CN116606815A - Klebsiella phage and application thereof - Google Patents
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- A—HUMAN NECESSITIES
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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Abstract
The application relates to the technical field of microorganisms, in particular to a Klebsiella phage and application thereof; the application discloses a Klebsiella phage vB_KpnP_PH42 which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 45257 in the year 08 and 12 of 2022; the Klebsiella phage vB_KpnP_PH42 has broad-spectrum sterilization effect, and can effectively kill the Klebsiella phage in the environment; the safety test shows that the Klebsiella phage vB_KpnP_PH42 has no toxic or side effect, high safety and convenient mass production; the Klebsiella phage vB_KpnP_PH42 has a good hydrophilic phase, is easy to prepare into spray liquid or injection, and can effectively kill Klebsiella pneumoniae in the environment.
Description
Technical Field
The application relates to the technical field of microorganisms, in particular to a Klebsiella phage and application thereof.
Background
Klebsiella is a conditional pathogenic bacterium which is mainly distributed in respiratory tracts, digestive tracts and the like of various animals and people, is a zoonotic disease in nature, and can lead to the decrease of animal resistance and the morbidity of various animals and the death of serious people especially when the external environment is changed. After the poultry is infected with Klebsiella pneumoniae, various diseases such as septicemia, pneumonia, bronchitis, meningitis, urinary system and the like are mainly manifested in clinic, the morbidity and mortality are high, and the serious patients cause acute death. In clinic, klebsiella pneumoniae is mainly treated by antibacterial drugs, and because the antibacterial drugs are unreasonably used, the klebsiella pneumoniae has wide drug resistance to the antibacterial drugs, and great difficulty is brought to the prevention and treatment of the klebsiella pneumoniae, and attention should be paid.
Phages are widely available in nature and are a virus that infects bacteria, and there are phages where bacteria are present. The phage has obvious specificity, only specific bacteria are lysed, normal flora of animal bodies is not affected, after specific bacteria are lysed, the phage stops propagating and gradually discharges outside the body, and drug resistance is not generated, so the phage is a gram of superbacteria. The phage has the characteristics of short research and development time, strong specificity, high proliferation speed, safety, effectiveness and no residue, and has great prospect when being used as a substitute of antibiotics.
At present, no safe and effective phage preparation with wide cleavage spectrum is found for preventing and treating avian Klebsiella disease, and therefore, further screening and research work of new phage are needed.
Disclosure of Invention
Aiming at the problems, the application provides a phage and phage preparation capable of effectively killing klebsiella and application thereof, a phage vB_KpnP_PH42 of the klebsiella, a broad cracking spectrum and a clinical strain of pathogenic klebsiella separated from a farm can be effectively cracked, and a safe, effective and pollution-free phage product is provided for the environmental disinfection of a poultry farm.
In a first aspect, the present application provides a klebsiella phage, which adopts the following technical scheme:
a Klebsiella phage (Klebsiella pneumoniae phage) selected from chicken house feces, designated as Klebsiella phage vB_KpnP_PH42, was deposited at the China general microbiological culture Collection center, accession number: china, beijing, the preservation number is CGMCC NO.45257; the phage is short-tail phage, and can form transparent plaque on a solid culture medium, and has consistent shape and size, clear and regular edge and diameter of 2mm.
Observed under an electron microscope: the form of Klebsiella phage vB_KpnP_PH42 has a regular hexahedral structure with 50-55nm head and 15-20nm tail length, and belongs to the family of short-tail phages.
The titer of the Klebsiella phage vB_KpnP_PH42 remains substantially unchanged after 1 hour of action at 40 ℃ and 50 ℃; is kept at 5.4X10 after 1 hour of action at 60 DEG C 8 Above PFU/mL, the thermal stability of Klebsiella phage vB_KpnP_PH42 was higher.
Klebsiella phage vB_KpnP_PH42 was subjected to a pH range of 5-12 for 1h with a titer falling within 1 gradient and a titer of 10 8 PFU/mL, 3h of action, 2 gradients of potency decrease, 10 7 PFU/mL or more; within 1 gradient of 1h potency decrease in pH4, potency is 10 8 PFU/mL, 3h of action, 3-4 gradient of potency decrease, 10 5 PFU/mL or more; indicating that vB_KpnP_PH42 can withstand certain weak acid and strong alkali environments.
The optimal multiplicity of infection of Klebsiella phage vB_KpnP_PH42 was 0.001, under conditions where phage infection of the host bacteria produced a titer of 8.62× of progeny phage10 10 PFU/mL, phage titers were highest among 8 multiplicity of infection.
In a second aspect, the application provides an application of klebsiella phage vb_kpnp_ph42 in preparing a medicament for preventing and/or treating klebsiella infection, which adopts the following technical scheme:
use of klebsiella phage vb_kpnp_ph42 in the manufacture of a medicament for the prevention and/or treatment of klebsiella infection.
In a third aspect, the present application provides a phage preparation, which adopts the following technical scheme:
a phage preparation comprising klebsiella phage vb_kpnp_ph42.
Preferably, the phage preparation further comprises one or more of mutants of klebsiella phage vb_kpnp_ph42; the mutant has no less than 90% homology with the corresponding phage.
Preferably, the content of Klebsiella phage vB_KpnP_PH42 in the phage preparation is not less than 5X 10 9 PFU/mL。
Preferably, the phage preparation further comprises a pharmaceutically acceptable carrier.
Preferably, the preparation form is an oral administration dosage form, an external administration dosage form or an parenteral administration dosage form; specifically, phage preparations of the present application can be administered via the percutaneous, oral, rectal, topical, intraperitoneal, intramuscular, intranasal, and inhalation routes.
In a fourth aspect, the application provides an application of a phage preparation in a medicine for preventing and treating avian klebsiella, which adopts the following technical scheme:
the phage preparation is applied to medicines for preventing and treating avian Klebsiella disease.
In a fifth aspect, the present application provides a feed additive, which adopts the following technical scheme:
a feed additive comprising klebsiella phage vb_kpnp_ph42 or a phage preparation as described above.
The Klebsiella phage vB_KpnP_PH42 has safety, and the protection rate of the phage is 90%, which indicates that the Klebsiella phage vB_KpnP_PH42 has good control effect on chicken Klebsiella disease.
In a sixth aspect, the present application provides a disinfectant, which adopts the following technical scheme:
a disinfectant comprising klebsiella phage vb_kpnp_ph42 or a phage preparation as described above.
Preferably, the concentration of phage in the disinfectant is 1X 10 9 PFU/mL or more.
Other active ingredients for inhibiting or destroying bacteria in the environment are also included.
The use of the above-described environmental disinfectant in the disinfection of a farm environment, wherein the farm environment comprises feed, water sources, tanks, cages, floors, walls, faeces and litter.
After the ground polluted by Klebsiella pneumoniae is disinfected by using Klebsiella phage vB_KpnP_PH42, the plate count result shows that the number of the ground Klebsiella pneumoniae is reduced to 10 after 1h 2 The number of the Klebsiella pneumoniae on the ground is reduced to about 10CFU after 1.5 hours below CFU, and the Klebsiella pneumoniae can not be detected on the ground after 2 hours, which indicates that phage vB_KpnP_PH42 can effectively kill the Klebsiella pneumoniae in the environment.
The Klebsiella phage vB_KpnP_PH42 has a good hydrophilic phase, is easy to prepare into spray liquid or injection, and can effectively kill Klebsiella pneumoniae in the environment.
In a seventh aspect, the present application provides a bactericide, which adopts the following technical scheme:
a bactericide comprising klebsiella phage vb_kpnp_ph42 or a phage preparation as described above.
Preferably, the concentration of phage in the bactericide is 1X 10 9 PFU/mL or more.
In an eighth aspect, the present application provides a kit, which adopts the following technical scheme:
a kit comprising klebsiella phage vb_kpnp_ph42 or a phage preparation as described above.
In summary, the present application includes at least one of the following beneficial technical effects:
1. the application screens a Klebsiella phage vB_KpnP_PH42 with strong cracking capability, and can be used for preventing and treating diseases caused by Klebsiella infection.
2. The klebsiella phage vB-KpnP-PH 42 has broad-spectrum sterilization effect, and can effectively kill the klebsiella phage in the environment.
3. The klebsiella phage vB_KpnP_PH42 disclosed by the application has no toxic or side effect through a safety test, is high in safety, and is convenient for large-scale production.
Drawings
FIG. 1 is a photograph of a Klebsiella phage vB_KpnP_PH42 plaque;
FIG. 2 is an electron micrograph of Klebsiella phage vB_KpnP_PH 42;
FIG. 3 is a graph showing the thermostability of Klebsiella phage vB_KpnP_PH 42;
FIG. 4 is a graph showing the pH stability of Klebsiella phage vB_KpnP_PH 42;
FIG. 5 is a graph showing the effect of the Klebsiella phage vB_KpnP_PH42 on sterilization.
Detailed Description
The following description of the embodiments of the present application will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to fall within the scope of the application. In the present application, the equipment, materials, etc. used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.
Example 1: isolation and purification culture of phages
1. Isolated culture of host bacteria
The host bacteria of the klebsiella phage in the examples were clinical isolates of klebsiella obtained from chicken farm infected with klebsiella.
Inoculating host bacteria on 2% nutrition agar, culturing overnight, inoculating single colony into 5mL nutrition broth culture medium, shaking culturing at 37deg.C in 170rpm shaker for 15 hr to obtain host bacteria culture.
2. Isolation and culture of phage
Feces obtained from chicken farm were placed in a bleb flask, 200. Mu.L of host bacteria were added to the bleb flask, and then appropriate amount of nutrient broth medium was added thereto, followed by shaking culture in a shaker at 37℃and 170rpm overnight. A portion of the overnight cultured liquid was placed in a 10mL centrifuge tube, centrifuged at 11000rpm for 10min, and the supernatant was collected and filtered with a 0.22 μm filter to form a phage stock solution.
Separating phage by double-layer plate method, diluting phage stock solution by 10 times, respectively taking 100 μl of the separated diluent, mixing with 100 μl of corresponding Klebsiella, incubating at 37deg.C for 5min, standing at 45deg.C, mixing with upper agar (agar mass concentration is 0.7%), spreading on lower agar (agar mass concentration is 1.5%), cooling, culturing for 6-8 hr at 37deg.C, observing plaque condition, picking single plaque, and repeatedly purifying for 3-5 times until plaque with uniform size and morphology appears on the culture dish.
100 mu L of host bacteria and phage spot-digging leaching liquid are respectively taken and added into 5mL of nutrient broth, 100 mu L of host bacteria are further taken and added into another 5mL of nutrient broth to serve as a control, and the mixture is simultaneously put into a shaking table at 37 ℃ and 170rpm for 2-3 hours, and the phage proliferation liquid is obtained after the mixture is clear. Phage proliferation liquid was diluted 10-fold, titers were measured by double-layer plate method, and 3 replicates were made for each dilution. The klebsiella phage formed transparent plaques, which were uniform in size and morphology, well-defined and regular in edge, with a 2mm diameter, and around which a halo was placed, and was designated PH42, as shown in fig. 1.
Example 2: morphological observation of phage experimental method: taking 1×10 9 20. Mu.L of the PFU/mL phage sample was dropped onto a microporous copper mesh, and the pellet was allowed to settle for 15min, and the excess liquid was aspirated off with filter paper. Dripping 15 mu L of 2% phosphotungstic acid (PTA) on a copper wire, dyeing for 5min, sucking excessive dye liquor with filter paper, and dryingAfter drying, the mixture was observed with a transmission electron microscope and photographed.
As shown in the electron microscope picture of FIG. 2, the head of the Klebsiella phage has a regular hexahedral structure of 50-55nm and a tail length of 15-20nm, and the shape of phage PH42 conforms to the characteristics of the family of Brevibacterium according to the definition of the International Commission for virus classification (ICTV), and is named as Brevibacterium phage vB_KpnP_PH42.
Example 3: determination of the cleavage spectrum of Klebsiella phage vB_KpnP_PH42 the cleavage spectrum of phage was determined by double-layer plate method as follows: the bacterial suspension of the bacteria was obtained in the same manner as in example 1, and 35 Klebsiella pneumoniae strains were derived from the lungs of birds such as chickens and ducks, which had been killed by Klebsiella pneumoniae strains. The klebsiella phage vb_kpnp_ph42 was plated in double-layered plates with 35 klebsiella pneumoniae strains, respectively, and cultured upside down overnight.
The result shows that 35 Klebsiella pneumoniae is used as host bacteria for determination of a lysis spectrum, 29 strains of the Klebsiella phage vB_KpnP_PH42 can be lysed, and the lysis rate reaches 82.86%.
TABLE 1 cleavage Spectrum of Klebsiella phage vB_KpnP_PH42
Example 4: study of the biological Properties of phages
1. Biological Property study of Klebsiella phage vB_KpnP_PH42
(1) Detection of the thermal stability of phages
Will be 1.0X10 10 PFU/mL Klebsiella phage vB_KpnP_PH42 proliferation solution is respectively subjected to water bath at 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃ for 20min, 40min and 60min, and two parallel groups are arranged at each temperature. Phage titers were determined by double-layer plate method.
As a result, the titer of the Klebsiella phage vB_KpnP_PH42 remained substantially unchanged after 1 hour of action at 40℃and 50 ℃; is kept at 5.4X10 after 1 hour of action at 60 DEG C 8 PFU/mL or more; basic inactivation of phage after 60min of action at 70 ℃ and specific junctionThe result is shown in FIG. 3, which illustrates that the thermal stability of Klebsiella phage vB_KpnP_PH42 is higher.
(2) PH stability detection of phages
Adding 4.5mL of NB broth with different pH values (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) into sterile test tubes, placing 3 in water bath at 37deg.C, and adding 500 μl of each 1.0X10 after temperature is stable 10 PFU/mL Klebsiella phage vB_KpnP_PH42 proliferation solution is mixed uniformly and subjected to water bath at 37 ℃ for 1h, 2h and 3h. After the completion of the reaction, a proper amount of HCl or NaOH was added to the mixture to give a pH of about 7, and the phage titer was measured by the double-plate method.
The specific results are shown in FIG. 4, and it can be seen that the Klebsiella phage vB_KpnP_PH42 acts for 1h within a pH range of 5-12, the titer is reduced within 1 gradient, and the titer is 10 8 PFU/mL, 3h of action, 2 gradients of potency decrease, 10 7 PFU/mL or more; within 1 gradient of 1h potency decrease in pH4, potency is 10 8 PFU/mL, 3h of action, 3-4 gradient of potency decrease, 10 5 PFU/mL or more; the activity was reduced or even completely inactivated at pH2-3 and pH 13. Indicating that vB_KpnP_PH42 can withstand certain weak acid and strong alkali environments.
(3) Determination of optimal multiplicity of infection (MOI) of phage
Klebsiella pneumoniae phage vB_KpnP_PH42 and host bacteria are propagated according to a conventional method, phage and host bacteria titers are determined, and the Klebsiella phage vB_KpnP_PH42 and host bacteria are diluted appropriately. 100. Mu.L of each of the Klebsiella phage vB_KpnP_PH42 and the host bacteria were added to the NB broth at a ratio of the multiplicity of infection of 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001. The culture was shaken at 37℃and 170rpm until the liquid became clear, and the liquid clear time was recorded. The phage titer was determined by double-layer plate method by centrifugation at 11000rpm for 5 min.
The optimal multiplicity of infection of Klebsiella phage vB_KpnP_PH42 is 0.001, and the titer of progeny phage produced by phage infection of the host bacteria under this condition is 8.62X10 10 PFU/mL, phage titers were highest among 8 multiplicity of infection.
(4) In vitro lysis assay of phages
Adding Klebsiella pneumoniae H021 and Klebsiella phage vB_KpnP_PH42 according to a certain proportion, wherein the final concentration of Klebsiella pneumoniae H021 is 1×10 8 CFU/mL, final concentration of Klebsiella phage vB_KpnP_PH42 was 1X 10, respectively 9 PFU/mL，1×10 8 PFU/mL，1×10 7 PFU/mL, control group added with Klebsiella pneumoniae phage vB_KpnP_PH42 of the same amount of sterile nutrient broth, the Klebsiella pneumoniae H021 and different final concentration of Klebsiella phage vB_KpnP_PH42 after mixing in 37 ℃ incubator culture. OD values are measured at regular intervals until the mixed solution becomes clear, and the residual quantity of each group of bacteria after a certain period of action is measured by a coating plate method.
The Klebsiella phage vB-KpnP-PH 42 has good effect of cracking Klebsiella pneumoniae, the cracking rate of 3 phages with different concentrations on Klebsiella pneumoniae strains can reach more than 99.9%, but the time is different, but the better killing effect can be achieved within 4 hours, as shown in figure 5.
Example 5: klebsiella phage vB_KpnP_PH42 disinfection test
Diluting klebsiella pneumoniae H021 bacterial solution to 1×10 4 CFU/mL, control group was diluted to 1X 10 4 CFU/mL Klebsiella pneumoniae H021 bacterial liquid 1mL is uniformly sprayed on 1m 2 Then uniformly spraying the PBS with equal volume; the experimental group was first diluted to 1X 10 4 CFU/mL Klebsiella pneumoniae H021 bacterial liquid 1mL is uniformly sprayed on 1m 2 Is then diluted to 1 x 10 9 PFU/mL of Klebsiella phage vB_KpnP_PH42 was sprayed evenly on the ground, 2 replicates per group. Every 0.5H, sampling on the ground with the same area, and detecting the number of Klebsiella pneumoniae H021 bacteria by using a plate counting method for 4H.
As a result, it was found that after the ground contaminated with Klebsiella pneumoniae H021 was sterilized with Klebsiella phage vB_KpnP_PH42, the number of Klebsiella pneumoniae on the ground after 1 hour was reduced to 10 as shown by the plate count result 2 Below CFU, after 1.5hThe number of the ground klebsiella pneumoniae is reduced to about 10CFU, the ground klebsiella pneumoniae can not be detected after 2 hours, and the number of the ground klebsiella pneumoniae in a control group is slightly increased, so that phage vB_KpnP_PH42 can effectively kill the klebsiella pneumoniae in the environment.
Example 6: safety test of klebsiella phage vb_kpnp_ph42 20 white feather chickens at 20 days old were selected, divided into 1 experimental group and 1 control group, and purified klebsiella phage vb_kpnp_ph42 proliferation solution and nutrient broth were respectively injected intraperitoneally 1mL each, and the injections were continued for 7 days to observe the behavior of chickens, and the changes of chicken organs were observed by section examination.
The results show that the chicken in the experimental group and the control group have no abnormal behaviors, and the organs such as liver, lung, heart, spleen, kidney and the like are normal in the section examination, and have no obvious difference from the control group.
Example 7: experimental method for preventing and treating chicken klebsiella infection by using klebsiella phage vB_KpnP_PH 42: 30 white feather broilers of 20 days of age were selected and divided into 3 groups of 10 broilers each. A group of intraperitoneal injections of 1mL of nutrient broth; two groups of intraperitoneal injections 1×10 8 CFU/Klebsiella H021 bacterial liquid; three groups of intraperitoneal injections 1X 10 8 CFU/Klebsiella bacterium H021 liquid simultaneous intraperitoneal injection of 1X 10 9 PFU/Klebsiella phage vB_KpnP_PH42 alone. The death of the chickens was observed and recorded, the chickens were continuously observed for 7 days, the chickens were examined by dissecting, and the death caused by Klebsiella pneumoniae H021 was verified, and the mortality and protection rate were counted (see Table 2).
TABLE 2 control effect of phage on Creutzfeldt-Jakob disease in chickens
The experimental results show that: the mortality rate of the chicken in the bacterial liquid control group is 80 percent, the mortality rate of the chicken in the phage group is 10 percent,
the protection rate of the phage is 90%, which shows that the Klebsiella phage vB_KpnP_PH42 has good control effect on chicken klebsiella.
It will be understood that equivalents and modifications will occur to those skilled in the art in light of the present teachings and concepts, and all such modifications and substitutions are intended to be included within the scope of the present application as defined in the accompanying claims.
Claims (10)
1. A klebsiella phage, characterized by: the Klebsiella phage (Klebsiell apneumoniae phage) is named as Klebsiella phage vB_KpnP_PH42, and the preservation number is CGMCC No.45257.
2. Use of the klebsiella phage vb_kpnp_ph42 of claim 1 for the preparation of a medicament for preventing and/or treating klebsiella infection.
3. A phage preparation characterized in that: comprising the klebsiella phage vb_kpnp_ph42 of claim 1.
4. A phage preparation according to claim 3, characterized in that: phage preparations also include one or more of mutants of klebsiella phage vb_kpnp_ph42; the mutant has no less than 90% homology with the corresponding phage.
5. A phage preparation according to claim 3, characterized in that: the phage preparation contains Klebsiella phage vB_KpnP_PH42 of 5×10 or more 9 PFU/mL。
6. A phage preparation according to claim 3, characterized in that: phage preparations also contain a pharmaceutically acceptable carrier.
7. The phage preparation of claim 6, wherein: the preparation is in the form of oral administration dosage form, external administration dosage form or parenteral administration dosage form.
8. Use of a phage preparation according to any one of claims 3-7 in a medicament for the control of avian klebsiella disease.
9. A feed additive, characterized in that: comprising the klebsiella phage vb_kpnp_ph42 of claim 1 or the phage preparation of any one of claims 3-7.
10. A disinfectant, characterized in that: comprising the klebsiella phage vb_kpnp_ph42 of claim 1 or the phage preparation of any one of claims 3-7.
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