Klebsiella phage and application thereof

Abstract

The invention discloses a Klebsiella phage and application thereof, and belongs to the technical field of life science. The Klebsiella bacteriophage is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.46000, the preservation date is July 22, 2024, and the Klebsiella bacteriophage is classified and named as FK9. The bacteriophage provided by the invention is a biological means capable of efficiently killing klebsiella pneumonia, has the characteristics of high efficiency, specificity and small side effect, achieves the effect of preventing and treating pneumonia caused by klebsiella pneumonia on the basis of not destroying in-vivo flora balance, is incomparable to antibiotics and other antibacterial drugs, and has a broad application prospect. And meanwhile, the method contributes to sustainable development of a health medical system.

Description

Technical Field

[0001] The invention belongs to the technical field of life sciences, and in particular relates to a Klebsiella phage and an application thereof. Background Art

[0002] Klebsiella pneumoniae is widely distributed in various environmental niches, such as water, soil, feces, hospitals, communities, animals and humans. Klebsiella pneumoniae can cause a range of clinical conditions, such as pneumonia, meningitis, endophthalmitis, pyogenic liver abscess, sepsis, urinary tract infection, and even non-alcoholic fatty liver disease. Klebsiella pneumoniae is considered the most common pathogen of hospital- and community-acquired pneumonia and one of the most common nosocomial pathogens in the world.

[0003] Klebsiella pneumoniae ( Klebsiella pneumoniae ) is a species of Klebsiella in the family Enterobacteriaceae. It is a Gram-negative bacterium, rod-shaped, and covered with a capsule formed by a large amount of sticky polysaccharides. Klebsiella pneumoniae can cause pneumonia, urinary tract infection, bacteremia and other infections in humans, especially individuals with low immunity. However, in addition to hospital-acquired infections, community-acquired infections have also gradually increased in recent years. The exudate of Klebsiella pneumoniae in the lesions is viscous and heavy, causing the interlobular spaces to fall. When the bacteria grow and multiply in the alveoli, they cause tissue necrosis, liquefaction, and the formation of single or multiple abscesses. When the lesions involve the pleura and pericardium, exudative or purulent effusions can be caused. The fibrous tissue in the lesions proliferates actively and is easy to organize; adhesions may appear early in fibrinous pleural effusions. In hospital-acquired sepsis, Klebsiella, Pseudomonas aeruginosa, and Serratia are all important pathogens with a high mortality rate.

[0004] KP is usually divided into classic Klebsiella pneumoniae according to its virulence and pathogenicity. Klebsiella pneumoniae , cKP) and hypervirulent Klebsiella pneumoniae Klebsiella pneumoniae , hvKP). hvKP is a highly invasive community-acquired Klebsiella pneumoniae originally isolated from capsular serotypes K1 or K2. Unlike cKP, hvKP usually infects young healthy individuals and causes invasive liver abscesses. It is worth noting that in recent years, hvKP strains resistant to multiple antibiotics have emerged, and the overlap of their high virulence and drug resistance will bring great challenges to clinical treatment.

[0005] The first case of hvKP causing liver abscess was reported in detail in 1986. In the following decades, hvKP gradually became the main pathogen of liver abscess in Asia. Compared with cKP, hvKP usually invades young and healthy individuals and is prone to sepsis, leading to severe multi-site invasive infection syndromes such as suppurative liver abscess, osteomyelitis, endophthalmitis, and necrotizing fasciitis. Since the first case of hvKP infection was reported in 1986, it has spread in Asia, especially in South Korea and Japan, and there have been sporadic reports in other countries and the trend is increasing.

[0006] The traditional view is that although hvKP exhibits a highly virulent phenotype, it is sensitive to commonly used clinical antibiotics except ampicillin. Clinically, patients infected with hvKP are often treated with corresponding effective antibiotics. However, in recent years, reports have been published on the emergence of hvKP-resistant strains and their high prevalence and outbreak trends. Compared with cKP, hvKP infection has a high mortality rate and can cause endogenous and metastatic infections, leading to serious complications. In particular, the widespread prevalence of carbapenem-resistant strains poses greater challenges to infection prevention and control and clinical treatment.

[0007] As a new approach to combat multi-drug resistant bacteria, phage therapy has shown positive clinical effects and therapeutic benefits, and increasing evidence shows that phages are effective as a synergistic or alternative strategy to antibiotics. In general, phages are abundant and highly strain-specific viruses that can precisely kill bacteria and automatically metabolize themselves after clearing host bacteria in vivo. Summary of the invention

[0008] In view of the above-mentioned prior art, the present invention provides a Klebsiella phage and its application, providing a biological means capable of efficiently killing Klebsiella pneumoniae, which has the characteristics of high efficiency, specificity and small side effects.

[0009] In order to achieve the above-mentioned purpose, the technical solution adopted by the present invention is: to provide a Klebsiella phage, which is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No. 46000 and a deposit date of July 22, 2024. It is classified and named as Klebsiella phage, named FK9.

[0010] Furthermore, Klebsiella phage is a short-tailed phage with a polyhedral head of 50±1 nm in diameter and a non-contractile tail of 15±1 nm in length.

[0011] Furthermore, the incubation period of Klebsiella phage was 40 min, the lysis period was 80 min, and the lysis amount was 407 PFU / cell.

[0012] Furthermore, the activity of Klebsiella phage is stable at pH 4-11.

[0013] Furthermore, the stable activity temperature of Klebsiella phage is -20~55 ℃.

[0014] Further, the use of Klebsiella phage, the use of Klebsiella phage for lysing Klebsiella pneumoniae ( Klebsiella pneumoniae )、Klebsiella michiganensis( Klebsiella michiganensis ) or Klebsiella oxytoca ( Klebsiella grimontii )middle.

[0015] Furthermore, the application of Klebsiella phage is to use Klebsiella phage to prepare drugs for treating lung infections caused by Klebsiella pneumoniae.

[0016] The beneficial effects of the above further technical scheme adopted by the present invention are: Klebsiella pneumoniae is a highly drug-resistant and highly transmissible pathogen that can cause serious infections such as pneumonia, urinary tract infections and sepsis, posing a major threat to public health; Klebsiella phage FK9 can be used in the treatment of infections caused by drug-resistant Klebsiella pneumoniae.

[0017] The deposit information of the Klebsiella phage of the present invention is as follows: Bacterial species name: Klebsiella phage, Latin for Studiervirinae teetrevirus ; Name: FK9; Depository: General Microbiology Center, China Microbiological Culture Collection Administration; Abbreviation of depository institution: CGMCC; Address of the depository institution: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Accession number: No.46000; Date of preservation: July 22, 2024.

[0018] The beneficial effects of the present invention are as follows: the Klebsiella phage FK9 provided by the present invention has strong lysis ability, a latent period of 40 min, a lysis period of 80 min, and a lysis amount of 407 PFU / cell; it has strong tolerance to temperature and pH, and can be used in the range of pH 4 to 11 and temperature of -20 to 55 ℃, can adapt to a variety of environments, and can effectively and specifically solve the problem of Klebsiella pneumoniae resistance to multiple antibiotics, which has great advantages in treating pneumonia caused by it; the advantages are as follows: (1) High specificity, phage specifically targets multidrug-resistant Klebsiella pneumoniae, Klebsiella michiganensis and Klebsiella acidophilus, and will not affect other beneficial bacteria; (2) It will not cause bacterial resistance and resistance; (3) It can rely on the host bacteria to proliferate, and a single dose can achieve the effect of multiple doses of other antibacterial drugs; (4) It has a short residence time in the body and is rapidly metabolized; (5) Short R&D and production time; (6) Low side effects, phage therapy has a lower risk of side effects because they only attack specific bacteria; (7) Biofilm destruction ability, phages can penetrate and destroy bacterial biofilms, which is a problem in antibiotic treatment; (8) Combination therapy potential, phages can be used in combination with antibiotics to enhance the therapeutic effect and overcome the limitations of single treatment. The bacteriophage provided by the present invention is a biological means capable of efficiently killing drug-resistant Klebsiella pneumoniae, has the characteristics of high efficiency, specificity and small side effects, and achieves the effect of killing pathogens without destroying the balance of the flora in the body, which is unmatched by antibiotics and other antibacterial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a transmission electron microscope observation of Klebsiella phage FK9; Figure 2 is the one-step growth curve of Klebsiella phage FK9; Figure 3 is the pH tolerance curve of Klebsiella phage FK9; Figure 4 is the temperature tolerance curve of Klebsiella phage FK9; Figure 5 This is the complete genome map of Klebsiella phage FK9; Figure 6 The plaques are produced by the lysis of bacteria by Klebsiella phage FK9, where a is Klebsiella michiganensis W14, b is Klebsiella grimontii 06D021. DETAILED DESCRIPTION

[0020] The specific implementation modes of the present invention are described in detail below with reference to the embodiments.

[0021] The Klebsiella pneumoniae used in Examples 1 to 4 isKlebsiella pneumoniae subsp.ozaenae ATCC11296.

[0022] Example 1: A Klebsiella phage FK9, the isolation method of which comprises the following steps: (1) Hospital sewage samples were filtered through a 0.22 μm filter membrane and graded diluted with SM buffer (0, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 ) and take 200 μL of each, and then mix with 200 μL of fresh Klebsiella pneumoniae in the logarithmic growth phase ( Klebsiella pneumoniae ) The bacterial solutions were mixed evenly and cultured using the double-layer plate method after infection for 10 to 20 min.

[0023] (2) After transparent plaques are observed on the plate, poke the entire agar with plaques with a sterile pipette tip (1 mL), pipette into 2 mL of SM buffer, and place at 4 °C overnight. The next day, filter the sample using a 0.22 μm pore size sterile filter membrane to remove agar fragments and separate from Klebsiella pneumoniae ( Klebsiella pneumoniae ) After the bacterial solution is mixed and infected, pour the plate using the double-layer plate method; this step is repeated 3 to 5 times until a single pure phage sample is obtained.

[0024] (3) Add 200 μL of pure phage FK9 sample to 20 mL of logarithmically growing host culture medium, culture for 48 h, and centrifuge at 8000×g for 45 min. Take the upper layer of liquid after centrifugation and filter it through a 0.22 μm filter membrane. Then use an ultrafiltration centrifuge tube with a molecular retention rate of 30 kDa to ultrafilter and concentrate it at 3800×g for 1 min to 1 mL. Then transfer it to 200 mL of host culture medium and culture it for 48 h. Centrifuge it at 8000×g for 45 min. Take the upper layer of liquid after centrifugation and filter it through a 0.22 μm filter membrane and ultrafilter and concentrate it for 1 min to 10 mL.

[0025] (4) Prepare CsCl gradient solution for gradient centrifugation: Add 2 mL of CsCl solution with a density of 1.40 g / mL (solvent is 20 mM Tris-HCl, pH 8.0), then slowly add 3 mL of CsCl solution with a density of 1.30 g / mL, and then add 5 mL of virus concentrate. Centrifuge at 20,000 rpm for 2 h at room temperature. Collect virus bands with densities between 1.30 g / mL and 1.40 g / mL into a dialysis bag (the dialysis bag was boiled with 10 mM EDTA-Na2 for 10 min before use). In dialysis buffer (50 g sucrose, 10 mL 1M Tris-HCl, pH 8.0, 2 mL 1M MgCl2 to 1L), stir and dialyze overnight at 4 °C, changing the dialysis solution once in the middle. Collect pure Klebsiella phage FK9.

[0026] Example 2: Characteristic Experiment of Bacteriophage FK9 1. Electron microscope observation test: 20 μL of phage enrichment band was added to the dry copper mesh. After waiting for 15 min, 2 wt% phosphotungstic acid (PTA) solution was added. After waiting for 10 min, the copper mesh was dried under a light and then placed under a transmission electron microscope for observation. The results of transmission electron microscopy are shown in the figure. Figure 1 As shown, morphological identification and observation showed that this phage is a short-tailed phage with a polyhedral head and a diameter of 50±1 nm. The tail is non-contractible and has a length of 15±1 nm. It was named Klebsiella phage FK9.

[0027] 2. One-step growth curve experiment: The specific steps are as follows: (1) Place 1 mL of pure phage sample FK9 (MOI = 0.1) into an equal volume of host bacterial solution and adsorb at 28 °C for 15-20 min.

[0028] (2) Pipette 1 mL of the above mixture, centrifuge at 13,000 × g for 1 min, discard the upper layer of liquid, add 1 mL of LB culture medium and vortex briefly to mix the precipitate; repeat this step 3 times to remove the unadsorbed phages.

[0029] (3) Place the final mixed solution back into 50 mL LB culture medium, incubate at 28 °C with constant temperature shaking and start timing; sample once every 5 min in the first hour, once every 10 min in the next hour, and once every 30 min in the last hour; sample 1 mL each time and set up three parallel groups.

[0030] (4) The sample was placed in 20 μL of glutaraldehyde, fixed in a dark environment for 15 min, and immediately frozen with liquid nitrogen. The sample was then thawed at room temperature and appropriately diluted with SM buffer (pH 8.0). The sample was stained with the DNA stain SYBR Green I at 80°C for 10 min.

[0031] (5) At the same time, take 1 mL of the host bacterial solution at the beginning of the timing and place it in 20 μL of glutaraldehyde. Fix it in the dark for 15 min and then immediately freeze it with liquid nitrogen. Thaw the sample at room temperature and dilute it appropriately with TE buffer (Tris-EDTA; pH 8.0). Stain the sample with the DNA stain SYBR Green I at 80 °C for 1 h.

[0032] (6) Flow cytometry is used to measure the abundance of phage and the abundance of infected host bacteria at the beginning of the timing, and a one-step growth curve of the phage is drawn.

[0033] The one-step growth curve of bacteriophage FK9 is as follows Figure 2 As shown, it can be seen that the incubation period of bacteriophage FK9 is about 40 minutes; then it enters the exponential outbreak stage, which takes about 120 minutes, and the lysis amount is 407 PFU / cell; indicating that bacteriophage FK9 has strong bacterial lysis ability and infectivity.

[0034] Example 3: pH stability experiment of bacteriophage FK9 Add 100 μL of phage FK9 sample (10 8 PFU / mL), 900 μL of SM buffer with pH values ​​of 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 were added to the samples, and cultured at 28 °C for 2 h. 200 μL of samples treated under different pH conditions were mixed with an equal volume of host bacterial solution, poured into double-layer plates after 15 min, and cultured at 28 °C for 12 h. The number of plaques was calculated and the pH effect trend was plotted; this was repeated three times.

[0035] The pH tolerance curve of bacteriophage FK9 is as follows Figure 3 As shown, phage FK9 maintains stable activity in the pH range of 4 to 11.

[0036] Example 4: Thermal stability experiment of bacteriophage FK9 Take 200 μL of phage FK9 sample (10 8PFU / mL; pH 7.0) was placed at -20 ℃, 4 ℃, 25 ℃, 35 ℃, 45 ℃, 55 ℃, 65 ℃, and 75 ℃ for 2 h; when the temperature in each tube returned to room temperature, 200 μL of host bacterial solution was added, and after infection for 15 min, double-layer plates were poured and cultured at 28 ℃ for 12 h. The number of plaques was calculated and a temperature effect trend chart was drawn; this was repeated three times.

[0037] The temperature tolerance curve of bacteriophage FK9 is as follows Figure 4 As shown, the titer of bacteriophage FK9 can be kept very stable between -20 and 55°C, and the activity starts to decline after 65°C.

[0038] Example 5: Purification and sequencing of bacteriophage FK9 genome The specific steps are as follows: (1) The genome of bacteriophage FK9 sample was extracted and purified using HP Viral DNA / RNA Kit (OMEGA) and DNeasy PowerClean pro Cleanup Kit (Qiagen) respectively.

[0039] (2) The purified DNA samples were broken into 350 bp in size by ultrasound, and the ends of the DNA fragments were then repaired, tailed, and folded. After PCR, the products were purified (AMPure-XP system) and a DNA library was constructed, which was then sequenced using Illumina NovaSeq PE150.

[0040] (3) RAST (https: / / topaz.gatech.edu / GeneMark / ) and GeneMarks (http: / / topaz.gatech.edu / GeneMark / ) software were used to determine the open reading frames (ORFs) in the phage sequence. The ORFs were then compared with the non-redundant (NR) protein database based on the BLASTP algorithm in NCBI (https: / / www.ncbi.nlm.nih.gov / ), the Pfam (https: / / pfam.xfam.org / search / sequence) database, and the PDB (http: / / www.rcsb.org / ) database. The alignment results with the highest scores (E-value≤10-5; id-entity>40%; coverage>40%) were selected for screening and integration to obtain the final annotation information of the phage genome.

[0041] The genome of bacteriophage FK9 is 39802 bp in length, and its complete genome map is as follows: Figure 5 As shown, there are 45 open reading frames (ORFs), of which 8 ORFs (18%) are annotated as hypothetical proteins, and the remaining 37 ORFs (82%) encode functional proteins. Among the 37 ORFs encoding functional proteins, there are genes that help phage FK9 complete the process of efficient host infection; ORF18 encodes N-acetylmuramic acid-L-alanine amidase, which specifically hydrolyzes the amide bond between muramic acid and L-alanine in peptidoglycan, dissolves the peptidoglycan structure, and can degrade the peptidoglycan of the bacterial host from the inside, promoting cell lysis and the release of progeny virus particles. ORF42 encodes type II phage perforin, whose main function is to regulate the entry of endolysin, thereby triggering the lysis of the host cell wall; it usually has two putative transmembrane domains, forming holes, allowing endolysin to enter the cell wall at the optimal time point; this process is crucial to the lysis cycle of the phage and helps release newly generated phage particles. ORF44 encodes an Rz-like transmembrane protein, which is a key phage protein that plays an important role in the lysis process of phage-infected host cells. It ensures complete cell lysis and the release of new phages by promoting the fusion of the outer and inner membranes of host cells. It works synergistically with endolysins and phage perforins to form an important link in the phage lysis mechanism.

[0042] Example 6: Host bacteria of bacteriophage FK9 Take 200 μL of phage FK9 sample (10 8 PFU / mL) was mixed with 200 μL of bacterial solution in logarithmic growth phase, and poured into double-layer plates after infection for 15 min. After incubation at 28 ℃ for 12 h, the plaque formation was observed. The bacterial solution included Klebsiella grimontii 06D021, Klebsiella michiganensis W14, Klebsiella quasivariicola KPN1705, Klebsiella quasipneumoniae subsp.similipneumoniae 07A044, Klebsiella pneumoniae subsp.pneumoniae DSM 30104 and Klebsiella pneumoniae subsp.ozaenae ATCC 11296.

[0043] Detected by plaque assay, Klebsiella grimontii 06D021, Klebsiella michiganensis W14, Klebsiella quasivariicola KPN1705, Klebsiella quasipneumoniae subsp.similipneumoniae 07A044, Klebsiella pneumoniae subsp.pneumoniae DSM 30104 and Klebsiella pneumoniae subsp.ozaenaeThe corresponding plates of ATCC 11296 all produce plaques, which are the host bacteria of phage FK9. Klebsiella michiganensis W14 and Klebsiella grimontii The plaque results produced by 06D021 are as follows Figure 6 a and Figure 6 As shown in b.

[0044] The above embodiment provides a method for efficiently degrading Klebsiella pneumoniae ( Klebsiella pneumoniae ) strain. Phage FK9 can specifically lyse Klebsiella pneumoniae , rapidly reducing the number of hosts in the sample in a short time. According to the one-step growth curve data, the incubation period of phage FK9 is only 40 min. After 40 min, a large number of phages complete replication and quickly lyse the host bacteria, making the originally turbid host bacterial solution clear. At the same time, according to the data of pH and temperature tolerance experiments, the phage can maintain a relatively stable activity between pH 4 and 11 and temperature between -20 and 55 ℃, indicating that phage FK9 can effectively adapt to a variety of harsh environments and achieve the purpose of efficient elimination of drug-resistant Klebsiella pneumoniae. After sequencing and analyzing the genome of FK9, it was found that in addition to the structural genes that maintain the basic morphology of the phage, the genome of phage FK9 contains a variety of coding genes such as N-acetylmuramic acid-L-alanine amidase, type II phage perforin, and Rz-like transmembrane protein, which can assist the phage in completing the process of efficiently infecting the host and improving the infection efficiency. In summary, phage FK9 is a phage that can efficiently lyse pathogenic bacteria. Klebsiella pneumoniae , a virulent bacteriophage with excellent adaptability to the complex human environment. Klebsiella pneumoniae The prevention and treatment of pneumonia caused by the disease has unparalleled advantages over traditional antibiotic treatments, and contributes to the sustainable development of the health care system.

[0045] Although the specific implementation of the present invention is described in detail in conjunction with the embodiments, it should not be understood as limiting the scope of protection of this patent. Within the scope described in the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. A bacteriophage of the genus Klebsiella, Features: The Klebsiella phage is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.46000 and the deposit date July 22, 2024, and is named FK9.

2. The Klebsiella phage according to claim 1, Features: The Klebsiella phage is a short-tailed phage, the head of which is a polyhedral structure with a diameter of 50±1 nm, and the tail is non-contractible and has a length of 15±1 nm.

3. The Klebsiella phage according to claim 1, Features: The incubation period of the Klebsiella phage is 40 min, the lysis period is 80 min, and the lysis amount is 407 PFU / cell.

4. The Klebsiella phage according to claim 1, Features: The Klebsiella phage activity is stable at pH 4-11.

5. The Klebsiella phage according to claim 1, Features: The Klebsiella phage activity stable temperature is -20~55°C.

6. Use of the Klebsiella phage according to any one of claims 1 to 5, Features: The Klebsiella phage is used to lyse Klebsiella pneumoniae, Klebsiella michiganensis or Klebsiella oxytoca.

7. Use of the Klebsiella phage according to any one of claims 1 to 5, Features: The Klebsiella phage is used for preparing medicine for treating lung infection caused by Klebsiella pneumoniae.

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