Bacteriophage for broad-spectrum lysis of klebsiella pneumoniae and application of bacteriophage
By developing the broad-spectrum lysing of Klebsiella pneumoniae phage Dhillonvirus KP02, the problem of treatment difficulty for high-virulence Klebsiella pneumoniae infection is solved, and effective inhibition and killing of multidrug-resistant strains is achieved, providing a safe and efficient new treatment option.
Patent Information
- Application Number
- CN202411693367.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-25
- Publication Date
- 2025-04-04
AI Technical Summary
High-virulent Klebsiella pneumoniae infection has increased the difficulty of treatment, and existing antibacterial preparations are difficult to effectively control drug-resistant strains, and a new treatment option is urgently needed.
A broad spectrum phage Dhillonvirus KP02 was developed, which can effectively kill Klebsiella pneumoniae with a wide spectrum of lyses.
This phage can quickly kill host bacteria, has high safety, a wide range of temperature and acid-base tolerance, and is easy to make sprays and injections. It has a good inhibitory and killing effect on multidrug-resistant Klebsiella pneumoniae, and is effectively used for treatment and protection.
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Figure CN119752812A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a bacteriophage capable of lysing Klebsiella pneumoniae with a broad spectrum and application thereof. Background Art
[0002] Klebsiella pneumoniae (KP) is a highly pathogenic Gram-negative bacterium belonging to the genus Klebsiella of the family Enterobacteriaceae. It is a common bacterium for bovine mastitis infection. In recent years, hypervirulent Klebsilla pneumoniae (hvKP) has been reported in various parts of the world. Compared with common Klebsilla pneumoniae ("classic" Klebsilla pneumoniae, cKP), hvKP can not only infect sick animals with low immunity, but also healthy animal groups: causing severe respiratory infections, pneumonia, liver abscesses, sepsis and mastitis. The bacteria are usually highly viscous, and the mucus threads produced by growth on agar plates can be stretched for more than 5 mm. Due to the production of mucus and the increase in drug-resistant hypervirulent Klebsilla pneumoniae, the difficulty of treatment has increased, posing a great threat to animal health, so a method is urgently needed to solve this problem.
[0003] Bacteriophages are the most abundant biological group in the biological world. They can infect bacteria and replicate. They have potential advantages such as strong bactericidal specificity, low development cost, easy isolation, self-limitation, few side effects, and the ability to kill drug-resistant strains. With the continuous emergence of drug-resistant strains and the inability of new antibacterial agents to meet the needs of drug-resistant bacteria control, bacteriophages have become one of the important options for the treatment of drug-resistant bacterial infections. In recent years, many successful cases have been reported, including bloodstream infections caused by pan-resistant Acinetobacter baumannii and complicated urinary tract infections caused by pan-resistant Klebsiella pneumoniae. At present, there are also animal studies on phage therapy, which show that the administration of bacterial preparations that meet good operating procedures will not produce observable harmful effects on the body. This suggests that bacteria have good safety as anti-infective biological agents, especially in the current situation where drug-resistant bacteria are extremely severe, and phage therapy has a relatively broad application potential. Summary of the invention
[0004] The technical problem to be solved by the present invention is to provide a new option for treating highly virulent Klebsiella pneumoniae infection.
[0005] The technical solution of the invention is a bacteriophage DhillonvirusKP02 capable of broadly lysing Klebsiella pneumoniae, and its preservation number is CCTCCNO: M20241154.
[0006] The present invention also provides the use of the bacteriophage DhillonvirusKP02 in preparing a preparation for preventing and treating Klebsiella pneumoniae.
[0007] Furthermore, the preparation is a spray or an injection.
[0008] Furthermore, the capsule type of the Klebsiella pneumoniae is K1, K2, K54 and / or K57.
[0009] The present invention also provides a method for culturing bacteriophage DhillonvirusKP02, comprising the following steps:
[0010] S1. Take the phage and host bacteria Klebsiella and react for 10 minutes, add LB liquid culture medium, culture at 37℃ for 6 hours, centrifuge, take the supernatant, filter, and the filtrate is the phage lysate that can be used directly;
[0011] S2. Add DNaseI and RNaseA to the phage lysate, incubate, add NaCl and place in an ice bath, centrifuge, take the supernatant and add PEG8000, incubate at 4°C overnight, centrifuge, discard the supernatant, add SM buffer to resuspend, add an equal volume of chloroform and gently shake for 30 seconds, centrifuge to separate the organic phase and the hydrophilic phase, recover the hydrophilic phase containing the phage particles, and obtain a purified bacterial suspension.
[0012] Among them, in step S1, the ratio of the number of live phages of bacteriophage DhillonvirusKP02 to the number of live bacteria of host bacteria Klebsiella pneumoniae is 10:1.
[0013] Furthermore, in step S1, the centrifugation conditions are 4°C and 12000 rpm for 20 min.
[0014] Particularly, in step S1, the filtration is performed using a 0.22 μm filter membrane.
[0015] Wherein, in step S2, DNase and RNaseA are added to the phage lysate to a final concentration of 14 g / mL each.
[0016] Specifically, in step S2, the incubation is 30 minutes.
[0017] Particularly, in step S2, NaCl was added and ice-bathed for 1 h.
[0018] Furthermore, in step S2, the first two centrifugation conditions are 4° C. and 12,000 rpm for 10 min.
[0019] Specifically, in step S2, the organic phase and the hydrophilic phase are separated by centrifugation at 4°C and 5000 rpm for 10 min.
[0020] Wherein, in step S2, the final concentration after adding PEG8000 is 10%.
[0021] The present invention also provides Klebsiella pneumoniae obtained by the culture method.
[0022] Beneficial effects of the invention: The invention discloses a Klebsiella pneumoniae phage KP02, which is a broad host spectrum bacterium, can lyse 10 different capsule types of Klebsiella pneumoniae, has a wide antibacterial spectrum, and can be used to prepare drugs for preventing and treating multidrug-resistant Klebsiella pneumoniae. Klebsiella pneumoniae phage KP02 can quickly kill host bacteria, has small toxic side effects, is highly safe, has a relatively wide temperature and acid-base tolerance range, has good hydrophilicity, and is easy to prepare into a spray and an injection; it has a good inhibitory and killing effect on the proliferation of Klebsiella pneumoniae, and has a good therapeutic and protective effect on the Caenorhabditis elegans model infected with multidrug-resistant Klebsiella pneumoniae.
[0023] The bacteriophage DhillonvirusKP02 for lysing K1 capsular Klebsiella pneumoniae of the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on June 6, 2024, with the deposit address: Wuhan University, Wuhan, China, Postal Code 430072; the deposit number is CCTCCNO: M20241154. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 , bacteriophage DhillonvirusKP02 plaque image.
[0025] Figure 2 , transmission electron micrograph of bacteriophage DhillonvirusKP02.
[0026] Figure 3 , pH stability diagram of bacteriophage Dhillonvirus KP02.
[0027] Figure 4 , temperature stability diagram of bacteriophage DhillonvirusKP02.
[0028] Figure 5 , diagram of optimal MOI determination of bacteriophage DhillonvirusKP02.
[0029] Figure 6 , one-step growth curve of bacteriophage DhillonvirusKP02.
[0030] Figure 7 , The therapeutic effect of different doses of bacteriophage DhillonvirusKP02 on GXNKP02-infected Caenorhabditis elegans. DETAILED DESCRIPTION
[0031] The technical solutions in the embodiments of the present application will be described in detail below in combination with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present application are within the scope of protection of the present application.
[0032] The host bacteria used in the experiment is a clinical strain of Klebsiella pneumoniae (GXNNKP02 in Table 1), which was isolated from mastitis milk in a cow farm in Nanning, Guangxi. It was deposited together with the Klebsiella pneumoniae phage DhillonvirusKP02 of the present invention in the China Center for Type Culture Collection (CCTCC), with the deposit address: Wuhan University, Wuhan, China, zip code 430072; the deposit number is CCTCCNO: M20241154.
[0033] LB (Luriabroth) liquid medium (1 L): peptone 10 g, yeast powder 5 g, NaCl 10 g, add ddH20 to 1 L, adjust pH to 7.0, and sterilize by high pressure at 121°C for 20 min.
[0034] 0.7% LB semisolid medium (1 L): peptone 10 g, yeast powder 5 g, NaCL 10 g, agar powder 70 g, add ddH20 to 1 L, adjust pH to 7.0, and sterilize by high pressure at 121°C for 20 min.
[0035] 1.5% LB solid medium (1L: peptone 10g, yeast powder 5g, NaCl 10g, agar powder 15g, add ddH2O to 1L, adjust pH to 7.0, sterilize at 121°C, 20min, cool to 50°C, pour the plate, cool and solidify, and then invert for use.
[0036] SM buffer (1 L): weigh 6.055 g, dilute to 100 mL with Tris-HCl (pH 7.5), add 5.800 g NaCL and 2.000 g MgS04, and add ddH2O to dilute to 1 L.
[0037] 1 mol / L sterile CaCl2 solution (1 L): Weigh 111 g of CaCl2 solid, pour into a beaker and add water to dissolve, pour the dissolved solution into a 1 L volumetric flask and make up to 1 L with ddH2O, sterilize at 121 ° C for 20 min, and cool for use.
[0038] NGM medium (1 L): 2.5 g peptone, 3.0 g sodium chloride, 0.111 g calcium chloride, 0.12 g magnesium sulfate, 0.005 g cholesterol, 3.4 g potassium dihydrogen phosphate, 17.0 g agar, autoclave at 121°C for 20 min.
[0039] Example 1 Isolation of bacteriophage Dhillonvirus KP02
[0040] The host bacteria were streaked onto 1.5% LB agar medium. After overnight culture, a single clone was picked and inoculated into 5 mL LB liquid medium. After shaking culture at 37° C. for 5 to 6 hours, the culture was used as the host bacteria culture for later use.
[0041] Wastewater was collected from a sewage treatment plant in Nanning, Guangxi, and centrifuged at 10,000 rpm for 20 min. The supernatant was co-cultured with the host bacterial solution overnight. The culture was then centrifuged at 10,000 rpm for 30 min, the supernatant was taken, and filtered with a 0.22 μm filter membrane to form a bacterial stock solution.
[0042] Pipette the bacterial stock solution, mix it with the host bacteria, pour it on the double-layer plate method, and culture it at 37°C overnight. On the second day, select the plate with plaques, select the larger and isolated plaques, dilute them in a gradient, mix them with the corresponding host bacteria solution, and pour them on the double-layer plate method. Repeat this step at least three times to obtain plaques of relatively uniform size.
[0043] Taking Klebsiella pneumoniae GXNKP02 as the host bacteria, the plaque morphology of DhillonvirusKP02 is as follows Figure 1 As shown, Dhillonvirus KP02 forms plaques on the plate with a clear center and a large halo around it.
[0044] The phage obtained after three purifications was named DhillonvirusKP02 and deposited in the China Center for Type Culture Collection (CCTCC) of Wuhan University, Wuhan, China.
[0045] Example 2 Amplification and purification of bacteriophage Dhillonvirus KP02
[0046] Take 0.01 mL of the phage purified three times in Example 1 and 0.1 mL of the host bacterial culture in Example 1 in a test tube for 10 min, add 10 ml of LB liquid culture medium, culture at 37°C for 6 h, centrifuge at 4°C and 12000 rpm for 20 min, take the supernatant, filter with a 0.22 μm filter membrane, and the filtrate is the phage lysate.
[0047] DNase and RNaseA were added to the phage lysate to a final concentration of 14 g / mL each, incubated for 30 min, NaCl was added to a final concentration of 1 mol / L and ice-bathed for 1 h, centrifuged at 4°C and 12000 rpm for 10 min, the supernatant was added with PEG8000 at a final concentration of 10%, incubated overnight at 4°C, then centrifuged at 4°C and 12000 rpm for 10 min, the supernatant was discarded, inverted for 5 min, SM buffer was added to resuspend, an equal volume of chloroform was added and gently shaken for 30 s, centrifuged at 4°C and 5000 rpm for 10 min to separate the organic phase and the hydrophilic phase, and the hydrophilic phase containing the phage particles was recovered to obtain a purified bacterial suspension.
[0048] Double-layer agar plates were used to detect bacterial titer: the purified phages were diluted 10 times in a gradient, 0.01 mL of the corresponding phage dilutions were taken and mixed with 0.1 mL of the host bacterial solution, and then spread on double-layer agar plates. The plates were cultured at 37°C for about 8 hours, and the plaques were counted on each plate. The plates with 100 to 200 plaques were selected, and the initial phage concentration was calculated according to the dilution multiple to obtain the phage titer. The bacterial titer was 3.1×10 9 PFU / mL.
[0049] Example 3 Transmission electron microscopy observation of bacteriophage Dhillonvirus KP02
[0050] Pipette a drop of phage stock solution (about 20 μL) onto the copper mesh, let it stand for 15 minutes, remove the excess liquid, let it stand for 15 minutes, remove the excess liquid, add a drop of 2% phosphotungstic acid solution, stain for 5 minutes, then immediately remove the excess staining liquid on the side, and observe the morphology under a transmission electron microscope after drying. The observation results are as follows: Figure 2 As shown, electron microscopy observation showed that the phage head had a regular icosahedral structure and the head diameter was about 55 nm.
[0051] Example 4 Acid-base and temperature tolerance experiment of bacteriophage Dhillonvirus KP02
[0052] pH tolerance: 12 portions of 0.1 mL of the isolated bacteria in Example 1 were placed in SM buffer (0.9 mL) at pH 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, respectively, at 37°C for 1 hour, and then the titer of the phages after the reaction was determined by the double-layer method (same as in Example 2). The test results are shown in Figure 2. Figure 3 As shown: The titer of Klebsiella pneumoniae phage KP02 changes little in an environment with a pH value of 3 to 11, and the activity remains basically unchanged. When the environmental pH is > 11 or pH < 3, the activity of the phage decreases with the increase of acidity and alkalinity. When pH = 12 or pH = 2, the phage titer is 0 and all are inactivated. Therefore, it can be known that the optimal pH of the phage is 3 to 11.
[0053] Temperature tolerance: 5 portions containing 1 mL of the phage stock solution in Example 1 were exposed to 4°C, 37°C, 40°C, 50°C, 60°C, 70°C and 80°C for 60 min and 90 min, respectively, and then the phage titer was determined. The test results are as follows: Figure 4 As shown: The phage loses its activity in an environment of 80°C.
[0054] Example 5 Optimal MOI and one-step growth curve determination of bacteriophage Dhillonvirus KP02
[0055] Take the host bacterial culture prepared in Example 1 and adjust the concentration to 1×10 9 CFU / mL, the bacteria prepared in Example 1 and the host bacteria culture prepared in Example 1 were added at a ratio of 1000, 100, 10, 1, 0.1, 0.01, and 0.001, respectively, and LB liquid culture medium was added to make the total volume of the culture system the same, and static culture was carried out at 37°C for 5h, and centrifuged at 10000rpm for 10min, and the supernatant was collected and diluted to an appropriate concentration, and the titer was determined by the double-layer method. The results are as follows Figure 5 As shown, the optimal infection multiplicity of phage KP02 is 10. When the ratio of phage to host bacteria is 10:1, the titer of the obtained progeny phage is the highest.
[0056] Mix the host bacteria with an excess of the bacteriophage in Example 1 (MOI>10), incubate at 37°C for 10 minutes, then centrifuge at 12000rpm for 1 minute, discard the supernatant, and wash the precipitate once with liquid culture medium. Resuspend the precipitate with 10mL of preheated LB liquid culture medium, and quickly place it in a 37°C shaker for shaking culture. Starting from 0min, take 100μL of culture every 10min, centrifuge at 4°C and 10000rpm for 2min to remove bacteria, take the supernatant and dilute it to an appropriate concentration (the appropriate concentration is the concentration that forms 30 to 300 plaques on the plate), and determine the phage titer by the double-layer method, monitoring for a total of 120min. If the sampling time is used as the horizontal axis and the logarithm of the phage titer is used as the vertical axis, a one-step growth curve is drawn to obtain the outbreak period and outbreak volume of the phage. The results of the one-step growth curve are as follows: Figure 6 As shown, the outbreak period of the host bacteria infection was 40 minutes and the outbreak volume was 405 PFU / Ce1l.
[0057] Example 6 Host spectrum analysis of bacteriophage Dhillonvirus KP02
[0058] This laboratory has collected and preserved 22 strains of Klebsiella pneumoniae from various places in Guangxi, and all are obtained by separating disease materials from various places. Carry out host spectrum analysis with the bacteriophage of embodiment 1, concrete operation is as follows: get 22 strains of Klebsiella pneumoniae overnight culture 0.1mL respectively, add 0.7%LB semi-solid medium 5mL of about 45 ℃, evenly spread on the solid 1.5%LB solid medium prepared in advance, above-mentioned bacteriophage is dripped on the surface, and another region drips and is physiological saline for contrast, treats that the droplet is inverted to 37 ℃ and cultivates 12h after drying, observation result, if there is bacterial plaque to produce, then be recorded as "+", then be "-". Result is as shown in table 1: Klebsiella pneumoniae phage KP02 can crack 10 strains of different capsule types (K1, K2, K54, K57) Klebsiella pneumoniae preserved.
[0059] Table 1 Bactericidal spectrum of bacteriophage KP02 against Klebsiella pneumoniae (the Klebsiella pneumoniae used in this study were all preserved by our laboratory)
[0060]
[0061]
[0062] Example 7 Experimental treatment of Caenorhabditis elegans infected with Klebsiella pneumoniae by bacteriophage Dhillonvirus KP02
[0063] The synchronized L4-stage Caenorhabditis elegans were placed in SK medium, with 20 worms per plate and 3 replicates per group. A layer of host bacteria GXNKP02 was evenly spread on the SK medium of the positive control group. After a layer of host bacteria GXNKP02 was spread on the experimental group, 20 μL of freshly prepared Klebsiella pneumoniae GXNKP02 (about 2×10 7 CFU / mL) were placed on nematode growth medium (NGM) agar plates at 37°C for 12 hours, and L4-stage Caenorhabditis elegans (N2 strain) were washed with M9 buffer and then transferred to the culture medium lawn, with 20 worms per plate. The treatment group was added with 10 7 PFU of bacteriophage KP02. The safety group was added only to the nematode growth medium. 7 PFU of bacteriophage KP02. The bacterial lawn of the negative group was Escherichia coli OP50, a bacterium that feeds Caenorhabditis elegans. The death of Caenorhabditis elegans was observed for 3 consecutive days.
[0064] The results are as follows Figure 7As shown, the survival rate of the treatment group (Group 2, i.e., GXNKP02+KP02) was significantly improved compared with the positive group (Group 1, i.e., GXNKP02), indicating that Klebsiella pneumoniae phage KP02 can better control the infection of highly virulent Klebsiella pneumoniae and reduce the mortality rate. The mortality rate of the third safety group and the fourth negative group was similar, indicating that phage KP02 had no obvious toxic side effects.
Claims
1. A bacteriophage Dhillonvirus KP02 that can lyse Klebsiella pneumoniae with a broad spectrum, and its deposit number is CCTCC NO: M20241154.
2. Use of the bacteriophage Dhillonvirus KP02 according to claim 1 in the preparation of a preparation for preventing and treating Klebsiella pneumoniae; Furthermore, the preparation is a spray or an injection; Furthermore, the capsule type of the Klebsiella pneumoniae is K1, K2, K54 and / or K57.
3. The method for culturing bacteriophage Dhillonvirus KP02 according to claim 1, characterized in that: The steps include: S1. Add bacteriophage Dhillonvirus KP02 and host bacteria Klebsiella pneumoniae into LB liquid culture medium at a ratio of 1:10, culture at 37°C for 6-12 hours, take the supernatant, filter, and the filtrate is the phage lysate that can be used directly; S2. Add DNase I and RNase A to the phage lysate, incubate, add NaCl and place in an ice bath, centrifuge, take the supernatant and add PEG8000, incubate at 4°C overnight, centrifuge, discard the supernatant, add SM buffer to resuspend, add an equal volume of chloroform and gently shake for 30 seconds, centrifuge to separate the organic phase and the hydrophilic phase, recover the hydrophilic phase containing the phage particles, and obtain a purified bacterial suspension.
4. The culture method according to claim 3, characterized in that: In step S1, the ratio of the number of live bacteriophages of bacteriophage Dhillonvirus KP02 to the number of live bacteria of host bacteria Klebsiella pneumoniae is 1:10; Further, in step S1, the centrifugation conditions are 4°C, 12000 rpm for 10 min; Particularly, in step S1, the filtration is performed using a 0.22 μm filter membrane.
5. The culture method according to claim 3, characterized in that: In step S2, DNase and RNase A were added to the phage lysate to a final concentration of 14 g / mL each; Specifically, in step S2, incubate for 30 min; Particularly, in step S2, NaCl was added and ice bathed for 1 h; Furthermore, in step S2, the first two centrifugation conditions are 4°C and 12000 rpm for 10 min; Particularly, in step S2, centrifugation is performed at 4°C and 5000 rpm for 10 min to separate the organic phase and the hydrophilic phase; Wherein, in step S2, the final concentration after adding PEG8000 is 10%.
6. A bacteriophage obtained by the culture method according to any one of claims 3 to 5.