Cross-species lysis bacteriophage RDP-EC-20146 and application thereof

Abstract

The invention provides a cross-species lysis bacteriophage RDP-EC-20146 and application thereof, and belongs to the technical field of biology. The cross-species lysis bacteriophage is an escherichia coli bacteriophage, the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.45351, and the cross-species lysis bacteriophage is preserved in China General Microbiological Culture Collection Center on October 28, 2022. The cross-species lysis bacteriophage RDP-EC-20146 provided by the invention has the characteristics of high lysis activity and good physical and chemical factor tolerance; the method is not influenced by the drug resistance of bacteria, does not have the problems of drug residues and the like, and has very high use safety; in addition, when the bacteriophage RDP-EC-20146 provided by the invention is combined with 8 low-dose antibiotics for use, a remarkable bacteriostatic effect can be shown, and the bacteriophage RDP-EC-20146 has a good application prospect in the aspect of preventing and treating salmonella, escherichia coli, staphylococcus aureus and / or pseudomonas aeruginosa diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically relates to a cross-species lytic phage RDP-EC-20146 and applications thereof. Background Art

[0002] Escherichia coli, a conditionally pathogenic bacterium, is widely present in aquaculture environments. Once infected, it can cause enteritis, peritonitis, and sepsis in animals, significantly affecting their weight gain and causing a sharp increase in mortality rates, resulting in significant losses for the aquaculture industry. Currently, farms primarily use antibiotics to prevent and control E. coli disease. However, antibiotic therapy has numerous side effects, such as disrupting the structure of the animal's intestinal microbiome and leaving drug residues. Furthermore, the extensive use of antibiotics accelerates the development and spread of severely drug-resistant and multidrug-resistant E. coli, further increasing the difficulty of preventing and controlling E. coli disease and severely impacting the healthy development of the aquaculture industry.

[0003] Bacteriophages are the most abundant group of organisms in the biological world, capable of infecting and replicating bacteria. They possess advantages such as strong bactericidal specificity, low development costs, ease of isolation, self-limiting properties, minimal side effects, and the ability to kill drug-resistant strains. Phages lyse bacteria differently from antibiotics, making them unaffected by bacterial resistance. They are highly species-specific for the bacteria they lyse, effectively and specifically killing target bacteria without disrupting the normal bacterial structure. They also have no side effects on animals and no potential for drug residues. Phages are widely available and inexpensive to produce, and numerous successful clinical applications have been reported for their effectiveness in preventing and controlling drug-resistant bacteria.

[0004] For example, Chinese patent CN115851617A discloses an Escherichia coli phage LHE83 and its applications. The deposit number of the Escherichia coli phage LHE83 is CCTCC NO: M 20221473, and the deposit date is September 22, 2022. The Escherichia coli phage LHE83 provided by this invention is a new phage isolated from nature, and has the characteristics of a wide host spectrum, high lytic activity, and good tolerance to physical and chemical factors. As a natural enemy of bacteria, it can specifically lyse pathogenic Escherichia coli without destroying the normal bacterial flora, and is not affected by bacterial resistance, and there is no problem of drug residue, etc., which has a high safety of use. In addition, the Escherichia coli phage LHE83 provided by this invention exhibits significant antibacterial effect when used in combination with low-dose antibiotics, and has good application prospects in the prevention and treatment of Escherichia coli disease.

[0005] For example, Chinese patent CN106929481A discloses a Pseudomonas aeruginosa phage. This Pseudomonas aeruginosa phage, with the strain name vB_PaeM_QKL1 and the deposit number CGMCC No. 13381, is classified as Pseudomonas aeruginosa phage and was deposited on December 8, 2016, at the General Microbiology Center of the China National Committee for the Collection of Microorganisms (CGMCC). The invention also provides applications for the aforementioned Pseudomonas aeruginosa phage. The Pseudomonas aeruginosa phage has enzymes that degrade extracellular polymers in its tail spines, which can lyse cell membranes and subsequently biofilm-forming bacteria, thereby completely removing bacterial biofilms. This Pseudomonas aeruginosa phage has a significant inhibitory effect on Pseudomonas aeruginosa biofilms, can destroy Pseudomonas aeruginosa biofilms, and can also be used in combination with antibiotics and common chemical detergents.

[0006] However, when existing phages are used in combination with antibiotics, the types of antibiotics are relatively single, and the lysis specificity and host dependence are high, which greatly limits the application of phages. Therefore, it is necessary to develop a cross-species lytic phage and its application that can be used in combination with multiple antibiotics and can simultaneously lyse multiple bacteria. Summary of the Invention

[0007] Based on the deficiencies of the existing technology, the present invention aims to provide a cross-species lytic phage RDP-EC-20146 and its application, which can be used in combination with multiple antibiotics and has inhibitory effects on multiple bacteria.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides a cross-species lytic phage RDP-EC-20146, whose Latin name is Escherichiacoli bacteriophage. The cross-species lytic phage RDP-EC-20146 has a preservation number of CGMCC No. 45351 and is classified as Escherichia coli phage. It was deposited in the "General Microbiology Center of China Culture Collection Administration", abbreviated as CGMCC, on October 28, 2022, and its address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.

[0010] Transmission electron microscopy showed that the phage RDP-EC-20146 has a polyhedral, three-dimensionally symmetrical head wrapped with nucleic acid, with a diameter of about 55nm, a tail about 90nm long, a tail sheath, and a neck connecting the head and tail. The phage can be classified as the Myoviridae family of the order Caudovirales, with a gene length of 52895bp, and double-plate plaques with a diameter of 2mm-3mm.

[0011] The bacteriophage RDP-EC-20146 has strong lysis properties against Salmonella, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa.

[0012] The present invention also provides a use of the cross-species lytic phage RDP-EC-20146, which is used to prepare drugs for resisting diseases caused by Salmonella, Escherichia coli, Staphylococcus aureus and / or Pseudomonas aeruginosa, or to prepare biological preparations for inhibiting Salmonella, Escherichia coli, Staphylococcus aureus and / or Pseudomonas aeruginosa, or to prepare feed additives or feed.

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

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

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

[0016] The present invention also provides a feed containing the feed additive or bacteriophage.

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

[0018] (1) The present invention discovered and isolated bacteriophage RDP-EC-20146, which has a strong lysis effect on poultry-derived Salmonella, Escherichia coli, Staphylococcus aureus and / or Pseudomonas aeruginosa in aquaculture environments, providing a good phage source for industrial production of phage for the prevention and treatment of Salmonella, Escherichia coli, Staphylococcus aureus and / or Pseudomonas aeruginosa in aquaculture environments;

[0019] (2) The titer of the bacteriophage RDP-EC-20146 in the present invention remains at 10 in the pH range of 5-9. 12 pfu / mL or above, it can be seen that bacteriophage RDP-EC-20146 can tolerate weak acid and weak base conditions and has good acid-base tolerance;

[0020] (3) The phage obtained by the present invention is treated at 30-50°C for 30-60 minutes, and the titer is still 10 12 pfu / mL or above, indicating that bacteriophage RDP-EC-20146 has good temperature tolerance. This characteristic is of great significance in the production, transportation and storage of bacteriophages.

[0021] (4) The bacteriophage RDP-EC-20146 provided by the present invention can exhibit significant antibacterial effects when used in combination with low doses of eight antibiotics, and has good application prospects in preventing and treating Salmonella, Escherichia coli, Staphylococcus aureus and / or Pseudomonas aeruginosa diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 Transmission electron microscopy image of the cross-species lytic phage RDP-EC-20146 provided by the present invention;

[0024] Figure 2 This is a graph showing the pH stability of the cross-species lytic phage RDP-EC-20146 provided by the present invention;

[0025] Figure 3 This is a graph showing the results of the thermal stability test of the cross-species lytic phage RDP-EC-20146 provided by the present invention;

[0026] Figure 4 These are the test results of the synergistic antibacterial effect of the cross-species lytic phage RDP-EC-20146 and antibiotics provided by the present invention. DETAILED DESCRIPTION

[0027] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0028] Example 1 Isolation and Identification of Pathogenic Escherichia coli

[0029] Sampling was done from the diseased farms, and the livers of diseased birds were aseptically removed and streaked onto a selective culture medium (i.e., MacConkey solid medium). After incubation at 37°C for 18-24 hours, round, flat, red colonies with neat edges, smooth and moist surfaces were formed on the culture medium. Typical colonies were picked and streaked and purified three times. Then, a single colony was picked and inoculated into 5 mL of LB broth and cultured at 37°C with shaking at 200 rpm for 6 hours to obtain a uniform turbid bacterial suspension. 16sRNA molecular identification and serotype identification were then used to identify the pathogenic Escherichia coli, named BEC-20261, and stored in a -80°C refrigerator.

[0030] Example 2 Isolation and identification of cross-species lytic phage RDP-EC-20146

[0031] (1) Feces treatment: Weigh 5 g of feces collected from the farm, add it to 10 mL of sterile water and soak it overnight. Centrifuge the leaching liquid at 10,000 rpm for 5 min, and then filter the supernatant with a 0.22 μm filter. Collect the filtrate for later use.

[0032] (2) Preparation of mixed bacterial suspension: 1 mL of bacterial suspension and 2 mL of filtrate were added to 27 mL of LB broth, cultured overnight at 37°C and 200 rpm, and then centrifuged at 10,000 rpm for 5 min. The supernatant was filtered through a 0.22 μm filter and the filtrate of the mixed bacterial suspension was collected for later use.

[0033] (3) Phage isolation: Phage isolation was performed using the double plate method. 0.1 mL of the mixed bacterial suspension filtrate and 0.1 mL of the Escherichia coli suspension were mixed evenly, incubated at 37°C for 10 minutes, and then spread on two plates. The plates were then placed in a 37°C incubator for 6-8 hours and observed. If phage was present, transparent spots would appear on the plates. The transparent spots were picked and placed in 1 mL of physiological saline in a 37°C water bath for 30 minutes. 0.1 mL of the leaching solution and 0.1 mL of the bacterial suspension were then taken and incubated at 37°C for 10 minutes. The plates were then spread on two plates and incubated at 37°C for 4-6 hours for purification. This procedure was repeated 2-3 times until the plaques were uniform in size. A phage strain was obtained and named RDP-EC-20146. The plaques formed by RDP-EC-20146 on the double plates were 2 mm to 3 mm in diameter.

[0034] Example 3 Transmission electron microscopic morphological observation of cross-species lytic phage RDP-EC-20146

[0035] Take 20 μL of liquid containing crude phage particles and drop it on a copper mesh. Let it precipitate naturally for 15 minutes, and use filter paper to absorb 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, observe the phage morphology using an electron microscope.

[0036] Depend on Figure 1The phage 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 sheath. The neck connects the head and tail. The phage is classified as belonging to the family Myoviridae, order Caudovirales. Its genome is 165,912 bp long. Full genome sequencing revealed the absence of virulence and lysogenic genes, demonstrating the phage's safety profile at the genetic level. The phage contains genes for multiple lytic enzymes, which are expressed in the cleavage products and can further lyse the associated Escherichia coli during use.

[0037] The phage titer was calculated based on the number of plaques, and the titer of the cross-species lytic phage RDP-EC-20146 was 1.26×10 12 pfu / mL.

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

[0039] Dilute hydrochloric acid and dilute NaOH solution to adjust the pH of physiological saline to prepare buffer solutions with pH values of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. Then use the prepared buffer solution to dilute the phage to 1*101 2 The dilution was placed in a water bath at 37°C for 1 hour, diluted 10-fold with normal saline and plated on two plates for titer determination. The results were counted after inverted incubation at 37°C for 4-6 hours.

[0040] Depend on Figure 2 It can be seen that under the condition of pH 5-9, after incubation at 37℃ for 1 hour, the titer only decreased by one order of magnitude and remained at 10 12 pfu / mL or higher, indicating 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 acid and alkali, which ensures that sufficient numbers of phages reach the intestines to exert their bactericidal effect.

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

[0042] The phage stock solution was divided into 50 ml sterilized centrifuge tubes and incubated at 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 80℃ and 90℃ for 30min and 60min respectively. Then, the phage stock solution was diluted 10-fold with normal saline and double-plated to determine its titer. Figure 3It can be seen that the phage titer gradually decreased with the increase of temperature and the extension of time. When treated at 50℃ for 30min and 60min, the phage titer only decreased by one order of magnitude and the titer was still around 10 12 pfu / mL, RDP-EC-20146 has good temperature tolerance. This characteristic is very important in the production, transportation and storage of bacteriophages.

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

[0044] The lysis spectrum experiment of the bacteriophage RDP-EC-20146 was carried out on pathogenic bacteria Salmonella, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa isolated from animal sources. The experiment found that the phage has cross-species lysis ability and has lysis properties against Salmonella, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. Lysis spectrum experiments were conducted on 17 strains of Salmonella, 15 strains of Escherichia coli, 11 strains of Staphylococcus aureus, and 17 strains of Pseudomonas aeruginosa. The results showed that the phage could lyse Salmonella, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, with lysis rates of 100% (17 / 17), 86.67% (13 / 15), 81.82% (9 / 11), and 88.24% (15 / 17), respectively. The phage showed a relatively broad 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 cleavable; “-” represents non-cleavable.

[0049] Example 7: Bacteriophage RDP-EC-20146-antibiotic combined antibacterial test

[0050] Experimental methods:

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

[0052] Minimum inhibitory concentration determination: Take two 96-well plates, add 100uL of sterile LB medium to the 96-well plates, add 100uL of the prepared drug solution to the first column of wells, dilute in series to the 11th column, discard, and the last column is the negative control. Add 100uL of bacterial suspension (activity 1×10 6cfu / mL), and 100uL of bacterial suspension (activity 1×10 6 cfu / mL) and phage (titer 1×10 8 pfu / mL), cultured in a 37°C constant temperature incubator overnight, and the results were recorded after 12-16 hours.

[0053] Depend on Figure 4 It can be seen that the bacteriophage RDP-EC-20146 has good synergistic effects with the eight antibiotics, and can reduce the MIC (minimum drug sensitivity concentration) to 1 / 32-1 / 4 of the original. This shows that the cross-species lytic phage RDP-EC-20146 has a good synergistic effect with the eight antibiotics on Escherichia coli, Salmonella, Pseudomonas aeruginosa and Staphylococcus aureus.

[0054] Test example: 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] 14-day-old white-feathered broilers were selected and challenged with 100 μml of toxic substance in the chest. The activity of E. coli was 1×10 8 cfu / mL. Following challenge, the experiment was conducted using the table below. Mortality was recorded and the condition of the flock was observed. After 7 days of treatment, internal organs were examined and symptoms recorded. The results are as follows.

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

[0059] Table 2

[0060] Serial number Fighting the virus treat Remark 1 1e6 cfu / unit / Positive 2 Normal saline / Negative 3 1e7 cfu / unit 0.256% lincomycin 0.1ml Antibiotic treatment 4 1e7 cfu / unit 0.1mL phage Phage therapy 5 1e7 cfu / unit 0.256% lincomycin 0.1ml + 0.1mL phage Antibiotics + phages 6 1e7 fcfu / unit 0.128% lincomycin 0.1ml + 0.1mL bacteriophage 1 / 2 antibiotics + bacteriophage 7 1e7 cfu / unit 0.064% lincomycin 0.1ml + 0.1mL bacteriophage 1 / 4 antibiotics + bacteriophage 8 1e7 cfu / unit 0.032% lincomycin 0.1ml + 0.1mL bacteriophage 1 / 8 antibiotics + bacteriophage 9 1e7 cfu / unit 0.016% lincomycin 0.1ml + 0.1mL bacteriophage 1 / 16 antibiotics + bacteriophage 10 1e7 cfu / unit 0.008% lincomycin 0.1ml + 0.1mL bacteriophage 1 / 32 antibiotics + bacteriophage

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

[0062] Table 3

[0063] Serial number Remark mortality rate(%) Autopsy symptoms 1 Positive 50.00 Severe pericarditis and air sacculitis 2 Negative 0.00 none 3 Antibiotic treatment 10.00 Mild pericarditis and air sacculitis 4 Phage therapy 15.00 Mild pericarditis and air sacculitis 5 Antibiotics + phages 0.00 none 6 1 / 2 antibiotics + bacteriophage 5.00 none 7 1 / 4 antibiotics + bacteriophage 5.00 none 8 1 / 8 antibiotics + bacteriophage 5.00 none 9 1 / 16 antibiotics + bacteriophage 10.00 Mild pericarditis and air sacculitis 10 1 / 32 antibiotics + bacteriophage 20.00 Severe pericarditis and air sacculitis

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

[0065] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of the present invention.

Claims

1. A cross-species lytic phage RDP-EC-20146, characterized by: This cross-species lytic phage is an Escherichia coli phage, with the deposit number CGMCC No. 45351. It was deposited in the General Microbiology Center of the China Culture Collection Administration on October 28, 2022.

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

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

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

5. The use of the cross-species lytic phage RDP-EC-20146 according to any one of claims 1 or 2, characterized in that: The cross-species lytic phage RDP-EC-20146 is used to prepare a feed additive for resisting Salmonella, Escherichia coli, Staphylococcus aureus and / or Pseudomonas aeruginosa.

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

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

8. 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 phage RDP-EC-20146 according to any one of claims 1 or 2.

9. A feed additive for preparing an anti-Salmonella, Escherichia coli, Staphylococcus aureus and / or Pseudomonas aeruginosa agent, characterized in that: The feed additive contains the cross-species lytic phage RDP-EC-20146 according to any one of claims 1 or 2.

10. A feed prepared for resistance to Salmonella, Escherichia coli, Staphylococcus aureus and / or Pseudomonas aeruginosa, characterized in that: The feed contains the cross-species lytic phage RDP-EC-20146 according to any one of claims 1 or 2.

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