Phage for splitting high-toxicity multi-drug-resistant klebsiella pneumoniae and application thereof

Abstract

The invention relates to the technical field of biological medicines, and provides a bacteriophage for splitting high-toxicity multi-drug-resistant klebsiella pneumoniae and application of the bacteriophage. The phage is a Klebsiella pneumoniae phage vBKpnPXY3, and the preservation number of the phage is CCTCC (China Center for Type Culture Collection) NO: M 2025022. The invention further discloses a preparation method of the phage. The bacteriophage can be used for cracking high-virulence multi-drug-resistant klebsiella pneumoniae 32416 carrying drug-resistant genes of blaTEM, blaSHV, phoQ and pmrB and virulence genes of rmpA, kfuBC, ybtA, iro-NB, fim-H, ureA, uge and wabG, has great potential in clinical application, and is a relatively ideal choice for replacing antibiotics to treat multi-drug-resistant bacteria. The bacteriophage vBKpnPXY3 disclosed by the invention not only widens the host spectrum and the splitting capacity, but also has wide application potential in clinical treatment of multi-drug-resistant klebsiella pneumoniae infection and environmental purification.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a phage capable of lysing highly virulent and multi-drug resistant Klebsiella pneumoniae and its applications. Background Art

[0002] Klebsiella pneumoniae (Kp), also known as pneumobacillus, is a Gram-negative facultative anaerobe with a relatively thick capsule. It can form large grayish-white mucoid colonies on ordinary agar culture dishes and is easy to draw into filaments. It often causes urinary tract, gastrointestinal tract, and respiratory tract infections in immunocompromised patients as well as biofilm-associated pneumonia. It is the main pathogenic bacterium of nosocomial infections and ranks second among clinically isolated strains, second only to Escherichia coli.

[0003] In recent years, due to the irrational use of antibacterial drugs, the number of antibiotic-resistant strains has gradually increased. At present, Klebsiella pneumoniae has developed resistance to a variety of antibiotics such as amoxicillin / clavulanate, tetracycline, polymyxin B, cefotiam hexetil, kanamycin, and cefquinome. The emergence of highly virulent and multi-drug resistant Klebsiella pneumoniae has brought many difficulties to clinical medication and seriously threatens the life and health of patients. Finding an alternative to antibiotic treatment has become a necessity.

[0004] Phages are a type of virus that parasitize within microbial cells such as bacteria, fungi, and spirochetes and cause their infection. The infection is highly specific and will not cause the destruction and imbalance of other flora. In recent years, due to the abuse of antibiotics, the continuous emergence of superbugs has made phage therapy a potential solution to antibiotic-resistant infections. For the highly virulent and multi-drug resistant Klebsiella pneumoniae Kpn32416, there is no specific phage yet. Therefore, it is of great significance to develop a phage that can efficiently lyse Klebsiella pneumoniae Kpn32416. Summary of the Invention

[0005] In view of this, the present invention provides a phage with a short latent period, strong lytic ability, not carrying virulence genes, and capable of efficiently lysing highly virulent and multi-drug resistant Klebsiella pneumoniae Kpn32416 and its applications in preventing and treating highly virulent and multi-drug resistant Klebsiella pneumoniae infections and environmental disinfection and purification.

[0006] The technical solution of the present invention is realized as follows: In the first aspect, the present invention provides a phage capable of lysing highly virulent and multi-drug resistant Klebsiella pneumoniae. The phage is Klebsiella pneumoniae phage vB_KpnP_XY3, and the deposit number is CCTCC NO: M2025022.

[0007] In the second aspect, the present invention provides the application of the phage in lysing Klebsiella pneumoniae.

[0008] Based on the above technical solutions, preferably, the Klebsiella pneumoniae 32416.

[0009] In a third aspect, the present invention provides the use of a phage in the preparation of a drug for treating Klebsiella pneumoniae infection.

[0010] A phage for lysing highly virulent multi-drug resistant Klebsiella pneumoniae and its application according to the present invention have the following beneficial effects compared with the prior art:

[0011] (1) In the present invention, a clinically isolated strain obtained from the clinical laboratory is used as an indicator bacterium, and a Klebsiella pneumoniae phage with strong lytic activity is isolated from the untreated sewage in the hospital. This phage has a strong inhibitory effect on the highly virulent multi-drug resistant Klebsiella pneumoniae Kpn32416, and has great reference significance for the treatment and prevention of clinical multi-drug resistant Klebsiella pneumoniae.

[0012] (2) The activity of the Klebsiella pneumoniae phage vB_KpnP_XY3 provided by the present invention can remain stable in an environment with a temperature of 4 - 60 °C and a pH of 4 - 11, and is insensitive to chloroform.

[0013] (3) The optimal multiplicity of infection of the Klebsiella pneumoniae phage vB_KpnP_XY3 provided by the present invention is 0.1.

[0014] (4) The latent period of the Klebsiella pneumoniae phage vB_KpnP_XY3 provided by the present invention for infecting the host bacterium is about 20 min, the lysis period is about 40 min, and it enters the plateau stable period after 60 min. The highest titer can reach 2.7×10 11 PFU / mL, and the average burst size is 340 PFU / cell. This shows that the phage can be amplified in large quantities in a short time and has high lytic activity.

[0015] (5) The Klebsiella pneumoniae phage provided by the present invention can specifically kill the highly virulent multi-drug resistant Klebsiella pneumoniae (Klebsiella pneumoniae) 32416 carrying 4 drug-resistant genes blaTEM, blaSHV, phoQ, pmrB and 8 virulence genes rmpA, kfuBC, ybtA, iro-NB, fim-H, ureA, uge, wabG.

[0016] (6) The Klebsiella pneumoniae phage provided by the present invention has broad application potential in the clinical treatment of Klebsiella pneumoniae infection and environmental pollution disinfection. Description of the Drawings

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0018] Figure 1 It is the plaque formation result diagram formed by the Klebsiella pneumoniae phage vB_KpnP_XY3 of the present invention.

[0019] Figure 2 It is the transmission electron microscope result diagram of the Klebsiella pneumoniae phage vB_KpnP_XY3 of the present invention.

[0020] Figure 3 It is the PCR result of identifying that the host bacterium Klebsiella pneumoniae (Klebsiella pneumoniae) 32416 of the present invention carries drug resistance and virulence genes. Among them, Figure A shows the drug resistance genes blaTEM, blaSHV, phoQ, and pmrB, and Figure B shows the virulence genes rmpA, kfuBC, ybtA, iro-NB, fim-H, ureA, uge, and wabG.

[0021] Figure 4 It is the optimal multiplicity of infection result diagram of the Klebsiella pneumoniae phage vB_KpnP_XY3 of the present invention.

[0022] Figure 5 It is the one-step growth curve result diagram of the Klebsiella pneumoniae phage vB_KpnP_XY3 of the present invention.

[0023] Figure 6 It is the thermal stability curve diagram of the Klebsiella pneumoniae phage vB_KpnP_XY3 of the present invention.

[0024] Figure 7 It is the pH stability curve diagram of the Klebsiella pneumoniae phage vB_KpnP_XY3 of the present invention.

[0025] Figure 8 It is the chloroform stability curve diagram of the Klebsiella pneumoniae phage vB_KpnP_XY3 of the present invention.

[0026] Figure 9 It is the in vitro antibacterial experiment result diagram of the Klebsiella pneumoniae phage vB_KpnP_XY3 of the present invention.

[0027] Figure 10 It is the biofilm inhibition rate result diagram of the Klebsiella pneumoniae phage vB_KpnP_XY3 of the present invention. Detailed implementation manners

[0028] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The main reagents and instruments used in the present invention: agar powder and tryptone were purchased from Saiguo Biotechnology Co., Ltd.; 0.22μm filter and bacterial culture dish were purchased from Biosharp; viral genome DNA / RNA extraction kit was purchased from Tiangen Biochemical Technology Co., Ltd.; 37°C constant temperature incubator was purchased from Shanghai Boxun Industrial Co., Ltd.; constant temperature shaking incubator was purchased from Shanghai Zhichu Instrument Co., Ltd.

[0030] 1. Source of strains

[0031] 66 strains of different types of test bacteria were provided by the Laboratory Department of Xiangyang First People's Hospital.

[0032] 2. Isolation and purification of bacteriophage

[0033] 50 mL of sewage obtained from the First People's Hospital of Xiangyang was mixed with 50 mL of LB liquid medium, inoculated with 200 μL of logarithmic phase host bacteria, and cultured overnight at 37°C and 200 rpm. The next day, centrifuged at 4°C and 10,000 rpm for 10 min and filtered with a 0.22 μm filter membrane. The filtered liquid was gradient diluted and cultured overnight on a double-layer agar plate. A single plaque was picked and immersed in LB medium containing host bacteria and cultured overnight in an oscillating incubator at 37°C and 200 rpm. After centrifugation at 4°C and 10,000 rpm for 10 min, the phage was filtered with a 0.22 μm filter membrane. After gradient dilution according to the above operation, the phage was purified 3-5 times using the double-layer agar plate method until the plaque size was uniform. The phage titer was determined by the double-layer plate method.

[0034] After culturing on the double-layer plate for 12 hours, the phage 32416 was able to form plaques with a transparent center and a translucent halo surrounding them, such as Figure 1 shown.

[0035] 3. Transmission electron microscopy observation of bacteriophage

[0036] Absorb the purified high titer (>10 9 PFU / mL) phage solution 20μL was dropped onto the copper mesh for natural adsorption for 5-10min. After drying, 20μL of 2% phosphotungstic acid solution was dropped onto the copper mesh and allowed to stand for 3-5min. Excess droplets were removed with filter paper strips. After drying under an incandescent lamp, the mesh was observed and photographed using a transmission electron microscope. The results are shown in Figure 2 .

[0037] Figure 2 As shown in Figure 2 , bacteriophage 32416 has an icosahedral head and a long non - contractile tail. According to the "Virus Taxonomy - The Eighth Report of the International Committee on Taxonomy of Viruses" published by the International Committee on Taxonomy of Viruses (ICTV) in 2015, bacteriophage 32416 belongs to the Siphoviridae family.

[0038] According to the bacteriophage nomenclature rules, it is named Klebsiella pneumoniae phage vB_KpnP_XY3, hereinafter referred to as phage vB_KpnP_XY3.

[0039] Klebsiella pneumoniae phage vB_KpnP_XY3 was deposited at the China Center for Type Culture Collection on January 6, 2025. The deposit address is: Wuhan University, Wuhan, China. The deposit number is: CCTCC NO: M 2025022.

[0040] IV. Detection of the host spectrum of phage vB_KpnP_XY3

[0041] A host spectrum lysis experiment was carried out on 66 test bacteria, including 26 strains of Klebsiella pneumoniae, 15 strains of Acinetobacter baumannii, and 25 strains of Staphylococcus aureus, using the spot - inoculation method. When the test bacteria were cultured to the logarithmic phase at about 10 8 CFU / mL, 100 μL of the bacterial suspension was evenly spread on an LB solid agar plate with an L - shaped spreader. Then, 20 μL of the purified phage vB_KpnP_XY3 solution was spotted on the plate and incubated overnight at 37 °C in an incubator for 12 h, and then the formation of plaques was observed.

[0042] Table 1 Results of the lysis spectrum test of phage vB_KpnP_XY3

[0043]

[0044]

[0045] Note: "+" indicates that it can be lysed, "++" indicates strong lysis ability, and "-" indicates that it cannot be lysed.

[0046] As can be seen from Table 1, phage vB_KpnP_XY3 specifically lyses the host bacterium Kpn 32416, that is, Klebsiella pneumoniae 32416.

[0047] V. The host bacterium Klebsiella pneumoniae 32416 (abbreviation for Klebsiella pneumoniae 32416) carries drug - resistant and virulence genes

[0048] The strain was resuscitated and cultured, and bacterial DNA was extracted using the Tiangen Bacterial Genomic DNA Extraction Kit. The primer sequences for drug resistance and virulence genes were referred to the literature and synthesized by Shanghai Sangon Biotech Co., Ltd.

[0049] The PCR system was 20 μL: 1 μL DNA template, 0.5 μL primer R, 0.5 μL primer F, 10 μL Premix Taq (TaKaRa Premix Taq Version 2.0), 8 μL ddH2O.

[0050] PCR conditions: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 30 s, annealing for 30 s, extension at 72 °C for 1 min, 30 cycles; maintenance at 72 °C for 10 min.

[0051] The PCR products were electrophoresed in a 1% concentration agarose gel (voltage 120 V, time 30 min), photographed and saved by an ultraviolet imager. The PCR identification results are shown in Figure 3 .

[0052] By PCR identification, the host bacterium Klebsiella pneumoniae Kpn32416 carried β-lactam resistance genes blaTEM, blaSHV and polymyxin resistance genes phoQ, pmrB (see Figure 3 A), which could lead to resistance to carbapenems and colistin antibiotics; the bacterium also carried capsule polysaccharide synthesis gene rmpA, iron uptake system genes kfuBC, ybtA, iroNB, fimbria synthesis genes fimH, ureA, lipopolysaccharide genes uge, wabG and other virulence genes (see Figure 3 B).

[0053] VI. Determination of the optimal multiplicity of infection (MOI) of phage vB_KpnP_XY3

[0054] The host bacterium Kpn 32416 was cultured to the logarithmic phase and the concentration was adjusted to 1×10 8 CFU / mL. Phage vB_KpnP_XY3 was added according to the ratios of MOI = 0.0001, 0.001, 0.01, 0.1, 1, 10, 100. After incubation at 37 °C for 15 min, the mixture was added to 1 mL of LB liquid medium, and cultured with shaking at 37 °C and 200 rpm for 6 h. Centrifuged at 4 °C and 1000 g for 10 min, the supernatant was filtered through a 0.22 μm filter, and the phage titer was determined by the double-layer agar plate method. The highest titer represented the optimal MOI. The experiment was repeated three times in parallel. The experimental results are shown in Figure 4 .

[0055] As Figure 4As shown, the optimal multiplicity of infection of phage vB_KpnP_XY3 is 0.1, and the titer can reach 3.1×10 10 PFU / mL.

[0056] VII. Determination of the one-step growth curve of phage vB_KpnP_XY3

[0057] Take the corresponding phage vB_KpnP_XY3 and host bacterium Kpn 32416, mix them according to the optimal MOI (0.1), incubate in a 37°C constant temperature incubator for 15 min, centrifuge at 4°C and 1000 rpm for 10 min, discard the supernatant, add 10 mL of LB liquid medium to resuspend the precipitate, and culture with shaking in a 37°C and 200 rpm incubator. Samples are taken at 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120 min of culture, and the phage titer is measured by the double-layer agar plate method. The experiment is repeated three times in parallel, and the results are shown in Figure 5 .

[0058] As Figure 5 shown, the latent period of phage vB_KpnP_XY3 lysing host bacterium Kpn 32416 is 20 min, the lysis period is 40 min, and there is a plateau period of about 60 min. This indicates that phage vB_KpnP_XY3 can be amplified in large quantities in a short time and has high lysis activity.

[0059] VIII. Detection of the temperature stability of phage vB_KpnP_XY3

[0060] Incubate the phage solution in a water bath at different temperatures of 4°C, 25°C, 37°C, 50°C, 60°C, and 70°C for 60 min. Immediately take it out and place it in an ice bath to cool, then dilute it stepwise and measure its titer by the double-layer plate method. The results are shown in Figure 6 .

[0061] As Figure 6 shown, phage vB_KpnP_XY3 is relatively stable in the environment with a temperature of 4 - 60°C, and the titer is 10 9 -10 10 PFU / mL. When the temperature reaches 70°C, phage vB_KpnP_XY3 is inactivated.

[0062] IX. Detection of the acid-base (pH) stability of phage vB_KpnP_XY3

[0063] Adjust the pH value of the LB liquid medium to different ranges from 2 to 12. Take 1 mL of the purified phage solution and add it to the LB medium with different pH ranges, incubate at 37°C for 60 min, then dilute it stepwise and measure the phage titer. The results are shown in Figure 7 .

[0064] AsFigure 7 As shown, phage vB_KpnP_XY3 is relatively stable in the environment with pH 4 - 11, and the titer is the highest at pH 3, approaching 3×10 9 PFU / mL; phage vB_KpnP_XY3 is inactivated at pH 3 and 12.

[0065] X. Detection of the chloroform stability of phage vB_KpnP_XY3

[0066] According to the volume ratios of chloroform to phage being 0.01, 0.05, 0.1, 0.5, and 1 respectively, 5 portions of phage were taken, with each portion being 500 μL. They were respectively mixed vigorously with 5 μL, 25 μL, 50 μL, 250 μL, and 500 μL of chloroform for 1 min, then incubated at room temperature for 30 min, centrifuged at 10000 rpm for 10 min, the supernatant was collected, and after gradient dilution, the titer was measured by the double-layer agar plate method. The results are shown in Figure 8 .

[0067] As Figure 8 shown, vB_KpnP_XY3 is not sensitive to chloroform. When the volume ratios of chloroform to phage are 0.01, 0.05, 0.1, 0.5, and 1 respectively, the titers are all higher than 10 10 PFU / mL.

[0068] XI. Detection of the in vitro antibacterial activity of phage vB_KpnP_XY3

[0069] After culturing the host bacterium Kpn 32416 to the exponential phase, the concentration was adjusted to 10 8 CFU / mL. It was infected with phage according to the MOIs of 10, 1, 0.1, 0.01, and 0.001 respectively. Using the LB liquid medium to infect the host bacterium Kpn 32416 as the positive control group, it was cultured at 37 °C and 200 rpm. The wavelength at 580 nm was detected at 0, 2, 4, 6, 8, 10, and 12 h respectively. The results are shown in Figure 9 .

[0070] As Figure 9 shown, phage vB_KpnP_XY3 has a significant inhibitory effect on the host bacterium Kpn 32416. As time prolongs, the activity of the host bacterium gradually weakens.

[0071] XII. Detection of the biofilm inhibition rate of phage vB_KpnP_XY3

[0072] The titer of the purified phage liquid was adjusted to 10 9 PFU / mL, and the concentration of the host bacterium Kpn 32416 was adjusted to 10 8CFU / mL. Add 180 μL of the bacterial liquid and 20 μL of the phage liquid mixture to the 96-well plate according to the ratios of MOI (phage concentration / bacterial concentration) of 1, 0.1, 0.01, and 0.001 respectively. Make 3 replicates for each titer. Use the mixture of 180 μL of the bacterial liquid and 20 μL of the SM buffer as the negative control, 200 μL of the LB liquid as the blank control, and add 200 μL of PBS to the outermost circle of the 96-well plate to prevent evaporation of the inner liquid. Incubate at 37 °C until 6, 12, 24, and 48 h, then discard the mixture. Gently rinse 3 times with PBS, add 200 μL of 99% formaldehyde to fix for 15 min, add 200 μL of 1% crystal violet staining solution to each well for 5 min, rinse with running water, and then add 160 μL of 33% glacial acetic acid to each well to dissolve. Detect the absorbance value at 570 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Biofilm inhibition rate = (OD570 of the negative control - OD570 at each MOI) ÷ OD570 of the negative control × 100%. The results are shown in Figure 10 .

[0073] As Figure 10 shown, the phage vB_KpnP_XY3 can effectively inhibit the growth of the host bacteria biofilm, and the inhibition rate at 48 h is close to 90%. This indicates that the above phage vB_KpnP_XY3 has antibacterial persistence and plays an important role in the future prevention and control of Klebsiella pneumoniae.

[0074] The above is only the preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A phage that lyses highly virulent and multi-drug resistant Klebsiella pneumoniae, characterized in that: The phage is Klebsiella pneumoniae phage vB_KpnP_XY3, and the deposit number is CCTCC NO: M2025022.

2. Use of the phage according to claim 1 in lysing Klebsiella pneumoniae.

3. The application according to claim 2, characterized in that: The Klebsiella pneumoniae 32416.

4. Use of the phage according to claim 1 in preparing a drug for preventing Klebsiella pneumoniae infection.

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