Carbapenem-resistant pseudomonas aeruginosa phage and application thereof
By developing carbapenem-resistant Pseudomonas aeruginosa phages, the problem of Pseudomonas aeruginosa to antibiotic resistance is solved, and an effective inhibition scheme for super-resistant strains is provided, thus achieving the use of Pseudomonas aeruginosa Effective control of cytobacteria.
Patent Information
- Application Number
- CN202411540729.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-31
- Publication Date
- 2025-01-03
AI Technical Summary
Pseudomonas aeruginosa shows inherent resistance to many antibiotics, resulting in a significant reduction in the therapeutic effect of traditional antibiotics. Most of the existing phages are targeted at ordinary or multidrug-resistant PA, and there is a lack of effective treatment plans for super-resistant Pseudomonas aeruginosa. .
Develop a carbapenem-resistant Pseudomonas aeruginosa phage numbered GDMCC No: 64594-B1, which has the inhibitory effect on carbapenem-resistant Pseudomonas aeruginosa and can be within different temperature and pH ranges Maintain activity and prepare phage preparations for the preparation of antibacterial agents.
This phage has good antibacterial properties against super resistant Pseudomonas aeruginosa CRPA190, can effectively inhibit its growth, and is easy to prepare, suitable as a potential disinfectant and therapeutic agent, alone or in combination with antibiotics, for prevention and Treatment of pathogenic Pseudomonas aeruginosa.
Smart Images
Figure CN119242596A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and specifically relates to a carbapenem-resistant Pseudomonas aeruginosa phage and an application thereof. Background Art
[0002] Pseudomonas aeruginosa (PA) is a Gram-negative opportunistic pathogen and one of the most common pathogens causing nosocomial infections. P. aeruginosa seriously affects immunocompromised patients, causing bacteremia and sepsis in patients, and is also frequently seen in various skin infections. Traditional antibiotics are the first choice for treating P. aeruginosa infections. However, P. aeruginosa exhibits intrinsic resistance to many antibiotic drugs, which greatly reduces the therapeutic effect of antibiotics. In recent years, due to the emergence of multidrug-resistant bacteria, phage therapy has attracted widespread attention because of its antibacterial mechanism of action that is completely different from antibiotic therapy.
[0003] Phages are viruses that specifically infect bacteria. They are an alternative treatment strategy to antibiotics and can be used as new therapeutic agents to treat bacterial infections, especially multidrug-resistant bacteria. Compared with the use of broad-spectrum antibiotics, phage therapy has obvious advantages. It is highly specific to the target bacterial pathogens and does not have adverse effects on the host and its microbiota. The combination of Pseudomonas aeruginosa, phages and antibiotics can effectively inhibit bacterial growth and biofilm formation. However, traditional antibiotics are usually used to treat Pseudomonas aeruginosa lung infections, and there are few reports on the use of phages and phage cocktail therapy to control Pseudomonas aeruginosa lung infections. In addition, in this process, a large number of different phages are required to study various aspects related to the application of phages. Therefore, the development of new Pseudomonas aeruginosa phages will be of great significance. Summary of the invention
[0004] In view of the above shortcomings, the present invention discloses a carbapenem-resistant Pseudomonas aeruginosa phage and an application thereof. The phage has an inhibitory effect on carbapenem-resistant Pseudomonas aeruginosa, and can solve the drug resistance problem of carbapenem-resistant Pseudomonas aeruginosa.
[0005] The present invention is achieved by adopting the following technical solutions: A carbapenem-resistant Pseudomonas aeruginosa phage, which is deposited in Guangdong Microbial Culture Collection Center on May 4, 2024, and the deposit number is GDMCC No: 64594-B1.
[0006] The optimal infection multiplicity of the carbapenem-resistant Pseudomonas aeruginosa phage is 0.01, the phage activity is maintained at a temperature of -20°C to 60°C, the phage stability is maintained at a pH of 4 to 11, and the growth of carbapenem-resistant Pseudomonas aeruginosa is significantly inhibited.
[0007] A phage preparation comprises the carbapenem-resistant Pseudomonas aeruginosa phage.
[0008] A bacteriostatic agent is prepared by using the above-mentioned carbapenem-resistant Pseudomonas aeruginosa phage or phage preparation.
[0009] The dosage form of the antibacterial agent is any one of a solution, a powder, a gel, a granule and a lyophilized agent.
[0010] The antibacterial agent is used for preparing products for inhibiting carbapenem-resistant Pseudomonas aeruginosa, and the products include medicines, feeds, bactericides, disinfectants and detergents.
[0011] Compared with the prior art, this technical solution has the following beneficial effects: The currently developed Pseudomonas aeruginosa phages are mostly targeted at common PA or multi-drug resistant PA, and the host bacteria of the phage provided by the present invention, in addition to common Pseudomonas aeruginosa, also include super-resistant Pseudomonas aeruginosa CRPA190 that is resistant to almost all commonly used antibiotics on the market (including ceftazidime / avibactam, polymyxin, etc.). After experimental verification, the carbapenem-resistant Pseudomonas aeruginosa phage of the present invention has good antibacterial properties against Pseudomonas aeruginosa, especially super-resistant Pseudomonas aeruginosa CRPA190, and is easy to prepare and store. It can be prepared into a phage preparation as a potential disinfectant and therapeutic agent, used alone or in combination with antibiotics, for the prevention and treatment of pathogenic Pseudomonas aeruginosa, especially multi-drug resistant Pseudomonas aeruginosa. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1 This is a plaque of the bacteriophage CRPAP1904 described in Example 1.
[0013] Figure 2 This is the genome circle map of bacteriophage CRPAP1904 described in Experimental Example 1.
[0014] Figure 3 This is the evolutionary tree diagram of the bacteriophage CRPAP1904 genome described in Experimental Example 1.
[0015] Figure 4 This is an electron microscope image of the bacteriophage CRPAP1904 particles described in Experimental Example 1.
[0016] Figure 5This is a test picture of the experimental group in Experimental Example 2 where bacteriophage CRPAP1904 was added.
[0017] Figure 6 This is a test picture of the control group in Experimental Example 2 where LB liquid was added.
[0018] Figure 7 This is a data graph of the optimal infection multiplicity detection results of bacteriophage CRPAP1904 described in Experimental Example 3.
[0019] Figure 8 This is a data graph showing the temperature sensitivity test results of bacteriophage CRPAP1904 described in Experimental Example 4.
[0020] Fig. 9 This is a data graph showing the results of the sensitivity test of bacteriophage CRPAP1904 to pH in Experimental Example 5.
[0021] Fig.10 This is a graph showing the in vitro bactericidal ability test results of bacteriophage CRPAP1904 in Experimental Example 6. DETAILED DESCRIPTION
[0022] The present invention is further described by the following examples, but is not intended to be limiting of the present invention. Specific experimental conditions and methods not specified in the following examples are conventional methods commonly known to those skilled in the art.
[0023] Example 1: The separation and purification of the carbapenem-resistant Pseudomonas aeruginosa phage of the present invention comprises the following steps: (1) 1000 mL of untreated domestic and medical wastewater was collected from the sewage treatment center of the People's Hospital of Guangxi Zhuang Autonomous Region (address: No. 6, Taoyuan Road, Qingxiu District, Nanning City, Guangxi Zhuang Autonomous Region) to obtain a sampling solution, and then solid CaCl2 was added to the sampling solution to fully dissolve and stand for 30 minutes, and then divided into 100 mL centrifuge tubes, each centrifuged at 8000 rpm for 40 minutes, and the supernatant was taken to obtain a pre-treated solution, and the pre-treated solution was stored at 4°C for later use; the amount of solid CaCl2 added was 0.6% of the weight of the sampling solution; (2) Take 100 mL of the pre-treated solution obtained in step (1) and transfer it into a sterile conical flask, add 25 mL of the CRPA190 strain cultured to the logarithmic growth phase and 100 mL of LB broth liquid culture medium in sequence, and then culture in a 37°C incubator for 24 h to obtain a culture suspension, then take 10 mL of the culture suspension, centrifuge at 8000 rpm for 20 min, take the supernatant and filter it with a 0.22 μm disposable filter. The filtrate obtained is the phage primary extract, and the phage primary extract is stored at 4°C for later use; (3) Use the plaque assay to detect phages. Pipette 100 μL of the CRPA190 strain in the logarithmic growth phase into a sterile conical flask, then add 5 mL of LB semi-solid medium cooled to 50±2°C, shake gently to mix, and then quickly pour it onto the surface of the LB agar plate. After the medium solidifies and cools to room temperature, add 10 μL of the phage primary extract on its surface, then let it stand at room temperature for 10 minutes. After the liquid on the surface of the plate is completely absorbed, place it at 37°C for 12 hours and observe whether there are plaques on the LB agar plate.
[0024] (4) Pick the plaques on the LB agar plate in step (3) and add them to 2 mL of the host bacterial solution in the logarithmic phase, then culture at 37°C for 12 h, and then centrifuge at 8000 rpm for 20 min. Collect the supernatant and filter it with a 0.22 μm disposable filter. The filtrate obtained is the phage stock solution; (5) The phage stock solution obtained in step (4) was diluted 10-fold to obtain a dilution solution. Then, 100 μL of the dilution solution was mixed with 100 μL of the host bacterial solution in the logarithmic growth phase. Then, 5 mL of LB semi-solid medium cooled to 50±2°C was added. The mixture was gently shaken and mixed. Then, the mixture was quickly and evenly poured onto the surface of the LB agar plate. The plate was allowed to stand at room temperature for 15 min until the upper layer of semi-solid medium was completely solidified. The plate was then cultured at 37°C overnight. The next day, a transparent and larger single phage plaque was picked. The above operation was repeated until the size of the plaque became uniform. Figure 1 As shown, phage plaques were observed on LB agar plates, and the purified phage (labeled as CRPAP1904) was stored in SM buffer at 4°C.
[0025] Experimental Example 1: The phage purified in Example 1 was amplified and DNA was extracted, and the extracted DNA was subjected to whole genome sequencing. The whole genome sequencing results are as follows: Figure 2 It was shown that the phage CRPAP1904 contained a double-stranded DNA of 62,430 bp in length, a GC content of 60.4%, 83 ORFs, and no tRNA. No virulence genes or drug resistance genes were found in the phage in the VFDB and CARD databases, indicating that this phage is safe and usable.
[0026] By comparing CRPA1904 with all known phages in NCBI by Blast, CRPA1904 is most closely related to phage YMC17 / 07 / R4900a, with a homology of 98.11% and a coverage of 95%. The results of phylogenetic tree analysis can be found in Figure 3 , it can be seen that this phage is a new type of phage.
[0027] At the same time, the phage particles purified and concentrated from the high-concentration phage CRPAP1904 lysate were treated by negative staining. 20 μL of phage particles were dripped on the copper grid, and then a drop of 2% phosphotungstic acid was added for staining for 1 minute. After drying at room temperature, the phage particles were placed in a transmission electron microscope at 80 kV for observation and photography. Figure 4 .
[0028] Experimental Example 2: Inhibitory effect of bacteriophage CRPAP1904 on fully drug-resistant Pseudomonas aeruginosa in semi-solid culture medium The phage purified in Example 1 was subjected to an antibacterial test, which specifically used a plaque test method to detect phages. 100 μL of fully resistant Pseudomonas aeruginosa CRPA190 in the logarithmic growth phase was respectively aspirated, and then 5 mL of LB semi-solid medium cooled to 50°C was added, gently shaken, mixed, and then quickly poured onto the surface of the LB agar plate. After the LB semi-solid medium solidified and cooled to room temperature, 10 μL of the purified and concentrated phage CRPAP1904 in Example 1 was dripped onto the surface, and 10 μL of LB liquid was dripped onto the control group. The results are shown in FIG. Figure 5 and Figure 6 .
[0029] The experimental results showed that obvious plaques were observed in the experimental group with the addition of bacteriophage CRPAP1904, and the fully resistant Pseudomonas aeruginosa CRPA190 could not grow. However, no plaques were observed in the control group, and the fully resistant Pseudomonas aeruginosa CRPA190 could grow normally, indicating that bacteriophage CRPAP1904 has a significant inhibitory effect on the growth of fully resistant Pseudomonas aeruginosa.
[0030] Experimental Example 3: Optimal multiplicity of infection of bacteriophage CRPAP1904 The phage solution of known titer was diluted, and 100µL of phage solution and 100µL of host were added to 1mL LB liquid culture medium according to different infection multiplicity (MOI) of phage to host bacteria concentration ratio of 100, 10, 1, 0.1, 0.01, and 0.001, respectively. The culture was incubated at 37°C in a constant temperature shaker (160rpm) for 12h, and the supernatant was filtered with a 0.22μm disposable filter. The titer of the filtrate was determined by the double-layer agar method. The infection multiplicity with the highest titer was the optimal infection multiplicity of the phage. The results are shown in Figure 7 As shown, the optimal infection multiplicity of phage CRPAP1904 is 0.01.
[0031] Experimental Example 4: Temperature sensitivity detection of bacteriophage CRPAP1904 The obtained CRPAP1904 phage suspension was revived by double-layer agar plate method and its titer was adjusted to 1×10 8PFU / mL, the initial titer of 1mL is 1×10 8 The phages with PFU / mL were incubated in a water bath at 4℃, 25℃, 37℃, 40℃, 50℃, 60℃, 70℃, and 80℃ for 1 h, and then the samples were taken out and the titer was measured by the double-layer agar method. The results are as follows: Figure 8 As shown, after the bacteriophage CRPAP1904 was exposed to -20℃~60℃ for 1 hour, the titer remained stable, after being exposed to 70℃ for 1 hour, the phage activity was significantly reduced, and the activity was completely inactivated after being exposed to 80℃ for 1 hour. Therefore, the bacteriophage described in the present invention can still maintain considerable activity at a high temperature of 60℃, indicating that the phage has a wide range of applications.
[0032] Experimental Example 5: pH sensitivity test of bacteriophage CRPAP1904 The obtained CRPAP1904 phage suspension was revived by double-layer agar plate method and its titer was adjusted to 1×10 8 PFU / mL, the initial titer of 1mL is 1×10 8 The phages with PFU / mL were incubated in pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 solutions for 1 h, and then the samples were taken out and the titers were measured by the double-layer agar method. The results are shown in Fig. 9 As shown, the titer of phage CRPAP1904 remained stable after being exposed to pH 5, 6, 7, 8, and 9 for 1 hour, and it still had a certain activity after being exposed to pH 4, 10, and 11 for 1 hour, indicating that the phage has a wide pH application range.
[0033] Experimental Example 6: In vitro bactericidal effect test of bacteriophage CRPAP1904: Carbapenem-resistant Pseudomonas aeruginosa CRPA190 was cultured in LB medium until the logarithmic growth phase, centrifuged at 9000g for 2 min, and the bacterial concentration was adjusted to 10 with PBS (pH 7.4). 6 CFU / mL, the bacterial suspension (2.5 mL) was mixed with bacteriophage (10 9 The positive control group was not added with phage, but with an equal amount of LB medium, and cultured at 37 °C for 12 h. The experimental results were checked and the OD620 values of the liquid in the experimental group and the positive control group were measured respectively.
[0034] The experimental results are as follows Fig.10As shown, in the case of no phage treatment in test tube B, after 12 hours, the bacterial solution in the positive control group was significantly turbid, and the measured OD620 was 0.282. However, after the test tube A was treated with phage CRPAP190 at 1000 MOI, it can be clearly found that the bacterial solution was clear and transparent, and the measured OD value was 0.063, which was significantly lower than 0.282 in the positive control group, and the antibacterial effect was obvious, indicating that the phage CRPAP1904 in the present invention has a significant inhibitory effect on the growth of carbapenem-resistant Pseudomonas aeruginosa in LB medium.
[0035] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.
Claims
1. A carbapenem-resistant Pseudomonas aeruginosa phage, characterized in that: The carbapenem-resistant Pseudomonas aeruginosa phage is deposited in Guangdong Microbiological Culture Collection Center on May 4, 2024, and the deposit number is GDMCC No: 64594-B1.
2. The carbapenem-resistant Pseudomonas aeruginosa phage according to claim 1, characterized in that: The optimal infection multiplicity of the carbapenem-resistant Pseudomonas aeruginosa phage is 0.01, the phage activity is maintained at a temperature of -20°C to 60°C, and the phage stability is maintained at a pH of 4 to 11.
3. A bacteriophage preparation, characterized in that: Including the carbapenem-resistant Pseudomonas aeruginosa phage.
4. A bacteriostatic agent, characterized in that: The antibacterial agent is prepared using the carbapenem-resistant Pseudomonas aeruginosa phage according to any one of claims 1 to 2 or the phage preparation according to claim 3.
5. The antibacterial agent according to claim 4, characterized in that: The dosage form of the antibacterial agent is any one of a solution, a powder, a gel, a granule and a lyophilized agent.
6. The use of the antibacterial agent according to claim 4 or 5, characterized in that: The antibacterial agent is used for preparing products for inhibiting carbapenem-resistant Pseudomonas aeruginosa, and the products include medicines, feeds, bactericides, disinfectants and cleaning agents.