A phage and its application in the preparation of a biological agent for inhibiting drug-resistant bacteria

Abstract

The present invention discloses a phage and its application in the preparation of a biological agent for inhibiting drug-resistant bacteria, belonging to the field of biomedicine. A strain of Escherichia coli phage Phage vB_EcoM_GZMU_E1004 was isolated from a mixture of lake water and sewage. The preservation number of this phage is GDMCC No: 64729-B1, and it has a significant function of inhibiting Escherichia coli. In addition, when the phage is used in combination with allicin, the antibacterial effect against Escherichia coli can be improved. The phage obtained in the present invention can reduce the use of antibiotics, enrich the natural phage resources of drug-resistant Escherichia coli, and provide an important basis and foundation for phage therapy of drug-resistant Escherichia coli.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to a phage and its application in the preparation of a biological agent for inhibiting drug-resistant bacteria. Background Art

[0002] Worldwide, with the irregular use and unreasonable abuse of antibiotics, the drug resistance of bacteria is increasing day by day. According to the World Health Organization ( WHO ), drug-resistant bacterial infections have become one of the top ten threats to global public health. Among the global antibiotic-resistant infections in 2019, Escherichia coli ( Escherichia coli,E.coli ) was the pathogenic bacterium that caused the largest number of deaths. Escherichia coli is commonly found in the lower digestive tract of warm-blooded organisms and mainly causes sepsis, urinary tract infections, and acute gastroenteritis in humans. It is reported that Escherichia coli resistant to the third-generation cephalosporins and Escherichia coli resistant to fluoroquinolones caused more than 100,000 deaths from drug-resistant infections. Moreover, Escherichia coli is also considered by the World Health Organization as a key pathogen for the research and development of new antibiotics. Therefore, there is an urgent need to develop new antibacterial drugs and antibacterial programs to combat drug-resistant Escherichia coli.

[0003] Phage therapy, due to its unique antibacterial mechanism, is regarded as a new antibacterial method with great potential. Phages are natural bacterial killers widely existing in nature. Compared with traditional antibacterial agents, they have the advantages of strong specificity, small side effects, not easily causing bacterial resistance, and can improve the effect through genetic modification, and are not affected by bacterial drug resistance. Basic and clinical studies have confirmed the safety and effectiveness of phage therapy for specific infections, and its natural lytic activity also makes it one of the effective alternative programs to antibiotics.

[0004] Drug-resistant bacterial infections have seriously endangered global public health safety. To reduce and replace the use of antibiotics and relieve the pressure brought by antibiotics, the research and development of new antibacterial drugs and antibacterial programs have become problems faced in the existing technology. Summary of the Invention

[0005] The object of the present invention is to provide a phage and its application in the preparation of a biological agent for inhibiting drug-resistant bacteria, so as to solve the problems existing in the above-mentioned prior art. By screening, a new Escherichia coli phage is obtained. This phage can significantly inhibit the growth of Escherichia coli, and in combination with allicin, it can also significantly improve the ability to inhibit Escherichia coli. The present invention enriches the natural phage resources of drug-resistant Escherichia coli, provides an important basis for phage therapy of drug-resistant Escherichia coli, and can reduce the use of antibiotics to a certain extent and relieve the pressure brought by the use of antibiotics.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an Escherichia Phage vB_EcoM_GZMU_E1004, abbreviated as E1004. The preservation number of the Escherichia Phage 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 on the 5th floor of Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences.

[0008] The present invention also provides the application of the above-mentioned Escherichia Phage in the preparation of a biological agent for inhibiting Escherichia coli or treating diseases caused by Escherichia coli.

[0009] Preferably, the Escherichia coli includes drug-resistant Escherichia coli.

[0010] Preferably, the biological agent includes Escherichia Phage with an effective concentration ≥ 1×10 3 PFU / mL.

[0011] The present invention also provides a biological agent for inhibiting Escherichia coli or treating diseases caused by Escherichia coli. The biological agent includes the above-mentioned Escherichia Phage, and the effective concentration of the Escherichia Phage is ≥ 1×10 3 PFU / mL. The above biological agent includes biological bacterial agents or drugs.

[0012] The present invention also provides the application of Escherichia Phage combined with allicin in the preparation of a biological agent for inhibiting Escherichia coli or treating diseases caused by Escherichia coli. The preservation number of the Escherichia Phage is GDMCC No: 64729-B1.

[0013] The present invention also provides the application of Escherichia Phage combined with allicin in the preparation of a biological agent for killing Escherichia coli by destroying the cell structure of Escherichia coli. The preservation number of the Escherichia Phage is GDMCC No: 64729-B1.

[0014] Preferably, the concentration of allicin is 0.875 mg / mL, and the concentration of the phage is 1×10 2 PFU / mL.

[0015] Preferably, the Escherichia coli includes drug-resistant Escherichia coli.

[0016] The present invention discloses the following technical effects:

[0017] A strain of Escherichia phage EscherichiaPhage vB_EcoM_GZMU_E1004 was isolated from the mixture of lake water and sewage. Observed by electron microscopy, it is a short-tailed phage. Through experiments, it was found that it has a broad-spectrum antibacterial effect when used alone, and has good stability in terms of pH, temperature and chloroform, and strong environmental adaptability.

[0018] In the present invention, phage E1004 was combined with allicin antibacterial agent. The results showed that it was confirmed that allicin had no effect on the titer of the phage itself, and the combination of the two had a synergistic antibacterial effect; when the concentration of allicin was 0.875 mg / mL and the phage titer was 10 2 PFU / mL, the combined effect was the best, and its absorbance at 600 nm was less than half of that of allicin or phage used alone.

[0019] The present invention also analyzed the antibacterial mechanism of the combination of phage E1004 and allicin, and found that the combined antibacterial agent had no damage to the DNA of Escherichia coli. Compared with the pure bacterial liquid group, the leakage of proteins and nucleic acids in the combined group increased significantly, indicating that this combined mode can destroy the cell structure of Escherichia coli and kill bacteria. The phage obtained in the present invention can enrich the natural phage resources of drug-resistant Escherichia coli, contribute to the understanding of phage biodiversity and evolutionary relationships, and also provide an important basis and foundation for phage treatment of drug-resistant Escherichia coli. Brief Description of the Drawings

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0021] Figure 1 It is the morphology and structure of the phage; A: Phage spotting plate; B: Phage infection or purification plate; C: Electron micrograph and size of phage particles;

[0022] Figure 2 It is the heat map of drug resistance information of 60 strains of Escherichia coli (the X-axis A-W represents drugs, and the Y-axis 1-60 represents strains);

[0023] Figure 3 It is the maximum likelihood method (ML) phylogenetic analysis based on the whole genome sequence; the phage E1004 isolated in this study is marked with a red frame, and the reference phages are from the NCBI database;

[0024] Figure 4 It is the result of pH stability determination; there is no significant difference between the same letters, and there is a difference between different letters. ab means no significant difference from group a and no significant difference from group b data;

[0025] Figure 5 are the results of temperature stability measurement; ns indicates no significant difference between groups;

[0026] Figure 6 are the results of chloroform sensitivity measurement; there is no significant difference between the same letters;

[0027] Figure 7 are the results of the optimal MOI measurement; there is no significant difference between the same letters, and there is a difference between different letters;

[0028] Figure 8 is the one-step growth curve;

[0029] Figure 9 are the results of lysis kinetics measurement;

[0030] Figure 10 is the effect of allicin on phage; Experimental group: phage treated with allicin; Control group: phage treated with an equal amount of LB; ns indicates no significant difference between groups;

[0031] Figure 11 are the results of allicin MIC measurement; there is no significant difference between groups with the same letters, and there is a difference between groups with non-identical letters;

[0032] Figure 12 is to determine the optimal combination mode by culturing Escherichia coli with allicin combined with phage;

[0033] Figure 13 are the results of DNA damage agarose gel electrophoresis detection; M: DL2000 DNA Marker, 1: allicin at MIC alone, 2: phage at 10 10 PFU / mL alone, 3: optimal combination of allicin at MIC, 4: phage at optimal combination concentration, 5: optimal combination mode of allicin and phage, 6: pure bacterial solution, 7: Blank (deionized water);

[0034] Figure 14 is the nucleic acid and protein leakage diagram, and OD at different times is measured respectively 260 (left of the figure) and OD 280 (right of the figure) to detect the leakage of nucleic acid and protein; the same letters represent no significant difference, and different letters represent a difference. Detailed implementation mode

[0035] Now, various exemplary implementation modes of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0036] It should be understood that the terms used in this invention are only for describing specific embodiments and are not intended to limit the invention. Additionally, for the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise specified, 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 invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0038] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the specification of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of this invention are obvious to those skilled in the art. The specification and examples of this invention are merely exemplary.

[0039] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0040] Example 1

[0041] 1. Experimental strains

[0042] Clinical patient pathological samples were obtained, and 60 strains of Escherichia coli were isolated from them. They were subjected to drug sensitivity tests, and EⅢ4 was selected as the main research object of this invention, while the others were only used for the host spectrum experiment.

[0043] 2. Isolation and purification of phages

[0044] Collect the lake water and sewage mixture within Xinzao Town, Panyu District, Guangzhou City, Guangdong Province, China. After centrifugation at low speed, take the supernatant and filter it through a 0.22 μm filter tip to obtain the sample solution. Take 10 mL of the sample solution, 5 mL of triple-concentration LB nutrient broth, and 1 mL of the target host bacteria, and co-culture them overnight in a constant-temperature shaker at 37°C. After centrifugation of the co-culture solution at low speed, take the supernatant and filter it through a 0.22 μm filter tip to obtain the lysate. Mix 100 μL of the host bacteria with 0.7% semi-solid LB agar and spread it evenly on the culture dish. Take 5 μL of the lysate and drop it on the semi-solid surface of the air-dried culture dish. After placing the culture dish in a constant-temperature incubator at 37°C for 8 h or overnight, if a clear halo can be observed at the dropping position, it means that the phage has been isolated. Use the LB plate to continue purification until the plaque sizes on the obtained plaque plate are consistent and round, indicating that the phage purification is basically completed.

[0045] 3. Preparation of phage stock and determination of titer

[0046] 3.1 Preparation of stock

[0047] Take 10 mL of liquid LB, add 5 mL of high-concentration host bacteria and 150 μL of phage, and culture them overnight in a shaker at 37°C and 220 rpm. Place it in a centrifuge and centrifuge at 5000 rpm and 4°C for 10 min. Filter the supernatant through a 0.22 μm filter tip to obtain the phage stock solution.

[0048] 3.2 Determination of phage titer

[0049] Use the double-layer plate method. Dilute the phage solution moderately with SM buffer (weigh 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, add deionized water to 1000 mL to prepare 1 L of SM buffer, and sterilize it at 121°C under high temperature and high pressure for 20 min). Mix 100 μL of the host bacteria and 100 μL of the phage release solution, and incubate them in an incubator at 37°C for 15 min. Mix the incubation solution with 0.7% semi-solid LB agar and spread it evenly above the 1.5% solid LB agar pre-laid on the culture dish. Place the plate upright in an incubator at 37°C and culture it for 8 h or overnight. After culturing, several translucent plaques can be seen on the culture dish. Select the plate with 30 - 300 plaques in the field of view for counting. The original titer (PFU / mL) = number × 10 × dilution factor.

[0050] 3.3 Observation of phage morphology by electron microscopy

[0051] Observation was carried out using phosphotungstic acid negative staining method. The operation steps were as follows: 10 μL of the activated phage LB solution was dropped onto a 400-mesh carbon film copper grid, and left standing for adsorption. After 2 min, the residual liquid around the copper grid was blotted with filter paper. A small drop of 2% phosphotungstic acid was dropped on the copper grid for staining for 1 min, and the staining solution was blotted with filter paper. It was washed twice with deionized water, left standing for drying, and then observed with a Hitachi electron microscope. The acceleration voltage was set at 80 kV, and the structural size of the phage was measured using Image J.

[0052] 3.4 Determination of phage host spectrum

[0053] 100 μL of the host bacteria was mixed with 0.7% semi-solid LB agar and spread on a culture dish to prepare a bacteria-containing plate. Different host bacteria were inoculated on different culture dishes. The lysate was obtained with reference to the phage isolation method. 5 μL of the phage lysate was dropped onto the semi-solid surface of the culture dish and cultured in a 37 °C constant temperature incubator for 8 h or overnight. If a clear zone or plaque appeared at the dropping position, it indicated that the phage infected the corresponding host bacteria.

[0054] 4. Results and analysis

[0055] 4.1 Plaque morphology and electron microscopy structure

[0056] Using EⅢ4-resistant Escherichia coli as the host, a phage with strong infectivity to the target Escherichia coli was isolated from lake water. As shown in B in Figure 1 , its plaque was transparent and large. As shown in A in Figure 1 , the clear zone obtained from the spot test was shown. Therefore, it was selected as the research object of the present invention and named Escherichia Phage vB_EcoM_GZMU_E1004, abbreviated as E1004. As shown in C in Figure 1 , the phage nucleocapsid was about 68 nm long, about 60 nm wide, the tail was about 17 nm long, about 34 nm wide, with an upper and lower error of no more than 2 nm, belonging to a short-tailed phage.

[0057] 4.2 Host spectrum and drug resistance information

[0058] For the 60 clinical Escherichia coli strains collected, the present invention tested their antibacterial spectra against different antibiotics (see Figure 2), Drugs A - W are amoxicillin / clavulanic acid, ampicillin (whether producing β - lactamase), etc., as shown in Table 1; The order of strains 1 - 60 is consistent with Table 2. Among them, the closer the color is to red, the stronger the drug resistance, and blue represents sensitivity to this antibiotic. It can be seen that the number of strains resistant to ampicillin is relatively large. And they are relatively sensitive to tetracycline - type and carbapenem - type antibiotics. The selected strain EⅢ4 of the present invention is resistant to co - trimoxazole, levofloxacin, ampicillin, and several cephalosporin drugs, and Escherichia coli can produce β - lactamase.

[0059] Table 1 Statistical information on drug resistance of 60 Escherichia coli strains (not every drug was tested on 60 strains)

[0060] To test the antibacterial spectrum of the obtained phage E1004, antibacterial tests were carried out on 60 clinically - sourced Escherichia coli strains collected, and the result was that the coverage rate = 33.33% (20 / 60), and the host spectrum was relatively wide (see Table 2).

[0061] Table 2 Host spectrum of phage E1004 against 60 Escherichia coli strains (× indicates non - infection, √ indicates infection) Example

[0062] The phage stock prepared in Example 1 was sent to Guangdong Megagene Technology Co., Ltd. for genome sequencing and analysis. After obtaining the phage genome sequence, first, the NCBI BLASTn was used to perform homology analysis on its whole genome. In order to more deeply analyze the possible classification of E1004, further, the Matcher in the EMBOSS suite and the ClustalW algorithm in the MEGA software were used to perform global pairwise sequence alignment on all known phages of the genus Kayfunavirus and E1004.

[0063] In addition, in order to further perform phylogenetic analysis on this phage and show its relationship with other phages of the genus Kayfunavirus, 1000 bootstrap replicates were established in the MEGA 11.0.13 version software, and the maximum likelihood method was used to construct a phylogenetic tree ( Figure 3 )

[0064] To identify potential tRNA genes in the phage genome, the present invention used tRNAscan - SE v. 2.0 for prediction and performed a search 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) web servers were respectively used to retrieve virulence factors and antibiotic resistance - encoding genes.

[0065] The results showed that the genome size of phage vB_EcoM_GZMU_E1004 was 39,250 bp and the GC content was 49.93%, indicating that this was a genome with a balanced GC content (Table 3). Using the Rapid annotation Subsystem Technology (RAST) annotation server for annotation, it was found that phage E1004 recognized a total of 47 open reading frames (ORFs), 46 of which were identified by gene prediction, and 32 of them were aligned with the UniProtKB database, including 6 unknown proteins. After analyzing the genomic sequence of phage E1004 with tRNAscan-SE, no RNA coding sequences were detected. The annotation data showed that the functions encoded by the genome were mainly concentrated in 3 subsystems, covering the phage lysis module and structural components, including endogenous lysozyme, endopeptidase, capsid protein, and tail fiber protein, all of which are necessary for host cell wall lysis and virion attachment during the phage infection cycle. Alignment with the virulence factor database and the Comprehensive Antibiotic Resistance Database showed that this phage did not carry resistance genes and virulence factors.

[0066] Table 3 General characteristics of phage genomes

[0067] Genome size (Kb) GC (%) Number of genes tRNA Species 39.25 49.93 46 0 Kayfunavirus

[0068] According to the criteria of the International Committee on Taxonomy of Viruses (ICTV), when the genomic sequence similarity between two phages is less than 95%, they should be classified as different species. As shown in Table 4, to evaluate the classification of phage E1004, first, the whole genome homology analysis of E1004 was performed using NCBI BLASTn. The results showed that the highest sequence similarity between E1004 and Escherichia coli phage vB_EcoP_F (Genbank accession number: NC_047808.1) was 95.99% (coverage rate was 86%). To more deeply analyze the possible classification of E1004, the Stretcher, Matcher, Water, and SSEARCH2SEQ in the EMBOSS suite, as well as the ClustalW algorithm in MEGA software, were further used to perform global pairwise sequence alignment of all known phages in the genus Kayfunavirus and E1004. The results showed that the whole genome homology range of phage E1004 was from 83.9% (with Escherichia coli phage ST31, using the EMBOSS Matcher method) to 92.9% (with Escherichia coli phage vB_EcoP_F, using the ClustalW method in MEGA). It should be noted that compared with BLASTn, the sequence similarity values obtained by the second method were all lower. This may be attributed to the fact that BLASTn mainly targets local sequence alignment, which may overestimate the true global similarity. Referring to the classification guidelines of BAVS, those with nucleotide sequence similarity exceeding 50% in the virus population can be classified into the same genus. Given that the similarity of the genome of E1004 with other phages in the genus Kayfunavirus mostly falls within the range of 50% - 95%, it is determined that E1004 represents a new species of the genus Kayfunavirus.

[0069] Table 4 Pairwise comparison of the whole genomes of phage genomes and members of the genus Kayfunavirus

[0070] In the phylogenetic tree constructed using the maximum likelihood method with 1000 bootstrap replicates in MEGA software version 11.0.13 (see Figure 3), Escherichia phage vB_EcoM_GZMU_E1004 and Escherichia phage ST31 were separated early on the evolutionary tree and were each located on independent branches. This indicates that these two phages may have specific evolutionary characteristics or unique genomic structures, thus supporting the classification of E1004 as a potential new species under the genus Kayfunavirus, which is consistent with the above results. On the other hand, Escherichia phage vB_EcoP_F and Escherichia phage ZG49 are located on the same branch in the phylogenetic tree, and the node bootstrap value obtained by ClustalW analysis is 85%, indicating that these two are highly related evolutionarily and closely related genetically.

[0071] Example 3 Stability Test

[0072] 1. pH Stability

[0073] Add 100 μL of phage to 900 μL of SM buffer with different pH values (the pH value was adjusted to 1 - 12 in advance by adding 1 mol / L HCl or 1 mol / L NaOH), let the mixture stand at 37°C for 1 h, and measure the titer with reference to the titer determination method in 3.2 of Example 1.

[0074] 2. Temperature Stability

[0075] Add 100 μL of phage to 900 μL of SM buffer at different temperatures (the buffer in different groups was pre-cooled or pre-heated at 4°C, 37°C, 50°C, 60°C, 70°C, 80°C respectively, and put back to the corresponding temperature after adding the phage), let it stand for 1 h, and measure the titer.

[0076] 3. Chloroform Sensitivity

[0077] Mix chloroform with 950 μL of phage at 0 μL, 10 μL, 20 μL, 50 μL respectively, and add 50 μL, 40 μL, 30 μL, 0 μL of LB liquid medium to make up a 1 mL system. After culturing in a shaker at 37°C and 220 rpm for 30 min, take 100 μL of the upper layer liquid from each and measure the titer.

[0078] 4. Optimal Multiplicity of Infection

[0079] Take the host bacteria in the logarithmic phase, dilute its concentration to 1×10 8 CFU / mL with LB liquid medium, pipette 200 μL into 5 groups of EP tubes, and dilute the phage concentration into 5 groups with LB, which are 1×10 10 PFU / mL, 1×109 PFU / mL, 1×10 8 PFU / mL, 1×10 7 PFU / mL, 1×10 6 PFU / mL. Pipette 200 μL into 5 groups of EP tubes in sequence, place the EP tubes in a shaker at 37 °C and 220 rpm for overnight culture. Take them out the next day, centrifuge at 5000 rpm and 4 °C for 10 min. After filtering the supernatant with a 0.22 μm filter tip, take the supernatant to measure the titer.

[0080] 5. One-step growth curve

[0081] Dilute the phage to 1×10 7 PFU / mL, take 0.1 mL and add it to 9.9 mL of Escherichia coli in the logarithmic phase (OD 600 = 0.5), place it in a shaker at 37 °C and 220 rpm for incubation for 15 min. Take it out, centrifuge at 13000 g for 15 min, discard the supernatant, resuspend the precipitate with 10 mL of liquid LB, place it in a shaker at 37 °C and 220 rpm for culture, sample at different time points, and measure the titer.

[0082] 6. Lysis kinetics

[0083] Take 75 μL of the host bacterial liquid in the logarithmic phase and mix it evenly with 75 μL of the phage suspension, prepare 6 groups with different multiplicity of infection (0.001, 0.01, 0.1, 1, 10, 100) and add them to a 96-well plate (the final titers of the corresponding phages are 10 2 、10 3 、10 4 、10 5 、10 6 、10 7 PFU / mL), at the same time, use an equal amount of sterile fresh MH broth and the bacterial liquid in the logarithmic phase as controls, culture in a shaker at 37 °C and 220 rpm, measure the OD 600 value of the mixture every 1 h, the experiment lasts for 13 h, and the experiment is repeated 3 times.

[0084] 7. Results and analysis

[0085] 7.1 pH stability

[0086] As Figure 4 shown, the titer of E1004 is the best when the pH is equal to 7. The titers at other pH values all decrease significantly, but it can be stable at a certain concentration between pH 4 - 10, maintaining more than 33%. When the pH is between 1 - 3 and 11 - 12, the phage is inactive.

[0087] 7.2 Temperature stability

[0088] As Figure 5 shown, the optimal temperature of E1004 is 37°C, and its titer is stable at 4 - 50°C, maintaining over 80%. At 60°C, the titer drops sharply, and the activity remains only 6.7%. When the temperature is greater than or equal to 70°C, the phage becomes inactive.

[0089] 7.3 Sensitivity to chloroform

[0090] As Figure 6 shown, E1004 is basically not affected by chloroform at a concentration of 5% or less. It is insensitive to chloroform, and it can be roughly inferred that it does not contain lipid substances such as envelope structures.

[0091] 7.4 Optimal multiplicity of infection

[0092] As Figure 7 shown, the optimal MOI of E1004 is 1, and it can maintain a titer close to the OMOI at 0.1 and 0.01. However, at 10 and 100, the titer is far from the OMOI, suggesting that its final culture concentration is greatly affected by the bacterial concentration.

[0093] 7.5 One-step growth curve

[0094] As Figure 8 shown, the lysis latent period is about 90 min, the rise period lasts for 150 min, and it reaches the plateau at 240 min. The burst size is 130 (41.67 / 0.32) PFU / infected cell.

[0095] 7.6 Lysis kinetics

[0096] As Figure 9 shown, the phages under each MOI condition all show an effective inhibitory effect on the growth of Escherichia coli. In addition, when the growth of Escherichia coli enters the stationary phase, the OD 600 value of Escherichia coli in each MOI group still remains at a low level.

[0097] Example 4 Antibacterial effect of allicin combined with phage

[0098] 1. Effect of allicin on phage activity

[0099] Take 200 μL each of 10 mg / mL allicin and phage liquid, and mix them. Another 200 μL each of fresh LB medium and phage liquid are mixed as a control group. After incubating them in a 37°C incubator for 1 h, measure the phage titer, with three replicates for each group.

[0100] 2. Minimum inhibitory concentration test

[0101] Determination of allicin MIC: Adjust the concentration of Escherichia coli in the logarithmic growth phase to 2×10 5CFU / mL. Take 75 μL of the bacterial solution and add it to several wells of a 96-well plate. Dilute allicin at 20 mg / mL two-fold to 10, 8.75, 7.5, 6.25, 5 mg / mL. Take 75 μL of each concentration gradient and add it to different wells pre-added with the bacterial solution, mix well. The control group is added with fresh LB medium to make the final concentration of the host bacteria 10 5 CFU / mL. The allicin concentration gradients are 10, 8.75, 7.5, 6.25, 5, 0 mg / mL. Measure the OD before cultivation 600 . Place the well plate in a shaker at 37 °C and 220 rpm for 12 h, and measure the OD600 after cultivation. Each group has 4 replicates.

[0102] Phage MIC determination: When performing the determination, follow the lysis kinetics method process in section 7.6 of Example 3. Each group has 3 replicates.

[0103] 3. Research on the best combination model

[0104] Take Escherichia coli in the logarithmic growth phase and dilute its concentration to 3×10 5 CFU / mL with LB liquid medium. Take 50 μL of the bacterial solution and add it to several wells of a 96-well plate. Dilute allicin at 20 mg / mL two-fold into 5 groups of concentrations: 3MIC, 3 / 10MIC, 3 / 100MIC, 3 / 1000MIC, 3 / 10000MIC. Take 50 μL of each gradient of allicin and add it to the corresponding wells with the bacterial solution. Dilute the phage gradient to 3×10 5 、3×10 4 、3×10 3 、3×10 2 、3×10 1 PFU / mL. Take 50 μL of each gradient of phage and add it to the corresponding above-mentioned wells. Add fresh LB liquid medium to each well to make the system up to 150 μL, mix well. The control group is added with fresh LB liquid medium to replace allicin and phage. Measure the OD before cultivation 600 . Place the well plate on a shaker and culture at 37 °C and 220 rpm for 12 h, and measure the OD after cultivation 600 . Repeat the experiment 3 times.

[0105] Judge whether there is a synergistic antibacterial effect between allicin and phage according to the fractional inhibitory concentration index (FIC). FIC index = MIC (combination of group A) / MIC (used alone in group A) + MIC (combination of group B) / MIC (used alone in group B). When the FIC index is less than 0.5, the two drugs have a synergistic effect; when the FIC index is 0.5 - 1, the two drugs have an additive effect; when the FIC index is greater than 1 and less than 2, the two drugs have an irrelevant effect; when the FIC index is greater than 2, the two drugs have an antagonistic effect.

[0106] 4. Observation of DNA damage

[0107] Prepare the bacterial solution, extract the genome using a gene extraction kit, and measure the DNA content using a NanoDrop spectrophotometer. Take the DNA and incubate it with 1 MIC of allicin, 1 MIC of phage, 1 MIC of allicin + 1 MIC of phage, the optimal combined concentration of allicin, the optimal combined concentration of phage, the optimal combined concentration of phage + the optimal combined concentration of allicin, and LB control at 37 °C for 30 min, and observe whether the bands are diffused by agarose gel electrophoresis.

[0108] 5. Detection of nucleic acid and protein leakage

[0109] Take the bacterial solution in the logarithmic growth phase, centrifuge it at 4000 rpm for 10 min, resuspend it with PBS to make the bacterial solution concentration 10 8 CFU / mL. Take the bacterial solution and incubate it with 1 MIC of allicin at the final concentration, 10 9 PFU / mL of phage, the optimal combined concentration of allicin, the optimal combined concentration of phage, the optimal combined concentration of phage + the optimal combined concentration of allicin, and LB control. After culturing at 37 °C for 3 h, centrifuge it at 6000 rpm for 5 min to precipitate the bacteria, take the supernatant and measure OD 260 and OD 280 .

[0110] 6. Results and analysis

[0111] 6.1 Effect of allicin on phage

[0112] As Figure 10 shown, there is no significant difference in the results of the two groups of experiments. It can be seen that allicin has basically no effect on the infectivity of phage, and the influence of allicin on phage itself can be ignored when conducting subsequent experiments.

[0113] 6.2 Minimum inhibitory concentration

[0114] Phage MIC: As Figure 9 shown, when cultured for 12 h, when MOI = 0.001 (corresponding phage titer is 10 2 PFU / mL), its OD 600 has an obvious fluctuation, indicating that at this MOI, the phage still fails to completely inhibit the host bacteria. Starting from MOI = 0.01 (corresponding phage titer is 10 3 PFU / mL), OD 600 is basically stable and close to 0, that is, the phage inhibition rate at an infection multiplicity of MOI = 0.01 and above is close to 100%. Therefore, it is considered that the phage titer 10 3PFU / mL is the minimum inhibitory concentration of phages.

[0115] The MIC of allicin: As Figure 11 shown, the minimum inhibitory concentration of allicin is 8.75 mg / mL, and the inhibition rate is about 76%.

[0116] 6.3 Optimal combination model

[0117] Combined with Figure 12 the data in 7 it can be known that the combined group has the highest inhibition rate when combined with 1 / 10 MIC allicin concentration (0.875 mg / mL) and 10 2 PFU / mL of phages. However, the combined group with 1 / 10 MIC allicin concentration (0.875 mg / mL) and 10 2 PFU / mL of phages can also achieve an antibacterial effect equivalent to that of the combination with the highest inhibition rate (it is at the 0 point). Considering that the drug dose should be reduced as much as possible, it is considered that the combination of 1 / 10 MIC allicin concentration (0.875 mg / mL) and 10

[0118] PFU / mL of phages is the optimal combination mode of allicin combined with phages. -3 Calculate the fractional inhibitory concentration (FIC). So FIC = 1 / 10 + 10

[0119] 6.4 DNA damage results

[0120] As Figure 13 shown, there is no DNA band diffusion phenomenon in each group compared with the pure bacterial liquid group. Therefore, allicin at MIC and lower concentrations and 10 10 PFU / mL concentration of phages have no DNA damage effect, and it can be basically determined that the antibacterial mechanism of this optimal combination mode does not include the DNA damage effect.

[0121] 6.5 Nucleic acid and protein leakage results

[0122] As Figure 14 shown, after 3 h of treatment, the release amounts of the contents of Escherichia coli in the drug group, phage group, and combined group are significantly higher than those in the pure bacterial liquid group, indicating that both allicin and phages can destroy the structure of Escherichia coli and make its contents flow out.

[0123] 10 9 PFU / mL concentration phage group and 10 7 PFU / mL concentration phage group have similar results, and even the results of the two are similar to those of the allicin MIC group and the combined group, indicating that at this time, most Escherichia coli are killed, so no greater release amount of contents is obtained under the action of high-concentration phages.

[0124] The release amount of the MIC allicin group was significantly higher than that of the 1 / 10 MIC allicin group, but both were higher than that of the pure bacterial liquid group, indicating that allicin has a certain ability to damage bacteria when its concentration does not reach MIC, but due to insufficient concentration, the killing ability is slightly weaker.

[0125] The combined use group could well damage Escherichia coli and release its contents, and the effect was significantly higher than that of the 1 / 10 MIC allicin used alone group and slightly higher than that of the phage used alone group. Combining the above results, it shows that this combined antibacterial mode can carry out antibacterial effects by damaging Escherichia coli and releasing its contents.

[0126] The embodiments described above are only descriptions of the preferred modes of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should all fall within the protection scope determined by the claims of the present invention.

Claims

1. The use of Escherichia coli phage combined with allicin in the preparation of biological preparations for inhibiting Escherichia coli or treating diseases caused by Escherichia coli, characterized in that: The deposit number of the Escherichia coli phage is GDMCC No: 64729-B1; The deposit number of the Escherichia coli phage is GDMCC No: 64729-B1; The concentration of the allicin is 0.875 mg / mL; The Escherichia coli includes drug-resistant Escherichia coli; The biological agent is a biological bacterial agent or a medicine.

2. The use according to claim 1, characterized in that The concentration of the phage was 1×10 2 PFU / mL.

3. A biological agent for inhibiting Escherichia coli or treating diseases caused by Escherichia coli, characterized in that: The biological preparation comprises Escherichia coli phage and allicin, and the effective concentration of the Escherichia coli phage is ≥1×10 3 PFU / mL, the deposit number of the Escherichia coli phage is GDMCC No: 64729-B1; The biological agent is a biological bacterial agent or a medicine.

4. The biological agent according to claim 3, characterized in that The concentration of the allicin is 0.875 mg / mL.

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