Klebsiella pneumoniae bacteriophage and application thereof

Abstract

The invention relates to a Klebsiella pneumoniae phage which is preserved in the China Center for Type Culture Collection (CCTCC) on January 13, 2025, and the preservation number of the Klebsiella pneumoniae phage is CCTCC NO: M 2025094. The invention also relates to application of the bacteriophage in preparation of drugs for resisting klebsiella pneumoniae infection and killing klebsiella pneumoniae in the environment. According to the invention, a bacteriophage capable of splitting the high-toxicity multi-drug-resistant klebsiella pneumoniae is separated, and the bacteriophage has a relatively strong inhibition effect on the high-toxicity multi-drug-resistant klebsiella pneumoniae Kpn31109, and has a wide application potential in the aspects of clinical treatment of klebsiella pneumoniae infection and environmental pollution disinfection; and the method has important reference significance on clinical treatment and prevention and control of multi-drug-resistant klebsiella pneumoniae.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a Klebsiella pneumoniae bacteriophage and application thereof. BACKGROUND

[0002] Klebsiella pneumoniae (Kp) is also called Klebsiella pneumoniae, which is a gram-negative facultative anaerobic bacteria with a thick capsule, can form a larger grayish white mucous colony on a general agar plate, and is easy to pull out a filament. It can often cause urinary tract, gastrointestinal tract, respiratory tract infections and biofilm-related pneumonia in patients with low immune function, and is the main pathogenic bacteria of nosocomial infection, ranking second in clinical isolated strains, only next to Escherichia coli.

[0003] In recent years, due to the irrational use of antibacterial drugs, antibiotic-resistant strains have gradually increased. At present, Klebsiella pneumoniae has developed resistance to amoxicillin / clavulanic acid, tetracycline, polymyxin B, cefotetan, kanamycin and cephalosporin. The emergence of high virulence and multidrug-resistant Klebsiella pneumoniae brings many difficulties to clinical drug use, and seriously threatens the life and health of patients. Finding an alternative method for antibiotic treatment has become a necessity.

[0004] Bacteriophage is a kind of virus that parasitizes in bacterial, fungal, spirochete and other microbial cells and causes infection, which has high specificity and will not cause destruction and disorder of other flora. In recent years, due to the abuse of antibiotics, superbugs have emerged, and bacteriophage therapy will become a potential solution to antibiotic-resistant infections.

[0005] For high virulence and multidrug-resistant Klebsiella pneumoniae Kpn31109, there is no specific bacteriophage, so it is of great significance to develop a bacteriophage that can efficiently lyse Klebsiella pneumoniae Kpn31109. SUMMARY

[0006] To solve the above problems, the application provides a Klebsiella pneumoniae bacteriophage, which was preserved in China Center for Type Culture Collection (CCTCC) on January 13, 2025, and the preservation number is CCTCC M2025094.

[0007] The application also provides application of the Klebsiella pneumoniae bacteriophage in lysis of Klebsiella pneumoniae.

[0008] The application also provides application of the Klebsiella pneumoniae bacteriophage in preparation of an anti-Klebsiella pneumoniae infection drug.

[0009] The application also provides application of the Klebsiella pneumoniae bacteriophage in killing Klebsiella pneumoniae in the environment.

[0010] The present invention also provides a method for killing Klebsiella pneumoniae, comprising the step of adding the Klebsiella phage according to claim 1 to an environment containing Klebsiella pneumoniae.

[0011] In a specific embodiment, the Klebsiella pneumoniae is one or more combinations of Klebsiella pneumoniae 31109, 31511 and 32406.

[0012] The present invention also provides a drug for resisting Klebsiella pneumoniae infection, which comprises the above-mentioned Klebsiella pneumoniae phage.

[0013] The present invention also provides an environmental disinfectant, which comprises the Klebsiella pneumoniae phage.

[0014] The present invention isolated a phage capable of lysing highly virulent and multidrug-resistant Klebsiella pneumoniae. The phage has a strong inhibitory effect on the highly virulent and multidrug-resistant Klebsiella pneumoniae Kpn31109, has broad application potential in the clinical treatment of Klebsiella pneumoniae infection and environmental pollution disinfection, and has important reference significance for the clinical treatment and prevention and control of multidrug-resistant Klebsiella pneumoniae.

[0015] Deposit of biological materials

[0016] The phage mentioned in the present invention was deposited in the China Type Culture Collection (CCTCC) of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province on January 13, 2025, with the deposit number: CCTCC NO: M 2025094, and the taxonomic name is Klebsiella pneumoniae phage vB_KpnP_XY4. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a photograph of a culture dish showing plaques formed by the Klebsiella pneumoniae phage vB_KpnP_XY4 of the present invention.

[0018] Figure 2 This is a transmission electron microscope photograph of the Klebsiella pneumoniae phage vB_KpnP_XY4 of the present invention.

[0019] Figure 3 The PCR results of drug resistance and virulence genes carried by the host bacteria Klebsiella pneumoniae 31109 were identified in the present invention, wherein Figure A shows the bl aTEM, bla SHV, bla KPC, phoQ, pmrB, and Cat1 resistance genes carried, and Figure B shows the kfuBC, ybtA, iro-NB, fim-H, iucA, ureA, uge, and wabG virulence genes carried.

[0020] Figure 4Figure for the optimal multiplicity of infection of Klebsiella pneumoniae bacteriophage vB_KpnP_XY4 of the present application.

[0021] Figure 5 One-step growth curve of Klebsiella pneumoniae bacteriophage vB_KpnP_XY4 of the present application.

[0022] Figure 6 Thermal stability curve of Klebsiella pneumoniae bacteriophage vB_KpnP_XY4 of the present application.

[0023] Figure 7 pH stability curve of Klebsiella pneumoniae bacteriophage vB_KpnP_XY4 of the present application.

[0024] Figure 8 Chloroform stability curve of Klebsiella pneumoniae bacteriophage vB_KpnP_XY4 of the present application.

[0025] Figure 9 In vitro antibacterial experiment result figure of Klebsiella pneumoniae bacteriophage vB_KpnP_XY4 of the present application.

[0026] Figure 10 Biofilm inhibition rate statistical graph of Klebsiella pneumoniae bacteriophage vB_KpnP_XY4 of the present application.

[0027] Figure 11 Whole genome map of Klebsiella pneumoniae bacteriophage vB_KpnP_XY4 of the present application.

[0028] Figure 12 Phylogenetic tree and comparative gene analysis of Klebsiella pneumoniae bacteriophage vB_KpnP_XY4 of the present application. DETAILED DESCRIPTION

[0029] The principles and features of the present application are described below in conjunction with the accompanying drawings, and the examples are only used to explain the present application and not to limit the scope of the present application.

[0030] 1. Strain source

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

[0032] 2. Isolation and purification of bacteriophage

[0033] 50 mL sewage from the First People's Hospital of Xiangyang was mixed with 50 mL LB liquid medium, inoculated with 200 μL of logarithmic phase host bacteria, and cultured at 37°C, 200 rpm overnight. The next day, centrifuge at 4°C, 10000 rpm for 10 min, then take the supernatant and filter it with a 0.22 μm filter. The filtered liquid was gradient diluted and cultured on double-layer agar overnight. A single plaque was picked and immersed in LB medium containing host bacteria and cultured in a 37°C, 200 rpm shaking incubator overnight. Then centrifuge at 4°C, 10000 rpm for 10 min and filter with a 0.22 μm filter. The filtrate was gradient diluted and purified by double-layer agar plate method for 3-5 times until the plaque size was uniform. It was numbered as phage 31109. The phage titer was determined by double-layer plate method.

[0034] After 12 hours of culture on double-layer plates, as shown in Figure 1 , phage 31109 can form plaques with a central transparent and a surrounding translucent halo.

[0035] 3. Transmission electron microscopy observation of phage

[0036] 20 μL of purified high-titer (>10 9 PFU / mL) phage solution was dropped onto a copper grid and naturally adsorbed for 5-10 min. After drying, 20 μL of 2% phosphotungstic acid solution was dropped onto the copper grid and left for 3-5 min. Excess liquid drops were absorbed with filter paper strips. After drying under an incandescent lamp, the sample was observed and photographed under a transmission electron microscope.

[0037] The results are shown in Figure 2 , phage 31109 has a icosahedral head and a long, non-contractile tail. According to the International Committee on Taxonomy of Viruses (ICTV) 2015 report, phage 31109 belongs to the long-tailed virus family.

[0038] According to the phage naming rules, it is named Klebsiella pneumoniae phage vB_KpnP_XY4, hereinafter referred to as phage vB_KpnP_XY4.

[0039] Phage vB_KpnP_XY4 was deposited at the China Center of Type Culture Collection (CCTCC) of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, on January 13, 2023, with the accession number CCTCC NO: M 2025094, and the taxonomic name Klebsiella pneumoniae phage vB_KpnP_XY4.

[0040] 4. Host range detection of phage vB_KpnP_XY4

[0041] The host range lysis experiment was performed on 26 strains of Klebsiella pneumoniae, 15 strains of Acinetobacter baumannii, and 25 strains of Staphylococcus aureus, using the spot method. The test bacteria were cultured to the logarithmic phase, about 10 8 CFU / mL, 100 μL of the bacterial solution was uniformly coated on LB solid agar plates using an L-shaped coating rod, 20 μL of the purified phage vB_KpnP_XY4 solution was spotted on the plates, and the plates were cultured overnight at 37°C in an incubator for 12 h, and then the plaque formation was observed.

[0042] The results are shown in Table 1. The phage vB_KpnP_XY4 specifically lysed the following three strains of Klebsiella pneumoniae: Kpn 31109, 31511, and 32406, and had strong lytic ability against Kpn 31109.

[0043] Table 1. Results of the phage vB_KpnP_XY4 lysis spectrum test

[0044]

[0045]

[0046] Note: "+" can be lysed, "++" has strong lytic ability, and "-" cannot be lysed.

[0047] 5. Drug resistance and virulence genes carried by the host bacteria Klebsiella pneumoniae 31109

[0048] The strains were resuscitated and cultured, and bacterial DNA was extracted using a bacterial genome DNA extraction kit. The primer sequences of the drug resistance and virulence genes were synthesized by Shanghai Sangon Biological Engineering Co., Ltd. according to the literature.

[0049] PCR system 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), and 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; and maintenance at 72°C for 10 min.

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

[0052] PCR identification, the host bacteria Klebsiella pneumoniae Kpn31109 is K57 serotype Figure 3 A), carrying beta-lactam drug resistance gene blaTEM, blaSHV, blaKPC and polymyxin resistance gene phoQ, pmrB, and chloramphenicol drug resistance gene Cat1 Figure 3 B), can cause resistance to carbapenems, colistin and chloramphenicol antibiotics; The bacteria also carry iron uptake system related genes kfuBC, ybtA, iroNB, iucA, pilus synthesis genes fimH, ureA, lipopolysaccharide genes uge, wabG and other virulence genes Figure 3 C).

[0053] 6. Determination of optimal multiplicity of infection (MOI) of phage vB_KpnP_XY4

[0054] The host bacteria Kpn31109 was cultured to the logarithmic phase and the concentration was adjusted to 1×10 8 CFU / mL, and phage vB_KpnP_XY4 was added at a ratio 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 incubated at 37°C, 200 rpm on a shaker for 6 h. After centrifugation at 4°C, 1000g for 10 min, the supernatant was filtered through a 0.22μm filter and the phage titer was determined by double-layer agar plate method. The highest titer represents the optimal MOI. The test was repeated three times in parallel.

[0055] The results are shown in Figure 4 The optimal multiplicity of infection of phage vB_KpnP_XY4 is 0.01, and the titer can reach 1.4×10 10 PFU / mL.

[0056] 7. Determination of one-step growth curve of phage vB_KpnP_XY4

[0057] The corresponding phage vB_KpnP_XY4 and host bacteria Kpn31109 were mixed at an optimal MOI (0.01) ratio, incubated at 37°C for 15 min, centrifuged at 4°C, 1000 rpm for 10 min, the supernatant was discarded, 10 mL of LB liquid medium was added to resuspend the precipitate, and the phage titer was determined by double-layer agar plate method at 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 min of incubation at 37°C, 200 rpm in an incubator. The test was repeated three times in parallel.

[0058] The results are shown in Figure 5The results showed that the latent period of phage vB_KpnP_XY4 to lyse host bacteria Kpn 31109 was about 5 min, the burst lasted about 35 min, and entered the plateau stable phase after 40 min, with the highest titer of 1.4 x 10 10 PFU / mL, and the average lysis amount was 126 pfu / cell. It showed that phage vB_KpnP_XY4 could amplify in a large amount in a short time and had high lytic activity.

[0059] 8. Temperature stability detection of phage vB_KpnP_XY4

[0060] The phage solution was incubated in a water bath at different temperatures of 4°C, 25°C, 37°C, 50°C, 60°C and 70°C for 60 min, and then immediately placed in an ice bath for cooling. The titer was determined by gradient dilution and double-layer plate method.

[0061] The results are shown in Figure 6 The results showed that phage vB_KpnP_XY4 was relatively stable at temperatures of 4-60°C, with a titer of 10 9 -10 10 PFU / mL, and the phage vB_KpnP_XY4 was inactivated when the temperature reached 70°C.

[0062] 9. Acid-base (pH) stability detection of phage vB_KpnP_XY4

[0063] The pH value of LB liquid medium was adjusted to different ranges of 2-12, and 1 mL of purified phage solution was added to the LB medium with different pH ranges and incubated at 37°C for 60 min. The titer of phage was determined by gradient dilution.

[0064] The results are shown in Figure 7 The results showed that phage vB_KpnP_XY4 was relatively stable at pH 4-11, with the highest titer of 3 x 10 9 PFU / mL at pH 3 and 12, and the phage vB_KpnP_XY4 was inactivated.

[0065] 10. Chloroform stability detection of phage vB_KpnP_XY4

[0066] According to the volume ratio of chloroform to phage solution of 0.01, 0.05, 0.1, 0.5 and 1, 500 μL of phage was mixed with 5 μL, 25 μL, 50 μL, 250 μL and 500 μL of chloroform respectively, and then incubated at room temperature for 30 min. After centrifugation at 10000 rpm for 10 min, the supernatant was collected, gradient diluted and the titer was determined by double-layer agar plate method.

[0067] The results are shown in Figure 8As shown, vB_KpnP_XY4 was not sensitive to chloroform, and the phage titers were all higher than 10 10 PFU / mL at MOI = 0.01, 0.05, 0.1, 0.5, and 1, respectively.

[0068] 11. In vitro bacteriophage vB_KpnP_XY4 inhibition detection

[0069] The host bacteria Kpn 31109 were cultured to the exponential phase, and the concentration was adjusted to 10 8 CFU / mL, and the phage was infected at MOI of 10, 1, 0.1, 0.01, and 0.001, respectively. The host bacteria Kpn 31109 were infected with LB liquid medium as a positive control group, and cultured at 37°C and 200 rpm. The absorbance at 580 nm was detected at 0, 2, 4, 6, 8, 10, and 12 h, respectively.

[0070] As shown in Table 1, the phage vB_KpnP_XY4 had a significant inhibitory effect on the host bacteria Kpn 31109, and the activity of the host bacteria gradually weakened with the extension of time. Figure 9

[0071] 12. Bacteriophage vB_KpnP_XY4 biofilm inhibition rate detection

[0072] The purified phage liquid titer was adjusted to 10 9 PFU / mL, and the host bacteria Kpn 31109 were adjusted to 10 8 CFU / mL. 180 μL of the bacterial liquid and 20 μL of the phage liquid mixture were added to the 96-well plate at MOI (phage concentration / bacterial concentration) of 1, 0.1, 0.01, and 0.001, respectively, and each titer was made in triplicate. The mixture of 180 μL of the bacterial liquid and 20 μL of the SM buffer was used as a negative control, 200 μL of LB liquid was used as a blank control, 200 μL of PBS was added to the outermost circle of the 96-well plate to prevent evaporation of the liquid inside, and the mixture was discarded at 6, 12, 24, and 48 h of incubation at 37°C. After being gently washed with PBS for 3 times, 200 μL of 99% formaldehyde was added for fixation for 15 min, 200 μL of 1% crystal violet staining solution was added to each well for 5 min, and then the wells were washed with flowing water. 160 μL of 33% glacial acetic acid was added to each well to dissolve, and the absorbance at 570 nm was detected by a microplate reader. The biofilm inhibition rate = (negative control OD570-OD570 at each MOI) ÷ negative control OD570 x 100%.

[0073] As shown in Table 2, the phage vB_KpnP_XY4 had a significant inhibitory effect on the biofilm of the host bacteria Kpn 31109, and the biofilm activity gradually weakened with the extension of time. Figure 10 ​As shown, the phage vB_KpnP_XY4 can effectively inhibit the growth of the host bacteria biofilm, and the inhibition rate at 48h is close to 95%. It is shown that the phage vB_KpnP_XY4 has bacteriostatic durability, and plays an important role in the future prevention and control of Klebsiella pneumoniae.

[0074] 13. Phage DNA extraction, whole genome sequencing and phylogenetic analysis

[0075] The genome was extracted from the purified and concentrated phage solution using the Tian Gen virus genome extraction kit and sent to Suzhou Jinweizhi Biological Technology Co., Ltd. for second-generation sequencing. After completing the whole genome sequencing and splicing, the SnapGene software was used for annotation and analysis of the whole genome information. The MEGA software was used to construct a phylogenetic tree.

[0076] As shown in the figure, Figure 11 The phage vB_KpnP_XY4 genome is 50,036 bp in length, with a GC content of 50.75%, and contains 74 open reading frames (ORFs). No virulence genes or drug resistance genes were detected in the two phages.

[0077] As shown in the figure, Figure 12 The phage vB_KpnP_XY4 has the closest genetic relationship with the Klebsiella pneumoniae phage LF20.

[0078] The above is only the preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A Klebsiella pneumoniae phage, characterized in that It was deposited in the China Center for Type Culture Collection (CCTCC) on January 13, 2025, with the deposit number CCTCC NO: M 2025094.

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

3. Use of the Klebsiella pneumoniae phage according to claim 1 in the preparation of a drug for treating Klebsiella pneumoniae infection.

4. Use of the Klebsiella pneumoniae phage according to claim 1 in killing Klebsiella pneumoniae in the environment.

5. A method for killing Klebsiella pneumoniae, characterized in that: The method comprises the step of adding the Klebsiella phage according to claim 1 to an environment containing Klebsiella pneumoniae.

6. The use according to any one of claims 2 to 4 or the method according to claim 5, characterized in that: The Klebsiella pneumoniae is one or more combinations of Klebsiella pneumoniae 31109, 31511 and 32406.

7. A drug for resisting Klebsiella pneumoniae infection, characterized in that: The invention comprises the Klebsiella pneumoniae phage according to claim 1.

8. An environmental disinfectant, characterized in that The invention comprises the Klebsiella pneumoniae phage according to claim 1.

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