CN118406662A - Salmonella phage vB_ SalP _LDW8 and application thereof - Google Patents

Abstract

The present invention belongs to the field of biotechnology, and in particular, relates to a Salmonella phage vB_SalP_LDW8 and its application. The technical solution adopted by the present invention is: a Salmonella phage, the deposit number is: CGMCC No.45253, the phage is named: vB_SalP_LDW8, the nucleotide sequence is as SEQ ID NO.1~SEQ ID NO.4, and the phage has a broad spectrum and high lysis power against Salmonella. The preparation made by the Salmonella phage of the present invention can effectively eliminate most of the Salmonella currently infected in poultry farms.

Description

A Salmonella phage vB_SalP_LDW8 and its application

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to a Salmonella phage vB_SalP_LDW8 and an application thereof.

Background technique

Salmonella is one of the main pathogens that causes disease in chickens in modern large-scale farms. Salmonellosis caused by it is common in farms of different sizes in various places, causing great trouble to the healthy development of the breeding industry and the sanitation and safety of animal-derived food. At present, the treatment of Salmonellosis in chickens is mainly antibiotics. The unscientific use of antibacterial drugs has brought a series of problems. Therefore, it is urgent to find efficient, safe and residue-free bactericidal preparations.

Bacteriophage is the general term for a class of viruses with the widest distribution range, the largest number and the largest variety in nature. According to Brussow's estimate, the number of bacteriophages on the earth is as high as 10 ^31. It can be said that bacteriophages are everywhere. At present, researchers have isolated many different Salmonella phages from a variety of samples, and have named them and studied their phage spectra. According to the interaction and life cycle between phages and host bacteria, phages can be divided into toxic phages and temperate phages. Toxic phages can rely on host bacteria to replicate and lyse the host bacteria, thereby releasing progeny phages and causing the host bacteria to die quickly; temperate phages can inject their own genomes into host bacteria, and during the proliferation of host bacteria, they can simultaneously replicate the phage genome and pass it to the progeny host bacteria, and only when the environment is stimulated can the host bacteria be lysed and die. Therefore, we generally use toxic, also known as virulent or lytic phages to prevent and treat bacterial diseases.

Summary of the invention

The purpose of the present invention is to provide a Salmonella phage which can effectively kill most of the Salmonella that infect poultry in farms.

Another object of the present invention is to provide an effective prevention and control means, which uses the Salmonella phage to prepare a liquid, freeze-dried powder or tablet form for killing Salmonella in poultry bodies, on the body surface, in feed, breeding tools or breeding environments.

In order to achieve the above technical objectives, the technical solution adopted by the present invention is: a Salmonella phage, the preservation number is: CGMCC No.45253, the preservation time is August 3, 2022, and the preservation location is the General Microbiology Center of China Culture Collection Administration (No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing). The phage is named: vB_SalP_LDW8, and its nucleotide sequence is shown in SEQ ID NO.1~SEQ ID NO.4, which has a broad spectrum and high lysis power against Salmonella.

Furthermore, the phage has a wide host spectrum and the plaque morphology is transparent. Through transmission electron microscopy , vB_SalP_LDW8 phage has a clear icosahedral head with a diameter of 56.4±3 nm and a thin and long cylindrical tail with a diameter of 10±3 nm and a length of 96.9±3 nm. The tail also has obvious fibrous structures, which can be judged that the phage belongs to the order Caudovirales and the family Longicaudophage.

A Salmonella phage preparation, comprising the above-mentioned Salmonella phage.

Furthermore, the Salmonella phage preparation is used in killing Salmonella.

Furthermore, the Salmonella phage preparation is used to kill Salmonella that infects poultry in farms.

Furthermore, the preparation is a liquid, a lyophilized powder, or a solid preparation.

The beneficial effects of the present invention are:

A Salmonella phage is provided to solve the problems of Salmonella infection in poultry in farms, rapid onset and untimely treatment, and ineffective medication due to drug resistance of pathogenic bacteria. The preparation made by the broad-spectrum and strong lytic Salmonella phage of the present invention can effectively eliminate most of the Salmonella infection in poultry in farms.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Comparison of lysis diagrams of bacteriophages against host bacteria

A and B show turbid plaques formed by the remaining phages, and C shows the lysis diagram of the WvB_SalP_LDW8 host bacteria;

FIG. 2 is an electron microscope image of the bacteriophage vB_SalP_LDW8 of the present invention

A is a schematic diagram of the morphology of a single bacteriophage of the bacteriophage vB_SalP_LDW8 of the present invention, and B is a schematic diagram of the morphology of multiple bacteriophages of the bacteriophage vB_SalP_LDW8 of the present invention;

FIG3A is a schematic diagram of the optimal infection multiplicity of bacteriophage vB_SalP_LDW8 of the present invention;

FIG3B is a schematic diagram of the growth curve of bacteriophage vB_SalP_LDW8 of the present invention;

FIG3C is a schematic diagram of the thermal stability of bacteriophage vB_SalP_LDW8 of the present invention;

FIG3D is a schematic diagram of the acid-base stability of bacteriophage vB_SalP_LDW8 of the present invention;

FIG. 4 is a nucleic acid sequence alignment diagram of the genome of bacteriophage vB_SalP_LDW8 of the present invention in the NCBI database.

Detailed ways

Example 1 Isolation and identification of pathogenic avian Salmonella

Sampling was carried out in the diseased farm. Liver samples were taken under sterile conditions and inoculated into BPW, and cultured at 37℃ constant temperature shaker at 180 r/min for 12 h for pre-enrichment. The pre-enrichment solution was inoculated into TTB, and cultured at 37℃ constant temperature shaker at 180 r/min for 12 h for selective enrichment of Salmonella. Then, streaks were made on Salmonella chromogenic culture medium and cultured in a 37℃ constant temperature incubator for 24 h. The colony morphology and growth were observed, and a single suspected colony was picked and streaked on MacConkey medium and LB solid culture medium. This was repeated 3 to 5 times for purification culture. The purified Salmonella was identified by PCR. The Salmonella that amplified invA (603 bp) and stn gene (260 bp) was Salmonella; the Salmonella that amplified sdf gene (203 bp) was Salmonella Enteritidis; the Salmonella Typhimurium that amplified STM 4497 gene (523 bp) was Salmonella Pullorum; the Salmonella Pullorum that amplified P1 gene (417 bp) was Salmonella Pullorum; the Salmonella that amplified both P1 (417 bp) and P2 (636 bp) genes was Salmonella Fowl Typhi. The isolated bacteria were named according to different serotypes and stored at -80℃ for future use.

Example 2 Isolation and identification of bacteriophage vB_SalP_LDW8

Separation: Take appropriate amount of sewage, bedding, etc. and put them into 5 mL LB liquid culture medium, culture them in a constant temperature shaker at 37℃ for 4h, centrifuge at 10000 r/min for 5 min, filter the supernatant with a 0.22 µm filter membrane to sterilize, and obtain the phage stock solution. Use the double-layer plate drop method to separate phages. Take 200 µL of laboratory-preserved Salmonella and 5 mL of LB semi-solid culture medium cooled to about 55℃ and mix them in a centrifuge tube, pour them on the LB solid culture medium, and let them stand until they solidify. Divide the back of the prepared double-layer plate into 8 equal areas, add 5 µL of a phage stock solution to each area, culture at 37℃ for 6 h, and observe whether plaques appear.

Purification: Use a sterile pipette tip to remove a single plaque, place it in SM buffer and let it stand for 12 hours, filter it with a 0.22 µm filter membrane, take the filtrate and prepare a double-layer plate with the host bacteria again, repeat the removal of a single plaque for purification 4 to 6 times to obtain the purified phage.

Results: A total of 22 phage strains were isolated from the clinic, among which phage vB_SalP_LDW8 had strong lytic activity and formed transparent phage plaques (see Figure 1C), while most of the other phages formed turbid plaques (see Figure 1A-B).

Example 3 Host spectrum determination of bacteriophage vB_SalP_LDW8

Take 200 µL of different serotypes of Salmonella stored in the laboratory and mix them with 5 mL of LB semi-solid medium cooled to about 55°C in a centrifuge tube, pour it on the LB solid medium, and let it stand until it solidifies. Divide the back of the prepared double-layer plate into 8 equal areas, add 5 µL of a purified phage stock solution to each area, and culture at 37°C for 6 hours to observe whether plaques appear.

The host spectrum was determined by cross-infection experiments between the 80 isolated Salmonella strains and the 22 Salmonella phages isolated and preserved in this laboratory. The host spectrum test results are shown in Table 1. Among the 22 phages isolated in this experiment, the phage with the highest coverage rate for the 80 Salmonella strains was vB_SalP_LDW8, with a coverage rate of 81.3%. The coverage rate of 18 phages (accounting for 63%) was above 60%.

Table 1 Information and host spectrum of isolated phages

Example 4 Determination of the titer of bacteriophage vB_SalP_LDW8

The phage titer was determined by the double-layer plate method. The purified phage filtrate was diluted 10 times in a gradient, 200 µL of phage dilution was mixed with 200 µL of host bacterial solution, incubated at 37°C for 5 min, 4-5 mL of LB semi-solid medium at about 55°C was poured into the mixture of phage dilution and host bacterial solution, and then immediately poured into a plate with LB solid medium at the bottom to prepare a double-layer plate, incubated at 37°C for 6 h, and the plaques were counted. Phage titer (PFU/mL) = number of plaques × 5 × dilution factor.

The titer of bacteriophage vB_SalP_LDW8 was measured by double-layer plate method and was 1.2×10 ¹⁰ PFU/mL.

Example 5 Morphological observation of bacteriophage vB_SalP_LDW8

The high-titer phage lysis solution was prepared by plate amplification method. 400 µL of host bacterial solution grown to the logarithmic phase was taken and mixed with the phage lysis solution at the optimal infection multiplicity ratio to prepare a double-layer plate, which was cultured at 37°C for 12 h. 10 mL of SM buffer was added to the culture dish, and the shaker was shaken at 100 r/min for 4 h. The eluate was collected, centrifuged at 12000 r/min for 5 min, and filtered with a 0.22 µm filter membrane to obtain the high-titer phage lysis solution for subsequent microscopic observation. The phage lysate was negatively stained with 2% phosphotungstic acid (w/v, pH7.0), observed using a transmission electron microscope, and micrographs were taken at an accelerating voltage of 80 kV to observe the morphology of the phage, and the head and tail length of the phage was measured using ImageJ software.

The vB_SalP_LDW8 phage lysate with a titer of 2×10 ¹² PFU/mL was obtained by plate amplification method. Through transmission electron microscopy, the vB_SalP_LDW 8 phage has a clear icosahedral head with a diameter of 56.4±3 nm and a thin and long cylindrical tail with a diameter of 10±3 nm and a length of 96.9±3 nm. The tail also has obvious fibrous structures (see Figure 2). According to the ninth report on virus classification of the International Virus Taxonomy Organization, the phage can be judged to be Caudovirales, Longicaudae.

Example 6 Determination of the optimal infection multiplicity of bacteriophage vB_SalP_LDW8

The multiplicity of infection (MOI) refers to the ratio of the number of phages to the number of host bacteria during infection. Adjust the host bacteria concentration to 10 ⁸ CFU/mL, and set the MOI to 0.1, 0.01, and 0.001, respectively. Take 500 µL of the phage dilution and host bacterial solution, mix them, and culture them at 37°C, 180 r/min, and shake for 3 h. Centrifuge the mixed culture at 12000 r/min for 2 minutes, filter with a 0.22 µm filter membrane, and obtain the lysate. The titer of the phage lysate was measured by the double-layer plate method, repeated three times, and the multiplicity of infection with the highest titer of the lysate was the optimal multiplicity of infection for the phage.

The results are shown in Figure 3A. When the infection multiplicity of phage vB_SalP_LDW8 was 0.01, the phage titer was the highest, which was 3×10 ⁹ PFU/mL. Therefore, the optimal infection multiplicity of phage vB_SalP_LDW8 was 0.01.

Example 7 Determination of the one-step growth curve of bacteriophage vB_SalP_LDW8

The host bacterial solution (10 ⁸ CFU/mL) and the phage lysis solution (10 ⁶ PFU/mL) were mixed at the optimal infection multiplicity ratio, incubated in a 37℃ water bath for 5 min, centrifuged at 12000 r/min for 5 min, the supernatant was discarded, washed twice with LB liquid medium preheated at 37℃, 1 mL of LB liquid medium preheated at 37℃ was added, the precipitate was resuspended, and it was added to 100 mL of LB liquid medium preheated at 37℃, and cultured in a constant temperature shaker at 37℃, sampling once every 10 min, and the phage titer was measured by the double-layer plate method, which lasted for 120 min and repeated three times. The one-step growth curve of the phage was drawn with time as the horizontal axis and phage titer as the vertical axis. The lysis amount was calculated according to the following formula: lysis amount = final phage titer/initial host bacterial number.

The results are shown in Figure 3B. The latent period of phage vB_SalP_LDW8 was 20 min, followed by a steady increase in titer. The outbreak period lasted for 50 min, followed by a plateau period, and the lysis amount was approximately 266 PFU/cell.

Example 8 Determination of temperature stability of bacteriophage vB_SalP_LDW8

Place 5 tubes of phage lysate (500 µL/tube) in water baths at 40°C, 50°C, 60°C, 70°C, and 80°C, respectively. Take samples once at 20 min, 40 min, and 60 min, and measure the phage titer using the double-layer plate method. Repeat three times.

The results are shown in Figure 3C. With the increase of temperature and the extension of time, the activity of the phage decreased to a certain extent. When the temperature was in the range of 40-50°C, the phage activity remained basically unchanged. The titer began to decrease after 20 min at 60°C. It was still active after 20 min and 40 min at 70°C. The titer was 0 after 60 min and at 80°C. This shows that the phage vB_SalP_LDW8 has a certain tolerance to temperature.

Example 9 Determination of acid-base stability of bacteriophage vB_SalP_LDW8

Use HCl (1 mol/L) and NaOH (1 mol/L) to adjust the pH value of LB liquid culture medium to 1-13, take 900 µL of LB culture medium with different pH values, add 100 µL of phage, mix well, and incubate at 37°C for 1 h. Use the double-layer plate method to determine the phage titer under different pH values, and repeat three times.

The results are shown in Figure 3D. The phage vB_SalP_LDW8 can maintain good activity in the pH range of 5-9, and the phage titer does not change significantly. Moreover, the phage titer is highest at pH 8. As the pH value increases or decreases, the phage titer decreases significantly, indicating that the phage vB_SalP_LDW8 has a certain acid and alkali resistance, and its acid resistance is stronger than its alkali resistance.

Example 10 Genome Sequencing of Bacteriophage vB_SalP_LDW8

Prepare high-titer phage lysis solution by plate amplification method. Take 400 µL of host bacterial solution grown to the logarithmic phase, mix it with phage lysis solution at the optimal infection multiplicity ratio, prepare double-layer plates, culture at 37°C for 12 h, add 10 mL of SM buffer to the culture dish, shake at 100 r/min for 4 h, collect the eluate, centrifuge at 12000 r/min for 5 min, and filter with 0.22 µm filter membrane to obtain high-titer phage lysis solution. Add 10% PEG8000 (Polyethyleneglycol 8000) to the pure phage culture, let it stand at 4°C for 6 h after PEG8000 is completely dissolved, centrifuge at 8000r/min for 20 min, discard the supernatant and resuspend the precipitate in 1 mL of SM buffer to concentrate the phage. Take 250 µL of phage concentrate and use the Viral DNA/RNA Kit (TIANGEN) to extract phage DNA according to the instructions. Submit the DNA sample to the sequencing company for completion. The whole genome of Salmonella phage vB_SalP_LDW8 was determined and bioinformatics analysis was performed to determine whether it contained virulence- or drug resistance-related genes.

The genome of the phage was compared with the NCBI database for nucleic acid sequence. The results are shown in FIG4 . The comparison results of the phage vB_SalP_LDW8 and the vB_SenS_SP8 with higher sequence similarity in the database were Query cover 93% and Ident 98%, indicating that the phage is a new type of phage.

In addition, the virulence or resistance-related genes of the test phage vB_SalP_LDW8 were analyzed, and the results showed that the phage did not contain genes encoding virulence or resistance, indicating that there is no potential safety risk in using the test phage for the prevention and control of Salmonella in aquaculture production.

Claims (5)

1. A Salmonella phage vB_SalP_LDW8, characterized in that the phage deposit number is: CGMCC No.45253, the deposit time is August 3, 2022, the deposit location is the General Microbiology Center of China National Microbiological Culture Collection Administration, and the phage is named: vB_SalP_LDW8. 2. A Salmonella phage preparation, characterized in that the preparation contains the Salmonella phage vB_SalP_LDW8 according to claim 1. 3. The preparation according to claim 2, characterized in that the preparation is a liquid, a freeze-dried powder, or a solid preparation. 4. Use of the Salmonella phage preparation according to claim 2 in killing Salmonella. 5. Use of the Salmonella phage preparation according to claim 4 in killing Salmonella, characterized in that it is used to kill Salmonella infecting poultry in farms.