CN115261339A - Multiple drug-resistant sequence 383 type Klebsiella pneumoniae phage and application thereof - Google Patents

Abstract

The invention discloses a multidrug-resistant sequence 383 Klebsiella pneumoniae bacteriophage and its application. Specifically, the present invention provides a phage whose deposit number is CGMCC No.45098. The present invention also provides the application of the phage in preparing a reagent for specifically and efficiently lysing Klebsiella pneumoniae with multidrug-resistant sequence type 383. The bacteriophage of the invention can proliferate in a large amount in a short time, has good tolerance to temperature and pH, and can be used to treat pneumonia caused by multidrug-resistant sequence 383 Klebsiella pneumoniae.

Description

A kind of multi-drug resistant sequence 383 Klebsiella pneumoniae phage and its application

technical field

The present invention relates to a kind of Klebsiella phage (Klebsiella phage) and application thereof, specifically, relates to a kind of multi-drug resistance sequence 383 type Klebsiella pneumoniae phage and application thereof, and the preparation of the phage The application of the medicine for treating pneumonia caused by multidrug-resistant (multidrug-resistant, MDR) sequence 383 Klebsiella pneumoniae (Klebsiellapneumoniae, Kpn) belongs to the field of biotechnology.

Background technique

Klebsiella pneumoniae is an encapsulated, lactose-fermenting, facultatively anaerobic, Gram-negative bacterium that can be found in the human gastrointestinal tract, respiratory tract, and on the skin of healthy individuals. Klebsiella pneumoniae is also ubiquitous in the environment and is an opportunistic pathogen that causes a variety of infectious diseases, including pneumonia, urinary tract infection, bacteremia, and liver abscess, among others. In recent years, Klebsiella pneumoniae has become one of the main causes of nosocomial infections in the world, and the susceptible populations are immunocompromised populations, newborns and the elderly. Studies have shown that sequence 383 type Kpn is one of the dominant types in multi-drug resistant Klebsiella pneumoniae causing pneumonia.

In recent years, the case fatality rate of Klebsiella pneumoniae has been rising, which may be related to the situation of antibiotic resistance of Klebsiella pneumoniae. Klebsiella pneumoniae can carry a variety of antibiotic resistance genes, including the production of extended-spectrum β-lactamase and carbapenemase, making the infection difficult to cure. Studies have shown that the resistance rate of Kpn in Asia to cephalosporins such as ceftazidime, cefotaxime, and cefepime is higher than 60%, and the resistance rate to carbapenems such as imipenem and meropenem is also higher than 50%. %. Numerous multidrug-resistant and even extremely drug-resistant Klebsiella pneumoniae strains have emerged over the past two decades. The blowout outbreak of multi-drug resistant strains and the extremely rapid global spread have brought great challenges to clinical treatment.

Phages are viruses that specifically kill bacteria, even those that are resistant to antibiotics. In recent years, a large number of research and clinical trials have been carried out on phage therapy. Compared with traditional antibacterial drugs, phages have many advantages, especially in terms of specificity and biological safety. Phages are mainly composed of proteins and nucleic acids, which can specifically lyse target bacteria without affecting other bacteria, viruses or host cells. They have little or no toxicity and have good clinical application prospects.

Therefore, if a phage capable of specifically cleaving sequence 383 type MDR sequence 383 type Kpn can be provided, it will be of great significance to the treatment of pneumonia caused by MDR sequence type 383 type Kpn.

Contents of the invention

One object of the present invention is to provide a Klebsiella pneumoniae phage with multi-drug resistance sequence 383.

Another object of the present invention is to provide related applications of the phage.

The inventors of the present case isolated a lytic multidrug-resistant Klebsiella pneumoniae phage, which was named pKp383 in the present invention. The bacteriophage pKp383 of the present invention has been preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee (Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on April 26, 2022. Date: April 26, 2022; Classification name: Klebsiella phage (Klebsiella phage), deposit number is CGMCC No.45098. This bacterium is also called phage pKp383 in the present invention.

Specifically, in one aspect, the present invention provides a bacteriophage whose deposit number is CGMCC No.45098.

On the other hand, the present invention provides a phage preparation, which includes: the phage whose deposit number is CGMCC No. 45098, and auxiliary materials.

According to a specific embodiment of the present invention, in the phage preparation of the present invention, the adjuvant includes nutritional components for maintaining the activity of the bacteria. In some specific embodiments, the auxiliary material can be LB medium. In some more specific embodiments, in the LB medium, tryptone 10g/L; yeast extract 5g/L; sodium chloride 10g/L.

According to some embodiments of the invention, the phage preparation of the invention is a medicament.

According to some embodiments of the invention, the bacteriophage preparation of the invention is a cleaning or disinfecting agent.

On the other hand, the present invention provides the application of the phage with deposit number CGMCC No. 45098 for lysing Klebsiella pneumoniae of multi-drug resistance sequence 383 type in vitro.

In some specific embodiments of the present invention, the present invention has determined the host spectrum of the phage pKp383, and the determination results show that the phage pKp383 can lyse 6 multi-drug resistant sequence 383 type Kpn strains.

In another aspect, the present invention provides the use of the phage with the deposit number CGMCC No. 45098 in the preparation of a preparation for lysing Klebsiella pneumoniae of multi-drug resistance sequence 383 type.

In another aspect, the present invention provides the use of the phage with the deposit number CGMCC No. 45098 in the preparation of medicines for preventing and treating pneumonia caused by multi-drug resistant sequence 383 Klebsiella pneumoniae.

According to a specific embodiment of the present invention, in the application of the phage of the present invention, the multidrug-resistant sequence 383 Klebsiella pneumoniae includes strain C6 with the deposit number of CGMCC No. 12540.

The phage pKp383 of the present invention is a long-tailed phage, has good tolerance to the environment, and can maintain a stable titer under the conditions of temperature 4-50°C and pH 6-10. The optimal multiplicity of infection for phage and bacteria is 0.001. The one-step growth curve shows that the incubation period of the bacteriophage pKp383 of the present invention is 10 minutes, the burst period is 110 minutes, and then enters the plateau period. The phage of the present invention can lyse the MDR sequence 383 type Kpn strain, especially the sequence 383 type strain C6 (Genebank number is JAJOVD000000000, which is an MDR Kpn strain in the prior art). The phage pKp383 of the present invention can effectively inhibit the growth of multidrug-resistant sequence 383 Klebsiella pneumoniae in vitro within the multi-infection range of 10 to ^10-5 , and is useful for the development and treatment of pneumonia caused by MDR sequence 383 Kpn Drugs matter. In addition, in order to verify the specificity of pKp383, we also tested the ability of other six phages to lyse strain C6, and the results showed that only pKp383 could lyse strain C6.

In summary, the present invention provides a phage pKp383, which can specifically lyse Klebsiella pneumoniae of multidrug-resistant sequence type 383, and has the characteristics of a wide range of hosts, high lysis efficiency, and good environmental tolerance. It has good inhibitory effect on Klebsiella pneumoniae type 383 multi-drug resistant sequence, and has a good application prospect.

Description of drawings

Figure 1 shows the transmission electron micrograph of phage pKp383.

Fig. 2 is a graph showing the temperature tolerance test of phage pKp383.

Fig. 3 is a graph showing the pH tolerance test of phage pKp383.

Figure 4 is a one-step growth curve of phage pKp383.

Fig. 5 shows the cleavage curve of phage pKp383 to host strain C6.

Fig. 6 is a graph showing the lysing ability of 7 strains of phages to the host bacterium C6.

Preservation of biological material for patent proceedings:

The bacteriophage pKp383 of the present invention (because of its ability to lyse the multidrug-resistant sequence 383 type Kpn strain, it was registered with the self-named pKp383 when it was submitted for deposit):

Deposit date: April 26, 2022;

Preservation unit: General Microbiology Center (CGMCC) of China Microbiological Culture Collection Management Committee;

Address of Preservation Unit: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing

Deposit number: CGMCC No.45098;

Classification name: Klebsiella phage (Klebsiella phage).

Detailed ways

In order to make the technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples, unless otherwise specified, are conventional biochemical reagents, which can be obtained commercially.

Example 1. Isolation, purification and preservation of phage pKp383

Preparation of phage isolation liquid: In June 2021, 50 mL of hospital sewage was collected from the Beijing area as a sample for phage isolation. The sewage was centrifuged at 4000 rpm for 20 minutes to remove large particles. The supernatant was filtered through a 0.22 μm millipore filter to remove bacteria. Obtain phage isolates.

Isolation of phage: take MDR sequence 383 type Klebsiella pneumoniae C6 as the host strain, add 50 μL of bacterial suspension and 200 μL of phage separation solution to 5 mL of LB medium, and shake at 220 rpm for 4 hours at 37°C. Centrifuge at 10,000 rpm for 2 minutes, and filter the supernatant with a 0.22 μm microporous filter. Take 100 μL of the supernatant, and use the double-layer agar plate method for phage plaque screening.

Purification of phage: After the double-layer agar plate was cultured statically in a 37°C incubator for 12-24 hours, pick a single phage plaque with an inoculation loop and put it into the LB liquid medium, and add 50 μL of the host bacteria C6 suspension at the same time. Shake culture at 37° C. at 220 rpm for 4 hours, centrifuge the culture solution at 10,000 rpm for 2 minutes, collect the supernatant, and filter with a 0.22 μm microporous filter. Take 100 μL of the filtered supernatant, and use the double-layer agar plate method for plaque purification. Repeat the above operation 4-5 times until uniform and consistent phage plaques appear, that is, purified phages are obtained.

In the present invention, a phage strain is obtained by using the above method, which is named pKp383 in the present invention.

Morphological observation of phage pKp383 under an electron microscope: 1% chloroform, DNase and RNase were added to the purified phage pKp383 culture solution. After the above solution was centrifuged, the supernatant was collected, 10% PEG8000 was added, and the precipitate was resuspended with SM buffer after centrifugation again. Add 1% chloroform again, take the upper aqueous phase after centrifugation, and obtain the phage suspension. After diluting the phage suspension to an appropriate concentration, settle on the surface of the copper grid, negatively stain the phage particles with 2% (wt./vol) uranyl acetate (pH 7.0), and observe with a transmission electron microscope at 80KV to find The field of view of the intact form of a single phage was photographed and recorded. As shown in Figure 1, phage pKp383 is a long-tailed phage with an icosahedral head, about 76 nm in diameter, and about 126 nm in tail length.

After testing, the phage pKp383 of the present invention has good thermal stability and pH stability, can maintain a stable titer under the conditions of temperature 4-50°C and pH 6-10, and the optimal multiplicity of infection between phage and bacteria is 0.001. The incubation period of phage pKp383 was 10 minutes, the burst time was 110 minutes, and then entered the plateau period.

The MDR Kpn strain C6 (Genebank No. JAJOVD000000000) that can be cleaved by phage pKp383 is sequence 383. Sequence 383 has been shown to be a prevalent sequence type among pneumonia-associated Klebsiella pneumoniae isolates. The bacteriophage pKp383 of the present invention has been preserved in the General Microbiology Center of China Microbial Culture Collection Management Committee (Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on April 26, 2022, and the date of preservation : On April 26, 2022, the classification was named: Klebsiella phage, and the preservation number was CGMCC No.45098. This bacterium is also called phage pKp383 in the present invention.

Embodiment 2, temperature tolerance test of bacteriophage pKp383

Take 1 mL of phage suspension (prepared with reference to Example 1) pKp383 and incubate at 4°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C and 80°C for 1 hour, use The titer of phage was determined by double-layer agar plate method.

The test results are shown in Figure 2, the phage pKp383 has good thermal stability, and can maintain a stable titer at a temperature of 4-50°C.

Example 3, pH tolerance test of bacteriophage pKp383

Phage pKp383 was inoculated in SM buffer with pH values of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14, and incubated at 37°C for 1 hour. Phage titers were determined using the double-layer agar plate method.

The test results are shown in Figure 3, the bacteriophage pKp383 has good pH stability, and can maintain a stable titer at a pH value of 6-10.

The best multiplicity of infection and one-step growth curve test of embodiment 4, phage pKp383

Determination of the optimum multiplicity of infection: The optimum multiplicity of infection (MOI) was determined by double-layer agar plate method. The MOI of adding phage pKp383 to the log phase host strain C6 were 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100, respectively. After mixing and shaking for 4 hours, the phage titer in the optimal MOI treatment group was the highest. The test results showed that when the multiplicity of infection was 0.001, the titer of phage pKp383 was the highest, that is, the optimal multiplicity of infection was 0.001.

Determination of one-step growth curve: Infect the host strain C6 with phage pKp383 under the optimal MOI condition, and measure the phage titer every 10 minutes during shaking culture for 150 minutes. The test results are shown in Figure 4, the incubation period of phage pKp383 was 10 minutes, the burst time was 110 minutes, and then entered the plateau period.

Embodiment 5, bacteriophage pKp383 is to the cracking ability of host bacterium C6 and host spectrum test

Determination of the lysing ability of the host strain C6: the phage pKp383 to the host strain C6 was determined under the conditions of the multiplicity of infection of 10, 1, 0.1, 0.01, 0.001, 0.0001, 10 ^-5 , 10 ^-6 , 10 ^-7 and 10 ^-8 Cleavage activity. During shaking culture for 7 hours, the OD ₆₀₀ value was detected once every hour with a microplate reader. The measurement results are shown in Figure 5. When the MOI was 10 to 10 ^-5 , pKp383 completely inhibited the host strain Kp C6, and the phage pKp383 showed good lytic ability to the host strain. It can be used as a candidate phage preparation for treating acute pneumonia caused by MDR Kpn.

Determination of host spectrum: phage pKp383 and 6 strains of sequence 383 type MDRKpn were mixed and shaken for 4 hours at the ratio of the optimal multiplicity of infection, and the culture solution was observed. The clarification of the culture medium means that the phage pKp383 can cleave the MDR Kpn. The assay results showed that phage pKp383 could cleave 6 strains of 383-type MDR Kpn with different sequences, and the specific information of the 6 strains of MDR Kpn is shown in Table 1.

Table 1

Strain number sequence type C6 383 Kp383-2 383 Kp383-3 383 Kp383-4 383 Kp383-5 383 Kp383-6 383

Embodiment 6, the lysing ability test of phage to host bacterium C6

Add 200 μL of bacteriophage pKp-1, pKp-2, pKp-3, pKp-4, pKp-5, pKp11 and pKp383 to 7 tubes of 5 mL LB medium containing 50 μL of host bacterial C6 suspension, shake at 37 °C Shake culture at 220 rpm. After culturing for 4 hours, observe whether the culture medium is clear. As shown in Figure 6, the culture fluid of the pKp-1, pKp-2, pKp-3, pKp-4, pKp-5 and pKp11 treatment groups was turbid, indicating that the 6 strains of phage could not lyse the host bacterium C6; while the pKp383 treatment group The culture medium was clarified, indicating that pKp383 could lyse the host strain C6.

It should be noted that the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (10)

1. A Klebsiella phage, the preservation number of which is CGMCC No.45098. 2. The phage according to claim 1, which maintains a stable titer under the conditions of temperature 4-50°C and pH 6-10. 3. A phage preparation, which comprises: the phage whose deposit number is CGMCC No.45098, and auxiliary materials. 4. phage preparation according to claim 3, wherein, described adjuvant comprises the nutrient component that is used to maintain thalline activity; Preferably, the auxiliary material is LB medium. 5. The phage preparation according to claim 3, which is a drug. 6. The phage preparation according to claim 3, which is a cleaning or disinfecting agent. 7. The application of the phage with deposit number CGMCC No. 45098 for lysing Klebsiella pneumoniae type 383 multi-drug resistant sequence in vitro. 8. The application of the phage with the deposit number CGMCC No.45098 in the preparation of a preparation for cracking Klebsiella pneumoniae of multi-drug resistance sequence type 383. 9. The use of the phage with the deposit number CGMCC No.45098 in the preparation of medicines for preventing and treating pneumonia caused by multi-drug resistant sequence 383 Klebsiella pneumoniae. 10. The application according to any one of claims 7-9, wherein the multidrug-resistant sequence type 383 Klebsiella pneumoniae includes a strain with deposit number CGMCC No.45098.

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