Salmonella longtail bacteriophage and application thereof

Abstract

The invention discloses a salmonella longtail bacteriophage and application thereof, the bacteriophage is salmonella bacteriophage YZA15 and is preserved in China Center for Type Culture Collection, the preservation number is CCTCC NO: M 20242878, the preservation date is December 23, 2024, and the preservation address is Wuhan. The salmonella bacteriophage YZA15 disclosed by the invention is good in stability, has no toxic drug-resistant genes, has splitting activity on main epidemic serotypes (mouse typhoid, enteritis and pullorum disease) of salmonella, is relatively specific in host range, short in incubation period and high in filial generation bacteriophage outbreak amount, can be applied to links of livestock breeding, slaughtering, processing and breeding, and has a broad application prospect. And a safe and efficient biological antibacterial product is provided for livestock and poultry environments and products thereof.

Description

Technical Field

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

[0002] Salmonella is a Gram-negative bacillus that is widely present in the natural environment, especially in livestock and poultry. It can cause a variety of diseases in humans and animals, including gastroenteritis, typhoid fever and sepsis. In recent years, the problem of Salmonella resistance has become increasingly serious, posing a huge challenge to public health. Studies have shown that Salmonella typhimurium, Salmonella enteritidis and Salmonella delbrueckii are the most common serotypes in my country, and the diversity of resistant strains is increasing. This resistance not only reduces the therapeutic effect of traditional antibiotics, but may also lead to more serious infection transmission.

[0003] The main route of Salmonella infection is through the food chain, especially contaminated livestock and poultry products. The presence of drug-resistant Salmonella makes infection prevention and treatment more difficult, increasing treatment costs and mortality. Therefore, developing new prevention and control methods to deal with the threat of drug-resistant Salmonella has become the focus of current research.

[0004] In the livestock and poultry breeding, slaughtering and processing links, the prevention and control of Salmonella mainly relies on good hygiene management, vaccination and the use of antibiotics. However, the abuse of antibiotics has led to the increase of drug-resistant strains. Therefore, non-antibiotic prevention and control methods, such as the use of bacteriophages, have gradually attracted attention. As a specific bacterial virus, bacteriophages can efficiently lyse target bacteria without negatively affecting the health of host animals or humans. Summary of the invention

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

[0006] Technical solution: The present invention provides a long-tailed Salmonella phage, which is Salmonella phage YZA15, preserved in China Center for Type Culture Collection, with a preservation number of CCTCC NO: M 20242878, and a preservation address of Wuhan.

[0007] Furthermore, the bacteriophage YZA15 is a long-tailed phage with a total length of 130±5 nm, of which the tail is 95±2 nm long.

[0008] Furthermore, the bacteriophage YZA15 has good acid-base tolerance, and the optimum pH value is 3-12.

[0009] Furthermore, the bacteriophage YZA15 has good thermal stability, and the optimum temperature is 40-70°C.

[0010] Furthermore, when the infection multiplicity of the bacteriophage YZA15 with the host bacteria was 0.0001, the titer was 1.57×10 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 mainly includes Salmonella typhimurium, Salmonella enteritidis, Salmonella pullorum, and Salmonella sanftenberg.

[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 YZA15, which has good stability, no toxicity and drug-resistant genes, has lytic activity against the main prevalent serotypes of Salmonella (typhoid, enteritis, and pullorum), has a relatively specific host range, a short incubation period, and a high explosive volume of progeny phages. It can be used in livestock and poultry breeding, slaughtering, processing and breeding, and provides a safe and efficient biological antibacterial product for livestock and poultry environments and their products. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the double-layer plate plaque image of bacteriophage YZA15;

[0020] Figure 2 It is a transmission electron microscopy schematic diagram of bacteriophage YZA15;

[0021] Figure 3 It is the circle map of the whole genome sequence of bacteriophage YZA15;

[0022] Figure 4 Schematic diagram of acid-base tolerance of bacteriophage YZA15;

[0023] Figure 5 Schematic diagram of the thermal stability of bacteriophage YZA15;

[0024] Figure 6Schematic diagram of the optimal infection multiplicity of bacteriophage YZA15;

[0025] Figure 7 Schematic diagram of the one-step growth curve of bacteriophage YZA15. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0027] Example 1 Phage Screening

[0028] A phage with high titer against Salmonella typhimurium HKY14 (Salmonella typhimurium isolated from a duck farm) isolated from animal feces (a pig farm in Yangzhou) was selected and named YZA15. It was deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20242878, a deposit date of December 23, 2024, a deposit name of Salmonella sp. phang YZA15, and a deposit address of Wuhan. The double-layer plate results are as follows Figure 1 shown.

[0029] Example 2 Transmission electron microscopy observation

[0030] 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 YZA15 is a long-tailed phage, with a regular icosahedron tail length of 95±2nm and a total length of about 130±5nm.

[0031] Example 3 Phage genome assembly and annotation

[0032] According to the manufacturer's instructions, phage genomic DNA was extracted using the Qiagen MinElute Virus Spin Kit and the library construction instructions were followed. The Universal Plus DNA library kit was used to construct a whole-genome sequencing library, and Illumina nextseq sequencer was used for whole-genome sequencing. To ensure the reliability of subsequent information analysis results, we used fastp to filter and quality-control the obtained raw sequencing data, trim the adapters, remove low-quality reads and reads with a high proportion of Ns, and obtain clean reads. The metaSPAdes software was used to perform de novo assembly on the clean reads. At the same time, different k-mer lengths were selected for testing to obtain the best assembly results. Then, the bwa software was used to align the clean reads to the assembled genome sequence to calculate the coverage. Prokka was used to annotate the coding genes and tRNAs in the genome. Then, blastp was used to align the protein sequences with the NR database to obtain the sequence information of genes with high similarity in the NR database.

[0033] Table 1 Quality control table of sequencing data

[0034]

[0035] Table 2 Statistics of genome assembly quality

[0036]

[0037] As shown in Table 1 and Table 2, phage YZA15 is a dsDNA phage. No drug-resistant genes and virulence genes were found in the whole-genome sequencing. The genome is in a circular state. After filtering and quality control, the number of raw sequencing data decreased by 19434; the data volume of sequencing decreased by 0.03G. There are no unknown bases, and at the same time, the GC base ratio decreased by 0.13%.

[0038] Example 4 Functional annotation of phage genome

[0039] The eggNOG-mapper was used to perform functional annotation on the genes, which included annotation information from the COG, GO, KEGG, CAZy, BiGG, and PFAM databases.

[0040] A total of 73 (42 kinds) CDSs were predicted in the genome of phage YZA15. Among them, 45 CDSs encoded known functions, and the other 28 encoded hypothetical proteins, such as Figure 3 , Table 3.

[0041] Table 3 Protein annotation information of YZA15

[0042]

[0043]

[0044] Lysis spectrum of phage YZA15 in Example 5

[0045] The double-layer plate spotting method was used to determine the lysis of phages against Salmonella of different serotypes. First, the fresh bacterial solutions shown in Table 4 were respectively mixed with semi-solid LB medium and poured onto solid LB agar plates. After standing and solidifying, 5 μL of phage was spotted, and cultured at 37 °C for 4 h. Whether plaques appeared in the spotting area was observed. According to the clarity of the plaques, the results were divided into three categories: clear and translucent plaques (++), relatively translucent plaques (+), and no plaques (-).

[0046] Table 4 Determination of phage lysis spectrum

[0047]

[0048]

[0049]

[0050] Example 6 Acid and alkali tolerance of phage

[0051] HCl and NaOH solutions were used to adjust the SM buffer to a pH of 2 - 13. 900 μL of the SM buffer at each pH value and 100 μL of the phage stock solution were taken and mixed in a sterile EP tube, and reacted in a metal bath at 37 °C for 1 h or 2 h. 100 μL of each was taken for gradient dilution, and the double-layer plate method was used to determine the titer.

[0052] It can be seen from Figure 4 that when the pH of YZA15 is 3 - 12, the titer is relatively stable.

[0053] Example 7 Thermal stability of phage

[0054] Phage YZA15 was divided into 20 portions (500 μL each), and they were respectively placed in a metal bath at 40 °C, 50 °C, 60 °C, 70 °C, and 80 °C and allowed to stand for 15 min, 30 min, 45 min, and 60 min. 100 μL of each sample was taken for gradient dilution, and the double-layer agar plate method was used to determine the titer of phages at each temperature.

[0055] It can be seen from Figure 5 that YZA15 has good stability at 40 - 70 °C. At 80 °C, the titer gradually decreases with the increase of time, and drops by about 6 log values after 60 min.

[0056] Example 8 Determination of the optimal multiplicity of infection

[0057] The bacterial solution of Salmonella typhimurium HKY14 was adjusted to 10 8 CFU / mL; 100 μL of phage YZA15 with different titers was mixed with an equal volume of the bacterial solution to obtain 101 , 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.

[0058] Depend on Figure 6 It can be seen that when the infection multiplicity of the phage and the host bacteria is 0.0001, the titer of YZA15 is the highest, which is 1.57×10 10 PFU / mL.

[0059] Example 9 Determination of one-step growth curve

[0060] Adjust the Salmonella typhimurium HKY14 bacterial solution (host bacteria) to 10 8 CFU / mL, phage YZA15 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 20min (a total of 150min) to measure the titer of the phage.

[0061] Depend on Figure 7 It can be seen that the incubation period of YZA15 is about 5 minutes, the burst period is about 115 minutes, and the lysis amount is 33.83 PFU / cell.

[0062] Example 10 Water Source Pollution

[0063] Salmonella typhimurium HKY14 was cultured in a shaking incubator (37°C, 180 r / min), and the bacterial solution concentration was adjusted to OD600 = 1.0 with PBS, and then diluted to 1 × 10 6 CFU / mL, take 100 μL of bacterial dilution and mix with an equal volume of bacteriophage YZA15 (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 mixed with bacterial dilution as a control; 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.

[0064] The results are shown in Table 5. The bacteriophage YZA15 had a significant bactericidal effect within 5 h, and no colonies were detected.

[0065] Table 5 Bactericidal efficiency of YZA15 against HKY14 strain (%)

[0066]

[0067]

[0068] Example 11 Solid Surface Contamination Application

[0069] 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 (about 1 mL), and observe whether there are colonies; if no colonies appear, continue to add phage stock solution and adjust the concentration of phage YZA15 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 Salmonella typhimurium HKY14 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.

[0070] The results are shown in Table 6. The bactericidal efficiency of bacteriophage YZA15 spray against Salmonella typhimurium HKY14 within 12 hours is >99.5%

[0071] Table 6 Bactericidal efficiency of bacteriophage YZA15 spray on HKY14 strain (%)

[0072]

[0073] Example 12 Particulate Solid Pollution Application

[0074] First 10 6 CFU / mL of Salmonella typhimurium HKY14 solution was mixed and sprayed on the granular bedding 40 times, and then bacteriophage YZA15 (5×10 9 PFU / mL), the 20th spraying was recorded as 0h, and the data before and after spraying were recorded; at 36h, the second spraying of the same amount of phage was carried out; at 72h, each group was sprayed with bacterial solution 5 times; at 84h, phage was sprayed 10 times, and the litter particles were weighed and counted before and after spraying at each time point. About 1mL was sprayed every 5 times, and repeated for 3 groups in total.

[0075] Table 7 Bactericidal efficiency of bacteriophage YZA15 spray on HKY14 strain (%)

[0076]

[0077] The results are shown in Table 7. The bactericidal efficiency of Salmonella typhimurium HKY14 within 12 hours after spraying phage and bacteria was >99.5%.

Claims

1. A Salmonella longicauda phage, characterized in that: The bacteriophage is Salmonella phage YZA15, which is deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 20242878, a deposit date of December 23, 2024, and a deposit address of Wuhan.

2. The Salmonella longicaudatus phage according to claim 1, characterized in that The bacteriophage YZA15 is a long-tailed phage with a total length of 130±5 nm, of which the tail is 95±2 nm long.

3. The Salmonella longicaudatus phage according to claim 1, characterized in that The bacteriophage YZA15 has good acid-base tolerance, and the optimum pH value is 3-12.

4. The Salmonella longicaudatus phage according to claim 1, characterized in that The bacteriophage YZA15 has good thermal stability, and the optimum temperature is 40-70°C.

5. The Salmonella longicaudatus phage according to claim 1, characterized in that When the infection multiplicity of the bacteriophage YZA15 with the host bacteria was 0.0001, the titer was 1.57×10 10 PFU / mL.

6. Use of the Salmonella longicaudatus 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 and salmonella sanftenberg.

8. A feed additive or drinking water additive containing 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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