Development method and application of phage atomization process

Abstract

The invention relates to the technical field of biology, in particular to a development method and application of a phage atomization process. The method comprises the following steps: atomizing a bacteriophage preparation of which the titer is 108PFU / mL and the liquid medium is a PBS (Phosphate Buffer Solution) by using an ultrasonic atomization device into an atomized dosage form of which the liquid drop particle size is 3 microns, atomizing in a closed space at the atomization amount of 57mL / m < 3 >, and naturally settling for 30 minutes, so as to achieve the purpose of reducing the bacterial load in the environment. The environment disinfectant based on the phage atomization dosage form has a good disinfection and sterilization effect on the environment in a hospital, the comprehensive bacteriostasis rate can reach 99% or above, and no disinfection dead angle exists. The method is of great significance to reduction of potential hospital cross infection risks in the clinical treatment process.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to a development method of a bacteriophage atomization process and its application. Background Art

[0002] With the continuous improvement of global medical and health levels, hospitals have become an important place for disease diagnosis, treatment and prevention. However, due to the particularity of the hospital environment, the problem of cross-infection of multidrug-resistant bacteria in hospitals has always been one of the major problems that the global medical and health system needs to solve urgently. Especially in high-risk areas such as intensive care, operating rooms, and emergency departments, patients are often prone to hospital infections due to low immunity or the presence of multidrug-resistant pathogens. These infections not only worsen the patient's condition, but may even cause a series of complications, affect the treatment effect, and even threaten life safety. Therefore, the prevention and control of hospital infections is extremely important, and it is necessary to continuously explore more efficient and sustainable disinfection and sterilization technologies.

[0003] As a natural virus, bacteriophages show great potential in bacterial infection control by specifically attacking and dissolving bacteria. In recent years, with the increasing antibiotic resistance, the study of bacteriophages as antibacterial alternatives has gradually become a hot topic. Bacteriophages have the advantages of high efficiency, specificity, and non-toxicity. They have a significant inhibitory effect on multidrug-resistant pathogens, and compared with traditional antibiotics, bacteriophages are less likely to cause drug resistance problems. Therefore, the application of bacteriophage technology in the disinfection of hospital environments, especially for the prevention and control of nosocomial infections, has a broad prospect.

[0004] However, the application of phages in clinical settings still faces many challenges, one of which is how to effectively and persistently deliver phages to every corner of the hospital environment. In particular, how to ensure that phages have a sustained antibacterial effect in air disinfection and hard-to-reach surfaces is an important topic of current research.

[0005] Atomization technology is a technology that converts liquid into small particles and evenly distributes them in the air. This technology is widely used in the fields of medicine and environmental disinfection. In hospital disinfection, atomization formulations have unique advantages: it can effectively cover a large area of ​​space, including airborne pathogens, various surfaces of objects, etc., and is especially suitable for air disinfection in high-risk areas.

[0006] The phage aerosol formulation combines the advantages of phage and aerosol technology, and can quickly disperse phage in the air through aerosol equipment to form tiny particles with high antibacterial effect. Because the aerosolized particles are extremely small, they can be suspended in the air for a long time and can penetrate into corners and dead ends that ordinary disinfection methods cannot reach, thereby providing continuous and extensive disinfection effects. Summary of the invention

[0007] The present invention provides a new environmental disinfection method to address the current threat of cross-infection of multi-drug resistant bacteria in hospitals. The present invention provides a phage atomization scheme, wherein the phage preparation that has been expanded and purified is atomized by ultrasonic atomization, preferably atomized into a liquid with a particle size of 3 μm, and the atomization volume is 57 mL / m 3 After atomization, it will naturally settle in a closed room for 30 minutes, which can effectively reduce the environmental bacterial load without any dead corners and will not cause corrosion to the surface of objects.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] The first aspect of the present invention provides a method for developing a bacteriophage atomization process, comprising the following steps:

[0010] After the phage was expanded and purified, the phage titer was diluted to 10 8 PFU / mL, and the phage preparation is obtained; the phage preparation is then placed in a closed space and atomized into droplets with a particle size of 2.8-4.5 μm, and then naturally settled for 20-40 minutes; the atomization volume is 43-71 mL / m 3 .

[0011] In the preferred embodiment, the liquid culture method is used for expansion, that is, the phage, host bacteria and LB liquid culture medium are mixed and inoculated at a ratio of 1:1:100, and then incubated at 37°C, 160rpm constant temperature shaker for 4 hours.

[0012] Under the preferred scheme, the atomized particle size is 3 μm and the atomized volume is 57 mL / m 3 , the sedimentation time is 30 minutes.

[0013] In a preferred embodiment, the phage is selected from one or more of Pseudomonas aeruginosa phage, Klebsiella pneumoniae phage, Acinetobacter baumannii phage, Staphylococcus aureus phage, Salmonella phage and Escherichia coli phage.

[0014] In a preferred embodiment, the atomization method is ultrasonic atomization.

[0015] In a preferred embodiment, the diluent used for dilution is PBS buffer.

[0016] A second aspect of the present invention provides an application of any of the above-mentioned methods for developing a phage atomization process to reduce the bacterial load in an environment.

[0017] In a preferred embodiment, the bacteria are selected from one or more of Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, Salmonella and Escherichia coli.

[0018] Under the preferred embodiment, it is used to prepare an environmental disinfectant for preventing and controlling cross infection within hospitals.

[0019] In a more detailed technical solution of the present invention, a method for developing a bacteriophage atomization process comprises the following steps:

[0020] S1. Based on the pathogens detected within the hospital, the corresponding bacteriophages are cultured using the liquid culture method.

[0021] S2. The cultured phages were purified using a tangential flow hollow fiber column and a "molecular exclusion + charge adsorption" combined chromatography column, and the liquid medium was replaced with PBS buffer.

[0022] S3, dilute the obtained phage preparation phage titer to 10 8 PFU / mL.

[0023] S4. Atomize the diluted phage preparation in a closed room.

[0024] S5. Allow it to settle naturally after atomization.

[0025] The present invention is mainly aimed at clinical multidrug-resistant bacteria. Such pathogenic bacteria in hospitals mainly include one or more of Pseudomonas aeruginosa phage, Klebsiella pneumoniae phage, Acinetobacter baumannii phage, Staphylococcus aureus phage, Salmonella phage and Escherichia coli phage.

[0026] In step S1, the bacteriophage is preferably one or more of Pseudomonas aeruginosa phage, Klebsiella pneumoniae phage, Acinetobacter baumannii phage, Staphylococcus aureus phage, Salmonella phage and Escherichia coli phage.

[0027] Step S2 uses the method provided by application number 2024112374409 to purify the expanded phage, and its patent name is a method for purifying a phage preparation and its application in industrial-scale production. Preferably, the molecular weight cutoff of the tangential flow hollow fiber column is 500 kDa, and the shear rate is 8000 s -1 , the system pressure is 1 psi; the chromatographic medium of the "molecular exclusion + charge adsorption" combination chromatography column is CaptoCore 400 TM , the loading buffer was PBS buffer.

[0028] In step S3, the diluent for dilution is preferably PBS buffer.

[0029] In step S4, preferably, the atomization method is ultrasonic atomization, and the phage preparation is atomized into 3 μm droplets, and the atomization volume should reach 57 mL / m 3 .

[0030] In step S5, the natural sedimentation time is preferably 30 minutes.

[0031] Next, an application of the aerosolized bacteriophage preparation prepared by the above-mentioned development method of the bacteriophage aerosolization process includes one or more of the following:

[0032] (1) Used to prepare aerosol formulations of bacteriophage disinfectants.

[0033] (2) Used to prepare disinfectant aerosol formulations with bacteriophage as the active ingredient.

[0034] Beneficial effects of the present invention:

[0035] 1. Strong antibacterial ability. The phage atomization scheme provided by the present invention can maximize the antibacterial rate of the phage preparation, and the environmental disinfection effect is better. The antibacterial rate can reach more than 99%, which is of great significance to reducing the potential risk of nosocomial cross-infection during clinical treatment.

[0036] 2. There are no dead corners for disinfection. The atomization solution provided by the present invention can disinfect any corner in a closed space and is suitable for disinfection in indoor environments.

[0037] 3. Compared with other atomization schemes, the phage atomization scheme provided by the present invention has stronger environmental disinfection ability and better diffusion ability of phage preparations, and is more suitable for the prevention and control of multidrug-resistant bacterial infections in hospitals. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the ability of phage preparations to resist aerosolization.

[0039] Figure 2 It is the orthogonal experimental result of optimizing the process parameters of bacteriophage atomization.

[0040] Figure 3 It is the response surface curve for optimization of phage atomization process parameters.

[0041] Figure 4 It is the antibacterial ability of phage aerosol formulation on the surfaces of different materials.

[0042] Figure 5 This is a verification chart of the diffusion ability of bacteriophage aerosol formulation. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0044] The present invention provides a method for developing a phage atomization process and its application. The preparation of the atomized dosage form includes the following steps: amplifying the phage preparation by liquid propagation method, purifying the phage preparation, and then atomizing the phage by ultrasonic atomization. The specific parameters of the atomization are: the atomized droplet size is 3 μm, the atomization volume is 57 mL / m 3 After atomization, the atomization solution is allowed to settle naturally in a closed space for 30 minutes. The atomization solution can effectively reduce the bacterial load in the environment, with an antibacterial rate of more than 99% and no dead corners for disinfection.

[0045] A method for developing a bacteriophage atomization process comprises the following steps:

[0046] S1. Based on the pathogens detected within the hospital, the corresponding bacteriophages are cultured using the liquid culture method.

[0047] S2. Referring to the method of the national invention patent "A method for purifying a bacteriophage preparation and its application in industrial-scale production" (application number 2024112374409), tangential flow hollow fiber column and "molecular exclusion + charge adsorption" combined chromatography were used to purify the cultured phage preparation and replace the medium.

[0048] S3, dilute the obtained phage preparation phage titer to 10 8 PFU / mL.

[0049] S4. Atomize the diluted phage preparation in a closed room.

[0050] S5. Allow it to settle naturally after atomization.

[0051] In a preferred embodiment, the phage preparation in step S1 is a clinical multi-drug resistant bacteria phage, selected from one or more of Pseudomonas aeruginosa phage, Klebsiella pneumoniae phage, Acinetobacter baumannii phage, Staphylococcus aureus phage, Salmonella phage and Escherichia coli phage.

[0052] In a preferred embodiment, the host bacteria in step S1 are clinical multidrug-resistant bacteria, selected from one or more of Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, Salmonella and Escherichia coli.

[0053] In the preferred embodiment, the molecular weight cut-off of the tangential flow hollow fiber column in step S2 is 500 kDa and the shear rate is 8000 s -1 , the system pressure is 1psi.

[0054] In a preferred embodiment, the chromatography filler used in the "molecular exclusion + charge adsorption" combined chromatography in step S2 is CaptoCore 400 TM .

[0055] In the preferred embodiment, the diluent in step S3 is PBS buffer.

[0056] In a preferred embodiment, the atomization method in step S4 is ultrasonic atomization.

[0057] In the preferred embodiment, the atomized droplet size in the atomization process of step S4 is 2.8-4.5 μm, and the atomization volume is 43-71 mL / m 3 .

[0058] In the preferred embodiment, the natural sedimentation time in step S5 is 30 minutes.

[0059] An application of the development method of any of the above-mentioned bacteriophage atomization processes for preparing an environmental disinfectant for preventing and controlling cross infection within hospitals.

[0060] In the following examples, unless otherwise specified, the phages before atomization were expanded and purified by the above method and then diluted. The phages used in the experiment can be obtained by screening and separation through conventional experimental operations. The host bacteria / pathogenic bacteria are obtained from the internal environment of the hospital. Other raw materials and experimental operations can be obtained through ordinary commercial channels or performed using conventional experimental methods.

[0061] Example 1 Verification of bacteriophage resistance to aerosolization

[0062] To investigate the effect of ultrasonic atomization on the titer of bacteriophages, three experimental groups and one control group (bacteriophage preparations without atomization) were set up. The specific operation is as follows:

[0063] In a sterile environment, 1 mL of Pseudomonas aeruginosa phage phipa 2 with known titer was placed in an ultrasonic atomizer. The atomized particle sizes were adjusted to 2.8 μm, 3 μm, and 4.5 μm, respectively, and a sterile glass slide was placed at the outlet of the atomizer. The atomized phage droplets were collected in a 1.5 mL centrifuge tube. The titer of the atomized phage preparation was measured and compared with the initial titer to verify its atomization resistance. The results are shown in Figure 1 shown.

[0064] Figure 1 It is the atomization resistance of bacteriophage under ultrasonic atomization. The results show that when the atomization particle size is 2.8μm, 3μm and 4.5μm, the ultrasonic atomization will not affect the phage titer, indicating that phage atomizer can be produced by ultrasonic atomization.

[0065] Example 2 Optimization of process parameters for bacteriophage atomization

[0066] The study on the effect of ultrasonic atomization process parameters on antibacterial ability includes the following steps:

[0067] S1. Put a mixed phage preparation (Pseudomonas aeruginosa phage phipa 2: Pseudomonas aeruginosa phage phipa 4: Pseudomonas aeruginosa phage phipa 10 = 1:2:1) into an ultrasonic atomization device.

[0068] S2, the concentration of phage mixture that can be lysed is 10 5 10 μL of the host bacteria mixed bacterial solution (PA35:PA16=1:1) with a CFU / mL was spread on an LB solid culture medium plate, and a control group was set up at the same time.

[0069] S3. Place the tablet in a 21L sealed transparent plastic box and connect it to the atomization port.

[0070] S4. Design a three-level orthogonal experiment with three factors (atomized particle size, atomized amount, and settling time).

[0071] S5. Set corresponding parameters according to the orthogonal experiment table (Table 1) to atomize the bacteriophage.

[0072] S6. After the atomization is completed, the plate is taken out and incubated in a 37°C constant temperature incubator for 12 hours together with the control group.

[0073] S7, take out and count, and determine the antibacterial rate respectively. Figure 2 As shown, the optimal solution is: atomization particle size 3μm, atomization volume 57mL / m 3 , sedimentation time 30min.

[0074] Table 1: Orthogonal test table for phage atomization process optimization

[0075]

[0076] like Figure 2 As shown in the figure, the three-factor three-level orthogonal test shows that under the atomization conditions of atomization particle size of 3μm and atomization volume of 57mL / m 3 , sedimentation time 30min and atomization conditions: atomization particle size 4.5μm, atomization volume 71mL / m 3 The highest inhibition rate was obtained when the former required less phage and took less time. Therefore, the preferred atomization conditions were atomization particle size of 3 μm and atomization volume of 57 mL / m 3 , sedimentation time 30min.

[0077] Example 3 Central combination design of process parameters for bacteriophage aerosol formulation

[0078] Further study on the effect of ultrasonic atomization process parameters on antibacterial ability includes the following steps:

[0079] S1. The model selected in Example 2 is further optimized by using a central composite design containing three variables, and a three-variable quadratic equation is established to associate the variables with the responses. The factor levels of the central composite design are shown in Table 2 below.

[0080] S2. Perform central response test design according to Design-Expert statistical software. The specific test process is consistent with Example 2.

[0081] S3. Analyze the experimental results, draw contour lines and response surface diagrams, and calculate the correlation equation:

[0082] Y=99.62+0.0619A+0.7316B+0.2494C–0.0441AB+0.0099AC+0.0596BC–0.0407A 2 -0.8189B 2 -0.2971C 2

[0083] Where, Y: predicted antibacterial rate (%); A: atomized particle size (μm); B: atomized volume (mL / m 3 ); C: sedimentation time (min).

[0084] Table 2: Distribution of factors at different levels in the central composite design

[0085]

[0086] Figure 3 It is the response surface of the atomization conditions. The steeper the slope of the result surface and the denser the contour lines are, the more significant the impact is. The image shows that the inhibition rate of atomization volume and sedimentation time gradually slows down with the increase of variables, and the effect of atomization particle size on the inhibition rate is small. The results confirm that the above atomization conditions are atomization particle size of 3μm and atomization volume of 57mL / m 3 , sedimentation time 30min, which is the objective optimal atomization conditions.

[0087] Example 4 Verification of antibacterial ability of phage aerosol on different material surfaces

[0088] The antibacterial ability test of aerosolized bacteriophage preparations on the surfaces of different materials is as follows:

[0089] S1. Put a mixed phage preparation (Pseudomonas aeruginosa phage phipa 2: Pseudomonas aeruginosa phage phipa 4: Pseudomonas aeruginosa phage phipa 10 = 1:2:1) into an ultrasonic atomization device.

[0090] S2, take 10 μL of sterile milk and spread it on 25 cm 2 Galvanized iron sheets, ceramic sheets, PVC plastic sheets and glass plates. Air dry naturally.

[0091] S3, the concentration of phage mixture that can be lysed is 10 5 10 μL of the host bacteria mixed solution (PA35:PA16=1:1) with CFU / mL was spread on 25 cm 2 Galvanized iron sheets, ceramic sheets, PVC plastic sheets and glass plates.

[0092] S4. Place different materials in a 21L sealed transparent plastic box and connect it to the atomization port.

[0093] S5, set the atomization particle size to 3μm and the atomization volume to 57mL / m 3 , sedimentation time 30min.

[0094] S6. After the atomization is completed, the plate is taken out and incubated in a 37°C constant temperature incubator for 12 hours together with the control group.

[0095] S7, take out and count, and determine the antibacterial rate ( Figure 4 ).

[0096] like Figure 4 As shown, the antibacterial ability of phage aerosol on different materials is not much different, and the antibacterial effect can reach more than 99%.

[0097] Example 5 Verification of the diffusion ability of bacteriophage aerosol

[0098] The diffusion capacity test of the aerosolized bacteriophage preparation includes the following steps:

[0099] S1. Put a mixed phage preparation (Pseudomonas aeruginosa phage phipa 2: Pseudomonas aeruginosa phage phipa 4: Pseudomonas aeruginosa phage phipa 10 = 1:2:1) into an ultrasonic atomization device.

[0100] S2, the concentration of phage mixture that can be lysed is 10 5 10 μL of the host bacteria mixed bacterial solution (PA35:PA16=1:1) with a CFU / mL was spread on an LB solid culture medium plate, and a control group was set up at the same time.

[0101] S3. Place the LB plates at the centers of the four corners of a 21L sealed transparent plastic box and connect them to the atomization port.

[0102] S4, set the atomization particle size to 3μm and the atomization volume to 57mL / m 3 , sedimentation time 30min.

[0103] S5. After the atomization is completed, the plate is taken out and incubated in a 37°C constant temperature incubator for 12 hours together with the control group.

[0104] S6, take out and count, and determine the antibacterial rate ( Figure 5 ).

[0105] like Figure 5 As shown, it shows that the phage aerosol has a strong diffusion ability and there is no difference in the antibacterial ability at various locations in the confined space.

[0106] The present invention involves a phage atomization process that can optimize the environmental disinfection ability of phage preparations. Compared with other atomization conditions, the antibacterial rate is higher and the diffusion ability is stronger. The preparation process of the phage atomization formulation is simple, providing a new idea for the development of phage environmental disinfectants.

[0107] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for developing a bacteriophage atomization process, characterized in that: The steps include: After the phage was expanded and purified, the phage titer was diluted to 10 8 PFU / mL, and the phage preparation is obtained; the phage preparation is then placed in a closed space and atomized into droplets with a particle size of 2.8-4.5 μm, and then naturally settled for 20-40 minutes; the atomization volume is 43-71 mL / m 3 .

2. The method for developing the bacteriophage atomization process according to claim 1, characterized in that: The liquid culture method was used for the expansion, which included mixing and inoculating the phage, the host bacteria and the LB liquid culture medium in a ratio of 1:1:100, and then incubating at 37°C and 160 rpm in a constant temperature shaker for 4 hours.

3. The method for developing the bacteriophage atomization process according to claim 1, characterized in that: The atomized particle size is 3μm and the atomized volume is 57mL / m 3 , the sedimentation time is 30 minutes.

4. The method for developing the bacteriophage atomization process according to claim 1, characterized in that: The phage is selected from one or more of Pseudomonas aeruginosa phage, Klebsiella pneumoniae phage, Acinetobacter baumannii phage, Staphylococcus aureus phage, Salmonella phage and Escherichia coli phage.

5. The method for developing the bacteriophage atomization process according to claim 1, characterized in that: The atomization method is ultrasonic atomization.

6. The method for developing the bacteriophage atomization process according to claim 1, characterized in that: The diluent used for dilution is PBS buffer.

7. An application of the development method of the bacteriophage atomization process according to any one of claims 1 to 6, characterized in that: Used to reduce bacterial load in the environment.

8. The use according to claim 7, characterized in that: The bacteria are selected from one or more of Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, Salmonella and Escherichia coli.

9. The use according to claim 7 or 8, characterized in that: Used to prepare environmental disinfectants to prevent and control cross infection within hospitals.

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