A carcinogenic Enterobacteriaceae phage RDP-QEC-24001 and its application

Abstract

The present invention relates to the field of microbial technology. The present invention provides a phage RDP-QEC-24001 that lyses oncogenic Enterobacteriaceae, which was deposited in the General Microbiology Center of the China Culture Collection Administration on September 23, 2024, with a deposit number of CGMCC No. 46191. The present invention also provides a phage composition. The present invention also provides the use of the phage RDP-QEC-24001 that lyses oncogenic Enterobacteriaceae or a phage composition in the preparation of products for preventing and / or treating livestock and poultry diseases caused by oncogenic Enterobacteriaceae, as well as the use of the phage composition in the preparation of products for killing and / or inhibiting oncogenic Enterobacteriaceae. The present invention also provides a phage pharmaceutical preparation, disinfectant or cleaning agent. The present invention provides a phage RDP-QEC-24001 that lyses oncogenic Enterobacteriaceae, which has high titer, good therapeutic effect and high lysis rate.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a phage RDP-QEC-24001 that lyses carcinogenic Enterobacteriaceae and applications thereof. Background Art

[0002] Carcinogenic Enterobacteriaceae ( Enterobacter cancerogenus strain ) is Enterobacter spp. ( Escherichia coli ) is a Gram-negative, short, thick rod with flagella, no spores, and facultative anaerobic activity. It is widely found in water, sewage, soil, and vegetables. It is a colonizer of the intestine and can cause pathogenic infections under various conditions, including bone and joint infections associated with severe trauma or crush injuries.

[0003] Furthermore, carcinogenic Enterobacteriaceae have been shown to be highly resistant to antibiotics such as amoxicillin. Due to their ability to produce bioemulsifiers and degrade azo dye pollutants and L-phosphinothricin herbicides, they are primarily used as pesticides and soil conditioners. Furthermore, as intestinal colonizers, carcinogenic Enterobacteriaceae can colonize the intestinal tract of chickens in large numbers, continuously excreting bacteria into the external environment throughout the breeding and slaughtering processes, ultimately spreading to humans and posing a threat to human life and health.

[0004] There are cases of wound-related infections of bones and joints following environmental exposure to carcinogenic Enterobacteriaceae.

[0005] Bacteriophages are viruses that target bacteria, mycoplasmas, and chlamydia, and are characterized by safety and specificity. First, bacteriophages are highly specific, lysing only the corresponding host bacteria without destroying the normal flora, making them widely applicable throughout the animal body. Second, the use of bacteriophages leaves no residue, as they are naturally excreted from the animal's body. Furthermore, bacteriophages do not cause pathogens to develop drug resistance and are effective against both Gram-positive and Gram-negative bacteria, as well as multidrug-resistant bacteria.

[0006] Therefore, it is urgent to develop a phage product with high titer, good therapeutic effect and high lysis rate for the prevention and treatment of carcinogenic Enterobacteriaceae. Summary of the Invention

[0007] The purpose of the present invention is to provide a phage RDP-QEC-24001 that lyses carcinogenic Enterobacteriaceae to replace the use of antibiotics in the prevention and treatment of livestock and poultry breeding, and to solve the problems of rapid onset of disease in livestock and poultry and untimely treatment, and ineffective medication due to drug resistance of pathogenic bacteria.

[0008] In order to solve the above problems, the present invention provides the following technical solutions:

[0009] In the first aspect, the present invention provides a phage that lyses carcinogenic Enterobacteriaceae ( Enterobacter cancerogenus strain phage) RDP-QEC-24001, isolated from chicken livers from a Shandong farmer, was deposited on September 23, 2024, at the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 46191. The oncogenic Enterobacter QT-24001 used in the present invention was deposited together with the oncogenic Enterobacter lytic phage RDP-QEC-24001 of the present invention.

[0010] Electron microscopic observation: The phage is a polyhedral, three-dimensionally symmetrical head encapsulating nucleic acid. Its size is approximately 90 nm, and the genome is 180,642 bp long. According to the definition of the International Committee on Taxonomy of Viruses (ICTV), the phage's morphology conforms to the characteristics of the Myotrophae family and belongs to the Myotrophae family. The titer of the oncogenic Enterobacteriaceae phage DP-QEC-24001 can reach 10 9 pfu / mL. This phage can be stored for a long time at -80°C with a stable titer. This phage can specifically lyse the oncogenic Enterobacter QT-24001 isolated in this experiment, with an optimal multiplicity of infection of 100:1.

[0011] In a second aspect, based on the same inventive concept, the present invention provides a phage composition comprising the oncogenic Enterobacteriaceae phage RDP-QEC-24001 as described above.

[0012] In a third aspect, based on the same inventive concept, the present invention provides the use of the above-mentioned oncogenic Enterobacteriaceae-lytic phage RDP-QEC-24001 or the above-mentioned phage composition in the preparation of products for preventing and / or treating livestock and poultry diseases caused by oncogenic Enterobacteriaceae.

[0013] In a fourth aspect, based on the same inventive concept, the present invention provides the use of the above-mentioned oncogenic Enterobacteriaceae-lysing phage RDP-QEC-24001 or the above-mentioned phage composition in the preparation of a product for killing and / or inhibiting oncogenic Enterobacteriaceae.

[0014] 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 above-mentioned oncogenic Enterobacteriaceae phage RDP-QEC-24001 or the above-mentioned phage composition.

[0015] The phage pharmaceutical preparation as described above, wherein the concentration of the lytic carcinogenic Enterobacteriaceae phage RDP-QEC-24001 is at least 10 9 pfu / mL.

[0016] In a sixth aspect, based on the same inventive concept, the present invention provides a disinfectant, the active ingredient of which includes the aforementioned oncogenic Enterobacteriaceae-lysing phage RDP-QEC-24001 or the aforementioned phage composition. Preferably, the disinfectant can be used to disinfect chicken farming environments against oncogenic Enterobacteriaceae by spraying or immersion.

[0017] In a seventh aspect, based on the same inventive concept, the present invention provides a cleaning agent, the active ingredients of which include the above-mentioned carcinogenic Enterobacteriaceae phage RDP-QEC-24001 or the above-mentioned phage composition.

[0018] Compared with the existing technology, the effects and advantages of the present invention are:

[0019] 1. The phage RDP-QEC-24001 provided by the present invention is capable of lysing carcinogenic Enterobacteriaceae. The phage has a high titer, and the titer of the phage can reach up to 10 9 pfu / mL or above, which provides a new source for phage products.

[0020] 2. The lysis rate of the oncogenic Enterobacteriaceae phage RDP-QEC-24001 provided by the present invention reaches 95.37%, which has a good lysis effect on the host and is suitable for use in the breeding process.

[0021] 3. The carcinogenic Enterobacteriaceae phage RDP-QEC-24001 provided by the present invention is safe and highly effective, and can replace the use of antibiotics in the prevention and treatment of livestock and poultry breeding, and can be highly efficient and economical in practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a picture of the plaques of the phage isolated by the present invention;

[0023] Figure 2 This is an electron micrograph of the bacteriophage RDP-QEC-24001 of the present invention;

[0024] Figure 3 This is a one-step growth curve of the bacteriophage RDP-QEC-24001 of the present invention. DETAILED DESCRIPTION

[0025] The following will be combined with the contents of the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described 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 making creative efforts are within the scope of protection of the present invention.

[0026] Unless otherwise defined, all technical and scientific terms used in this specification have the same meanings as those commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are intended solely for the purpose of describing specific embodiments and are not intended to limit the invention. The term "and / or" as used in this specification includes any and all combinations of one or more of the associated listed items.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Hereinafter, the oncogenic Enterobacteriaceae lysing bacteriophage RDP-QEC-24001 is referred to as "phage RDP-QEC-24001" or "RDP-QEC-24001."

[0029] Example 1 Isolation and identification of pathogenic bacteria and bacteriophages

[0030] (1) Isolation and identification of carcinogenic Enterobacteriaceae:

[0031] The chicken liver used in the present invention was collected from a farmer in Shandong in 2024 as a sample for isolation of pathogens and bacteriophages.

[0032] From chicken liver, aseptically streak onto CHROMagar colorimetric medium for colon development and incubate at 37°C for 18-24 hours. Select blue or blue-green colonies with a diameter of approximately 1-2 mm and neat edges. Then, streak onto LB medium and incubate at 37°C for 18-24 hours. Select a representative colony and continue streaking and purification three times. A single colony was inoculated into 5 mL of LB medium and incubated at 37°C with shaking at 200 rpm for 4 hours to obtain a uniform, turbid bacterial suspension. 16sRNA molecular identification confirmed the strain as Enterobacter carcinogenicus, named QT-24001, and stored at -80°C.

[0033] (2) Phage isolation and identification:

[0034] Phage enrichment: Grind an appropriate amount of chicken liver with saline solution, centrifuge at 10,000 rpm for 10 minutes to remove large impurities and most bacteria, and sterilize by filtration through a 0.22 μm filter. Add 0.1 ml of the filtrate and 0.1 ml of the host bacterial suspension to 5 ml of LB broth and incubate at 37°C on a shaker for 6 hours. After incubation, remove 3-4 ml of the aliquot, centrifuge at 10,000 rpm for 5 minutes, and sterilize by filtration through a 0.22 μm filter. Reserve the filtrate.

[0035] Phage isolation: Take 0.1 ml of the suspension of Enterobacter carcinogenicus QT-24001 and spread it on a double-layer plate. After cooling, add 0.1 ml of the filtrate of the mixed bacterial suspension. After incubation at 37°C for 6-8 hours, remove the transparent spot and soak it in 1 mL of normal saline for 10 minutes. Dilute it tenfold to 10 -6 , take 10 -4 , 10 -5 , 10 -6 Add 0.1 mL of the bacterial suspension to each of the three dilution gradients, add to 3 mL of 0.4% nutrient agar heated to 45-54°C, spread on two plates, and incubate at 37°C for 4-6 hours for purification. Repeat this procedure 2-3 times until the plaque size is uniform.

[0036] Thus, a phage strain was obtained and named RDP-QEC-24001. The plaque picture of the phage isolated in this example is shown in FIG. Figure 1 shown.

[0037] Depend on Figure 1 It can be seen that the plaques formed by phage RDP-QEC-24001 on the double plates are 2 mm in diameter, round and transparent.

[0038] (3) Determination of phage titer

[0039] Purified phage was plated on a double-layer plate. A single plaque was then harvested and propagated in LB liquid medium containing a host bacterial suspension. The propagated medium was centrifuged at 10,000 rpm for 5 minutes and sterilized by filtration through a 0.22 μm filter. The plaque was diluted tenfold to 10⁻⁶. 0.1 mL of the last three dilutions of phage solution and 0.1 mL of the host bacterial suspension were plated on a double-layer plate and the phage titer was determined.

[0040] Phage titer (pfu / mL) = number of plaques * dilution factor ÷ 0.1.

[0041] The results showed that the plaques had the same morphology, size and clarity, and had typical lytic phage characteristics; the titer of the phage could reach up to 10 9 pfu / mL or above.

[0042] (4) Preservation of bacteriophages

[0043] The phage proliferation fluid filtrate was mixed with 60% glycerol at a ratio of 1:1 and stored in a -80°C refrigerator.

[0044] Example 2 Electron microscopic observation of bacteriophage

[0045] 1. Experimental methods

[0046] Take higher than 10 5Drop 50 μL of the PFU / mL phage sample onto a microporous copper mesh and let it settle for 1 minute. Remove excess liquid with filter paper. Add 50 μL of 1% phosphotungstic acid (PTA) onto the mesh and stain for 5 minutes. Remove excess stain with filter paper. After drying, observe and photograph using a transmission electron microscope.

[0047] 2. Experimental results and analysis

[0048] Depend on Figure 2 As shown, under an electron microscope, this phage has a polyhedral, three-dimensionally symmetrical head encapsulating nucleic acid, approximately 90 nm in size and with a tail approximately 110 nm long. According to the International Committee on Taxonomy of Viruses (ICTV), this phage's morphology conforms to the Myoviridae family, making it a member of the Myoviridae family.

[0049] Example 3 Phage Genome Sequencing

[0050] 1. Experimental methods

[0051] After enrichment and culture of a single phage, centrifuge at 8000 r for 15 minutes at 4°C, add 10% PEG8000 and 0.5 M NaCl and let it stand overnight. Then, add an equal amount of chloroform and mix well. After standing and stratification, centrifuge at 5000 r for 10 minutes. After removing the chloroform layer and PEG layer, add restriction endonuclease for digestion, and suspend the phage under gradient density cesium chloride conditions. Later, dialyze with TM buffer three times, each time for 30 minutes, and send it to BGI Biological Sequencing Company for whole genome sequencing.

[0052] 2. Experimental results and analysis

[0053] The results are shown in the sequence listing. The full length of the RDP-QEC-24001 genome is 180642 bp.

[0054] Example 4: Determination of the optimal multiplicity of infection

[0055] 1. Experimental methods

[0056] Phage propagation medium and host cells were added to LB at multiplicities of infection (MOIs) of 100:1, 10:1, 1:1, 1:10, 1:100, and 1:1000, ensuring the same total volume. After shaking at 37°C and 160 rpm for 6 hours, the cells were centrifuged at 12,000 rpm for 5 minutes at room temperature. The supernatant was plated on two plates for titer determination. The results are shown in Table 1.

[0057] 2. Experimental results and analysis

[0058] Table 1 Results of optimal multiplicity of infection

[0059]

[0060] As shown in Table 1, when the multiplicity of infection was 100:1, the proliferation fluid was relatively clear after 6 h of culture, and the titer was the highest, reaching 1.92×10 9 cfu / mL, indicating that the optimal infection multiplicity of the oncogenic Enterobacteriaceae phage RDP-QEC-24001 of the present invention is 100:1.

[0061] Example 5: Phage lysis curve of host

[0062] 1. Experimental methods

[0063] A bacterial suspension in the logarithmic growth phase was inoculated with Enterobacter oncogenicus QT-24001 and bacteriophage RDP-QEC-24001 at the optimal multiplicity of infection (MOI). The suspension was incubated at 37°C in a water bath for 10 minutes, followed by centrifugation at 12,000 rpm for 5 minutes at room temperature. The supernatant was discarded to remove free phage not adsorbed to the host. The suspension was then washed twice with liquid culture medium. The pellet was then resuspended in liquid culture medium at 37°C and quickly placed in a 37°C incubator. Samples were taken at designated locations and their titers were determined. A growth curve was plotted with time as the horizontal axis and the logarithm of the phage titer as the vertical axis.

[0064] 2. Experimental results and analysis

[0065] The results are as follows Figure 3 As shown, the incubation period and lytic period of the phage can be determined, and the average lytic yield can be calculated. Average lytic yield = phage titer at the end of the outbreak / host bacteria concentration at the beginning of infection.

[0066] Incubation period: After inoculation with the oncogenic Enterobacter phage RDP-QEC-24001, the titer remained unchanged for 3 hours, indicating an incubation period of approximately 3 hours. Within 3-20 hours of infection, the phage population rapidly increased, indicating a lytic phase of approximately 17 hours. Over the following 3 hours, the phage population remained essentially constant, entering a stable growth phase. The average lytic yield was calculated to be 32 PFU / infected cell.

[0067] Example 6: Lysis rate experiment

[0068] 1. Experimental methods

[0069] Under sterile conditions, dilute the freshly grown host bacterial suspension to 1×10 6 CFU / mL, 10 8 , 10 7 , 10 6Phage at a fixed concentration of PFU / mL was mixed evenly with a fixed concentration of bacterial solution and incubated at 37°C and 170 rpm for 15 minutes. After incubation, 100 μL of each gradient was spread onto nutrient agar plates and incubated at 37°C for 24 hours. Each gradient was repeated twice. Plates with 30–300 colonies were selected for colony counting. The interaction trends of phage titers with host bacteria were plotted, and the phage lysis efficiency was calculated.

[0070] Phage lysis rate = (1-number of colonies in the treatment group / number of colonies in the control group) × 100%.

[0071] The results are shown in Table 2.

[0072] 2. Experimental results and analysis.

[0073] Table 2 Results of lysis rate experiment

[0074]

[0075] Calculations show that the lysis rate of the bacteriophage RDP-QEC-24001 reaches 95.37%, which has a good lysis effect on the host and is suitable for use in the breeding process.

[0076] Example 7: In vivo efficacy study of the carcinogenic Enterobacteriaceae phage RDP-QEC-24001 in chickens

[0077] 1. Experimental methods

[0078] (1) Carcinogenic Enterobacteriaceae and their phage activation

[0079] Streak the oncogenic Enterobacter QT-24001 into LB medium and incubate at 37°C for 24 hours. Streak the culture again into LB medium and incubate at 37°C for 24 hours. Inoculate the shaken oncogenic Enterobacter QT-24001 into 2% inoculum of the culture. Incubate at 37°C for 24 hours, centrifuge, and filter.

[0080] (2) Activity detection of carcinogenic Enterobacter QT-24001

[0081] Aseptically scrape a colony of Enterobacter oncogenic QT-24001 from LB medium and place it in 20 mL of normal saline. Shake to disperse the colony. Then, dilute the colony 10-fold with normal saline. Select the appropriate concentration and spread it on a plate. Incubate at 37°C for 24 hours and calculate the colony count. Refrigerate the sample at 4°C until ready for use.

[0082] (3) Chicken embryo inoculation

[0083] The chicken embryos were hatched to 14 days old and divided into groups, with 15 in each group. The inoculation was performed by air chamber inoculation with a dose of 1.0×10 4CFU / piece (inoculation volume is 0.1mL), simulating the process of chicken breeders being exposed to pathogens in the farm.

[0084] In a sterile laminar flow hood, thoroughly wipe the chick embryos with alcohol cotton balls to disinfect them. Then, using sterile surgical forceps, drill a small hole in the top of the chick embryo's air chamber. Using a sterile 1mL syringe, inject 0.1mL of the appropriate concentration of bacterial solution into the chick embryo's air chamber. After disinfection with alcohol cotton balls, seal the hole with liquid paraffin and return the chicks to the fully automatic incubator for incubation. Grouping is shown in Table 3. The QT-24001 challenge group received 1.0×10 5 cfu pathogenic bacteria QT-24001 0.1mL, RDP-QEC-24001 treatment group inoculated 1.0×10 5 Each piece of the negative control group was inoculated with 0.1 mL of 0.9% NaCl.

[0085] Table 3 Grouping of chicken embryo challenge vaccination

[0086]

[0087] (4) Observation after vaccination

[0088] After the chicks hatched, they were raised in cages according to groups. The mental state and disease incidence of the chicks in each group were observed every 12 hours. The RDP-QEC-24001 treatment group was protected by oral administration of carcinogenic Enterobacteriaceae phage, and the number of chick deaths was counted for 7 consecutive days.

[0089] (5) Protection of carcinogenic Enterobacteriaceae phage RDP-QEC-24001

[0090] After 24 hours of incubation, each mouse in the QT-24001 challenge group was gavaged with 0.1 mL of 0.9% NaCl, and each mouse in the RDP-QEC-24001 treatment group was gavaged with 10 mL of 0.9% NaCl. 9 PFU / mL RDP-QEC-24001 0.1mL, and the negative control group was gavage-administered with 0.1mL of 0.9% NaCl for three consecutive days.

[0091] Table 4 Grouping of chicken embryo administration

[0092]

[0093] 2. Experimental results and analysis

[0094] Survival rate testing: The effectiveness of the phage was determined by the survival rate of treated and untreated chicks. The initial number of chicks in the QT-24001 challenge group was 11, the RDP-QEC-24001 treatment group was 11, and the negative control group was 14. The results are shown in Tables 5 and 6.

[0095] Table 5 Hatching rate results

[0096]

[0097] Table 6 Results of seven-day mortality

[0098]

[0099] In summary, it can be seen from Table 5 that the hatching rate of 73.3% in the RDP-QEC-24001 treatment group was the same as that in the QT-24001 challenge group, and was lower than the 93.3% hatching rate in the negative control group, indicating that the carcinogenic Enterobacterium QT-24001 had a significant lethal effect on chicken embryos. At the same time, the influence of the quality of the chicken embryos on the next experiment can also be ruled out. The remaining chicken embryos were subjected to the next experiment according to the group.

[0100] Combined with Table 6, it can be seen that the negative control group did not get sick, the chickens were in a healthy mental state, and their eating, drinking, and defecation were normal; the QT-24001-challenged group had a depressed mental state, and the umbilical cords of the sick chicks were red and swollen, the abdomen was swollen, and the feces were thin and yellow-white; after phage treatment, the mental state improved, the incidence rate decreased, and the mortality rate decreased by about 18.2%, which proves that the phage RDP-QEC-24001 is very effective and greatly reduces the mortality rate of chicks.

[0101] It should be noted that the specific embodiments are only representative examples of the present invention. Obviously, the technical solutions of the present invention are not limited to the above embodiments and may be subject to many variations. Those skilled in the art who are clear about the disclosure of the present invention or who can unambiguously derive the invention from the written description of the document should be considered to be within the scope of protection of this patent.

Claims

1. A lytic carcinogenic Enterobacteriaceae phage ( Enterobacter cancerogenus strain phage ) RDP-QEC-24001, characterized in that, The lytic oncogenic Enterobacteriaceae phage RDP-QEC-24001 was deposited in the General Microbiology Center of the China Culture Collection Administration on September 23, 2024, with the deposit number CGMCC No.46191.

2. A phage composition, characterized in that: The invention comprises the oncogenic Enterobacteriaceae phage RDP-QEC-24001 as claimed in claim 1.

3. Use of the oncogenic Enterobacteriaceae-lysing phage RDP-QEC-24001 according to claim 1 or the phage composition according to claim 2 in the preparation of a product for preventing and / or treating poultry diseases caused by oncogenic Enterobacteriaceae.

4. Use of the oncogenic Enterobacteriaceae-lysing bacteriophage RDP-QEC-24001 according to claim 1 or the bacteriophage composition according to claim 2 in the preparation of a product for killing and / or inhibiting oncogenic Enterobacteriaceae.

5. A bacteriophage pharmaceutical preparation, characterized in that: The active ingredient comprises the carcinogenic Enterobacteriaceae phage RDP-QEC-24001 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 lytic carcinogenic Enterobacteriaceae phage RDP-QEC-24001 is at least 10 9 pfu / mL.

7. A disinfectant, characterized in that The active ingredient comprises the carcinogenic Enterobacteriaceae phage RDP-QEC-24001 as claimed in claim 1 or the phage composition as claimed in claim 2.

8. A cleaning agent, characterized in that The active ingredient comprises the carcinogenic Enterobacteriaceae phage RDP-QEC-24001 as claimed in claim 1 or the phage composition as claimed in claim 2.

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