A Klebsiella phage and its application

Abstract

The present invention discloses a Klebsiella phage and its application, belonging to the field of life science and technology. The Klebsiella phage is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 46000, the deposit date being July 22, 2024, and the taxonomic name being Klebsiella phage, named FK9. The phage provided by the present invention is a biological means capable of efficiently killing Klebsiella pneumoniae, featuring high efficiency and specificity and small side effects. It can prevent and treat pneumonia caused by Klebsiella pneumoniae without disrupting the balance of the body's flora, which is incomparable to antibiotics and other antibacterial drugs. At the same time, it contributes to the sustainable development of the healthy medical system.

Description

Technical Field

[0001] The present invention belongs to the technical field of life sciences, and particularly relates to a Klebsiella phage and its application. 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 series of clinical diseases, such as pneumonia, meningitis, endophthalmitis, pyogenic liver abscess, sepsis, urinary tract infection, and even non-alcoholic fatty liver. Klebsiella pneumoniae is considered the most common pathogen of hospital- and community-acquired pneumonia and is also one of the most common hospital infection pathogens in the world.

[0003] Klebsiella pneumoniae ( Klebsiella pneumoniae ) is a species of the genus Klebsiella in the family Enterobacteriaceae, belonging to Gram-negative bacteria, rod-shaped, and covered with a large amount of viscous polysaccharide-formed capsule. Klebsiella pneumoniae can cause infections such as pneumonia, urinary tract infection, and bacteremia in humans, especially individuals with low immunity. However, in addition to nosocomial infections, community-acquired infections have gradually increased in recent years. The exudate in the lesions caused by Klebsiella pneumoniae is viscous and heavy, resulting in the downward drop of the interlobar fissure. When the bacteria grow and multiply in the alveoli, it causes tissue necrosis, liquefaction, and forms single or multiple abscesses. When the lesions involve the pleura and pericardium, it can cause exudative or purulent effusion. The fibrous tissue proliferation in the lesions is active and easy to organize; fibrous pleural effusion can show adhesions at an early stage. In nosocomial septicemia, Klebsiella, as well as Pseudomonas aeruginosa and Serratia, are all important pathogenic bacteria, and the fatality rate is relatively high.

[0004] Generally, according to the differences in virulence and pathogenic characteristics, KP is divided into Classic Klebsiella pneumoniae , cKP) and Hypervirulent Klebsiella pneumoniae , hvKP). hvKP is a community-acquired Klebsiella pneumoniae with high invasiveness initially isolated from capsular serotypes K1 or K2. Different from cKP, hvKP usually infects young and healthy individuals and causes invasive liver abscess. It is worth noting that in recent years, hvKP strains resistant to multiple antibiotics have emerged, and the overlap of its high virulence and drug resistance will pose a great challenge to clinical treatment.

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

[0006] According to the traditional view, although hvKP shows a highly virulent phenotype, it is sensitive to commonly used clinical antibiotics except ampicillin. Clinically, appropriate and effective antibiotics are often used to treat patients infected with hvKP. However, in recent years, the emergence of drug-resistant strains of hvKP and its trend of high epidemic outbreaks have been successively reported. Compared with cKP, the mortality rate after hvKP infection is high, and it can cause endogenous and metastatic infections, leading to serious complications. In particular, the widespread prevalence of carbapenem-resistant strains has brought greater challenges to infection prevention and control and clinical treatment.

[0007] As a new method to combat multi-drug-resistant bacteria, phage therapy has shown positive clinical effects and therapeutic benefits. More and more evidence indicates that phages are effective as a synergistic or alternative strategy to antibiotics. Generally speaking, phages are abundant and highly strain-specific viruses that can precisely kill bacteria after clearing host bacteria in vivo and automatically carry out self-metabolism. 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 is characterized by high efficiency, specificity, and low side effects.

[0009] To achieve the above object, 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 Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 46000, the deposit date is July 22, 2024, and the taxonomic name is Klebsiella phage, named FK9.

[0010] Furthermore, the Klebsiella phage is a short-tailed phage, its head has a polyhedral structure with a diameter of 50 ± 1 nm, and the tail is non-retractable with a length of 15 ± 1 nm.

[0011] Furthermore, the latent period of the Klebsiella phage is 40 min, the lysis period is 80 min, and the burst size is 407 PFU / cell.

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

[0013] Furthermore, the stable temperature for the activity of Klebsiella phage is -20 - 55 °C.

[0014] Furthermore, for the application of Klebsiella phage, Klebsiella phage is used to lyse Klebsiella pneumoniae ( Klebsiella pneumoniae ), Klebsiella michiganensis ( Klebsiella michiganensis ), or Klebsiella oxytoca ( Klebsiella grimontii ).

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

[0016] The beneficial effects of the present invention adopting the above further technical solutions are as follows: Klebsiella pneumoniae is a pathogen with high drug resistance and strong transmissibility, which 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 preservation information of the Klebsiella phage of the present invention is as follows:

[0018] Species name: Klebsiella phage, Latin name is Studiervirinae teetrevirus ;

[0019] Named: FK9;

[0020] Depositary institution: General Microbiology Center of China Committee for Culture Collection of Microorganisms;

[0021] Abbreviation of depositary institution: CGMCC;

[0022] Address of depositary institution: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0023] Deposit number: No. 46000;

[0024] Deposit date: July 22, 2024.

[0025] The beneficial effects of the present invention are as follows: The Klebsiella phage FK9 provided by the present invention has strong lysis ability, with a latent period of 40 min, a lysis period of 80 min, and a burst size of 407 PFU / cell; it has strong tolerance to temperature and pH, maintains stable activity between pH 4 - 11 and temperature -20 - 55 °C, can adapt to various environments, and can effectively and specifically solve the problem of drug resistance of Klebsiella pneumoniae to various antibiotics, having great advantages in the treatment of pneumonia caused by it; the specific advantages are: (1) High specificity, the phage specifically targets multidrug-resistant Klebsiella pneumoniae, Klebsiella michiganensis, and Klebsiella oxytoca, without affecting other beneficial bacteria; (2) It does not cause bacterial drug resistance and resistance; (3) It can proliferate depending on the host bacteria, and a single administration can achieve the effect of multiple administrations of other antibacterial drugs; (4) It has a short residence time and fast metabolism in the body; (5) The R & D and production time is short; (6) Low side effects, phage therapy has a low risk of side effects because they only attack specific bacteria; (7) Biofilm disruption ability, phages can penetrate and disrupt bacterial biofilms, which is a difficult problem in antibiotic treatment; (8) Potential for combination therapy, phages can be used in combination with antibiotics to enhance the therapeutic effect and overcome the limitations of single therapy. The phage provided by the present invention is a biological means capable of efficiently killing drug-resistant Klebsiella pneumoniae, with the characteristics of high efficiency and specificity and low side effects, achieving the effect of killing pathogenic bacteria without disrupting the balance of the body's flora, which is incomparable to antibiotics and other antibacterial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a transmission electron microscope observation diagram of Klebsiella phage FK9;

[0027] Figure 2 It is a one-step growth curve of Klebsiella phage FK9;

[0028] Figure 3 It is a pH tolerance curve of Klebsiella phage FK9;

[0029] Figure 4 It is a temperature tolerance curve of Klebsiella phage FK9;

[0030] Figure 5 It is a complete genome map of Klebsiella phage FK9;

[0031] Figure 6 It is a plaque produced by Klebsiella phage FK9 lysing bacteria, where a is Klebsiella michiganensis W14, b is Klebsiella grimontii 06D021. DETAILED DESCRIPTION OF THE INVENTION

[0032] The following is a detailed description of the specific implementation manners of the present invention in conjunction with embodiments.

[0033] The Klebsiella pneumoniae used in Examples 1 to 4 was Klebsiella pneumoniae subsp.ozaenae ATCC11296.

[0034] Example 1: A Klebsiella phage FK9, and its isolation method includes the following steps:

[0035] (1) The hospital sewage sample filtered through a 0.22 μm filter membrane was serially 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 200 μL of each dilution was taken and mixed evenly with 200 μL of fresh Klebsiella pneumoniae ( Klebsiella pneumoniae ) bacterial liquid in the logarithmic growth phase. After infection for 10 - 20 min, the double-layer plate method was used for cultivation.

[0036] (2) After observing transparent plaques on the plate, a sterile pipette tip (1 mL) was used to pierce the whole agar block with the plaque, and it was blown into 2 mL of SM buffer and placed at 4 °C overnight. The next day, the sample was filtered through a sterile filter membrane with a pore size of 0.22 μm to remove agar fragments, and then mixed with Klebsiella pneumoniae ( Klebsiella pneumoniae ) bacterial liquid for infection and then poured the double-layer plate; this step was repeated 3 - 5 times until a single and pure phage sample was obtained.

[0037] (3) 200 μL of the pure phage FK9 sample was added to 20 mL of the host bacterial liquid in logarithmic growth. After culturing for 48 h, it was centrifuged at 8000×g for 45 min; the supernatant after centrifugation was filtered through a 0.22 μm filter membrane, and then a ultrafiltration centrifugal tube with a molecular cut-off rate of 30 kDa was used. After ultrafiltration and concentration at 3800×g for 1 min to 1 mL, it was transferred to 200 mL of the host culture medium and cultured for 48 h, and then centrifuged at 8000×g for 45 min; the supernatant after centrifugation was filtered through a 0.22 μm filter membrane and then ultrafiltered and concentrated for 1 min to 10 mL.

[0038] (4) Equip with CsCl gradient solution for gradient centrifugation: Add 2 mL of CsCl solution with a density of 1.40 g / mL (the 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 20000 rpm at room temperature for 2 h. Collect the virus band with a density between 1.30 g / mL and 1.40 g / mL into a dialysis bag (the dialysis bag is boiled with 10 mM EDTA-Na2 for 10 min before use). Dialyze overnight with stirring at 4 °C in a dialysis buffer (50 g sucrose, 10 mL 1M Tris-HCl, pH 8.0, 2 mL 1M MgCl2 made up to 1 L), and change the dialysis solution once in the middle. The pure Klebsiella phage FK9 is collected.

[0039] Example 2: Characteristic experiments of phage FK9

[0040] 1. Electron microscopy observation test:

[0041] Pipette 20 μL of the phage enrichment band and drop it on a dry copper mesh. After waiting for 15 min, drop 2 wt% phosphotungstic acid (PTA) solution. Wait for another 10 min, dry it under the lamp, and then place the copper mesh under a transmission electron microscope for observation. The observation results of the transmission electron microscope are as Figure 1 shown. Through morphological identification and observation, this phage belongs to the short-tailed phage. Its head is in a polyhedral structure with a diameter of 50 ± 1 nm, and the tail is non-retractable with a length of 15 ± 1 nm. It is named Klebsiella phage FK9.

[0042] 2. One-step growth curve experiment:

[0043] The specific steps are as follows:

[0044] (1) Put 1 mL of the pure phage sample FK9 (MOI = 0.1) into an equal volume of the host bacterial solution and adsorb it at 28 °C for 15 - 20 min.

[0045] (2) Pipette 1 mL of the above mixture, centrifuge at 13000×g for 1 min, discard the upper liquid, add 1 mL of LB culture medium and briefly vortex to mix the precipitate; repeat this step 3 times to remove the unadsorbed phages.

[0046] (3) Put the last mixture back into 50 mL of LB culture medium, incubate at a constant temperature of 28 °C with shaking and start timing; sample every 5 min in the first 1 h, then sample every 10 min in the next 1 h, and finally sample every 30 min in the last 1 h; take 1 mL of each sample and set up three parallel groups.

[0047] (4) The sample was placed in 20 μL of glutaraldehyde and fixed in the dark for 15 min, then immediately frozen with liquid nitrogen; afterwards, the sample was thawed at room temperature and appropriately diluted with SM buffer (pH 8.0), and the sample was stained with the DNA stain SYBR Green I at 80 °C for 10 min.

[0048] (5) At the same time, 1 mL of the host bacterial solution at the initial time of timing was placed in 20 μL of glutaraldehyde, fixed in the dark for 15 min and then immediately frozen with liquid nitrogen; the sample was thawed at room temperature and appropriately diluted with TE buffer (Tris-EDTA; pH 8.0), and the sample was stained with the DNA stain SYBR Green I at 80 °C for 1 h.

[0049] (6) Flow cytometry was used to measure the abundance of phages and the abundance of infected host bacteria at the initial time of timing, and a one-step growth curve of phages was plotted.

[0050] The one-step growth curve of phage FK9 is as Figure 2 shown. It can be seen that the latent period of phage FK9 is about 40 min; then it enters the exponential burst phase, which takes about 120 min, and the burst size is 407 PFU / cell; indicating that phage FK9 has strong bacterial lysis ability and infectivity.

[0051] Example 3: pH stability experiment of phage FK9

[0052] 900 μL of SM buffer with pH values of 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 were respectively added to 100 μL of phage FK9 sample (10 8 PFU / mL), and cultured at 28 °C for 2 h. 200 μL of the samples treated under different pH conditions were respectively mixed with an equal volume of the host bacterial solution, and after 15 min, double-layer plates were poured. After culturing at 28 °C for 12 h, the number of plaques was calculated and a pH influence trend graph was plotted; repeated three times.

[0053] The pH tolerance curve of phage FK9 is as Figure 3 shown. Phage FK9 maintains stable activity within the pH range of 4 - 11.

[0054] Example 4: Thermal stability experiment of phage FK9

[0055] 200 μL of phage FK9 samples (10 8PFU / mL; pH 7.0) was incubated at -20 °C, 4 °C, 25 °C, 35 °C, 45 °C, 55 °C, 65 °C, and 75 °C for 2 h; after the temperature in each tube dropped back to room temperature, 200 μL of the host bacterial solution was added, and after 15 min of infection, a double-layer plate was poured. After culturing at 28 °C for 12 h, the number of plaques was counted and a temperature influence trend graph was drawn; the experiment was repeated three times.

[0056] The temperature tolerance curve of phage FK9 is as Figure 4 shown. Phage FK9 can maintain a very stable titer between -20 and 55 °C, and its activity only starts to decline after 65 °C.

[0057] Example 5: Purification and sequencing of the phage FK9 genome

[0058] The specific steps are as follows:

[0059] (1) The phage FK9 sample was successively used with the HP Viral DNA / RNA Kit (OMEGA) and the DNeasy PowerClean pro Cleanup Kit (Qiagen) for genomic extraction and purification.

[0060] (2) The purified DNA sample was fragmented to 350 bp in size by sonication, and then the DNA fragment ends were repaired, tailed, and folded. After PCR, the product was purified (AMPure-XP system) and a DNA library was constructed, and then sequenced using Illumina NovaSeq PE150.

[0061] (3) The 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; then the ORFs were respectively 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 result with the highest score (E-value ≤ 10-5; identity > 40%; coverage > 40%) was selected for screening and integration to obtain the final annotation information of the phage genome.

[0062] The full-length genome of bacteriophage FK9 is 39,802 bp, and its whole-genome map is as Figure 5 shown. It has a total of 45 open reading frames (ORFs), among 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 encoding for assisting bacteriophage FK9 to complete the efficient infection of the host; ORF18 encodes N-acetylmuramoyl-L-alanine amidase, which specifically hydrolyzes the amide bond between muramic acid and L-alanine in peptidoglycan, dissolving 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 a type II phage holin, 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 pores to allow endolysin to enter the interior of the cell wall at the optimal time point; this process is crucial for the phage lysis cycle and helps release newly generated phage particles. ORF44 encodes an Rz-like transmembrane protein, which is a key phage protein and plays an important role in the lysis process after the phage infects the host cell; by promoting the fusion of the outer and inner membranes of the host cell, it ensures the complete lysis of the cell and the release of newly formed phages; it works in cooperation with endolysin and phage holin, constituting an important part of the phage lysis mechanism.

[0063] Example 6: Host bacteria of bacteriophage FK9

[0064] Take 200 μL of bacteriophage FK9 sample (10 8 PFU / mL) and mix it with 200 μL of bacterial liquid in the logarithmic growth phase, pour a double-layer plate after 15 minutes of infection, and observe the formation of plaques after culturing at 28 °C for 12 h; among which the bacterial liquid includes 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.

[0065] After detection by the plaque experiment, Klebsiella grimontii 06D021, Klebsiella michiganensis W14, Klebsiella quasivariicola KPN1705, Klebsiella quasipneumoniae subsp.similipneumoniae 07A044, Klebsiella pneumoniae subsp.pneumoniae DSM 30104 and Klebsiella pneumoniae subsp.ozaenaePlaques were formed on the plates corresponding to ATCC 11296, which is the host bacterium of phage FK9. Among them Klebsiella michiganensis W14 and Klebsiella grimontii The plaque formation results of 06D021 are shown in Figure 6 a in Figure 6 and b in

[0066] The above embodiments provide a phage FK9 that can efficiently degrade Klebsiella pneumoniae ( Klebsiella pneumoniae ). Phage FK9 can specifically lyse Klebsiella pneumoniae , and rapidly reduce the number of hosts in the sample in a short time. According to the one-step growth curve data, the latent period of phage FK9 is only 40 min. After 40 min, a large number of phages complete replication and rapidly lyse the host bacteria, making the originally turbid host bacteria broth clear. At the same time, according to the data of pH and temperature tolerance experiments, this phage can maintain relatively stable activity between pH 4 - 11 and temperature -20 - 55 °C, indicating that phage FK9 can effectively adapt to a variety of harsh environments and achieve the purpose of efficiently clearing 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 in the genome of phage FK9, there are various coding genes such as N-acetylmuramic acid-L-alanine amidase, type II phage holin, and Rz-like transmembrane protein, which can assist this phage to complete the process of efficiently infecting the host and improve the infection efficiency. In summary, phage FK9 is a virulent phage that can efficiently lyse pathogenic bacteria Klebsiella pneumoniae and has excellent adaptability to the complex human environment. It has advantages over traditional antibiotic treatments in the prevention and treatment of pneumonia caused by Klebsiella pneumoniae , contributing to the sustainable development of the health care system.

[0067] Although the specific implementation manners of the present invention have been described in detail in combination with the embodiments, it should not be construed as a limitation on the protection scope of this patent. Within the scope described in the claims, various modifications and deformations that can be made by those skilled in the art without creative efforts still fall within the protection scope of this patent.

Claims

1. A Klebsiella phage, characterized in that: The Klebsiella phage is deposited in the China General Microbiological Culture Collection Center, with the deposit number CGMCC No. 46000, the deposit date being July 22, 2024, and it is named FK9; the Klebsiella phage is a short-tailed phage, with its head presenting a polyhedral structure, a diameter of 50 ± 1 nm, and the tail being non-contractile, with a length of 15 ± 1 nm; the latent period of the Klebsiella phage is 40 min, the lysis period is 80 min, and the burst size is 407 PFU / cell.

2. The Klebsiella phage according to claim 1, wherein: The stable pH for the activity of the Klebsiella phage is 4 - 11.

3. The Klebsiella phage according to claim 1, characterized in that: The stable temperature for the activity of the Klebsiella phage is -20 - 55 °C.

4. Use of the Klebsiella phage according to any one of claims 1 to 3, characterized in that: The Klebsiella phage is used for lysing Klebsiella pneumoniae, Klebsiella michiganensis or Klebsiella oxytoca.

5. Use of the Klebsiella phage according to any one of claims 1 to 3, characterized in that: The Klebsiella phage is used for preparing a drug for treating lung infections caused by Klebsiella pneumoniae.

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