CN115261339A - Multiple drug-resistant sequence 383 type Klebsiella pneumoniae phage and application thereof - Google Patents
Multiple drug-resistant sequence 383 type Klebsiella pneumoniae phage and application thereof Download PDFInfo
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Abstract
The invention discloses a multiple drug-resistant sequence 383 type Klebsiella pneumoniae phage and application thereof. Specifically, the invention provides a bacteriophage, the preservation number of which is CGMCC No.45098. The invention also provides application of the phage in preparation of a reagent for specifically and efficiently cracking Klebsiella pneumoniae with multiple drug-resistant sequences 383. The phage can be massively proliferated in a short time, has good tolerance to temperature and pH value, and can be used for treating pneumonia caused by multiple drug-resistant sequence 383 type Klebsiella pneumoniae.
Description
Technical Field
The invention relates to a Klebsiella pneumoniae (Klebsiella phage) and application thereof, in particular to a Klebsiella pneumoniae (Klebsiella pneumoniae) with a multiple drug-resistant sequence 383 and application thereof, and application of the phage in preparation of a drug for treating pneumonia caused by Klebsiella pneumoniae (Kpn) with a multiple drug-resistant (MDR) sequence 383, belonging to the field of biotechnology.
Background
Klebsiella pneumoniae is a capsulated, fermented lactose, facultative anaerobic, gram-negative bacterium that can be found in the gastrointestinal tract, respiratory tract, and skin of healthy individuals. Klebsiella pneumoniae is also ubiquitous in the environment, is an opportunistic pathogen, and can cause various infectious diseases including pneumonia, urinary tract infection, bacteremia, liver abscess 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 people with low immune function, newborns and the elderly. Research shows that the sequence 383 type Kpn is one of the dominant types in the multiple drug-resistant Klebsiella pneumoniae causing pneumonia.
The mortality rate of Klebsiella pneumoniae has increased in recent years, which may be related to the antibiotic resistance situation of Klebsiella pneumoniae. Klebsiella pneumoniae can carry various antibiotic resistance genes, including production of extended-spectrum beta-lactamase and carbapenemase, and the like, so that infection is difficult to cure. Research shows that the drug resistance rate of Kpn to cephalosporins such as ceftazidime, cefotaxime and cefepime in Asia region is higher than 60%, and the drug resistance rate to carbapenems such as imipenem and meropenem is also higher than 50%. Over the last two decades, numerous multidrug-resistant and even extremely resistant strains of klebsiella pneumoniae have emerged. The outbreak of the multi-drug resistant strains in a well-sprayed manner and the extremely rapid global spread of the situation present a great challenge to clinical treatment.
Bacteriophage is a virus that specifically kills bacteria, and also acts against antibiotic-resistant bacteria. In recent years, a great deal of research and clinical trials have been conducted on phage therapy. Bacteriophages have many advantages over traditional antibacterial drugs, especially in terms of specificity and biosafety. The phage mainly comprises protein and nucleic acid, can specifically crack target bacteria under the condition of not influencing other bacteria, viruses or host cells, has little or no toxicity, and has good clinical application prospect.
Therefore, if a phage capable of specifically cleaving the 383 type Kpn of MDR sequence 383 is provided, it would be of great significance to the treatment of pneumonia caused by the 383 type Kpn of MDR sequence 383.
Disclosure of Invention
An object of the present invention is to provide a multiple drug resistant sequence 383 type Klebsiella pneumoniae phage.
It is another object of the invention to provide related uses of the bacteriophage.
The inventor separates and obtains a lytic multi-drug-resistant Klebsiella pneumoniae phage, which is named as pKp in the invention. The phage pKp383 of the present invention has been deposited in the general microbiological culture collection center of the China Committee for culture Collection of microorganisms at 26.4.2022 (address: west Lu No.1 Hospital No. 3, institute of microbiology, china academy of sciences), and its deposition date: 26 months 4 in 2022; the classification is named as: klebsiella phage (Klebsiella phase) with the preservation number of CGMCC No.45098. The bacterium is also called phage pKp383 in the invention.
Specifically, in one aspect, the invention provides a bacteriophage with the preservation number of CGMCC No.45098.
In another aspect, the invention provides a phage preparation comprising: bacteriophage with the preservation number of 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 phage. In some embodiments, the adjuvant may be LB medium. In some more specific embodiments, 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 pharmaceutical.
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 the bacteriophage with the preservation number of CGMCC No.45098 in-vitro cracking of multiple drug-resistant sequence 383 type Klebsiella pneumoniae.
In some embodiments of the invention, the host spectrum of phage pKp383 is determined, and the determination result shows that phage pKp can crack 6 multi-drug-resistant sequence 383 type Kpn strains.
On the other hand, the invention provides the application of the bacteriophage 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 bacteriophage with the preservation number of CGMCC No.45098 in preparing the medicine for preventing and treating pneumonia caused by the multiple drug-resistant sequence 383 type Klebsiella pneumoniae.
According to a specific embodiment of the invention, in the application of the bacteriophage of the invention, the multiple drug-resistant sequence 383 type klebsiella pneumoniae comprises a strain C6 with the preservation number of CGMCC No. 12540.
The phage pKp383 belongs to long-tail phage, has good tolerance to the environment, and can keep stable titer at the temperature of 4-50 ℃ and the pH value of 6-10. The optimal multiplicity of infection of phage and bacteria is 0.001. One-step growth curves show that phage pKp383 of the present invention had a 10 minute latency, a 110 minute burst phase, and then a plateau phase. The phage can crack MDR sequence 383 type Kpn strain, especially for sequence 383 type strain C6 (Genebank number is JAJOVD000000000, and is MDR Kpn strain in the prior art). Phage pKp383 of the invention had a multiplicity of infection of 10 to 10 -5 Can effectively inhibit the growth of the multiple drug-resistant sequence 383 type Klebsiella pneumoniae in vitro, and has important significance for developing the medicine for treating pneumonia caused by the MDR sequence 383 type Kpn. In addition, to verify the specificity of pKp383, we also tested the lytic capacity of other 6 phage on strain C6, and the result showed that only pKp383 could lyse strain C6.
In conclusion, the invention provides the phage pKp383 capable of specifically cracking the multiple drug-resistant sequence 383 type Klebsiella pneumoniae, and the phage pKp has the characteristics of wide host spectrum, high cracking efficiency and good environmental tolerance, has a good inhibition effect on the multiple drug-resistant sequence 383 type Klebsiella pneumoniae, and has a good application prospect.
Drawings
FIG. 1 shows a transmission electron micrograph of phage pKp383.
FIG. 2 shows a graph of the temperature tolerance test of phage pKp.
FIG. 3 shows a pH tolerance test chart of phage pKp.
FIG. 4 is a graph showing the one-step growth of phage pKp383.
FIG. 5 shows the lysis profile of phage pKp383 against host bacterium C6.
FIG. 6 shows a graph for testing the lytic ability of 7 phage strains against host bacterium C6.
Preservation of biological material for patent procedure:
phage pKp383 of the invention (which, because of its lytic capacity against multiple drug resistant sequence 383 type Kpn strains, was filed on deposit under its self-designation registration at pKp):
the preservation date is as follows: 26 months 4 in 2022;
the preservation unit: china general microbiological culture Collection center (CGMCC);
the address of the depository: xilu No.1 Hospital No. 3, the institute of microbiology, china academy of sciences, beijing, chaoyang
The preservation number is: CGMCC No.45098;
and (3) classification and naming: klebsiella phage (Klebsiella phase).
Detailed Description
In order to make the technical solutions and advantages of the present invention clearer, the present invention will be further described in detail with reference to the accompanying drawings and examples.
The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples, unless otherwise specified, were all conventional biochemical reagents and were commercially available.
Example 1 isolation, purification and preservation of phage pKp383
Preparation of phage isolate: in 6 months 2021, 50mL of hospital wastewater was collected from Beijing area as a sample for phage isolation. The wastewater was centrifuged at 4000rpm for 20 minutes to remove large particles. The supernatant was filtered through a 0.22 μm millipore filter to remove bacteria. Obtaining the phage separating medium.
Separation of phage: the MDR sequence 383 type Klebsiella pneumoniae C6 is used as a host strain, 50 mu L of bacterial suspension and 200 mu L of phage separation liquid are added into 5mL LB culture medium, the rotating speed is 220rpm, the temperature is 37 ℃, the culture liquid is cultured by shaking for 4 hours, the rotating speed is 10000rpm, the culture liquid is centrifuged for 2 minutes, and the supernatant is filtered by using a 0.22 mu m microporous filter. Taking 100 mu L of supernatant, and performing plaque screening by adopting a double-layer agar plate method.
And (3) purifying the phage: after the double-layer agar plate is statically cultured for 12-24 hours in an incubator at 37 ℃, a single plaque is picked by using an inoculating loop and put into an LB liquid culture medium, 50 mu L of host bacterium C6 suspension is added at the same time, the culture medium is cultured in a shaker at 37 ℃ for 4 hours under the oscillation of the rotating speed of 220rpm, the culture medium is centrifuged for 2 minutes at the rotating speed of 10000rpm, and a supernatant is collected and filtered by using a 0.22 mu m micropore filter. Taking 100 mu L of supernatant after filtration, and performing plaque purification by a double-layer agar plate method. Repeating the above operations for 4-5 times until uniform and consistent plaques appear, and obtaining the purified phage.
In the invention, the method is adopted to obtain a phage, and the phage is named as pKp383 in the invention.
Morphological observation of phage pKp under electron microscope: 1% chloroform, DNase and RNase were added to the culture broth of the purified phage pKp383. After centrifugation of the solution, the supernatant was collected, 10% PEG8000 was added, and after centrifugation again, the pellet was resuspended in SM buffer. Adding 1% chloroform again, centrifuging, and collecting the upper water phase to obtain phage suspension. After the phage suspension is diluted to a proper concentration, the phage suspension is settled on the surface of a copper mesh, phage particles are negatively dyed by 2% (wt./vol) uranium acetate (pH is 7.0), the phage particles are observed by a transmission electron microscope at 80KV, and a visual field with a complete shape of a single phage is found and photographed and recorded. As shown in FIG. 1, phage pKp383 was a long-tailed phage with an icosahedron head, about 76nm in diameter and a tail about 126nm long.
Through detection, the phage pKp383 provided by the invention has good thermal stability and pH stability, can keep stable titer under the conditions of temperature of 4-50 ℃ and pH of 6-10, and has the optimal complex infection number of 0.001 for the phage and bacteria. Phage pKp383 had a latency of 10 minutes, a burst time of 110 minutes, and then entered the plateau phase.
The MDR Kpn strain C6 (Genebank number JAJOVD 000000000) which phage pKp383 can be cleaved is of sequence 383 type. Type 383 has been shown to be a universal sequence for pneumonia-associated Klebsiella pneumoniae isolates. The phage pKp383 of the present invention has been deposited in the general microbiological culture collection center of the China Committee for culture Collection of microorganisms (address: west Lu No.1 Hospital No. 3, institute of microbiology, china academy of sciences) at 26.4.26.2022 years, and the deposition date is: 26/4/2022, classified and named as: the Klebsiella phage has a preservation number of CGMCC No.45098. The bacterium is also called phage pKp383 in the invention.
Example 2 temperature tolerance test of bacteriophage pKp383
Phage suspensions (prepared in reference example 1) pKp383 mL were each incubated at 4 ℃, 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ for 1 hour, and phage titer was determined using the double-layer agar plate method.
The test result is shown in figure 2, the phage pKp383 has good thermal stability, and can keep stable titer under the condition of the temperature of 4-50 ℃.
Example 3 pH tolerance test of phage pKp383
Phage pKp383 were inoculated in SM buffer 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 titer was determined using the double-layer agar plate method.
The test result is shown in FIG. 3, the bacteriophage 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 bacteriophage pKp383
Determination of optimal multiplicity of infection: the optimal multiplicity of infection (MOI) was determined using a double agar plate method. The MOI of phage pKp added to log phase host C6 was 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100, respectively. After mixed shaking culture for 4h, the optimal MOI treated phage titer was highest. The test results showed that the titer of phage pKp383 was the highest when the multiplicity of infection was 0.001, i.e. the optimal multiplicity of infection was 0.001.
Measurement of one-step growth curve: phage pKp383 was infected into host strain C6 under optimal MOI conditions and phage titer was determined every 10 min during shake culture for 150 min. The results of the test are shown in FIG. 4, phage pKp383 had a latency of 10 minutes, a burst time of 110 minutes, and then entered the plateau phase.
Example 5 testing of the lytic ability and host spectra of phage pKp on host bacteria C6
Determination of the lytic capacity of host bacterium C6: at a multiplicity of infection 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 pKp on host strain C6 was determined under conditions. The OD was measured with a microplate reader every hour during shaking culture for 7 hours 600 Value 1 times. The results are shown in FIG. 5, and the MOI is 10 to 10 -5 While pKp completely inhibited host strain Kp C6, phage pKp showed good lytic ability against the host strain. Can be used as a candidate phage preparation for treating acute pneumonia caused by MDR Kpn.
And (3) determining a host spectrum: phage pKp and 6 strain 383 type MDR Kpn were mixed and cultured for 4 hours at the optimal multiplicity of infection ratio, and the culture solution was observed. The culture solution is clarified to obtain the phage pKp which can crack the MDR Kpn. The determination result shows that the bacteriophage pKp can crack the 6 strains of different sequences 383 type MDR Kpn, and the specific information of the 6 strains of MDR Kpn is shown in the 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 lysis of phage against host bacterium C6
200. Mu.L of phages pKp-1, pKp-2, pKp-3, pKp-4, pKp-5, pKp and pKp were added to 5mL of LB medium in 7 tubes containing 50. Mu.L of host strain C6 suspension, and cultured with shaking at a rotation speed of 220rpm in a shaker at 37 ℃. After 4 hours of culture, the culture broth was observed for clarification. As shown in FIG. 6, the culture broth of the pKp-1, pKp-2, pKp-3, pKp-4, pKp-5 and pKp treatment groups was turbid, indicating that the 6 phage could not lyse host bacterium C6; while pKp the broth of the treated group was clear, indicating that pKp could lyse host C6.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention, and those skilled in the art will appreciate that various modifications and changes can be made to the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A Klebsiella phage (Klebsiella phase) with preservation number of CGMCC No.45098.
2. The bacteriophage of claim 1, which maintains a stable titer under conditions of a temperature of 4-50 ℃ and a pH of 6-10.
3. A phage preparation, comprising: bacteriophage with the preservation number of CGMCC No.45098 and auxiliary materials.
4. The phage preparation of claim 3, wherein said adjuvant comprises nutritional components for maintaining bacterial viability;
preferably, the auxiliary material is an LB culture medium.
5. The phage preparation of claim 3, which is a medicament.
6. The phage preparation of claim 3 which is a detergent or disinfectant.
7. The application of the bacteriophage with the preservation number of CGMCC No.45098 in vitro cracking of multiple drug resistant sequence 383 type Klebsiella pneumoniae.
8. Application of bacteriophage with the preservation number of CGMCC No.45098 in preparing a preparation for cracking multiple drug-resistant sequence 383 type Klebsiella pneumoniae.
9. The application of the bacteriophage with the preservation number of CGMCC No.45098 in preparing the medicine for preventing and treating pneumonia caused by Klebsiella pneumoniae with multiple drug-resistant sequences 383 is disclosed.
10. The use of any one of claims 7-9, wherein the multiple drug resistant sequence 383 type klebsiella pneumoniae comprises a strain with a collection number of CGMCC No.45098.
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| CN106434489A (en) * | 2016-11-18 | 2017-02-22 | 中国人民解放军疾病预防控制所 | High-wine-yield Klebsiella pneumoniae and application thereof |
| CN114381436A (en) * | 2022-02-09 | 2022-04-22 | 首都儿科研究所 | High-ethanol-yield Klebsiella pneumoniae phage and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434489A (en) * | 2016-11-18 | 2017-02-22 | 中国人民解放军疾病预防控制所 | High-wine-yield Klebsiella pneumoniae and application thereof |
| CN114381436A (en) * | 2022-02-09 | 2022-04-22 | 首都儿科研究所 | High-ethanol-yield Klebsiella pneumoniae phage and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| RONGRONG ZHANG: "Biological characteristics and genome analysis of a novel phage vB_KpnP_IME279 infecting Klebsiella pneumoniae", 《FOLIA MICROBIOLOGICA》, vol. 65, pages 925 - 936 * |
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