Salmonella bacteriophage and application thereof

Abstract

The invention discloses a salmonella bacteriophage and application thereof, the salmonella bacteriophage is salmonella bacteriophage TZCZ65 and is preserved in China Center for Type Culture Collection, CCTCC NO: M 20242879, the preservation date is December 23, 2024, and the preservation address is Wuhan. The salmonella bacteriophage TZCZ65 disclosed by the invention is good in stability, free of non-toxic drug-resistant genes, high in safety, wide in host range, short in incubation period and high in filial generation bacteriophage outbreak amount, has splitting activity on salmonella from different sources, can be applied to livestock and poultry slaughtering, processing and breeding links, and has broad application prospects. A safe and efficient biological antibacterial product is provided for livestock and poultry and products thereof.

Description

Technical Field

[0001] The invention relates to a salmonella phage and application thereof, belonging to the technical field of microorganisms. Background Art

[0002] Salmonella is an important facultative intracellular bacterium that is common to both humans and animals. It has a wide range of hosts and is commonly found in animals such as pigs, chickens, and ducks. Salmonella typhimurium is one of the important serotypes of Salmonella and can easily infect humans through the food chain.

[0003] In the farming and slaughtering environment, antibiotics are mostly used to control and prevent pathogenic bacteria infections in animals and maintain animal health. However, excessive and inappropriate use of antibiotics can lead to a series of problems, including antibiotic residues and the emergence of multidrug-resistant bacteria.

[0004] As an alternative treatment for bacterial infections, phage therapy is highly specific compared to antibiotics and can avoid imbalance of normal bacterial flora. It also has strong lytic properties, is environmentally friendly, can achieve self-replication in clinical use, and has a short R&D cycle. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a Salmonella phage and its application.

[0006] Technical solution: The present invention provides a Salmonella phage, which is Salmonella phage TZCZ65, deposited in China Center for Type Culture Collection, CCTCC NO: M 20242879, deposited on December 23, 2024, and deposited in Wuhan.

[0007] Furthermore, the bacteriophage TZCZ65 is a myotail phage, and the head is a regular icosahedron with an edge length of 20±0.5nm and a diameter of 46±0.8nm.

[0008] Furthermore, the bacteriophage TZCZ65 has good acid-base tolerance, and the optimum pH value is 3-11.

[0009] Furthermore, the bacteriophage TZCZ65 has good thermal stability, and the optimum temperature is 40-70°C.

[0010] Furthermore, when the infection multiplicity of the bacteriophage TZCZ65 with the host bacteria was 0.1, the titer was 3.34×10 10 PFU / mL.

[0011] The present invention also provides the use of the above-mentioned Salmonella phage in any of the following items:

[0012] (1) Salmonella fungicide;

[0013] (2) Drugs for preventing / treating diseases caused by Salmonella.

[0014] Furthermore, the Salmonella includes Salmonella typhimurium, Salmonella enteritidis, Salmonella pullorum, Salmonella Kentucky, Salmonella paratyphi A, Salmonella Sanftenberg, Salmonella Indiana, Salmonella Derby, Salmonella Corvallis, Salmonella Rosen, Salmonella Thompson, Salmonella Mbandaka, Salmonella bovis, Salmonella infantis, Salmonella London, Salmonella Aberdeen, and Salmonella California.

[0015] The present invention also provides a feed additive or a drinking water additive with the above-mentioned Salmonella phage as an effective ingredient.

[0016] The present invention also provides a cleaning agent or disinfectant for breeding and slaughtering environment, which contains the above-mentioned Salmonella bacteriophage as an effective ingredient.

[0017] Furthermore, the concentration of the Salmonella phage is not less than 10 9 PFU / mL

[0018] Beneficial effects: Compared with the prior art, the present invention has the following outstanding advantages: The present invention discloses for the first time a Salmonella phage TZCZ65, which has good stability, no virulence and drug-resistant genes, high safety, has lytic activity against Salmonella from different sources, has a wide host range, a short incubation period, and a high explosive volume of progeny phages. It can be used in the slaughtering, processing and breeding of livestock and poultry, providing a safe and efficient biological antibacterial product for livestock and poultry and their products. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the double-layer plate plaque image of bacteriophage TZCZ65;

[0020] Figure 2 is a transmission electron microscopy schematic diagram of bacteriophage TZCZ65;

[0021] Figure 3 Schematic diagram of the optimal infection multiplicity of bacteriophage TZCZ65;

[0022] Figure 4 Schematic diagram of the one-step growth curve of bacteriophage TZCZ65;

[0023] Figure 5 Schematic diagram of acid-base tolerance of bacteriophage TZCZ65;

[0024] Figure 6 Schematic diagram of the thermal stability of bacteriophage TZCZ65;

[0025] Figure 7 Circle map of the whole genome sequence of bacteriophage TZCZ65;

[0026] Figure 8 This is a schematic diagram of the changes in organ weights of BALB / c mice after tail vein injection of bacteriophage TZCZ65;

[0027] Fig. 9 Schematic diagram of IL-1β content in BALB / c mice after tail vein injection of bacteriophage TZCZ65;

[0028] Fig.10 Schematic diagram of pathological sections of BALB / c mice after tail vein injection of bacteriophage TZCZ65. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0030] Example 1 Isolation and characterization of bacteriophage

[0031] Feces (collected from Lvyuan Pig Slaughterhouse, Yangzhou, Jiangsu) were picked up with the blunt end of a 10 μL long gun tip and placed directly in a 1.5 mL centrifuge tube filled with SM buffer. After the feces suspension was allowed to stand, the supernatant was collected and incubated at 4 °C and 12,000 rp. m Centrifuge for 10 minutes. Then filter the supernatant through a 0.22 μm filter to obtain a sterile phage stock solution, take 5 μL and spot-drip on a double-layer plate, then invert and culture in a 37°C constant temperature incubator.

[0032] Dilute the sample with spots in the above double-layer plate in a gradient, find a suitable gradient, mix it with Salmonella D6 solution, and add it to the semi-solid culture medium to observe whether there is a single plaque. If a single plaque appears, pick it with a 10μL small pipette tip and place it in SM buffer for washing and soaking, dilute it again in a gradient to find a suitable diluent and repeat the above steps several times until the size and transparency of the plaques are basically the same.

[0033] The isolated phage was named TZCZ65, CCTCC NO: M 20242879, and the double-layer plate results were as follows: Figure 1 shown.

[0034] Example 2 Lysis spectrum of bacteriophage TZCZ65

[0035] The double-layer plate spot method was used to determine the lysis of phages against different serotypes of Salmonella. First, the fresh bacterial solution shown in Table 1 was mixed with the semi-solid LB medium and poured onto the solid LB agar plate. After standing and solidifying, 5 μL of phage was spotted and cultured at 37°C for 4 h. Whether plaques appeared in the spotted area was observed. According to the clarity of the plaques, the results were divided into three categories: clear and transparent plaques (++), relatively transparent plaques (+), and no plaques (-).

[0036] Table 1 Phage lysis spectrum determination

[0037]

[0038]

[0039]

[0040] Example 3 Transmission electron microscopy observation

[0041] The phage stock solution was dropped onto a 200-mesh carbon-coated copper mesh, and the liquid was dried after 15 minutes. The mesh was then stained with 2% phosphotungstic acid solution for 1 minute. After the liquid was dried, the mesh was inverted and dried under a drying lamp for 30 minutes. The magnification was increased to 800,000 times, and the phage image was selected and saved ( Figure 2 ). Bacteriophage TZCZ65 is a muscle-tailed phage, whose head is a regular icosahedron with an edge length of about 20±0.5nm, a diameter of about 46±0.8nm, and a contractile muscle sheath.

[0042] Example 4 Determination of the Optimal Multiplicity of Infection

[0043] Adjust the D6 bacterial solution to 10 8 CFU / mL; 100 μL of phages of different titers were mixed with an equal volume of bacterial solution to make 10 1 , 10 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 The total volume was 5 mL, and the mixture was incubated at 180 rpm for 4 h in a 37°C constant temperature oscillator. The mixture was centrifuged at 10,000 rpm for 15 min, and the supernatant was filtered with a 0.22 μm filter to remove the bacterial precipitate. After gradient dilution with SM, the titer of the phage was determined by the double-layer plate method.

[0044] Depend on Figure 3 It can be seen that when the infection multiplicity of phage and host bacteria is 0.1, the titer of TZCZ65 is the highest, which is 3.34×10 10 PFU / mL.

[0045] Example 5 Determination of one-step growth curve

[0046] Adjust the D6 bacterial solution to 10 8CFU / mL, phage TZCZ65 and host bacteria were mixed at MOI = 0.01, and the mixture was placed at 37°C, 180r / min shaking culture for 15min; then centrifuged at 4°C, 4000rpm for 15min, the supernatant was discarded, the precipitate was washed twice with LB liquid culture medium to remove free phages, and finally the precipitate was resuspended with LB liquid culture medium preheated at 37°C, and then incubated in a 37°C metal bath; a gradient dilution was taken every 5min in the first 20min, and a gradient dilution was taken every 10min after 30min (a total of 150min) to measure the titer of the phage.

[0047] Depend on Figure 4 It can be seen that the incubation period of TZCZ65 is short and was not detected within 5 minutes; the burst period is about 135 minutes, and the lysis amount is 45.52 PFU / cell.

[0048] Example 6 Acid-base tolerance of bacteriophage

[0049] Use HCL and NaOH solution to adjust the SM buffer to a pH of 2-13, take 900 μL of SM buffer of each pH value and 100 μL of phage stock solution in a sterile EP tube and mix well, incubate at 37°C in a constant temperature incubator (metal bath), take 100 μL of gradient dilution after 1h and 2h of action, and determine the titer using the double-layer plate method.

[0050] Depend on Figure 5 It can be seen that the potency of TZCZ65 is relatively stable when the pH is 3-11, and it can persist for 1 hour at pH = 2, and is generally more acid-resistant.

[0051] Example 7 Thermal stability of bacteriophage

[0052] The phage was divided into 20 portions (500 μL each), and placed in a constant temperature water bath or metal bath at 40°C, 50°C, 60°C, 70°C and 80°C for 15 min, 30 min, 45 min and 60 min respectively. 100 μL of each sample was taken for gradient dilution, and the titer of the phage at each temperature was determined using the double-layer agar plate method.

[0053] Depend on Figure 6 It can be seen that TZCZ65 has good stability at 40-70℃; at 80℃, the titer gradually decreases, and it decreases by about 2 log values ​​within 60 minutes. In general, the thermal stability of the phage is good.

[0054] Example 8 Phage genome assembly and annotation

[0055] According to the manufacturer's instructions, Qiagen MinElute Virus Spin Kit was used to extract phage genomic DNA, and the size of genomic DNA was determined by 0.8% agarose gel electrophoresis using Equalbit 1×dsDNA HS Assay Kit (Vazyme Biotech Co.). The library construction instructions were followed and the library was constructed using Illumina (Vazyme Biotech Co., Ltd.) The Universal Plus DNA library kit was constructed and the whole genome sequencing was performed using the Illumina nextseq sequencer. In order to ensure the reliability of the subsequent information analysis results, we used fastp to filter and quality control the obtained raw sequencing data, cut the adapter, remove low-quality reads and reads with a high N ratio, and obtain clean reads. The clean reads were de novo assembled using metaSPAdes software, and different k-mer lengths were selected for testing and the best assembly results were obtained. Then, the clean reads were aligned to the assembled genome sequence for statistical coverage using bwa software. Prokka was used to annotate the coding genes and tRNAs of the genome, and then blastp was used to align the protein sequence with the NR library to obtain the sequence information with high similarity of each gene in the NR library.

[0056] TZCZ65 phage is a dsDNA phage. No drug resistance genes or virulence genes were found in the whole genome sequencing. The genome is in a circular state. The raw data of sequencing was reduced by 12536 after filtering and quality control; the amount of sequencing data was reduced by 0.01G. There are no unknown bases, and the GC base ratio is also reduced by 0.13%.

[0057] Table 2 Sequencing data quality control table

[0058]

[0059] Table 3 Genome assembly quality statistics

[0060]

[0061] Example 9 Functional annotation of bacteriophage genomes

[0062] The eggNOG-mapper was used to perform functional annotation of genes, which contains annotation information from the COG, GO, KEGG, CAZy, BiGG, and PFAM databases.

[0063] A total of 121 CDSs and 24 tRNAs were predicted in the bacteriophage TZCZ65 genome ( Table 4 ), of which 73 (64 species) CDSs encode known functions and the other 48 encode hypothetical proteins, e.g. Figure 7 .

[0064] Table 4 TZCZ65 protein annotation information

[0065]

[0066]

[0067]

[0068] Example 10 Phage Safety Evaluation

[0069] Twenty BALB / c mice (7 weeks old, provided by the Center for Comparative Medicine of Yangzhou University; all experimental procedures for experimental animals were approved by the Animal Welfare and Use Committee of Yangzhou University (SYXK[Su]2012-0029) and in accordance with the experimental and animal management procedures) were randomly divided into four groups (5 mice in each group) and injected with different doses of phage (1.0×10 8 , 1.0×10 9 , 1.0×10 10 PFU / mouse), and the control group was injected with 100 μL of sterile SM buffer. The mice were observed once in the morning and evening every day, and the survival status of the mice was recorded.

[0070] 14 days after the injection, the eyeballs of the four groups of mice were removed and blood was collected in 1.5 mL EP tubes. After standing at 37°C for 12 h, the tubes were centrifuged at 4000 r / min for 15 min, and the upper serum was collected. The serum, capture microspheres and PE detection reagent were mixed in a 1:1 ratio using CBA technology and incubated at room temperature in the dark. After 2 h, 500 μL of washing solution was added, and the tubes were resuspended with 300 μL of washing solution at 200 g × 5 min.

[0071] Mice were killed and some organs were taken out, which were fully immersed in 4% paraformaldehyde tissue fixative. After being well fixed, they were trimmed, dehydrated, embedded, sliced, stained, and sealed according to the SOP procedures of pathological laboratory detection. Finally, the samples that passed the microscopic examination were subjected to microscopic examination / panoramic scanning analysis.

[0072] Table 5 Phage safety test - 14 days

[0073]

[0074] like Figure 8 The diagram of the changes in the weight of mouse organs shows that compared with the control group, after injection of bacteriophage TZCZ65, 10 10The spleen weights of the following groups showed no significant changes, 10 10 There was no significant change in liver weight in the groups below.

[0075] like Fig. 9 The diagram of IL-1β content in mice showed that there was no significant difference between the three phage dose groups and the control group.

[0076] Depend on Fig.10 It can be seen that after 14 days, there were no obvious abnormalities in the liver and spleen of mice in the TZCZ65 group compared with those in the control group.

[0077] Example 11 Simulated water source pollution

[0078] The Salmonella typhimurium D6 suspension was adjusted to OD600 = 1 with PBS and then diluted to 1 × 10 6 CFU / mL, take 100 μL of bacterial dilution and mix with an equal volume of phage (5×10 9 PFU / mL) and mix well, and use PBS to make up the system to 5mL. At the same time, use SM buffer and bacterial dilution solution as controls; take samples every 1 hour, and drip 10μL into the culture dish after gradient dilution. After drying, invert it in a 37℃ incubator, and count the colonies the next day.

[0079] The results are shown in Table 6. The bacteriophage TZCZ65 has the best bactericidal effect within 3 hours of action, and the minimum reduction is 4 log values ​​within 5 hours.

[0080] Table 6 Bactericidal efficiency of TZCZ65 against D6 strain (%)

[0081]

[0082]

[0083] Example 12 Spray method to simulate solid surface contamination

[0084] First, sterilize the spray bottle: soak and wipe it with 75% ethanol in a clean bench, dry it and irradiate it with ultraviolet light for 4 hours, add a certain amount of SM buffer, spray it into the culture dish 5 times, and observe whether there are colonies; if no colonies appear, continue to add phage stock solution and adjust the concentration of phage TZCZ65 to 5×10 9 PFU / mL, spray the culture dish again for 5 times to observe whether there are colonies; after confirming sterility, dilute the adjusted bacterial suspension to 1×10 6 CFU / mL, 100 μL of bacterial dilution was spread on the culture dish, and after drying, it was sprayed with bacteriophage five times, and placed at room temperature for 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, and 12 h. It was sent to a 37 °C incubator, and the colonies were counted the next day. SM buffer was used as a control.

[0085] The results are shown in Table 7. The bacteriophage spray has a good disinfecting effect on bacteria within 0-1.5h and 4-12h, and the minimum reduction is 4 log values ​​within 12h.

[0086] Table 7 Bactericidal efficiency of bacteriophage TZCZ65 spray on D6 strain (%)

[0087]

Claims

1. A Salmonella phage, characterized in that: The bacteriophage is Salmonella phage TZCZ65, which is deposited in the China Center for Type Culture Collection, CCTCC NO: M 20242879, the deposit date is December 23, 2024, and the deposit address is Wuhan.

2. The Salmonella phage according to claim 1, characterized in that The bacteriophage TZCZ65 is a muscle-tailed phage, and the head is a regular icosahedron with an edge length of 20±0.5nm and a diameter of 46±0.8nm.

3. The Salmonella phage according to claim 1, characterized in that The bacteriophage TZCZ65 has good acid-base tolerance, and the optimum pH value is 3-11.

4. The Salmonella phage according to claim 1, characterized in that The bacteriophage TZCZ65 has good thermal stability, and the optimum temperature is 40-70°C.

5. The Salmonella phage according to claim 1, characterized in that When the infection multiplicity of the bacteriophage TZCZ65 with the host bacteria was 0.1, the titer was 3.34×10 10 PFU / mL.

6. Use of the Salmonella phage according to claims 1 to 5 in any of the following: (1) Salmonella fungicide; (2) Drugs for preventing / treating diseases caused by Salmonella.

7. The use according to claim 6, characterized in that: The salmonella include Salmonella typhimurium, Salmonella enteritidis, Salmonella pullorum, Salmonella Kentucky, Salmonella paratyphi A, Salmonella Sanftenberg, Salmonella Indiana, Salmonella Derby, Salmonella Corvallis, Salmonella Rosen, Salmonella Thompson, Salmonella Mbandaka, Salmonella bovis, Salmonella infantis, Salmonella London, Salmonella Aberdeen, and Salmonella California.

8. A feed additive or drinking water additive comprising the Salmonella phage according to claim 1 as an effective ingredient.

9. A cleaner or disinfectant for aquaculture and slaughtering environments, comprising the Salmonella bacteriophage according to claim 1 as an effective ingredient.

10. The use according to claim 9, characterized in that: The concentration of the Salmonella phage is not less than 10 9 PFU / mL.

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