Phage and use thereof in preparing biological formulation for inhibiting drug-resistant bacteria

Abstract

Provided are a phage and a use thereof in preparing a biological formulation for inhibiting drug-resistant bacteria, relating to the field of biomedicine. An Escherichia coli phage Phage vB_EcoM_GZMU_E1004 is isolated from a mixture of lake water and sewage. The phage is deposited under the accession number GDMCC No: 64729-B1, and has the function of significantly inhibiting Escherichia coli. In addition, the combined use of the phage and allicin can further enhance the antibacterial effect against Escherichia coli. The obtained phage can reduce the use of antibiotics, enrich the natural phage resources of drug-resistant Escherichia coli, and provide an important foundation and basis for the phage treatment of drug-resistant Escherichia coli.

Description

Phage and application thereof in preparation of biological preparation for inhibiting drug-resistant bacteria TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a phage and application thereof in preparation of biological preparation for inhibiting drug-resistant bacteria. BACKGROUND

[0002] In the world, with the non-standard use and unreasonable abuse of antibiotics, the drug resistance of bacteria is increasing. According to the report of the World Health Organization (WHO), drug-resistant bacterial infection has become one of the top ten threats to global public health. In 2019, Escherichia coli (E. coli) was the pathogen that caused the most deaths in global antibiotic-resistant infections. E. coli is commonly found in the lower digestive tract of warm-blooded organisms and mainly causes septicemia, urinary tract infection and acute gastroenteritis in humans. It is reported that E. coli resistant to the third generation cephalosporin and E. coli resistant to fluoroquinolones caused more than 100,000 drug-resistant infection deaths, not only that, E. coli is also considered by the World Health Organization as a key pathogen for the development of new antibiotics. Therefore, there is an urgent need to develop new antibacterial drugs and antibacterial programs to combat drug-resistant E. coli.

[0003] Phage therapy is considered as a potential new antibacterial method due to its unique antibacterial mechanism. Phage is a natural bacterial killer widely existing in nature, compared with traditional antibacterial agents, it has the advantages of strong specificity, small side effects, not easy to cause bacterial resistance and can improve the effect through genetic modification, and is not affected by bacterial drug resistance. Basic and clinical research has confirmed the safety and effectiveness of phage therapy for specific infections, and its natural lytic activity also makes it one of the effective alternatives to antibiotics.

[0004] Drug-resistant bacterial infection has seriously endangered global public health safety, in order to reduce the use of antibiotics and alleviate the pressure brought by antibiotics, the development of new antibacterial drugs and antibacterial programs has become a problem faced by the prior art.

[0005] SUMMARY

[0006] The purpose of the present application is to provide a phage and application thereof in preparation of biological preparation for inhibiting drug-resistant bacteria, to solve the problems existing in the prior art, a new E. coli phage is obtained by screening, which can significantly inhibit the growth of E. coli, and combined with allicin can significantly improve the ability to inhibit E. coli. The present application enriches the natural phage resources of drug-resistant E. coli, provides an important basis for phage therapy of drug-resistant E. coli, and to some extent can reduce the use of antibiotics and alleviate the pressure brought by the use of antibiotics.

[0007] To achieve the above object, the present application provides the following scheme:

[0008] The present application provides an Escherichia Phase vB_EcoM_GZMU_E1004, referred to as E1004, the preservation number of the Escherichia Phase is GDMCC No: 64729-B1, the preservation time is June 7, 2024, the preservation unit is Guangdong Microbial Culture Collection Center, and the preservation address is the 5th floor of No. 59 Building, Institute of Microbiology, Guangdong Academy of Sciences, 100, Martyrs' Road, Guangzhou.

[0009] The present application also provides application of the Escherichia Phase in preparation of a biological preparation for inhibiting Escherichia coli or treating diseases caused by Escherichia coli.

[0010] Preferably, the Escherichia coli includes drug-resistant Escherichia coli.

[0011] Preferably, the biological preparation includes an effective concentration of greater than or equal to 1*10 3 PFU / mL of the Escherichia Phase.

[0012] The present application also provides a biological preparation for inhibiting Escherichia coli or treating diseases caused by Escherichia coli, wherein the biological preparation includes the Escherichia Phase, and the effective concentration of the Escherichia Phase is greater than or equal to 1*10 3 PFU / mL. The biological preparation includes a biological agent or a medicine.

[0013] The present application also provides application of the Escherichia Phase in preparation of a biological preparation for inhibiting Escherichia coli or treating diseases caused by Escherichia coli, and the preservation number of the Escherichia Phase is GDMCC No: 64729-B1.

[0014] The present application also provides application of the Escherichia Phase in preparation of a biological preparation for killing Escherichia coli by destroying the cell structure of the Escherichia coli, and the preservation number of the Escherichia Phase is GDMCC No: 64729-B1.

[0015] Preferably, the concentration of the allicin is 0.875 mg / mL, and the concentration of the bacteriophage is 1*10 2 PFU / mL.

[0016] Preferably, the Escherichia coli includes drug-resistant Escherichia coli.

[0017] The present application discloses the following technical effects:

[0018] The application obtains an Escherichia Phase vB_EcoM_GZMU_E1004 from a mixture of lake water and sewage, and the bacteriophage is observed as a short tail bacteriophage by electron microscopy, and it is found through experiments that the bacteriophage has a broad-spectrum antibacterial effect when used alone, and has good pH, temperature and chloroform stability, and strong environmental adaptability.

[0019] The bacteriophage E1004 is combined with the garlicin antibacterial agent in the application, and the results show that the garlicin has no effect on the titer of the bacteriophage itself, and the combination has a synergistic antibacterial effect; the combination has the best effect when the concentration of the garlicin is 0.875 mg / mL and the titer of the bacteriophage is 10 2 PFU / mL, and the absorbance at 600 nm is less than half of that of the garlicin or the bacteriophage used alone.

[0020] The application also analyzes the antibacterial mechanism of the combination of the bacteriophage E1004 and the garlicin, and finds that the combination of the antibacterial agents does not damage the DNA of the Escherichia coli, and the protein and nucleic acid leakage of the combination group is significantly increased compared with the pure bacterial liquid group, which shows that the combination mode can destroy the cell structure of the Escherichia coli and kill the bacteria. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below are only some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative effort.

[0022] Figure 1 is the morphology and structure of the bacteriophage; A: bacteriophage drop plate; B: bacteriophage infection or purification plate; C: electron microscope image and size of the bacteriophage particle;

[0023] Figure 2 is a heat map of 60 strains of Escherichia coli resistance information (X-axis A-W represents the drug, and Y-axis 1-60 represents the strain);

[0024] Figure 3 is a maximum likelihood method (ML) phylogenetic analysis based on whole genome sequence; the red box identifies the bacteriophage E1004 obtained in the study, and the reference bacteriophage is derived from the NCBI database;

[0025] Figure 4 is the pH stability determination result; there is no significant difference between the same letters, and there is a difference between different letters, and ab represents no significant difference between a group and no significant difference between b group data;

[0026] Figure 5 is the result of temperature stability determination; ns means no significant difference between groups.

[0027] Figure 6 is the result of chloroform sensitivity determination; no significant difference between groups with the same letter.

[0028] Figure 7 is the result of optimal MOI determination; no significant difference between groups with the same letter, and difference between groups with different letters.

[0029] Figure 8 is a one-step growth curve.

[0030] Figure 9 is the result of lysis kinetics determination.

[0031] Figure 10 is the effect of allicin on bacteriophage; experimental group: bacteriophage treated with allicin; control group: bacteriophage treated with the same amount of LB; ns means no significant difference between groups.

[0032] Figure 11 is the result of allicin MIC determination; no significant difference between groups with the same letter, and difference between groups with different letters.

[0033] Figure 12 is the determination of the optimal combination mode of allicin and bacteriophage culture of E. coli.

[0034] Figure 13 is the result of DNA damage agarose gel electrophoresis detection; M: DL2000 DNA Marker, 1: MIC allicin alone, 2: 10 10 PFU / mL bacteriophage alone, 3: optimal combination of MIC allicin, 4: optimal combination of bacteriophage concentration, 5: optimal combination mode of allicin and bacteriophage, 6: pure bacteria solution, 7: Blank (deionized water).

[0035] Figure 14 is a nucleic acid and protein leakage chart, respectively measuring OD 260 (figure left) and OD 280 (figure right) to detect the leakage of nucleic acid and protein; no significant difference between groups with the same letter, and difference between groups with different letters. DETAILED DESCRIPTION

[0036] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0037] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, concentration, intensity, and time, and the like, every intermediate value of the

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In the case of conflict between the present specification and any incorporated document, the present specification controls.

[0039] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0040] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional elements or steps.

[0041] Example 1

[0042] 1. Experimental strains

[0043] Sixty strains of E. coli were isolated from pathological samples of clinical patients and subjected to drug sensitivity test. EIII4 was selected as the main research object of the present application, and the rest were only used for host spectrum experiment.

[0044] 2. Isolation and purification of phage

[0045] The sample liquid was obtained by filtering the supernatant of the mixture of lake water and sewage collected from Xinzao Town, Panyu District, Guangzhou City, Guangdong Province, China, through a 0.22 μm filter after low-speed centrifugation. 10 mL of the sample liquid, 5 mL of three-fold concentration LB nutrient broth, and 1 mL of the target host bacteria were incubated at 37°C overnight on a constant temperature shaker. The lysate was obtained by filtering the supernatant of the co-culture liquid through a 0.22 μm filter after low-speed centrifugation. 100 μL of the host bacteria was mixed with 0.7% semi-solid LB agar to uniformly spread on a culture dish. 5 μL of the lysate was dropped on the semi-solid surface of the dried culture dish, and the culture dish was placed in a 37°C constant temperature incubator for 8 hours or overnight. If a transparent circle was observed at the dropping position, it indicated that the bacteriophage was isolated. The purification was continued using LB plates until the size of the plaques on the obtained plaque plate was uniform and round, indicating that the bacteriophage was basically purified.

[0046] 3. Preparation of bacteriophage stock and determination of titer

[0047] 3.1 Preparation of stock

[0048] 10 mL of liquid LB, 5 mL of high concentration host bacteria, and 150 μL of bacteriophage were placed in a 37°C, 220 rpm shaker for overnight culture. The supernatant was obtained by centrifuging the culture at 5000 rpm and 4°C for 10 min, and then filtering the supernatant through a 0.22 μm filter to obtain the bacteriophage stock.

[0049] 3.2 Determination of bacteriophage titer

[0050] The double-layer plate method was used to dilute the bacteriophage liquid with SM buffer (0.97 g of anhydrous magnesium sulfate, 5.8 g of sodium chloride, 50 mL of 1M Tris-Cl (pH=7.5), 0.1 g of gelatin powder, and deionized water to 1000 mL to make 1L of SM buffer, and autoclaved at 121°C for 20 min). 100 μL of host bacteria and 100 μL of bacteriophage release liquid were mixed and incubated at 37°C for 15 min. The mixture was uniformly spread on 1.5% solid LB agar pre-coated on the culture dish using 0.7% semi-solid LB agar. The plate was placed in a 37°C incubator for 8 hours or overnight. After incubation, several transparent plaques were observed on the culture dish. Plates with 30-300 plaques in the field of view were selected for counting. The original titer (PFU / mL) = number x 10 x dilution factor.

[0051] 3.3 Electron microscope observation of bacteriophage morphology

[0052] The phosphotungstic acid negative staining method was used for observation, and the operation steps were as follows: 10 μL of activated bacteriophage LB solution was added dropwise to a 400-mesh carbon film copper mesh, and was allowed to adsorb, 2 min later, the residual liquid around the copper mesh was absorbed with filter paper, a drop of 2% phosphotungstic acid was added to the copper mesh for 1 min of staining, the staining solution was absorbed with filter paper, and the copper mesh was washed twice with deionized water, and then was observed under a Hitachi electron microscope after standing and drying, the acceleration voltage was set to 80 kV, and the structure size of the bacteriophage was measured by using Image J.

[0053] 3.4 Determination of bacteriophage host spectrum

[0054] 100 μL of host bacteria was mixed with 0.7% semi-solid LB agar to prepare a bacterium-containing plate, and different host bacteria were inoculated in different culture dishes. The lysis solution was obtained according to the separation method of the bacteriophage, 5 μL of the bacteriophage lysis solution was added dropwise to the semi-solid surface of the culture dish, and was incubated in a 37°C constant temperature incubator for 8 h or overnight. If a transparent circle or a bacteriophage plaque appeared at the drop position, it indicated that the bacteriophage infected the corresponding host bacteria.

[0055] 4. Results and analysis

[0056] 4.1 Bacteriophage plaque morphology and electron microscope structure

[0057] EcoM_GZMU_E1004, abbreviated as E1004. As shown in Fig. 1C, the bacteriophage nucleocapsid was about 68 nm long, about 60 nm wide, the tail was about 17 nm long, and about 34 nm wide, with an error of not more than 2 nm, and belonged to a short-tailed bacteriophage.

[0058] 4.2 Host spectrum and drug resistance information

[0059] For the 60 strains of Escherichia coli collected from the clinic, the present application tested their antibacterial spectrum against different antibiotics (see Fig. 2), and the drugs A-W were amoxicillin / clavulanic acid, ampicillin (whether β-lactamase was produced) and the like, as shown in Table 1; the strains 1-60 were in the same order as Table 2, and the closer the color was to red, the stronger the drug resistance was, and the blue color represented sensitivity to the antibiotic. It can be seen that the number of strains resistant to ampicillin was relatively large. However, the strains were relatively sensitive to tetracycline and carbapenem antibiotics. The strain EIII4 selected by the present application was resistant to cotrimoxazole, levofloxacin and ampicillin, and a number of cephalosporin drugs, and the Escherichia coli could produce β-lactamase.

[0060] Table 1 Drug resistance information statistics of 60 strains of E. coli (not every drug is full of 60 strains of bacteria)

[0061] In order to test the antibacterial spectrum of the obtained bacteriophage E1004, antibacterial tests were carried out on 60 strains of clinically derived E. coli, and the results were coverage = 33.33% (20 / 60), and the host spectrum was wide (see Table 2).

[0062] Table 2 Host spectrum of E1004 bacteriophage on 60 strains of E. coli (x represents not infected, and represents infected)

[0063] Example 2

[0064] The phage stock prepared in Example 1 was sent to Guangdong Meige Gene Technology Co., Ltd. for genome sequencing and analysis. After obtaining the bacteriophage genome sequence, first, the whole genome was subjected to homology analysis using NCBI BLASTn, and in order to further analyze the possible classification of E1004, the Matcher in the EMBOSS suite and the ClustalW algorithm in the MEGA software were used for global double-sequence alignment of all known bacteriophages of the Kayfunavirus genus and E1004.

[0065] In addition, in order to further analyze the phylogenetic relationship of the bacteriophage with other bacteriophages of the Kayfunavirus genus, 1000 bootstrap repeats were established in the MEGA 11.0.13 version software, and a phylogenetic tree was constructed using the maximum likelihood method (Figure 3).

[0066] In order to identify the potential tRNA genes in the bacteriophage genome, the present application uses tRNAscan-SE v.2.0 for prediction, and searches are performed with default parameters. The virulence factor database (VFDB, http: / / www.mgc.ac.cn / VFs / main.htm) and the comprehensive antibiotic resistance database (CARD, https: / / card.mcmaster.ca / analyze / rgi) network servers are used to retrieve virulence factors and antibiotic resistance coding genes.

[0067] The results showed that the genome size of phage vB_EcoM_GZMU_E1004 was 39250 bp, and the GC content was 49.93%, indicating that it was a balanced genome in GC content (Table 3); using the Rapid annotation Subsystem Technology (RAST) annotation server for annotation, it was found that the phage E1004 identified 47 open reading frames (ORF) in total, 46 of which were identified by gene prediction, 32 of which were aligned to the UniProtKB database, of which 6 were unknown proteins, and no RNA coding sequence was detected after tRNAscan-SE analysis of the genome sequence of the E1004 phage. The annotation data showed that the functions encoded by the genome were mainly concentrated in three subsystems, covering phage lysis modules and structural components, including endogenous lyase, endopeptidase, capsid protein and tail fiber protein, which are essential for host cell wall lysis and virus particle attachment in the phage infection cycle. The comparison of virulence factor database and comprehensive antibiotic resistance database showed that the phage did not carry drug resistance genes and virulence factors.

[0068] Table 3 General characteristics of phage genome

[0069] According to the International Committee on Taxonomy of Viruses (ICTV) standard, two phages should be classified as different species when their genome sequence similarity is below 95%. As shown in Table 4, to evaluate the classification of phage E1004, first, the homology analysis of its whole genome was performed using NCBI BLASTn, which showed that the sequence similarity between E1004 and E. coli phage vB_EcoP_F (Genbank No: NC_047808.1) was the highest, 95.99% (with 86% coverage). To further analyze the possible classification of E1004, Stretcher, Matcher, Water and SSEARCH2SEQ in EMBOSS suite, and ClustalW algorithm in MEGA software were further used to perform global pairwise alignment of all known phages in Kayfunavirus genus and E1004, which showed that the whole genome homology of E1004 phage ranged from 83.9% (with E. coli phage ST31, using EMBOSS Matcher method) to 92.9% (with E. coli phage vB_EcoP_F, using ClustalW method in MEGA). It is worth noting that the sequence similarity values obtained by the second method were all lower compared with BLASTn. This might be attributed to the fact that BLASTn mainly targets local sequence alignment, thus might overestimate the true global similarity. Referring to the classification guideline of BAVS, viruses with more than 50% nucleotide sequence similarity in a viral population can be classified as the same genus. Given that the genome of E1004 has most of the similarity with other phages in Kayfunavirus genus falling within the range of 50%-95%, it was determined that E1004 represents a new species of Kayfunavirus genus.

[0070] Table 4 Whole genome pairwise comparison of phage genome with Kayfunavirus genus members

[0071] In the phylogenetic tree constructed by MEGA 11.0.13 software using maximum likelihood method with 1000 bootstrap repeats (see Figure 3), Escherichia phage vB_EcoM_GZMU_E1004 and Escherichia phage ST31 were early separated in the evolutionary tree and each located in an independent branch. This indicates that the two phages may have specific evolutionary characteristics or unique genomic structure, thereby supporting the classification of E1004 as a potential new species under the genus Kayfunavirus, consistent with the above results. On the other hand, Escherichia phage vB_EcoP_F and Escherichia phage ZG49 were located in the same branch in the phylogenetic tree, and the ClustalW analysis obtained a bootstrap value of 85%, indicating that the two have a high degree of proximity in evolution and tightness in genetic relationship.

[0072] Example 3 Stability test

[0073] 1. pH stability

[0074] 100 μL of phage was added to 900 μL of SM buffer with different pH values (pH values were adjusted to 1-12 by adding 1 mol / L HC1 or 1 mol / L NaOH in advance), and the mixture was incubated at 37°C for 1 h. The titer was determined according to the titer determination method in Example 1.

[0075] 2. Temperature stability

[0076] 100 μL of phage was added to 900 μL of SM buffer with different temperatures (different groups of buffer were pre-cooled or pre-heated at 4°C, 37°C, 50°C, 60°C, 70°C, and 80°C, respectively, and then returned to the corresponding temperature after adding the phage), and incubated for 1 h. The titer was determined.

[0077] 3. Chloroform sensitivity

[0078] Chloroform was mixed with 950 μL of phage at 0 μL, 10 μL, 20 μL, and 50 μL, respectively, and 50 μL, 40 μL, 30 μL, and 0 μL of LB liquid medium were added to make up 1 mL system, respectively. After incubation at 37°C and 220 rpm for 30 min, 100 μL of supernatant was taken for titer determination.

[0079] 4. Optimal multiplicity of infection

[0080] The logarithmic phase host bacteria were diluted to 1 × 10 8CFU / mL, 200 μL was taken into 5 EP tubes, and the phage concentration was diluted into 5 groups, 1 x 10 10 PFU / mL, 1 x 10 9 PFU / mL, 1 x 10 8 PFU / mL, 1 x 10 7 PFU / mL, 1 x 10 6 PFU / mL, 200 μL was taken into 5 EP tubes, and the phage concentration was diluted into 5 groups, 1 x 10

[0081] 5, One-step growth curve

[0082] The phage was diluted into 1 x 10 7 PFU / mL, 0.1 mL was taken and added into 9.9 mL of logarithmic phase E. coli (OD 600 = 0.5), and was placed in a 37°C, 220 rpm shaker for 15 min, taken out, centrifuged at 13000 g for 15 min, the supernatant was discarded, 10 mL of liquid LB was used to resuspend the precipitate, and was placed in a 37°C, 220 rpm shaker for culture, and samples were taken at different time points, and the titer was determined.

[0083] 6, Lysis kinetics

[0084] 75 μL of logarithmic phase host bacteria solution was mixed with 75 μL of phage suspension to prepare 6 groups of different infection multiplicity (0.001, 0.01, 0.1, 1, 10, 100), which were added into a 96-well plate (the final titer of the phage was 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 PFU / mL, respectively), and an equal amount of sterile fresh MH broth and logarithmic phase bacteria solution were used as controls, and the mixture was cultured in a 37°C, 220 rpm shaker, and the OD 600 value of the mixture was determined every 1 h, the experiment lasted for 13 h, and the experiment was repeated 3 times.

[0085] 7, Results and analysis

[0086] 7.1 pH stability

[0087] As shown in Figure 4, the titer of E1004 was best when the pH was equal to 7, and the titer decreased significantly at other pH values, but the phage could be stably maintained at a certain concentration between pH 4-10, and could maintain more than 33%, and the phage had no activity when the pH was 1-3 and 11-12.

[0088] 7.2 Temperature stability

[0089] As shown in Figure 5, the optimum temperature of E1004 is 37°C, and the titer is stable at 4-50°C, maintaining more than 80%. At 60°C, the titer decreases sharply, and the activity is only 6.7%. When the temperature is greater than or equal to 70°C, the phage is not active.

[0090] 7.3 Chloroform sensitivity

[0091] As shown in Figure 6, E1004 is not sensitive to chloroform at a concentration of 5% or less, and it does not contain lipid substances such as a capsular structure.

[0092] 7.4 Optimum multiplicity of infection

[0093] As shown in Figure 7, the optimum MOI of E1004 is 1, and it can maintain a titer close to OMOI at 0.1 and 0.01, and a titer far from OMOI at 10 and 100. It is speculated that the final culture concentration is greatly affected by the bacterial concentration.

[0094] 7.5 One-step growth curve

[0095] As shown in Figure 8, the latent period of lysis is about 90 min, the rising period maintains for 150 min, and the plateau is reached at 240 min, with a lysis amount of 130 (41.67 / 0.32) PFU / infectious cell.

[0096] 7.6 Lysis kinetics

[0097] As shown in Figure 9, the phage under each MOI condition exhibits effective inhibition of the growth of E. coli. In addition, when the growth of E. coli enters the stable phase, the OD 600 values of E. coli in each MOI group remain at a low level.

[0098] Example 4 Allicin combined with phage antibiosis

[0099] 1. Effect of allicin on phage activity

[0100] Take 10 mg / mL allicin and 200 μL of phage solution to mix, and take 200 μL of fresh LB medium and phage solution to mix as a control group. Incubate them in a 37°C incubator for 1 h, and then determine the phage titer, with three repeats in each group.

[0101] 2. Minimum inhibitory concentration test

[0102] Allicin MIC determination: Adjust the concentration of E. coli in the logarithmic growth phase to 2 x 10 5CFU / mL, 75 μL of the bacterial solution was added to several wells of a 96-well plate, and 20 mg / mL garlicin was diluted twice to obtain concentrations of 10, 8.75, 7.5, 6.25, and 5 mg / mL, and 75 μL of each concentration was added to different wells of the 96-well plate to which the bacterial solution had been added, and mixed well. The control group was added with fresh LB medium, so that the final concentration of the host bacteria was 10 5 CFU / mL, and the concentration gradient of garlicin was 10, 8.75, 7.5, 6.25, 5, and 0 mg / mL. The OD 600 was measured before culture. The 96-well plate was placed in a 37°C, 220 rpm shaker for 12 h, and the OD600was measured after culture. Each group had 4 replicates.

[0103] Phage MIC determination: when the determination was performed, the lysis kinetics method procedure in Section 7.6 of Example 3 was followed. Each group had 3 replicates.

[0104] 3. Study of the optimal combination model

[0105] The logarithmic growth phase E. coli was diluted with LB liquid medium to a concentration of 3 x 10 5 CFU / mL, and 50 μL of the bacterial solution was added to several wells of a 96-well plate. 20 mg / mL garlicin was diluted twice to obtain concentrations of 3 MIC, 3 / 10 MIC, 3 / 100 MIC, 3 / 1000 MIC, and 3 / 10000 MIC, and 50 μL of each concentration of garlicin was added to the corresponding wells to which the bacterial solution had been added. The phage was diluted to concentrations of 3 x 10 5 , 3 x 10 4 , 3 x 10 3 , 3 x 10 2 , and 3 x 10 1 PFU / mL, and 50 μL of each concentration of the phage was added to the corresponding wells. Fresh LB liquid medium was added to each well to make up the system to 150 μL, and mixed well. The control group was added with fresh LB liquid medium instead of garlicin and the phage. The OD 600 was measured before culture. The 96-well plate was placed in a shaker at 37°C and 220 rpm for 12 h, and the OD 600 was measured after culture. The experiment was repeated 3 times.

[0106] The FIC (fractional inhibitory concentration) index was used to determine whether there was a synergistic antibacterial effect between garlicin and the phage. The FIC index = MIC (A group in combination) / MIC (A group alone) + MIC (B group in combination) / MIC (B group alone). When the FIC index is less than 0.5, the two drugs are synergistic; when the FIC index is 0.5-1, the two drugs are additive; when the FIC index is greater than 1 and less than 2, the two drugs are irrelevant; and when the FIC index is greater than 2, the two drugs are antagonistic.

[0107] 4. DNA damage observation

[0108] Prepare bacterial solution, extract genome with gene extraction kit, and use NanoDrop spectrophotometer to determine DNA content. Take DNA and mix with 1 MIC allicin, 1 MIC phage, 1 MIC allicin + 1 MIC phage, combined optimal concentration allicin, combined optimal concentration phage, optimal combined concentration phage + optimal combined concentration allicin, LB control, incubate at 37°C for 30 min, and observe band dispersion on agarose gel electrophoresis.

[0109] 5. Nucleic acid and protein leakage detection

[0110] Take logarithmic growth phase bacterial solution, centrifuge at 4000 rpm for 10 min, resuspend with PBS, and make the bacterial solution concentration 10 8 CFU / mL, take the bacterial solution and mix with 1 MIC allicin, 10 9 PFU / mL phage, combined optimal concentration allicin, combined optimal concentration phage, optimal combined concentration phage + optimal combined concentration allicin, LB control, centrifuge at 6000 rpm for 5 min after 3 h culture at 37°C, precipitate bacteria, and use NanoDrop spectrophotometer to determine OD 260 , OD 280 .

[0111] 6. Results and analysis

[0112] 6.1 Effect of allicin on phage

[0113] As shown in Figure 10, there is no significant difference between the two experimental results, indicating that allicin has little effect on the infectivity of phage, and the effect of mixing allicin and phage on phage itself can be ignored in subsequent experiments.

[0114] 6.2 Minimum inhibitory concentration

[0115] Phage MIC: As shown in Figure 9, when cultured for 12 h, at MOI = 0.001 (corresponding to phage titer 10 2 PFU / mL), the OD 600 has obvious fluctuation, indicating that at this MOI, the phage has not yet completely inhibited the host bacteria. Starting from MOI = 0.01 (corresponding to phage titer 10 3 PFU / mL), the OD 600 is basically stable and close to 0, i.e. the phage inhibition rate at MOI = 0.01 and above is close to 100%, so the phage titer 10 3 PFU / mL corresponding to this MOI is considered to be the minimum inhibitory concentration of phage.

[0116] Garlicin MIC: As shown in Figure 11, the minimum inhibitory concentration of garlicin is 8.75 mg / mL, and the inhibitory rate is about 76%.

[0117] 6.3 Optimal combination model

[0118] In combination with the data in Figure 12, it can be seen that the combination of 1 / 10 MIC garlicin concentration (0.875 mg / mL) combined with 10 7 PFU / mL of phage has the highest inhibitory rate, but 1 / 10 MIC garlicin concentration (0.875 mg / mL) combined with 10 2 PFU / mL of phage can also achieve an inhibitory effect comparable to the highest combination (which is at 0 point). Considering that the drug dosage should be reduced as much as possible, it is believed that 1 / 10 MIC garlicin concentration (0.875 mg / mL) combined with 10 2 PFU / mL of phage is the optimal combination mode of garlicin combined with phage.

[0119] Calculate the inhibitory concentration index (FIC), so FIC = 1 / 10 + 10 -3 = 0.101 < 0.5, which can be judged as synergistic effect.

[0120] 6.4 DNA damage results

[0121] As shown in Figure 13, each group did not appear DNA band diffusion phenomenon compared with the pure bacteria solution group, so MIC and lower concentration of garlicin and 10 10 PFU / mL concentration of phage have no damage effect on DNA, which can basically determine that the antibacterial mechanism of the optimal combination mode does not include the damage effect on DNA.

[0122] 6.5 Nucleic acid and protein leakage results

[0123] As shown in Figure 14, after 3h treatment, the release amount of E. coli content in the drug group, phage group and combination group is significantly higher than that in the pure bacteria solution group, indicating that garlicin and phage can destroy the structure of E. coli and make its content flow out.

[0124] 10 9 PFU / mL concentration of phage group and 10 7 PFU / mL concentration of phage group have similar results, and even both of them are similar to the results of MIC garlicin group and combination group, indicating that at this time most of the E. coli are killed, so there is no greater content release amount under the action of high concentration of phage.

[0125] The release amount of MIC garlicin group is significantly higher than that of 1 / 10 MIC garlicin group, but both of them are higher than that of the pure bacteria solution group, indicating that garlicin has a certain ability to destroy bacteria when the concentration is less than MIC, but the killing ability is weak due to insufficient concentration.

[0126] The combination group can well destroy E. coli to release its contents, and the effect is significantly higher than that of the 1 / 10 MIC garlicin single group, and slightly higher than that of the phage single group. The above results show that the combined antibacterial mode can destroy E. coli to release its contents to perform antibacterial action.

[0127] The above-described embodiments are only to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements of the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. An Escherichia coli bacteriophage, characterized in that, The preservation number of the Escherichia coli bacteriophage is GDMCC No: 64729-B1.

2. Use of the Escherichia coli bacteriophage according to claim 1 in the preparation of a biological preparation for inhibiting Escherichia coli or treating a disease caused by Escherichia coli.

3. Use according to claim 2, wherein the compound is ###0002### The Escherichia coli includes drug-resistant Escherichia coli.

4. The use according to claim 2, wherein the compound is ###0002### The biological agents include E. coli bacteriophages at an effective concentration of > 1 x 10 3 PFU / mL.

5. A biological preparation for inhibiting Escherichia coli or treating a disease caused by Escherichia coli, characterized by, The biological agent comprises the E. coli bacteriophage of claim 1 at an effective concentration of > 1 x 10 3 PFU / mL.

6. The use of an Escherichia coli bacteriophage in combination with allicin in the preparation of a biological preparation for inhibiting Escherichia coli or treating a disease caused by Escherichia coli, characterized in that, The preservation number of the Escherichia coli bacteriophage is GDMCC No: 64729-B1.

7. Use of an Escherichia coli bacteriophage in combination with allicin for the preparation of a biological agent for killing Escherichia coli by destroying its cell structure, characterized in that, The preservation number of the Escherichia coli bacteriophage is GDMCC No: 64729-B1.

8. Use according to claim 6 or 7, wherein the compound is ###0002### The concentration of the allicin is 0.875 mg / mL, and the concentration of the phage is 1 x 10 2 PFU / mL.

9. Use according to claim 6 or 7, wherein the compound is ###0002### The Escherichia coli includes drug-resistant Escherichia coli.

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