Highly lytic shigella phage and uses thereof

Abstract

This invention provides a highly lytic Shigella phage and its applications, belonging to the field of bioengineering technology. The phage is named vB-SsoS-Q1, classified as Shigella sonnei phage, and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 45414. This phage is a double-stranded DNA phage with an icosahedral head approximately 75 nm long and 70 nm wide, possessing a retractable tail. It belongs to the class Tailed Phages, with a full-length gene of 50326 bp. It does not contain virulence or drug resistance genes, exhibits highly efficient lytic ability, and can lyse broadly against Shigella and Escherichia coli. This phage can be applied to prevent and treat bacterial diarrhea in livestock and poultry, for the biopreservation of fresh meat, dairy products, and other foods, effectively controlling contamination by multidrug-resistant Shigella, and providing a safe and efficient new solution to replace antibiotics.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, and specifically relates to a highly lytic Shigella phage and its applications. Background Technology

[0002] Shigella is a type of Gram-negative pathogenic bacterium. It is not only one of the core pathogens causing bacterial diarrhea in livestock and poultry, but also widely prevalent in poultry and livestock farming environments, leading to stunted growth and reduced survival rates, causing huge economic losses to the livestock industry. At the same time, this bacterium is also a common foodborne pathogen, widely distributed in dairy products, chicken, fruits and other foods. It can be transmitted through the fecal-oral route, easily contaminating fresh meat, dairy products and other foods as well as the farming environment, posing a serious threat to public food safety and human health.

[0003] Currently, antibiotics remain the primary means of preventing and controlling Shigella infection in the aquaculture industry and controlling Shigella contamination in food and the environment. However, the long-term irrational use and even abuse of antibiotics has led to the continuous emergence of multidrug-resistant Shigella strains and a continuously expanding resistance spectrum. Clinical isolates have shown high resistance to many commonly used antibiotics, resulting in a significant decline in the effectiveness of traditional antibiotics and becoming a prominent problem in the aquaculture industry and public health.

[0004] Bacteriophages are a class of viruses capable of specifically infecting and lysing bacteria. They utilize the self-replication of host bacteria to produce a large number of progeny phages, which then lyse the bacteria to achieve a killing effect. Their lytic ability is unaffected by bacterial drug resistance or the environment, and they possess advantages such as strong host specificity, low likelihood of cross-resistance, and minimal impact on the host flora and environment. They are considered potential biological agents for the prevention and control of bacterial diseases and bacterial contamination, serving as alternatives to or adjuncts to antibiotics. Therefore, developing a highly efficient, broad-spectrum, safe, and stable Shigella phage is of significant practical importance for ensuring the development of the aquaculture industry and food safety. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of the prior art by providing a highly lytic Shigella phage and its applications.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: The first aspect of this invention provides a highly lytic Shigella phage, characterized in that: the phage is named vB-SsoS-Q1, classified as Shigella sonnei phage, and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 45414, and the deposit date is February 10, 2023.

[0007] Furthermore, this Shigella phage vB-SsoS-Q1 is a double-stranded DNA phage with an icosahedral head and a retractable tail. It belongs to the class Tailed Phages and has a full-length gene of 50326 bp. It does not possess virulence genes or drug resistance genes.

[0008] A second aspect of the present invention provides a microbial preparation comprising the above-described highly lytic Shigella phage vB-SsoS-Q1.

[0009] The third aspect of the present invention provides any of the following applications of the above-described highly lytic Shigella phage or the above-described microbial preparation: 1) To prepare drugs for the prevention and / or treatment of diseases caused by Shigella in livestock and poultry; 2) Prepare additives for the prevention and / or treatment of diseases caused by Shigella in livestock and poultry; 3) Prepare products for killing or inhibiting Shigella; 4) Prepare products for killing or inhibiting Escherichia coli.

[0010] Furthermore, the Shigella is a multidrug-resistant Shigella.

[0011] A fourth aspect of the present invention provides an environmental disinfectant comprising the above-mentioned highly lytic Shigella phage vB-SsoS-Q1 or the above-mentioned microbial preparation.

[0012] The fifth aspect of the present invention provides a food preservative comprising the above-mentioned highly lytic Shigella phage vB-SsoS-Q1 or the above-mentioned microbial preparation.

[0013] The sixth aspect of the present invention provides a bacteriophage drug formulation, the active ingredient of which includes the above-mentioned highly lytic Shigella phage vB-SsoS-Q1 or the above-mentioned microbial preparation.

[0014] The beneficial effects of this invention are as follows: 1. The bacteriophage vB-SsoS-Q1 of this invention does not carry any known virulence genes or antibiotic resistance genes, fundamentally ensuring that when this bacteriophage is used as a drug, food additive, or environmental disinfectant, it will not mediate the horizontal transfer of resistance genes, nor will it produce additional toxic side effects on the body. It has high biosafety and fundamentally avoids problems such as gene contamination and excessive residues. 2. The bacteriophage vB-SsoS-Q1 of this invention possesses stable and high titer, ultra-low optimal multiplicity of infection, short latency and rapid outbreak period, and high lysis rate, among other biological characteristics. In practical applications, only a trace amount is needed to rapidly induce an exponential lysis reaction in complex environments, efficiently lysing the host bacteria in a short time, blocking toxin production and transmission, and ensuring the stability and sustainability of the bactericidal control effect; 3. The bacteriophage vB-SsoS-Q1 of this invention can not only efficiently lyse multidrug-resistant Shigella, but also lyse various types of Escherichia coli, thus simultaneously solving the problem of dual contamination by Shigella and Escherichia coli. Therefore, this bacteriophage preparation is applicable to the prevention and control of livestock and poultry diseases caused by various pathogens and the control of various types of bacterial contamination, with a wide range of applications. Furthermore, bacteriophage vB-SsoS-Q1 exhibits strong lytic activity against multidrug-resistant Shigella, indicating that the lytic effect of the bacteriophage is completely unaffected by bacterial resistance. It can effectively kill multidrug-resistant Shigella, which is currently a difficult problem in the current breeding industry and clinical treatment, and provides a direct and effective solution to overcome the problem of antibiotic resistance. 4. The bacteriophage vB-SsoS-Q1 of this invention has good tolerance to temperature, pH and light. Its good physicochemical stability enables it to adapt to different practical application conditions such as livestock and poultry breeding environment, food processing and storage, and environmental disinfection. It can play a role without complicated protection measures, which reduces the operating cost of practical application. 5. The bacteriophage and microbial preparations containing the bacteriophage of the present invention can be prepared as drugs and feed / drinking water additives for the prevention and treatment of Shigella diseases in livestock and poultry, effectively solving the problem of bacterial diarrhea in livestock and poultry, improving the clinical intervention effect, and reducing breeding losses; it can be used as an environmental disinfectant for the control of Shigella and Escherichia coli contamination in breeding and food processing environments, reducing the risk of pathogen contamination from the source and strengthening the biosafety defense line in the breeding and processing links; it can also be used as a food preservative for the biological preservation of fresh meat, dairy products and other foods, realizing the whole-chain control of multidrug-resistant Shigella from the breeding end to the food processing end; the bacteriophage is adapted to the actual production and disease diagnosis and treatment needs of the breeding industry, has no special restrictions on use, can be seamlessly integrated with existing processes, is easy to promote and apply, and provides practical technical support for the comprehensive prevention and control of Shigella. Attached Figure Description

[0015] Figure 1 Transmission electron microscopy (TEM) image of bacteriophage vB-SsoS-Q1; Figure 2 This is a complete genome diagram of bacteriophage vB-SsoS-Q1. Detailed Implementation

[0016] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above description. In the following embodiments, reagents and instruments not specifically mentioned are commercially available, and experimental operations not specifically mentioned are performed according to the manufacturer's instructions or conventional techniques in the art. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the present invention; the endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered as specifically disclosed herein; unless otherwise specified in the following embodiments of the present invention, the temperature is at room temperature. Normal temperature refers to the natural room temperature conditions in all four seasons, without any additional cooling or heating treatment. Normal temperature is generally controlled between 10-30℃, and preferably between 15-25℃.

[0017] This invention provides a highly lytic Shigella phage, named vB-SsoS-Q1, classified as Shigella sonnei phage, and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 45414, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. This phage is a double-stranded DNA phage with an icosahedral head approximately 75 nm long and 70 nm wide, possessing a retractable tail. It belongs to the class Tailed Phages, with a full-length gene of 50326 bp, and does not contain virulence or drug resistance genes.

[0018] The present invention also provides a microbial preparation comprising the highly lytic Shigella phage vB-SsoS-Q1; the microbial preparation further comprises pharmaceutically, food or feed-acceptable excipients.

[0019] The present invention also provides that the above-mentioned highly lytic Shigella phage or the above-mentioned microbial preparation can be used to prepare drugs for the prevention and / or treatment of diseases caused by Shigella in livestock and poultry, to prepare additives for the prevention and / or treatment of diseases caused by Shigella in livestock and poultry (such as feed additives, drinking water additives and meat product cleaners, etc.), to prepare products for killing or inhibiting Shigella (such as environmental disinfectants, food preservatives, etc.), and to prepare products for killing or inhibiting Escherichia coli.

[0020] In some preferred embodiments, the Shigella is a multidrug-resistant Shigella.

[0021] The present invention also provides an environmental disinfectant comprising the above-mentioned highly lytic Shigella phage vB-SsoS-Q1 or the above-mentioned microbial preparation.

[0022] The present invention also provides a food preservative comprising the above-mentioned highly lytic Shigella phage vB-SsoS-Q1 or the above-mentioned microbial preparation.

[0023] The present invention also provides a phage drug formulation, wherein the active ingredient of the drug formulation includes the above-mentioned highly lytic Shigella phage vB-SsoS-Q1 or the above-mentioned microbial preparation.

[0024] In this invention, the phage drug formulation further includes the aforementioned pharmaceutically acceptable excipients, and the dosage form of the phage drug formulation includes injections, oral preparations, or sprays, wherein the "active ingredient" refers to the key ingredient for treating, alleviating, or preventing a target effect or disease.

[0025] The culture medium and reagent formulations and preparation methods involved in this invention are as follows: LB liquid medium: 10g trypsin, 5g yeast extract, 10g NaCl, add H2O to... 1000 mL, pH 7.4, autoclaved at 121°C for 20 min; LB semi-solid medium: Add 7g of agar powder to every 1000mL of liquid LB medium and autoclave at 121℃ for 20min.

[0026] LB solid medium: Add 15g of agar powder to every 1000mL of liquid LB medium and autoclave at 121°C for 20min.

[0027] SS medium: 5.0g beef meal, 5.0g peptone, 10.0g lactose, 8.5g bile salt No. 3, 8.5g sodium citrate, 8.5g sodium thiosulfate, 1.0g ferric citrate, 0.025g neutral red, 0.00033g brilliant green, 17.0g agar, dissolved in 1000ml distilled water, heated to boiling, no need for autoclaving, cooled to 45-50℃, poured into plates under aseptic conditions, and allowed to solidify completely before use; SM buffer: 8.5g sodium chloride, 2g magnesium sulfate, 50mL 1mol / L Tris-HCl, 0.25g gelatin, 1000mL distilled water, autoclaved at 121℃ for 20min; PBS buffer: 85.61g Na2HPO4·2H2O, 43.41g KH2PO4, 8.0g NaCl, 0.2g KCl. Dissolve the above substances in 800ml distilled water and bring the volume to 1L with ultrapure water.

[0028] It should be noted that: This application sampled the internal organs of sick animals with diarrhea in farms in Binzhou, Qingdao, Weifang, Linyi and other places in Shandong Province. Fifteen Shigella isolates were obtained by existing isolation technology and 16sRNA sequencing (the results are shown in Table 1). The pathogenicity of the 15 Shigella isolates was then determined by experiments (the results are shown in Table 2). Finally, the isolate with the strongest pathogenicity was selected as the host bacterium of this application embodiment and named Y12. It can be preserved in an environment of -80℃ using the glycerol suspension preservation method.

[0029] Table 1. Length and homology of 16S RNA amplification gene fragments from fifteen Shigella isolates. The pathogenicity assay for Shigella was performed as follows: 15 Shigella isolates were inoculated into LB liquid medium and cultured overnight at 37°C. The cultures were then centrifuged at 4000g and 4°C for 10 min. The supernatant was discarded, and the precipitate was resuspended by pipetting with sterile PBS buffer. The concentration of the bacterial suspension was calculated using the plate count method. The suspension was then aliquoted and stored at 4°C for later use. Thirty-day-old Kunming mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into several experimental groups, with five mice in each group. Water and feed were withheld the night before the experiment. The mice were divided into groups of 1 x 10-1. 8 Each mouse was inoculated with CFU via gavage. After inoculation, normal feeding and management were resumed. Finally, the mice's mental state and fecal condition were continuously monitored for 24 hours, and the following evaluation criteria were used: Pathogenicity (+++): Severe diarrhea, with watery or bloody stools, and no tendency for spontaneous recovery within 24 hours; Pathogenicity (++): Mild diarrhea symptoms, but basically recovered spontaneously within 24 hours; Pathogenicity (+): Basically no diarrhea symptoms.

[0030] Table 2. Results of pathogenicity testing of fifteen Shigella strains. The Shigella host strain Y12 isolated above was subjected to drug resistance testing using the antimicrobial susceptibility disc method. The antimicrobial susceptibility discs included: gentamicin, spectinomycin, amikacin, streptomycin, ampicillin, florfenicol, norfloxacin, enrofloxacin, ciprofloxacin, doxycycline, lincomycin, clindamycin, cefotaxime, cefadroxil, vancomycin, trimethoprim-sulfamethoxazole, polymyxin, tetracycline, neomycin, azithromycin, erythromycin, and penicillin G. The results are shown in Table 3 below.

[0031] Table 3 Results of drug resistance assay for Shigella host strain Y12 The criteria for judging the results in Table 3 are as follows: d > 20 mm is highly sensitive (S); 10 < d ≤ 20 mm is moderately sensitive (I); d ≤ 10 mm is resistant (R). As shown in Table 3, Shigella host strain Y12 is resistant to ampicillin, doxycycline, trimethoprim-sulfamethoxazole, lincomycin, clindamycin, trimethoprim-sulfamethoxazole, vancomycin, tetracycline, polymyxin, erythromycin, and penicillin G, but sensitive to spectinomycin, amikacin, florfenicol, norfloxacin, enrofloxacin, ciprofloxacin, and cefotaxime. This indicates that the host strain in the examples of this application has acquired multidrug resistance. Example 1

[0032] Isolation and purification of Shigella phage vB-SsoS-Q1 Sample collection Wastewater was collected from manure pits in farms in Shandong Province. The supernatant was collected after centrifugation at 12,000 r / min for 10 min. The supernatant was filtered through a 0.22 μm microporous membrane, and the filtrate was collected for the isolation of bacteriophages.

[0033] 2. Enrichment of bacteriophages Add 10 mL of the above filtrate to 10 mL of sterilized LB liquid medium, then add a logarithmic growth phase host bacterial suspension (Shigella Y12) at a 1% inoculation rate and mix thoroughly. Incubate overnight at 37°C with shaking at 180 r / min. Centrifuge the culture at 8000 r / min for 15 min, and filter the supernatant through a 0.22 μm filter membrane to obtain the phage enrichment stock solution. Store at 4°C for later use.

[0034] 3. Isolation of bacteriophages Spread 100 μL of host bacteria Y12 suspension onto an LB agar plate, then add 10 μL of phage enrichment stock solution. Incubate the plate overnight at 37°C and observe for plaque formation. Using sterile ophthalmic forceps, pick up a single plaque and place it in 10 mL of LB liquid medium, then add 100 μL of host bacteria Y12 suspension. Incubate overnight at 37°C with shaking at 180 rpm. Filter the culture medium through a 0.22 μm filter membrane, then serially dilute the filtrate 10-fold using SM buffer. Mix 0.1 mL of each concentration gradient phage dilution with 0.2 mL of host bacteria Y12 suspension, add 4 mL of LB semi-solid medium cooled to 55°C, mix well, and immediately spread onto a plate with a bottom layer of LB solid medium. After solidification, invert the plate and incubate at 37°C for 4-6 hours, observing plaque growth.

[0035] 4. Purification of bacteriophages On a bilayer agar plate containing phage plaques, pick a single plaque with a sterile pipette tip and place it in SM buffer. Incubate overnight at 4°C to allow the phage to fully dissolve in the SM buffer. Perform a 10-fold serial dilution of the SM buffer containing the phage using sterile physiological saline. Take 0.1 mL of each dilution and mix it thoroughly with 0.2 mL of the host bacterium Y12 suspension. Incubate the mixture at 37°C for 5 min, then add 4 mL of LB semi-solid medium cooled to 55°C. Mix well and immediately pour the mixture onto a plate with LB solid medium at the bottom. After solidification, invert the plate and incubate at 37°C for 6-8 h. Once the phage plaques are clearly visible, pick a single plaque with the same morphology and repeat this step 3-5 times until the phage plaques observed on the bilayer agar plate are completely uniform in morphology and size, thus obtaining purified phage.

[0036] Bacteriophage proliferation Pick 20-30 purified phage plaques with a sterilized pipette tip and place them in 2 mL of SM buffer. Incubate overnight at 4°C to allow the phages to fully dissolve in the SM buffer, preparing purified phage solution. Then, mix 1 mL of host bacterium Y12 suspension with LB liquid medium at a 1:5 ratio and incubate at 37°C with shaking until the bacteria reach the logarithmic growth phase. Add 1 mL of the purified phage solution and incubate overnight at 37°C in a shaker for phage amplification. Centrifuge the overnight culture at 8000 rpm for 15 min, and filter the supernatant through a 0.22 μm filter to obtain the purified phage stock solution, which should be stored at 4°C for later use. Example 2

[0037] This embodiment uses phosphotungstic acid negative staining to observe the morphology of bacteriophage vB-SsoS-Q1. Take 15µL of the phage vB-SsoS-Q1 stock solution prepared in Example 1 and drop it onto a copper grid. Let it stand for 15 min, blot off the excess liquid with filter paper, then add a drop of 2% phosphotungstic acid to the copper grid and stain for 10 min. After drying, observe its morphology under an electron microscope. Figure 1 As shown.

[0038] Depend on Figure 1 The electron micrograph shows that the bacteriophage vB-SsoS-Q1 virus particle consists of an icosahedral head and a retractable tail. The head is about 75 nm long and 70 nm wide, and the tail is about 140 nm long. According to the latest classification system of the International Committee on Taxonomy of Viruses, this bacteriophage belongs to the class Tailed bacteriophages, and its morphological characteristics are consistent with the morphological description of myotail bacteriophages in the traditional classification. Example 3

[0039] Whole genome sequencing and analysis of bacteriophage vB-SsoS-Q1 1) Extract and purify the genomic DNA of the bacteriophage, and use a hybrid sequencing strategy combining Illumina and PacBio to construct short-read and long-read libraries for dual-platform sequencing to obtain high-precision and cross-repetitive region data simultaneously; 2) After quality filtering of the obtained raw sequencing data (subreads), the long read data after quality control was assembled from scratch using Flye software, and high-precision short read data was used for correction to obtain the complete genome sequence. QUAST was used to evaluate the final genome sequence to confirm the integrity and reliability of the assembly results. 3) Use Prodigal software to predict open reading frames (ORFs) in the genome sequence, set the screening criteria to nucleic acid length ≥100bp, and translate the predicted ORFs into the corresponding amino acid sequences; 4) The amino acid sequences encoded by the ORF were BLASTP aligned in databases such as NCBINr and Swiss-Prot, and functional classification and annotation were performed based on homology. All ORF sequences were also compared with virulence factor databases and antibiotic resistance gene databases. Analysis confirmed that the genome of bacteriophage vB-SsoS-Q1 does not carry any known virulence genes or antibiotic resistance genes.

[0040] Gene sequencing results showed that the bacteriophage vB-SsoS-Q1 is a double-stranded linear DNA with a full genome length of 50326 bp (as shown in SEQ ID: 1 in the sequence listing) and a GC content of 45.21%; it contains 87 open reading frames (ORFs) and does not have any known related virulence genes or drug resistance genes.

[0041] The whole genome sequence of the bacteriophage was submitted to the non-redundant nucleic acid database of the National Center for Biotechnology Information (NCBI) for BLASTN alignment. The alignment results showed that no completely matching sequence with 100% coverage and 100% identity to the bacteriophage vB-SsoS-Q1 of this invention was found in the whole genome of the database.

[0042] Table 4. ORF gene annotation table for phage vB-SsoS-Q1 Example 4

[0043] Biological characteristics analysis of bacteriophage vB-SsoS-Q1 Determination of phage titer Experimental method: The phage vB-SsoS-Q1 stock solution prepared in Example 1 was serially diluted 10-fold with LB liquid medium, and 10 μL of each solution was taken. 5 10 6 10 7 10 8 10 9 10 10 0.1 mL of phage dilution was mixed with 0.2 mL of host bacterium Y12 suspension. After incubating at 37°C for 5 min, 4 mL of LB semi-solid medium cooled to 55°C was added to the mixture. The mixture was then immediately spread onto LB solid medium plates. After solidification, the plates were inverted and incubated at 37°C for 8 h. The number of phage plaques was then counted. Three replicates were performed for each dilution, and the average of the three replicates for each dilution was used for counting.

[0044] The formula for calculating phage titer (PFU / mL) is: number of phage plaques × dilution factor × 10.

[0045] Experimental results: After three independent replicate experiments, the titer of bacteriophage vB-SsoS-Q1 remained stable at 5.42 × 10⁻⁶. 10 The concentration of PFU / mL or higher indicates that bacteriophage vB-SsoS-Q1 has extremely high proliferative capacity and infectious activity.

[0046] Optimal Multiple of Infection Determination Experimental methods: The multiplicity of infection (MOI) in the phage-host assay was calculated as phage concentration / bacterial concentration; the concentration of the host bacterium Y12 cultured to the exponential phase was adjusted to 1.0 × 10⁻⁶. 8 cfu / mL, with multiples of infection (MOI) set at 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100, i.e., a concentration of 10. 4 10 5 10 6 10 7 10 8 10 9 10 10 PFU / mL phage solution was prepared by mixing equal volumes of phage vB-SsoS-Q1 dilution and host bacterial Y12 culture, and incubating at 37℃ with shaking at 180 rpm for 8 h. The culture was then centrifuged at 8000 rpm for 15 min, and the supernatant was collected. The supernatant was filtered through a 0.22 μm filter membrane, and the filtrate was serially diluted 10-fold with SM solution. The phage titers at different multiplicity of infection (MLI) were determined using the double plate method. The MII that produced the highest titer was the optimal MII of infection for the phage. The results are shown in Table 5 below. Table 5. Determination of the optimal multiplicity of infection for phage vB-SsoS-Q1 As shown in Figure 5, when the multiplicity of infection is 0.01, the phage production titer is 4.82 × 10⁻⁶. 10 The PFU / mL reached its peak, thus determining the optimal multiplicity of infection (MOI) for phage vB-SsoS-Q1 to be 0.01. The ultra-low MOI allows for highly efficient control of Shigella with the addition of very small amounts of phage preparations, significantly reducing the amount of phage used and making the cost of large-scale application of phage preparations controllable. Furthermore, the low dose of exogenous phages will not disrupt the normal microbial community balance in the breeding and food processing environments.

[0047] Determination of one-step growth curve of bacteriophage Experimental method: The host bacteria were cultured for about 5 hours to achieve a concentration of 1×10⁻⁶. 7 With a concentration of approximately cfu / mL, 500 μL each of phage liquid and host bacterium Y12 bacterial solution were mixed at the optimal multiplicity of infection (0.01). The mixture was incubated in a 37°C water bath for 5 min, then centrifuged at 12000 rpm for 1 min. The supernatant was discarded, and the precipitate was washed three times with sterile LB liquid medium. 100 mL of LB liquid medium was added to the washed precipitate and mixed thoroughly. The mixture was then incubated in a 37°C shaker. Sampling was performed every 20 min starting from 0 min to determine the phage titer. Three replicates were performed, and the average value was taken. The results are shown in Table 6.

[0048] Table 6. One-step growth curve of phage vB-SsoS-Q1 As shown in Table 6, the titer of bacteriophage vB-SsoS-Q1 remained stable for the first 20 minutes after infection. Then, from 20 to 60 minutes, the titer increased sharply by about four orders of magnitude, reaching its peak at about 80 minutes. After that, it remained relatively stable until 120 minutes. Therefore, the latency period of this bacteriophage is 20 minutes, the outbreak period is 20-60 minutes, and the lysis rate is 6086 pfu / cell.

[0049] Wherein, the amount of cleavage = peak potency / initial potency (potency at 80 minutes / potency at 0 minutes).

[0050] The lysis efficiency of bacteriophages is determined by their incubation period, outbreak period, and lysis rate. A short incubation period of 20 minutes means that after adsorbing Shigella, the bacteriophage can quickly enter the bacterial cell to complete genome replication and progeny assembly. The lysis process is initiated before Shigella has completed a single proliferation cycle, thus preventing pollution or livestock diseases caused by the massive proliferation of pathogens in the environment / food. The short outbreak period indicates that progeny bacteriophages can be released rapidly and in a concentrated manner. The high lysis rate of 6086 pfu / cel means that after a single Shigella is infected, it will quickly release 6086 progeny bacteriophages. These progeny bacteriophages can immediately recognize and adsorb surrounding Shigella, restarting the lysis process and forming an exponential cascade lysis reaction. This reaction mode causes a cliff-like drop in the number of viable Shigella bacteria, which can quickly suppress the number of pathogens in a short period of time and achieve the goal of "rapid bacterial control".

[0051] Determination of bacteriophage temperature stability Experimental method: 500 μL of a solution with a concentration of 1×10⁻⁶ was prepared. 10 Centrifuge tubes containing PFU / mL phage were placed in constant temperature water baths at 40℃, 50℃, 60℃, and 70℃ for 30 min and 60 min, respectively, and samples were taken. The changes in phage titer were determined using the double-layer plate method. The results are shown in Table 7. Table 7. Heat tolerance of bacteriophage vB-SsoS-Q1 As shown in Table 7, the titer of this bacteriophage remained almost unchanged after being exposed to 30℃ and 40℃ for 30 min and 60 min, respectively; after being exposed to 50℃ for 30 min and 60 min, the titer decreased by 2 orders of magnitude; after being exposed to 60℃ for 30 min and 60 min, the titer decreased by 4 orders of magnitude; after being exposed to 70℃ for 30 min, the titer decreased by 6 orders of magnitude; and after being exposed to 70℃ for 60 min, the titer decreased by 7 orders of magnitude. This indicates that the bacteriophage of this application can maintain good activity in the temperature range of 30℃-70℃.

[0052] Determination of pH stability of bacteriophages Experimental method: The pH of LB liquid medium was adjusted to 3, 4, 5, 6, 7, 8, 9, 10, and 11 respectively using concentrated hydrochloric acid and NaOH solution. For each pH value, 200 μL of LB liquid medium was taken, and then 200 μL of a 10% concentration was added. 8 After mixing the phage solution at pfu / mL and incubating at 37℃ for 1 h, 200 μL of phage was taken from each tube and the titer change of phage was determined by the double-layer plate method. Three replicates were set for each pH. The results are shown in Table 8. Table 8 pH tolerance of bacteriophage vB-SsoS-Q1 As shown in Table 8, bacteriophage vB-SsoS-Q1 can maintain its activity in the pH range of 4 to 10, and its activity is most stable in the pH range of 6 to 9, with its titer maintained at a high level. Among them, the bacteriophage exhibits the highest stability under neutral and weakly alkaline conditions (pH 7-8), indicating that its optimal environment range is relatively wide. Example 5

[0053] Detection of lysis profile of bacteriophage vB-SsoS-Q1 In this embodiment, the droplet method was used to determine the lysis effect of bacteriophages on 26 strains of Escherichia coli and 15 strains of Shigella isolated in the laboratory. The strains were obtained from farms in Binzhou, Qingdao, Weifang and Linyi, Shandong Province.

[0054] Experimental method: After taking a sterile cotton swab to collect the bacterial suspension of the test bacteria in the logarithmic growth phase, spread it evenly on an LB solid medium plate. After drying, take 5 μL of phage stock solution and drop it onto the plate. Incubate at 37℃ for 6 hours under inverted conditions and observe whether there are empty spots. The results are shown in Table 9 below.

[0055] Table 9. Lysis spectrum of bacteriophage vB-SsoS-Q1 Note: + indicates that it can be split, and - indicates that it cannot be split.

[0056] As shown in Table 9, bacteriophage vB-SsoS-Q1 can effectively lyse 10 strains of Shigella and 17 strains of Escherichia coli, with a total lysis rate of 65.85% (27 / 41), indicating that bacteriophage vB-SsoS-Q1 is a broad-spectrum bacteriophage with potential application value. Example 6

[0057] Stability testing of bacteriophage vB-SsoS-Q1 under light conditions This embodiment uses a method of directly exposing bacteriophages to sunlight to evaluate the effect of light on bacteriophage activity; Experimental method: The stock solution of phage vB-SsoS-Q1 prepared in Example 1 was dispensed into transparent EP tubes and placed under sunlight for 6 hours (10:00 am to 4:00 pm). Samples were taken every 2 hours during this period, and the titer changes of the sampled phages were determined by the double-layer plate method. The results are shown in Table 10 below. Table 10 Effects of direct sunlight on phage titer As shown in Table 10, the phage titer gradually decreased with increasing illumination time. After 6 hours of irradiation, the titer decreased from the initial 5.2 × 10⁻⁶. 10 PFU / mL decreased to 2.5 × 10⁻⁶9 The concentration of PFU / mL was maintained, but the activity level remained considerable. This result indicates that phage vB-SsoS-Q1 has a certain tolerance to ambient light. However, in practical applications, it is advisable to apply it during periods of weaker sunlight, such as early morning or late evening, to help maintain its optimal activity. Example 7

[0058] Experiment on the bactericidal effect of bacteriophage vB-SsoS-Q1 on a carrier (feces) Sample preparation: The concentration of the host bacterium Y12, cultured to the logarithmic growth phase, was adjusted to 1.0 × 10⁻⁶ using LB liquid medium. 6 cfu / mL; the phage vB-SsoS-Q1 stock solution was diluted with SM dilution buffer to prepare 1×10⁻⁶ solutions. 5 pfu / mL, 1×10 6 pfu / mL and 1×10 7 Three concentrations of PFU / mL dilution were prepared; 10g of fresh rabbit feces were placed in a sterile penicillin bottle, and sterilized by autoclaving before use.

[0059] Experimental methods: The experiment consisted of 4 groups, with 3 replicates in each group; Control group: 1 mL of host bacteria suspension was added to sterile rabbit feces; Experimental group 1: 1 mL of host bacteria suspension and 1 mL of 1×10⁻⁶ bacteria solution were added to sterile rabbit feces. 5 phage dilution at pfu / mL; Experimental group 2: 1 mL of host bacterial suspension and 1 mL of 1×10 pfu / mL phage dilution were added to sterile rabbit feces. 6 phage dilution at pfu / mL; Experimental group 3: 1 mL of phage solution with a concentration of 1×10 pfu / mL was added to sterile rabbit feces. 7 Phage dilution buffer (pfu / mL); after thoroughly mixing all samples, incubate them in a 25°C incubator. Take samples at 0h, 2h, 4h, and 6h after treatment. Dilute the rabbit feces suspension 10-fold, and spread 1ml of the diluted solution onto the surface of LB solid medium. After complete absorption, invert the medium and incubate overnight in a 37°C incubator. Determine the bacterial count using the bacterial plate count method. Repeat the experiment three times to determine the number of viable host bacteria in the rabbit feces. The results are shown below. Table 11. Elimination effect of bacteriophage vB-SsoS-Q1 against Shigella in feces. Table 11 shows that bacteria proliferated significantly in the control group, while all phage treatment groups exhibited significant bactericidal effects. Specifically, when the ratio of phage count to host bacteria in feces was 1:10, after 6 hours of disinfection, the number of viable Y12 host bacteria in the feces decreased from 2.5 × 10⁻⁶. 6 CFU / mL dropped sharply to 4.5 × 10⁻⁶. 2The cfu / mL concentration decreased by four orders of magnitude, indicating that phage vB-SsoS-Q1 has efficient and rapid bactericidal ability in complex organic carriers, and is also low in cost. Example 8

[0060] Experiment on the bactericidal effect of bacteriophage vB-SsoS-Q1 in contaminated fresh meat Chicken breast, beef, and shrimp purchased from a reputable supermarket were used as experimental samples. The fresh meat was stored at 4°C before the experiment. Experimental methods: Sample preparation: Under aseptic conditions, chicken breast, beef, and shrimp were cut into 3×3cm pieces using a sterile knife. 2 Meat slices weighing approximately 2g were placed in sterile petri dishes and irradiated with ultraviolet light for 60 minutes, turning them occasionally to ensure even irradiation and eliminate bacteria on the surface of the fresh meat to obtain sterile meat slices. The sterile meat slices were then immersed in a solution with a concentration of 2×10⁻⁶. 8 Artificially contaminated meat slices were prepared by 5 min of a CFU / mL host bacterium Y12 suspension and then dried in a clean bench for 30 min. Phage treatment and culture: The artificially contaminated meat slices were randomly divided into a treatment group and a control group. Treatment group: 1 mL of phage with a titer of 1×10⁻⁶ was evenly added to the surface of each meat slice. 8 A suspension of bacteriophage vB-SsoS-Q1 at pfu / mL was prepared and evenly spread; the control group was prepared by using an equal volume of sterile physiological saline instead of bacteriophage suspension. Samples were taken and homogenized after 0h, 6h, 12h, and 24h of culture, and the viable cell count was determined. Sampling and bacterial counting: All samples were placed in sterile petri dishes and incubated at 37°C to simulate normal temperature storage and transportation conditions. After 0h, 6h, 12h, and 24h of incubation, one sample was randomly taken from each group and homogenized with phosphate-buffered saline (PBS). Then, 1mL of homogenate was taken and serially diluted with PBS buffer. 100μL of the diluted solution was spread onto SS medium and incubated overnight at 37°C. The bacterial count was determined by the bacterial plate count method. The experiment was repeated three times, and the results are shown in Table 12. Table 12. Current bactericidal effects of bacteriophages against *Rhes* contamination. As shown in Table 12, the number of bacteria in the control group continued to increase within 24 hours, while the number of viable Y12 host bacteria on the surface of all meat samples treated with bacteriophage vB-SsoS-Q1 was significantly inhibited. This indicates that bacteriophage vB-SsoS-Q1 has a rapid and sustained killing effect on Shigella attached to the surface of different types of fresh meat under simulated room temperature storage conditions, demonstrating the potential of this bacteriophage in food preservation. Example 9

[0061] Experiment on the bactericidal effect of bacteriophage vB-SsoS-Q1 in contaminated dairy products This experiment selected three different types of commercial dairy products from legal entities: fresh pure milk, fermented milk, and modified milk as experimental samples. Experimental methods: For each dairy product, a control group and an experimental group were set up; 100 μL of a 1.0 × 10⁻⁶ solution was added to each control and experimental group. 8 CFU / ml host bacteria Y12 suspension was used. Then, based on the determined optimal multiplicity of infection (MOI=0.01), 100 μL of phage vB-SsoS-Q1 suspension was added to the phage treatment group, while an equal volume of sterile SM buffer was added to the control group. The above mixture was added to fresh pure milk, fermented milk, or modified milk, and the volume was adjusted to 10 mL and thoroughly shaken. All samples were placed in a 37℃ incubator for static incubation. At 0 h, 6 h, 12 h, and 24 h of incubation, 500 μL of samples were aseptically taken from each group and serially diluted with PBS buffer. Bacterial counting was performed on SS selective medium using the plate plating method. The experiment was repeated three times, and the results are shown in Table 13. Table 13. Bactericidal effect of bacteriophages on dairy products contaminated with *Rhesus chinensis* Table 13 shows that after culturing at 37℃ for 24 hours, the host strain Y12 in all dairy product control groups proliferated significantly, indicating good growth under these conditions. In contrast, all experimental groups treated with bacteriophage vB-SsoS-Q1 exhibited significant antibacterial effects, with the most prominent effect observed in fermented milk, where the viable bacterial count was reduced to the lowest level. Significant bacterial reduction was also achieved in fresh pure milk and formulated milk, demonstrating that bacteriophage vB-SsoS-Q1 can adapt to different dairy substrates and effectively control Shigella contamination.

[0062] In summary, it should be noted that the above description is only a preferred embodiment of the present invention and should not be used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still make some simple deductions, substitutions, or equivalent substitutions of some technical features for the technical solutions described in the foregoing embodiments without departing from the concept of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A highly lytic Shigella phage, characterized in that: The bacteriophage was named vB-SsoS-Q1, classified as Shigella sonnei bacteriophage, and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 45414.

2. The highly lytic Shigella phage according to claim 1, characterized in that: The Shigella phage vB-SsoS-Q1 is a double-stranded DNA phage with an icosahedral head and a retractable tail. It belongs to the class Tailed Phages and has a full-length gene of 50,326 bp. It does not contain virulence genes or drug resistance genes.

3. A microbial preparation, characterized in that: Includes the highly lytic Shigella phage vB-SsoS-Q1 as described in claim 1 or 2.

4. Any of the following applications of the highly lytic Shigella phage according to any one of claims 1 to 2 or the microbial preparation according to claim 3: To prepare drugs for the prevention and / or treatment of diseases caused by Shigella in livestock and poultry; To prepare additives for the prevention and / or treatment of diseases caused by Shigella in livestock and poultry; To prepare products for killing or inhibiting Shigella sonnei; Prepare products for killing or inhibiting Escherichia coli.

5. The application according to claim 4, characterized in that: The Shigella mentioned is a multidrug-resistant Shigella.

6. An environmental disinfectant, characterized in that: The preparation comprises the highly lytic Shigella phage vB-SsoS-Q1 as described in any one of claims 1 to 2 or the microbial preparation as described in claim 3.

7. A food preservative, characterized in that: The preparation comprises the highly lytic Shigella phage vB-SsoS-Q1 as described in any one of claims 1 to 2 or the microbial preparation as described in claim 3.

8. A bacteriophage drug formulation, characterized in that: The active ingredient of the phage drug formulation includes the highly lytic Shigella phage vB-SsoS-Q1 as described in any one of claims 1 to 2.

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