Cross-species lytic bacteriophage RDP-EC-20146 and applications thereof

Abstract

The application provides a cross-species lytic bacteriophage RDP-EC-20146 and an application thereof, and belongs to the technical field of biotechnology. The cross-species lytic bacteriophage is an Escherichia coli bacteriophage, the preservation number of which is CGMCC No. 45351, and the bacteriophage is preserved in the China General Microbiological Culture Collection Center on October 28, 2022. The cross-species lytic bacteriophage RDP-EC-20146 provided by the application has high lytic activity and good physical and chemical factor tolerance characteristics; the cross-species lytic bacteriophage is not affected by bacterial drug resistance, and does not have problems such as drug residues, and has high use safety; in addition, the bacteriophage RDP-EC-20146 provided by the application can all show significant bacteriostatic effects when being used in combination with low-dose 8 kinds of antibiotics, and has a good application prospect in the prevention and treatment of salmonellosis, colibacillosis, staphylococcosis and / or pseudomonas aeruginosa disease.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, more particularly relates to a cross-species lytic bacteriophage RDP-EC-20146 and its application. BACKGROUND

[0002] As a conditional pathogen, E. coli widely exists in the breeding environment, and once infected, it can cause enteritis, peritonitis, septicemia and other diseases in animals, significantly affecting the weight gain of the animal body, and dramatically increasing the mortality rate of the animal population, causing huge losses to the breeding industry. At present, the breeding farm mainly prevents and controls E. coli disease by using antibiotics, but antibiotic therapy has many side effects, such as destroying the structure of animal intestinal microbial flora, drug residues, etc., and the large-scale use of antibiotics accelerates the production and spread of serious drug-resistant and multi-drug-resistant E. coli, thereby increasing the difficulty of preventing and controlling E. coli disease and seriously affecting the healthy development of the breeding industry.

[0003] Bacteriophages are the most abundant biological population in the biological world, which can infect bacteria and replicate, and have the advantages of strong bactericidal specificity, low development cost, easy isolation, self-limitation, few side effects, and the ability to kill drug-resistant strains. The mechanism of bacteriophage lysis of bacteria is different from that of antibiotics, so it is not affected by bacterial drug resistance, and has high species specificity for lysed bacteria, can efficiently and specifically kill target bacteria without destroying the normal flora structure, has no side effects on the animal body, and there is no drug residue problem. Bacteriophages are widely sourced and have low production costs, and there have been many successful reports on the prevention and control of clinically drug-resistant bacteria.

[0004] For example, Chinese patent CN115851617A discloses an E. coli bacteriophage LHE83 and its application. The E. coli bacteriophage LHE83 has a preservation number of CCTCC NO: M 20221473 and was preserved on September 22, 2022. The E. coli bacteriophage LHE83 provided by the present application is a new bacteriophage isolated from nature, which has the characteristics of wide host spectrum, high lytic activity, and good physical and chemical factor tolerance. As a natural enemy of bacteria, it can specifically lyse pathogenic E. coli without destroying the normal flora structure, and is not affected by bacterial drug resistance, and there is no problem of drug residue, etc., and has high safety in use. In addition, the E. coli bacteriophage LHE83 provided by the present application shows significant bacteriostatic effect when combined with a low dose of antibiotic, and has good application prospects in the prevention and control of E. coli disease.

[0005] For example, a Pseudomonas aeruginosa bacteriophage is disclosed in Chinese Patent CN106929481A. The Pseudomonas aeruginosa bacteriophage, strain name vB_PaeM_QKL1, accession number CGMCC No.13381, classified as Pseudomonas aeruginosa bacteriophage, was deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 8, 2016. The application also provides the use of the above-mentioned Pseudomonas aeruginosa bacteriophage. The Pseudomonas aeruginosa bacteriophage has extracellular polymerase-degrading enzymes in the tail spine, which can lyse the cell envelope and further lyse the biofilm-producing bacteria, thereby completely removing the bacterial biofilm; the Pseudomonas aeruginosa bacteriophage has obvious inhibitory effect on Pseudomonas aeruginosa biofilm, can destroy Pseudomonas aeruginosa biofilm, and can be used in combination with antibiotics and common chemical cleaning agents.

[0006] However, the existing bacteriophage is combined with a single type of antibiotic, and has high lysis specificity and host dependence, which greatly limits the application of the bacteriophage, and therefore a cross-species lysis bacteriophage capable of being combined with multiple antibiotics and capable of simultaneously lysing multiple bacteria and its application need to be developed. SUMMARY

[0007] Based on the deficiencies of the prior art, the present application aims to provide a cross-species lysis bacteriophage RDP-EC-20146 capable of being combined with multiple antibiotics and having inhibitory effect on multiple bacteria and its application.

[0008] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0009] The present application provides a cross-species lysis bacteriophage RDP-EC-20146, the Latin name of which is Escherichia coli bacteriophage. The accession number of the cross-species lysis bacteriophage RDP-EC-20146 is CGMCC No.45351, the classification name is Escherichia coli bacteriophage, and it was deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 28, 2022, the address of which is No.3, Yikuangli, Beichenxi Road, Chaoyang District, Beijing, China, and the postal code is 100101.

[0010] Transmission electron microscopy shows that the bacteriophage RDP-EC-20146 has a polyhedral head with symmetry, encapsulating nucleic acids, with a diameter of about 55nm, a tail about 90nm long, a sheath, and a neck connecting the head and the tail. The bacteriophage can be classified as a Myoviridae virus with a gene length of 52895bp, and a double-plate forming diameter of 2mm-3mm of bacteriophage plaques.

[0011] The bacteriophage RDP-EC-20146 has strong lytic activity on Salmonella, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa.

[0012] The application also provides the use of the cross-species lytic bacteriophage RDP-EC-20146 for preparing a medicine for resisting diseases caused by Salmonella, Escherichia coli, Staphylococcus aureus and / or Pseudomonas aeruginosa, or preparing a biological preparation for inhibiting Salmonella, Escherichia coli, Staphylococcus aureus and / or Pseudomonas aeruginosa, or preparing a feed additive or feed.

[0013] The application also provides a medicine for resisting diseases caused by Salmonella, Escherichia coli, Staphylococcus aureus and / or Pseudomonas aeruginosa, which contains the bacteriophage.

[0014] The application also provides a biological preparation for inhibiting Salmonella, Escherichia coli, Staphylococcus aureus and / or Pseudomonas aeruginosa, which contains the bacteriophage.

[0015] The application also provides a feed additive, which contains the bacteriophage.

[0016] The application also provides a feed, which contains the feed additive or bacteriophage.

[0017] Compared with the prior art, the application has the following beneficial effects:

[0018] (1) The application finds and separates the bacteriophage RDP-EC-20146, which has strong lytic activity on Salmonella, Escherichia coli, Staphylococcus aureus and / or Pseudomonas aeruginosa in a breeding environment, and provides a good bacteriophage source for industrial production of the bacteriophage for preventing and treating Salmonella, Escherichia coli, Staphylococcus aureus and / or Pseudomonas aeruginosa in the breeding environment;

[0019] (2) The titer of the bacteriophage RDP-EC-20146 in the application still remains above 10 12 pfu / mL in the pH range of 5-9, and the bacteriophage RDP-EC-20146 can resist weak acid and weak base conditions and has good acid and alkali resistance;

[0020] (3) The titer of the bacteriophage obtained in the application still remains above 10 12 pfu / mL after being treated at 30-50℃ for 30-60min, and the bacteriophage RDP-EC-20146 has good temperature resistance. This characteristic is very important in the production, transportation and storage of the bacteriophage;

[0021] (4) The bacteriophage RDP-EC-20146 provided by the application can exhibit significant bacteriostatic effect when combined with low-dose 8 kinds of antibiotics, and has a good application prospect in preventing and treating salmonellosis, colibacillosis, staphylococcosis and / or pseudomonas aeruginosa disease. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0023] Figure 1 A transmission electron microscope picture of the cross-species lytic bacteriophage RDP-EC-20146 provided by the present application is provided.

[0024] Figure 2 A pH stability result picture of the cross-species lytic bacteriophage RDP-EC-20146 provided by the present application is provided.

[0025] Figure 3 A thermal stability determination result picture of the cross-species lytic bacteriophage RDP-EC-20146 provided by the present application is provided.

[0026] Figure 4 A cross-species lytic bacteriophage RDP-EC-20146 and antibiotic synergistic bacteriostatic effect test result provided by the present application is provided. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions suggested by the manufacturer are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased in the market.

[0028] Example 1 Isolation and identification of pathogenic E. coli

[0029] Samples were taken from the sick breeding farm, and the liver of the sick bird was taken aseptically. The selective medium (i.e. MacConkey solid medium) was streaked, and after 18-24h culture at 37℃, round, flat, edge neat, smooth and wet surface, red colonies were formed on the culture medium. The typical colonies were picked and streaked for purification for 3 times, and then a single colony was picked and inoculated in 5mL LB broth, which was cultured at 37℃ with 200rpm shaking for 6h to obtain a uniform turbid bacterial suspension. The 16sRNA molecular identification and serotype identification were performed to determine that it was pathogenic E. coli, which was named as BEC-20261 and stored in a-80℃ refrigerator.

[0030] Example 2: Isolation and Identification of Transspecies Lysing Phage RDP-EC-20146

[0031] (1) Fecal treatment: Weigh 5g of feces collected from the farm, add 10mL of sterile water and soak overnight. Centrifuge the leachate at 10000rpm for 5min, then filter the supernatant through a 0.22μm filter and collect the filtrate for later use.

[0032] (2) Preparation of mixed bacterial suspension: Take 1 mL of bacterial suspension and 2 mL of filtrate and add them to 27 mL of LB broth. Incubate overnight at 37°C and 200 rpm with shaking. Then centrifuge at 10000 rpm for 5 min. Take the supernatant and filter it through a 0.22 μm filter. Collect the mixed bacterial suspension filtrate for later use.

[0033] (3) Phage isolation: Phages were isolated using the double-plate method. 0.1 mL of the mixed bacterial suspension filtrate and 0.1 mL of E. coli suspension were mixed thoroughly and incubated at 37°C for 10 min. The mixture was then plated onto double plates and incubated at 37°C for 6-8 h. The results were observed; if phages were present, clear spots would appear on the plates. Clear spots were picked and placed in 1 mL of physiological saline and incubated at 37°C for 30 min. Then, 0.1 mL of the extract and 0.1 mL of the bacterial suspension were incubated at 37°C for 10 min, plated onto double plates, and incubated at 37°C for 4-6 h for purification. This purification process was repeated 2-3 times until the phage plaques were uniform in size, yielding a phage strain named RDP-EC-20146. The phage plaques formed by RDP-EC-20146 on the double plates had a diameter of 2 mm-3 mm.

[0034] Example 3: Transmission electron microscopy morphology observation of cross-species lystic phage RDP-EC-20146

[0035] Take 20 μL of liquid containing crude phage particles and drop it onto a copper grid. Allow it to settle naturally for 15 min. Then, use filter paper to absorb the excess liquid from the side. Add one drop of 2% phosphotungstic acid (PTA) to the copper grid to stain the phage for 10 min. Then, use filter paper to absorb the staining solution from the side. After the sample dries, observe the phage morphology using an electron microscope.

[0036] Depend on Figure 1The bacteriophage RDP-EC-20146 has a polyhedral, three-dimensionally symmetrical head encapsulating nucleic acid, approximately 55 nm in diameter, and a tail approximately 90 nm long with a tail sheath. The neck connects the head and tail. This bacteriophage can be classified as belonging to the order Twinovirales, family Myotailviridae. Its gene length is 165912 bp. Sequencing of the entire genome revealed the absence of virulence and lysogenic genes, indicating good safety at the genetic level. The bacteriophage's genome contains multiple lysin genes, and its lysis products contain the lysins expressed by these genes, allowing for further lysis of related *E. coli* bacteria during product use.

[0037] Phage titer was calculated based on the number of plaques, and the titer of the cross-species lysing phage RDP-EC-20146 was determined to be 1.26 × 10⁻⁶. 12 pfu / mL.

[0038] Example 4: Determination of the acid-base stability of cross-species lysing phage RDP-EC-20146

[0039] The pH of the physiological saline was adjusted using dilute hydrochloric acid and dilute NaOH solution to prepare buffer solutions with pH values ​​of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. The bacteriophages were then diluted to a concentration of 1 × 10¹ using these buffer solutions. 2 The pfu / mL dilution was incubated in a water bath at 37℃ for 1 hour, then diluted 10 times with physiological saline and plated on double plates to determine the potency. The plate was incubated upside down at 37℃ for 4-6 hours before the results were statistically analyzed.

[0040] Depend on Figure 2 It can be seen that under pH 5-9 conditions, after culturing at 37℃ for 1 hour, its potency only decreased by one order of magnitude, and the potency remained at 10. 12 A pfu / mL concentration above this indicates that RDP-EC-20146 has good acid and alkali resistance. This property has great application value in the treatment of diseases. RDP-EC-20146 itself has high activity and good tolerance to acids and alkalis, which can ensure that a sufficient number of bacteriophages reach the intestine to exert a bactericidal effect.

[0041] Example 5: Determination of the thermal stability of cross-species lysing phage RDP-EC-20146

[0042] The phage stock solution was aliquoted into 50 mL sterile centrifuge tubes and incubated at 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 80℃, and 90℃ for 30 min and 60 min, respectively. Afterward, it was serially diluted 10-fold with physiological saline, and the titer was determined by plating on double plates. Figure 3It can be seen that with the increase of temperature and time, the phage titer gradually decreases. The phage titer only decreases by one order of magnitude at 50°C for 30 min and 60 min, and the titer is still above 10 12 The RDP-EC-20146 has good tolerance to temperature, which is of great significance in the production, transportation and storage of the phage.

[0043] Example 6: Determination of the lysis spectrum of the cross-species lytic phage RDP-EC-20146

[0044] The pathogenic bacteria Salmonella, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa isolated from animal sources were selected for the lysis spectrum experiment of the phage RDP-EC-20146. It was found through the experiment that the phage had cross-species lytic ability and could lyse Salmonella, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. Seventeen strains of Salmonella, fifteen strains of Escherichia coli, eleven strains of Staphylococcus aureus and seventeen strains of Pseudomonas aeruginosa were selected for the lysis spectrum experiment. The experimental results showed that the phage could lyse Salmonella, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, and the lysis rates were 100% (17 / 17), 86.67% (13 / 15), 81.82% (9 / 11) and 88.24% (15 / 17), respectively. The phage had a wide lysis range for Salmonella, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. The final experimental results are shown in Table 1.

[0045] Table 1

[0046]

[0047]

[0048] Note: "+" represents lysis; "-" represents no lysis.

[0049] Example 7: Antibacterial test of the phage RDP-EC-20146 combined with antibiotics

[0050] Experimental method:

[0051] The antibiotics were prepared into a 1.024% antibiotic stock solution, and doxycycline and colistin were diluted by 10 times, and the stock solution concentration was 0.1024%.

[0052] Minimum inhibitory concentration determination: two 96-well plates were taken, 100 uL of sterilized LB culture medium was added to the 96-well plates, 100 uL of prepared drug solution was added to the first column of wells, and then it was diluted by 10 times in turn to the 11th column, and then it was discarded. The last column was the negative control. 100 uL of bacterial suspension (activity 1 x 10 6Add 100 μL of bacterial suspension (1 × 10⁻⁶ cfu / mL) to another 96-well plate. 6 cfu / mL) and bacteriophage (titer 1×10⁻⁶) 8 The samples (pfu / mL) were incubated overnight at 37°C, and the results were recorded 12-16 hours later.

[0053] Depend on Figure 4 It can be seen that phage RDP-EC-20146 has a good synergistic effect with 8 antibiotics, which can reduce the MIC (minimum susceptibility concentration) to 1 / 32-1 / 4 of the original value. This indicates that the cross-species lysing phage RDP-EC-20146 has a good synergistic effect with 8 antibiotics on Escherichia coli, Salmonella, Pseudomonas aeruginosa and Staphylococcus aureus.

[0054] Experimental Case: Application of Bacteriophage RDP-EC-20146 in Clinical Treatment

[0055] Design experiments to verify the synergistic effect of bacteriophage RDP-EC-20146 and antibiotics.

[0056] Experimental steps:

[0057] Fourteen-day-old broiler chickens were selected and challenged with the virus via thorax injection at a dose of 100 μml, resulting in an E. coli activity of 1 × 10⁻⁶. 8 The cfu / mL concentration was determined, and the following table was used for experiments after challenge. Mortality rates and flock condition were recorded during the treatment. Seven days after treatment, internal organs were examined and symptoms were recorded. The results are as follows.

[0058] The grouping and medication details are shown in Table 2 below, and the corresponding treatment results are shown in Table 3 below.

[0059] Table 2

[0060] SEQ ID NO Challenge Treatment Notes 1 1e6 cfu / animal / Positive 2 Saline / Negative 3 1e7 cfu / animal 0.256% lincomycin 0.1 ml Antibiotic treatment 4 1e7 cfu / animal 0.1 mL phage Phage treatment 5 1e7 cfu / animal 0.256% lincomycin 0.1 ml + 0.1 mL phage Antibiotic + phage 6 1e7 cfu / animal 0.128% lincomycin 0.1 ml + 0.1 mL phage 1 / 2 antibiotic + phage 7 1e7 cfu / animal 0.064% lincomycin 0.1 ml + 0.1 mL phage 1 / 4 antibiotic + phage 8 1e7 cfu / animal 0.032% lincomycin 0.1 ml + 0.1 mL phage 1 / 8 antibiotic + phage 9 1e7 cfu / animal 0.016% lincomycin 0.1 ml + 0.1 mL phage 1 / 16 antibiotic + phage 10 1e7 cfu / animal 0.008% lincomycin 0.1 ml + 0.1 mL phage 1 / 32 antibiotic + phage

[0061] Note: The minimum susceptibility concentration for lincomycin is 0.256%; the phage titer is 1.06*10. 10 pfu / mL.

[0062] Table 3

[0063] SEQ ID NO Notes Mortality (%) Necropsy symptoms 1 Positive 50.00 Severe pericardial and pneumonic inflammation 2 Negative 0.00 None 3 Antibiotic treatment 10.00 Mild pericardial and pneumonic inflammation 4 Phage treatment 15.00 Mild pericardial and pneumonic inflammation 5 Antibiotic + phage 0.00 None 6 1 / 2 antibiotic + phage 5.00 None 7 1 / 4 antibiotic + phage 5.00 None 8 1 / 8 antibiotic + phage 5.00 None 9 1 / 16 antibiotic + phage 10.00 Mild pericardial and pneumonic inflammation 10 1 / 32 antibiotic + phage 20.00 Severe pericardial and pneumonic inflammation

[0064] According to the test results in Table 3 above, when phage RDP-EC-20146 is used in combination with antibiotics, reducing the antibiotic susceptibility concentration to 1 / 8 or 1 / 16 can still achieve a good therapeutic effect.

[0065] The above merely illustrates the preferred embodiments of the present application, and is not intended to limit the patent scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the patent protection scope of the present application.

Claims

1. A cross-species lytic bacteriophage, RDP-EC-20146, characterized by: The cross-species lytic bacteriophage is an Escherichia coli bacteriophage, and the preservation number is CGMCC No.45351, which was preserved in the China General Microbiological Culture Collection Center on October 28, 2022; the cross-species lytic bacteriophage RDP-EC-20146 can be combined with eight antibiotics, including amoxicillin, apramycin, doxycycline, neomycin, florfenicol, lincomycin, mucobacterin and single kanamycin, to lyse Salmonella, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, and the concentration of the antibiotics is 1 / 8 of the minimum drug sensitivity concentration.

2. The cross-species lytic phage according to claim 1, characterized in that: The bacteriophage RDP-EC-20146 can lyse Salmonella, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa.

3. Use of the cross-species lytic bacteriophage RDP-EC-20146 according to any one of claims 1 or 2, characterized in that: The cross-species lytic bacteriophage RDP-EC-20146 is used to prepare a medicine for resisting diseases caused by Salmonella, Escherichia coli, Staphylococcus aureus and / or Pseudomonas aeruginosa.

4. Use of the cross-species lytic bacteriophage RDP-EC-20146 according to any one of claims 1 or 2, characterized in that: The cross-species lytic bacteriophage RDP-EC-20146 is used to prepare a biological preparation for inhibiting Salmonella, Escherichia coli, Staphylococcus aureus and / or Pseudomonas aeruginosa.

5. A medicament against diseases caused by Salmonella, Escherichia coli, Staphylococcus aureus and / or Pseudomonas aeruginosa, characterized in that: The medicine contains the cross-species lytic bacteriophage RDP-EC-20146 according to any one of claims 1 or 2.

6. A biological agent for inhibiting Salmonella, Escherichia coli, Staphylococcus aureus and / or Pseudomonas aeruginosa, characterized in that: The biological preparation contains the cross-species lytic bacteriophage RDP-EC-20146 according to any one of claims 1 or 2.

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