Broad-spectrum salmonella enteritidis bacteriophage and application thereof

Abstract

The invention discloses a salmonella enteritidis bacteriophage, which is named as salmonella bacteriophage PAC / QD23120510, and the preservation number is CGMCC No.45998. The form of the bacteriophage is as follows: the head part is of a 24-face structure, the length of the head part is 750-800nm, and the length of the tail part is 300-350nm; the infection complex number is 0.001 to 100; the strain has good survival characteristics at 60 DEG C or below, and shows high temperature resistance. In the bacteriophage disclosed by the invention, the genome of the bacteriophage does not carry virulence genes, drug-resistant genes and fusion source genes, so that the transmission of the virulence genes and the drug-resistant genes is not caused, and the use is safe. The salmonella enteritidis bacteriophage PAC / QD23120510 is strong in infection ability, can quickly kill host bacteria in a culture medium, is wide in temperature and acid-base tolerance range, can be prepared into a spraying agent, has good inhibition and killing effects on proliferation of salmonella enteritidis, and has good treatment and protection effects on chicken breast infected with salmonella enteritidis; the bacteriophage has wide spectrum, can kill 98.77% of salmonella enteritidis, and has obvious treatment effect advantages.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a broad-spectrum Salmonella enteritidis phage and application thereof. Background Art

[0002] Since Fleming discovered penicillin, the widespread use of antibiotics has achieved great success in the fields of medicine, agriculture, animal husbandry and aquaculture. However, the rapid development of bacterial resistance, especially the resistance of common pathogens such as Salmonella, has brought serious challenges to human health. In this context, the development of new food additives, especially bacteriophages, is particularly urgent and important.

[0003] As a new type of biological antibacterial agent, bacteriophage has great potential and prospects. First, its lysis spectrum is wide and can act on a variety of pathogens including Salmonella, which provides the possibility of treating a variety of bacterial infections. Secondly, the lysis rate of bacteriophage is fast, which can quickly kill bacteria in a short time and reduce the risk of infection. In addition, compared with antibiotics, bacteriophage has lower side effects and less risk of drug resistance, which makes it an ideal alternative product. The study of bacteria will also be combined with fields such as immunology and microecology to provide new ideas and methods for the treatment and prevention of bacterial infections. For example, by combining bacteriophages with immunotherapy, more efficient and safe vaccines and immune preparations can be developed to reduce the risk of bacterial infections.

[0004] In short, as a new type of food additive product, bacteriophage has great potential and prospects. With the deepening of research and the advancement of technology, I believe we will be able to develop more efficient and broad-spectrum bacteriophages that are beneficial to food preservation and antiseptic treatment, provide strong support for the treatment and prevention of bacterial infections, and make greater contributions to human health. Summary of the invention

[0005] The present invention aims to solve the above problems. In a first aspect, a Salmonella enteritidis phage is provided, which is named as Salmonella phage PAC / QD23120510 and has a deposit number of CGMCC No.45998.

[0006] Salmonella phage PAC / QD23120510 is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit date of July 17, 2024 and the deposit code of CGMCCNo.45998.

[0007] Furthermore, the morphology of the Salmonella phage PAC / QD23120510 is as follows: the head is a 24-hedron structure, the head length is 750-800nm, and the tail length is 300-350nm.

[0008] Furthermore, the multiplicity of infection of the Salmonella phage PAC / QD23120510 is 0.001-100.

[0009] Furthermore, the Salmonella phage PAC / QD23120510 has good survival characteristics at 60°C and below, showing high temperature resistance.

[0010] In a second aspect, the present invention provides a Salmonella phage composition as described in the first aspect, wherein the composition contains Salmonella phage PAC / QD23120510.

[0011] In a third aspect, the present invention provides the use of the Salmonella phage PAC / QD23120510 as described in the first aspect in a biological preparation. The pH value of the biological preparation is 4-11. Furthermore, the biological preparation may also include one or more auxiliary materials selected from stabilizers, dispersants, solvents and fillers.

[0012] In a fourth aspect, the present invention provides use of the Salmonella phage PAC / QD23120510 as described in the first aspect in environmental disinfection.

[0013] In a fifth aspect, the present invention provides use of the Salmonella phage composition as described in the second aspect in a biological preparation and / or a kit.

[0014] The present invention has the following advantages / benefits:

[0015] Compared with the prior art, the present invention has at least the following advantages and positive effects:

[0016] (1) Among the bacteriophages of the present invention, the genome of the Salmonella phage PAC / QD23120510 does not carry virulence genes, drug-resistant genes, and fusion genes, and will not cause the spread of virulence genes and drug-resistant genes, and is safe to use.

[0017] (2) The Enteritidis Salmonella phage PAC / QD23120510 has a strong infection ability and can quickly kill the host bacteria in the culture medium. It has a wide range of temperature and acid-base tolerance and can be made into a spray. It has a good inhibitory and killing effect on the proliferation of Enteritidis Salmonella and has a good therapeutic and protective effect on chicken breast infected with Enteritidis.

[0018] (3) The Salmonella Enteritidis phage PAC / QD23120510 has a broad spectrum. As shown in Table 1, it can kill 98.77% of Salmonella Enteritidis. The therapeutic effect of this phage is obviously superior. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0020] Figure 1 : Schematic diagram of plaque morphology and electron microscopic morphology of Salmonella phage PAC / QD23120510 of the present invention, wherein A is the plaque morphology and B is the electron microscopic image of the phage;

[0021] Figure 2 : A schematic diagram of the genome of the Salmonella phage PAC / QD23120510 of the present invention;

[0022] Figure 3 : Schematic diagram of the optimal multiplicity of infection of the Salmonella phage PAC / QD23120510 of the present invention;

[0023] Figure 4 : Schematic diagram of the thermal stability of the Salmonella phage PAC / QD23120510 of the present invention;

[0024] Figure 5 : Schematic diagram of pH stability of Salmonella phage PAC / QD23120510 of the present invention;

[0025] Figure 6 : Schematic diagram of the bactericidal effect of the Salmonella phage PAC / QD23120510 of the present invention on the surface of Salmonella Enteritidis of chicken breast at 4°C, wherein A is MOI=100, B is MOI=1000, and C is MOI=10000;

[0026] Figure 7 : Schematic diagram of the bactericidal effect of the Salmonella phage PAC / QD23120510 of the present invention on Enteritidis Salmonella on the surface of chicken breast at 25°C, wherein A is MOI=100, B is MOI=1000, and C is MOI=10000. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. The following embodiments are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. The experimental methods used in the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the present invention, unless otherwise specified, can be obtained from commercial channels. In addition, other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0028] Example 1: Propagation of Salmonella Enteritidis phage PAC / QD23120510

[0029] Salmonella phage PAC / QD23120510 was isolated from the wastewater of a farm in Qingdao.

[0030] The obtained sewage samples were added to the bubble sample bottle, and 500 μL of bacterial solution was added to each strain of bacteria, and then an appropriate amount of NB liquid culture medium was added, and the samples were soaked and incubated overnight at 37°C. For the samples incubated overnight, 8 mL was extracted from each sample using a sterile syringe and dispensed into a 10 mL centrifuge tube. After centrifugation at 11000 rpm for 10 minutes, the supernatant was aspirated with a new sterile syringe, filtered using a 0.22 μm filter, and the filtrate was transferred to a new sterile 10 mL centrifuge tube. 100 μL of the selected strains were added to the NB upper culture medium, and the test tubes were quickly rubbed to mix, and then poured into the culture dish containing the specific lower culture medium, and the filtrate was diluted 10 times to the appropriate gradient. 1.5 μL of the filtrate and diluent of each sample were aspirated and dripped onto the surface of the culture medium according to the pre-marked position. After the filtrate was absorbed, the culture dish was inverted and cultured in a 37°C incubator overnight to culture transparent plaques. Use sterile tweezers to pick out a single plaque together with the attached upper culture medium, put it into a centrifuge tube containing 1 mL of specific culture medium, and crush the attached culture medium with tweezers; place the centrifuge tube in a shaker at 37°C and 170 rpm for at least 30 minutes for later use.

[0031] Dilute the phage extract 10 times to a suitable gradient, take 120 μL of phage extract and 120 μL of bacterial solution and mix them evenly, incubate at 37°C for 5 minutes, place in a specific upper culture medium (agar concentration is 0.7%) at about 60°C, mix well and quickly pour into a culture dish containing a specific lower culture medium (agar concentration is 1.5%), shake well, place flat until the culture medium solidifies, place at 37°C and invert for an appropriate time, and obtain a double-layer plate with transparent plaques again. Until the plaques are consistent in shape, size, and transparency.

[0032] Take out the Enteritidis Salmonella CICC21482 stored at -80°C, provided by Qingdao Noan Biotech Co., Ltd., which is a standard strain of Enteritidis Salmonella. After streaking on the NB non-antibiotic plate at 37°C for 10 hours, pick a single colony and culture it in 10mL NB culture medium at 37°C and 220rpm overnight; after determining the concentration of the bacterial solution, take the overnight bacterial solution and add the PAC / QD23120510 phage solution; in this embodiment, the ratio of phage to host bacteria is 0.01:1, and the solution is cultured at 37°C and 220rpm for 4 hours until the bacterial solution becomes clear, and the phage proliferation solution is obtained after sterilization by filtering with a 0.22μm filter membrane.

[0033] Example 2: Morphological observation of bacteriophage

[0034] Phages were observed by TEM using the phosphotungstic acid (PTA) negative staining method. The phage proliferation solution was dropped onto the Formvar carbon support film, left to dry for 10 minutes, and excess liquid was removed with paper. 10 μL of PTA was added to the film for staining for 10 minutes, and deionized water was added to wash away excess PTA, excess liquid was removed with paper, and the sample was dried again for 10 minutes.

[0035] The morphology of the phage was observed under TEM. Figure 1 As shown in the electron microscope image, the morphology of the Salmonella phage PAC / QD23120510 is: the head is a 24-hedron structure, the head length is 750-800nm, and the tail length is 300-350nm. It belongs to the muscle-tail phage. According to the latest classification and naming of phages, the whole genome sequence analysis was compared in the NCBI Uniprot database, and the phage PAC / QD23120510 belongs to the Caudoviricetes bacteriophage family.

[0036] Example 3: Phage genome extraction and sequencing

[0037] Take 1mL of phage proliferation liquid, add DNase I (PROMEGA) with a final concentration of 6U / mL and RNase (TAKARA) with a final concentration of 50μg / mL to remove exogenous nucleic acids, and react at 37℃ for 1h. Add 20μL 2M ZnCl2, mix well by inversion, react at 37℃ for 5min, centrifuge at 10000g for 1min, and discard the supernatant. Extract with TIANGEN virus genome extraction kit, and finally dissolve nucleic acids with 20μL sterile water, and send to Beijing Liuhe BGI Gene Technology Co., Ltd. for whole genome sequencing. The results are as follows Figure 2 The full length of the genome of bacteriophage PAC / QD23120510 is 83275 bp, and it does not carry drug resistance genes, virulence genes, or lysogeny genes.

[0038] Example 4: Determination of optimal infection multiplicity of phage

[0039] 1 mL (1×10 9 pfu / mL) was diluted 10-fold with NB broth, and 0.1 mL of each dilution and 0.1 mL of host bacterial proliferation solution (10 6 pfu / mL) was added to 5 mL of NB broth and cultured at 37°C and 200 rpm until the liquid was clear. The culture was centrifuged at 10,000 rpm for 5 min and the titer was measured using the double-layer plate method. The combination with the highest titer was the optimal multiplicity of infection. Figure 3 shown.

[0040] Depend on Figure 3 It can be seen that phage PAC / QD23120510 has a strong lysis ability from MOI 100 to 0.001.

[0041] Example 5: Determination of bacteriophage thermal stability

[0042] Take several sterile 2mL EP tubes and add 500μL (1.21×10 11 PFU / mL), incubated at 4°C, 25°C, 40°C, 50°C, 60°C, 70°C and 80°C for 30 min, and the phage titer after incubation at different temperatures was determined using the double-layer agar method. Figure 4 shown.

[0043] Depend on Figure 4 It can be seen that the Salmonella phage PAC / QD23120510 has good survival characteristics at 60°C and below, showing high temperature resistance.

[0044] Example 6: Determination of pH stability of bacteriophage

[0045] The pH of the NB liquid medium was adjusted with HCl and NaOH to obtain a NB solution with a pH of 2-12, which was then filtered with a 0.22 μm microporous filter membrane. 100 μL of phage was mixed with 900 μL of NB liquid medium with different pH values, respectively, and incubated in a 37°C water bath for 2 h. The titer of phage at different pH values ​​was determined using the double-layer agar method. The results are shown in Table 1. Figure 5 shown.

[0046] Depend on Figure 5 It can be seen that bacteriophage PAC / QD23120510 still maintains high titer when acting in the pH range of 3-11 for 2 hours, indicating that bacteriophage PAC / QD23120510 has a wide acid-base tolerance range.

[0047] Example 7: Determination of phage host spectrum

[0048] A total of 81 strains of clinical enteritis Salmonella from Qingdao Noan Biotech Co., Ltd. were tested. A small amount of liquid specific lower agar medium at about 60°C was poured into a culture dish (covering the bottom of the culture dish) and waited for solidification; the name of the strain to be tested was marked on the bottom of the culture dish, 0.2mL of the corresponding bacterial solution was added to a specific upper culture medium at about 50°C, mixed and quickly poured into a specific lower culture medium culture dish, and a double-layer agar culture dish with bacteria was obtained after solidification. When the double-layer agar culture dish is completely solidified and there is basically no water vapor in the plate, 0.15mL of phage liquid is placed in the corresponding square. After the filtrate is absorbed, it is inverted and cultured at 37°C for 8h. The results are shown in Table 1. After the host spectrum was determined, it was found that phage PAC / QD23120510 can form transparent plaques for 98.77% of clinical enteritis Salmonella, indicating that phage PAC / QD23120510 has a good broad spectrum.

[0049] Table 1

[0050]

[0051]

[0052]

[0053]

[0054] Note: "C, B, N" are used to evaluate the degree of phage lysis of bacteria. "C" means that the plaque is completely clear, "B" means that the plaque is fuzzy, and "N" means that the phage has no lysis effect on bacteria.

[0055] Example 8: Phage modeling test on chicken breast surface

[0056] At 4℃ and room temperature (25℃), 1×10 5 CICC21482 with a concentration of CFU / ml of bacterial solution was colonized in 1cm 2 The surface of chicken breast meat was exposed to the sun for 20 minutes (this time was recorded as 0h), and the MOI was adjusted to 100, 1000, and 10000, respectively. Samples were taken at 0h, 2h, 4h, 6h, and 8h. The samples were fully mixed with 5mL of saline and the concentration of Salmonella was determined. The experimental results are shown in Figure 6 and Figure 7 As shown, Figure 6 As shown in C, the best bactericidal effect against CICC21482 was achieved at 4°C and MOI = 10000.

[0057] Example 9: Phage-related applications

[0058] Application of Salmonella phage PAC / QD23120510 in biological preparations.

[0059] Optionally, the pH value of the phage preparation is 4-11.

[0060] Optionally, the phage preparation further comprises the above acceptable excipients, wherein the above acceptable excipients comprise one or more of a stabilizer, a dispersant, a solvent and a filler.

[0061] The bacteriophage preparation of the present invention can be administered via external route.

[0062] Optionally, the bacteriophage preparation includes a composition preparation or an environmental disinfectant, wherein the bacteriophage composition preparation is in the form of a liquid preparation, a freeze-dried preparation or a solid preparation, and is used to improve the environment and reduce the content of Salmonella enteritidis on the surface of the medium by spraying.

[0063] It should be noted that the phage preparation provided by the present invention can use the above phage as the only active ingredient of the composition. It can also contain other phages in addition to the phage of the present invention, which can be used in combination with the phage of the present invention to obtain an effect of preventing and treating Salmonella enteritidis that is equivalent to or better than that of the phage of the present invention.

[0064] It should be noted that in the environmental disinfectant provided by the present invention, the concentration of each bacteriophage is not less than 10 3 pfu / mL, and the environmental disinfectant also contains other active ingredients for inhibiting or eliminating Salmonella Enteritidis in the environment.

[0065] The present invention also provides the use of the environmental disinfectant in the disinfection of supermarket cold counters, farm environments and object surfaces. The application method of the environmental disinfectant includes but is not limited to the disinfection and decontamination of water distribution systems, medical facilities, aquaculture facilities, public and private facilities or other environmental surfaces in the form of liquid immersion, spraying, and combined use with aqueous carriers, which can effectively control the activity and growth of Salmonella enteritidis.

[0066] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A Salmonella Enteritidis phage, characterized in that: It was named Salmonella phage PAC / QD23120510, and its deposit number was CGMCC No.45998.

2. The Salmonella Enteritidis phage according to claim 1, characterized in that: The morphology of the Salmonella phage PAC / QD23120510 is as follows: the head is a 24-hedron structure, the head length is 750-800nm, and the tail length is 300-350nm.

3. The Salmonella Enteritidis phage according to claim 1, characterized in that: The infection multiplicity of the Salmonella phage PAC / QD23120510 is 0.001-100.

4. The Salmonella Enteritidis phage according to claim 1, characterized in that: The Salmonella phage PAC / QD23120510 has good survival characteristics at 60°C and below, showing high temperature resistance.

5. A Salmonella phage composition comprising any one of claims 1 to 4, characterized in that: The composition contains Salmonella phage PAC / QD23120510.

6. Use of the Salmonella phage according to any one of claims 1 to 4 in biological preparations.

7. Use of the Salmonella phage according to any one of claims 1 to 4 in environmental disinfection.

8. Use of the Salmonella phage composition according to claim 5 in biological preparations and / or kits.

Images