Broad-spectrum pseudomonas aeruginosa bacteriophage QM01 resistant to biofilm and application thereof
By using Pseudomonas aeruginosa phage QM01, the drug resistance problem of Pseudomonas aeruginosa infection was solved, effective inhibition and destruction of biofilms were achieved, and new treatment plans were provided.
Patent Information
- Application Number
- CN202410171237.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-02-06
- Publication Date
- 2025-08-08
AI Technical Summary
The effectiveness of existing antibiotics in treating Pseudomonas aeruginosa infection has weakened, and bacterial biofilms have led to increased drug resistance, making it difficult to cure chronic infections.
Using Pseudomonas aeruginosa phage QM01, inhibitors and drugs are prepared to treat infection by lysing host bacteria and inhibiting biofilm formation.
Effectively inhibit and kill Pseudomonas aeruginosa, significantly destroying the formed biofilms, providing new options for treating Pseudomonas aeruginosa infection.
Smart Images
Figure CN120442559A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a broad-spectrum anti-biofilm Pseudomonas aeruginosa phage QM01 and an application thereof. Background Art
[0002] Pseudomonas aeruginosa, also known as Pseudomonas aeruginosa, is a Gram-negative bacterium that is widely present in the environment. It is an opportunistic pathogen that can cause surgical site infections, urinary tract infections, burn wound infections, keratitis, and otitis media. Pseudomonas aeruginosa can evade innate and adaptive immune defenses through adhesion, colonization, and biofilm formation, and produces various virulence factors, leading to sepsis. Antibiotics are used to treat P. aeruginosa infections, but the overuse of antibiotics has led to the gradual development of bacterial resistance and a weakening of the antibacterial efficacy of antibiotics, resulting in the failure of antibiotic treatment. Bacterial biofilms (biofilms) are the primary cause of antibiotic treatment failure. According to statistics, 80% of bacterial infections are caused by biofilms. Biofilms are 10-1000 times more resistant to antimicrobial drugs than free bacteria, making them a major contributor to bacterial resistance. Biofilm formation by Pseudomonas aeruginosa often leads to chronic, difficult-to-treat infections. Pseudomonas aeruginosa biofilms are primarily composed of bacteria and an extracellular matrix (EPS). The main components of EPS are exopolysaccharides (Psl polysaccharides, Pel polysaccharides, and alginate), proteins, extracellular DNA, RNA, and lipids. These EPS components significantly increase drug resistance and stress tolerance in Pseudomonas aeruginosa. Bacteria in biofilms can also increase their tolerance to antibiotics by upregulating certain genes. Therefore, there is an urgent need to develop drugs that prevent and treat bacterial biofilms.
[0003] Bacteriophages are viruses that infect bacteria. They were first discovered in Staphylococcus and Shigella in the early 20th century. Phages are highly specific for bacterial infection, infecting only specific species while sparing others. Phage therapy can avoid the damage to normal bacterial flora caused by broad-spectrum antibiotics. Phages multiply and lyse host bacteria through three stages: adsorption and incorporation, biosynthesis, and maturation and release. Phages differ from antibiotics in their bactericidal mechanisms, making their use in therapy significantly less prone to bacterial resistance and the side effects of antibiotics. In the 1990s, phages were used in some European countries to treat bacterial infections. Phage therapy has demonstrated the ability to reduce biofilm formation by pathogens. Phages can significantly disrupt biofilms, killing bacteria on the surface and within the biofilm, and inhibiting biofilm formation. The depolymerase synthesized and secreted by phages can degrade the biofilm of sensitive bacteria to achieve the effect of sterilization and destruction of biofilms. In addition, the combination of phages with antibiotics, nanoparticles, antimicrobial peptides, etc. can greatly improve the effect of destroying bacterial biofilms. Phage cocktails composed of multiple phages have also been proven to be an effective means of destroying bacterial biofilms and treating bacterial infections. Summary of the Invention
[0004] The technical problem to be solved by the present invention is to provide a new option for treating Pseudomonas aeruginosa infection.
[0005] The technical solution of the present invention is Pseudomonas aeruginosa phage QM01, with a deposit number of CCTCC NO: M20231388. It was deposited in the China Center for Type Culture Collection on August 1, 2023, and the depository address is Wuhan University, Wuhan, China, zip code 430072.
[0006] The present invention also provides use of the Pseudomonas aeruginosa phage QM01 in preparing a Pseudomonas aeruginosa inhibitor.
[0007] Furthermore, the present invention also provides a Pseudomonas aeruginosa inhibitor, the main component of which is Pseudomonas aeruginosa phage QM01, or the culture, secretion or extract of QM01.
[0008] The present invention also provides the use of the Pseudomonas aeruginosa phage QM01 in preparing a medicine for treating Pseudomonas aeruginosa infection.
[0009] The present invention also provides a drug for treating Pseudomonas aeruginosa infection, the drug comprising Pseudomonas aeruginosa phage QM01, or the culture, secretion or extract of QM01.
[0010] The present invention also provides a preparation for inhibiting the formation or regeneration of Pseudomonas aeruginosa biofilm, the ingredients of which include Pseudomonas aeruginosa phage QM01, or the culture, secretion or extract of QM01.
[0011] Specifically, the ingredients of the medicine also include pharmaceutically acceptable auxiliary ingredients.
[0012] Furthermore, in the above application, Pseudomonas aeruginosa is PA uk 001, ATCC27853, PA dw 001, PA uk 002, PA dw 002, PA uk 003, PA uk 004, PA dw 003, PA dw 004, PA uk 005, PA dw 005, PA dw 006, PA uk 006, PA01.
[0013] The present invention also provides a method for inhibiting Pseudomonas aeruginosa, which comprises the following steps: treating a target object with a culture, secretion and / or extract of QM01.
[0014] Beneficial effects of the present invention: The present invention isolates a virulent bacteriophage, Pseμdomonasaerμginosa Phage QM01 (deposit number CCTCC NO: M20231388), from the natural environment, which can effectively inhibit the growth of and kill Pseudomonas aeruginosa. The host lysis spectrum of the phage shows that it has a wide host spectrum and can lyse multiple Pseudomonas aeruginosa strains; it can effectively inhibit the growth of Pseudomonas aeruginosa biofilms and significantly destroy existing biofilms. Therefore, the present invention provides a new option for the treatment and prevention of Pseudomonas aeruginosa infections and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 , bacteriophage QM 01 kills Pseudomonas aeruginosa and produces plaques.
[0016] Figure 2 , determination of the optimal multiplicity of infection of bacteriophage QM 01.
[0017] Figure 3 , one-step growth curve of bacteriophage QM01.
[0018] Figure 4 , ATCC 27853 growth curve.
[0019] Figure 5 , pH tolerance of Pseudomonas aeruginosa phage QM01.
[0020] Figure 6 , temperature sensitivity of Pseudomonas aeruginosa phage QM01.
[0021] Figure 7 , electrophoresis results of phage QM01 genome.
[0022] Figure 8 , circle map of the bacteriophage QM01 genome.
[0023] Figure 9 , ATCC27853 biofilm formation curve.
[0024] Figure 10 , Effects of different MOI phage QM01 on ATCC27853 biofilm.
[0025] Figure 11 , effects of QM01 on ATCC 27853 biofilm.
[0026] Figure 12 , QM01 host spectrum determination.
[0027] The Pseudomonas aeruginosa phage QM01 of the present invention was deposited in the China Center for Type Culture Collection on August 1, 2023, with the deposit number CCTCC NO: M20231388; the deposit address is Wuhan University, Wuhan, China, postal code 430072. DETAILED DESCRIPTION
[0028] The technical solution of the present invention is further illustrated by describing specific implementation methods below.
[0029] Example 1 Isolation of Pseudomonas aeruginosa (ATCC27853) phage
[0030] Samples for isolating Pseudomonas aeruginosa phages were collected from natural environments, and the host bacteria used by the present inventors to isolate phages have been identified as Pseudomonas aeruginosa ATCC27853.
[0031] Wastewater samples were collected from a slaughterhouse in Jinyang County, Liangshan Yi Autonomous Prefecture. 50 mL of the sample was centrifuged at 9000 g for 10 min at 4°C. The supernatant was filtered through a 0.45 μm filter membrane and the filtrate was collected and added to 2× LB broth (20 g / L NaCl, 20 g / L tryptone, and 10 g / L yeast extract) at a volume ratio of 1:1. 10 9Add ATCC27853 bacterial suspension containing 100 CFU (CFU) of ATCC27853 to the aforementioned culture medium and incubate at 37°C with shaking at 180 rpm for 24-48 hours to enrich for phage. Centrifuge the enriched solution at 9000g for 10 minutes at 4°C. Filter the supernatant through a 0.22 μm filter and store at 4°C. Use the supernatant filtrate in a spot test to verify the presence of phage capable of killing Pseudomonas aeruginosa.
[0032] The spot test is performed as follows: 300 μL of an overnight ATCC27853 bacterial culture is added to 5 mL of 0.8% semisolid agar (10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract, and 8 g / L agar powder). Mix thoroughly and pour onto a bottom agar plate (10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract, and 16 g / L agar powder) to create a double-layer plate. Air-dry the plate on a clean bench for 15 minutes. 10 μL of the supernatant filtrate is then spotted onto the double-layer plate. After air-drying for 15 minutes, the plate is incubated upside down at 37°C for 16-20 hours and inspected for clear or turbid lysis zones.
[0033] After confirming that the phage filtrate that can kill Pseudomonas aeruginosa is obtained by the above method, the phage is separated and purified using the plate method. The specific operation is as follows: the presence of phage plaques on the bacterial lawn plate is determined to determine whether the phage to be separated and purified exists. The supernatant is diluted 10 times to 10 -9 100 μL of each dilution and 300 μL of ATCC27853 bacterial suspension were added to 5 mL of 0.8% semi-solid agar, mixed thoroughly, and poured onto the surface of the solid medium. After solidification, the mixture was transferred to a 37°C incubator and inverted for overnight incubation. Plates with a single plaque were selected for phage purification. A single plaque was picked with a sterile toothpick or sterilized pipette tip and stored at 4°C in SM buffer (5.8 g NaCl, 2 g MgSO4·7H2O in 750 mL ddH2O. 50 mL Tris·HCl (1 M) was added, the pH was adjusted to 7.4, and the ddH2O was filled to 1 L). To purify phage, the phage stock solution was serially diluted and purified using the double-layer plate method 3-5 times until the resulting plaques were of similar size and morphology, indicating that the isolated phage was pure.
[0034] The host bacteria were cultured to the logarithmic phase (10 8Phages were then added to 50 mL of SM buffer (50:1) containing phages. The culture was then incubated for 6-8 hours until host cell lysis was observed. The culture medium was collected and centrifuged at 4000g for 10 minutes at 4°C in a refrigerated centrifuge. The supernatant was filtered through a 0.22 μm filter and purified using PEG precipitation. NaCl (final concentration 0.5 M) was added to the supernatant to dissolve and mix thoroughly, followed by the addition of 10% w / v PEG8000. The mixture was incubated at 4°C overnight to allow phage particles to precipitate. The mixture was then centrifuged at 11000g for 20 minutes at 4°C, and the supernatant was carefully removed to obtain the phage pellet. The pellet was dissolved in 5 mL of SM buffer, and chloroform was added at a 1:1 volume ratio to remove the PEG. The mixture was thoroughly mixed and centrifuged, and the supernatant was removed. This material was the phage solution. Figure 1 The invented bacteriophage QM 01 kills Pseudomonas aeruginosa and produces plaques with a proliferation titer of 10 11 PFU / mL.
[0035] Example 2 Study on Physiological Characteristics of Bacteriophage
[0036] 1) Determination of the optimal phage infection multiplicity of infection (MOI)
[0037] a. Cultivate the host Pseudomonas aeruginosa to the logarithmic growth phase (10 8 CFU / mL), and the phage solution was diluted in a gradient manner according to the multiplicity of infection of 0.001, 0.01, 0.1, 1, 10, 100, etc.
[0038] b. Pipette 100 μL of bacterial solution and 100 μL of phage dilution solution respectively, keep warm for 5 minutes, then add to LB broth preheated at 37℃, and culture at 37℃ and 180 rpm for 4 hours.
[0039] c. Centrifuge the culture medium at 4000g for 15 minutes at 4°C, collect the supernatant, filter through a 0.22μm filter, and determine the phage titer using the double plate method. The highest MOI in each group is the optimal multiplicity of infection.
[0040] Figure 2 The optimal multiplicity of infection image shows that the optimal multiplicity of infection of Pseudomonas aeruginosa phage QM01 is MOI=0.01, at which point the number of progeny phages produced is the largest.
[0041] 2) Phage one-step growth curve determination
[0042] The one-step growth curve of phage was determined according to the multiplicity of infection (MOI) = 1. The specific operation is as follows:
[0043] a. Transfer the overnight bacterial suspension into fresh LB broth and adjust to OD 600 =0.1, continue to culture at 37℃ 600=0.6(10 8 CFU / mL);
[0044] b. The phage solution was diluted in a gradient manner and added to the above-mentioned Pseudomonas aeruginosa culture medium at an MOI of 1.
[0045] c. Place the mixture in a 37°C incubator for 10 minutes.
[0046] d. Centrifuge at 13000g at 4℃ for 2 min.
[0047] e. Discard the supernatant and wash the sample twice with 10 mL of LB broth.
[0048] f. Resuspend the cells in LB broth preheated at 37°C and incubate in a shaker at 37°C. Samples were collected every 5 minutes and centrifuged at 13,000 g for 1 minute at 4°C.
[0049] d. Use the double-layer plate method to determine the phage titer and OD 600 absorbance.
[0050] Depend on Figure 3 The one-step growth curve of phage showed that the incubation period of Pseudomonas aeruginosa phage QM01 was 10 min, the rise time was 20 min, and the average burst size was 4.83×10 2 PFU / Cell. Figure 4 OD 600 The data showed that Pseudomonas aeruginosa phage QM01 can not only inhibit the growth of Pseudomonas aeruginosa but also effectively kill Pseudomonas aeruginosa.
[0051] 3) Phage pH tolerance
[0052] SM buffer with a pH range of 2 to 13 was prepared using NaOH and HCl.
[0053] 100 μL of phage solution (10 10 PFU / mL) were added to 900 μL of SM buffer for each pH gradient and incubated at 37°C for 1 h.
[0054] The mixed solution was taken and the phage titer was determined by double-layer plate method.
[0055] Depend on Figure 5 The pH tolerance results show that Pseudomonas aeruginosa phage QM01 is relatively stable at pH 3-11.
[0056] 4) Temperature sensitivity determination
[0057] a. Add 100 μL of phage solution (10 10PFU / mL) were added to 900 μL SM buffer, and the mixed solution was placed in a water bath at 40, 50, 60, 70, or 80°C for 3 h.
[0058] b. Take samples every 20 minutes for a total of 60 minutes.
[0059] c. Determine the phage titer by double-layer plate.
[0060] Depend on Figure 6 The temperature sensitivity results showed that Pseudomonas aeruginosa phage QM01 was stable at 50°C.
[0061] 5) Phage genome extraction and sequencing annotation
[0062] The extraction of phage genome is as follows:
[0063] a. By adding 20 μL RNase and 20 μL DNase (200 U) to 10 mL phage solution to remove residual DNA and RNA from cell lysis, the mixture was stored at 37°C for 30 min.
[0064] b. After the insulation is completed, place the enzyme in an 80℃ metal bath for 15 minutes to inactivate the enzyme and then cool to room temperature.
[0065] c. Extract the phage genome using the Aidlab Biotechnologies Co., Ltd. lambda phage genomic DNA rapid extraction kit. Verify the phage genome extraction is complete by gel electrophoresis ( Figure 7 ) and sent to Chengdu Life Baseline Co., Ltd. (Gene Help) for sequencing.
[0066] The complete genome DNA sequence of QM01 phage was obtained by sequencing and splicing. The full length of QM01 phage genome is 72280bp, the CG content is 55.11%, and the genome circle map is shown in Figure 8 . Figure 8In the figure, BLAST alignment shows the 91 ORFs encoded by the QM01 phage genome, totaling 68,052 base pairs. The outermost circle (I) represents 71 positive-sense ORFs, while the inner circle (III) represents the G+C content. The green and purple circles (III) represent the G+C bias and GC / G+C, respectively. Analysis of drug resistance and virulence factors revealed that QM01 does not contain genes with homology to either drug resistance or virulence genes, indicating that QM01 is non-toxic. A BLAST alignment of the QM01 phage genome sequence at NCBI revealed that QM01 is most similar to Pseudomonas phage LP14 (GeneBank: MH356729.1), sharing 99% genome coverage and 96% sequence identity with LP14. It belongs to the genus Litunavirus, family Schitoviridae.
[0067] Example 3 Phage host spectrum determination
[0068] The host spectrum of the phage of the present invention against Pseudomonas aeruginosa was determined by the spot test method according to the above-mentioned operation method. The Pseudomonas aeruginosa strains used in the determination included ATCC 27853, PA01 and PA14, as well as 16 laboratory-preserved Pseudomonas aeruginosa strains. 500 μL of overnight cultured host bacterial solution and 5 mL of 0.8% semi-solid agar (10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract, 8 g / L agar powder) melted and incubated at 40-50°C were mixed evenly and poured onto the bottom agar plate (10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract, 16 g / L agar powder) to prepare a host bacterial lawn. The mixture was placed on a clean bench and air-dried for 15 minutes. The supernatant filtrate was diluted to obtain 10 -1 ~10 -8 PFU / mL Take 2μL and spot it on the plate containing Pseudomonas aeruginosa and let it air dry for 15 minutes. Then place it in a 37℃ incubator and culture it overnight. Check whether there is a clear or turbid lysis zone. The results of the spotting test are shown in Figure 12 and Table 1.
[0069] Table 1 QM 01 host spectrum determination
[0070]
[0071]
[0072] Note: -: strain does not grow; +: strain grows.
[0073] The above results indicate that Pseudomonas aeruginosa phage QM01 can kill a variety of host bacteria and is a polyvalent broad-spectrum phage.
[0074] Example 4 Inhibition of Pseudomonas aeruginosa biofilm formation
[0075] The bacteriophage of the present invention can effectively kill Pseudomonas aeruginosa. To study whether it can inhibit the formation of Pseudomonas aeruginosa biofilm, the following method was used: Pseudomonas aeruginosa biofilm was cultured and its growth curve was measured. The amount of biofilm was determined using the crystal violet method.
[0076] a. Inoculate 50 mL of LB broth with an overnight culture of ATCC 27853 and incubate at 37°C and 180 rpm until the OD 600 =0.4~0.6.
[0077] b. Use 96-well cell culture plates to make Pseudomonas aeruginosa biofilm. Dilute the above bacterial solution with LB broth to 1×10 6 CFU / mL. The diluted bacterial suspension was added to a 96-well plate. 100 μL of bacterial suspension and 100 μL of QM01 phage diluted in LB broth (MOI = 10) were added to each well and incubated at 37°C. 100 μL of LB broth was used as a negative control. Biofilm content was measured after 0, 6, 12, 24, and 48 hours of incubation.
[0078] c. After the incubation period, the culture medium was discarded and the biofilm was washed 2-3 times with PBS buffer to remove suspended cells and waste liquid. The biofilm was then fixed with 200 μL of methanol for 15 minutes.
[0079] d. After fixation, wash away the methanol with PBS (1×) and add 200 μL of 0.1% crystal violet dye for staining for 15-20 minutes.
[0080] e. After staining, rinse with ddH2O 2 to 3 times and place at room temperature to dry.
[0081] f. Add 200 μL of 33% acetic acid solution, extract and dissolve the crystal violet dye for 15 minutes, and measure the absorbance at 570 nm.
[0082] Depend on Figure 9 It can be seen that the bacteriophage of the present invention can effectively inhibit the formation of Pseudomonas aeruginosa biofilm.
[0083] Example 5 Pseudomonas aeruginosa phage destroys Pseudomonas aeruginosa biofilm
[0084] The bacteriophage of the present invention can effectively kill Pseudomonas aeruginosa. To study its ability to destroy the biofilm of Pseudomonas aeruginosa, the following method was used: the crystal violet method was used to determine the amount of biofilm, and the slide culture method was used to observe the effect of the bacteriophage on the biofilm.
[0085] Cultivate Pseudomonas aeruginosa biofilms using the same method as above. The crystal violet method is performed as follows:
[0086] a. After the Pseudomonas aeruginosa biofilm is formed, the culture waste liquid is discarded and the cells are washed 2-3 times with PBS (1×) to remove suspended cells and culture waste liquid.
[0087] b. Add 200 μL of phage solution to the wells where biofilm has formed at MOI = 100, 10, and 1, incubate at 37°C, and take samples at 0, 6, 8, 12, and 24 hours to determine the amount of biofilm.
[0088] Slide culture method:
[0089] a. Sterilize the slide at 121℃ for 20min and place it in a sterile culture dish. Add 1mL of 10 8 CFU / mLATCC27853 bacterial solution was added with 15 mL LB broth and cultured at 37°C for 72 h, with the LB broth replaced every 24 h.
[0090] b. After the mature biofilm is formed, add 10 9 After 8 h of phage solution treatment, the slides were washed 2-3 times with PBS (1×) to remove suspended cells and culture waste liquid.
[0091] c. After fixing the slides with methanol for 20 minutes, stain with 0.1% crystal violet for 30 minutes and observe under a 40× microscope.
[0092] Depend on Figure 10 It can be seen that after adding bacteriophage, the biofilm formed by Pseudomonas aeruginosa can be destroyed. Figure 11 It can be seen that the addition of phages can significantly destroy the biofilm formed by Pseudomonas aeruginosa and inhibit the re-formation of the biofilm.
Claims
1. Pseudomonas aeruginosa phage QM01, deposit number is CCTCC NO: M20231388.
2. Use of the Pseudomonas aeruginosa phage QM01 according to claim 1 in the preparation of a Pseudomonas aeruginosa inhibitor.
3. A Pseudomonas aeruginosa inhibitor, characterized in that: The main component is Pseudomonas aeruginosa phage QM01, or the culture, secretion or extract of QM01.
4. Use of the Pseudomonas aeruginosa phage QM01 according to claim 1 in the preparation of a medicament for treating Pseudomonas aeruginosa infection.
5. A drug for treating Pseudomonas aeruginosa infection, characterized in that: The composition includes Pseudomonas aeruginosa phage QM01, or the culture, secretion or extract of QM01.
6. The medicine according to claim 5, characterized in that: The components of the medicine also include pharmaceutically acceptable auxiliary components.
7. A preparation for inhibiting the formation or regeneration of Pseudomonas aeruginosa biofilm, characterized in that: The composition includes Pseudomonas aeruginosa phage QM01, or the culture, secretion or extract of QM01.
8. The use according to claim 2 or 4, characterized in that: The Pseudomonas aeruginosa is PA uk 001, PA uk 002, PA uk 003, PA uk 004, PA uk 005, PA uk 006, PA dw 001, PA dw 002, PA dw 003, PA dw 004, PA dw 005, PA dw 006, ATCC27853 and / or PA01.
9. A method for inhibiting Pseudomonas aeruginosa, characterized in that: The target object is treated with culture, secretions and / or extracts of QM01.