CN115261339B

CN115261339B - Klebsiella pneumoniae phage with multiple drug-resistant sequences 383 and application thereof

Info

Publication number: CN115261339B
Application number: CN202210997923.3A
Authority: CN
Inventors: 袁静; 甘霖; 赵汉青; 冯燕玲; 崔晓虎; 范政; 崔晶花; 薛冠华; 闫超; 冯俊霞
Original assignee: Capital Institute of Pediatrics
Current assignee: Capital Institute of Pediatrics
Priority date: 2022-08-19
Filing date: 2022-08-19
Publication date: 2024-02-06
Anticipated expiration: 2042-08-19
Also published as: CN115261339A

Abstract

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

Description

Klebsiella pneumoniae phage with multiple drug-resistant sequences 383 and application thereof

Technical Field

The invention relates to a Klebsiella phage (Klebsiella phage) and application thereof, in particular to a Klebsiella pneumoniae phage with a multi-drug-resistant sequence 383 and application thereof, and application of the phage in preparing a medicament for treating pneumonia caused by Klebsiella pneumoniae (Klebsiella pneumoniae, kpn) with a multi-drug-resistant (MDR) sequence 383, belonging to the field of biotechnology.

Background

Klebsiella pneumoniae is a capsular, fermented lactose, facultative anaerobic gram-negative bacterium that can be found in the human gastrointestinal tract, respiratory tract, and skin of healthy individuals. Klebsiella pneumoniae is also ubiquitous in the environment and is an opportunistic pathogen that can cause a variety of infectious diseases including pneumonia, urinary tract infections, bacteremia, liver abscesses and the like. In recent years, klebsiella pneumoniae has become one of the main causes of nosocomial infections in the world, and susceptible people are immunocompromised people, newborns and the elderly. Studies show that in Klebsiella pneumoniae with multiple drug resistance, which causes pneumonia, kpn of the sequence 383 type is one of dominant types.

In recent years, the death rate of klebsiella pneumoniae has been increasing, which may be related to the antibiotic resistance situation of klebsiella pneumoniae. Klebsiella pneumoniae can carry multiple antibiotic resistance genes, including beta-lactamase and carbapenemase which produce an ultra-broad spectrum, so that infection is difficult to cure. Studies show that Kpn in Asian areas has a drug resistance rate of higher than 60% for cephalosporins such as ceftazidime, cefotaxime and cefepime and a drug resistance rate of higher than 50% for carbapenem drugs such as imipenem and meropenem. Over the last two decades, numerous strains of klebsiella pneumoniae have emerged that are multi-resistant, even extremely resistant. The outbreak of multiple resistant strains of well-blown and extremely rapid global spread situation presents a great challenge for clinical treatment.

Phage is a virus that specifically kills bacteria and also acts on bacteria that are resistant to the antigen. In recent years, phage therapy has been extensively studied and clinically tested. Phages have many advantages over traditional antibacterial agents, especially in terms of specificity and biosafety. The phage mainly comprises protein and nucleic acid, can specifically lyse target bacteria without affecting other bacteria, viruses or host cells, has little or no toxicity, and has good clinical application prospect.

Therefore, if a phage which specifically lyses type 383 Kpn of type 383 MDR sequence can be provided, it would be of great importance for the treatment of pneumonia caused by type 383 Kpn of MDR sequence.

Disclosure of Invention

It is an object of the present invention to provide a multiple drug resistant sequence 383 Klebsiella pneumoniae bacteriophage.

It is a further object of the present invention to provide related uses of said phage.

The inventor separates and obtains a splitting multi-drug resistant Klebsiella pneumoniae bacteriophage, which is named pKp383 in the invention. The phage pKp383 of the invention was deposited at the China general microbiological culture Collection center (address: north West Lu No. 1, north Sei Lu No. 3, national academy of sciences of China) at 4 months 26 of 2022, and the date of the deposition: 2022, 4, 26; the classification is named: klebsiella phage (Klebsiella phage) with a preservation number of CGMCC No.45098. The bacterium is also called phage pKp383 in the present invention.

In particular, in one aspect, the invention provides a phage with a collection number of CGMCC No.45098.

In another aspect, the present invention provides a phage preparation comprising: phage with preservation number of CGMCC No.45098 and auxiliary materials.

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

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

According to some embodiments of the invention, the phage preparation of the invention is a detergent or disinfectant.

On the other hand, the invention provides the application of phage with the preservation number of CGMCC No.45098 in the in-vitro cracking of the multiple drug resistant sequence 383 type Klebsiella pneumoniae.

In some embodiments of the invention, the invention provides an assay for the host profile of phage pKp383, which shows that phage pKp383 can lyse 6 multi-drug resistant sequence 383-type Kpn strains.

On the other hand, the invention provides the application of the phage with the preservation number of CGMCC No.45098 in preparing a preparation for cracking multiple drug resistant sequence 383 type Klebsiella pneumoniae.

On the other hand, the invention provides the application of the phage with the preservation number of CGMCC No.45098 in preparing the medicine for preventing and treating the pneumonia caused by the Klebsiella pneumoniae with the multiple drug-resistant sequence 383.

The phage pKp383 belongs to long-tail phage, has good tolerance to environment and can keep stable titer under the conditions of temperature of 4-50 ℃ and pH of 6-10. The optimal multiplicity of infection of phage and bacteria was 0.001. The one-step growth curve shows that the phage pKp383 of the invention had a latency of 10 minutes, a burst of 110 minutes, and then a plateau. The phage of the invention can lyse MDR sequence 383 type Kpn strain, especially for sequence 383 type strain C6 (Genebank number is JAJOVD000000000, a MDR Kpn strain in the prior art). Phage pKp of the invention3 at a multiplicity of infection of 10 to 10 ^-5 Can effectively inhibit the growth of Klebsiella pneumoniae with multiple drug resistant sequences 383 in vitro within the range, and has important significance for developing medicaments for treating pneumonia caused by MDR sequence 383 type Kpn. Furthermore, to verify the specificity of pKp383, we also examined the lytic capacity of the other 6 phages against strain C6, which showed only pKp383 to lyse strain C6.

In summary, the invention provides a phage pKp383 which can specifically lyse Klebsiella pneumoniae with a multi-drug resistant sequence 383, has the characteristics of wide host spectrum, high lysis efficiency and good environmental tolerance, has a good inhibition effect on the Klebsiella pneumoniae with the multi-drug resistant sequence 383, and has a good application prospect.

Drawings

FIG. 1 shows a transmission electron microscope image 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.

FIG. 4 shows a one-step growth plot of phage pKp383.

FIG. 5 shows a graph of the lysis of phage pKp383 against host C6.

FIG. 6 is a graph showing the lytic ability test of 7 phages against host bacterium C6.

Biological material preservation for patent procedures:

phage pKp383 of the invention (registered under the self-designation pKp383 at the time of submission to storage because of its lytic ability to a Kpn strain of the type 383 of the multidrug resistance sequence):

preservation date: 2022, 4, 26;

preservation unit: china general microbiological culture Collection center (CGMCC);

deposit unit address: beijing city, the region of Chaoyang, north Chen Xili, no. 1, 3, china academy of sciences microbiological institute

Preservation number: CGMCC No.45098;

classification naming: klebsiella phage (Klebsiella phage).

Detailed Description

In order to make the technical scheme and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings and examples.

The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the examples described below, unless otherwise specified, are all conventional biochemical reagents and are commercially available.

EXAMPLE 1 isolation purification and preservation of phage pKp383

Preparation of phage isolates: 50mL of hospital wastewater was collected from Beijing area as phage isolated samples, 6 months 2021. The wastewater was centrifuged at 4000rpm for 20 minutes to remove large particles. The supernatant was filtered through a 0.22 μm microporous filter to remove bacteria. Obtaining phage separating liquid.

Isolation of phages: with MDR sequence 383 Klebsiella pneumoniae C6 as a host strain, 50. Mu.L of a bacterial suspension and 200. Mu.L of a phage isolate were added to 5mL of LB medium, the culture was shake-cultured at 220rpm and 37℃for 4 hours, the culture was centrifuged at 10000rpm for 2 minutes, and the supernatant was filtered using a 0.22 μm microporous filter. 100. Mu.L of the supernatant was used for plaque screening by double-layer agar plate method.

Purification of phage: after the double-layer agar plates were allowed to stand for 12-24 hours in a 37℃incubator, individual plaques were picked up with an inoculating loop into LB liquid medium, 50. Mu.L of host bacterium C6 suspension was added at the same time, the culture was shake-cultured in a shaking table at 220rpm for 4 hours at 37℃and centrifuged at 10000rpm for 2 minutes, and the supernatant was collected and filtered using a 0.22 μm microporous filter. 100. Mu.L of the filtered supernatant was used for plaque purification by double-layer agar plate method. Repeating the above operation for 4-5 times until uniform plaque appears, to obtain purified phage.

In the present invention, a phage, named pKp383 in the present invention, was obtained by the above method.

Morphological observations under electron microscope of phage pKp 383: 1% chloroform, DNase and RNase were added to the purified phage pKp383 broth. The supernatant was collected after centrifugation of the solution, 10% PEG8000 was added, and the pellet was resuspended in SM buffer after centrifugation again. Again 1% chloroform was added and after centrifugation the upper aqueous phase was taken to obtain phage suspension. After dilution of the phage suspension to the appropriate concentration, sedimentation on the copper mesh surface, negative staining of phage particles with 2% (wt./vol) uranium acetate (pH 7.0) and observation with a transmission electron microscope at 80KV, photographic recording after finding the complete form of the individual phage. As shown in FIG. 1, phage pKp383 is a long tail phage with an icosahedron head, a diameter of about 76nm and a tail length of about 126nm.

According to detection, the phage pKp383 has good heat stability and pH stability, can keep stable titer under the conditions of temperature of 4-50 ℃ and pH of 6-10, and has an optimal complex infection of phage and bacteria of 0.001. Phage pKp383 had a latency of 10 minutes and a burst time of 110 minutes, followed by entry into the plateau.

MDR Kpn strain C6 (Genebank number JAJOVD 000000000) from which phage pKp383 can lyse is of the type sequence 383. Sequence 383 has been shown to be a common sequence form of Klebsiella pneumoniae isolates associated with pneumonia. The phage pKp383 of the invention was deposited at the China general microbiological culture Collection center (address: north West Lu No. 1, north Sei Lu No. 3, china academy of sciences of the area of facing Yang) at 4 months 26 of 2022, and the date of deposit: 2022, 4, 26, classification designation: klebsiella phage with preservation number of CGMCC No.45098. The bacterium is also called phage pKp383 in the present invention.

Example 2, temperature tolerance test of phage pKp383

Phage titers were determined by a double-layer agar plate method by incubating pKp383 1mL of phage suspension (prepared in reference example 1) at 4 ℃, 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ for 1 hour, respectively.

As shown in FIG. 2, phage pKp383 has good heat stability and can maintain stable titer at 4-50deg.C.

Example 3, PH tolerance test of phage pKp383

Phage pKp383 were inoculated into SM buffers at pH 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14, respectively, incubated at 37℃for 1 hour, and phage titers were determined using the double-layer agar plate method.

As shown in FIG. 3, phage pKp383 has good pH stability and can maintain stable titer under the condition of pH value of 6-10.

Example 4, optimal multiplicity of infection and one-step growth Curve test for phage pKp383

Determination of optimal multiplicity of infection: the optimal multiplicity of infection (MOI) was determined using a double-layer agar plate method. The MOI of phage pKp383 added to log-phase host bacterium C6 was 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100, respectively. After 4h of mixed shaking culture, the phage titers of the optimal MOI treatment group were highest. The test results showed that phage pKp383 had the highest titer, i.e., the optimal multiplicity of infection was 0.001, when the multiplicity of infection was 0.001.

Measurement of one-step growth curve: phage pKp383 was infected to host strain C6 under optimal MOI conditions and phage titers were determined every 10 minutes in 150 minutes of shaking culture. The test results are shown in FIG. 4, with phage pKp383 having a latency of 10 minutes and a burst time of 110 minutes, followed by entry into the plateau.

EXAMPLE 5 lytic ability of phage pKp383 against host bacterium C6 and test of host Spectrum

Determination of the lytic ability of the host bacterium C6: at a complex number of infections of 10, 1, 0.1, 0.01, 0.001, 0.0001, 10 ^-5 、10 ^-6 、10 ^-7 And 10 ^-8 The lytic activity of phage pKp383 against host strain C6 was determined under the conditions. Shake culturing for 7 hr, and detecting OD per hr with enzyme labeling instrument ₆₀₀ The value was 1 time. As a result of measurement, as shown in FIG. 5, MOI was 10 to 10 ^-5 When pKp383 completely inhibited the host strain Kp C6, phage pKp383 exhibited good lytic ability against the host strain. Can be used as a candidate phage preparation for treating acute pneumonia caused by MDR Kpn.

Host profile determination: phage pKp383 and 6 lines of sequence 383 MDR Kpn were mixed and shake cultured at the ratio of optimal multiplicity of infection for 4 hours, respectively, and the culture broth was observed. The culture medium was clarified to give phage pKp383 which lyses the MDR Kpn. The results of the assay show that phage pKp383 can cleave 6 different sequences 383 of the type MDR Kpn and that specific information for 6 MDR Kpn is shown in Table 1.

TABLE 1

Strain numbering	Sequence type
		C6	383
Kp383-2	383
		Kp383-3	383
Kp383-4	383
		Kp383-5	383
Kp383-6	383

Example 6 test of the lytic ability of phage against host bacterium C6

200. Mu.L of phages pKp-1, pKp-2, pKp-3, pKp-4, pKp-5, pKp11 and pKp383 were added to 7 tubes of 5mL LB medium containing 50. Mu.L of host bacterium C6 suspension, respectively, and cultured by shaking at 220rpm in a shaking table at 37 ℃. After 4 hours of incubation, the culture broth was observed for clarity. As shown in FIG. 6, the culture broths of the treatment groups pKp-1, pKp-2, pKp-3, pKp-4, pKp-5 and pKp11 were cloudy, indicating that the 6 phages were unable to lyse the host bacterium C6; whereas the culture of the pKp383 treated group was clear, indicating pKp383 could lyse host bacteria C6.

It should be noted that the above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, and that various modifications and variations of the present invention will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, 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 Klebsiella phage, the deposit number of which is CGMCC No. 45098.

2. The phage according to claim 1, which maintains stable titer under the conditions of temperature 4-50°C and pH 6-10.

3. A phage preparation, which includes: the phage deposited as CGMCC No. 45098, and auxiliary materials.

4. The phage preparation according to claim 3, wherein the auxiliary materials include nutritional components for maintaining bacterial activity.

5. The phage preparation according to claim 4, wherein the auxiliary material is LB culture medium.

6. The phage preparation according to claim 3, which is a drug.

7. The phage preparation according to claim 3, which is a cleaning agent or disinfectant.

8. Application of phage deposited with CGMCC No. 45098 for in vitro lysis of multidrug-resistant sequence 383 Klebsiella pneumoniae.

9. Use of phage deposited with CGMCC No. 45098 in the preparation of preparations for lysing Klebsiella pneumoniae type 383, a multidrug-resistant sequence.

10. Application of the phage deposited as CGMCC No. 45098 in the preparation of drugs for the prevention and treatment of pneumonia caused by Klebsiella pneumoniae type 383, a multidrug-resistant sequence.