A highly toxic Klebsiella pneumoniae phage and its application

Abstract

The present invention discloses a highly virulent Klebsiella pneumoniae phage and its application. Specifically, the present invention provides a phage, and its deposit number is CGMCC No.46178. The present invention also provides the application of the phage in the preparation of a reagent for specifically and efficiently lysing highly virulent Klebsiella pneumoniae. The phage of the present invention exhibits a specific lysis ability against highly virulent Klebsiella pneumoniae, and the lysis effect is significant, the host range is wide, and the activity can be maintained stably under a variety of temperature and pH conditions, showing excellent environmental adaptability.

Description

Technical Field

[0001] The present invention relates to a bacteriophage and its application, in particular to a highly virulent Klebsiella pneumoniae phage and the application of the bacteriophage in the preparation of medicines for treating a series of diseases such as liver abscess, pneumonia and bacteremia caused by highly virulent Klebsiella pneumoniae (hvKp), belonging to the field of biotechnology. Background Art

[0002] Klebsiella pneumoniae (Kp) is a highly virulent Gram-negative bacterium. A key factor in the continuous evolution of Klebsiella pneumoniae is its strong ability to acquire new genetic material. According to its pathogenicity, it can be divided into classical Klebsiella pneumoniae (cKp) and hypervirulent Klebsiella pneumoniae (hvKp). Klebsiella pneumoniae can cause a variety of clinical diseases, such as pneumonia, abdominal infection, liver abscess, urinary tract infection and bacteremia. It is reported that Klebsiella pneumoniae is a common cause of Gram-negative bacteremia.

[0003] The high mucus phenotype is one of the strain-specific traits of hvKp, and hvKp has acquired many virulence genes present in large virulence plasmids (such as pK2044 and pLVPK) and integrated chromosomal elements, which give it the characteristics of high virulence. Unlike other members of the Enterobacteriaceae family, hvKp can infect healthy or immunocompromised people of any age, and infected people often have multiple infection sites or metastasis at the same time. Among hvKp that cause liver abscesses, the most representative model strain is NTUH-K2044.

[0004] Serotype is a classification method that distinguishes bacterial types by bacterial surface antigens. It is of great significance in clinical diagnosis and treatment, vaccine development, and epidemiological research. Studies have confirmed that Klebsiella pneumoniae serotype K1 mostly belongs to hvKp, which can cause a series of invasive diseases in the body. Strain NTUH-K2044 also belongs to type K1. In addition, hvKp of sequence type 23 (ST23) is often associated with community-acquired infections and may evolve into new "super bacteria" (MDR-hvKp) in hospital environments, posing great challenges to clinical diagnosis and treatment. It is worth noting that strain NTUH-K2044, as a representative strain of Klebsiella pneumoniae of type K1 / ST23, is of particular clinical significance.

[0005] The situation of drug resistance of Klebsiella pneumoniae in recent years is severe. The strain can acquire a multidrug-resistant phenotype through horizontal transfer of antibiotic resistance genes carried by transposons or plasmids. At present, the number of β-lactam-resistant Kp has increased dramatically, and with the discovery and spread of the new carbapenemase New Delhi metallo-β-lactamase, the situation has become more serious. This has significantly increased the difficulty of treating bacterial diseases in clinical practice.

[0006] Faced with the treatment challenges of pathogens such as hvKp, especially the increasingly prominent limitations of antibiotic therapy, it is crucial to explore and develop new antibacterial strategies. As an innovative method, phage therapy is gradually showing its unique charm and potential. As the natural enemy of bacteria in nature, phages have extraordinary antibacterial capabilities in their unique way - adsorbing and invading specific bacteria, then replicating and releasing lytic enzymes in them, ultimately leading to the lysis of host bacteria. Compared with traditional antibiotics, phage therapy has several significant advantages: (1) High specificity: phages can accurately identify and attack target bacteria without harming non-target bacteria, viruses and other microorganisms and normal human cells, ensuring the safety and effectiveness of treatment; (2) Applicability to antibiotic-resistant groups: Even in the context of widespread antibiotic resistance, phage therapy can still provide these patients with hope for treatment and broaden the range of treatment options; (3) Small and flexible doses: The dose required for phage therapy is much lower than that of antibiotics, and it can be used as an independent treatment or in combination with antibiotics to enhance efficacy and reduce side effects; (4) No risk of resistance: A key advantage of phage therapy is that it almost never causes bacterial resistance, which is in stark contrast to the frequent resistance problems in antibiotic treatment; (5) Self-proliferation and targeted enrichment: phages can replicate in large quantities at the site of infection and automatically enrich in areas where pathogens are most concentrated, thereby achieving efficient and precise antibacterial effects, which is a feature that traditional antibiotics cannot achieve. Summary of the invention

[0007] An object of the present invention is to provide a new Klebsiella pneumoniae phage.

[0008] Another object of the present invention is to provide related applications of the bacteriophage.

[0009] The inventors of this case discovered a highly virulent Klebsiella pneumoniae phage with significant lytic activity in their research, and named it phiK2044 in the present invention. The phage phiK2044 of the present invention has been deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on September 5, 2024, with a preservation date of September 5, 2024; the classification name is: Klebsiella phage (Klebsiella virus), and the preservation number is CGMCC No. 46178. The bacterium is also referred to as phage phiK2044 in the present invention.

[0010] The bacteriophage phiK2044 of the present invention, as a tailed phage, exhibits excellent environmental adaptability and can maintain its relatively efficient lysis activity under a wide range of temperatures (4°C to 50°C) and pH conditions (pH 4 to 10), ensuring its application potential in different environments. Its unique infection characteristics are reflected in the extremely low optimal multiplicity of infection (MOI) of 0.0001, which means that even at an extremely low ratio of phage to bacteria, phiK2044 can effectively infect and lyse hvKp. Furthermore, the lysis cycle of phiK2044 has been precisely measured, and its incubation period is extremely short (0-20 minutes), which is almost negligible. It then quickly enters the outbreak period (20-90 minutes), releases a large number of progeny phages, and finally enters the stable period. This efficient lysis cycle gives phiK2044 a significant advantage in controlling highly virulent Klebsiella pneumoniae infections. More importantly, phiK2044 exhibited a strong lysis ability against hvKp, especially the model strain NTUH-K2044. In the range of infection multiplicity from 10 to an extremely low 0.0001, phiK2044 can effectively inhibit the growth of these highly virulent strains, providing a solid scientific basis for the development of new antibiotic alternative therapies, especially for severe infections such as bacteremia caused by highly virulent Klebsiella pneumoniae. The bacteriophage phiK2044 of the present invention can not only broaden the application field of phage therapy, but also provide new ideas and hopes for solving the current problem of antibiotic resistance.

[0011] Specifically, in one aspect, the present invention provides a bacteriophage, whose deposit number is CGMCC No.46178.

[0012] On the other hand, the present invention provides a phage preparation, comprising: a phage with a deposit number of CGMCC No. 46178, and auxiliary materials.

[0013] According to a specific embodiment of the present invention, in the phage preparation of the present invention, the auxiliary material includes a nutrient component for maintaining the activity of the bacteria. In some specific embodiments, the auxiliary material can be LB culture medium. In some more specific embodiments, the LB culture medium contains 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride.

[0014] According to some specific embodiments of the present invention, the bacteriophage preparation of the present invention is a drug.

[0015] According to some specific embodiments of the present invention, the bacteriophage preparation of the present invention is a cleaning agent or a disinfectant.

[0016] On the other hand, the present invention provides the use of the bacteriophage with the deposit number of CGMCC No.46178 for lysing highly virulent Klebsiella pneumoniae in vitro.

[0017] In some specific embodiments of the present invention, the present invention measured the host spectrum of bacteriophage phiK2044, and the measurement results showed that bacteriophage phiK2044 can lyse multiple strains of hvKp.

[0018] On the other hand, the present invention provides the use of a bacteriophage with a deposit number of CGMCC No. 46178 in preparing a preparation for lysing highly virulent Klebsiella pneumoniae.

[0019] In another aspect, the present invention provides the use of a bacteriophage with a deposit number of CGMCC No. 46178 in the preparation of a medicament for preventing and treating diseases caused by highly virulent Klebsiella pneumoniae, specifically, diseases such as pneumonia, abdominal infection, liver abscess, urinary tract infection and bacteremia.

[0020] According to a specific embodiment of the present invention, in the application of the bacteriophage of the present invention, the highly virulent Klebsiella pneumoniae includes NTUH-K2044.

[0021] In summary, the present invention provides a bacteriophage phiK2044, which exhibits specific lysis ability against highly virulent Klebsiella pneumoniae, and the lysis effect is significant and the host range is wide. In particular, phiK2044 can maintain stable activity under a variety of temperature and pH conditions, showing excellent environmental adaptability. These characteristics make phiK2044 have significant advantages in inhibiting highly virulent Klebsiella pneumoniae and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Shown is a transmission electron micrograph of bacteriophage phiK2044.

[0023] Figure 2Shown is a graph showing the temperature tolerance test of bacteriophage phiK2044.

[0024] Figure 3 Shown is a graph showing the pH tolerance test of bacteriophage phiK2044.

[0025] Figure 4 Shown is a one-step growth curve of bacteriophage phiK2044.

[0026] Figure 5 Shown is the lysis curve of bacteriophage phiK2044 against host bacteria NTUH-K2044.

[0027] Deposit of biological material for patent procedure:

[0028] Bacteriophage phiK2044 of the present invention:

[0029] Deposit date: September 5, 2024;

[0030] Depository: China General Microbiology Center (CGMCC);

[0031] Address of the depository: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Deposit number: CGMCC No. 46178;

[0032] Classification name: Klebsiella phage (Klebsiella virus). DETAILED DESCRIPTION

[0033] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention.

[0034] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0035] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the technical field.

[0036] In the examples, all reagents and materials used, unless otherwise specified, are conventional biochemical reagents and can be obtained commercially.

[0037] Example 1. Isolation, purification and preservation of bacteriophage phiK2044

[0038] Preparation of phage isolation fluid: In June 2023, 50 ml of sewage samples were collected from the sewage of the Capital Institute of Pediatrics to obtain samples that may contain rich phages. Subsequently, the samples were placed in a centrifuge and centrifuged at 4000 rpm for 20 minutes. The supernatant after centrifugation was filtered using a 0.22 micron microporous filter. Finally, the supernatant was identified as phage isolation fluid.

[0039] Isolation of phages: In the co-culture experiment of phages and host bacteria, 5 ml of LB medium was first used as the basic environment, and then 50 microliters of host bacteria NTUH-K2044 suspension and 200 microliters of previously prepared phage isolation liquid were added. This mixture was placed in a shaker set at 37°C and cultured at 220 rpm. After 4 hours of full cultivation, the culture solution was centrifuged at high speed, with the speed set to 10,000 rpm for 2 minutes. Subsequently, the supernatant was finely filtered using a 0.22-micron microporous filter. Finally, 100 microliters of sample was taken from the filtered supernatant, and the plaques were screened using the classic double-layer agar plate method.

[0040] Purification of phages: During the phage purification process, the double-layer agar plate containing potential plaques was first placed in an incubator at 37°C for static culture for 12 to 24 hours. Subsequently, a single, independent plaque was accurately picked up using an inoculation loop and transferred to a test tube containing LB medium, and 50 microliters of the host bacteria NTUH-K2044 suspension was added at the same time. This mixture was then placed in a shaker at 37°C and cultured at 220 rpm for 4 hours. After the culture was completed, the cell debris and unlysed host bacteria in the culture medium were removed by centrifugation at 10,000 rpm for 2 minutes, and the supernatant was then filtered using a 0.22 micron microporous filter to further purify the phage suspension. Next, 100 μl of sample was taken from the filtered supernatant and the double-layer agar plate method was used again to screen the plaques. In order to obtain completely purified phages, the above operation needed to be repeated 4 to 5 times, and each time the phages were picked and amplified from the newly formed plaques.

[0041] In the present invention, the above method is used to obtain a bacteriophage, which is named phiK2044 in the present invention.

[0042] Morphological observation of bacteriophage phiK2044 under electron microscope: First, a series of treatment agents were added to the purified culture medium, and 1% chloroform was added with DNase and RNase at the same time. Subsequently, the supernatant containing the phage was collected by centrifugation. 10% PEG8000 was added to the supernatant, and after centrifugation again, it was resuspended with SM buffer, and 1% chloroform was added again. The upper aqueous phase was separated by centrifugation, and the phage suspension was diluted to an appropriate concentration and added dropwise to the surface of the copper mesh. The contrast of the phage was enhanced by counterstaining technology. Subsequently, a transmission electron microscope was used for observation. By adjusting the focal length and field of view of the microscope, a clear field of view showing the complete morphology of a single phage phiK2044 was found, and photographed and recorded. Figure 1 As shown, phage phiK2044 exhibits typical short-tailed phage characteristics with a diameter of approximately 50 nanometers.

[0043] According to the test, the bacteriophage phiK2044 of the present invention has good tolerance to temperature and pH, and can maintain a stable titer under the conditions of temperature 4-50°C and pH 4-10, and the optimal multiplicity of infection (MOI) of bacteriophage and bacteria is 0.001. The incubation period of bacteriophage phiK2044 is 0-20 minutes, the outbreak period is 20-80 minutes, and then enters the stable period.

[0044] The hvKp strain that can be lysed by bacteriophage phiK2044 is the model strain NTUH-K2044 (Genebank number is GCA_000009885.1), which has strong virulence and has been proven to cause a series of invasive diseases such as liver abscess in the body. The bacteriophage phiK2044 of the present invention has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on September 5, 2021, with a preservation date of September 5, 2024, and is classified and named as: Klebsiella phage (Klebsiella virus), and the preservation number is CGMCC No. 46178. In the present invention, the bacterium is also referred to as bacteriophage phiK2044.

[0045] Example 2: Temperature tolerance test of bacteriophage phiK2044

[0046] In order to evaluate the stability of bacteriophage phiK2044 under different temperature conditions, an equal amount (1 ml) of the phage suspension phiK2044 prepared in Reference Example 1 was placed in a series of preset temperature environments (4°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C and 80°C) and incubated for 1 hour at each temperature. After the incubation, the double-layer agar plate method, a classic phage activity assay technique, was used. The experimental results are shown in Figure 2. Figure 2 As shown, phiK2044 exhibited a wide tolerance to temperature, especially in the relatively mild temperature range of 4°C to 50°C, where its activity remained stable.

[0047] Example 3: pH tolerance test of bacteriophage phiK2044

[0048] The phage phiK2044 was exposed to SM buffer at pH 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 and incubated at 37°C for 1 hour. The survival and activity of the phage were evaluated by double-layer agar plate technique. Figure 3 The experimental results showed that the phiK2044 phage exhibited good acid-base tolerance. Specifically, in the relatively wide range of pH 4 to pH 10, its titer remained relatively stable and its activity was not significantly affected.

[0049] Example 4. Optimal multiplicity of infection and one-step growth curve test of bacteriophage phiK2044

[0050] In order to find out the optimal efficiency of phage phiK2044 infecting host bacteria W14, different infection multiplicities (MOI) were used in the experiment, specifically 10, 1, 0.1, 0.01, 0.001 and 0.0001. After the mixture of phage and host bacteria in these ratios was shaken and cultured for 4 hours, the titer of the phage was evaluated by the double-layer agar plate method. The experimental results showed that when the MOI was set to 0.0001, the titer of phage phiK2044 reached the highest, so 0.0001 was determined to be the optimal infection multiplicity under this condition.

[0051] Based on the optimal infection multiplicity determined above, the phage and host bacteria were mixed at an MOI ratio of 0.0001 and cultured with shaking. The phage titer was measured every 10 minutes over the next 2 hours. A one-step growth curve (such as Figure 4 (As shown) The incubation period of bacteriophage phiK2044 is 0-20 minutes, the burst period is 20-80 minutes, and then it enters the stable period.

[0052] Example 5. Lysis ability of bacteriophage phiK2044 against host bacteria NTUH-K2044 and host spectrum test

[0053] To evaluate the lytic efficiency of phage phiK2044 against host bacteria NTUH-K2044, multiple infection multiplicity (MOI) conditions were used, including 10, 1, 0.1, 0.01, 0.001 and 0.0001. Phage and host bacteria were mixed at these ratios and cultured with shaking. During the 6-hour culture period, the growth of host bacteria was monitored by measuring the OD600 value every 1 hour.

[0054] like Figure 5 The experimental results shown reveal the broad lytic ability of phage phiK2044: the phage can effectively inhibit the growth of host bacteria NTUH-K2044 (i.e., hvKp) in a wide range of MOI from 10 to 0.000001. In view of its significant lytic effect and potential application value, phage phiK2044 is proposed as a candidate phage preparation for the treatment of diseases caused by hvKp.

[0055] Host spectrum determination: Phage phiK2044 and 22 strains of Kp were mixed and cultured at the optimal infection multiplicity ratio, and the culture fluid was observed after 2 hours of culture. If the culture fluid was clear, it meant that phage phiK2044 could lyse the Kp. The determination results showed that phage phiK2044 could lyse 22 strains of Kp. The specific information of the 22 strains of Kp is shown in Table 1.

[0056] Table 1

[0057] Strain number Sequence type (ST) Kp1 QD70 Kp2 Q22 Kp3 14896 Kp4 QD52 Kp5 10430 Kp6 DT189 Kp7 QD51 Kp8 D053 Kp9 11796 Kp10 SY7 Kp11 12333 Kp12 2-5 Kp14 C26 Kp15 C46 Kp16 4-26 Kp17 12605 Kp18 4-25 Kp19 4-11 Kp20 10836 Kp21 DT220 Kp22 16205

[0058] It should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A bacteriophage, whose deposit number is CGMCC No.46178.

2. The bacteriophage according to claim 1, which maintains a stable titer under the conditions of a temperature of 4-40°C and a pH of 5-10.

3. A bacteriophage preparation comprising: The bacteriophage with the deposit number of CGMCC No.46178, and auxiliary materials.

4. The bacteriophage preparation according to claim 3, wherein The auxiliary materials include nutritional components for maintaining bacterial activity; Preferably, the auxiliary material is LB medium. The bacteriophage preparation according to claim 3 , which is a medicine. The bacteriophage preparation according to claim 3 , which is a cleaning agent or a disinfectant.

7. Use of the bacteriophage with a deposit number of CGMCC No. 46178 for in vitro lysis of highly virulent Klebsiella pneumoniae.

8. Use of a bacteriophage with a deposit number of CGMCC No. 46178 in the preparation of a preparation for lysing highly virulent Klebsiella pneumoniae.

9. Use of a bacteriophage with a deposit number of CGMCC No. 46178 in the preparation of a drug for preventing and treating non-alcoholic fatty liver disease caused by highly virulent Klebsiella pneumoniae.

10. The use according to any one of claims 7 to 9, wherein: The highly virulent Klebsiella pneumoniae includes strain NTUH-K2044.

Images