL. 10 Mice with CFU / mL exhibited symptoms such as quiescence, bloody discharge from the corners of the eyes, and piloerection, ultimately leading to death. Intraperitoneal injection of 1×10 10 All mice treated with CFU / mL died within 24 hours. Meanwhile, mice treated with 1×10⁶ CFU / mL... 9 Although mice at CFU / mL showed symptoms briefly after infection, they gradually recovered and did not die. Therefore, this experiment determined that 1×10⁻⁶ CFU / mL was the optimal concentration for infection. 10CFU / mL is the challenge concentration used when phage therapy is employed.

[0126] 4. Evaluation of the efficacy of phage therapy for bacteremia

[0127] From the survival curve Figure 12 As can be seen, the results in the infection group were the same as those in the previously established bacteremia model; mice died within 24 hours after intraperitoneal injection of a certain amount of bacterial solution. Following infection, if antibiotics or bacteriophages were administered alone 1 hour later, the survival rates of the mice were 40% and 60%, respectively. However, if antibiotics and bacteriophages were used in combination, the survival rate of the mice was 80%, indicating that the combined use of bacteriophages and antibiotics was beneficial in improving the survival rate of the mice.

[0128] Figure 13 The data includes the mice's scores and daily weight fluctuations. Although there are individual differences among the mice, it can be seen that the mice lose weight in a short period of time after being infected with bacteremia. This may be related to the mice's resting state, failure to consume food and water, and abnormal defecation. However, the mice's weight slowly increases after treatment.

[0129] Figure 14 As shown in Figure A, 9 hours after infection, the bacterial count in the liver and spleen of both the phage group and the phage-Lev group decreased, indicating that phages have a certain clearance effect on bacteria. We selected 30 bacterial colonies from the diluted and spread plates for phage susceptibility testing. We found that 20 of the 30 strains remained sensitive, while 10 were resistant. This suggests that phage-resistant strains are easily generated during treatment. If phage therapy is used, using a phage "cocktail" or "engineered" phages should be considered to improve this issue; otherwise, treatment failure may occur. Figure 14 As can be seen from Figure B, when mice are infected, the bacteriophages proliferate by adsorbing onto Ef 10-17 in the mouse body, thereby lysing the host bacteria.

[0130] Based on pathological section results Figure 15 It can be seen that in the infection group, the liver vessels were congested and edematous, and the hepatocellular damage was more obvious, showing a map-like change. The spleen was significantly congested, and the kidneys showed glomerular damage and renal vascular congestion. In the groups using phage or antibiotics alone, the liver vessels showed slight congestion, and the glomerular damage was milder. In contrast, the combined treatment group showed almost no liver vessel congestion, and the renal vessels were more orderly and almost without congestion. Figure 16 and Figure 17It can be seen that inflammation occurred in the mice 9 hours after infection. Bacterial infection reduced the number of neutrophils, which were quickly depleted and disordered. Phage treatment relatively enhanced the neutrophil count. Therefore, it is speculated that phage therapy first stimulates and restores the number and function of neutrophils, and then works with neutrophils to clear bacterial pathogens.

[0131] Comparing the effects of different treatment durations on mouse survival rates, by Figure 18 The survival curves show that, compared with treatment at intervals of 1 hour or 6 hours, immediate combined treatment resulted in a 100% survival rate for mice after infection, significantly improving their survival rate. This indicates that early treatment helps improve the survival rate of mice.

[0132] We administered bacteriophages intraperitoneally 12 hours and 24 hours prior to infection for prophylaxis. Upon reinfection, all mice died without treatment. However, with prophylactic phage injection followed by treatment 1 hour later, the survival rate was 80%, consistent with the result of treatment 1 hour after infection. Based on previous experimental results, phage titers were undetectable in organs around 24 hours after infection, suggesting that the 80% survival rate was likely due to the effect of the 1-hour injection. However, if we administered phage 24 hours prior to infection and treated 6 hours after infection, the survival rate increased to 50%, indicating that prophylactic phage administration may delay death, providing more time for treatment and improving survival. Figure 19 This is a graph showing the mouse's score and weight fluctuation.

[0133] 5. Retention of phage in mice after intraperitoneal injection

[0134] To investigate the survival time of bacteriophages after injection and whether phage injection improves survival by influencing mouse immunity, we administered 10 doses of phage intraperitoneally. 9 After phage concentration of PFU / mL, mouse organs were dissected, and the homogenate was immediately serially diluted after homogenization. The remaining titer of phage was then determined. Figure 20 It was observed that phage titers decreased in the liver, kidneys, and blood one hour after injection, but remained abundant. Six hours after injection, a significant number of phages remained in the kidneys, while blood phage levels decreased dramatically. After 12 hours, titer measurements showed no phages in the liver or blood. At 24 hours, no phages were detected in the liver, spleen, or blood using the double-layer plate method. This is similar to the literature report that in the absence of host bacteria, phage titers rapidly decrease and are cleared from organs (especially the liver), while phages can be detected in the spleen. Figure 21It can be concluded that comparing the number of white blood cells and neutrophils in the blood of mice at different time points after phage injection using routine blood tests revealed no difference. Regardless of the time, concentration, or temperature, blood samples taken at regular intervals showed that white blood cells could bind to and internalize the phage under various conditions. While a brief increase in white blood cell and neutrophil counts was observed after phage injection, the difference was not statistically significant. Figure 22 As shown, flow cytometry was used to measure the number of CD3 and NK cells. CD3 is involved in activating T lymphocytes, and NK cells are an important component of white blood cells. As substances used to recognize and clear foreign substances, they can stimulate an immune response under appropriate conditions. However, no differences were observed between the groups. Therefore, it is inferred that after intraperitoneal injection of phage, the phage will quickly enter the systemic circulation and will not trigger a strong innate immune response in the host. This also proves the safety of phage injection.

[0135] In conclusion, it can be determined that bacteriophage vB_EfaS-1017 has in vivo and in vitro safety. Furthermore, the combined use of bacteriophage vB_EfaS-1017 and the antibiotic levofloxacin can effectively treat bacteremia in mice, and preventative use of bacteriophages can prolong the treatment time after infection.

[0136] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. A type of Enterococcus faecalis bacteriophage, characterized in that, The bacteriophage, named vB_EfaS-1017, was screened from untreated medical wastewater and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNO.46182 on September 6, 2024. This bacteriophage exhibits strong lytic activity against Enterococcus faecalis.

2. The Enterococcus faecalis phage according to claim 1, characterized in that, The bacteriophage vB_EfaS-1017 exhibited good tolerance under conditions of 4℃ to 80℃ and pH values ​​of 4 to 13.

3. The Enterococcus faecalis phage according to claim 1, characterized in that, The phage vB_EfaS-1017 had a titer of 2.0 × 10⁻⁶ under the condition of MOI = 0.

001. 10 pfu / mL, after 5 min of incubation, it enters the lysis phase and the burst reaches 97 pfu / cell.

4. The Enterococcus faecalis phage according to claim 1, characterized in that, The bacteriophage vB_EfaS-1017 belongs to the class Caudoviricetes, which are tailed bacteriophages.

5. The Enterococcus faecalis phage according to any one of claims 1 to 4 has any one of the following uses: (a) Use in the inhibition of Enterococcus faecalis in non-disease treatment; (b) Use in the preparation of formulations that broadly inhibit Enterococcus faecalis; (c) Use in the preparation of products for the prevention or treatment of diseases caused by Enterococcus faecalis; (d) Use in the preparation of products that inhibit / eliminate biofilms produced by Enterococcus faecalis.

6. The use according to claim 5, characterized in that, This bacteriophage is used to inhibit Enterococcus faecalis Ef 10-17.

7. The use according to claim 5, characterized in that, The diseases mentioned include urinary tract infections, abdominal infections, pelvic infections, soft tissue infections, endocarditis infections, and bacteremia caused by Enterococcus faecalis.

8. The use according to claim 5, characterized in that, In the preparation of products for preventing biofilm formation by Enterococcus faecalis, the products include antibacterial agents, pharmaceuticals, disinfectants, or cleaning agents.

9. A bactericidal composition for preventing and controlling Enterococcus faecalis, characterized in that, The composition uses Enterococcus faecalis phage vB_EfaS-1017 as the active ingredient according to any one of claims 1 to 4, and the composition may or may not contain a carrier or excipient.

10. The bactericidal composition for preventing and controlling Enterococcus faecalis according to claim 9, characterized in that, The bactericidal composition is used in combination with antibiotics when used to prevent or treat diseases or contamination caused by Enterococcus faecalis.

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