Klebsiella oxytoca phage RDP-EC-23031 and application thereof

Abstract

The invention relates to the technical field of microorganisms. The invention provides a Klebsiella oxytoca bacteriophage RDP-EC-23031 strain, and the Klebsiella oxytoca bacteriophage RDP-EC-23031 strain is preserved in the China General Microbiological Culture Collection Center on April 22, 2024, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.45963. The Klebsiella oxytoca bacteriophage RDP-EC-23031 strain has the advantages that the Klebsiella oxytoca bacteriophage RDP-EC-23031 strain can be used for preparing the Klebsiella oxytoca bacteriophage; the invention also provides a phage composition. The invention also provides application of the klebsiella oxytoca phage RDP-EC-23031 or the phage composition in preparation of a product for preventing and / or treating livestock and poultry diseases caused by klebsiella oxytoca and application of the klebsiella oxytoca phage RDP-EC-23031 or the phage composition in preparation of a product for killing and / or inhibiting klebsiella oxytoca. The invention also provides a bacteriophage pharmaceutical preparation, a drinking water additive, a feed additive, a disinfectant or a cleaning agent. The Klebsiella oxytoca bacteriophage RDP-EC-23031 provided by the invention is used for replacing antibiotics to be applied to prevention and treatment of livestock and poultry breeding, and solves the problems that diseases of livestock and poultry animals are acute and cannot be treated in time, and drug use is invalid due to drug resistance of pathogenic bacteria.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a strain of Klebsiella oxytoca phage RDP-EC-23031 and application thereof. Background Art

[0002] Klebsiella genus (K.lebsiella) of Enterobacteriaceae can be divided into 6 types, namely K.oxytoca, K.pneumoniae, K.planticola, K.terrigena and K.mobilis. Among them, K.oxytoca has a strong ability to release endotoxins. It is a Gram-negative bacterium and a major conditional pathogen. It exists in the intestines of humans and animals and can cause diseases in humans and animals, causing liver abscesses, peritonitis, enteritis and diarrhea. With the continuous expansion of the scale of the breeding industry, the probability of animal diseases caused by K.oxytoca has greatly increased, causing great economic losses to the breeding industry.

[0003] Currently, antibiotics are relatively cheap and easy to use, but the abuse of antibiotics in the production process has caused bacteria to develop resistance to antibiotics, which has become a difficult problem in the prevention, control and treatment of animal diseases. Animal disease prevention and control faces huge challenges.

[0004] At present, there is no high-titer and stable-titer bacteriophage product that can effectively prevent and treat oxytocin Klebsiella disease. Therefore, it is urgent to develop an alternative to antibiotics and a high-titer and stable-titer bacteriophage product for the prevention and treatment of oxytocin Klebsiella disease to solve the problems of rapid onset of the disease in livestock and poultry and untimely treatment, and ineffective medication due to drug resistance of pathogenic bacteria. Summary of the invention

[0005] The purpose of the present invention is to provide a cross-species lytic Klebsiella oxytoca phage RDP-EC-23031 to replace the application of antibiotics in the prevention and treatment of livestock and poultry breeding, and to solve the problems of acute disease in livestock and poultry and untimely treatment, and ineffective medication due to drug resistance of pathogenic bacteria.

[0006] To solve the above problems, the present invention provides the following technical solutions:

[0007] In the first aspect, the present invention provides a strain of Klebsiella oxytocabacteriophage RDP-EC-23031, which was isolated from the chicken liver of a farmer in Jiugu River, Tai'an, Liaoning Province, and was deposited in the General Microbiological Center of China Microbiological Culture Collection Committee on April 22, 2024. The deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 45963. The present invention uses the chicken liver of a farmer in Jiugu River, Tai'an, Liaoning Province to isolate the acid-producing Klebsiella EC-2401015, and experimentally determines the pathogenicity and drug resistance of EC-2401015. Using EC-2401015 as a host, a lytic acid-producing Klebsiella phage is isolated from the chicken liver, and the genotype of RDP-EC-23031 is determined by the whole genome sequence. The Klebsiella oxytoca EC-2401015 used in the present invention is isolated from the chicken liver of a farmer in Jiugu River, Tai'an, Liaoning Province, and is preserved together with the Klebsiella oxytoca bacteriophage RDP-EC-23031 of the present invention.

[0008] Through electron microscopic observation: the phage RDP-EC-23031 is a short-tailed phage under electron microscopy, with a regular icosahedron head and an overall size of 75nm, with a head of about 50nm and a tail of about 25nm. According to the classification standards of the International Committee on Taxonomy of Viruses (ICTV), the phage is classified as Caudovirales, Brachyviridae.

[0009] The full genome length of Klebsiella oxytoca EC-2401015 is 39503 bp, and its optimal infection multiplicity is 10 -6 The optimum growth temperature is 37°C, and the titer can reach 3.4×10 10 pfu / mL.

[0010] In a second aspect, based on the same inventive concept, the present invention provides a phage composition, comprising the Klebsiella oxytoca phage RDP-EC-23031 as described above.

[0011] In a third aspect, based on the same inventive concept, the present invention provides the use of the Klebsiella oxytoca phage RDP-EC-23031 as described above or the phage composition as described above in the preparation of products for preventing and / or treating livestock and poultry diseases caused by Klebsiella oxytoca.

[0012] In a fourth aspect, based on the same inventive concept, the present invention provides the use of the above-mentioned Klebsiella oxytoca phage RDP-EC-23031 or the above-mentioned phage composition in the preparation of a product for killing and / or inhibiting Klebsiella oxytoca.

[0013] In a fifth aspect, based on the same inventive concept, the present invention provides a phage pharmaceutical preparation, the active ingredient of which includes the Klebsiella oxytoca phage RDP-EC-23031 as described above or the phage composition as described above.

[0014] The phage pharmaceutical preparation as described above, wherein the concentration of the Klebsiella oxytoca phage RDP-EC-23031 is at least 10 6 pfu / mL.

[0015] In a sixth aspect, based on the same inventive concept, the present invention provides a drinking water additive, the active ingredient of which includes the Klebsiella oxytoca phage RDP-EC-23031 as described above or the phage composition as described above.

[0016] In a seventh aspect, based on the same inventive concept, the present invention provides a feed additive, the active ingredient of which includes the Klebsiella oxytoca phage RDP-EC-23031 as described above or the phage composition as described above.

[0017] In an eighth aspect, based on the same inventive concept, the present invention provides a disinfectant, wherein the active ingredient comprises the above-mentioned Klebsiella oxytoca phage RDP-EC-23031 or the above-mentioned phage composition. Preferably, the disinfectant can be used to disinfect Klebsiella oxytoca in a poultry breeding environment by spraying or soaking.

[0018] In a ninth aspect, based on the same inventive concept, the present invention provides a cleaning agent, the active ingredients of which include the Klebsiella oxytoca phage RDP-EC-23031 as described above or the phage composition as described above.

[0019] Compared with the existing technology, the effects and advantages of the present invention are:

[0020] 1. The Klebsiella oxytoca phage RDP-EC-23031 provided by the present invention has a strong lysis efficiency of Klebsiella oxytoca, has broad application prospects in the prevention and control of pathogenic Klebsiella oxytoca in livestock and poultry, and provides a phage source for industrial application.

[0021] 2. The Klebsiella oxytoca phage RDP-EC-23031 provided by the present invention has good thermal stability within a certain range and can withstand a high temperature of 60°C. At this temperature, the phage still maintains a high activity and titer, which is of great significance for the transportation and storage of phages in practical applications.

[0022] 3. The oxytocin-producing Klebsiella phage RDP-EC-23031 provided by the present invention is diluted 10 6After the reaction was repeated for 2-3 times, the bacteriophage RDP-EC-23031 still had a good lysis effect on the host bacteria, indicating that the phage RDP-EC-23031 was highly efficient and economical in practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the electron micrograph of bacteriophage RDP-EC-23031;

[0024] Figure 2 This is a graph showing the results of the infection multiplicity experiment of bacteriophage RDP-EC-23031;

[0025] Figure 3 It is the lysis curve of bacteriophage RDP-EC-23031 to host bacteria EC-2401015;

[0026] Figure 4 This is a graph showing the results of a thermal stability experiment of bacteriophage RDP-EC-23031;

[0027] Figure 5 This is a graph showing the lysis rate of bacteriophage RDP-EC-23031;

[0028] Figure 6 Schematic diagram of the RTD experiment of bacteriophage RDP-EC-23031. DETAILED DESCRIPTION

[0029] The following will combine the contents in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in this specification includes any and all combinations of one or more of the related listed items.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0032] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0033] Example 1 Isolation and identification of pathogenic bacteria and bacteriophages

[0034] (1) Isolation and identification of Klebsiella oxytoca EC-2401015

[0035] This host was sampled from farmers in Jiugu River, Tai'an, Liaoning Province. From chicken livers, the aseptic method was used to separate the strains by streaking on the CHROMAGA colorimetric medium. After culturing at 37°C for 18-24 hours, blue or blue-green colonies with neat edges appeared on the colorimetric plate of the large intestine, with a diameter of about 1-2 mm. Then, typical colonies were streaked on the MacConkey medium. After culturing at 37°C for 18-24 hours, white colonies with pink centers and white edges appeared on the MacConkey medium. Typical colonies were streaked and purified three times, and then single colonies were inoculated in 5 mL of LB broth medium, and cultured at 37°C and 200 rpm for 4 hours to obtain a uniform turbid bacterial suspension. Then, the strains were identified as acid-producing Klebsiella by 16sRNA molecular identification, and one of them was named EC-2401015 and stored in a -80°C refrigerator.

[0036] (2) Phage isolation and identification

[0037] The chicken liver samples used in the experiment of the present invention were collected from farmers in Jiugu River, Tai'an, Liaoning Province, and used as samples for phage isolation.

[0038] Sample processing: Take an appropriate amount of chicken liver and add an appropriate amount of physiological saline to grind it, centrifuge it at 10000rpm for 10 minutes to remove larger impurities and most bacteria, and then filter it with a 0.22μm microporous filter membrane for sterilization.

[0039] Prepare mixed bacterial suspension: Take 2 mL of filtrate and 1 mL of host bacterial suspension, add them together into 27 mL of autoclaved LB broth medium, and then place in a 37°C incubator for overnight culture. After culture, take 3 mL, centrifuge at 10,000 rpm for 5 minutes, and then filter and sterilize with a 0.22 μm microporous filter. Collect the filtrate for use. The filtrate is the original solution to contain the bacteriophage.

[0040] Phage isolation: The double plate method is used to isolate phages. Take 0.1mL of the mixed bacterial suspension filtrate and 0.1mL of the acid-producing Klebsiella suspension and mix them evenly, then add them to 3mL of 0.4% nutrient agar heated to 45-54℃, then spread on double plates, and place them in a 37℃ incubator for 6-8h. Observe the results. If there are phages, there will be transparent plaques on the plates. Pick the plaques and soak them in 1mL of physiological saline for 10min, then take 0.1mL of the leaching liquid and 0.1mL of the bacterial suspension, add them to 3mL of 0.4% nutrient agar heated to 45-54℃, spread on double plates, and culture them in a 37℃ incubator for 4-6h for purification. Follow this step for 2-3 times until the plaque size is uniform, thus obtaining a phage strain, which is named RDP-EC-23031. The plaques formed by RDP-EC-23031 on double plates are 1.5mm-2mm in diameter, round and transparent.

[0041] (3) Determination of bacterial titer

[0042] The purified phage was spread on a double-layer plate, and then a single phage plaque was taken and added to LB liquid culture medium, and the host bacterial suspension was added at the same time. After the phage was proliferated, the proliferated liquid was centrifuged at 10000rpm for 5 minutes and then filtered with a 0.22μm microporous filter. The filtrate was diluted 10 times to 10 -6 Take 0.1mL of the last three dilutions of phage dilution and 0.1mL of host bacterial suspension, spread on double-layer plates, and measure the phage titer. Phage titer (pfu / mL) = number of plaques * dilution factor ÷ 0.1. The results showed that the phage plaques had the same morphology, size and clarity, and had typical lytic phage characteristics; the phage titer could reach up to 10 8 pfu / mL or above.

[0043] (4) Preservation of bacteriophages

[0044] The phage proliferation solution was mixed with 60% glycerol in a ratio of 1:1 and stored in liquid nitrogen.

[0045] Example 2 Electron microscopic observation of bacteriophage

[0046] 1. Experimental methods:

[0047] Take 20 μL of liquid containing crude phage particles and drop it on the copper mesh. Let it settle naturally for 15 minutes, and then use filter paper to absorb the excess liquid from the side. Add a drop of 2% phosphotungstic acid (PTA) on the copper mesh to stain the phage for 10 minutes, then use filter paper to absorb the staining liquid from the side. After the sample is dry, use an electron microscope to observe the phage morphology. The results are as follows: Figure 1 shown.

[0048] 2. Experimental results:

[0049] Depend on Figure 1 It can be seen that the phage RDP-EC-23031 is a short-tailed phage under an electron microscope, with a regular icosahedron head and an overall size of 75nm, with the head being about 50nm and the tail being about 25nm. According to the classification standards of the International Committee on Taxonomy of Viruses (ICTV), the phage is classified as Caudovirales, Brachyviridae.

[0050] Example 3 Phage genome sequencing

[0051] After enrichment and cultivation of a single phage, centrifuge at 8000r for 15 minutes at 4°C, add 10% PEG8000 and 0.5M NaCl and let stand overnight, then add an equal amount of chloroform and mix well, let stand and separate, centrifuge at 5000r for 10 minutes, remove the chloroform layer and PEG layer, add restriction endonuclease digestion, and suspend the phage under gradient density cesium chloride conditions, then dialyze 3 times with TM buffer, 30 minutes each time, and send to BGI Biological Sequencing Company for whole genome sequencing. The results are shown in the sequence table, and the full length of the genome is 39503bp.

[0052] Example 4: Determination of the optimal multiplicity of infection

[0053] 1. Experimental methods:

[0054] According to the multiplicity of infection: 100:1, 10:1, 1:1, 1:10, 1:100, 1:1000, 1:10 -4 , 1:10 -5 , 1:10 -6 , 1:10 -7 , 1:10 -8 The phage proliferation solution and host were added to LB in the same ratio, and the total volume of the culture system was ensured to be the same. After shaking culture at 37℃ and 200rpm for 6h, centrifuged at 12000r / min for 5min at room temperature, and the supernatant was spread on two plates to determine its titer. The results are shown in Table 1 and Figure 2 shown.

[0055] Table 1 Determination results of optimal infection multiplicity

[0056]

[0057] 2. Experimental results:

[0058] From the data in Table 1, we can see that when the multiplicity of infection is 10 -6 After 6 hours of culture, the proliferation fluid was relatively clear and the titer was the highest, reaching 3.4×10 10 pfu / mL, indicating that the optimal infection multiplicity of the Klebsiella oxytoca phage RDP-EC-23031 of the present invention is 10 -6 .

[0059] Example 5: Phage lysis curve of host

[0060] 1. Experimental methods:

[0061] The host bacterial suspension cultured for 4 hours and the phage were inoculated into 5 mL LB medium at an optimal infection multiplicity of 2%. Figure 3 Marked as 10 6 :1, and set up a blank control group ( Figure 3marked as "LB broth"), host bacteria control group ( Figure 3 marked as "BE"), phage control group ( Figure 3 The samples were labeled as "EC-23031" in the growth curve detector and cultured at 37°C with shaking at 160 rpm. The sampling interval was 10 minutes and the culture time was 12-36 hours. Figure 3 shown.

[0062] 2. Experimental results:

[0063] The host bacteria EC-2401015 and the bacteriophage RDP-EC-23031 were infected at an optimal multiplicity of infection of 10 6 :1. Place in growth curve detector for 36 hours and the results are as follows Figure 3 As shown. Bacteriophage RDP-EC-23031 was added to the host bacterial suspension of EC-2401015, and a certain lysis effect appeared after about 1.8 hours of action. After about 1.8 hours, the OD600 value began to decrease. The action lasted for 8 hours. After 8 hours, the mixed bacterial solution began to become turbid, and the OD value gradually increased, but it was still lower than the OD value of the host bacteria. Since the phage was in the stage of adsorption and infection of the host bacteria in the early stage, only a small number of phages began to lyse the host. Therefore, the amount of host lysed was far less than the growth of the host. Therefore, OD600 maintained a certain upward trend in the first 1.8 hours, accompanied by the exponential increase in the number of phages. After 1.8 hours, the number of hosts lysed was greater than the proliferation rate of the host, so the OD600 value began to gradually decrease, and the mixed bacterial solution reached the clearest. After 8 hours of continuous action, the mixed bacterial solution became turbid again, and the original sensitive bacteria were lysed by the phage and replaced by the mutant tolerant bacteria.

[0064] Example 6: Determination of phage thermal stability

[0065] 1. Experimental methods:

[0066] The phage stock solution was divided into EP tubes and divided into 3 groups: a, b, and c. Group a was incubated at 50°C for 30 minutes; group b was incubated at 60°C for 30 minutes; and group c was incubated at 70°C for 30 minutes. Then, after 10-fold dilution with physiological saline, double plates were spread to determine the titers of the 3 groups and the initial titer of phage RDP-EC-23031. The results are shown in Table 2 and Figure 4 shown.

[0067] Table 2 Thermal stability results

[0068]

[0069] 2. Experimental results:

[0070] As shown in Table 2, bacteriophage RDP-EC-23031 can tolerate a high temperature of 60°C, while the phage titer dropped to 10 pfu / mL after treatment at 70°C for 0.5 h.

[0071] Example 7: Lysis rate experiment

[0072] 1. Experimental methods:

[0073] Under sterile conditions, 1 mL of sample and 1 mL of host bacterial solution (1×10 6 CFU / mL), incubate at 37°C for 15 minutes, mix well and dilute to 10 with saline. -1 -10 -3 Each gradient was plated with 100 μL of each gradient on a nutrient agar plate and incubated at 37°C for 24 hours. Each gradient was repeated twice ( Figure 5 Marked as EC, see Figure 5 Left side). At the same time, take 1 mL of normal saline and 1 mL of host bacterial solution (1×10 6 CFU / mL) as blank control ( Figure 5 Marked as BE, see Figure 5 The above steps were repeated. Plates with 30-300 colonies were selected for counting. The test was repeated 3 times and the average value was taken. Phage lysis rate = (1-number of colonies in the treatment group / number of colonies in the control group) × 100%. The results are shown in Figure 5 shown.

[0074] 2. Experimental results:

[0075] According to calculation, the lysis rate of RDP-EC-23031 is 99.9%, which has a good lysis effect on the host and is suitable for use in breeding.

[0076] Example 8: RTD Experiment

[0077] 1. Experimental methods:

[0078] Divide the plate into areas, add a drop (about 5 μL) of host bacteria EC-2401015 in the center of the area, dry it, and add different dilutions (10 -1 -10 -8 , Figure 6 A drop (about 5 μL) of phage solution (marked as -1 to 8) was added to the spot of the host bacteria. Two groups of blank controls were set up: host bacteria EC-2401015 ( Figure 6 In the group marked as E2401015 in the middle, only one drop (about 5 μL) of host bacteria EC-2401015 was added at the center of the area; bacteriophage RDP-EC-23031 ( Figure 6In the group marked as RDP-EC-23031 in the middle, only one drop (about 5 μL) of bacteriophage RDP-EC-23031 was added to the center of the area. After drying, it was cultured at 37°C overnight. According to the growth status of the host bacteria EC-2401015, those that could not grow in a continuous piece were called CL concentration (CL concentration is the concentration that can completely lyse the host bacteria in the environment, which can be used to guide the dilution plan of production practice). The results are as follows Figure 6 shown.

[0079] 2. Experimental results:

[0080] like Figure 6 As shown, RDP-EC-23031 was diluted 10 6 times ( Figure 6 -6 region in the host bacteria) still has a good lysis effect on the host bacteria.

[0081] It should be pointed out that the specific implementation methods are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above-mentioned embodiments, and there are many variations. Those skilled in the art who can obtain the invention clearly disclosed in the present invention or without objection based on the written description of the document should be considered as the scope of protection of this patent.

Claims

1. A Klebsiella oxytoca bacteriophage RDP-EC-23031, characterized in that: The acid-producing Klebsiella phage RDP-EC-23031 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 22, 2024, with the deposit number CGMCC No.45963.

2. A bacteriophage composition, characterized in that: It comprises the Klebsiella oxytoca phage RDP-EC-23031 as claimed in claim 1.

3. Use of the Klebsiella oxytoca phage RDP-EC-23031 according to claim 1 or the phage composition according to claim 2 in the preparation of products for preventing and / or treating livestock and poultry diseases caused by Klebsiella oxytoca.

4. Use of the Klebsiella oxytoca phage RDP-EC-23031 according to claim 1 or the phage composition according to claim 2 in the preparation of a product for killing and / or inhibiting Klebsiella oxytoca.

5. A bacteriophage pharmaceutical preparation, characterized in that: The active ingredient comprises the Klebsiella oxytoca phage RDP-EC-23031 as claimed in claim 1 or the phage composition as claimed in claim 2.

6. The bacteriophage pharmaceutical preparation according to claim 5, characterized in that The concentration of the Klebsiella oxytoca phage RDP-EC-23031 is at least 10 6 pfu / mL.

7. A drinking water additive, characterized in that: The active ingredient comprises the Klebsiella oxytoca phage RDP-EC-23031 as claimed in claim 1 or the phage composition as claimed in claim 2.

8. A feed additive, characterized in that: The active ingredient comprises the Klebsiella oxytoca phage RDP-EC-23031 as claimed in claim 1 or the phage composition as claimed in claim 2.

9. A disinfectant, characterized in that: The active ingredient comprises the Klebsiella oxytoca phage RDP-EC-23031 as claimed in claim 1 or the phage composition as claimed in claim 2.

10. A cleaning agent, characterized in that: The active ingredient comprises the Klebsiella oxytoca phage RDP-EC-23031 as claimed in claim 1 or the phage composition as claimed in claim 2.

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