A pharmaceutical composition for inhibiting Enterococcus faecalis and its application

Abstract

The invention discloses a drug combination for inhibiting enterococcus faecalis and application thereof. The drug composition comprises enterococcus faecalis bacteriophage and amoxicillin or ampicillin trihydrate. Amoxicillin with a concentration of 70 μg / mL or ampicillin trihydrate with a volume ratio of 1:1 is added to the bacteriophage to prepare a drug for treating diseases caused by enterococcus faecalis infection, and the disease caused by enterococcus faecalis infection is inhibited by synergistic effect, thereby reducing the use of antibiotics.

Description

Technical Field

[0001] The invention belongs to medical technology, and particularly relates to a pharmaceutical composition for inhibiting Enterococcus faecalis, and more particularly to an application of the pharmaceutical composition in preparing a drug for resisting Enterococcus faecalis infection. Background Art

[0002] After suffering from apical periodontitis, root canal treatment is usually performed. The process is as follows: the patient takes a dental X-ray before the operation, and after understanding the root canal morphology, adjustments are made (so that the affected tooth and the opposing tooth are not in contact), routine pulp opening, and pulp roof exposure. The root canal opening is fully exposed, an unobstructed straight path is established, the working length of the root canal is measured, and the root canal is prepared with a large-tapered nickel-titanium file for root canal dredging. The root canal is rinsed with 0.5% sodium hypochlorite and saline in turn. After rinsing, the root canal is flushed with ultrasound. After the root canal is dried, calcium hydroxide in a sesame paste state is introduced into the root canal, and the dental strip temporary sealant is used to seal it tightly and observe for 7 days.

[0003] With the development and progress of science and technology, root canal treatment equipment and technology have been partially improved, and the success rate after root canal treatment has also increased. However, relevant literature points out that many patients still fail the initial root canal treatment, and apical periodontitis will still recur, requiring root canal retreatment. Endodontic treatment failure may be caused by different reasons, such as incomplete root canal cleaning leading to bacterial residues, improper coronal sealing (leakage), and untreated root canals (missed root canals). However, the main reason for the failure of endodontic treatment is that there are certain bacteria in the root canal system, which are more resistant to disinfectants, leading to persistent intra-root or extra-root infections, such as Enterococcus faecalis. A study counted the relevant cases of apical periodontitis at home and abroad, and included 11 studies. Through analysis, it was found that the prevalence of Enterococcus faecalis in the root canal system was very high. And Enterococcus faecalis can affect the size of periapical lesions and the microbial balance in the root canal system during endodontic treatment. In addition, Enterococcus faecalis can survive by expressing some survival genes (such as biofilm-related genes, etc.) and relying on metabolites. In conclusion, this systematic study highlights that E. faecalis is the main pathogen causing periapical periodontitis and that E. faecalis is resistant to conventional disinfectants and to the presence of adverse environmental conditions.

[0004] The inherent characteristics of Enterococcus faecalis combined with the tooth structure can easily form biofilms. Biofilms refer to groups of bacteria wrapped in polysaccharides, proteins, and lipids produced by the bacteria themselves. They can be adsorbed on medical devices or damaged tissues for treatment. It is a way of survival for bacteria to resist threatening living environments. Some doctors prescribe antibiotics during treatment to minimize the symptoms and signs of asymptomatic apical periodontitis or acute apical abscesses, and to treat or prevent severe oral and facial swelling and other inflammations of the body. However, antibiotics or conventional disinfectants not only cannot remove biofilms, but also produce drug-resistant bacteria. The health problems caused by multidrug-resistant bacteria have caused huge waves worldwide, with approximately 700,000 deaths each year. Therefore, finding new antibacterial agents, reducing the use of antibiotics, and alleviating the pressure caused by the abuse of antibiotics have become problems that are being faced in the existing technology. Summary of the invention

[0005] In view of the problems existing in the prior art, the present application provides a drug combination for inhibiting Enterococcus faecalis and its application. The specific technical scheme is as follows:

[0006] A drug combination for inhibiting Enterococcus faecalis, comprising: an Enterococcus faecalis phage and amoxicillin or ampicillin trihydrate; the Enterococcus faecalis phage is a virulent Enterococcus faecalis phage PEf771, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2019276 and a deposit date of April 19, 2019.

[0007] Application of a pharmaceutical composition for inhibiting Enterococcus faecalis in preparing a drug for resisting Enterococcus faecalis infection.

[0008] Furthermore, the pharmaceutical composition for inhibiting Enterococcus faecalis can inhibit the expression of Enterococcus faecalis biofilm-related genes ebpC, gelE, and esp, and remove the biofilm.

[0009] Furthermore, the pharmaceutical composition for inhibiting Enterococcus faecalis is a combination of Enterococcus faecalis phage and amoxicillin, wherein the Enterococcus faecalis phage has a potency of 10 10 PFU / mL suspension, the amoxicillin concentration is 70 μg / mL, and the added volume ratio of Enterococcus faecalis phage and amoxicillin is 1:1.

[0010] Furthermore, the pharmaceutical composition for inhibiting Enterococcus faecalis is a combination of Enterococcus faecalis phage and ampicillin trihydrate, wherein the Enterococcus faecalis phage has a potency of 10 10 PFU / mL suspension, the concentration of ampicillin trihydrate is 32 μg / mL, and the added volume ratio of Enterococcus faecalis phage and ampicillin trihydrate is 1:1.

[0011] Furthermore, the pharmaceutical composition for inhibiting Enterococcus faecalis is used in the preparation of drugs for treating diseases caused by Enterococcus faecalis infection.

[0012] Furthermore, the drug combination for inhibiting Enterococcus faecalis is used in the preparation of a drug for improving inflammatory bowel disease caused by Enterococcus faecalis.

[0013] Furthermore, the drug combination for inhibiting Enterococcus faecalis is used in the preparation of a drug for treating tooth periapical periodontitis.

[0014] Furthermore, amoxicillin or ampicillin trihydrate in the drug acts simultaneously with the Enterococcus faecalis bacteriophage PEf771 to inhibit Enterococcus faecalis.

[0015] The mechanism of synergistic effect of the Enterococcus faecalis phage and antibiotics of the present invention: Amoxicillin and ampicillin trihydrate have inhibitory effects on both Enterococcus faecalis and Enterococcus faecalis phages. By setting the experimental system to 4 μL, the study found that the inhibition zone of 2 μL of combined phage PEf771 and antibiotics was significantly larger than the inhibition zone of the antibiotics or phage groups alone, proving that the antibiotics and phages have synergistic effects, and the combined use of antibiotics and phages can significantly inhibit the expression of Enterococcus faecalis biofilm-related genes ebpC, gelE, and esp, and the biofilm removal rate reaches 68%. Adding antibiotics and phages at the same time can achieve a better synergistic effect, and phages can enhance the effect of antibiotics in removing biofilms.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) Synergistic combinations can reduce the use of antibiotics and reduce or avoid the emergence of drug-resistant bacteria;

[0018] (2) The combined treatment of phage and antibiotics was applied to the in vitro tooth model of apical periodontitis and the rat model, providing a new treatment direction for apical periodontitis and providing a basis for clinical trials in order to promote phage therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Oxford cup method inhibition zone; Figure A is 70μg / mL amoxicillin, 10 10 PFU / mL phages were used alone or in combination, and the blank control group was supplemented with PBS; Figure B was 32 μg / mL ampicillin trihydrate, 10 10 PFU / mL of phage alone or in combination, P<0.05, there are significant differences, indicated by *.

[0020] Figure 2 Disk method inhibition zone

[0021] Figure 3Synergy effect diagram for different addition orders

[0022] Figure 4 Line graph showing the changes in OD values ​​of bacteria in the logarithmic growth phase during synergistic treatment

[0023] Figure 5 This is a line chart showing the changes in bacterial OD value and viable count in the late growth period of synergistic treatment

[0024] Among them, Figure a and Figure c show the changes in the OD value of bacteria in the late growth stage of synergistic treatment, and Figure b and Figure d show the changes in the number of live bacteria in the late growth stage of synergistic treatment.

[0025] Figure 6 This is the SEM electron micrograph after 4 hours of synergistic treatment;

[0026] Figure A shows Enterococcus faecalis treated with PBS, and Figure B shows Enterococcus faecalis treated with PBS for 10 10 PFU / mL phage treatment, Figure C is after 10 10 PFU / mL phage and 4μg / mL ampicillin trihydrate treatment, Figure D is after 10 10 PFU / mL phage and 9 μg / mL amoxicillin treatment.

[0027] Figure 7 The OD value bar graph of the biofilm cultured for three days with different culture methods. Among them, A is the culture method of using BHI medium on the first day, and adding 2% glucose on the second and third days; B is the BHI medium with 2% glucose added for three days; C is the BHI medium without any glucose added for three days.

[0028] Figure 8 This is the effect diagram of biofilm removal after 0.5h of synergistic treatment;

[0029] Fig. 9 This is the effect diagram of biofilm removal after 1 h of synergistic treatment;

[0030] Fig.10 This is the effect diagram of biofilm removal after 2h of synergistic treatment;

[0031] Fig.11 This is the effect diagram of biofilm removal after 4h of synergistic treatment;

[0032] Fig.12 To investigate the gene expression of biofilms after synergistic treatment for different durations;

[0033] Among them, A is the biofilm gene expression after 0.5h of synergistic treatment; B is the biofilm gene expression after 2h of synergistic treatment; C is the biofilm gene expression after 4h of synergistic treatment.

[0034] Fig.13This is a diagram showing the effects of different durations of collaborative treatment on an in vitro periapical periodontitis model;

[0035] Among them, A is the effect of synergistic treatment of the in vitro apical periodontitis model for 24 hours; B is the effect of synergistic treatment of the in vitro apical periodontitis model for 48 hours; C is the effect of synergistic treatment of the in vitro apical periodontitis model for 72 hours.

[0036] Fig.14 Abdominal dissection results of SD rats;

[0037] Fig.15 Species distribution map of intestinal flora at the phylum level in SD rats.

[0038] Among them, BL in the figure is the blank control group, CO is the Enterococcus faecalis control group, P is the phage group, PAM is the phage-amoxicillin combined medication group, and PAN is the phage-ampicillin trihydrate group.

[0039] Fig.16 Heat map of the phylum level of intestinal flora in SD rats.

[0040] Fig.17 Species distribution map of the intestinal flora at the genus level in SD rats.

[0041] Fig.18 Heat map of species composition at the genus level in the intestinal flora of SD rats.

[0042] Fig.19 Species Circos diagram of the intestinal flora of SD rats at the species level.

[0043] Fig. 20 Alpha diversity analysis based on the total number of OTUs.

[0044] Fig.21 are the species richness and uniformity curves in the reaction sample; A is the dilution curve; B is the Rank abundance curve.

[0045] Fig. 22 UPGMA clustering tree.

[0046] Fig.23 This is a significant analysis diagram of PICRUSt2 function prediction after synergistic action of different durations;

[0047] Among them, A is the significance analysis of PICRUSt2 function prediction at 24 hours; B is the significance analysis of PICRUSt2 function prediction at 48 hours; C is the significance analysis of PICRUSt2 function prediction at 72 hours.

[0048] Fig.24 The phenotypic abundance is stacked.

[0049] Fig.25It is a stacked graph of contribution; A is the phenotype with the ability to form biofilm; B is the phenotype with the anaerobic type; C is the phenotype with the pathogenicity. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present invention. In addition, in the following invention, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0051] Note: 4A is 4μg / mL ampicillin trihydrate, 8A is 8μg / mL ampicillin trihydrate; 9a is 9μg / mL amoxicillin, 18a is 18μg / mL amoxicillin; 70a is 70μg / mL amoxicillin, 32A is 32μg / mL ampicillin trihydrate; C is the control group, with PBS solution added (PBS is phosphate buffered saline); P is a titer of 10 10 PFU / mL phage; + sign in front means adding this reagent first, such as +4A+P means adding ampicillin trihydrate for 15 minutes before adding phage, and the total reaction time is 4 hours

[0052] Example 1: Selection of antibiotics

[0053] 1. Experimental Materials

[0054] The experimental strain YN771 was isolated from the root canal of the dental department in the laboratory, and was deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China in 2019. The deposit number is CCTCC NO: M 2019275. The bacteria are round or oval, arranged in chains, and the diameter is 0.9-1.1 μm. The virulent Enterococcus faecalis bacteriophage PEf771 was deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the deposit number: CCTCC NO: M2019276, and the deposit date is April 19, 2019. It uses E. faecalis YN771 as the host bacteria and is a virulent phage of the Myocaudaaceae family isolated from dental sewage.

[0055] 2. Experimental reagents

[0056] Gentomycin sulfate, ampicillin sodium, cefotaxime, ampicillin trihydrate, amoxicillin, brain heart infusion (BHI) medium, PBS buffer, sterile water purchased from the laboratory

[0057] 3. Choice of antibiotics

[0058] Weigh 2 mg of antibiotics and dissolve them in 1 mL of sterile water to prepare 2 mg / mL stock solution. Use the Oxford cup method to perform the inhibition zone drug sensitivity test. After culturing at 37°C for 12 h, measure the size of the inhibition zone. The antibacterial ability is determined based on the diameter of the inhibition zone. Select the two antibiotics with the best antibacterial effect. The results are shown in Table 1.

[0059] Table 1 Inhibition zone size of different antibiotics

[0060]

[0061] By adjusting the ratio of antibiotics to water, it was found that amoxicillin had the best antibacterial effect, followed by thiophanate-methyl and ampicillin trihydrate. After verification, it was found that the use of thiophanate-methyl would have great side effects. It may cause various blood disease complications after use, leading to hemolytic anemia and thrombocytopenia. There may be risks in the use of medication in root canal treatment. Therefore, thiophanate-methyl was excluded. Amoxicillin and ampicillin trihydrate were selected for subsequent experiments.

[0062] 4. Minimum inhibitory concentration of antibiotics

[0063] The selected two antibiotic stock solutions were diluted to 20 μg / mL in a gradient manner, and the added dosage was gradually reduced in a 2-fold gradient. The minimum inhibitory dose of antibiotics against Enterococcus faecalis was determined by the Oxford cup method. After activating the culture of YN771, 100 μL OD 600 The plate is evenly coated with a bacterial solution of 1.0. The Oxford cup is placed on the plate, and different antibiotic solutions diluted in multiple ratios are added, dried, and cultured overnight in a 37°C incubator. The minimum inhibitory dose is the critical value when there is no inhibition zone.

[0064] When the antibiotic concentration was 20 μg / mL, the minimum inhibitory dose of amoxicillin was 8 μL, and the minimum inhibitory dose of ampicillin trihydrate was 4 μL (Table 2).

[0065] Table 2 Minimum inhibitory dose experimental results

[0066]

[0067] 5. Concentration selection for synergistic treatment

[0068] The pre-activated YN771 was selected with a pipette and inoculated into BHI liquid medium, and cultured at 37°C and 150rpm until OD 600 When the OD value is about 0.7, transfer to new liquid culture medium at 2% inoculation volume and culture at 37℃150rpm until the OD value is 600When the value is about 1.0, take 100 μL of Enterococcus faecalis YN771 bacterial solution and evenly spread it on the BHI medium plate. Set the experimental system to 4 μL. Adjust the antibiotic concentration with the minimum inhibitory dose as a reference, and explore the antibiotic concentration that can produce synergistic effects with phages.

[0069] The experiment was divided into 4 groups: (1) blank control group: 4 μL PBS buffer was added; (2) antibiotic group: 4 μL antibiotics of various concentrations were added; (3) bacteriophage PEf771 group: 4 μL 10 10 (4) 2 μL of PEf771 and antibiotics were added to the group with three replicates. The Oxford cup method and the paper strip method were used to compare the diameters of the inhibition zones to observe whether phage PEf771 and antibiotics had a synergistic effect.

[0070] When the experimental system was 4 μL, the combination of amoxicillin and ampicillin trihydrate with phage had a synergistic effect when the concentration was 70 μg / mL and 32 μg / mL using the Oxford cup method, and the inhibition zone of the combined drug was significantly greater than that of the antibiotic alone (P < 0.05) (Table 3 and Figure 1 As shown). This indicates that the combined use of drugs significantly enhanced the antibacterial effect on Enterococcus faecalis YN771, and the total addition amount was 4 μL, and the inhibition zone of phage PEf771 and antibiotic 2 μL each was significantly larger than that of the antibiotic or phage group alone, so the combined application group (phage + antibiotic) had a synergistic antibacterial effect.

[0071] Table 3 Diameter of inhibition zone (long diameter × short diameter)

[0072]

[0073] When the experimental system was 4 μL, the paper strip method showed a synergistic effect when the concentration of amoxicillin was 9 μg / mL and the concentration of ampicillin trihydrate was 4 μg / mL. The inhibition zone of the combined drug was significantly greater than that of the antibiotics alone. There was no inhibition zone when the antibiotics were used alone ( Figure 2 )(In Figure A, 4A is 4 μg / mL ampicillin trihydrate, 8A is 8 μg / mL ampicillin trihydrate, and P is the potency of 10 10 PFU / mL phage, PBS is phosphate buffered saline, 9a in Figure B is 9μg / mL amoxicillin, 18a is 18μg / mL amoxicillin, P is the potency of 10 10 PFU / mL phage. ).

[0074] 6. Study on the sequence of collaborative treatment

[0075] Prepare OD as above 600The concentration of E.faecalis YN771 was about 1.0. 100 μL YN771 bacterial solution was taken into a 2 mL EP tube. The experimental system was also 4 μL. The experiment was divided into 6 groups: (1) blank control group: 4 μL PBS buffer was added, (2) bacteriophage PEf771 group: 4 μL 10 10 PFU / mL PEf771, (3) Phage PEf771 and antibiotic combination group: 2 μL of phage PEf771 and antibiotic were added at the same time, (4) 2 μL 10 10 PFU / mL PEf771 phage was added after 2 h of action, and then 2 μL of antibiotics was added. (5) 2 μL of antibiotics was added after 2 h of action, and then 2 μL of 10 10 PFU / mL PEf771 phage. The above operations were all reacted in a 37°C incubator. After a total of 4 hours, the reaction was terminated by taking out the incubator. Samples were taken from each group, diluted and spread on the plate, and the viable bacteria were counted. Each group had three parallels. By comparing the number of viable bacteria, we explored whether the order of synergistic treatment would affect the treatment effect. The results are as follows Figure 3 As shown, whether it is 70μg / mL amoxicillin and 32μg / mL ampicillin trihydrate obtained by the Oxford cup method or 9μg / mL amoxicillin and 4μg / mL ampicillin trihydrate obtained by the paper disk method and the potency of 10 10 When PFU / mL phage was used in combination, the effect of simultaneous use was not significantly different from adding antibiotics first and then phages or adding phages first and then antibiotics ( Figure 3 4A in A is 4μg / mL ampicillin trihydrate, 9a is 9μg / mL amoxicillin, and P is 10 10 PFU / mL phage, PBS is phosphate buffered saline, Figure 3 In B, 70a is 70 μg / mL amoxicillin, 32A is 32 μg / mL ampicillin trihydrate, and P is 10 10 PFU / mL phage, + sign in front means adding this reagent first, such as +4A+P means adding ampicillin trihydrate for two hours and then adding phage for 2 hours. ).

[0076] 7. Effects of synergistic treatment on bacteria at different stages, SEM observation

[0077] Similar to the above experiment, the experiment was divided into a blank control group, a phage group, and a phage and antibiotic combination group, with three replicates in each group. E. faecalis YN771 was cultured to OD 600When the value is 0.7 (logarithmic phase) and 1.0 (stable phase), take 1mL of bacterial solution in 2mL EP tubes, add 1mL of reagents in each experimental group according to the experimental system, and act for 16 hours, and take samples every 4 hours. Perform plate dilution and live bacteria count, and observe the therapeutic effect of synergistic treatment on bacteria in different stages through the number of live bacteria. The specific results are as follows:

[0078] Effects of synergistic treatment on bacteria in logarithmic phase: Figure 4 As shown, the synergistic combination of four different concentrations had an effect on the OD of Enterococcus faecalis in the logarithmic phase for 4h. 600 The value decreased from 0.7 to about 0.2, while the control group had an OD 600 The value increased, indicating that the combination of four different concentrations of antibiotics and phages had a good inhibitory effect on bacteria in the logarithmic phase.

[0079] Effects of synergistic therapy on bacteria in the stable phase: Figure 5 As shown in the figure, when the combined drug combination obtained by the paper disc method treats bacteria in the stable phase, its OD 600 The value and the number of viable bacteria decreased with the extension of time ( Figure 5 a), 4 μg / mL ampicillin trihydrate was used in combination with phage for 4 h, OD 600 The value dropped from 1 to 0.7, and from 8h to 0.4. When 9μg / mL amoxicillin and phage were used together for 4h, OD 600 The value dropped from 1 to 0.5, 8h to 0.4, 12h to 0.3, and after 16h of treatment, the two groups dropped by about 0.4 compared with the control group. Figure 5 ), 4h OD 600 It dropped to about 0.9, to 0.6 in 8 hours, and to 0.5 in 16 hours, which was 0.2 lower than the control group overall. The number of viable bacteria in the combined use of 4μg / mL ampicillin trihydrate and bacteriophage decreased by about 1 logarithmic level in 4 hours, and the number of viable bacteria in the combined use of 9μg / mL amoxicillin and bacteriophage decreased by about 2 logarithmic levels in 4 hours. Both groups decreased by about 4 logarithmic levels in 16 hours. The number of viable bacteria in the phage group decreased by about 1 logarithmic level in 8 hours and by about 3 logarithmic levels in 16 hours, both of which had obvious antibacterial effects.

[0080] When the drug combination obtained by the Oxford cup method was used to treat bacteria in the late logarithmic growth period, its OD 600 The value and viable count also decreased with time ( Figure 5 c), 32 μg / mL ampicillin trihydrate was used in combination with phage for 4 h, OD 600 The value dropped from 1 to about 0.5, and then to 0.2 after 8 h. When 70 μg / mL amoxicillin was used in combination with phage for 4 h, the OD 600It dropped from 1 to 0.6, and then to about 0.2 after 12 hours. After 16 hours of treatment, the two groups were about 0.6 lower than the control group. The number of live bacteria in the combination of 32μg / mL ampicillin trihydrate and bacteriophage decreased by 2 logarithmic levels in 4 hours and 5 logarithmic levels in 12 hours. The number of live bacteria in the combination of 70μg / mL amoxicillin and bacteriophage decreased by 1 logarithmic level in 4 hours and about 4 logarithmic levels in 12 hours. After 16 hours of treatment, the two groups were about 6 logarithmic levels lower than the control group ( Figure 5 d). Therefore, several synergistic combinations have extremely strong antibacterial effects.

[0081] Effects of synergistic treatment on E.faecalis YN771 bacteria observed by SEM electron microscopy

[0082] The same experimental operation was performed, and samples were taken from the experimental group after 4 hours of action for scanning electron microscopy observation: first, the samples were fixed, and each sample was centrifuged at low temperature and high speed. The supernatant was discarded to collect the bacteria, and 2.5% glutaraldehyde with a pH of about 6.8 was added, and stored in a refrigerator at 4°C overnight. Then rinse three times with PB buffer with a pH of about 6.8, and stand at room temperature for 30 minutes each time. Note that after each rinse, use a pipette to slowly aspirate the residual rinse solution from the previous rinse. Next, ethanol dehydration, replacement, CO2 drying, sticking, gold coating, and finally sampling, observation and photography are carried out. The results are as follows Figure 6 As shown, the bacteria were obviously lysed when the phage was used alone, while the bacteria treated with both drugs in combination had cell wall shrinkage.

[0083] In summary, four synergistic combinations were successfully screened, 32μg / mL ampicillin trihydrate (1.6MIC) + PEf771, 70μg / mL amoxicillin (1.75MIC) + PEf771, 4μg / mL ampicillin trihydrate (0.2MIC) + PEf771, and 9μg / mL amoxicillin (0.225MIC) + PEf771. When exploring the effect of the sequence on the antibacterial effect, it was found that adding them simultaneously had a better antibacterial effect; the SEM electron microscope scanning image showed that the bacterial cell wall shrank due to the synergistic effect, and no cell filamentation was found. The synergistic mechanism is that the phage cooperates with the antibiotic.

[0084] Example 2: Study on the removal of Enterococcus faecalis biofilm by combined use of bacteriophage and antibiotics

[0085] 1. Optimal biofilm culture method

[0086] Add 200 μL BHI liquid medium to a 96-well plate and inoculate 10 μL OD 600The YN771 bacterial liquid with a concentration of 0.6-0.8 was divided into three experimental groups. The first group was cultured with BHI medium on the first day, and 2% glucose was replaced every 8 hours on the second and third days. The second group used BHI medium supplemented with 2% glucose for three days, and the third group used a single BHI medium for three consecutive days. The second and third groups also changed the culture medium every 8 hours. After three days of culture, the liquid culture medium and floating objects in the 96-well plate were discarded, and the plate was rinsed twice with PBS buffer. The thickness of the biofilm was determined by crystal violet staining: 200μL 100% methanol was used to fix it for 30 minutes, and the methanol was aspirated and then inverted at room temperature to dry, and then 0.1% crystal violet stain was used to stain at room temperature for 30 minutes, rinsed twice with sterile water, inverted and placed in a 37°C constant temperature box to dry, dissolved in 95% ethanol for 30 minutes, and placed in an enzyme reader to measure the OD 595 .

[0087] The results are as follows Figure 7 As shown, when the culture method is to use BHI medium on the first day and replace 2% glucose every 8 hours on the second and third days, the biofilm thickness is the thickest, which is 1.8 times the thickness of the biofilm using BHI medium alone. Therefore, the former is selected as the best way to culture biofilm.

[0088] 2. Study on the effect of synergistic treatment on biofilm removal and the order of addition

[0089] According to the above experimental operation, the most suitable method for biofilm growth was selected to culture the biofilm, and the biofilm was removed according to different experimental groups. The experiment was divided into 6 groups: (1) blank control group (P): 200 μL PBS buffer was added; (2) antibiotic group: 200 μL antibiotics of different concentrations were added; (3) bacteriophage PEf771 group: 200 μL 10 10 PFU / mL PEf771; (4) Phage PEf771 and antibiotic combination group: 100 μL of phage PEf771 and antibiotic were added simultaneously; (5) 100 μL of 10 10 PFU / mL PEf771 phage was added after 15 min of action, and then 100 μL of antibiotics was added; (6) 100 μL of antibiotics was added after 15 min of action, and then 100 μL of 10 10 PFU / mL PEf771 phage, for a total of 4 hours, the biofilm removal effect was measured after 30 minutes, 1 hour, 2 hours, and 4 hours of action, with three replicates in each group. (Note: Figure 1-Figure 25 In the diagram, 4A is 4 μg / mL ampicillin trihydrate, 9a is 9 μg / mL amoxicillin, 32A is 32 μg / mL ampicillin trihydrate, 70a is 70 μg / mL amoxicillin, and P is a potency of 10 10PFU / mL phage, C is the control group with PBS buffer, + sign in front means adding this reagent first, such as +4A+P means adding ampicillin trihydrate for 15 minutes and then adding phage, and the total action time is 4 hours. )(When P<0.1, there is a significant difference, indicated by *, when P<0.05, there is an extremely significant difference, indicated by **, and ns indicates no significant difference)

[0090] The results are as follows Figure 8 As shown in the figure, after 0.5h of treatment, except for the combination of 32μg / mL ampicillin trihydrate and bacteriophage, which had no significant effect on removing biofilm, the other three combined drug combinations all had a significant effect on removing biofilm, and compared with the control group, they could remove about 29% of the biofilm. Among them, there was no significant difference between the order of adding 4μg / mL ampicillin trihydrate, 32μg / mL ampicillin trihydrate and bacteriophage and adding them at the same time, while the combination of 9μg / mL amoxicillin, 70μg / mL amoxicillin and bacteriophage had a better effect on removing biofilm when used at the same time (such as Figure 8 B, 8D), and if Figure 8 As shown in D, a titer of 10 10 There was a significant difference between the phage PEf771 and 70μg / mL amoxicillin groups and the control group; there was a significant difference between the simultaneous addition group and the group that added 70μg / mL amoxicillin for 15 minutes and then added phages for a total of 0.5h; there was no significant difference between the simultaneous addition group and the group that added phages for 15 minutes and then added an equal volume of 70μg / mL amoxicillin for a total of 0.5h. Figure 8 As shown in C, a titer of 10 10 There was a significant difference between the PFU / mL of phage PEf771 and 9μg / mL amoxicillin compared with the control group; there was a significant difference between the simultaneous addition group and the addition of 9μg / mL amoxicillin for 15 minutes and then the addition of phage for a total of 0.5 hours; there was no significant difference between the simultaneous addition group and the addition of phage for 15 minutes and then the addition of an equal volume of 70μg / mL amoxicillin for a total of 0.5 hours. In summary, the simultaneous addition group had a better effect in removing biofilms.

[0091] After 1 hour of treatment, the combination of drugs can significantly remove biofilm, and can remove about 44% of biofilm. There is no significant difference between the order of adding antibiotics and phages and adding them simultaneously (such as Fig. 9 ).

[0092] After 2 h of treatment, 58% of biofilms were removed. The effect of adding both antibiotics and phages at the same time was better than that of adding antibiotics first and then phages, or adding phages first and then antibiotics. Fig.10 ).

[0093] 68% of biofilms can be removed after 4 hours of treatment, and the effect of removing biofilms is the best when adding both drugs at the same time. In general, the effect of removing biofilms by the phage group in the first 2 hours is slightly better than that of the combination drug compared with the single phage, but there is no significant difference. ( Fig.11 )

[0094] 3. Effects of synergistic treatment on the expression of biofilm-related genes

[0095] Six genes related to the biofilm formation of Enterococcus faecalis, such as ebpB, fsrA, and esp, were selected, and primers were designed using Primer5.0 software. After the target gene was amplified, 1% gel was used for nucleic acid electrophoresis to observe the results, and the gene amplification products showing a single band and matching size were sent to a sequencing company for sequence determination. Primer sequences for the biofilm genes ebpC, gelE, and esp related to E. faecalis YN771 were designed using Primer5.0 software and sent to Qingke Biotechnology Co., Ltd. for primer synthesis.

[0096] Add 3 mL of fresh BHI liquid medium to a 6-well plate and inoculate 100 μL of OD 600 The YN771 bacterial solution was about 0.7, and the biofilm was cultured according to the above experimental method. After the biofilm was cultured, the liquid culture medium and floating matter were discarded, and the biofilm was rinsed twice with PBS buffer. The experiment was divided into three groups according to different experimental groups: blank control group: 2mL PBS buffer was added; phage group: 2mL 10 10 PFU / mL PEf771; phage antibiotic combination group: 10 10 PFU / mL of phage PEf771 and antibiotics, 1 mL each. Act for 4 hours, take samples after 30 minutes, 2 hours, and 4 hours, discard the liquid culture medium and floating matter, rinse twice with PBS buffer, rinse the six-well plate repeatedly with sterile water, transfer the rinsed liquid to a 1.5 mL EP tube, suspend the bacteria with 100 μL of lysozyme at a final concentration of 10 mg / mL for pretreatment, let stand at room temperature for 10 minutes (Note: centrifugation is not allowed in this step), and then extract total bacterial RNA according to the Bioflux Bacterial Genomic RNA Extraction kit.

[0097] Specific results such as Fig.12 As shown in the figure, compared with the control group, the expression levels of the three biofilm-related genes were significantly reduced 0.5 h after combined use (there was a very significant difference when P < 0.01, indicated by *), indicating that combined use can effectively inhibit the expression of biofilm-related genes ( Fig.12A). After 2 hours of treatment, the combination of 4 μg / mL ampicillin trihydrate and bacteriophage had no significant inhibitory effect on the expression of biofilm-related genes, while the other three combinations significantly inhibited the expression of biofilm-related genes ( Fig.12 B). After 4 h of treatment, only the combination of 32 μg / mL ampicillin trihydrate and 70 μg / mL amoxicillin could significantly inhibit the expression of biofilm-related genes ( Fig.12 C). Compared with the control group, the expression of biofilm-related genes increased in the combination of 4μg / mL ampicillin trihydrate and 9μg / mL amoxicillin, especially in the combination of 9μg / mL amoxicillin, where the expression was about 2 times that of the control group. The effect of phage acting alone will reduce the inhibition of biofilm-related gene expression over time.

[0098] In summary, both the phage group and the synergistic combination have strong ability to remove biofilm, with a 4h removal rate of 68%. YN771 biofilm-related genes include gelE, ebpC, and esp, and qPCR verification shows that phage therapy can indeed inhibit biofilm-related expression. As time goes on, the combined use of 32μg / mL ampicillin trihydrate and 70μg / mL amoxicillin has a stronger effect on biofilm-related inhibition, followed by the phage group, while the combined use of 4μg / mL ampicillin trihydrate and 9μg / mL amoxicillin for 4h has a weaker ability to inhibit biofilm gene expression than the former two.

[0099] Example 4: Exploring the effect of synergistic treatment in an in vitro tooth model

[0100] An in vitro tooth model was established and several drug combinations were applied to it. The specific results are as follows Fig.13 As shown:

[0101] After 24 hours of action, the combination of 4μg / mL ampicillin trihydrate and bacteriophage had a good antibacterial effect, and no live bacteria appeared. The other several drugs also had a good antibacterial effect. Compared with the control group, the number of live bacteria decreased by 6 logarithmic levels ( Fig.13 A).

[0102] After 48 hours of action, no live bacteria were detected in any combination of drugs ( Fig.13 B).

[0103] After 72 hours of action, only the combination of 70 μg / mL amoxicillin and phage did not detect the presence of live bacteria. Compared with the control group, the number of live bacteria in the rest of the groups decreased by about 6 logarithmic levels. The antibacterial effect of the combination was better than that of the phage alone ( Fig.13 C).

[0104] Therefore, after comprehensive consideration, the subsequent animal experiments selected 70μg / mL amoxicillin and 32μg / mL ampicillin trihydrate combined with phage for use in rat abdominal infection experiments. P<0.1 represents a significant difference, indicated by *, and P<0.05 represents an extremely significant difference, indicated by **.

[0105] Example 5: Establishment of rat abdominal infection model to explore synergistic therapeutic effects

[0106] Prepare YN771 bacterial suspension with an OD600 of about 1.0 and a titer of 10 10 PFU / mL bacteriophage PEf711, 70μg / mL amoxicillin, 32μg / mL ampicillin trihydrate, SD rats were adaptively fed for 3 days, starved for 12 hours, and fasted for 6 hours. Then, 3% sodium pentobarbital (injection calculated at a dose of 1mL / kg body weight) was injected into the abdominal cavity to anesthetize the rats. Note: The puncture site during surgery is prone to infection and needs to be disinfected with iodine. The reagents in the experimental group were injected 4cm below the xiphoid process of the rat and 0.5cm to the left of the center of the abdomen. After the operation, the diet and drinking water of the SD rats were supplied normally, and the changes in the rats' drinking water, diet, hair and other conditions were observed daily. The fecal samples of 42 SD rats after surgery were divided into groups:

[0107] 1) Control group (Enterococcus faecalis group) (n=5): 0.5 mL of Enterococcus faecalis was injected intraperitoneally, and the mice were killed and samples were collected after 72 hours.

[0108] 2) Blank group (n=10): No reagent was injected into the peritoneal cavity, and the mice were killed immediately for sampling (n=5); No reagent was injected into the peritoneal cavity, and the mice were killed 72 hours later for sampling (n=5).

[0109] 3) 0.5 mL Enterococcus faecalis + 0.5 mL phage group (n=9): sampling was performed 24 hours after surgery (n=3), 48 hours after surgery (n=3), and 72 hours after surgery (n=3).

[0110] 4) 0.5 mL Enterococcus faecalis + 0.25 mL ampicillin + 0.25 mL bacteriophage (n=9), sampling was performed 24 hours after surgery (n=3), 48 hours after surgery (n=3), and 72 hours after surgery (n=3).

[0111] 5) 0.5 mL Enterococcus faecalis + 0.25 mL amoxicillin + 0.25 mL bacteriophage (n=9), sampling was performed 24 hours after surgery (n=3), 48 hours after surgery (n=3), and 72 hours after surgery (n=3).

[0112] Fecal samples were collected 72 hours after injection of Enterococcus faecalis YN771 and 24 hours, 48 ​​hours and 72 hours after surgery, and the feces were subjected to 16SrRNA detection and sequencing analysis; dissection was performed 72 hours later, and the physiological status of various tissues of the rats was observed under a surgical microscope.

[0113] After 72 hours of treatment, all SD rats in each group survived and were killed and dissected to observe the following: Fig.14 As shown: The whole abdominal cavity of normal SD rats is clean, the ascites is clear and without mucus, the intestines are bright in color, the liver is bright red and smooth, the liver lobe is clearly marked, and the spleen is reddish brown. The abdominal tissues of rats in the E. faecalis control group were red, swollen and bleeding. The liver of one phage group mouse (P group) was dark red, with a small amount of mucus, mesenteric abscess and gastrocolic ligament abscess. The stomach, intestine and spleen of the other two rats were stained normally, and the liver was bright red. Phage PEf771 and amoxicillin combined group: 1 case of mesenteric abscess, 2 normal; phage PEf771 and ampicillin combined group: 3 normal. From the observation of the physiological state of each organ and tissue of the rat alone, the effect of phage PEf771 + ampicillin was stronger than that of phage PEf771 + amoxicillin, followed by the phage group.

[0114] It can be seen that bacteriophage PEf771+ampicillin and bacteriophage PEf771+amoxicillin can improve inflammatory bowel disease caused by Enterococcus faecalis, and the effect is more obvious than that of the phage group alone.

[0115] Example 6: The V3-V4 region of 16S rDNA was amplified using specific primers, the library was constructed, and the Illumi-na MiSeq sequencing platform was used for sequencing. The sample sequencing was completed by Kidio Biosequencing. Usearch software was used for clustering to obtain OTU abundance and OTU representative sequences. Qiime software was used for alpha diversity index analysis, and R language and other software were used for species composition analysis, Beta diversity analysis, etc.

[0116] From the door level (such as Figure 15-16 ), the dominant bacterial phyla are mainly three categories: Bacteroidetes, Firmicutes, and Verrucomicrobia. After 24 hours of action, the relative abundance of Firmicutes in the phage group (P-24h) decreased from 55.81% to 52.12% compared with the Enterococcus faecalis control group (CO-24h), and decreased to 41.37% in the phage-ampicillin trihydrate group (PAN-24). After 48 hours of action, the phage group (P-48h) decreased to 40.97%, the phage-ampicillin trihydrate group (PAN-48) decreased to 34.59%, the phage group (P-72h) decreased to 40.77%, and the phage-ampicillin trihydrate group (PAN-72) decreased to 39.28%. That is, the relative abundance of Firmicutes in both the phage group (P) and the phage-ampicillin trihydrate group (PAN) decreased, and the change in the phage-ampicillin trihydrate group was more significant.

[0117] Overall, the level Figure 17-18 ) were Akkermansia, Prevotella_9, Lactobacillus, Lachnospiraceae_NK4A136_group, Ruminococcaceae_UCG-014 (beneficial bacteria) and Phascolarctobacterium. The dominant genera in the blank group were mainly Prevotella (19%), Lachnospiraceae_NK4A136_group (11%), and Lactobacillus (10%). The dominant genera in the Enterococcus faecalis group were Akkermansia (19%), Lactobacillus (17%), and Lachnospiraceae_NK4A136_group (10%).

[0118] The dominant genera in the 24h phage group were Lactobacillus (21%), Akkermansia muciniphila (18%), and Lachnospiraceae NK4A136 (8%); the dominant genera in the 48h phage group were Akkermansia muciniphila (24%), Ruminococcaceae UCG-014 (10%), and Lachnospiraceae NK4A136 (6%); the dominant genera in the -72h phage group were Akkermansia muciniphila (25%), Lactobacillus (11%), and Ruminococcaceae UCG-014 (8%).

[0119] The dominant genera in the phage and amoxicillin 24h group were Akkermansia muciniphila (16%), Lactobacillus (16%), and UCG-014 of the family Ruminococcaceae (8%). The dominant genera in the phage and amoxicillin 48h group were Akkermansia muciniphila (23%), UCG-014 of the family Ruminococcaceae (13%), and Lactobacillus (8%). The dominant genera in the phage and amoxicillin 72h group were Akkermansia muciniphila (19%), Prevotella (8%), and NK4A136 of the family Lachnospiraceae (5%).

[0120] The dominant bacterial genera in the 24h group of bacteriophage and ampicillin trihydrate were Akkermansia muciniphila (25%), Lactobacillus (10%), and Prevotella (9%); the dominant bacterial genera in the 48h group of bacteriophage and ampicillin trihydrate were Akkermansia muciniphila (34%), Lactobacillus (9%), and Lachnospiraceae NK4A136 (5%); the dominant bacterial genera in the 72h group of bacteriophage and ampicillin trihydrate were Akkermansia muciniphila (20%), Lactobacillus (11%), and Lachnospiraceae NK4A136 (7%).

[0121] In general, compared with the blank group, the genus Prevotella in the other groups decreased. The genus Akkermansia muciniphila increased in all experimental groups compared with the control group. The relative abundance of Akkermansia muciniphila in the control group was only 3%. Akkermansia muciniphila is associated with the alleviation of inflammatory bowel disease (IBD).

[0122] like Fig.19 As shown, the dominant bacterial species in the blank group were: Lactobacillus murinus; Lactobacillus reuteri; the dominant bacterial species in the Enterococcus faecalis control group were: Lactobacillus reuteri; Lactobacillus murinus; Lactobacillus gasseri; the abundance of L. murinus in the phage group increased at 24 hours, and the abundance of the dominant bacterial species decreased at 48 hours and 72 hours; compared with the control group, the abundance of Bacteroides massiliensis dnLKV3 in the phage group, phage-amoxicillin group, and phage-ampicillin trihydrate group increased, and the abundance of Phascolarctobacterium faecium decreased.

[0123] α diversity analysis: Simpson and Shannon jointly reflect the richness and uniformity of species, so the level of the index will also be affected by the uniformity. The more uniform the species distribution in the sample, the higher the diversity. There are many forms of the Simpson index calculation formula. The formula is Σ(Pi)2. The smaller the value, the higher the diversity; the formula is 1-Σ(Pi)2 or 1 / Σ(Pi)2. The larger the value, the higher the diversity. The formula used in this analysis is 1-Σ(Pi)2. As can be seen from the figure, there is no significant difference in the Simpson index and Shannon index between the phage group and the phage-antibiotic combination group and the blank control group. Only the Simpson index and Shannon index of the phage and ampicillin trihydrate 48h group are slightly lower than those of the blank group, but overall phage therapy does not affect the richness and diversity of species ( Fig. 20 ).

[0124] The calculation method of the dilution curve is to extract n test substance samples multiple times, calculate the expected value of each test substance, and then draw a curve based on the set of n values ​​and the corresponding exponential expected value. If the trend of the curve is finally close to a flat state, it means that increasing the sequencing depth will not affect the species diversity, and the sequencing amount is sufficient. The dilution curve of this experiment eventually tends to be flat, indicating that the sequencing amount is sufficient. The blank group has the highest richness, followed by the phage group. The lowest richness is the Enterococcus faecalis control group and the phage and amoxicillin combined 24h group ( Fig.21 A).

[0125] The rank curve can directly reflect the richness and uniformity of species in the sample. In the horizontal direction, the length of the curve represents the diversity of the sample species. The longer the curve on the horizontal axis, the higher the diversity. In the vertical direction, the smoothness of the curve represents the uniformity of the species. The flatter the curve, the more uniform the species distribution. Fig.21 As shown in B, all the curves have the same trend, but the curve of the blank control group is longer, flatter, and has the largest span. The curves of the other groups are almost the same length, and the length of the phage and ampicillin trihydrate 72h is the shortest.

[0126] Beta diversity analysis: Each terminal branch in the graph represents an individual. Usually, individuals from the same group form a large branch, and different groups form different branches. Individuals on the same branch have more similar group structures. The distance between two individuals is equal to the sum of the branch lengths from the ends of these individuals to the common node. Fig. 22 It can be seen that the blank control group is closest to one sample in the E. faecalis control group, and the other two are farther away, and the sample distances are relatively scattered. The next closest is the two combined application groups, and the farthest is the phage group, but the separation of several samples in the control group is relatively scattered. In general, the distance between the phage and amoxicillin combination and the blank group is relatively close, indicating that their flora structures are relatively similar, while the phage group is farther away, indicating that the phage may have changed the flora structure.

[0127] PICRUSt2 function prediction analysis: This analysis mainly uses Class B classification to perform significance analysis on the phenotypic abundance of each group of PICRUSt2 function predictions, such as Fig.23 As shown in A, at 24h, compared with the blank group, the Metabolism of other amino acids, Replication and repair (RPA, DNA replication and repair), Lipid metabolism, and Translation increased in the Enterococcus faecalis group, the phage group, and the other two synergistic groups. At 48h, Amino acid metabolism, Metabolism of terpenoids and polyketides, RPA, and Glycan biosynthesis and metabolism also increased compared with the control group ( Fig.23B). At 72h, except for the phage-amoxicillin group, the carbohydrate metabolism, other amino acid metabolism, RPA, and lipid metabolism of the other groups increased compared with the blank group, and the metabolic pathway of the phage-amoxicillin group was closer to that of the blank group ( Fig.23 C).

[0128] Bugbase function prediction analysis

[0129] The software integrates the gene information of species in the three major databases of IMG, KEGG and PATRIC, and performs predictive analysis from nine phenotypes. The results showed that the abundance of biofilm generated in the phage-amoxicillin group at 24 hours was relatively low compared with other experimental groups, followed by the phage-ampicillin trihydrate group at 72 hours and the phage group at 24 hours. The abundance of pathogenic bacteria in the phage group and the phage-ampicillin group was low. In addition, the abundance of Gram-negative bacteria in each experimental group increased compared with the control group, while the abundance of Gram-positive bacteria decreased relatively, such as Fig.24 shown.

[0130] The contribution stacking diagram is based on the phenotype of being able to form biofilm ( Fig.25 A) found that the Enterococcus faecalis control group contributed the most, followed by the phage-ampicillin trihydrate group, and the most abundant of these two groups was Verrucomicrobia, a Gram-negative bacterium that is commonly found in soil, water, and animal intestines, and often exists in the intestinal mucosal lining. Contribution diagram with anaerobic type as the phenotype ( Fig.25 B) found that the phage group and the phage-ampicillin trihydrate group contributed the most, and found that in the bacterial phyla they composed, the phage-ampicillin group had Proteobacteria at 72 hours, most of which were pathogenic bacteria, and the blank group and the phage-amoxicillin 24h group had TM7, sugar bacteria, which are obligate epiphytes parasitic on the surface of host bacteria. They are closely related to probiotics in periodontitis and other inflammatory diseases and are putative pathogens. Contribution diagram based on pathogenicity as the phenotype ( Fig.25 C) It was found that TM7 also appeared in the E. faecalis control group and the phage-ampicillin group. Except for the control group, the relative abundance of Verrucomicrobia was higher in the other experimental groups.

[0131] In summary, the combination of 70μg / mL amoxicillin or 32μg / mL ampicillin trihydrate and phage has a better effect in the in vitro tooth apical periodontitis model. Combined with the biofilm gene expression, these two concentrations were selected in the subsequent rat peritoneal infection model. The results showed that these phage therapies had a good therapeutic effect after being applied to the rat peritoneum, had no effect on the diversity of the rat's intestinal flora, changed the structure of the rat flora, and increased some beneficial bacterial genera. Overall, the synergistic use of 70μg / mL amoxicillin and phage has the potential to become a clinical treatment for apical periodontitis.

[0132] It should be understood that the above specific embodiments of the present invention are only used for illustrative inventions or to explain the principles of the present invention, and do not constitute limitations on the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention. In addition, the appended claims of the present invention are intended to cover all changes and modifications that fall within the scope and boundaries of the appended claims, or the equivalent forms of such scope and boundaries.

Claims

1. A pharmaceutical composition for inhibiting Enterococcus faecalis, characterized in that: The pharmaceutical composition comprises the Enterococcus faecalis phage PEf771 and also comprises amoxicillin or ampicillin trihydrate.

2. Use of the pharmaceutical composition for inhibiting Enterococcus faecalis according to claim 1 in the preparation of a drug for resisting Enterococcus faecalis infection.

3. The use according to claim 2, characterized in that: The pharmaceutical composition for inhibiting Enterococcus faecalis can inhibit the biofilm-related genes of Enterococcus faecalis. ebpC, gelE, esp expression and removal of biofilm.

4. The use according to claim 3, characterized in that: The pharmaceutical composition for inhibiting Enterococcus faecalis is a combination of Enterococcus faecalis phage and amoxicillin, wherein the Enterococcus faecalis phage has a potency of 10 10 PFU / mL suspension, the amoxicillin concentration is 70 μg / mL, and the added volume ratio of Enterococcus faecalis phage and amoxicillin is 1:

1.

5. The use according to claim 3, characterized in that: The pharmaceutical composition for inhibiting Enterococcus faecalis is a combination of Enterococcus faecalis phage and ampicillin trihydrate, wherein the Enterococcus faecalis phage has a potency of 10 10 PFU / mL suspension, the concentration of ampicillin trihydrate is 32 μg / mL, and the added volume ratio of Enterococcus faecalis phage and ampicillin trihydrate is 1:

1.

6. The use according to claim 4 or 5, characterized in that: The pharmaceutical composition for inhibiting Enterococcus faecalis is used in preparing medicines for treating diseases caused by Enterococcus faecalis infection.

7. The use according to claim 4 or 5, characterized in that: The drug combination for inhibiting Enterococcus faecalis is used in preparing a drug for improving inflammatory bowel disease caused by Enterococcus faecalis.

8. The use according to claim 4 or 5, characterized in that: Application of the drug combination for inhibiting Enterococcus faecalis in the preparation of a drug for treating tooth periapical periodontitis.

9. The use according to claim 2, characterized in that: Amoxicillin or ampicillin trihydrate in the drug acts simultaneously with the Enterococcus faecalis bacteriophage PEf771 to inhibit Enterococcus faecalis.

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