Phage for efficiently splitting pseudomonas aeruginosa and application thereof

Abstract

The invention relates to separation and application of a bacteriophage BD815 with a high splitting rate on pseudomonas aeruginosa, and particularly relates to a bacteriophage BD815 with a high splitting rate on pseudomonas aeruginosa. The bacteriophage belongs to the long-tail bacteriophage family, and the host of the bacteriophage is pseudomonas The bacteriophage BD815 is composed of a polyhedral stereosymmetric head with the length of 66.9 + / -1.5 nm and the width of 45.8 + / -1.5 nm and a tail with the length of about 159.8 + / -1.9 nm. The bacteriophage can efficiently split host bacteria and inhibit the growth of the host bacteria, the titer can reach 3.46 * 10 < 14 > PFU / mL, the optimal infection complex number is 0.001, and the bacteriophage can be applied to the fields of production, research and development of medicines, health-care products, foods and feeds and the like.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to the separation and application of a bacteriophage BD815 with a high lysis rate for Pseudomonas aeruginosa. Background Art

[0002] Pseudomonas aeruginosa, also known as Pseudomonas aeruginosa, is a Gram-negative bacterium and one of the main pathogens causing hospital-acquired infections (Jangra V, et al. Microbes Infect, 2022, 24(4): 104950.). Pseudomonas aeruginosa can infect multiple sites in immunocompromised individuals, including burns or trauma sites, urinary tract, cornea, gastrointestinal tract, etc., causing urinary tract infections, chronic obstructive pulmonary disease (COPD), cystic fibrosis, cancer, sepsis and ventilator-associated pneumonia (VAP, including covid-19) and other diseases (Jurado-Martin I, et al. Int J Mol Sci, 2021, 22(6); Cendra M, et al. Biotechnol Adv, 2021, 49: 107734; Rossi E, et al. Nat Rev Microbiol, 2021, 19(5): 331-342.). Pseudomonas aeruginosa is a multidrug-resistant (MDR) conditional pathogen that can develop resistance to antibiotics such as aminoglycosides, quinolones and β-lactams (Hancock RE, et al. Drug Resist Updat, 2000, 3(4): 247-255.), and is clinically highly resistant to treatment with antibiotics including penicillin, cephalosporin, atranetron, cefazolin-tazobactam, etc. (Paterson DL, et al. Clin Microbiol Rev, 2005, 18(4): 657-686.; Rawat D, et al. J Glob Infect Dis, 2010, 2(3): 263-274; Poirel L, et al. Antimicrob Agents Chemother, 2019, 63(1).). Research on inhibiting and destroying drug-resistant bacteria is of great significance.

[0003] Phage therapy is a treatment method for pathogenic bacterial infections by lysing bacteria with phages. Compared with antibiotics, phages do not develop secondary drug resistance and often co-evolve with bacterial hosts, thereby delaying the development of drug-resistant bacteria (Koskella B, et al. Annu Rev Virol, 2022, 9(1): 57-78.). Gram-negative bacterial resistance is mainly due to the low antibiotic permeability of the cell membrane (Poole K, et al. Curr Pharm Biotechnol, 2002, 3(2): 77-98.). The combination of phages and antibiotics can improve the penetration of biofilms and enhance the ability to inhibit bacteria (Gu LC, et al. MBio, 2020, 11(4); Tagliaferri TL, et al. Front Cell Infect Microbiol, 2019, 9: 22.). Studies have shown that phage therapy is one of the effective methods to destroy Pseudomonas aeruginosa biofilms (Chegini Z, et al. Ann Clin Microbiol Antimicrob, 2020, 19(1): 45.), and can be used to combat the high drug resistance of Pseudomonas aeruginosa. At the same time, the types and numbers of phages found so far are still relatively small, which is an important factor limiting related research and widespread application. Therefore, it is necessary to further enrich the phage library to deal with the problem of high drug resistance. Summary of the invention

[0004] We screened Pseudomonas aeruginosa phages to inhibit drug-resistant bacteria and reduce the emergence of new drug-resistant bacteria, and promote the development of phage therapy. The present invention screened out a phage against Pseudomonas aeruginosa, and conducted detection and identification of morphological, genomic and biological characteristics.

[0005] The object of the present invention is to provide a bacteriophage that can efficiently lyse Pseudomonas aeruginosa. The bacteriophage is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit date of January 22, 2024, a deposit number of CGMCC No. 45873, and a classification name of Pseudomonas aeruginosa phage BD815.

[0006] The present invention provides the use of the bacteriophage in lysing Pseudomonas aeruginosa, which can be used in the fields of medicine, health products, food, feed manufacturing and research and development, provides a solution for the prevention and control of pathogens, and enriches the bacteriophage library.

[0007] The present invention identifies and sequences the entire genome of bacteriophage BD815, deeply analyzes its functional genes and proteins, and provides an enzyme substance that can lyse pathogens, enriches the field of phage therapy and biological control, expands the host spectrum, and improves the breadth and effectiveness of phage applications.

[0008] The beneficial effect of the present invention is that the bacteriophage BD815 is isolated from Pseudomonas aeruginosa as a host, and the bacteriophage has a strong lysis effect on Pseudomonas aeruginosa, providing a basis for the research and development of new antibacterial products.

[0009] The features, properties and advantages of the present invention are further described below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The plaque morphology of the bacteriophage of the present invention;

[0011] Figure 2 The transmission electron microscopic morphology of the bacteriophage of the present invention, the scale bar is 50 nm;

[0012] Figure 3 is a circle map of the phage genome of the present invention;

[0013] Figure 4 is a phylogenetic tree of the phage genome of the present invention;

[0014] Figure 5 Schematic diagram of the optimal infection multiplicity experiment of bacteriophage of the present invention;

[0015] Figure 6 Schematic diagram of the one-step growth curve experiment of the bacteriophage of the present invention;

[0016] Figure 7 Schematic diagram of the bacteriophage pH stability experiment of the present invention;

[0017] Figure 8 Schematic diagram of the bacteriophage thermal stability experiment of the present invention;

[0018] Fig. 9 Schematic diagram of the host lysis experiment of the bacteriophage of the present invention;

[0019] Fig.10 This is a schematic diagram of the phage RTD experimental results of the present invention; DETAILED DESCRIPTION

[0020] The technical scheme of the present invention is described below in conjunction with embodiments. The selected embodiments are only a part of the present invention, not all, and do not limit the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0021] Example 1 Phage Isolation and Purification

[0022] 0.1 g of Pseudomonas aeruginosa CGMCC 1.10274 freeze-dried powder was dissolved in 1 mL of LB medium buffer, cultured using the plate streak method, and a single colony was picked and cultured in LB medium for three generations to obtain a bacterial suspension with good activity and stability.

[0023] About 5 mL of sewage sample was collected from the natural environment in Fangshan District, Beijing. 100 mL of Pseudomonas aeruginosa LB culture medium suspension in the logarithmic growth phase was added and incubated at 37°C and 150 r / min for 24 hours. The suspension was filtered through a 0.22 μm microporous membrane to obtain a sterile phage stock solution, which was stored at 4°C. The obtained phage suspension was identified by the spot-test method. A layer of solid culture medium was placed in the culture dish, and a semi-solid culture medium containing bacterial solution and phage dilution was placed on the upper layer. It was cultured at 37°C for 6-8 hours to observe whether plaques appeared. If clear and transparent plaques appear on the plate, it indicates that the Pseudomonas aeruginosa phage has been isolated from the above operation, otherwise it needs to be re-collected and isolated. See the results. Figure 1 .

[0024] Select the phage spots with regular shapes and add them to the suspension of Pseudomonas aeruginosa. Incubate at 37°C and 150r / min for 12h, and filter through a 0.22μm microporous membrane to obtain the phage suspension. Repeat the above operation 3 times to obtain a purified phage suspension. The phage BD815 has been deposited in the China General Microbiological Culture Collection Center (CGMCC) with the number CGMCC No. 45873.

[0025] Example 2 Phage morphology observation

[0026] BD815 was centrifuged at 30,000 r / min for 2 h, and the precipitate was resuspended in ultrapure water. 10 μL of phage suspension was placed on a carbon support film (Formvar / Carbon Film) and allowed to stand for 10 min. Excess liquid was absorbed with filter paper, and a drop of uranyl acetate (pH 4.5, 1% w / v) was dropped onto the grid surface. After leaving it in the air for 90 s, it was blotted dry with filter paper and left in the air for 3 h. Finally, the morphology of BD815 at an accelerating voltage of 120 kV was observed using a JEM-1400Plus transmission electron microscope, and imaging was performed using iTEM and KeenView soft imaging systems. The results are shown in Figure 2. Figure 2 The phage has a regular polyhedral head and a long non-contractile tail sheath. The head is 66.9±1.5nm long, the head is 45.8±1.5nm wide, and the tail is 159.8±1.9nm long. According to the virus classification report of the International Taxonomy of Viruses (ICTV), this phage can be preliminarily classified as a long-tailed phage family.

[0027] Example 3 Phage Identification

[0028] The genome of bacteriophage BD815 was extracted using the Solarbio DNA virus genome extraction kit and whole genome sequencing was performed. The data were processed using Megahit software (v1.1.2), and the utilization of read data was calculated using BWA (v0.7.17) software. The protein sequence corresponding to each ORF in the genome was compared with the protein sequence corresponding to the COG database using blastp (v2.9.0+) software. The results are shown in Figure 3 The comparison results with e < 1e5 were screened to obtain the functional information of phage genes. The whole genome sequences were compared in the NCBI database, and the phylogenetic tree was constructed using MEGA 10.2.6. The results are shown in Figure 4 .

[0029] Example 4 Optimal multiplicity of infection determination

[0030] Pseudomonas aeruginosa was cultured to the logarithmic growth phase and the bacterial solution concentration was adjusted to 3.4×10 8 CFU / mL and perform gradient dilution. Adjust the concentration of phage solution to 3.4×10 4 PFU / mL. The phage BD815 suspension was mixed with the above gradient diluted bacterial suspension at a multiplicity of infection (MOI) (BD815 concentration / bacterial concentration) of 0.0001, 0.001, 0.01, 0.1, 1, 10, and incubated at 37°C and 150 r / min for 12 h. The phage titers at different multiplicity of infection were determined by the double-layer plate method. The results are shown in Figure 5 The optimal infection multiplicity is 0.001, and the titer can reach 3.46×10 14 PFU / mL.

[0031] Example 5 One-step growth curve drawing

[0032] The optimal infection multiplicity was 0.001, and Pseudomonas aeruginosa in the logarithmic growth phase (final concentration 1.2×10 8 CFU / mL) was mixed with the phage BD815 suspension, and the suspension was incubated at 37°C and 150r / min for 10min. The suspension was centrifuged at 12000r / min for 5min, and the supernatant was discarded. The precipitate was resuspended in LB medium and 500μL was collected at 0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 and 110min, respectively. After filtering with a 0.22μm microporous membrane, the phage titer in the filtrate was determined by the double-layer plate method, and the one-step growth curve of the phage was drawn. The results are shown in Figure 6 The incubation period of bacteriophage is 0-5 minutes, and the outbreak period is 5-90 minutes.

[0033] Example 6 Phage stability experiment

[0034] Prepare 12 phage BD815 suspensions, adjust their pH values ​​to 1-12, incubate at 37°C for 2 hours, and determine the phage titer using the double-layer plate method. Prepare 6 phage BD815 suspensions, incubate in a water bath at 30, 40, 50, 60, 70, and 80°C for 1 hour. After incubation, determine the phage titer using the double-layer plate method. The results are shown in Figure 7 , Figure 8 The optimum temperature range of bacteriophage is between 30-60℃. After 60℃, the activity of bacteriophage decreases significantly with the increase of temperature and time. The optimum pH of bacteriophage is 7-10. The titer of bacteriophage decreases with the increase or decrease of pH value.

[0035] Example 7 In vitro bacteriophage bactericidal activity test

[0036] Pseudomonas aeruginosa was cultured to the logarithmic growth phase, and the concentration of the bacterial solution was 1.2×10 8 The phage BD815 suspension was mixed with the above bacterial culture medium at different MOI values, and the OD600 was tested at different time points to reflect the in vitro lysis ability of the phage. The results are as follows: Fig. 9 After inoculation with phages at different MOI values, the OD600 values ​​of the bacterial solutions with MOI of 10 and 1 decreased significantly at 120 min, and the OD600 values ​​of the bacterial solutions with MOI of 0.1, 0.01 and 0.001 showed a trend of first increasing and then decreasing. The OD600 values ​​of the bacterial solutions with MOI of 0.0001 and the bacterial solutions without phages showed a continuous upward trend, but the OD600 values ​​of the bacterial solutions with MOI of 0.0001 increased significantly slower than that of the control group. The experimental results show that phages can effectively inhibit the growth of host bacteria.

[0037] Example 8 Routine test dilution test RTD (Routine test dilution)

[0038] The host bacteria in the logarithmic growth phase (about 100 μL) were added to the plate and dried, and then different dilutions of phage solution (10 9 PFU / mL, 10 8 PFU / mL, 10 7 PFU / mL, 10 6 PFU / mL, 10 5 PFU / mL, 10 4 PFU / mL, 10 3 PFU / mL, 10 2 PFU / mL, 10PFU / mL) and dried, and cultured at 37°C for 16-24h. If the host bacteria in the dripping area cannot grow in a continuous piece, it is the CL concentration, that is, the concentration that can completely lyse the host bacteria in the environment. The results are as follows Fig.10BD815 has an RTD value of 10 7 PFU / mL(10 -8 dilution).

[0039] The above description enables those skilled in the art to use or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of a Pseudomonas aeruginosa phage and its preparation in the manufacture and research and development of medicines, health products, food and feed.

2. The bacteriophage according to claim 1 is named BD815 and has been deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with a deposit number of CGMCC No.45873.

3. The bacteriophage according to claim 1 and 2, characterized in that The bacteriophage has a strong lytic effect on Pseudomonas aeruginosa.

4. The bacteriophage according to claims 1-3, characterized in that The phage has a strong lytic effect on Pseudomonas aeruginosa CGMCC 1.10274.

5. Use of the bacteriophage and preparation thereof according to claims 1-4 in lysing Pseudomonas aeruginosa.

6. Use of the bacteriophage and preparation thereof according to claims 1-4 in the manufacture and research and development of medicines, health products, food and feed.

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