Long tail bacteriophage and use thereof
By using the novel long-tailed bacteriophage WHSATYSB-2501, the problem of low killing efficiency of existing bacteriophages against Salmonella typhimurium has been solved, achieving highly efficient killing and inhibition of Salmonella and expanding the scope of application.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- WUHU INST OF TECH
- Filing Date
- 2026-03-31
- Publication Date
- 2026-05-29
AI Technical Summary
Existing long-tailed bacteriophages have low killing or lysis efficiency against Salmonella typhimurium, which is difficult to meet the needs of practical antibacterial applications.
A novel long-tailed bacteriophage, WHSATYSB-2501 (CCTCC No.: M2026038), was used to kill and inhibit Salmonella bacteria by adding it to wastewater containing Salmonella Typhimurium under conditions where the multiplicity of infection was greater than 0.001, the culture temperature was ≥4℃, the culture time was at least 30 minutes, and the pH value was 5-9.
Bacteriophage WHSATYSB-2501 achieved a titer of 6.8 × 10¹¹ PFU/mL at the optimal multiplicity of infection (MOF) of 0.01, significantly improving its lysis and inhibition effects against Salmonella Typhimurium. Furthermore, it maintained high activity under conditions of 30-50℃ and pH 5-10, thus broadening its application scenarios.
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Abstract
Description
Technical Field
[0001] This invention relates to a bacteriophage and its application, and more particularly to a long-tailed bacteriophage and its application in inhibiting or killing Salmonella typhimurium. Background Technology
[0002] Salmonella is a large genus in the family Enterobacteriaceae, widely found in the intestines and viscera of various animals, including pigs, cattle, sheep, poultry, birds, and rodents. The vast majority of Salmonella bacteria are pathogenic to humans and animals, causing septicemia and gastroenteritis, even miscarriage, and can cause food poisoning in humans, making it one of the most common pathogens of bacterial food poisoning in humans. However, there are numerous Salmonella serotypes; currently, over 2000 serotypes have been identified.
[0003] Regarding Salmonella, Chinese patent CN118638743A discloses a long-tailed bacteriophage, SPYS-1. While this bacteriophage is effective in killing Salmonella typhimurium, exhibits strong tolerance to temperature and pH, adapts to various environments, and proliferates rapidly, it also has a high multiple of infection (MOI), with a minimum MOI of only 0.1, and low titers, with all phage titers not exceeding 10. 10 The PFU / mL level indicates that the phage has low efficiency in killing or lysing Salmonella, making it difficult to meet the needs of practical antibacterial applications. Therefore, obtaining a phage with better antibacterial effects against Salmonella has become an urgent technical problem to be solved. Summary of the Invention
[0004] Objectives of this invention: The objective of this invention is to provide a long-tailed bacteriophage to address the problem of how to efficiently inhibit or kill *Salmonella typhimurium* in contaminated environments. A second objective is to propose the application of the long-tailed bacteriophage in the preparation of bactericides or disinfectants, addressing the problem of how to prepare such agents. A third objective is to propose the application of the long-tailed bacteriophage in inhibiting or lysing *Salmonella typhimurium*, addressing the problem of how to inhibit or kill *Salmonella typhimurium* in contaminated environments. A fourth objective is to provide a formulation for inhibiting or killing *Salmonella typhimurium*.
[0005] Technical solution: The present invention discloses a long-tailed bacteriophage, the preservation name of which is WHSATYSB-2501 (Salmonella Typhimurium phage WHSATYSB-2501), the preservation number of which is CCTCC No: M2026038, and which was deposited at the China Center for Type Culture Collection on January 8, 2026, at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.
[0006] The second aspect of this invention discloses the application of the above-mentioned long-tailed bacteriophage in the preparation of bactericides or disinfectants.
[0007] The third aspect of this invention discloses the application of the above-mentioned long-tailed bacteriophage in inhibiting or lysing Salmonella typhimurium.
[0008] Specifically, the method for inhibiting or lysing Salmonella typhimurium using the above-mentioned long-tailed bacteriophage includes the following steps: The long-tailed phage was added to wastewater containing Salmonella typhimurium with a multiplicity of infection of at least 0.001, and then cultured after mixing.
[0009] Preferably, the concentration of Salmonella typhimurium in the wastewater is at least 10. 6 CFU / mL.
[0010] In some embodiments, the multiplicity of infection is preferably ≥0.01.
[0011] Preferably, the culture conditions are a culture temperature ≥ 4℃ and a culture time of at least 30 min.
[0012] Preferably, the culture temperature is 10-50℃ and the culture time is 30-80 min.
[0013] Preferably, the pH of the wastewater is 5-9.
[0014] The fourth aspect of this invention discloses an agent for inhibiting or killing Salmonella typhimurium, comprising the above-mentioned long-tailed bacteriophage.
[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The long-tailed bacteriophage WHSATYSB-2501 isolated in this invention can effectively kill Salmonella typhimurium in sewage, achieving a titer of 6.8 × 10⁻⁶ at the optimal multiplicity of infection (MOI) of 0.01. 11 The PFU / mL phage showed significantly higher lysis and inhibitory effects on Salmonella Typhimurium than existing bacteriophages.
[0016] The bacteriophages of this invention maintain high activity under conditions of 30-50 °C and pH 5-10, and their bactericidal activity is also stably maintained in normal environments as well as in extreme environments of acidity, alkalinity, and high temperature. This invention significantly broadens the application scenarios and scope of bacteriophages in Salmonella control, providing an efficient and stable new technical means for the biocontrol of Salmonella. Attached Figure Description
[0017] Figure 1 A genome circle diagram of bacteriophage WHSATYSB-2501; Figure 2The phylogenetic tree of bacteriophage WHSATYSB-2501; Figure 3 Electron micrograph of bacteriophage WHSATYSB-2501; Figure 4 The one-step growth curve of bacteriophage WHSATYSB-2501; Figure 5 Results of thermal stability test for bacteriophage WHSATYSB-2501; Figure 6 The results show the pH stability test results for bacteriophage WHSATYSB-2501. Figure 7 The inhibition curves of bacteriophage WHSATYSB-2501 under different multiplicity of infection; Figure 8 The antibacterial effect of bacteriophage WHSATYSB-2501 against Salmonella at 4℃; Figure 9 The antibacterial effect of bacteriophage WHSATYSB-2501 against Salmonella at 25℃; Figure 10 The results show the salt tolerance test results for bacteriophage WHSATYSB-2501. Detailed Implementation
[0018] The technical solution of the present invention will be further described below with reference to the accompanying drawings.
[0019] Example 1: A long-tailed phage isolated from slaughterhouse wastewater, with the preservation name WHSATYSB-2501 and accession number CCTCC No: M 2026038, was deposited on January 8, 2026, at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.
[0020] like Figure 3 As shown, bacteriophage WHSATYSB-2501 has a polyhedral head and a tail with distinct fibrous structures, exhibiting a slender and elongated shape. It belongs to the order Caudataphages and the family Longtailphages. WHSATYSB-2501 has a diameter of approximately 68 nm and a length of approximately 148 nm.
[0021] The genome map obtained after whole-genome sequencing of Salmonella phage WHSATYSB-2501 is shown below. Figure 1 As shown, Figure 1In the diagram, from the inside out, the first ring represents the scale; the second ring represents the GC skew; the third ring represents the GC content; and the fourth and fifth rings represent the positions of CDS, tRNA, and rRNA on the genome. Phage WHSATYSB-2501 carries double-stranded DNA genetic material, with a total genome length of 47243 bp. Its base composition is: A (27.57%), G (22.96%), C (22.83%), T (26.64%), with a GC content of 45.78% and an AT content of 54.22%. The genome encodes 74 open reading frames (ORFs), with a total ORF length of 43254 bp, accounting for 91.55% of the total gene length, and an intergenic region length of 3989 bp. The ORF density is 1.566 genes / kb. The average length of the ORF is 584.51 bp, with the longest ORF being ORF60, which is 3,294 bp in length.
[0022] The complete nucleotide sequence of bacteriophage WHSATYSB-2501 was obtained through BLASTn search results in the NCBI database, as shown in Table 1. Table 1. Comparison of similarity between bacteriophage WHSATYSB-2501 and other bacteriophages.
[0023] As shown in Table 1, bacteriophage WHSATYSB-2501 and Salmonella bacteriophage LPST10 exhibit the highest overall DNA sequence homology (80.00% coverage and 98.96% similarity). According to the International Committee on Taxonomy of Viruses (ICTV), overall DNA sequence homology is defined as coverage multiplied by similarity. Therefore, the overall DNA sequence homology between WHSATYSB-2501 and Salmonella bacteriophage LPST10 is 79.16%. According to the International Committee on Taxonomy of Viruses (ICTV), the primary species classification criterion for bacterial viruses and archaea is 95% genomic similarity. The similarity between these two bacteriophages is below this standard, confirming that bacteriophage WHSATYSB-2501 is a novel bacteriophage.
[0024] Based on BLASTn analysis, 30 phage sequences with over 96% homology to the target phage genome were screened. A phylogenetic tree was constructed using the whole genome sequences (e.g., ...). Figure 2As shown in the figure, Salmonella phage LPST10 (Query Cover=80%, Per. Ident=98.96%) is closely related to bacteriophage WHSATYSB-2501, which belongs to the order Tailphages and family Longtailphages.
[0025] Example 2: Determination of the antibacterial parameters and effects of bacteriophage WHSATYSB-2501: (1) Optimal multiplicity of infection for bacteriophage WHSATYSB-2501 First, determine the titer of the phage fluid, then adjust the host bacterial concentration to 1 × 10⁻⁶. 8 CFU / mL was used to set MOIs of 10, 1, 0.1, 0.01, and 0.001. The phage and host bacteria were thoroughly mixed, and LB broth was added. The mixture was incubated at 37°C with shaking at 160 rpm for 6 h. The culture was then centrifuged at 8000 rpm for 5 min, and the supernatant was filtered through a 0.22 µm filter to obtain the lysis buffer. The titer of the phage lysis buffer was measured using the double-layer plate method; the highest titer was considered the optimal multiple of infection. The results are as follows: Table 2 Optimal Multiplicity of Infection for Bacteriophage WHSATYSB-2501
[0026] Table 2 shows that the titer of bacteriophage WHSATYSB-2501 was 6.8 × 10⁻⁶ at MOI = 0.01. 11 The PFU / mL titer was the highest among the five multiples of infection, therefore the optimal multiple of infection for phage WHSATYSB-2501 was 0.01.
[0027] (2) One-step growth curve of bacteriophage Mix 1 mL each of host bacterial culture and phage lysis buffer at the optimal multiple of infection ratio, incubate at 37 °C for 15 min, centrifuge at 8000 r / min for 2 min, discard the supernatant, and wash the precipitated bacterial cells twice with preheated LB broth at 37 °C to remove unadsorbed host bacteria. Resuspend the precipitate in 5 mL of LB broth and incubate at 37 °C with shaking at 180 r / min. Start timing, and take 100 μL samples every 10 min from time 0 to determine phage titer using the double-layer plate method. Plot a one-step growth curve with infection time on the x-axis and phage titer on the y-axis; calculate the phage latency, lysis period, and lysis volume.
[0028] The results are as follows Figure 4As shown. By Figure 4 As can be seen, the latency period is 20 min, the lysis period is 20-80 min, and the plateau phase occurs after 80 min. The lysis rate is approximately 158 PFU / cell.
[0029] (3) Thermal stability of bacteriophages Take 1 mL of phage suspension in 5 portions and incubate them at constant temperatures of 0, 10, 20, 30, 40, 50, 60 and 70℃ for 60 min. Then, take samples and use the double-layer plate method to determine the potency of each sample. Perform 3 replicates under each condition.
[0030] The results are as follows Figure 5 As shown, Figure 5 As shown in the figure, thermal stability tests demonstrate that WHSATYSB-2501 exhibits outstanding low-temperature resistance. Within the temperature range of 10-50℃, its potency remains essentially unchanged at 10. 9 The potency was above PFU / mL; after incubation at 0℃ or 60℃ for 60 min, the potency decreased by about 3 orders of magnitude; after incubation at 70℃ for 60 min, the potency decreased significantly, to only 10. 2 The activity level is approximately PFU / mL. It is evident that WHSATYSB-2501 maintains high activity between 10-50℃, but its potency begins to decrease below 10℃ or above 50℃, and temperatures of 70℃ and above severely affect its activity.
[0031] (4) Stability of bacteriophages at different pH levels The pH of LB liquid medium was adjusted to 2-15 using HCl (1 mol / L) and NaOH (1 mol / L). 900 µL of LB medium at different pH values was added to 100 µL of phage, mixed thoroughly, and incubated at 37℃ for 1 h. The phage titer under different pH conditions was determined using the double-layer plate method, and the experiment was repeated three times.
[0032] The results are as follows Figure 6 As shown, after incubation at different pH levels for 1 hour, WHSATYSB-2501 exhibited the highest and essentially unchanged activity at pH 5-9, with decreased activity at pH > 9 and pH < 5. Under strongly acidic (pH < 3) or strongly alkaline (pH > 12) conditions, phage activity decreased significantly. Furthermore, compared to acidic environments, WHSATYSB-2501 showed stronger tolerance to alkaline environments, maintaining a titer of 10 at pH above 13. 6 Approximately PFU / mL.
[0033] (5) The antibacterial effect of bacteriophages against Salmonella typhimurium under different infection multiples Salmonella typhimurium was cultured to the logarithmic phase, and the concentration was approximately 10. 7CFU / mL. Take an appropriate amount of WHSATYSB-2501 phage and mix it with the host bacteria at MOI ratios of 0.01, 0.1, 1, and 10. For the control group, add an equal volume of SM buffer to the phage culture medium. Incubate at 37℃ and 180 rpm with shaking. Samples are taken every 1 hour, and the colony count of the host bacteria is measured using the plate colony method. Measurements are performed continuously for 10 hours, with three replicates.
[0034] The results are as follows Figure 7 As shown, by Figure 7 As can be seen, when MOI=0.01, the bacterial concentration increases slowly, and the curve is generally above other MOI values, although the rate is significantly slower compared to the control group. When MOI=10, the curve shows a slight decreasing trend in the 3rd-4th hour, followed by a slow increase. When MOI=0.1, the bacterial concentration decreases slightly in the 2nd-3rd hour, followed by a slow increase. When MOI=1, the bacterial concentration remains almost unchanged in the 1st-3rd hour, maintaining a low trend, reaching its lowest point compared to other MOI values, and then increases slowly.
[0035] (6) Inhibitory effect of bacteriophages on Salmonella Typhimurium at different temperatures Wastewater was filtered to initially remove large solid particles, and then centrifuged at 7000×g for 3 minutes to remove most of the suspended particles such as bacteria and microbial residues. The supernatant was then collected and sterilized in an autoclave at 121℃ for 20 minutes to prepare wastewater samples.
[0036] Salmonella typhimurium solution was added to the sewage sample to achieve a bacterial concentration of approximately 10. 7 CFU / mL was aliquoted into ten 50 mL centrifuge tubes, 30 mL per tube. Bacteriophage WHSATYSB-2501 was added to the centrifuge tubes at MOI ratios of 0.1, 1, and 10, respectively. Bacteriophage SPYS-1 (CCTCC NO: M2024984) was added to the centrifuge tubes at an MOI of 10. Each group had three replicates. The last centrifuge tube contained an equal volume of SM buffer as a blank control. The centrifuge tubes were incubated at 25°C and 4°C, respectively. Samples were taken every 1 hour, and the colony count of the host bacteria was measured using the plate colony method for 10 consecutive hours.
[0037] The results are as follows Figure 8 and Figure 9 As shown, Figure 8 The figure shows the antibacterial curve of bacteriophage WHSATYSB-2501 at 4℃. Figure 8It can be seen that when MOI=0.1, the curve rises slowly from hour 2 to hour 5. When MOI=1, the bacterial concentration decreases slightly from hour 3 to hour 4, and then rises slowly. When MOI=10, the bacterial concentration remains almost unchanged from hour 2 to hour 6, maintaining a low trend, reaching its lowest point compared to other MOI values, and then rises slowly. All curves of bacteriophage WHSATYSB-2501 are below the curve of bacteriophage SPYS-1, indicating that the antibacterial effect of bacteriophage WHSATYSB-2501 is better than that of bacteriophage SPYS-1 at 4℃.
[0038] Figure 9 The figure shows the antibacterial curve of bacteriophage WHSATYSB-2501 at 25℃. Figure 9 It can be seen that when MOI=0.1, the curve rises slowly from hour 1 to 3, and then tends to stabilize. When MOI=1, the bacterial concentration fluctuates significantly from hour 1 to 4, and then tends to stabilize from hour 6. When MOI=10, the bacterial concentration fluctuates erratically from hour 1 to 6, and then tends to stabilize from hour 7. All curves of bacteriophage WHSATYSB-2501 are below the curve of bacteriophage SPYS-1, indicating that the antibacterial effect of bacteriophage WHSATYSB-2501 is also better than that of bacteriophage SPYS-1 at 25℃.
[0039] (7) Inhibitory effect of bacteriophages on Salmonella Typhimurium at different salt concentrations Take 500 µL of saline solutions with concentrations of 0.9%, 3%, 7%, 15%, and 30%, respectively, and mix them with 500 µL of phage. Incubate at 37 °C for 1 h. Use the double-layer plate method to determine the phage titer under different saline concentrations. Repeat three times.
[0040] The results are shown in Figure 10. After incubation for 1 hour in salt solutions of different concentrations, WHSATYSB-2501 maintained an initial potency of 10. 9 At PFU / mL, the potency decreases with increasing salt concentration, but the decrease is small. Even at saline saturation, the potency of WHSATYSB-2501 remains at 10. 8 PFU / mL or higher. Figure 10 This indicates that WHSATYSB-2501 has good salt resistance.
Claims
1. A long-tailed bacteriophage, characterized in that, The phage is named WHSATYSB-2501 and has the accession number CCTCC No: M 2026038. It was deposited at the China Center for Type Culture Collection on January 8, 2026.
2. The application of the long-tailed bacteriophage according to claim 1 in the preparation of bactericides or disinfectants.
3. The application of the long-tailed bacteriophage according to claim 1 in inhibiting or lysing Salmonella typhimurium.
4. The application according to claim 3, characterized in that, Includes the following steps: The long-tailed phage was added to wastewater containing Salmonella typhimurium with a multiplicity of infection of at least 0.001, and then cultured after mixing.
5. The application according to claim 4, characterized in that, The concentration of Salmonella typhimurium in the wastewater is at least 10. 6 CFU / mL.
6. The application according to claim 4, characterized in that, The cultivation conditions are: cultivation temperature ≥ 4℃ and cultivation time at least 30 min.
7. The application according to claim 4, characterized in that, The culture temperature is 10-50℃ and the culture time is 30-80 min.
8. The application according to claim 4, characterized in that, The pH of the wastewater is 5-9.
9. An agent for inhibiting or killing Salmonella typhimurium, characterized in that, It includes the long-tailed bacteriophage as described in claim 1.