Klebsiella pneumoniae bacteriophage vBKpnPGZMUVR2301 and application thereof

Abstract

The invention discloses a klebsiella pneumoniae bacteriophage vBKpnPGZMUVR2301 and application thereof, and relates to the technical field of biology. The klebsiella pneumoniae bacteriophage vBKpnPGZMUVR2301 is preserved in Guangdong Microbial Culture Collection Center on October 14, 2024, the preservation address is the 5th floor of the building 59, No.100 Courtyard, Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC No: 65268-B1. The bacteriophage keeps stable titer in a wide pH range (4-10) and a temperature interval (4-50 DEG C), and the highest titer can reach more than 109 pfu / mL. The bacteriophage can efficiently split klebsiella pneumoniae. The invention provides powerful technical support for developing novel antibacterial drugs and treatment schemes for klebsiella pneumoniae, and shows wide application prospect and potential clinical value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a Klebsiella pneumoniae bacteriophage vB_KpnP_GZMU_VR2301 and application thereof. BACKGROUND

[0002] In recent years, the drug resistance problem of Klebsiella pneumoniae has gradually intensified, especially the emergence of multiple drug-resistant strains resistant to carbapenem antibiotics, which has greatly reduced the efficacy of traditional antibiotics. Bacteriophage therapy is a promising alternative treatment method. Unlike broad-spectrum antibiotics, bacteriophages can precisely target specific bacteria, reducing damage to normal flora. Therefore, bacteriophage therapy is considered as one of the feasible paths to solve the problem of antibiotic resistance.

[0003] Although bacteriophage therapy has shown certain therapeutic effects in theoretical and experimental studies, its promotion in clinical application still faces many technical challenges. First, it is necessary to screen bacteriophages with high lytic effect on bacteria, second, to explore effective treatment often requires the use of high concentrations of bacteriophages, high concentration of bacteriophages application not only increases the cost and complexity of large-scale production, but also may increase the potential risk in certain cases, and how to combine bacteriophages with other treatment methods, etc. Problems, further in-depth research is needed.

[0004] Baicalin is the main active component of traditional Chinese medicine Scutellaria baicalensis Georgi, which has broad-spectrum antibacterial and immunomodulatory effects. The present application aims to explore the antibacterial effect of baicalin combined with bacteriophages, and to provide a new idea for reducing the concentration of bacteriophages. SUMMARY

[0005] The purpose of the present application is to provide a Klebsiella pneumoniae bacteriophage vB_KpnP_GZMU_VR2301 and its application, in order to solve the problems existing in the prior art. The bacteriophage can lyse Klebsiella pneumoniae, providing technical support for the development of new antibacterial drugs and antibacterial schemes for inhibiting Klebsiella pneumoniae, and has good application prospect.

[0006] To achieve the above purpose, the present application provides the following scheme:

[0007] The present application provides a Klebsiella pneumoniae bacteriophage (Klebsiella phage) vB_KpnP_GZMU_VR2301, which was deposited in the Guangdong Microbial Culture Collection Center on October 14, 2024, and the deposit address is No. 59 Building, 5th Floor, Guangzhou Xianlie Middle Road 100 Courtyard, Guangzhou, China, and the deposit number is GDMCC No: 65268-B1.

[0008] The application further provides application of the Klebsiella pneumoniae bacteriophage vB_KpnP_GZMU_VR2301 in the above aspect in the preparation of a medicine for preventing and / or treating Klebsiella pneumoniae infection.

[0009] The application further provides application of the Klebsiella pneumoniae bacteriophage vB_KpnP_GZMU_VR2301 in the above aspect in the preparation of a Klebsiella pneumoniae fungicide.

[0010] Further, the Klebsiella pneumoniae is carbapenem-resistant Klebsiella pneumoniae.

[0011] The application further provides a medicine for preventing and / or treating Klebsiella pneumoniae infection, wherein an active ingredient comprises the Klebsiella pneumoniae bacteriophage vB_KpnP_GZMU_VR2301 in the above aspect.

[0012] Further, the medicine further comprises a pharmaceutically acceptable excipient.

[0013] Further, the medicine is in the form of an injection, a powder, a gel, a granule or a lyophilized agent.

[0014] Further, the medicine further comprises other active ingredients having a bacteriostatic effect on Klebsiella pneumoniae.

[0015] The application further provides a Klebsiella pneumoniae fungicide, wherein an active ingredient comprises the Klebsiella pneumoniae bacteriophage vB_KpnP_GZMU_VR2301 in the above aspect.

[0016] Further, the Klebsiella pneumoniae fungicide is in the form of a spray, a powder, a gel, a granule or a lyophilized agent.

[0017] The application further provides a composition having a bacteriostatic effect, comprising baicalin and a lytic bacteriophage with a bacterium as a host.

[0018] Further, the lytic bacteriophage is the Klebsiella pneumoniae bacteriophage vB_KpnP_GZMU_VR2301 in the above aspect.

[0019] The application further provides application of the composition in the above aspect in the preparation of an antibacterial medicine.

[0020] The application discloses the following technical effects:

[0021] The application successfully finds a novel Klebsiella pneumoniae bacteriophage vB_KpnP_GZMU_VR2301, and the bacteriophage shows typical lysis characteristics. 9The phage can effectively lyse Klebsiella pneumoniae, especially carbapenem-resistant Klebsiella pneumoniae (CRKP).

[0022] The phage is combined with baicalin, and a synergistic effect is generated, so that the inhibitory effect on Klebsiella pneumoniae is effectively improved.

[0023] The present application provides strong technical support for developing new antibacterial drugs and treatment plans for Klebsiella pneumoniae, and has broad application prospects and potential clinical value. BRIEF DESCRIPTION OF DRAWINGS

[0024] 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. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0025] Figure 1 It is a phage morphology diagram;

[0026] Figure 2 It is a phylogenetic analysis diagram based on distance method of whole genome sequence;

[0027] Figure 3 It is a phage particle electron microscope diagram;

[0028] Figure 4 It is a thermal stability determination result diagram;

[0029] Figure 5 It is a pH stability determination result diagram;

[0030] Figure 6 It is a lysis kinetics determination result diagram;

[0031] Figure 7 It is a bacteriostatic effect detection result diagram of different MOI after 7h;

[0032] Figure 8 It is a bacteriostatic effect detection result diagram of different MOI after 12h;

[0033] Figure 9 It is a phage inhibition effect result diagram on biofilm;

[0034] Figure 10 It is a determination result diagram of bacteriostatic effect of phage on Klebsiella pneumoniae combined with baicalin after 14h of culture;

[0035] Figure 11The results of the determination of the bacteriostatic effect of the combination of E. coli bacteriophage and baicalin after 14 h of incubation are shown in the figure. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of the application and not as limiting the application. It will be understood that the detailed description and specific examples, while indicating certain aspects of the application, are given by way of illustration only, since various changes and modifications within the spirit and scope of the application will become apparent to those skilled in the art from this detailed description.

[0037] It should be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the application. Further, for a range of values, it is to be understood that each intervening value, to the upper or lower limit of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is also specifically disclosed. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in detail the methods and / or materials which are described in them. In the case of conflict between the present specification and any document incorporated herein by reference, the present specification will control.

[0039] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0040] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional, unrecited elements or method steps.

[0041] The baicalin referred to in the present application has a CAS number of 21967-41-9, a molecular formula of C 21 H 18 O 11 and a structural formula as follows:

[0042]

[0043] Example 1 Isolation and identification of bacteriophage

[0044] 1. Isolation and purification of bacteriophage

[0045] The lake water and sewage mixture was collected from Guangzhou Medical University and the Second Affiliated Hospital of Guangzhou Medical University in Guangdong, China. After centrifugation, the larger impurities and most bacteria were removed by 0.22 μm microporous filter membrane. Klebsiella pneumoniae was used as the host bacteria, and the strain was inoculated in LB liquid medium and incubated at 37°C on a shaking table until the OD 600 = 0.6. 10 mL of the filtrate and 1 mL of the host bacteria were taken, 5 mL of three times concentrated LB nutrient broth was added, and they were co-cultured overnight at 37°C on a constant temperature shaking table. After centrifugation, the supernatant was filtered through a 0.22 μm microporous filter to obtain the first enrichment liquid, and the above operation was repeated to obtain the second enrichment liquid. The presence of phage plaques was detected by spot test. The phage was purified on the plate by double-layer agar plate method until the plaque size was uniform. After purification, a lytic phage Klebsiella phage vB_KpnP_GZMU_VR2301 (hereinafter referred to as VR2301) was obtained, which had a circular transparent plaque and a large plaque, as shown in Figure 1 .

[0046] 2. Bacteriophage titer determination

[0047] The double-layer plate method was used to determine the titer of the phage. The phage liquid was gradiently diluted, 100 μL of the diluted phage liquid was mixed with the logarithmic phase host bacteria at a ratio of 1:1, and then incubated at 37°C for 15 min. Then 0.7% semi-solid LB agar was uniformly spread on the top of 1.5% solid LB agar pre-poured on a culture dish, and then placed in a 37°C incubator for overnight culture. Several transparent plaques were observed on the plate, and the plate with 30-300 plaques in the field of view was selected for counting. The titer (PFU / mL) = plaque number x 10 x dilution factor. The results showed that the phage VR2301 had typical lytic phage characteristics, and the titer of the phage could be as high as more than 10 9 pfu / mL.

[0048] 3. Whole genome analysis of the phage

[0049] The phage was subjected to single viral genome sequencing, and whole genome sequencing of the phage was performed using the Illumina sequencing platform. After obtaining the sequencing data, the quality of the sequencing data was controlled, that is, the low-quality data was evaluated and removed to ensure the reliability of the subsequent analysis results. After removing the host contamination, genome assembly was performed using Megahit, and Checkv was used to evaluate the assembly effect. The virulence factors and antibiotic resistance genes in the phage genome were compared with the VFDB database (http: / / www.mgc.ac.cn / VFs / ) and the CARD database (https: / / card.mcmaster.ca / ). The results showed that the genome size of the phage VR2301 was 40600 bp, the GC content was 53.4%, no virulence genes and antibiotic resistance genes were found in the phage genome, indicating the safety of the phage at the genetic level.

[0050] According to the standard of the International Committee on Taxonomy of Viruses (ICTV), when the genome sequence similarity of two phages is less than 95%, they should be classified as different species. The phage genome sequences similar to VR2301 were downloaded from the Genbank database, and the similarity between genomes was calculated using VIRIDIC. The results showed that VR2301 had the highest similarity of 94.626% with Klebsiella_phage_phi1_146027 (Genbank No: PP889477.1). Using MEGA 11.0.13 software, a phylogenetic tree was constructed by 1000 bootstrap repeats using the distance method, as shown in Figure 2 According to the classification guidelines of BAVS, viruses with nucleotide sequence similarity of more than 50% in the virus population can be classified as the same genus, and new species have more than 5% difference at the nucleotide level from existing species. Given that the genome of VR2301 has most of the similarity with other phages in the Przondovirus genus falling within the range of 50%-95%, VR2301 is determined to be a new phage species of the Przondovirus genus.

[0051] 4. Preservation of the phage

[0052] The phage VR2301 was preserved in the Guangdong Microbial Culture Collection Center on October 14, 2024, at the address of No. 59, Building 5, Guangzhou Xianlie Middle Road 100, and the preservation number is GDMCC No: 65268-B1.

[0053] Example 2 Determination of phage host spectrum

[0054] The host range of phage VR2301 was determined by detecting different strains through spot test. The logarithmic phase bacterial solution was mixed with 0.7% semi-solid LB agar to prepare a bacterial plate. 5-10 μL of phage droplet was added to the surface of the inoculated agar plate. The droplet position was noted to avoid multiple droplets crossing and to ensure that the range of each phage could be clearly observed. The plate was incubated in a 37°C incubator overnight, and the results on the agar plate were observed. If the phage could attack and lyse the host, a transparent lysed area or light circle would form at the droplet position. The results are shown in Table 1, and the lysis rate of phage VR2301 was 63.46% (33 / 52).

[0055] Table 1 Determination of the host spectrum of 52 strains of Klebsiella pneumoniae by phage ("++" for strong lysis, "+" for lyable, "-" for non-lyable)

[0056]

[0057] Example 3 Biological properties of the phage

[0058] 1. Morphological observation of the phage

[0059] Phosphotungstic acid negative staining method was used for observation. Activated phage VR2301 was attached to a 400-mesh carbon film copper grid, allowed to adsorb, and after 2 min, the residual liquid around the copper grid was absorbed with filter paper. 2% phosphotungstic acid was dropped on the copper grid for 1 min, the dye was absorbed with filter paper, and deionized water was used for washing twice. The sample was allowed to dry. Hitachi electron microscope was used for observation, the acceleration voltage was set to 80 kV, and ImageJ was used to measure the structure size of the phage. As shown in Figure 3 , VR2301 is a short-tailed phage, the tail is not retractable, the tail length is 16 ± 0.4 nm, the tail width is 12 ± 3 nm, the head is a icosahedral structure, and the head length is 60 ± 1.5 nm.

[0060] 2. Thermal stability

[0061] In order to evaluate the effect of different temperatures on the titer of the phage, 100 μL of phage solution was added to 900 μL of sterile LB liquid medium pretreated at different temperatures, and the temperature conditions were 4°C, 37°C, 50°C, 60°C, and 70°C, respectively, and the sample was allowed to stand for 1 hour. Double-layer agar plate method was used for titer determination, and the experimental results are shown in Figure 4 . The phage VR2301 remained relatively stable in the temperature range of 4°C to 50°C, and the titer could reach 10 8 PFU / mL or more. At 60°C, the titer of the phage decreased to 10 6 PFU / mL or less, and the phage was inactivated at 70°C or above.

[0062] 3. pH stability

[0063] To evaluate the effect of different pH on the titer of phage, hydrochloric acid solution or sodium hydroxide solution was added in advance to sterile LB liquid medium to adjust the pH value to different ranges from 1 to 12, and the bacteria in the medium were removed by 0.22 μm microporous filter. 100 μL of phage solution was added to 900 μL of LB liquid medium with different pH values, and incubated at 37°C for 1 hour. The titer was determined by double-layer agar plate method, and the experimental results are shown in Figure 5 As shown in the figure, the phage VR2301 can maintain a relatively stable titer in the environment with pH value of 4 to 10, and the titer can reach 10 8 PFU / mL or more, indicating that it has good stability and activity in this pH range.

[0064] 4. Phage lysis kinetics

[0065] The MOI was adjusted to 1000, 100, 10, 1, 0.1, 0.01, and 0.001, respectively, to evaluate the efficiency of phage infection. By comparing the OD 600 to evaluate the inhibition of bacterial growth, which lasted for 12 h, and the experiment was repeated 3 times. The logarithmic phase host bacteria were mixed with phage solution 1:1 and added to the 96-well plate, and the same amount of logarithmic phase bacteria solution was used as the control group, which was incubated at 37°C on a shaking bed with 220 rpm shaking. The results are shown in Figure 6-8 As shown in the figure, at 7 h, the bacterial solution of the control group entered the logarithmic phase (OD 600 = 0.6), while the OD 600 of the phage group remained at about 0.1. At 12 h, all phage-treated groups still showed good antibacterial activity.

[0066] 5. Ability to inhibit biofilm formation

[0067] To evaluate the inhibitory effect of phage on the biofilm formation ability of host bacteria, crystal violet staining method was used. MOI was set to 100, 10, and 1, respectively; the positive control group (PC) only added host bacteria solution (without phage) to detect the natural biofilm formation of the strain; the blank control group (NC) added the same amount of LB medium (without host bacteria) to each well to exclude the background interference that the medium might produce. Each group had three parallel repeated wells to improve the reliability of the experimental data.

[0068] The logarithmic phase host bacteria liquid and the phage liquid were mixed in a ratio of 1:1 according to the predetermined MOI, and were added to a 96-well plate. The 96-well plate was placed in a 37°C constant temperature incubator for 24 hours to form a biofilm. The culture solution in each well was discarded, and the bacteria were washed twice with 200 μL of PBS to remove the planktonic bacteria. The bacteria were fixed with a methanol solution for 15 minutes, and then were discarded and air-dried. Then, 100 μL of 1% crystal violet solution was added for staining for 30 minutes, and then was discarded and washed twice. After being air-dried, 100 μL of anhydrous ethanol was added for decolorization for 5 minutes, and then the eluent was transferred to a new sterile 96-well plate. The OD value at a wavelength of 595 nm was measured using a multifunctional microplate reader, and the higher the value, the greater the amount of biofilm formed. The results are shown in Table 1. Figure 9 As shown in Table 1, the biofilm inhibition rates of the phage VR2301 at MOIs of 100, 10 and 1 were 61.3%, 66.3% and 58.3%, respectively. The OD values of all the phage groups and the PC group were statistically significantly different, indicating that the phage VR2301 effectively inhibited the formation of a host biofilm.

[0069] Example 4: Combination of the phage with baicalin

[0070] 1. Combination of the phage VR2301 with baicalin

[0071] The chessboard method was used, and 50 μL of each of an exponential growth phase Klebsiella pneumoniae bacteria liquid (adjusted to 10 5 CFU / mL) and 50 μL of a baicalin working solution and 50 μL of a phage liquid were sequentially added to each well of a sterile 96-well plate. The baicalin working solution was set at three gradient concentrations: 0.78125 mg / mL (B1), 1.5625 mg / mL (B2) and 3.125 mg / mL (B3). The phage liquid was set at three gradient concentrations: 10 1 PFU / mL (P1), 10 2 PFU / mL (P2) and 10 3 PFU / mL (P3). Three single-drug groups (B1, B2 and B3), three single-phage groups (P1, P2 and P3), a pure bacteria control group (PC) and a blank control group were also set. After mixing, the 96-well plate was incubated at 37°C on a shaking table, and the OD value was measured using a microplate reader. The results are shown in Table 2. 600 Figure 10

[0072] After 14 hours of culture, the OD values of the phage combined drug groups were statistically significantly different from the OD value of the pure bacteria control group, except for the B3P3 group (phage concentration of 10 3 PFU / mL + baicalin concentration of 3.125 mg / mL) and the B3P1 group (phage concentration of 10 3 PFU / mL + baicalin concentration of 0.78125 mg / mL). The OD value of the B1P1 group (phage concentration of 10 1 ​​The OD value of the group with PFU / mL+baicalin concentration of 0.78125 mg / mL was the lowest, showing better antibacterial effect, and there were significant statistical differences compared with the pure bacteria group, single phage group, and single drug group. At this concentration combination, the antibacterial effect of phage combined with baicalin was better than that of phage alone or baicalin alone.

[0073] 2. Combination of Escherichia Phage vB_EcoM_GZMU_E1004 and Baicalin

[0074] The deposit number of bacteriophage vB_EcoM_GZMU_E1004 is GDMCC No: 64729-B1, the deposit date is June 7, 2024, the depository is Guangdong Provincial Microbial Culture Collection Center, and the deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0075] The checkerboard method was used in a sterile 96-well plate, and 50 μL of exponential growth phase Escherichia coli solution (adjusted to 10 5 CFU / mL) and 50 μL baicalin working solution and 50 μL phage solution; among them, baicalin working solution was set to three gradient concentrations: 0.78125 mg / mL (B1), 1.5625 mg / mL (B2), 3.125 mg / mL (B3); phage solution was set to three gradient concentrations: 10 1 PFU / mL(P1), 10 2 PFU / mL(P2), 10 3 PFU / mL (P3). At the same time, three single drug groups (B1, B2, B3), three single phage groups (P1, P2, P3), a pure bacteria control group (MH) and a blank control group were set up; after mixing, they were cultured on a shaker at 37°C for 14 hours and the OD was measured with a microplate reader. 600 Value, see the result Figure 11 .

[0076] After 14 hours of culture, the concentration of E. coli phage was 10 3 PFU / mL, the resistance of the pure phage group began to develop, and the concentration of baicalin was 1.5625 mg / mL + the concentration of phage was 10 2 PFU / mL is the best combination group. This combination can significantly delay the development of phage resistance and show better antibacterial effect. At this concentration combination, the antibacterial effect of phage combined with baicalin is better than that of phage alone or baicalin alone.

[0077] The above described embodiments are only to illustrate the preferred modes of the present application, and are not intended to limit the scope of the present application. Any modification and improvement made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the protection scope of the present application as defined by the claims.

Claims

1. A Klebsiella pneumoniae phage vB_KpnP_GZMU_VR2301, characterized in that: The Klebsiella pneumoniae phage vB_KpnP_GZMU_VR2301 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 14, 2024. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, and the deposit number is GDMCC No: 65268-B1.

2. Use of the Klebsiella pneumoniae phage vB_KpnP_GZMU_VR2301 according to claim 1 in the preparation of a medicament for preventing and / or treating Klebsiella pneumoniae infection.

3. Use of the Klebsiella pneumoniae phage vB_KpnP_GZMU_VR2301 according to claim 1 in the preparation of a Klebsiella pneumoniae bactericide.

4. A drug for preventing and / or treating Klebsiella pneumoniae infection, characterized in that: The active ingredient comprises the Klebsiella pneumoniae phage vB_KpnP_GZMU_VR2301 described in claim 1.

5. The drug according to claim 4, characterized in that The drug also includes pharmaceutically acceptable excipients.

6. The drug according to claim 5, characterized in that The dosage form of the medicine is injection, powder, gel, granule or lyophilized agent.

7. A Klebsiella pneumoniae bactericide, characterized in that The active ingredient comprises the Klebsiella pneumoniae phage vB_KpnP_GZMU_VR2301 described in claim 1.

8. A composition having an antibacterial effect, characterized in that: Including baicalin and lytic bacteriophages with bacteria as hosts.

9. The composition according to claim 8, characterized in that The lytic phage is the Klebsiella pneumoniae phage vB_KpnP_GZMU_VR2301 described in claim 1.

10. Use of the composition according to claim 8 or 9 in the preparation of antibacterial drugs.

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