CN118028251A - A bacteriophage specifically targeting ST447 type Klebsiella pneumoniae and its application - Google Patents

Abstract

The invention discloses a specific targeting ST447 type klebsiella pneumoniae phage and application thereof. In particular discloses phage YJ-pK3-24, which has a preservation number of CGMCC No 45875 in the China general microbiological culture Collection center. The phage of the invention has good tolerance to temperature and pH value, and the optimal multiplicity of infection (MOI) is 0.01; latency is 0-50 minutes, burst is 50-150 minutes, and the average burst size is about 50 plaque forming units/cell. The phage of the invention can specifically lyse ST447 type Klebsiella pneumoniae, has the characteristics of large burst quantity, good temperature and pH tolerance, has good inhibition effect on Klebsiella pneumoniae, has good application prospect, and lays a foundation for developing antibiotic substitution therapy for treating lung infection caused by Klebsiella pneumoniae.

Description

A bacteriophage specifically targeting ST447 type Klebsiella pneumoniae and its application

Technical Field

The invention belongs to the field of biotechnology, and in particular relates to a phage specifically targeting ST447 type Klebsiella pneumoniae and an application thereof.

Background technique

Klebsiella pneumoniae is a facultative anaerobic Gram-negative bacterium of the genus Klebsiella in the family Enterobacteriaceae. It is an important conditional pathogen and often carries a drug-resistant phenotype, accounting for 66.7% of carbapenem-resistant Enterobacteriales infections. Klebsiella pneumoniae is a common Enterobacteriaceae bacterium in nosocomial infections. It can form biofilms on the surface of medical devices or in pipelines, thereby causing ventilator-associated pneumonia, which is a potential threat to life safety, especially for patients with low immunity or in intensive care. Klebsiella pneumoniae is divided into different multilocus sequence typing (MLST) types according to the differences in the sequences of its seven housekeeping genes (rpo B, gapA, mdh, pgi, pho E, inf B, ton B). In recent years, due to the widespread use of various antimicrobial drugs, Klebsiella pneumoniae has become resistant to multiple antimicrobial drugs. The emergence of multidrug-resistant Klebsiella pneumoniae caused by the irrational use of antibiotics has caused great difficulties in the selection of clinical treatment drugs and has become a major issue threatening global public health. Therefore, the development of new antibacterial drugs is urgent.

Bacteriophage (phage) is a virus that can infect bacteria. It is highly specific to bacteria, can be adsorbed to the surface of bacteria, and can produce proteases to degrade bacterial surface polysaccharides. Phages have high efficiency and high specificity in killing bacteria, and do not act on animal and plant cells, so they are highly safe. The development cycle of phages is short, and they can replace antibiotics as a treatment for multidrug-resistant bacterial infections. In recent years, they have been used to treat a variety of intractable infectious diseases. At present, clinical practice has emerged of using phages to treat pneumonia, osteomyelitis, urinary tract infections and other diseases caused by multidrug-resistant bacteria. Phage therapy is a treatment method for pathogenic bacterial infections by lysing bacteria with phages. The invasion of phages will cause bacterial cell lysis, destroy bacterial metabolism, and cause bacterial self-destruction. After lysing specific bacterial targets, phages will stop reproducing immediately. Therefore, phages will not develop secondary resistance similar to antibiotics. However, the emergence of phage-resistant bacteria also occurs from time to time, which is a major limitation of phage therapy. Therefore, it is of great clinical significance to continuously isolate and purify new phages targeting Klebsiella pneumoniae and study their biological, morphological, and genomic characteristics, which will provide new ideas and methods for the clinical treatment of multidrug-resistant Klebsiella pneumoniae infections.

Summary of the invention

One of the purposes of the present invention is to provide a bacteriophage having a good inhibitory effect on ST447 type Klebsiella pneumoniae and its application in inhibiting and killing multi-drug resistant Klebsiella pneumoniae. The technical problems to be solved are not limited to the described technical themes, and those skilled in the art can clearly understand other technical themes not mentioned in this article through the following description.

To achieve the above object, the present invention first provides a bacteriophage, which can be Klebsiella phage YJ-pK3-24, whose deposit number in the General Microbiological Center of China National Committee for Microbiological Culture Collection is CGMCC No.45875.

The present invention also provides a culture containing the bacteriophage YJ-pK3-24.

The present invention also provides a method for preparing the culture, which comprises: culturing the bacteriophage YJ-pK3-24 in a culture medium to obtain the culture.

The term "culture" refers to a general term for liquid or solid products (all substances in the culture container, i.e., fermentation products) that have a microbial community after artificial inoculation and culture. That is, the product obtained by growing and/or amplifying the microorganisms, which can be a biologically pure culture of the microorganisms, or it can contain a certain amount of culture medium, metabolites, or other components produced during the culture process. The term "culture" also includes a subculture obtained by subculturing the microorganisms, which can be a culture of a certain generation or a mixture of several generations.

The culturing is to culture the bacteriophage YJ-pK3-24 under conditions suitable for culturing the bacteriophage. Those skilled in the art are familiar with a variety of methods that can be used to culture the bacteriophage strain of the present invention, such as batch methods or continuous methods such as fed-batch methods or repeated fed-batch methods, but the present invention is not limited thereto.

The present invention also provides a preparation, which comprises the bacteriophage YJ-pK3-24 and/or the culture.

Furthermore, the preparation can be used to inhibit or kill Klebsiella pneumoniae, or to prevent Klebsiella pneumoniae contamination.

The dosage form of the preparation may include powders (including lyophilized powders and wet powders), liquids, solids, ointments, creams, emulsions, gels, water-dispersible granules, suspensions, suspoemulsions, aqueous emulsions or microemulsions, etc., but is not limited thereto.

The preparation may be a bacteriophage preparation.

The preparation method of the phage preparation is well known to those skilled in the art. For example, a solid bacterial agent can be obtained by mixing the phage YJ-pK3-24 with a solid carrier and/or an auxiliary agent. The solid carrier may include one or more of kaolin, light calcium carbonate, diatomaceous earth, medical stone, calcite, zeolite, white carbon black, talcum powder, fine sand and clay; the auxiliary agent may include one or more of sodium dodecylbenzene sulfonate, trehalose, glycerol, sodium lignin sulfonate, sodium alkylnaphthalene sulfonate polycondensate, nicotinic acid, glucose, amino acids, and vitamins. Methods for preparing phage YJ-pK3-24 into freeze-dried preparations, liquid preparations and other dosage forms are well known to those skilled in the art.

The present invention also provides an antibacterial pharmaceutical composition against Klebsiella pneumoniae, wherein the antibacterial pharmaceutical composition comprises the bacteriophage YJ-pK3-24 and/or the culture, and a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier is selected from diluents, excipients, fillers, binders, wetting agents, disintegrants, stabilizers, absorption promoters, adsorption carriers, surfactants, lubricants, preservatives, aerosolizers, suspending agents, plasticizers and dispersants.

The active ingredient of the antibacterial pharmaceutical composition may be the bacteriophage YJ-pK3-24. The antibacterial pharmaceutical composition may be used to prevent or treat Klebsiella pneumoniae infection or diseases caused by Klebsiella pneumoniae.

The present invention also provides the use of the bacteriophage YJ-pK3-24 or the culture in preparing a product for inhibiting or killing Klebsiella pneumoniae.

The present invention also provides the use of the bacteriophage YJ-pK3-24 or the culture in preparing a product for preventing or treating Klebsiella pneumoniae infection or a disease caused by Klebsiella pneumoniae.

The products described in this article include reagents (such as cleaners, disinfectants or preservatives, etc.), drugs (such as antibacterial drugs), feed, additives, health products, daily chemical products, cosmetics, toiletries, disinfectants (such as environmental disinfectants, medical device disinfectants), etc. but are not limited to these.

The present invention also provides the use of the bacteriophage YJ-pK3-24 or the culture in killing Klebsiella pneumoniae in the environment.

The environment may include a breeding environment, a medical environment, a food processing environment, etc. but is not limited thereto.

Furthermore, the environment may include soil, water, air, objects (such as medical equipment, ground, walls, sinks, containers, packaging, fabrics, food, feed, etc.), etc. but is not limited thereto.

Herein, the Klebsiella pneumoniae may be ST447 type Klebsiella pneumoniae.

Furthermore, the Klebsiella pneumoniae may be multi-drug resistant ST447 type Klebsiella pneumoniae.

The ST447 type is multilocus sequence typing (MLST).

The present invention also provides a method for killing Klebsiella pneumoniae in an environment, the method comprising applying the bacteriophage YJ-pK3-24, the culture and/or the preparation to the environment.

Further, the administering may include contacting the bacteriophage, the culture and/or the preparation with an environment.

Further, the application method may include spraying, atomizing, atomizing, spraying, flooding, pouring, washing and/or rinsing but is not limited thereto.

After extensive and in-depth research, the inventors of the present application discovered a lytic Klebsiella pneumoniae phage, named YJ-pK3-24. Experiments have shown that phage YJ-pK3-24 is a tailed phage with good tolerance to temperature and pH, and can maintain a stable titer at a temperature of 25-50°C and a pH of 6-10. The optimal multiplicity of infection (MOI) of phage and bacteria is 0.01. The incubation period of phage YJ-pK3-24 is 0-50 minutes, the burst period is 50-150 minutes, and the average burst volume is about 50 plaque-forming units/cell. The bacteriophage of the present invention can specifically lyse ST447 type Klebsiella pneumoniae (such as Klebsiella pneumoniae strain YJ-Kpn32-X), and has the characteristics of large burst volume and good temperature and pH tolerance, has a good inhibitory effect on Klebsiella pneumoniae, has good application prospects, and lays a foundation for the development of antibiotic alternative therapies for treating lung infections caused by Klebsiella pneumoniae.

Collection Instructions

Bacterial species: Klebsiella phage

Latin name: Klebsiella phage

Suggested taxonomic name: Klebsiella phage

Strain ID: YJ-pK3-24

Depository: China National Microbiological Culture Collection Administration General Microbiology Center

Abbreviation of depository unit: CGMCC

Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

Date of deposit: February 4, 2024

CGMCC Registration Number: CGMCC No.45875

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a transmission electron micrograph of bacteriophage YJ-pK3-24.

FIG. 2 is a graph showing the temperature tolerance test of bacteriophage YJ-pK3-24.

FIG. 3 is a graph showing the pH tolerance test of bacteriophage YJ-pK3-24.

FIG. 4 shows the optimal infection multiplicity of bacteriophage YJ-pK3-24.

FIG5 is a one-step growth curve of bacteriophage YJ-pK3-24.

FIG. 6 shows the experimental results of the lysis effect of bacteriophage YJ-pK3-24 on YJ-Kpn32-X in G. mellonella.

Detailed ways

The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

The quantitative experiments in the following examples were repeated three times unless otherwise specified, and the results were averaged.

The Klebsiella phage YJ-pK3-24 CGMCC No. 45875 in the following examples has been deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee (referred to as CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on February 4, 2024, with a deposit registration number of CGMCC No. 45875. Klebsiella phage YJ-pK3-24 is referred to as phage YJ-pK3-24.

The composition of the LB liquid culture medium in the following examples is: 10 g/L tryptone; 5 g/L yeast extract; and 10 g/L sodium chloride.

The composition of the LB solid medium in the following examples is: 10 g/L tryptone; 5 g/L yeast extract; 10 g/L sodium chloride, and 15 g/L agar powder.

The composition of the SM buffer in the following examples is: 100 mmol/L sodium chloride; 8 mmol/L magnesium sulfate; 0.01% gelatin;

The DNase in the following examples is a product of Beijing Solebow Technology Co., Ltd., item number D8071-25 mg; the RNase is a product of Beijing Solebow Technology Co., Ltd., item number R8020-25 mg.

The host bacteria YJ-Kpn32-X in the following examples is Klebsiella pneumoniae, which is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with a deposit number of CGMCC No.23190.

Example 1. Isolation, purification and preservation of bacteriophage YJ-pK3-24

Preparation of phage isolation solution: In October 2021, 50 mL of sewage was collected from the sewers in Beijing as samples for phage isolation. The sewage was centrifuged at 5000 rpm for 10 minutes, and the supernatant after centrifugation was filtered through a 0.22 μm microporous filter to obtain the phage isolation solution.

Preparation of host bacteria YJ-Kpn32-X suspension: streak the stored host bacteria on LB solid culture medium, invert and culture in a 37°C incubator for 12-16 hours, pick a single clone into LB liquid culture medium, shake and culture at 37°C in a shaker at 220 rpm for 12-16 hours to obtain the host bacteria YJ-Kpn32-X suspension.

Isolation of phage: Add 50 μL of host bacteria YJ-Kpn32-X suspension and 200 μL of phage isolation solution to 5 mL of LB liquid culture medium, and culture at 37°C in a shaker at 220 rpm. After 4 hours of culture, centrifuge the culture at 10,000 rpm for 2 minutes, and filter the supernatant after centrifugation using a 0.22 μm microporous filter. Take 100 μL of the supernatant and perform plaque screening using the double-layer agar plate method.

Purification of phage: After the double-layer agar plate has been incubated in a 37°C incubator for 6-10 hours, use a pipette tip to take a single plaque and place it in 1mL SM buffer, shake and incubate for 1 hour, then take 100μL of liquid and add it to 5mL LB liquid culture medium, and add 50μL of logarithmic phase host bacteria YJ-Kpn32-X suspension, shake and culture at 220rpm in a 37°C shaker. After 4 hours of incubation, centrifuge the culture at 10000rpm for 2 minutes, and filter the supernatant after centrifugation with a 0.22μm microporous filter. Take 100μL of supernatant and use the double-layer agar plate method for plaque screening. Repeat the above operation 6-8 times until plaques with uniform and consistent morphology appear, and the purified phage is obtained.

In the present invention, the above method is used to obtain a phage strain, which is named YJ-pK3-24 in the present invention.

Preparation of purified phage YJ-pK3-24 culture medium: Add 100 μL of host bacteria YJ-Kpn32-X suspension and a purified YJ-pK3-24 plaque to 5 mL of fresh LB liquid culture medium, and culture at 37°C in a shaker at 220 rpm for 4 hours. Centrifuge the culture medium at 12,000 rpm for 5 minutes, and filter the supernatant after centrifugation with a 0.22 μm microporous filter to obtain phage YJ-pK3-24 culture medium.

Morphological observation of bacteriophage YJ-pK3-24 under electron microscope: After centrifugation of the purified bacteriophage YJ-pK3-24 culture solution, the supernatant was collected, 10% PEG8000 was added, and the precipitate was resuspended in SM buffer after centrifugation again to obtain a phage suspension. After diluting the phage suspension to an appropriate concentration, it was precipitated on the surface of a copper mesh, and the phage was stained with a 1% uranyl acetate solution. After transmission electron microscopy observation, a single phage with a complete morphology was found and photographed. The results are shown in Figure 1. Bacteriophage YJ-pK3-24 is a short-tailed phage with a regular hexagonal head with a head diameter of about 45nm; it has several short tail filaments.

According to the test, the bacteriophage YJ-pK3-24 of the present invention has good tolerance to temperature and pH, and can maintain a stable titer under the conditions of temperature 25-50°C and pH 6-10, and the optimal MOI of the bacteriophage and bacteria is 0.01. The incubation period of bacteriophage YJ-pK3-24 is 0-50 minutes, the burst period is 50-150 minutes, and then enters the stable period, and the average burst volume is about 50 plaque forming units/cell.

Phage YJ-pK3-24 can lyse the Klebsiella pneumoniae strain YJ-Kpn32-X, with a deposit number of CGMCC No. 23190. The bacteria can cause severe lung infection in a mouse model, specifically manifested as: pulmonary congestion and consolidation, alveolar wall thickening, lung inflammatory cell infiltration, and destruction of lung structure. The bacteriophage YJ-pK3-24 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 February 4, 2024, and its classification name is: bacteriophage, and the deposit number is CGMCC No. 45875. The bacteria are also referred to as bacteriophage YJ-pK3-24 in the present invention.

Example 2: Temperature tolerance test of bacteriophage YJ-pK3-24

1 mL of phage suspension (prepared in reference to Example 1) was incubated at 25°C, 37°C, 50°C, 60°C, 70°C and 80°C for 100 min, samples were taken at intervals of 10-15 minutes, and the phage activity was determined using the double-layer agar plate method.

The test results are shown in Figure 2. The bacteriophage YJ-pK3-24 has good temperature tolerance and can maintain a stable titer at temperatures between 25 and 50°C.

Example 3, pH tolerance test of bacteriophage YJ-pK3-24

Phage YJ-pK3-24 was inoculated into SM buffer with pH values of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13, respectively, incubated at 37°C for 1 hour, and the phage activity was determined using the double-layer agar plate method.

The test results are shown in Figure 3. The bacteriophage YJ-pK3-24 has good tolerance to pH and can maintain a stable titer at a pH value of 6-10.

Example 4. Optimal multiplicity of infection and one-step growth curve test of bacteriophage YJ-pK3-24

The multiplicity of infection (MOI) refers to the ratio of the number of phages to the number of host bacteria at the time of initial infection. The one-step growth curve is a curve that quantitatively describes the growth law of lytic phages, from which the incubation period, lytic period (outbreak period) and stable period of phages can be clearly seen.

Determination of the optimal MOI: The ratio of phage YJ-pK3-24 to host bacteria YJ-Kpn32-X, i.e., the MOI was 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, and 0.00001, was mixed and cultured with shaking, and the phage titer was determined by the double-layer agar plate method after 4 hours of culture. The highest titer was the optimal MOI. The test results (Figure 4) showed that when the MOI was 0.01, the titer of phage YJ-pK3-24 was the highest, i.e., the optimal MOI was 0.01.

Determination of one-step growth curve: Phage YJ-pK3-24 and host bacteria YJ-Kpn32-X were mixed and cultured at the optimal infection multiplicity ratio for 150 minutes, during which the phage titer was measured every 10 minutes. The test results are shown in Figure 5. The incubation period of phage YJ-pK3-24 is 0-50 minutes, and the outbreak period is 50-150 minutes. Calculation of the outbreak volume: Average outbreak volume = (average titer of phage in the stable period - average titer of phage at the initial stage) / initial bacterial concentration.

Example 5. Analysis of the lysis range of bacteriophage YJ-pK3-24

The Klebsiella pneumoniae 2-5, 2-6, 2-7, 2-8, 2-10, 2-11, 3-21, 3-22, and BAA-2146 in this embodiment are preserved in this laboratory. The public can obtain the above biological materials from the applicant. The biological materials are only used to repeat the experiments of the present invention and cannot be used for other purposes.

Preparation method of bacteriophage YJ-pK3-24 liquid: add 100 μL of host bacteria YJ-Kpn32-X suspension and 100 μL of bacteriophage YJ-pK3-24 purified liquid to 5 mL of fresh LB liquid culture medium, and culture at 37°C in a shaker at 220 rpm for 4 hours. Centrifuge the culture liquid at 12000 rpm for 5 minutes, and filter the supernatant after centrifugation with a 0.22 μm microporous filter to obtain bacteriophage YJ-pK3-24 culture liquid.

The bacteriophage YJ-pK3-24 was adjusted to a titer of 10 ^9- well plate forming units/ml for use. Multiple strains of Klebsiella pneumoniae were selected as the subjects to perform lysis range analysis on bacteriophage YJ-pK3-24. The specific operation was as follows:

Plaque test determination: Take 100 μL of the overnight culture of the strain to be tested, drop it on the LB solid culture medium plate, and spread it evenly with a coating rod. After the surface is dry, take 10 μL of phage YJ-pK3-24 droplets and add them on the surface of the plate; after the droplets are dry, invert them and culture them in a 37°C incubator for 4 to 6 hours, observe the results, if plaques are produced, record them as "+", otherwise "-", the results are shown in Table 1. The results show that phage YJ-pK3-24 has strong lysis specificity and high lysis rate (100%) for Klebsiella pneumoniae with MLST typing ST447, and has efficient lysis activity.

Table 1. Analysis of the lysis range of bacteriophage YJ-pK3-24

Example 6: Experiment on the effect of bacteriophage YJ-pK3-24 on lysis of YJ-Kpn32-X in G. mellonella

In the phage treatment group, YJ-Kpn32-X (2×10 ⁵ CFU) was injected into the first right posterior abdomen of G. mellonella. Two hours later, different concentrations of phage YJ-pK3-24 or LB medium were injected into the first left posterior abdomen of G. mellonella. In the phage control group, the highest concentration (2×10 ⁶ PFU) of phage YJ-pK3-24 was injected into the first left posterior abdomen of untreated G. mellonella to evaluate the safety of phage treatment. In the bacterial liquid control group, YJ-Kpn32-X (2×10 ⁵ CFU) was injected into the first left posterior abdomen of untreated G. mellonella to observe the mortality of G. mellonella. The number of G. mellonella deaths in each group was recorded every 12 hours for 72 hours for survival analysis. The results are shown in Figure 6. After the highest concentration of phage treatment, the number of deaths of G. mellonella decreased and the median death time was delayed, which proved that the phage YJ-pK3-24 had a good lysis effect in G. mellonella and the safety of YJ-pK3-24 was also very good.

The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims (10)

1. A bacteriophage, characterized in that the bacteriophage is Klebsiella phage YJ-pK3-24, and its deposit number in the General Microbiological Center of China National Microbiological Culture Collection is CGMCC No.45875. 2. A culture containing the bacteriophage according to claim 1. 3. A method for preparing the culture according to claim 2, characterized in that the method comprises: culturing the bacteriophage according to claim 1 in a culture medium to obtain the culture. 4. A preparation, characterized in that it comprises the bacteriophage according to claim 1 and/or the culture according to claim 2. 5. An antibacterial pharmaceutical composition against Klebsiella pneumoniae, characterized in that the antibacterial pharmaceutical composition comprises the bacteriophage according to claim 1 and/or the culture according to claim 2, and a pharmaceutically acceptable carrier. 6. Use of the bacteriophage according to claim 1 or the culture according to claim 2 in preparing a product for inhibiting or killing Klebsiella pneumoniae. 7. Use of the bacteriophage according to claim 1 or the culture according to claim 2 in the preparation of a product for preventing or treating Klebsiella pneumoniae infection or a disease caused by Klebsiella pneumoniae. 8. Use of the bacteriophage according to claim 1 or the culture according to claim 2 in killing Klebsiella pneumoniae in the environment. 9. The antibacterial pharmaceutical composition according to claim 5 or the use according to any one of claims 6 to 8, characterized in that the Klebsiella pneumoniae is ST447 type Klebsiella pneumoniae. 10. A method for killing Klebsiella pneumoniae in an environment, characterized in that the method comprises applying the bacteriophage according to claim 1, the culture according to claim 2 and/or the preparation according to claim 4 to the environment.