Bacteriophage for specifically lysing high-virulence capsular klebsiella pneumoniae, bacteriophage liquid formulation, and use thereof

Abstract

A bacteriophage for specifically lysing high-virulence capsular Klebsiella pneumoniae, pertaining to the field of microorganisms. The deposit number of the Klebsiella pneumoniae bacteriophage vB_kpnP_D39 is CCTCC NO: M 2024690. In the bacteriophage liquid formulation prepared on the basis of the bacteriophage, the working titer of the bacteriophage is greater than 1 × 109 PFU / mL. The bacteriophage has high specificity and can kill all high-virulence multidrug-resistant Klebsiella pneumoniae of K1, K2, and K57 capsular serotypes, with an adsorption efficiency of 99.60%. The bacteriophage exhibits a biofilm clearance rate of 47.4%-63.2% and a capsule clearance rate of 42.1%-60.6% against the high-virulence Klebsiella pneumoniae. The bacteriophage is expected to become a safe, non-toxic agent for the prevention, control, and treatment of high-virulence multidrug-resistant Klebsiella pneumoniae infections, or an antibiotic adjuvant.

Description

Phage specifically lysing high virulence capsular klebsiella pneumoniae, phage liquid preparation and application

[0001] The present application claims priority to the Chinese patent application No. CN202410817046.6, filed on June 24, 2024, and entitled "Phage specifically lysing high virulence capsular klebsiella pneumoniae and phage preparation", the entire content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application belongs to the field of biotechnology, and specifically relates to a phage specifically lysing high virulence capsular klebsiella pneumoniae, a phage liquid preparation and application. BACKGROUND

[0003] Klebsiella pneumoniae (KP) is a conditional pathogenic gram-negative enterobacterium, which is often colonized in the gastrointestinal tract, skin and nasopharynx of the human body, and can cause pneumonia, liver abscess, urinary tract infection, wound infection, sepsis, etc. when the immunity of the body is low. Klebsiella pneumoniae has become an important pathogen of infection. Capsular (K antigen) serotype is one of the most important evidence for evaluating the pathogenicity of KP strains. Studies have shown that the high virulence capsular serotypes of Klebsiella pneumoniae are mainly K1, K2, K5, K16, K20, K54, K57 and KN1, among which K1, K2 and K57 are the most important. The cell surface of KP of these three types is more prone to form extracellular polysaccharide, thereby showing higher strain toxicity and tolerance to antibiotics, serum antibodies and the like, and thus leading to great difficulty in clinical treatment. Therefore, effective prevention and treatment of KP high virulence drug-resistant strains has become a difficult problem to be solved in the food industry and clinical practice.

[0004] With the abuse of antibiotics leading to the emergence of bacteria resistant to "super bacteria", phages are expected to become an important strategy to solve the problem of bacterial resistance. As a natural bactericidal substance, phages have great potential in bactericidal treatment and prevention. Compared with antibiotics, phage treatment has high specificity and effectiveness for target bacteria, and phages do not produce drug resistance. The depolymerase encoded by phages is responsible for stripping bacterial polysaccharides, including extracellular polysaccharide (EPS), capsular polysaccharide (CPS) and lipopolysaccharide (LPS), so that bacteria after phage infection are more susceptible to host immune attack and treatment with antibacterial drugs. Currently, phages have been particularly concerned as an alternative to antibiotics in the fields of agriculture, environment and food.

[0005] SUMMARY

[0006] In view of the lack of effective prevention and treatment means for high virulence drug-resistant Klebsiella pneumoniae (KP) strains, the application provides a bacteriophage capable of specifically lysing high virulence capsular KP, and simultaneously provides a preparation method of the bacteriophage.

[0007] To solve the above technical problems, the application provides the following technical solutions:

[0008] The application provides a bacteriophage capable of specifically lysing high virulence capsular KP, which is named bacteriophage vB_kpnP_D39, and the bacteriophage vB_kpnP_D39 is preserved in the China Center for Type Culture Collection, with a preservation number of CCTCC NO: M 2024690 and a preservation date of April 15, 2024.

[0009] The bacteriophage is a short tail bacteriophage with a size of 40,628 bp, belongs to the Autographiviridae family, and the Przondovirus genus.

[0010] The application provides an application of the bacteriophage vB_kpnP_D39 in specifically lysing K1, K2 and K57 high virulence capsular serotype multidrug-resistant KP strains.

[0011] The application provides an application of the bacteriophage vB_kpnP_D39 in degrading K1, K2 and K57 high virulence capsular serotype KP capsular polysaccharides and biofilms.

[0012] Preferably, in the application, the multiplicity of infection of the bacteriophage vB_kpnP_D39 is (1-10):(1-100000).

[0013] The application provides a preparation method of a bacteriophage liquid preparation of the bacteriophage vB_kpnP_D39 capable of specifically lysing high virulence capsular KP, and the operation steps are as follows:

[0014] (1) The logarithmic phase KP type K57, the bacteriophage vB_kpnP_D39 and LB broth medium are mixed in a mass ratio of 1:1:50, and are cultured at 37°C for 6 hours to obtain a culture solution;

[0015] The logarithmic phase KP and the bacteriophage vB_kpnP_D39 are both obtained by culturing in the LB broth medium.

[0016] (2) The culture solution is centrifuged, the supernatant is taken, and the supernatant is filtered to obtain a bacteriophage vB_kpnP_D39 proliferation liquid; the titer of the bacteriophage vB_kpnP_D39 proliferation liquid is higher than 10 9 PFU / mL.

[0017] (3) in 100 mL of the phage vB_kpnP_D39 proliferation liquid, 0.5 M of sodium chloride (NaCl) and 20% (w / v) of polyethylene glycol 8000 with a final mass-volume concentration were added to obtain a mixture, the mixture was mixed uniformly, incubated overnight at 4°C, centrifuged to obtain a precipitate;

[0018] (4) 5 mL of SM buffer was added to the precipitate and mixed uniformly to obtain a Klebsiella pneumoniae phage concentrated liquid, i.e., a phage liquid preparation;

[0019] The working titer of the Klebsiella pneumoniae phage in the phage liquid preparation is greater than 1×10 9 PFU / mL.

[0020] Preferably, comprising:

[0021] (1) logarithmic phase Klebsiella pneumoniae Kp214, the phage vB_kpnP_D39 described in the above technical solution and LB broth medium were mixed uniformly at a mass ratio of 1:1:50, and cultured at 37°C for 6 h to obtain a culture liquid;

[0022] The logarithmic phase Klebsiella pneumoniae Kp214 and the phage vB_kpnP_D39 are both obtained by culturing in LB broth medium;

[0023] (2) the culture liquid was centrifuged, and the supernatant was taken and filtered to obtain a phage vB_kpnP_D39 proliferation liquid; the titer of the phage vB_kpnP_D39 proliferation liquid is greater than 10 9 PFU / mL;

[0024] (3) in 100 mL of the phage vB_kpnP_D39 proliferation liquid, 0.5 M of sodium chloride (NaCl) and 20% (w / v) of polyethylene glycol 8000 with a final mass-volume concentration were added to obtain a mixture, the mixture was mixed uniformly, incubated overnight at 4°C, centrifuged to obtain a precipitate;

[0025] (4) 5 mL of SM buffer was added to the precipitate and mixed uniformly to obtain a phage vB_kpnP_D39 concentrated liquid, i.e., a phage liquid preparation;

[0026] The working titer of the phage vB_kpnP_D39 in the phage liquid preparation is greater than 1×10 9 PFU / mL.

[0027] Preferably, in step (2), the centrifugation condition is: speed of 12000 rpm, time of 10 min.

[0028] Preferably, in step (3), the centrifugation conditions are: 12000 rpm for 20 min.

[0029] Preferably, the time for the phage liquid preparation to lyse K. pneumoniae or remove the biofilm of K. pneumoniae is 5-6 h.

[0030] The application provides a K. pneumoniae bactericide, which comprises the phage vB_kpnP_D39 or the phage preparation obtained by the preparation method.

[0031] Preferably, the working titer of the K. pneumoniae bactericide is ≥1×10 9 PFU / mL.

[0032] The application provides an application of a culture solution in preventing and controlling biofilm and / or capsular polysaccharide of K. pneumoniae, and the preparation method of the culture solution comprises the following steps: uniformly mixing logarithmic phase K. pneumoniae K57, the phage vB_kpnP_D39 or the phage preparation obtained by the preparation method with LB broth medium at a mass ratio of 1:1:50, and culturing at 37 DEG C for 24 h to obtain the culture solution.

[0033] The phage vB_kpnP_D39 and the preparation can not only effectively kill high virulence K. pneumoniae, but also efficiently degrade the capsule and remove and control the biofilm, and are expected to become a new technology for preventing and treating high virulence K. pneumoniae infection.

[0034] The beneficial technical effects of the application are embodied in the following aspects:

[0035] 1. The phage vB_kpnP_D39 provided by the application can not only specifically lyse high virulence K. pneumoniae of K1, K2 and K57 capsular serotypes, but also has a high inhibitory and removing effect on the capsular polysaccharide and biofilm produced by the K1, K2 and K57 serotype strains. There is no related technical report on the above-mentioned specific action mode of the high virulence K antigen type strain.

[0036] 2. The phage preparation of the application shows a short latent period, a fast outbreak and a high outbreak amount (50 min and 163.6 pfu / cell) through one-step growth curve, and can prepare a large amount of phage preparation in a short period of time; biological genomic analysis shows that the phage does not carry any virulence or drug resistance related genes, and is relatively safe in application.

[0037] 3. The bacteriophage of the present application is highly specific and can lyse 15 of the 29 strains of Klebsiella pneumoniae with high virulence. The optimal multiplicity of infection (MOI) of the bacteriophage is 0.0001, the maximum adsorption efficiency is 99.60% (adsorption time is 6 min), the latent period is 10 min, the burst period is 40 min, and the maximum burst size is 163.64 pfu / cell.

[0038] 4. The bacteriophage of the present application does not contain bacterial drug resistance and virulence genes, and is safe to use.

[0039] Biological preservation instructions

[0040] Klebsiella phage vB_kpnP_D39, deposited on April 15, 2024, at the China Center for Type Culture Collection, with the deposit number CCTCC NO: M 2024690, and the address of the deposit unit being 299, Baoyi Road, Wuchang District, Wuhan, Hubei Province, China. BRIEF DESCRIPTION OF DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below.

[0042] Fig. 1 is a plaque diagram of Klebsiella pneumoniae bacteriophage vB_kpnP_D39 of the present application;

[0043] Fig. 2 is a transmission electron microscope diagram of Klebsiella pneumoniae bacteriophage vB_kpnP_D39 of the present application;

[0044] Fig. 3 is a genome circle diagram of Klebsiella pneumoniae bacteriophage vB_kpnP_D39 of the present application;

[0045] Fig. 4 is a schematic diagram of the genome ANI analysis results of Klebsiella pneumoniae bacteriophage vB_kpnP_D39 of the present application;

[0046] Fig. 5 is a schematic diagram of the phylogenetic tree analysis results of Klebsiella pneumoniae bacteriophage vB_kpnP_D39 of the present application;

[0047] Fig. 6 is a schematic diagram of the genome visualization analysis results of Klebsiella pneumoniae bacteriophage vB_kpnP_D39 of the present application;

[0048] Fig. 7 is a host spectrum and strain characteristic diagram of Klebsiella pneumoniae bacteriophage vB_kpnP_D39 of the present application;

[0049] Fig. 8 is an adsorption curve diagram of Klebsiella pneumoniae bacteriophage vB_kpnP_D39 of the present application;

[0050] Figure 9 is a one-step growth curve of Klebsiella pneumoniae bacteriophage vB_kpnP_D39 of the application.

[0051] Figure 10 is a microscope image of Klebsiella pneumoniae bacteriophage vB_kpnP_D39 of the application digesting capsular polysaccharide.

[0052] Figure 11 is a quantitative detection result image of Klebsiella pneumoniae bacteriophage vB_kpnP_D39 of the application digesting capsular polysaccharide.

[0053] Figure 12 is a quantitative detection result image of Klebsiella pneumoniae bacteriophage vB_kpnP_D39 of the application removing biofilm.

[0054] Figure 13 is a quantitative detection result image of Klebsiella pneumoniae bacteriophage vB_kpnP_D39 of the application controlling biofilm. DETAILED DESCRIPTION

[0055] In order to make the content described in the application more convenient to understand, the technical solutions described in the application will be further described below in combination with specific embodiments and drawings, but the application is not limited thereto.

[0056] In order to be consistent with the preservation certificate, the bacteriophage vB_kpnP_D39 of the application is uniformly named Klebsiella phage vB_kpnP_D39, and the expression "Klebsiella pneumoniae vB_kpnP_D39" in the application is equivalent to "Klebsiella phage vB_kpnP_D39".

[0057] The strains, reagents and media used in the following examples are as follows:

[0058] The host bacteria used in the experiment were Klebsiella pneumoniae Kp214 (K57 type).

[0059] LB (Luria broth) liquid medium (1 L): 10 g of proteose peptone, 5 g of yeast powder, 10 g of sodium chloride, and ultrapure water to 1 L, adjust pH to 7.0, 121℃, 20 min high pressure sterilization.

[0060] 0.4% LB semi-solid medium (1 L): 10 g of proteose peptone, 5 g of yeast powder, 10 g of sodium chloride, 6 g of agar powder, and ultrapure water to 1 L, adjust pH to 7.0, 121℃, 20 min high pressure sterilization.

[0061] 1.5% LB solid medium (1 L): Tryptone 10 g, yeast extract 5 g, NaCl 10 g, agar powder 15 g, add ultrapure water to 1 L, adjust pH to 7.0, 121℃, 20 min high pressure sterilization.

[0062] Double LB medium (1 L): yeast extract 10 g, tryptone 20 g, NaCl 10 g, add ultrapure water to 1 L, 121℃, 20 min high pressure sterilization.

[0063] SM buffer (1 L): weigh 6.055 g Tris-HCl (pH 7.5) to 100 mL, add 5.8 g NaCl, 2.0 g MgSO4, then add 5 mL of 2% gelatin solution, finally add ultrapure water to 1 L.

[0064] Eluent: mix beef extract, Tween 80 and NaCl according to the final concentration of 1-3% (w / v), 3% (v / v) and 50 mM respectively, filter sterilization with 0.45 μm microporous membrane.

[0065] Maneval solution: including 3 g FeCl3, 3.9 mL phenol, 5 mL acetic acid and 0.5 g congo red, add water to 100 ml.

[0066] Crystal violet (0.1% w / v), acid fuchsin (1% w / v), PEG8000, phosphotungstic acid (PTA, 2% w / v) are commercially available.

[0067] Example 1

[0068] Isolation and purification of bacteriophages in the present application

[0069] 1.1 Treatment and enrichment of water samples

[0070] The water sample used in the experiment for isolating bacteriophages in the present application was collected from the sewage of Hefei Wangtang Sewage Treatment Plant in 2023.

[0071] Centrifuge 2 L of water sample at 10,000 rpm for 10 min, collect the supernatant. Vacuum suction filter the supernatant through 0.45 μm microporous membrane in succession, then transfer it to a clean sterile container. Add MgSO4 to a final concentration of 50 mM. Stir well, stand for 15 min. Vacuum suction filter the mixture, discard the supernatant. Cut the filter membrane into pieces and put it into a clean sterile beaker, add an appropriate amount of eluent, stir well. Transfer the beaker to an ultrasonic cleaner, ultrasonic elution for 5 min. It is the bacteriophage mixture.

[0072] Inoculate host bacteria Kp214 on agar medium by streaking, incubate overnight, then pick single colonies and inoculate in 5 mL of LB liquid medium, incubate at 37℃ for 8 h as host bacteria culture for standby.

[0073] Under sterile conditions, one loop of Klebsiella pneumoniae was taken and streaked onto a chromogenic plate and incubated at 37°C overnight. Single colonies were picked from the plate and inoculated into 5 mL LB medium and incubated at 37°C overnight. The activated strain was inoculated into 5 mL LB medium at 2% inoculation and incubated at 37°C until early exponential growth phase (OD 600 =0.2). An equal volume of phage mixture was mixed with double volume of LB medium and then added with the bacterial solution grown to early exponential growth phase and incubated at 37°C, 200 rpm for about 6 hours. Centrifugation was performed at 4000 rpm for 15 min and the supernatant was filtered through a microporous filter into a new sterile tube, which was the phage sample.

[0074] 1.2 Purification and propagation, concentration of phage

[0075] Each 100 μL of phage and overnight activated strain was mixed and then mixed with 5 mL 0.4% LB soft agar and spread onto the surface of 1.5% LB agar. Incubation was performed at 37°C for 6 hours. Single phage plaques were picked in SM buffer solution and the purification step was repeated at least three times to obtain single clear plaques with translucent halos around them, as shown in Figure 1. The present application named it as: vB_kpnP_D39.

[0076] Sodium chloride at a final concentration of 0.5 M and 20% polyethylene glycol were added to the phage sample and mixed well, and the phage particles were precipitated by overnight treatment at 4°C. The supernatant was removed after centrifugation of the suspension at 12000 rpm for 20 min. The obtained precipitate was resuspended in SM buffer solution. It was stored in a refrigerator at 4°C for standby use.

[0077] 1.3 Electron microscope observation of phage

[0078] The purified phage suspension was dropped onto a carrier net covered with film, and after 2 min, the filter paper was used to absorb it. One drop of 2% phosphotungstic acid was dropped, and after 1 min of staining, the excess liquid was absorbed. Drying was performed in air, and then transmission electron microscope observation was performed. As shown in Figure 2, the vB_kpnP_D39 phage belongs to short-tailed phage, with a head of 50±2 nm and a tail of 11±2 nm.

[0079] Example 2

[0080] Phage whole genome analysis

[0081] The phage DNA obtained using the viral genome extraction kit was sequenced using Ion Torrent S5 (Thermo Fisher Scientific, USA). The assembled whole genome sequence and amino acid sequence of the encoded protein were obtained. First, the whole genome circle diagram of the phage and the encoded gene product were drawn by the Proksee software, as shown in Figure 3, the functional proteins of the phage vB_kpnP_D39 in the application were divided into several functional modules: lysis module, packaging module, structural module, DNA replication and regulation module, and other functional modules. Second, sequence comparison analysis was performed in the NCBI database and downloaded. ANIm was used for analysis. The data obtained by ANIm was drawn into a heat map by TBtools, as shown in Figure 4, it was found that the phage vB_kpnP_D39 in the application was a new species of phage compared with other phages, the maximum coverage was 93% and the similarity was 95%. Finally, 20 strains of Klebsiella pneumoniae phage were downloaded from the ICTV database, which all contained RNA polymerase, and the related phylogenetic tree was constructed by MEGA and TBtools, as shown in Figure 5. The RNA polymerase phylogenetic tree based on the Klebsiella pneumoniae phage of different genera in the Autographiviridae family showed that the phage vB_kpnP_D39 in the application was in the Autographiviridae family and the Przondovirus genus. As shown in Figure 6, the phage genome visualization analysis result was the same as Figure 3, the functional proteins of the phage vB_kpnP_D39 in the application were divided into five functional modules, and there were 48 ORFs, of which 38 were functional ORFs and 10 were annotated as hypothetical proteins.

[0082] Example 3

[0083] Determination of the host spectrum of the phage

[0084] After 0.1 mL of the logarithmic phase bacterial culture solution of the 45 strains in Table 1 was gently mixed with LB soft agar medium (5 mL) containing 0.4% agar, it was poured onto a general LB bottom plate and dried. Then, 5 μL of the gradient dilution solution of the phage vB_kpnP_D39 propagation liquid was spotted onto the solidified upper plate, naturally dried and cultured at 37°C for 6 h. The appearance of the phage plaques indicated that the bacterial strain was a sensitive host bacterium. As can be seen from Table 1 and Figure 7, the phage of the application efficiently and specifically lysed Klebsiella pneumoniae of K1, K2 and K57 serotypes, and had no effect on Klebsiella pneumoniae of other capsule serotypes.

[0085] Referring to FIG. 7, the lysis effect, the capsular polysaccharide production, and the drug resistance of the bacteria to antibiotic drugs, including: meropenem (MEM), ceftazidime (CAZ), cefotaxime (CTX), tobramycin (TOB), cephalothin (CEP), ceftriaxone (CRO), cefepime (FEP), cefazolin (CZO), trimethoprim-sulfamethoxazole (SXT), ampicillin (AMP), amikacin (AMK), ciprofloxacin (CIP), tetracycline (TCY), chloramphenicol (CHL), nalidixic acid (NAL), aztreonam (ATM), gentamicin (GEN), kanamycin (KAN), streptomycin (STR), ampicillin-sulbactam (SAM), of the bacteriophage vB_kpnP_D39 in the present application were shown. The results showed that the host bacteria of the bacteriophage vB_kpnP_D39 in the present application all showed multiple drug resistance, thereby indicating that the vB_kpnP_D39 is expected to become a good antibiotic substitute.

[0086] Table 1. Host spectrum of the bacteriophage vB_kpnP_D39

[0087] Note: "+" in Table 1 represents positive, and the lysis intensity of the bacteriophage is divided into strong, moderate and low results according to +++, ++, +.

[0088] Example 4

[0089] Optimal multiplicity of infection (MOI) of the bacteriophage (MOI is the ratio of the number of bacteriophages to the number of host bacteria at the initial stage of infection)

[0090] Strains (K57 type) in the host bacteria culture prepared in Example 1 were taken, and the concentration was adjusted to 1x10 8 cfu / mL, 0.1 mL of the bacteriophage prepared in Example 1 and the host bacteria culture prepared in Example 1 were added in the ratio of 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, respectively, and 4.8 mL of LB liquid medium was added. After incubation at 37°C for 6 h on a shaker, centrifugation was performed at 7000 rpm for 10 min, the supernatant was collected and diluted to an appropriate concentration, and the titer was determined by double-layer agar plate method. The results are shown in Table 2, and the optimal MOI of the Klebsiella pneumoniae bacteriophage vB_kpnP_D39 is 0.0001.

[0091] Table 2. Optimal multiplicity of infection (MOI) of the Klebsiella pneumoniae bacteriophage vB_kpnP_D39

[0092] Example 5

[0093] Determination of the adsorption curve of the Klebsiella pneumoniae bacteriophage vB_kpnP_D39

[0094] The strain (K57 type, ~10) from the host bacterial culture prepared in Example 1 was used. 8 A mixture of cfu / mL and an equal volume of phage (MOI = 0.1) prepared in Example 1 was placed in a sterile Erlenmeyer flask and incubated at 37°C in a water bath. 500 μL of the mixture was collected at 0, 2, 4, 6, 8, 10, 12, 14, 16, and 18 min, and centrifuged at 7000 rpm for 10 min. The supernatant phage lysate was then obtained by filtration. The supernatant was diluted to an appropriate concentration, and the phage titer was determined using a two-layer chromatography method. Adsorption curves were plotted with sampling time on the x-axis and phage titer on the y-axis. The adsorption curve results are shown in Figure 8, with a maximum adsorption efficiency of 99.60% and an adsorption time of 6 min.

[0095] Example 6

[0096] Determination of one-step growth curve of Klebsiella pneumoniae bacteriophage vB_kpnP_D39

[0097] The K57 strain from the host bacterial culture prepared in Example 1 was mixed with an excess of the bacteriophage prepared in Example 1 (MOI = 0.1). After incubating at 37°C for 6 min, the mixture was centrifuged at 12000 rpm for 2 min, and the supernatant was discarded. The precipitate was washed twice with LB-Ca liquid medium (containing 4 mM CaCl2). The precipitate was resuspended in 30 mL of preheated LB liquid medium and quickly placed in a shaker at 37°C for incubation. Before incubation, the amount of free bacteriophage in the discarded supernatant was counted to calculate the amount of bacteriophage adsorbed. Then, starting from 0 min, 1 mL of culture was taken every 5 min in the early stage and every 10 min in the later stage. The culture was centrifuged at 12000 rpm for 30 s at 4°C to remove bacteria. The supernatant was diluted to an appropriate concentration, and the bacteriophage titer was determined by the two-layer method. The test was conducted for 90 min, and a total of 12 samples were taken. A one-step growth curve was plotted with the sampling time as the x-axis and the bacteriophage titer as the y-axis to obtain the phage incubation period, outbreak period, and outbreak amount. The one-step growth curve results are shown in Figure 9. The incubation period for infection with the host bacteria was 10 min, the outbreak period was 40 min, the outbreak amount was 163.64 pfu / cell, and then it entered the stationary phase.

[0098] Example 7: Klebsiella pneumoniae phage vB_kpnP_D39 preparation

[0099] This application also provides a method for preparing a liquid phage formulation based on the precise control of Klebsiella pneumoniae and its capsular polysaccharide and biofilm. The method involves culturing the phage in a culture medium containing the host bacteria, removing the host bacteria to obtain phage lysate. The removal of the host bacteria can be achieved through filtration or centrifugation. The host bacteria is Klebsiella pneumoniae, and the culture medium can be LB broth.

[0100] The preparation procedure of the bacteriophage preparation based on the bacteriophage of the present application is as follows:

[0101] (1) The logarithmic phase Klebsiella pneumoniae, Klebsiella pneumoniae bacteriophage and LB broth medium were mixed uniformly at a mass ratio of 1:1:50, and cultured at 37°C for 6h to obtain a culture solution; the logarithmic phase Klebsiella pneumoniae and Klebsiella pneumoniae bacteriophage were both obtained by culturing in LB broth medium; the titer of the culture solution was 10 9 PFU / mL or more.

[0102] (2) The culture solution was centrifuged at a speed of 12000 rpm for 10 min. The supernatant was taken, and the supernatant was filtered to obtain a Klebsiella pneumoniae bacteriophage proliferation solution.

[0103] (3) 0.5M sodium chloride (NaCl) and 20% (w / v) polyethylene glycol 8000 were added to 100mL of the Klebsiella pneumoniae bacteriophage proliferation solution to obtain a mixture, and the mixture was mixed uniformly and centrifuged at a speed of 12000 rpm for 15 min to obtain a precipitate.

[0104] (4) 5mL of SM buffer was added to the precipitate and mixed uniformly to obtain a Klebsiella pneumoniae bacteriophage concentrate, i.e., a bacteriophage liquid preparation.

[0105] The working titer of the Klebsiella pneumoniae bacteriophage in the bacteriophage liquid preparation was greater than 1×10 9 PFU / mL.

[0106] Example 8

[0107] Microscopic observation of the digestion of capsular polysaccharide by Klebsiella pneumoniae bacteriophage preparation

[0108] Three strains (including K1, K2, and K57 type each 1 strain) in the host bacteria culture prepared in Example 1 were mixed with the bacteriophage preparation (>10 9 PFU / mL) prepared in Example 7 and LB broth medium at a mass ratio of 1:1:50, and cultured at 37°C on a shaker for 24h, then centrifuged at 4000 rpm for 10 min, and the precipitate was washed with sterile normal saline (the operation was repeated twice). Subsequently, the culture was streaked on LB plates, and single colony bacteria were picked (10 strains were picked from each plate), and the resistance to the bacteriophage vB_kpnP_D39 was verified. The above operation was repeated three times to subculture the bacteriophage-resistant strains.

[0109] Take 100 μL of bacterial solution (wild-type and resistant strains) and mix with an equal volume of 1% aqueous solution of Congo red for 1 min. Take 5 μL of the mixture and evenly spread on a glass slide to form a thin film. Then use 5 μL of Maneval solution to gently spread on the film area and dry thoroughly; use an optical microscope imaging system to observe and take pictures; see Figures 10 A, B and C for serotypes K1, K2, K57 strains, respectively, with the capsule structure clearly visible (white halo) around the bacterial body, while Figures 10 D, E and F show that the capsule around the bacterial body of serotypes K1, K2, K57 treated with phage is significantly reduced.

[0110] Example 9

[0111] Quantitative detection of the digestion of capsular polysaccharide by Klebsiella pneumoniae phage preparation

[0112] Crude capsular polysaccharide

[0113] Take 100 μL of bacterial solution from the culture of the three strains (including one each of K1, K2, K57) of the host bacteria prepared in Example 1 and the culture of the phage-resistant strains prepared in Example 8 and evenly spread on LB agar plates, and incubate at 37°C for 48 h. Add 3 mL of normal saline to each plate, repeatedly blow and scrape the bacteria on the surface of the plate, centrifuge at 4000 rpm to collect the precipitate, wash with normal saline for 2-3 times, add bacterial lysis solution to the precipitate at 10% of the original volume, mix well, and lyse the bacteria by ultrasonic treatment at 200 W for 20 min. Then, centrifuge at 10000 rpm for 10 min to collect the supernatant. Add 10% CTAB solution to the supernatant to a final concentration of 1%, mix well and stand to form a precipitate, centrifuge at 10000 rpm for 10 min to collect the precipitate. Add 1 M CaCl2 solution to a final concentration, shake on a shaker for 1 h, centrifuge at 10000 rpm for 10 min to collect the supernatant. Add 95% ethanol to a final concentration of 25%, and stand at 4°C overnight. Centrifuge at 10000 rpm for 10 min to collect the supernatant. Then, add pre-cooled 95% ethanol to a final concentration of 80%, mix well, and stand at room temperature until the capsular polysaccharide is completely dissolved, centrifuge at 10000 rpm for 10 min to collect the precipitate. Wash the precipitate with anhydrous ethanol once to obtain the capsular polysaccharide extract.

[0114] Quantification of capsular polysaccharide

[0115] Weigh 200 mg of glucose, add ultrapure water to make up to 100 mL, mix well by inverting, and prepare for use. Add excess phenol to water, heat and stir to dissolve, cool to room temperature, and take the supernatant as the saturated phenol solution.

[0116] Take 500 μL saturated phenol solution, add deionized water to 10 mL, upside down and mix well, this is the volume fraction of 5% phenol solution. The phenol solution is placed in an ice water bath, and concentrated sulfuric acid is added to the 5% phenol solution at a ratio of 5:1 and mixed well, which is the chromogenic solution, and is stored at room temperature in the dark.

[0117] Take 0.0, 200, 400, 600, 800, 1000 μL of glucose standard solution respectively and dilute in 5 mL water, add 200 μL of glucose diluent, 200 μL of 5% phenol solution and 700 μL of concentrated sulfuric acid to 1.5 mL of EP tube, mix well and react at 80°C for 10 min, and then use a cold water bath for 5 min. Then take 200 μL of the mixture from each EP tube and add it to a 96-well plate, make three parallels, detect the optical density at 490 nm by automatic enzyme marker, take the glucose concentration in the mixture as the horizontal coordinate, OD 490 as the vertical coordinate, and make a sugar standard curve. The final sugar standard curve is y=0.0032x+0.476, R 2 =0.9963.

[0118] Take the optical density of the sample to be tested as the y value and bring it into the standard curve to calculate the x value, which is the sugar concentration, and then calculate the polysaccharide content.

[0119] As shown in FIG. 11, the results show that the digestion rates of the phage vB_kpnP_D39 on the capsular polysaccharides of K. pneumoniae K1, K2 and K57 type strains are 60.6%, 42.1% and 59.8%, respectively.

[0120] Example 10

[0121] Quantitative detection of Klebsiella pneumoniae phage preparation for removing Klebsiella pneumoniae biofilm

[0122] Three strains (including one K1, one K2 and one K57 type) in the host bacterial culture prepared in Example 1 were diluted with physiological saline to the same bacterial concentration, and inoculated into a 96-well cell culture plate containing 200 μL of LB medium at a bacterial amount of 2%, and cultured in a 37°C incubator for 36 h.

[0123] After 36 h of culture, the cell culture plate was taken out, the culture medium was discarded with a pipette gun, and the free bacteria were removed by washing twice with sterilized PBS buffer to obtain mature biofilm.

[0124] The phage vB_kpnP_D39 preparation (>10 9PFU / mL) was added to each well of the 96-well cell culture plate containing the biofilm and an equal amount of normal saline (as a negative control). After incubation in a 37°C incubator for 6 h, the liquid was discarded and the wells were gently washed twice with sterile PBS buffer to remove free biofilm. Then 200 μL of methanol was added to each well and allowed to fix for 30 min. After the fixing solution was discarded and allowed to air dry, the biofilm was stained with 200 μL of 0.1% crystal violet at room temperature for 20 min. After the staining was completed, the staining solution was discarded and the wells were washed twice with PBS buffer to remove free staining solution. The cell culture plate was placed in a 65°C oven to dry, and after removal, 200 μL of a 33% acetic acid solution was added to each well and placed in a 37°C incubator for 30 min to release the crystal violet from the biofilm. Finally, the absorbance at 590 nm was measured using a microplate reader. The amount of biofilm remaining was determined based on the size of the OD 590 value.

[0125] Referring to FIG. 12, the clearance rate of the bacteriophage vB_kpnP_D39 lysate on the biofilm formed by Klebsiella pneumoniae K1, K2, and K57 type strains was 59.3%, 47.4%, and 63.2%, respectively.

[0126] Example 11

[0127] Quantitative detection of Klebsiella pneumoniae bacteriophage for controlling biofilm formation by Klebsiella pneumoniae

[0128] The three strains (including one K1, one K2, and one K57 type strain) in the host bacterial culture prepared in Example 1 were diluted to 10 4 PFU / mL with LB broth medium, and then 200 μL of the mixture was taken to a 96-well cell culture plate. After 24 h of incubation, the cell culture plate was removed, the culture medium was discarded with a pipette, and the wells were washed twice with sterile PBS buffer to remove free bacteria and bacteriophage solution. Then 200 μL of methanol was added to each well and allowed to fix for 30 min. After the fixing solution was discarded and allowed to air dry, the biofilm was stained with 200 μL of 0.1% crystal violet at room temperature for 20 min. After the staining was completed, the staining solution was discarded and the wells were washed twice with PBS buffer to remove free staining solution. The cell culture plate was placed in a 65°C oven to dry, and after removal, 200 μL of a 33% acetic acid solution was added to each well and placed in a 37°C incubator for 30 min to release the crystal violet from the biofilm. Finally, the absorbance at 590 nm was measured using a microplate reader. The amount of biofilm remaining was determined based on the size of the OD 590 value.

[0129] Referring to Fig. 13, the inhibition rates of the phage vB_kpnP_D39 lysate on the biofilm formed by Klebsiella pneumoniae K1, K2 and K57 type strains were 71.1%, 69.0% and 77.1%, respectively.

[0130] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which all belong to the protection scope of the present application.

Claims

1. A bacteriophage vB_kpnP_D39 that specifically lyses highly virulent capsular Klebsiella pneumoniae, with accession number CCTCC NO: M 2024690.

2. The application of the bacteriophage vB_kpnP_D39 according to claim 1 in lysing highly virulent capsular serotypes of multidrug-resistant Klebsiella pneumoniae (K1, K2, K57).

3. The application of the bacteriophage vB_kpnP_D39 according to claim 1 in the degradation of capsular polysaccharides and biofilms of highly virulent capsular serotypes Klebsiella pneumoniae K1, K2, and K57.

4. The application according to claim 2 or 3, characterized in that, When the application is described, the infection multiplicity of bacteriophage vB_kpnP_D39 is (1-10):(1-100000).

5. A method for preparing a liquid phage formulation based on phage vB_kpnP_D39 according to claim 1, characterized in that, include: (1) Klebsiella pneumoniae K57 in logarithmic phase, bacteriophage vB_kpnP_D39 as described in claim 1, and LB broth medium were mixed evenly at a mass ratio of 1:1:50 and cultured at 37°C for 6 hours to obtain the culture medium. The Klebsiella pneumoniae Kp214 and bacteriophage vB_kpnP_D39 in the logarithmic phase were both obtained by culturing in LB broth medium. (2) Centrifuge the culture medium, collect the supernatant, filter the supernatant to obtain the phage vB_kpnP_D39 proliferation solution; the titer of the phage vB_kpnP_D39 proliferation solution is 10. 9 PFU / mL or higher; (3) Add sodium chloride with a final concentration of 0.5M and polyethylene glycol 8000 with a final mass volume concentration of 20% (w / v) to 100mL of phage vB_kpnP_D39 proliferation solution to obtain a mixture. Mix the mixture evenly, incubate overnight at 4℃, and centrifuge to obtain the precipitate. (4) Add 5 mL of SM buffer to the precipitate and mix well to obtain phage vB_kpnP_D39 concentrate, i.e., phage liquid preparation; The working potency of bacteriophage vB_kpnP_D39 in the liquid bacteriophage formulation is greater than 1×10⁻⁶. 9 PFU / mL.

6. The preparation method according to claim 5, characterized in that, include: (1) Klebsiella pneumoniae Kp214 in logarithmic phase, bacteriophage vB_kpnP_D39 as described in claim 1, and LB broth medium were mixed evenly at a mass ratio of 1:1:50 and cultured at 37°C for 6 hours to obtain a culture solution. The Klebsiella pneumoniae Kp214 and bacteriophage vB_kpnP_D39 in the logarithmic phase were both obtained by culturing in LB broth medium. (2) Centrifuge the culture medium, collect the supernatant, filter the supernatant to obtain the phage vB_kpnP_D39 proliferation solution; the titer of the phage vB_kpnP_D39 proliferation solution is 10. 9 PFU / mL or higher; (3) Add sodium chloride with a final concentration of 0.5M and polyethylene glycol 8000 with a final mass volume concentration of 20% (w / v) to 100mL of phage vB_kpnP_D39 proliferation solution to obtain a mixture. Mix the mixture evenly, incubate overnight at 4℃, and centrifuge to obtain the precipitate. (4) Add 5 mL of SM buffer to the precipitate and mix well to obtain phage vB_kpnP_D39 concentrate, i.e., phage liquid preparation; The working potency of bacteriophage vB_kpnP_D39 in the liquid bacteriophage formulation is greater than 1×10⁻⁶. 9 PFU / mL.

7. The preparation method according to claim 5 or 6, characterized in that, In step (2), the centrifugation conditions are 12,000 rpm and 10 min.

8. The preparation method according to claim 5 or 6, characterized in that, In step (3), the centrifugation conditions are 12,000 rpm and 20 min.

9. The preparation method according to claim 5 or 6, characterized in that, The phage liquid preparation is used to lyse Klebsiella pneumoniae or remove the biofilm of Klebsiella pneumoniae for 5 to 6 hours.

10. A bactericide against Klebsiella pneumoniae, characterized in that, The bacteriophage liquid preparation includes the bacteriophage vB_kpnP_D39 as described in claim 1 or the bacteriophage liquid preparation obtained by the preparation method described in any one of claims 5 to 9.

11. The Klebsiella pneumoniae bactericide according to claim 10, characterized in that, The potency of the Klebsiella pneumoniae bactericide is ≥1×10⁻⁶. 9 PFU / mL.

12. The application of a culture medium in controlling the growth of biofilms and / or capsular polysaccharides of Klebsiella pneumoniae, wherein the preparation method of the culture medium includes: At a mass ratio of 1:1:50, Klebsiella pneumoniae K57 in logarithmic phase, bacteriophage vB_kpnP_D39 as described in claim 1, or the liquid bacteriophage preparation obtained by the preparation method described in any one of claims 5 to 9, are mixed evenly with LB broth medium and incubated at 37°C for 24 hours to obtain the culture medium.

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