Carbapenem-resistant klebsiella pneumoniae bacteriophage and application thereof

Abstract

According to the carbapenem-resistant klebsiella pneumoniae bacteriophage and the application, biological characteristic research, morphological analysis and other analysis are carried out on the bacteriophage, and it is found that the bacteriophage has a wide application prospect as a novel potential therapeutic agent and bactericide; meanwhile, the invention also provides a new feasible method for infection control and related application of clinical carbapenem-resistant klebsiella pneumoniae by researching the in-vitro synergistic effect of the klebsiella pneumoniae after the klebsiella pneumoniae is combined with carbapenem antibiotic meropenem.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a carbapenem-resistant Klebsiella pneumoniae phage and an application thereof. Background Art

[0002] Klebsiella pneumoniae is a common opportunistic pathogen in clinical practice. It can cause infections in various systems and parts of the human body, or colonize in the patient's surrounding environment and various surfaces, causing nosocomial transmission and cross-infection. With the rapid development and widespread use of antibiotics, the infection rate and drug resistance of Klebsiella pneumoniae have been rising year by year in clinical practice. Among them, the infection caused by carbapenem-resistant Klebsiella pneumoniae has become increasingly difficult to treat due to its very limited selection of targeted antibiotics. There are many existing bacteriophages that have demonstrated excellent bactericidal ability. As natural catchers of bacteria, they have been important in early anti-infection treatment in the medical community. They are highly specific to bacteria and have strong lethality. As a potential auxiliary means of conventional antibiotic application, the value of phage therapy has been redefined. However, since bacteria can quickly develop anti-phage mechanisms, it is increasingly important to isolate more new phages that can be used in clinical practice.

[0003] The present invention obtains this strain of phage through large-scale laboratory screening, and has the following advantages: first, it is extensive and has a strong killing ability against multiple strains of Klebsiella pneumoniae; second, after being used in combination with antibiotics, the antibiotic MIC value is converted from resistance to sensitive concentration, and at the same time, the emergence of phage-resistant bacteria can be inhibited, thereby achieving a rapid and effective bactericidal effect. Summary of the invention

[0004] In view of this, the present invention aims to overcome the defects in the prior art and proposes a carbapenem-resistant Klebsiella pneumoniae phage and its application. By studying the biological characteristics and morphological analysis of the phage, it is found that the phage has broad application prospects as a new potential therapeutic agent and bactericidal agent. At the same time, the present invention also provides a new feasible method for the infection control and related applications of clinical carbapenem-resistant Klebsiella pneumoniae by studying the in vitro synergistic effect after combined use with the carbapenem antibiotic meropenem.

[0005] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0006] In the first aspect, a carbapenem-resistant Klebsiella pneumoniae phage phK-203 is classified and named: Klebsiella pneumoniae bacteriophage, which was deposited in the China General Microbiological Culture Collection Center (CGMCC) on August 8, 2024, with the storage address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the storage number is CGMCC No. 46098.

[0007] The carbapenem-resistant Klebsiella pneumoniae phage phK-203 of the present invention has a regular polyhedral head with a diameter of about 55nm and a non-contractible long tail with a tail length of about 200nm. According to the classification standards of the International Committee on Taxonomy of Viruses (ICTV), the morphology conforms to the characteristics of the Siphoviridae family.

[0008] After the carbapenem-resistant Klebsiella pneumoniae phage phK-203 of the present invention is adsorbed to bacteria, it enters the outbreak period 10 minutes later, the outbreak time lasts for more than 30 minutes, and the titer can reach 10 in 1 hour. 9 PFU / mL, with strong lysis and replication ability.

[0009] The carbapenem-resistant Klebsiella pneumoniae phage phK-203 of the present invention maintains a stable titer under 4-50°C conditions, and after being treated at 60°C for 1 hour, the survival rate is still greater than 50%, and it is not completely inactivated under 80°C conditions; the titer remains stable in the pH range of 6 to 12, and it is not completely inactivated under pH ≥ 14 conditions, indicating that the phage has good tolerance to temperature and environmental pH, and can still maintain a certain lysis effect on host bacteria under high temperature and strong alkaline conditions.

[0010] The bacterial lysis spectrum of the carbapenem-resistant Klebsiella pneumonia phage phK-203 of the present invention was measured, and its lysis rate for carbapenem-resistant Klebsiella pneumonia (CRKP) reached 40% (4 out of 10 strains could be lysed), and the lysis rate for non-multidrug resistant Klebsiella pneumonia was 66.7% (8 out of 12 strains could be lysed).

[0011] The carbapenem-resistant Klebsiella pneumoniae phage phK-203 of the present invention can reduce the meropenem MIC value from the original 128 μg / mL to ≤2 μg / mL under the condition of MOI=0.01; under the conditions of MOI=0.1 and 1, the meropenem MIC value can be reduced to 1 μg / mL. Phage phK-203, when used in combination with 2 μg / mL meropenem under the condition of MOI=0.01, can reduce the concentration of drug-resistant bacteria in the culture system by 4×log within 8 hours. 10 CFU / mL. After adding the same proportion of phages to the bacterial solution for pretreatment 1 hour in advance, combined with 2μg / mL meropenem, the bacteria in the system can be completely killed within 7 hours. And no bacteria were observed to grow again in the phage and antibiotic combination group after 24 hours of observation.

[0012] In a second aspect, the present invention also provides the use of the bacteriophage phK-203 in the preparation of a preparation for preventing and treating carbapenem-resistant Klebsiella pneumoniae.

[0013] As a preferred embodiment, the preparation is an antibacterial drug and / or an environmental disinfectant.

[0014] In a third aspect, the present invention provides the use of the bacteriophage phK-203 in combination with a carbapenem antibiotic in the preparation of a preparation for preventing and treating carbapenem-resistant Klebsiella pneumoniae.

[0015] As a preferred implementation manner, the carbapenem antibiotic is meropenem.

[0016] As a preferred embodiment, the preparation is an antibacterial drug and / or an environmental disinfectant.

[0017] In a fourth aspect, the present invention further provides a pharmaceutical composition, the main components of which include bacteriophage phK-203 and carbapenem antibiotics.

[0018] As a preferred implementation manner, the carbapenem antibiotic is meropenem.

[0019] In a fifth aspect, the present invention also provides the use of the above-mentioned pharmaceutical composition in the preparation of a preparation for preventing and treating carbapenem-resistant Klebsiella pneumoniae.

[0020] As a preferred implementation manner, the carbapenem antibiotic is meropenem.

[0021] As a preferred embodiment, the preparation is an antibacterial drug and / or an environmental disinfectant.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] The bacteriophage phK-203 provided by the present invention has the advantages of strong lysis and proliferation ability, a wide range of lysis host bacteria, good tolerance to the living environment, and the ability to reduce the MIC value of carbapenem-resistant Klebsiella pneumoniae to meropenem, and has a strong killing ability against pan-resistant Klebsiella pneumoniae, laying a research foundation for bacteriophages as a supplement or alternative therapy for antibiotics for infections caused by Klebsiella pneumoniae, and can also be used to develop and manufacture environmental surface disinfectants, and has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The plaque picture is of phage phK-203 lysing CRKP203;

[0025] Figure 2 The plaque image is of phage phK-203 lysing CRKP1467;

[0026] Figure 3 This is the electron microscope morphology of bacteriophage phK-203;

[0027] Figure 4 is the one-step growth curve of bacteriophage phK-203;

[0028] Figure 5 This is a graph showing the temperature tolerance test results of bacteriophage phK-203;

[0029] Figure 6 This is a test result diagram of the pH tolerance of bacteriophage phK-203;

[0030] Figure 7 It is the time killing curve of lysing bacteria at different concentration ratios of bacteriophage phK-203;

[0031] Figure 8 This is a graph showing the effect of different concentration ratios of bacteriophage phK-203 on reducing the MIC value of meropenem;

[0032] Fig. 9 This is the time-killing curve of the combined use of bacteriophage phK-203 and meropenem;

[0033] Fig.10 This is the genome map of bacteriophage phK-203. DETAILED DESCRIPTION

[0034] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

[0035] The present invention is described in detail below in conjunction with embodiments.

[0036] Example 1

[0037] Isolation method of bacteriophage phK-203:

[0038] Step 1: Collect sewage from the sewage well behind the inpatient building of the Second Hospital of Tianjin Medical University for 4 consecutive weeks, totaling 200×4=800 ml, and store it in sterile flasks in a refrigerator for later use.

[0039] Step 2: Take 30 mL of sewage sample each time, centrifuge at 6000×g for 15 min, and take the supernatant. Resuspend the sediment at the bottom of the centrifuge tube with SM solution, centrifuge at 6000×g for 15 min at 4°C overnight, and take the supernatant. Collect and combine the two supernatants, and filter through a 0.45μm microporous filter membrane to obtain the initial sewage filtrate.

[0040] Step 3: Resuscitate the host bacteria CRKP203, inoculate the pure colonies in liquid LB medium, and culture overnight at 37°C and 160 rpm. The next day, transfer the culture until the OD 600 The value of 0.5-0.6 is used as the host bacterial solution in the logarithmic phase.

[0041] Step 4: Add 20 mL of the initial sewage filtrate, 3 mL of the logarithmic host bacterial solution, 200 μL of 1 M calcium chloride solution, 200 μL of 1 M magnesium chloride solution and 15 mL of liquid LB medium to the centrifuge tube in sequence, shake and mix, and culture overnight at 37°C and 160 r / min in a shaking incubator. The next day, centrifuge the mixture at 6000 × g for 20 min, take the supernatant, and filter through a 0.22 μm sterile microporous filter membrane to obtain the phage stock solution.

[0042] Purification method of bacteriophage phK-203:

[0043] Step 1. Add 300 μL of phage stock solution and logarithmic host bacterial solution to a centrifuge tube, place at room temperature for 10 minutes, add 10 mL of 55°C semi-solid LB medium containing 0.4% agar, vortex and mix thoroughly, and pour on the lower LB medium plate containing 1.2% agar. When the upper semi-solid medium solidifies, invert and culture at 37°C incubator for 8-10 hours until plaques form on the plate.

[0044] Step 2: Pick independent, large and bright single plaques, crush them and soak them in 1 mL SM solution at 4°C overnight. Centrifuge at 10,000 × g for 1 min the next day, filter the supernatant with a 0.22 μm sterile filter membrane, repeat step 1 after ten-fold gradient dilution, and purify the phage again using the double-layer plate method. Repeat 4 times until the plaque size and morphology are consistent to obtain the purified phage solution.

[0045] Amplification method of bacteriophage phK-203:

[0046] Using the liquid amplification method, 300 μL of bacterial solution was mixed with 100 μL of purified phage solution. After standing for 10 min, 15 mL of LB broth was added. The mixture was cultured in a shaking incubator at 37°C and 160 rpm for 6 to 10 h until the mixture was completely clarified. The mixture was centrifuged at 6000 × g for 15 min, the supernatant was filtered through a 0.22 μm filter membrane, and stored at 4°C for later use.

[0047] The purified and amplified phage stock solution can be stained with 2% phosphotungstic acid solution and its morphology can be photographed under a 100 kV TEM.

[0048] Electron micrograph of bacteriophage Figure 3 As shown, the phage phK-203 has a regular polyhedral head with a diameter of about 55nm and an inextractable long tail with a length of about 200nm. According to the classification standards of the International Committee on Taxonomy of Viruses (ICTV), the morphology conforms to the characteristics of the long-tailed bacteriophage family (Siphoviridae).

[0049] Example 2

[0050] One-step growth curve assay of bacteriophage phK-203:

[0051] Resuscitate and prepare the logarithmic phase host bacterial solution, mix 500μL logarithmic phase host bacterial solution with 500μL phage solution at MOI = 0.1, incubate at room temperature for 10min, centrifuge at 10000×g for 1min, and discard the supernatant. Gently wash the precipitate twice with 1mL LB broth, centrifuge and discard the supernatant, resuspend the precipitate and add 100mL LB liquid culture medium to the mixture, mix well, and culture on a shaking table at 37℃ and 160r / min. Sampling 200μL every 5min from 0min, centrifuge at 10000×g for 30s, collect the supernatant, dilute and determine the titer by double-layer plate sandwich method, continue for 90min, set up 3 groups in parallel.

[0052] The results are as follows Figure 4 As shown in the figure, it can be seen that the incubation period of bacteriophage phK-203 is about 10 minutes, after which it enters the outbreak period, which lasts for more than 30 minutes, and the titer can reach 10 in 1 hour. 9 PFU / mL, with strong lysis and replication ability.

[0053] Example 3

[0054] Determination of optimal infection multiplicity of bacteriophage phK-203:

[0055] Dilute the phage stock solution tenfold to 10 4 ~10 9 PFU / mL different titers, respectively with 0.5 McFarland turbidity (about 10 8CFU / mL) host bacterial liquid, 200μL of each was mixed to obtain different ratios of 0.0001, 0.001, 0.01, 0.1, 1 and 10. Add 5mL of liquid LB medium, culture at 37℃, 160r / min shaking for 6h, centrifuge at 8000×g for 10min, collect the supernatant, and determine the phage titer by double-layer plate sandwich method after ten-fold dilution. The titer of phage phK-203 is the highest when MOI is 1, that is, the optimal multiplicity of infection (OMOI) is 1. The experiment was set up in 3 parallel groups. The results are shown in Table 1.

[0056] Table 1 Determination of optimal infection multiplicity of phage

[0057]

[0058] Example 4

[0059] Bacteriophage phK-203 temperature tolerance test:

[0060] Take the phage liquid of known titer and dispense it into each EP tube with 1 mL, place it in a metal bath heater at 4, 37, 50, 60, 70, 80, and 90℃ for 1 hour, then put it to room temperature, dilute it and measure the titer. The experiment is set up in 3 parallel groups, and the tolerance curve is drawn with temperature as the horizontal axis and phage titer as the vertical axis.

[0061] Example 5

[0062] Bacteriophage phK-203 acid and base tolerance test:

[0063] Use 1M HCl and 1M NaOH to adjust the LB broth to pH 2-14, add 900μL broth and 100μL phage dilution to each EP tube, incubate at 37℃, 160r / min shaker for 1h, and measure the titer after gradient dilution. The experiment was set up in 3 parallel groups, and the tolerance curve was drawn with pH as the horizontal axis and phage titer as the vertical axis.

[0064] The present invention evaluates the survival stability of bacteriophage phK-203 through Examples 4 and 5. The temperature tolerance test of bacteriophage phK-203 is as follows: Figure 5 As shown in the figure, it can be seen that phage phK-203 has maintained a high stability at 4-60℃, and the titer remains at 10 at 70-80℃. 2 ~10 3 PFU / mL, and completely loses activity above 90°C. The results of the acid-base tolerance test are as follows Figure 6 As shown in the figure, it can be seen that phage phK-203 maintains stable activity in the range of pH 6 to 12, and the titer remains at 10 at pH = 14. 2PFU / mL, and loses activity when pH ≤ 4.

[0065] Example 6

[0066] Bacteriophage phK-203 split spectrum determination:

[0067] Twelve sensitive strains of Klebsiella pneumoniae and all 10 carbapenem-resistant Klebsiella pneumoniae strains within half a year were randomly selected from the Second Hospital of Tianjin Medical University, and pure colonies were revived. Using the phage spot method, 200 μL of the logarithmic test bacterial solution was mixed with 2 mL of semi-solid LB medium and poured into a 6-well plate. After solidification, 2 μL of phage solution was dripped in the center of the well. After absorption, it was inverted and cultured in a 37°C incubator for 10 hours to observe whether large and bright plaques appeared (the diameter of the plaques was not less than 5 mm). The plaque pictures of CRKP203 and CRKP1467 were shown below. Figure 1 and Figure 2 The results of the bacterial lysis spectrum are shown in Table 2. According to statistics, bacteriophage phK-203 can lyse 4 of 10 resistant bacteria and 8 of 12 sensitive bacteria.

[0068] Table 2 Bacteriophage phK-203 bacterial lysis spectrum

[0069]

[0070]

[0071] Note: “+” indicates lysable / colony drawing test>5mm; “-” indicates non-lysable / colony drawing test≤5mm.

[0072] Example 7

[0073] Determination of the lysis ability of bacteriophage phK-203 on host bacteria at different concentration ratios:

[0074] 20 μL of logarithmic phase host bacterial solution (OD 600 ≈0.6), 20 μL of phage solution with different concentrations (MOI=0, 0.0001, 0.001, 0.01, 0.1, 1, 10), and LB broth to make up the volume to 200 μL. Incubate at 37°C, and measure OD with a microplate reader every 30 minutes from 0 min. 600 The value was set to 240min. A positive control well with only bacterial solution (MOI = 0) and a negative control well with only broth were set up, and the experiment was set up in 3 parallel groups. Figure 7 As shown, it can be seen that bacteriophage phK-203 can effectively inhibit the growth of host bacteria in the infection multiplicity range of 0.01 to 10.

[0075] Example 8

[0076] Determination of the effect of bacteriophage phK-203 on meropenem MIC value at different concentration ratios:

[0077] The host bacterial suspension was calibrated to 0.5 McFarland turbidity by turbidimeter and diluted to 5 × 10 5 CFU / mL for later use; SM solution was used to dilute the phage solution; meropenem was prepared with sterile distilled water to prepare a 1280μg / mL stock solution, and MH broth was diluted in multiples for later use. Drugs were added in the horizontal and vertical rows of the 96-well plate from low to high concentrations, and the chessboard method was cross-combined. 100μL of bacterial solution, 50μL of phage solution (concentration ratio was MOI=0.001, 0.01, 0.1, 1, 10), and 100μL of meropenem (concentration reached 0.125-256μg / mL) were added to each well. Negative and positive control wells were set on each plate, and the plate was placed in a 37°C incubator for 20h to observe the results and record the MIC value. The experiment was set up in 3 parallel groups.

[0078] The results are as follows Figure 8 As shown, it can be seen that the phage phK-203 can reduce the meropenem MIC value from the original 128μg / mL to (13.3±3.8)μg / mL under the condition of MOI=0.001; under the condition of MOI=0.01, the meropenem MIC value can be reduced from the original 128μg / mL to (1.7±0.2)μg / mL; under the conditions of MOI=0.1 and 1~10, the meropenem MIC value can be reduced to 1μg / mL.

[0079] Example 9

[0080] Determination of the bactericidal effect of combined use of bacteriophage phK-203 and meropenem:

[0081] The host bacterial suspension in the logarithmic growth phase was adjusted to a concentration of 10 5 CFU / mL for later use. SM solution was used to dilute the phage solution to an MOI of 0.01; MH broth was used to dilute the meropenem stock solution to a final concentration of 2μg / mL in the reaction wells. Samples were added to a sterile 6-well plate and divided into the following six groups: bacterial growth control group, broth negative control group, antibiotic + bacterial solution group, phage + bacterial solution group, phage + antibiotic + bacterial solution group, and bacterial solution and phage mixed pretreatment for 1h before adding antibiotics. After mixing, the mixture was cultured in a 37℃ incubator for 8 consecutive hours starting from the initial 0h. 100μL of the mixture was drawn from each well every hour. After ten-fold gradient dilution, 50μL was taken and evenly spread on the MH agar plate with a coating rod. All plates were placed in a 37℃ incubator for 20h. The colonies were counted the next day and a time-kill curve (TKC) was drawn.

[0082] Each group was sampled and plated for counting at 24h to obtain the final number of surviving bacteria. 10 CFU / mL, the group is considered to have bactericidal effect; the final bacterial concentration of the combined drug group is reduced by ≥2×log compared with the final bacterial concentration of the strongest single drug group 10 CFU / mL, it is believed that the combination therapy has a synergistic effect.

[0083] The results are as follows Fig. 9 As shown in the figure, it can be seen that when bacteriophage phK-203 was used in combination with 2 μg / mL meropenem at MOI = 0.01, the concentration of resistant bacteria in the culture system could be reduced by 4×log within 8 hours. 10 CFU / mL or more. After adding the same proportion of phages to the bacterial solution 1 hour in advance for pretreatment, and then combined with 2μg / mL meropenem, the bacteria in the system can be completely killed within 7 hours. The combination of the two has a synergistic effect. And after 24 hours of observation, no bacteria were observed to grow again in the group treated with phages and antibiotics, which was 5×log higher than the initial bacterial concentration. 10 CFU / mL and above, the bactericidal and antibacterial effects are ideal.

[0084] Example 10

[0085] Phage whole genome sequencing and bioinformatics analysis:

[0086] 500 μL of phage stock solution was taken, and nucleic acid was extracted according to the instructions using the viral DNA / RNA universal extraction kit (TIANamp, DP315). The product was sequenced by Genewiz (Jinweizhi, Suzhou) using the IIIumina MiSeq system for whole genome sequencing and genomic library construction. The sequencing results were cut and assembled using SPAdes 3.15.3 software. The genome sequence was annotated using RAST and PHASTER software, and the hypothetical protein sequence was further annotated and function predicted using BLAST. The resistance gene and virulence factor databases were compared to predict the carrying of related genes. The genome circle image generator (BlastRing Image Generator, BRIG) was used to create visualization of the phage genome.

[0087] The results are as follows Fig.10 As shown, it can be seen that the genome sequence size of phK-203 is 176062bp, and the (G+C)% content is 40.6%. It contains 87 open reading frames (ORFs), and the coding region accounts for more than 90% of the genome, which encodes phage structure and packaging genes, DNA replication and regulatory genes, host conjugation and lysis functional genes, and other unknown functional genes. Fig.10It can be seen that genes related to the structure of phage include head, neck protein, tail protein, base plate, etc.; genes related to DNA replication and regulatory function include endonucleases, RNA enzymes, DNA enzymes, translation and transcriptase, tRNA and various regulatory enzymes; lysozyme genes related to phage lysis function, etc. There are no drug resistance genes and virulence genes, and no lysogeny-related genes.

[0088] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A carbapenem-resistant Klebsiella pneumoniae phage phK-203, characterized in that: The bacteriophage was deposited in the China General Microbiological Culture Collection Center (CGMCC) on August 8, 2024, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC No. 46098.

2. Use of the carbapenem-resistant Klebsiella pneumoniae phage phK-203 according to claim 1 in the preparation of a preparation for preventing and treating carbapenem-resistant Klebsiella pneumoniae.

3. The use according to claim 2, characterized in that: The preparation is an antibacterial drug and / or an environmental disinfectant.

4. Use of the carbapenem-resistant Klebsiella pneumoniae phage phK-203 according to claim 1 in combination with a carbapenem antibiotic in the preparation of a preparation for preventing and treating carbapenem-resistant Klebsiella pneumoniae.

5. The use according to claim 4, characterized in that: The carbapenem antibiotic is meropenem.

6. The use according to claim 4, characterized in that: The preparation is an antibacterial drug and / or an environmental disinfectant.

7. A pharmaceutical composition, characterized in that: The effective ingredients include the carbapenem-resistant Klebsiella pneumoniae phage phK-203 according to claim 1 and a carbapenem antibiotic.

8. The pharmaceutical composition according to claim 7, characterized in that: The carbapenem antibiotic is meropenem.

9. Use of the pharmaceutical composition according to claim 7 or 8 in the preparation of a preparation for preventing and treating carbapenem-resistant Klebsiella pneumoniae.

10. The use according to claim 9, characterized in that: The carbapenem antibiotic is meropenem. Preferably, the preparation is an antibacterial drug and / or an environmental disinfectant.

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