Application of lysing bacteriophage Myy9 in preparation of product for inhibiting or killing escherichia coli

Abstract

The invention discloses application of a lysing bacteriophage Myy9 in preparation of a product for inhibiting or killing escherichia coli, and belongs to the technical field of microorganisms. The bacteriophage keeps good activity in a temperature range of 4-50 DEG C, still keeps relatively high activity in a pH range of 3-11, still can grow after ultraviolet irradiation for 1 hour, and the activity is reduced along with the prolonging of irradiation time, so that the bacteriophage can adapt to multi-scene application requirements. The bacteriophage has the potential of being developed into a novel antibacterial preparation, and is expected to provide a new choice for prevention and treatment of drug-resistant escherichia coli infection.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to the application of lysing bacteriophage Myy9 in the preparation of products that inhibit or kill Escherichia coli. Background Technology

[0002] In the field of microbial prevention and disease treatment, bacteriophages, as viruses that specifically infect bacteria and archaea, have high specificity, infecting only one type or class of bacteria. They are one of the most abundant biological entities in nature, widely existing in environments such as soil, water bodies, and the human gut, playing an important role in maintaining environmental ecological stability, and have been extensively studied and applied.

[0003] Currently reported Vequintavirus species of bacteriophages differ in physicochemical properties and host range. Discovering new bacteriophages with excellent characteristics can provide key materials for the development of novel antibacterial agents, and has potential application value, especially in solving the problem of drug-resistant Escherichia coli infection. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the lysing phage Myy9 in the preparation of products that inhibit or kill Escherichia coli, so as to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is the application of the lysing phage Myy9 in the preparation of products that inhibit or kill Escherichia coli.

[0006] The second technical solution of the present invention is an anti-Escherichia coli preparation, the active ingredient of which includes the lysing phage Myy9.

[0007] Based on the above technical solution, the present invention has the following technical effects: 1. Environmental adaptability: The bacteriophage maintains good activity in the temperature range of 4-50℃, and can still maintain high activity in the pH range of 3-11. It can still grow after 1 hour of ultraviolet irradiation, and its activity decreases with the extension of irradiation time, so it can adapt to the application needs of multiple scenarios.

[0008] 2. Transmission electron microscopy morphological observation was completed: it was identified as a tailed phage (tail length approximately 85 nm).

[0009] 3. Lysis characteristics: The bacteriological killing effect of lysed bacteriophage Myy9 on bacteria under different MOI conditions was determined; one-step growth curve determination (latency period, outbreak size) was completed.

[0010] 4. Broad application prospects: This bacteriophage has the potential to be developed into a novel antibacterial agent, which is expected to provide a new option for the prevention and control of drug-resistant Escherichia coli infections. Attached Figure Description

[0011] Figure 1 The lytic ability of lysing phage Myy9 was demonstrated using the double-layer agar plate method.

[0012] Figure 2 For titer testing of lysed bacteriophage Myy9, dilute lysed bacteriophage Myy9 to 10⁻⁶. -5 The data was repeated three times to count the number of plaques on each plate.

[0013] Figure 3 Thermal stability test for lysed bacteriophage Myy9. Each data point represents the average of three replicates, and the error bar represents the standard deviation (n = 3).

[0014] Figure 4 Stability of lysed bacteriophage Myy9: (A) UV stability and (B) pH stability. Each data point represents the mean of three replicates, and the error bar represents the standard deviation (n = 3).

[0015] Figure 5 To monitor the growth trend of lysing phage Myy9 under different MOI conditions, time-kill assays were performed at MOIs of 0, 100, 10, 1.0, 0.1, 0.01, 0.0001, and 0.00001. Each data point represents the average of three replicates, and the error bar represents the standard deviation.

[0016] Figure 6 This is a one-step growth curve for lysed bacteriophage Myy9. Each data point represents the average of three replicates, and the error bars represent the standard deviation.

[0017] Figure 7 The image shows the lysed bacteriophage Myy9 (100 nm) as observed by transmission electron microscopy. Detailed Implementation

[0018] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0019] This invention provides the application of the lysing phage Myy9 in the preparation of products that inhibit or kill Escherichia coli.

[0020] In some specific implementations, the *E. coli* includes *E. coli* SHBCC D25054.

[0021] In some specific implementations, the titer of the lysing phage Myy9 is 1.52 × 10⁻⁶. 9 PFU / mL.

[0022] In some specific implementations, the pH value of the lysing phage Myy9 for inhibiting or killing Escherichia coli is 3-11, and the temperature is 4℃~40℃.

[0023] This invention also provides an anti-Escherichia coli formulation, the active ingredient of which includes the lysing phage Myy9.

[0024] The technical solution of this invention revolves around the core characteristics of the lysing bacteriophage Myy9 (including physicochemical properties, lysis characteristics, and morphological observation).

[0025] Application potential: Based on the lysis characteristics of this bacteriophage against Escherichia coli, it is expected to be used for the prevention and treatment of Escherichia coli infection.

[0026] The lysing phage Myy9 exhibits lytic activity against *E. coli*, with a head diameter of 76 nm and a tail length of approximately 85 nm. Compared to the control group (MOI=0), Myy9 showed strong lytic activity at MOIs of 100, 10, 1.0, 0.1, 0.01, 0.0001, and 0.00001, but the ability to lyse the host bacteria increased with increasing MOI. After infecting *E. coli*, Myy9 efficiently lyses the host and releases progeny viruses after an incubation period of approximately 30 minutes, directly demonstrating its efficient and rapid lytic activity.

[0027] Example 1 (i) The lysing phage Myy9 used in this invention comes from the Wenzhou Institute of the University of Chinese Academy of Sciences. This phage has been disclosed in the literature “Novel Lytic Phages Protect Cells and Mice against Pseudomonas aeruginosa Infection”.

[0028] (II) Physicochemical property testing 1. Phage titer (PFU): 1.1 Dilute the lysed phage Myy9 10-fold serially to 10 -5 .

[0029] 1.2 Pour the mixed semi-solid agar into the LB solid plates prepared in 1.1. Repeat the process for three plates using the same phage dilution gradient. After cooling and solidification, incubate overnight at 37°C.

[0030] 1.3 Phage titer = Count result (average number of phage plaques on three plates) × (1 ml / 10 ul) × dilution gradient = Count result × 100 × dilution gradient.

[0031] The PFU of the lysing bacteriophage Myy9 was 1.52 × 10⁻⁶. 9 PFU / mL.

[0032] In vitro testing of the lysed bacteriophage Myy9 requires calculation of the bacteriophage titer (pfu) (see [link]). Figure 2 ).

[0033] Formula: PFU = Average number of plaques / Volume (mL) * Dilution factor; PFU=(168+138+152) / 3 / (10*10 -3 )*10 5 =1.52×10 9 pfu / mL.

[0034] 2. Temperature tolerance test: 2.1 Prepare the host bacterium Escherichia coli bacterial suspension, LB solid medium plates, and liquid semi-solid medium.

[0035] 2.2 Set the PFU to 1.52 × 10 9 Phage diluted to 1×10⁻⁶ PFU / mL 7 PFU / mL, then aliquot 1ml into several sterile 1.52mL EP tubes, label them with numbers and incubate at temperatures of 4℃, 25℃, 37℃, 40℃, 50℃, 60℃, 70℃, and 80℃ for 1 hour.

[0036] 2.3 After the time is up, remove the EP tube, invert and mix well. Take 10 uL of phage solution and 100 uL of overnight host bacterial solution for each temperature, mix well, add 5-6 ml of semi-solid culture medium and mix well. Pour into double-layer agar plates, repeat three plates for each condition, wait for cooling and solidification, and incubate overnight in a 37°C incubator.

[0037] 2.4 The next day, count the PFU of phages after treatment under different temperature conditions and take the average value.

[0038] The thermal stability of the lysed bacteriophage Myy9 was tested, with a PFU of 1.52 × 10⁻⁶. 9 pfu / mL diluted to 1×10 7 Phage lysates in SM buffer were incubated for 1 hour at 4°C, 25°C, 37°C, 50°C, 60°C, 70°C, and 80°C, respectively. Phage titers (PFU) were measured using the host bacterium (E. coli SHBCC D25054) on double-layer agar plates, with three sets of data repeated at each temperature (see [link to data]). Figure 3 ).

[0039] 3. pH tolerance test: 3.1 Prepare the materials as in 2.1.

[0040] 3.2 Using sterile SM buffer solution and hydrochloric acid and 10% sodium hydroxide solution, SM buffer solutions with different pH values ​​were prepared under pH meter monitoring. The pH values ​​were adjusted to 3, 4, 5, 6, 7 and 11.

[0041] 3.3 Adjust the phage titer to 10 9 PFU / ml, take 10μL of phage fluid + 990μL of SM buffer of various pH values, vortex mix, place in a 4℃ refrigerator for 1h, and set up a control group (i.e.

[0042] 3.4 After the time is up, remove the EP tube, vortex to mix evenly, take 10 μL of phage solution for each pH value and mix with 100 μL of overnight bacterial solution, add 5-6 ml of semi-solid culture medium and mix well, pour into double-layer agar plates, repeat three plates for each condition, wait for cooling and solidification, and incubate overnight in a 37°C incubator.

[0043] 3.5 The next day, count the PFU of phages treated under different pH conditions and take the average value.

[0044] 4. Ultraviolet tolerance test: 4.1 Prepare the materials as in 2.1.

[0045] 4.2 Prepare 1×10 7 Pour 2 mL of PFU / ml phage solution (refer to experimental requirements) into a sterile disposable culture dish with a diameter of 90 mm, spread it evenly on the bottom plate, and place it in a clean bench, 30 cm away from the ultraviolet lamp.

[0046] 4.3 Turn on the UV lamp and take samples at 0, 10, 20, 30, 40, 50 and 60 min respectively. Take 200 μL of the sample at each time point. Dilute the phage at each time point 100 times and take 10 μL + 100 μL of overnight host bacterial culture and mix them. Add 5-6 mL of semi-solid culture medium and pour it into a double layer agar plate.

[0047] 4.4 Place the double-layer agar plates in a 37℃ incubator overnight, count the plaques the next day to calculate the phage titer, and plot the curve.

[0048] pH stability and UV tolerance tests were performed on the lysed bacteriophage Myy9, with a PFU of 1.52 × 10⁻⁶. 9 pfu / mL diluted to 1×10 7Phage lysates in SM buffer at different pH values ​​(3, 5, 6, 7, 11) were incubated at 4°C for 1 hour. Under the same PFU conditions, phages were collected for different UV irradiation times (0–60 min), and phage titers (PFU) were measured using a host cell (E. coli SHBCC D25054) on double-layer agar plates. Three sets of data were repeated for each condition (see [link to data]). Figure 4 ).

[0049] The results are as follows Figure 3-4 As shown, the phage maintains good activity at 37℃; exhibits certain tolerance within the pH range of 3–11; and demonstrates good physicochemical stability under UV irradiation: its activity decreases with prolonged irradiation time, but it can still survive after 60 minutes of irradiation.

[0050] (III) Study on pyrolysis characteristics 1. Optimal MOI determination: 1.1 Bacterial preparation: Take bacterial suspension in the logarithmic growth phase (OD600 ≈ 0.4~0.6), and adjust the concentration to 2×10⁻⁶ with fresh culture medium. 6 CFU / mL.

[0051] 1.2 Setting the MOI Gradient: A series of MOI values ​​are planned for testing: 100, 0.1, 1.0, 0.1, 0.01, 0.0001, 0.00001. Based on the formula, the PFU is set to 1.52 × 10⁻⁶. 9 PFU / mL phage was diluted to 2×10⁻⁶. 8 2×10 7 2×10 6 2×10 5 2×10 4 2×10 3 2×10 2 2×10 1 PFU / mL.

[0052] 1.3 In a sterile 96-well plate, add 100 μL of diluted 2×10⁻⁶ solution to the corresponding well. 6 Prepare a bacterial suspension at CFU / mL, then add 100 μL of phages of different titers to the wells containing the bacterial suspension. Repeat this process for three wells for each titer, and shake horizontally to thoroughly mix the phages and bacterial suspension.

[0053] 1.4 Set the microplate reader to the correct conditions (37℃, 220 rpm, OD measurement every 10 minutes). 600 The data was continuously monitored for 16 hours. A curve was plotted using the obtained data.

[0054] The growth trend of lysed bacteriophage Myy9 was monitored under different MOI conditions using an ELISA reader to determine the optimal MOI for lysed bacteriophage Myy9. Three sets of data were repeated for each condition (see [link to ELISA reader]). Figure 5 ).

[0055] The results are as follows Figure 5 As shown, at MOI values ​​of 100, 10, 1.0, 0.1, 0.01, 0.0001, and 0.00001, the lysing activity was stronger than that of the blank control group (MOI=0). However, the ability to lyse the host bacteria increased with the increase of MOI value.

[0056] 2. One-step growth curve: 2.1 Prepare LB solid medium plates and liquid semi-solid medium. Take the bacterial suspension in the logarithmic growth phase (OD600 ≈ 0.4~0.6), and adjust the concentration to 2×10⁻⁶ with fresh medium. 6 CFU / mL.

[0057] 2.2 Selecting a suitable MOI for the phage. The condition used in this invention is MOI=10, that is, the phage is diluted to a titer of 2×10. 7 PFU / mL, mix equal volumes of phage suspension and Escherichia coli bacterial suspension according to the set multiplicity of infection (MOI=10), and let stand at 37℃ for 10 min to allow for full adsorption.

[0058] 2.3 After the time is up, centrifuge at 10000×g for 3 min, discard the supernatant, resuspend in preheated LB, and incubate at 37℃ with shaking at 220 rpm / min. Take 100 μL samples at 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 40 min, 50 min, 60 min, and 90 min, respectively, and immediately centrifuge at 4 °C at 10000×g for 2 min, and store in a 4℃ refrigerator.

[0059] 2.4 After sampling at all time points, the supernatant was collected to determine the phage titer (two-sided agar plate method, repeated on three plates). A one-step growth curve was plotted with sampling time as the x-axis and the logarithm of phage titer as the y-axis to determine the incubation period and lysis period.

[0060] At MOI=10, lysed bacteriophage Myy9 was co-cultured with bacteria at 220 rpm. Phage-bacteriophage mixtures were collected at different time points (5 / 10 / 15 / 20 / 25 / 30 / 40 / 50 / 60 / 90 min), centrifuged at low speed, and the supernatant (i.e., bacteriophage) was collected and the phage titer (PFU) was measured using a double-layer agar plate with the host cell. Three sets of data were repeated for each condition (see [link to data]). Figure 6 ).

[0061] The results are as follows Figure 6 As shown, after infecting E. coli, the lysing phage Myy9 can efficiently lyse the host and release progeny viruses after an incubation period of about 30 minutes, directly confirming its efficient and rapid lysing activity.

[0062] (iv) Observation of bacteriophage morphology 1. Phage amplification and collection: Amplify and collect phages according to the methods described above.

[0063] 2. Phage concentration: The amplified phage solution was centrifuged and ultrafiltered for an appropriate time at 4℃ and 4000×g using a 100kD pore size ultrafiltration tube (do not over-concentrate) to obtain a concentrated phage solution.

[0064] 3. Use a micropipette to draw 5 μL of concentrated phage solution and drop it onto a 200-mesh carbon support membrane. After allowing it to settle naturally for 5–6 minutes, use absorbent paper to blot away any excess liquid.

[0065] 4. On the carbon support membrane that has been adsorbed with bacteriophages, drop 5 μL of 2% phosphotungsten solution for negative staining for 3 min, and then use absorbent paper to blot away the excess stain.

[0066] 5. Send the samples to the transmission electron microscope room to observe the morphology and structure of the bacteriophages at 80kV, and take pictures and record them in a clear field of view.

[0067] Bacteriophage Myy9 was lysed using a 200-mesh copper mesh and stained with 2% phosphotungstic acid. Its morphology was observed using transmission electron microscopy (see [link to original text]). Figure 7 The results showed that the head diameter was 76 nm and the tail length was approximately 85 nm.

[0068] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. Application of lysing phage Myy9 in the preparation of products that inhibit or kill Escherichia coli.

2. The application according to claim 1, characterized in that, The Escherichia coli includes Escherichia coli SHBCCD25054.

3. The application according to claim 1, characterized in that, The titer of the lysing phage Myy9 was 1.52 × 10⁻⁶. 9 PFU / mL.

4. The application according to claim 1, characterized in that, The pH range of Myy9 lysing phage for inhibiting or killing Escherichia coli is 3-11, and the temperature range is 4℃-40℃.

5. An anti-Escherichia coli preparation, characterized in that, The active ingredient includes the lysing phage Myy9 as described in claim 1.

Images