Shigella sonnei bacteriophage vBSsPLW and application thereof

Abstract

The invention provides a Shigella sonnei bacteriophage vBSsPLW and application thereof, and belongs to the technical field of microorganisms, the bacteriophage is preserved in Guangdong Microbiological Culture Collection Center, the address is 59 building, No.100 courtyard, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, the preservation date is June 17, 2025, and the preservation number is GDMCC No: 66526-B1. The phage vBSsPLW provided by the invention is high in titer, short in incubation period and good in environmental stability, and still has relatively high activity when the temperature reaches 70 DEG C; the recombinant shigella sonnei strain has the advantages that the recombinant shigella sonnei strain is stable in titer change within the pH range of 6.0-11.0, has a very strong bacteriostatic effect on shigella sonnei under different infection complex conditions, and can achieve the bacteriostatic purpose by inhibiting the formation of a shigella sonnei biofilm. The method has strong application potential in food preservation, biology, medicine and other aspects.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and in particular relates to a Shigella sonnei bacteriophage vB_SsP_LW and an application thereof. Background Art

[0002] Modern bacillary dysentery is primarily caused by Shigella pathogens, but the predominant Shigella species prevalent in countries with different levels of economic development vary. Shigella sonnei is the primary cause of dysentery in industrialized regions such as Europe and North America, and its prevalence is also increasing in countries such as Asia and Latin America. The disease caused by Shigella sonnei is characterized by bloody diarrhea, fever, and abdominal pain, which can hinder growth and development in children and even be life-threatening.

[0003] Shigella sonnei is primarily transmitted through contaminated water, food, flies, and fingers. There is no specific food vector; any food can cause infection. Large-scale outbreaks of Shigella sonnei infections have occurred in most countries. Antimicrobial drugs remain the primary means of preventing and controlling Shigella sonnei. Ampicillin, streptomycin, tetracycline, sulfonamides, azithromycin, and quinolones are the primary drugs used to inhibit Shigella sonnei. However, over time, due to long-term and irregular use of antibiotics, Shigella sonnei has developed resistance to these drugs. In 2015, Ma et al. first reported the emergence of Shigella sonnei strains highly resistant to azithromycin and third-generation cephalosporins. Ayele et al. isolated Shigella sonnei in Ethiopia harboring multiple resistance genes, with all isolates being completely resistant to amoxicillin and erythromycin. The emergence of drug-resistant bacteria poses a significant threat to human health. Therefore, there is a desire to find alternatives to antimicrobial drugs for pathogen control.

[0004] Bacteriophages are becoming an alternative to antibiotics due to their specificity, low cost, and low drug resistance. Studies have been conducted using bacteriophages to control the growth of pathogenic bacteria in food. Zhou et al. demonstrated that the bacteriophage vB-LmoM-SH3-3 has a strong inhibitory effect on Listeria monocytogenes in salmon and orange juice. Some bacteriophages have also been successfully used as commercial preparations to inhibit food pathogens, such as the bacteriophage preparation EcoShield launched by Intralytix in the United States. TM 、EcolicidePX TM 、SalmoFresh TM PhageGuard S TMetc., which are used to control Escherichia coli O157:H7 on the surface of vegetables, meat and food processing equipment, and Salmonella in foods such as chicken and beef, but there is no bacteriophage specifically for Shigella sonnei. Summary of the Invention

[0005] The object of the present invention is to provide a bacteriophage vB_SsP_LW that can lyse Shigella sonnei.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a Shigella sonnei phage vB_SsP_LW, which is deposited in Guangdong Provincial Microbial Culture Collection Center, with the address being: 5th Floor, Building 59, Compound 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, with a deposit date of June 17, 2025, and a deposit number of GDMCC No: 66526-B1.

[0008] The present invention provides the use of the Shigella sonnei bacteriophage vB_SsP_LW in preparing a bactericidal product for inhibiting Shigella sonnei.

[0009] Preferably, the infection multiplicity of the Shigella sonnei phage vB_SsP_LW to the host bacteria is 0.1 to 0.01, and the titer of the phage solution is ≥10 9 PFU / mL.

[0010] Preferably, the growth pH of the Shigella sonnei phage vB_SsP_LW is 6.0-11.0, and the growth temperature is 4-60°C.

[0011] The invention provides an antibacterial agent with Shigella sonnei bacteriophage vB_SsP_LW as a main active ingredient.

[0012] The present invention also provides applications of the Shigella sonnei bacteriophage vB_SsP_LW, including the following aspects: food preservation, biology and medicine, animal breeding and animal feed.

[0013] Beneficial effects:

[0014] The bacteriophage vB_SsP_LW of the present invention has the characteristics of high titer and short incubation period. Under the optimal MOI conditions, the titer of bacteriophage vB_SsP_LW is ≥10 9PFU / mL. The bacteriophage vB_SsP_LW of the present invention has good environmental stability. The potency remains stable in the range of 4 to 60°C. When the temperature reaches 70°C, the phage still has high activity. In the range of pH 6.0 to 11.0, its potency changes steadily. The bacteriophage vB_SsP_LW of the present invention has a strong antibacterial effect on Shigella sonnei under different MOI conditions, and can achieve the antibacterial purpose by inhibiting the formation of biofilm of Shigella sonnei. It has strong application potential in food preservation, biology and medicine, animal husbandry and animal feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 These are the plaques formed by phage vB_SsP_LW on double-layer agar plates.

[0016] Figure 2 This is a transmission electron micrograph of bacteriophage vB_SsP_LW.

[0017] Figure 3 This is the genotype identification diagram of bacteriophage vB_SsP_LW.

[0018] Figure 4 This is the one-step growth curve of bacteriophage vB_SsP_LW.

[0019] Figure 5 This is the stability result of bacteriophage vB_SsP_LW under different temperature and pH conditions.

[0020] Figure 6 The results of chloroform sensitivity (left) and UV stability (right) of bacteriophage vB_SsP_LW are shown.

[0021] Figure 7 This is a diagram showing the inhibitory effect of bacteriophage vB_SsP_LW on biofilm.

[0022] Figure 8 This is a diagram showing the in vitro antibacterial ability of bacteriophage vB_SsP_LW.

[0023] Figure 9 This is a diagram showing the inhibitory effect of bacteriophage vB_SsP_LW on Shigella sonnei in milk.

[0024] Preservation Instructions

[0025] Shigella sonnei phage vB_SsP_LW, the taxonomic name of the phage is: Shigella sonneiphage, deposited in Guangdong Microbial Culture Collection Center, address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, the deposit date is: June 17, 2025, the deposit number is: GDMCC No: 66526-B1. DETAILED DESCRIPTION

[0026] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1 Phage Isolation and Screening

[0028] Water samples were collected from a sewage treatment plant. CaCl2 solution was added dropwise to 40 mL of the liquid sample to a final concentration of 1 mmol / L. The sample was allowed to stand overnight. The supernatant was collected and filtered through a 0.45 μm filter membrane at 4°C. The filtrate was stored at 4°C for later use. 5 mL of the filtrate was mixed with 100 μL of the host bacteria (Shigella sonnei standard strain ATCC 29930, OD 600nm ≈0.6), add an equal volume of 2×LB liquid medium, mix thoroughly, and culture at 37°C overnight. The culture solution was centrifuged at 4°C and 8000r / min for 10min, and the supernatant was filtered with a 0.22μm filter membrane to obtain a titer of about 10 9 The phage enrichment solution with a concentration of PFU / mL was stored at 4°C for future use.

[0029] Phages were isolated using the double-layer plate method. The phage enrichment solution was diluted 10-fold with SM buffer, and 200 μL of phage enrichment solution with different dilution multiples was taken and incubated with 200 μL of host bacteria solution at room temperature for 10 minutes, and then mixed with 10 mL of LB semi-solid medium and poured into the prepared LB solid medium. After solidification, it was inverted and cultured at 37°C for 8 hours. Observe whether there are plaques. Pick a single plaque and place it in 20 mL of LB liquid medium, then add 200 μL of host bacteria (OD 600nm ≈0.6), shake culture overnight, centrifuge at 4°C, 8000r / min for 10min, filter the supernatant with a 0.22μm filter membrane to obtain phage purification solution, and observe the morphology of phage plaques using the double-layer plate method. Repeat this operation until the morphology of the phage plaques is consistent (size and shape). The results show that (such as Figure 1 ), phage vB_SsP_LW formed clear and transparent circular plaques with a diameter of about 9.98 mm on the double-layer plate.

[0030] Example 2 Determination of the optimal multiplicity of infection

[0031] 200 μL of purified phage solution and 2 mL of Shigella sonnei culture (OD ≈ 0.6) were added to 20 mL of LB liquid medium and cultured until the liquid clarified. The cells were centrifuged at 8000 rpm for 10 min at 4°C. The supernatant was filtered through a 0.22 μm filter to obtain a phage enrichment solution. The phage enrichment solution was mixed with Shigella sonnei culture (OD ≈ 0.6) at different multiplicities of infection (MOI = 100, 10, 1, 0.1, 0.01, 0.001, 0.0001) and incubated for 10 min. The titer at different MOI conditions was determined using the double-layer plate method (phage titer = number of plaques × dilution factor × 5).

[0032] The optimal multiplicity of infection of phage vB_SsP_LW is shown in Table 1. When MOI of phage vB_SsP_LW is 0.1, the phage titer reaches the maximum, so the optimal multiplicity of infection is 0.1.

[0033] Table 1

[0034]

[0035] Example 3: Phage morphology observation

[0036] 1) Phage Concentration: To 30 mL of phage solution, DNase I and RNase A were added to a final concentration of 1 μg / mL each. The mixture was thoroughly mixed and placed on ice for 30 min. NaCl solution was then added to a final concentration of 1 mol / L. The mixture was thoroughly mixed and placed on ice for 1 h. PEG-8000 was then added to a final concentration of 10% (w / v). The mixture was allowed to stand at 4°C overnight. The mixture was centrifuged at 8000 rpm at 4°C for 20 min. The pellet was resuspended in 500 μL of SM buffer to obtain the phage concentrate.

[0037] 2) Phage negative staining with phosphotungstic acid: 10 μL of phage concentrate was added dropwise to a copper grid, allowed to dry for 10 min, excess liquid was removed with filter paper, 10 μL of 2% phosphotungstic acid was added dropwise and stained for 3 min, excess stain was removed with filter paper, and the grid was dried overnight. The phage morphology was observed using a transmission electron microscope.

[0038] like Figure 2 As shown, the head of the phage is a typical icosahedral structure, about 60 nm long, and the tail is a non-contractible structure about 40 nm long. Based on the morphology, the phage vB_SsP_LW belongs to the Podoviridae family of the Caudovirales order.

[0039] Example 4 Identification of the Genome Type of Bacteriophage vB_SsP_LW

[0040] According to the extraction of genes from the viral genome, the extracted DNA solution was treated with DNase I enzyme, RNase A enzyme and MungBean Nuclease enzyme in a water bath at 37°C for 30 minutes. The control group was not treated and the band results were observed by 1% nucleic acid agarose gel electrophoresis. Figure 3 As shown (Note: M: Maker, 1: control group, 2: Mung Bean Nuclease group, 3: RNA group, 4: DNA group), bacteriophage vB_SsP_LW is double-stranded DNA.

[0041] Example 5 Whole genome sequencing and analysis of bacteriophage vB_SsP_LW

[0042] The phage genome was sequenced by Beijing Novogene Technology Co., Ltd. The phage vB_SsP_LW is a linear double-stranded DNA (dsDNA) genome measuring 40,084 base pairs (SEQ ID NOs. 1 to 5, respectively). The genome contains 49% G+C content and contains no repetitive regions. The phage vB_SsP_LW genome encodes 48 open reading frames and lacks virulence or resistance genes.

[0043] Example 6: One-step growth curve assay

[0044] Take 2 mL of host bacterial solution (OD 600nm ≈0.6) and phage solution at the optimal MOI (0.1) were incubated at room temperature for 10 min, the supernatant was discarded, and the precipitate was resuspended three times with LB liquid medium. The supernatant was discarded to remove the phage that was not adsorbed to the host bacteria, and 20 mL of LB liquid medium was added. The culture was shaken and cultured at 37°C. 1 mL of the culture solution was centrifuged every 10 min, and the supernatant was filtered through a 0.22 μm filter membrane to determine the phage titer in the supernatant.

[0045] The one-step growth curve of bacteriophage vB_SsP_LW is shown in the figure below. Figure 4 As shown in the figure, the incubation period of this phage is short, 10 minutes, with an explosive period from 10 to 70 minutes, with a high burst volume of 232 PFU / mL. After 70 minutes, it enters a plateau period.

[0046] Example 7: Temperature and pH stability

[0047] 1) Take 1 mL of phage solution and treat it in a water bath at 4℃, 24℃, 37℃, 50℃, 60℃, 70℃, and 80℃ for 1 hour, and determine its titer using the double-layer plate method. Figure 5 As shown in a, the titer of the phage remains stable at 4-60°C. When the temperature is >60°C, the titer of the phage begins to decrease and the stability weakens.

[0048] 2) Add 900 μL of LB liquid medium with different pH values ​​(pH 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0) to 100 μL of phage solution, let it stand at room temperature for 1 hour, and measure its titer. Figure 5 As shown in Figure b, the optimal pH range of the phage is pH 6.0-11.0. When the pH is less than 6.0, as the acidity increases, the phage activity decreases until it loses its biological activity. When the pH is greater than 11.0, the phage quickly loses its activity.

[0049] Example 8: Chloroform Sensitivity

[0050] In the chloroform group, the phage solution was mixed with chloroform at a ratio of 1:100 (v / v), allowed to stand for 30 minutes, and the aqueous phase was taken and diluted with SM buffer in a gradient manner. Chloroform was not added to the blank group, and the titers of the two groups were determined using the double-layer plate method. Figure 6 As shown on the left, after chloroform treatment, the phage titer is still >10 9 PFU / mL, indicating that bacteriophage vB_SsP_LW is not sensitive to chloroform.

[0051] Example 9: UV sensitivity

[0052] Five experiments were set up. 10 mL of phage solution was spread on a culture dish and irradiated at a distance of 30 cm from the ultraviolet lamp for 0 min, 15 min, 30 min, 45 min and 60 min, respectively. The titer was measured using the double-layer plate method. The results are as follows. Figure 6 As shown on the right, after 15 minutes of UV irradiation, the phage titer decreased slightly, from 9.8 lg (PFU / mL) to 7.7 lg (PFU / mL). After 30 minutes, the titer dropped to 4.3 lg (PFU / mL). After 60 minutes of continuous irradiation, the phage still had biological activity, with the titer remaining at around 3.4 lg (PFU / mL). This indicates that the phage has good tolerance to UV light and can survive for a long time under UV irradiation.

[0053] Example 10: Inhibitory effect on biofilm

[0054] The ability of phage to inhibit biofilm was verified using a 96-well plate. 100 μL of LB liquid medium was added to the 96-well plate, and the host bacterial culture was added to the phage-treated group and cultured to an OD of 0. 600nm≈0.6, then add 50 μL of phage solution according to the optimal MOI (0.1), the control group only adds bacterial solution, and the blank group does not add bacterial solution and phage solution, and culture for 12h, 24h, 36h and 48h, carefully aspirate the liquid with a 1mL sterile syringe, repeatedly wash three times with physiological saline, add 200 μL of glutaraldehyde to fix for 30min after drying, aspirate the excess glutaraldehyde, add 200 μL of 1% (w / v) crystal violet solution to stain for 30min, wash three times with sterile PBS buffer, dry, add 200 μL of 33% (v / v) glacial acetic acid solution to dissolve for 30min, and measure its OD 600nm value.

[0055] The results of biofilm inhibition ability are as follows Figure 7 As shown in the figure, compared to the control group, the phage-treated group inhibited bacterial biofilm formation by approximately 78%, 83%, 88%, and 93% at different time points, respectively. Once formed, bacterial biofilms enhance their resistance to adverse environments and allow them to stubbornly adhere to surfaces, making them difficult to eradicate and complicating bacterial control. These results demonstrate that phages can effectively inhibit bacterial biofilm formation.

[0056] Example 11: In vitro antibacterial ability

[0057] The antibacterial ability of phages in LB liquid medium was verified using a 96-well plate method. 100 μL of LB liquid medium was added to each well. 50 μL of bacterial solution and 50 μL of phage solution were added to the phage treatment group at different MOIs (MOI = 10, 1, 0.1, 0.01, 0.001). 50 μL of bacterial solution was added to the control group, and only LB liquid medium was added to the blank group. Their OD values ​​were measured every 1 hour. 600nm The value was measured continuously for 12 hours.

[0058] The results are as follows Figure 8 As shown, as the culture time increases, the OD 600nm The concentration of Shigella sonnei bacteria continued to increase. At 12h, OD 600nm When the MOI was higher than 1.2, the phages at each MOI could significantly inhibit the growth of Shigella sonnei. The OD values ​​of all phage-treated groups in the first 9 hours were 600nm The phage treatment group under the best MOI=0.1 condition showed a slight increase in 9-10h, but the OD value of the bacterial solution was still 600nm The results showed that the value of β-actin was controlled at about 0.2, which provided a strong basis for the antibacterial application of bacteriophage vB_SsP_LW.

[0059] Example 12: Freshness preservation of milk

[0060] Add 100 μL of Shigella sonnei culture solution (OD 600nm ≈0.6), 100 μL phage solution was added according to different MOI (MOI=10, 1, 0.1, 0.01), the blank group only had sterile milk, and the control group was added with Shigella sonnei solution in milk, and 100 μL was spread on LB solid medium every day for colony counting.

[0061] The results are as follows Figure 9 As shown in the results, compared with the control group, the phage-treated groups at different MOIs all had an inhibitory effect on Shigella sonnei in milk. The best inhibitory effect was achieved at MOI = 10, which provides experimental evidence for the preservation effect of phage in milk.

[0062] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A Shigella sonnei phage (Shigella sonnei phage) vB_SsP_LW, which was deposited in Guangdong Microbial Culture Collection Center, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on June 17, 2025, with the deposit number: GDMCC No: 66526-B1.

2. Use of the Shigella sonnei phage vB_SsP_LW according to claim 1 in the preparation of a bactericidal product for inhibiting Shigella sonnei.

3. The use according to claim 2, characterized in that The infection multiplicity of the Shigella sonnei phage vB_SsP_LW to the host bacteria is 0.1 to 0.01, and the titer is ≥10 9 PFU / mL.

4. The use according to claim 3, characterized in that The growth pH of the Shigella sonnei phage vB_SsP_LW is 6.0-11.0, and the growth temperature is 4-60°C.

5. An antibacterial agent comprising the Shigella sonnei phage vB_SsP_LW according to claim 1 as a main active ingredient.

6. Use of the Shigella sonnei phage vB_SsP_LW according to claim 1 or the antibacterial agent according to claim 5, characterized in that: The applications include the following: food preservation, biology and medicine, animal breeding and animal feed.

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