Listeria bacteriophage and application thereof

Abstract

The invention discloses a Listeria monocytogenes phage and application thereof, the Listeria monocytogenes phage is Listeria monocytogenes phage LP4H1, the Listeria monocytogenes phage LP4H1 is preserved in the China Center for Type Culture Collection, the preservation date is April 22, 2025, and the preservation number is CCTCC (China Center for Type Culture Collection) NO: M 2025849. The listeria bacteriophage disclosed by the invention can specifically split various serotypes of food-borne monocyte hyperplasia listeria including Lm high-virulence serotype strains for 4 hours, so that the generation and aggravation of the drug resistance of the listeria are prevented; the bacteriophage strain provided by the invention has a hydrophilic phase, and can be easily prepared into spraying liquid or leacheate through a traditional method to disinfect the environment or appliances. The bacteriophage strain has no virulence factor coding gene and antibiotic resistance gene, can be amplified by harmless Listeria monocytogenes, and can be used as a food additive for specifically cracking Listeria monocytogenes to prevent the Listeria monocytogenes from polluting food. Large-scale production can be realized, and a safe and efficient new way is provided for industrially producing the bacteriophage, applying the bacteriophage to food and processing equipment thereof and sterilizing the environment.

Description

Technical Field

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

[0002] Foodborne listeriosis has become a global priority for food safety prevention and control due to its high mortality rate and widespread transmission. In its "Global Burden of Foodborne Disease Estimates," the World Health Organization lists Lm as the top priority foodborne pathogen for prevention and control. Listeria monocytogenes (Lm) is a highly resilient Gram-positive foodborne pathogen that can survive in a variety of food processing environments, including low temperatures and dry conditions, and can prolong its survival by forming biofilms. Consuming food contaminated with Lm can cause invasive listeriosis and non-invasive gastroenteritis. Mild cases can result in abdominal pain and diarrhea, while more severe cases can lead to serious illnesses such as sepsis, meningitis, and miscarriage, with a mortality rate as high as 20-30%.

[0003] Multiple international studies have revealed a high detection rate of this pathogen in poultry products. A microbiological survey of raw chicken sold in Shaanxi Province by Zhang Liping's team revealed a 54.17% positive rate for Lm isolation. Foodborne pathogen surveillance conducted by Zhao Jin and other researchers in Sichuan Province found that frozen poultry had a significantly higher Lm carriage rate than other food categories, with a positive detection rate of 47.5%. A study by Ristori and other researchers on the Brazilian frozen chicken supply chain even documented 58% Lm-positive samples. This high rate of pathogen carriage has directly led to numerous human listeriosis outbreaks. The US Centers for Disease Control and Prevention (CDC) reported clusters of infections associated with turkey and chicken products in 2008, 2021, and 2022.

[0004] Currently, Lm is classified into 14 serotypes based on specific combinations of flagellar antigens and bacterial antigens: serotypes 1 / 2a, 1 / 2b, 1 / 2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, 4h, and 7. There is a correlation between Lm serotype and virulence. Serotypes 1 / 2a, 1 / 2b, and 4b cause over 85% of human listeriosis worldwide. Serotype 4h is a newly designated serotype. This serotype is highly virulent and has a unique genetic evolutionary position, forming a new sublineage of lineage II (HSL-II). Its colonization capacity in the host is 400 times greater than that of the internationally recognized virulent strain, LmEGD-e. Serotype 4h Listeria has caused outbreaks and epidemics of listeriosis in sheep on multiple farms, causing economic losses to the livestock industry. The high level of virulent serotype 4h Listeria monocytogenes hinders the healthy development of the entire industry chain and threatens food safety. Strengthening the prevention and control of pathogenic Listeria such as serotype 4h in the livestock and poultry industry chain is of great significance to the breeding industry, slaughtering industry and public safety.

[0005] Under national policies, bacteriophages, as viruses that specifically lyse bacteria, offer unique control advantages. Their high specificity, rapid proliferation, and short development time have opened up new avenues for the biocontrol of foodborne pathogens. Phages are categorized as lytic and lysogenic based on their lytic properties. After infecting bacteria, lytic phages utilize nutrients within the host bacteria to replicate their own genomes and release progeny phages by lysing the infected bacteria. Listeria phages can specifically infect and lyse Lm, offering broad application prospects in the livestock and food industries, and possess high development value.

[0006] Currently, enriching broad-spectrum phage resources and identifying new phages with strong lytic activity against emerging serotype 4h strains are pressing challenges in the prevention and control of Lm pathogens. Screening for potent phages from wastewater is an effective approach for developing novel antibacterial agents and promoting the development of phage therapy. Summary of the Invention

[0007] Purpose of the invention: The purpose of the present invention is to provide a Listeria phage and its application.

[0008] Technical solution: The present invention provides a Listeria phage, which is Listeria monocytogenes phage LP4H1, deposited in the China Center for Type Culture Collection, with a preservation date of April 22, 2025, and a preservation number of CCTCC NO: M 2025849.

[0009] Furthermore, the phage LP4H1 is a muscle-tailed phage with a symmetrical head and a diameter of approximately 72±2 nm, a tail length of approximately 80±2 nm, and a tail diameter of 16±1 nm.

[0010] Furthermore, the bacteriophage LP4H1 has good acid and alkali tolerance, with an optimal pH value of 3-11.

[0011] Furthermore, the bacteriophage LP4H1 has good thermal stability, with an optimum temperature of 30-50°C.

[0012] Furthermore, when the infection multiplicity of the bacteriophage LP4H1 with the host bacteria is 1, the titer is 6×10 9 PFU / mL.

[0013] The present invention also provides the use of the Listeria phage in any of the following:

[0014] (1) Listeria fungicide;

[0015] (2) Drugs for preventing / treating diseases caused by Listeria.

[0016] Furthermore, the serotypes of Listeria include 1 / 2a, 1 / 2b, 4b and 4h.

[0017] The present invention also provides a feed additive or food additive containing the Listeria phage as an effective ingredient.

[0018] The present invention also provides a cleaner or disinfectant for aquaculture and slaughtering environments, which contains the Listeria phage as an effective ingredient.

[0019] Furthermore, the concentration of the Listeria phage is not less than 10 9 PFU / mL.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the Listeria phage of the present invention can specifically lyse various serotypes of Listeria, including the highly virulent Lm serotype 4h strain, to prevent the generation and aggravation of Listeria resistance; the phage strain of the present invention has a hydrophilic phase and can be easily prepared into a spray liquid or rinse liquid by traditional methods to disinfect the environment or utensils. The phage strain of the present invention does not have virulence factor encoding genes and antibiotic resistance genes, and can be amplified using harmless Listeria. It can be used as a food additive to specifically lyse Listeria and prevent Listeria from contaminating food. Large-scale production can be achieved, providing a safe and efficient new way for the industrial production of phages and their application in food, its processing equipment and environmental sterilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Results of phage double-layer plate assays; A: Double-layer plate image of LP4H1 formed on the Lm XYSN host bacteria; B: Double-layer plate image of LP4H1 formed on the L. innocua host bacteria;

[0022] Figure 2 Electron micrograph of bacteriophage;

[0023] Figure 3 bacteriophage genome maps;

[0024] Figure 4 Phage evolutionary tree analysis;

[0025] Figure 5 One-step growth curve results of phage;

[0026] Figure 6 Results of bacteriophage temperature sensitivity test;

[0027] Figure 7 Phage pH sensitivity test results;

[0028] Figure 8 Results of phage optimal MOI determination;

[0029] Figure 9 Determination of phage antibacterial ability in vitro;

[0030] Figure 10 Pathological sections and scoring results of the liver and spleen of mice gavaged with phage; A: liver pathological section of the control group; B: spleen pathological section of the control group; C: gavage 10 8 PFU / ml phage liver pathological section; D: gavage 10 8 PFU / ml phage spleen pathological section; E: gavage 10 9 PFU / ml phage liver pathological section; F: gavage 10 9 PFU / ml phage spleen pathological section; G: liver pathological scoring results of the control group and experimental group; H: spleen pathological scoring results of the control group and experimental group;

[0031] Figure 11 Results of bacteriophage sterilization test in chicken (4°C);

[0032] Figure 12 Results of bacteriophage sterilization test in chicken (25°C);

[0033] Figure 13 Results of bacteriophage sterilization test in milk (4°C);

[0034] Figure 14 Qualitative detection results of bacteriophage Lm in chicken by spraying bacteriophage; DETAILED DESCRIPTION

[0035] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0036] Example 1. Phage morphology observation, phage genome sequencing, and similarity analysis between genomes

[0037] 1. Acquisition of bacteriophage

[0038] 1) Collect 50 mL of sewage samples from the chicken slaughterhouse and sewage, and centrifuge them at 8000 r / min for 15 minutes at 4°C. Take the supernatant after centrifugation and filter it with a 0.22 μm filter membrane. Add 1 mL of Lm XYSN (CCTCC NO: M 2019765) culture in the logarithmic growth phase to the filtered supernatant, add 1.25 mmol / L sterile CaCl2 solution, and mix thoroughly. Subsequently, add 50 mL of TSB-YE medium, incubate the mixture at 37°C for 1 hour, and culture it at 30°C overnight. Finally, take the above culture and centrifuge it at 10000 r / min for 15 minutes at 4°C, take the supernatant, and filter it with a 0.22 μm filter membrane to obtain the phage stock solution.

[0039] 2) Take 0.1 ml of phage stock solution and dilute it 10 times. -2 , 10 -4 and 10 -6 0.1 ml of each diluent was mixed with 0.1 ml of overnight cultured host bacterial solution. After incubation at room temperature for 15 minutes, about 5 ml of TSA semi-solid medium containing 0.6% agar was added. After mixing, the mixture was quickly poured onto the top layer of a TSA-YE solid medium plate. Shake and let it stand for 5 minutes to solidify. Place it in a 30°C incubator and observe after 12 hours to obtain a double-layer plate with plaque formation.

[0040] 3) Use a Pasteur pipette or a 10 μL pipette tip to aspirate a single plaque from the plate where plaques appear; add 500 μL of SM buffer to a 1.5 mL centrifuge tube; add the aspirated plaque to the centrifuge tube and pipette evenly with SM buffer; dissociate at room temperature for 4-6 hours; dilute the dissociation solution 10-fold to 10 -2 , add each concentration of dissociation solution to an inverted double-layer agar plate covered with bacteria, culture overnight at 30℃, pick a single phage plaque, continue to dissociate, continuously dilute 10 times in a gradient, culture on a double-layer agar plate, repeat 5-7 times to purify a single phage.

[0041] 2. Observation of plaque morphology using the phage double-layer plate method

[0042] After the phage LP4H1 isolated from sewage was amplified and diluted to an appropriate titer, 100 μL of phage, 100 μL of Lm XYSN (CCTCC NO: M 2019765) (OD 600 =0.6) and 6 mL TSA semi-solid medium were mixed thoroughly, poured onto a BHI solid plate, and cultured at 30°C overnight. Figure 1 As shown, the diameter of the plaques formed by the phage on the host bacteria Lm XYSN and Listeria innocua is about 1-2 mm.

[0043] 3. Phage transmission electron microscopy detection

[0044] The morphological characteristics of the purified phage LP4H1 were analyzed using transmission electron microscopy. A 20 μL phage sample was dripped onto a copper grid. After standing at room temperature for 10 minutes, excess liquid was removed from the surrounding area using filter paper. 5 μL of a 2% phosphotungstic acid solution was dripped onto the grid and stained for 1 minute in the dark. The stain was then removed using filter paper. The grid was then dried under a heat lamp (55°C, 30 minutes) and the phage morphology was observed using transmission electron microscopy.

[0045] like Figure 2 As shown, phage LP4H1 belongs to the family Myocaudatus, with a symmetrical head and a diameter of approximately 72 ± 2 nm, a tail length of approximately 80 ± 2 nm, and a tail diameter of 16 ± 1 nm.

[0046] 4. Phage genome sequencing and similarity analysis between genomes

[0047] The phage LP4H1 genome consists of a double-stranded circular DNA consisting of 141,473 bases with a GC content of 36.3%. The LP4H1 genome contains 199 genes, totaling 125,502 bases in length, representing 88.7% of the entire genome. The genome contains 199 open reading frames (ORFs), including 73 known ORFs and 126 ORFs of unknown function. Bioinformatics prediction revealed that the LP4H1 genome does not harbor known drug resistance genes or genes encoding virulence factors.

[0048] The Mash v2.3 tool based on the MinHash algorithm was used to evaluate the similarity between LP4H1 and known Listeria phages at the genomic level and to reconstruct their phylogenetic relationships. The tool estimates the Jaccard distance by sharing k-mers and generates two indicators for each genome pair: shared genome content (SGC) and average nucleotide identity (ANI). Based on these two indicators, the evolutionary relationship between phages LP4H1 and LP4H4 was analyzed. Figure 4 Analysis revealed that phage LP4H1 shares 54.3% of its genome with its closest relative, Lm phage MT4387610. While LP4H1 is related to known Listeria phages, the genomes differ significantly. Therefore, the phage was named Listeria monocytogenes phage LP4H1 and deposited with the China Center for Type Culture Collection on April 22, 2025, under the CCTCC No. M 2025849. The location of the deposit is Wuhan.

[0049] Example 2: Determination of phage biological characteristics

[0050] 1. Phage titer determination

[0051] Phage titer was determined using a double-layer plate. The specific steps were as follows: Lm XYSN was cultured in a 37°C incubator with shaking at 220 rpm until the logarithmic growth phase. 200 μL of freshly cultured bacterial solution was added to 6 mL of TSB medium containing 0.6% agar (heated in a microwave oven and then cooled to about 50°C), shaken well, and poured onto a pre-prepared BHI agar plate to dry. The phage sample to be tested was diluted to 10 in SM buffer using a 10-fold serial dilution method. -7 For each gradient, 10 μL was added dropwise to a double-layer plate. After the added phage dilution solution was naturally air-dried in a clean bench, the double-layer plate was placed in a 30°C constant temperature incubator for 12 hours. The number of phage plaques formed on the plate by each gradient solution was counted to calculate the phage titer. The measured LP4H1 titer was approximately 6×10 9 PFU / mL.

[0052] 2. One-step growth curve of phage

[0053] Pick a single colony of Listeria XYSN and culture it to the logarithmic growth phase. Centrifuge it at 8000 rpm for 5 min and discard the supernatant. Resuspend the pellet with an appropriate amount of BHI liquid medium and calculate the OD value of the bacterial solution. 600 The value was adjusted to 0.78. The adjusted bacterial solution was inoculated into a conical flask containing 100 mL of BHI liquid medium at a ratio of 1:10. The phage LP4H1 was then inoculated into the conical flask at an MOI of 1 and mixed evenly. The mixture was then incubated in a 37°C constant temperature shaker at 220 rpm for 120 minutes. 1 mL of the culture medium was aspirated every 10 minutes, and the titer of LP4H1 was determined after gradient dilution. Figure 5 As shown, there was no significant increase in phage titer in the culture medium between 0 and 20 minutes, indicating a 20-minute incubation period for LP4H1. From 20 to 100 minutes, the phage titer in the supernatant increased exponentially and plateaued after 100 minutes, indicating an 80-minute burst period for LP4H1 in XYSN cells, with a burst volume of 25 pfu / cell. From 100 to 120 minutes, the LP4H1 titer plateaued, indicating a plateau in LP4H1 growth during this period.

[0054] 3. Effects of temperature and pH on bacteriophage

[0055] (1) Add 1 mL of phage LP4H1 stock solution to five sterile 1.5 mL centrifuge tubes and incubate in a constant temperature water bath at 30°C, 40°C, 50°C, 60°C, and 70°C for 1 hour. Every 30 minutes, 100 μL of the incubation solution was diluted serially, and the phage titer was determined using the double-layer plate method.

[0056] The results are as follows Figure 6 As shown, after the phage was exposed to temperatures of 30-50°C for 1 hour, its activity did not change significantly; after the phage was exposed to temperatures of 70-80°C for 1 hour, the phage activity was below the detectable level.

[0057] (2) 900 μL of SM buffer with pH values of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 were added to 12 sterile 1.5 mL centrifuge tubes, and 100 μL of phage LP4H1 stock solution was added and mixed evenly. After incubation of all treatment groups in a 37°C constant temperature water bath for 2 hours, the incubation solution was serially diluted with the corresponding SM buffer, and the phage titer was detected by double-layer plate method.

[0058] The results are as follows Figure 7 As shown in the figure, the curve of phage LP4H1 is relatively stable at pH 3-11, and the original titer can be basically maintained; while the titer drops below the detection line after incubation for 2 hours at pH 2, 12 and 13, indicating that the phage has been inactivated.

[0059] 4. Phage host lineage detection

[0060] Divide the TSA-YE plate into several areas. Pipette 15 μL of the overnight culture of each serotype of Listeria monocytogenes isolated, identified, and preserved in our laboratory and drop it in the center of each area of the TSA-YE plate and let it dry. Then, take 7 μL of the LP4H1 phage stock solution and drop it on the plate coated with different host bacteria. After leaving it upright to dry naturally, place it in a 30°C incubator for overnight culture to observe the lysis effect of the phage on different host bacteria.

[0061] Table 1

[0062]

[0063]

[0064] Note: Determination of cracking ability: EOP ≥ 0.5 is "+++" 0.1 < EOP < 0.5 is "++"

[0065] 0.0001≤EOP≤0.1 is "+" and EOP<0.0001 is "-". EOP=test bacteria titer / 4h strain XYSN titer

[0066] Table 2

[0067] Host bacteria Host strain number Cracking capacity Listeria monocytogenes ATCC33090 + Escherichia coli BL21(DE3) - Salmonella ATCC13076 - Staphylococcus aureus ATCC25923 -

[0068] The results are shown in Tables 1 and 2. Bacteriophage LP4H1 has good lysis properties against pathogenic serotypes (1 / 2a, 1 / 2b, and 4b) Lm strains, 4h strains, and harmless Listeria monocytogenes that cause more than 90% of human listeriosis cases, but has no lysis effect on other bacteria, and no clear spots were found in the plates.

[0069] 5. Phage optimal MOI determination

[0070] 500 μL of phage LP4H1 was mixed with Lm XYSN at MOIs of 0.01, 0.1, 1, 10, and 100, respectively, and then added to 10 mL of LB medium. The culture was shaken at 220 rpm in a 37°C incubator for 5 hours. The culture was then centrifuged at 10,000 rpm for 15 minutes at 4°C. After centrifugation, the phage titer in the supernatant was determined using a double-layer plate assay. The optimal MOI was determined to be the MOI with the highest phage titer.

[0071] The results are as follows Figure 8 As shown in the figure, the phage titer obtained by mixing phage LP4H1 and Lm XYSN at an MOI of 1 and culturing for 5 hours was the highest, which was 6.1×10 9 PFU / mL, so the optimal multiplicity of infection for LP4H1 is 1.

[0072] 6. Determination of phage antibacterial ability in vitro

[0073] Pick a single colony of Lm XYSN and culture it until the logarithmic growth phase. Centrifuge at 8000 rpm for 5 minutes in a high-speed centrifuge and discard the supernatant. Resuspend the pellet in an appropriate amount of BHI liquid medium. Co-incubate phage LP4H1 with LmXYSN at an MOI of 10, 1, and 0.1. Place the culture plate in a 37°C incubator and incubate for 12 hours. Measure the OD value of the culture medium every hour using a microplate reader. 600 The values were calculated and bacterial growth curves were generated. Under the same culture conditions, an equal volume of SM buffer was added to the phage as a control group. Each experimental group was cultured in triplicate.

[0074] The results are as follows Figure 9 As shown, the OD of phage LP4H1 600 The values remained stable or slightly decreased over the experimental period. LP4H1 exhibited strong antibacterial activity, and its antibacterial effect was in stark contrast to that of the control group. This indicates that LP4H1 can effectively inhibit the growth of target bacteria in vitro.

[0075] Example 3, Phage Safety Experiment

[0076] 6-week-old female SPF BALB / c mice, 18 g ± 2 g, a total of 24, were purchased from Nanjing Weitonglihua Company. The mice were randomly divided into 3 groups, 8 in each group: one experimental group was orally administered with phage LP4H110 8 PFU / each: Another experimental group was orally administered with bacteriophage LP4H110 9 PFU / mouse. The control group received an equal volume of SM buffer orally. Each dose was administered orally for 7 consecutive days, followed by sacrifice by cervical dislocation. The livers and spleens of the mice were compared with those of the control group, and organ lesions were observed visually. The livers and spleens of the deceased mice were simultaneously removed, cut into small pieces, and fixed with tissue fixative. Pathological sections were then prepared and observed.

[0077] The results are as follows Figure 10 As shown, oral 10 8 PFU / phage group and 10 9 There was no significant difference in PFU / phage between the group and the control group, indicating that phage LP4H1 was safe.

[0078] Example 4: Bacteriophage sterilization in solid food

[0079] (1) Preparation of sterile chicken nuggets: Cut fresh chicken breast into 1 cm × 1 cm cubes (about 1 g) in a sterile operating table, soak in sodium hypochlorite disinfectant for 5 min, rinse with sterile dd H2O three times, place on autoclaved tin foil and sterilize under ultraviolet light for 30 min (turn over every 10 min), and test for sterility before use.

[0080] (2) Preparation of bacterial suspension: Select Lm XYSN and culture at 37℃ overnight. Adjust the OD value of the bacterial suspension. 600 to 0.78 (about 1×10 9 CFU / mL), and diluted to 10 4 CFU / mL and 10 2 CFU / mL.

[0081] (3) Artificial contamination of chicken: Sterile chicken pieces were soaked in two concentrations of bacterial suspension for 15 min, so that the inoculation volume reached 1×10 4 -5×10 4 CFU / g and 1×10 2 -5×10 2 CFU / g and then transferred to sterile 24-well plates.

[0082] (4) Phage preparation and processing: Preparation 10 9 PFU / mL phage stock solution was diluted to 10 with SM buffer. 8 PFU / mL. 200μL 10 8A phage solution (PFU / mL) was applied to the surface of contaminated chicken nuggets. A control group (blank control) was dripped with 200 μL of SM buffer. Three replicates were set up for each group, sealed, and incubated at 4°C and 25°C. Samples were collected for testing at 0, 2, and 12 hours. The chicken nuggets were weighed, added with 1 mL of PBS buffer, and homogenized using a tissue grinder. Ten-fold serial dilutions were made using sterile PBS, and the bacterial load (CFU / g) per gram of chicken was calculated using a standard plate count method.

[0083] Test results such as Figure 11 and Figure 12 As shown, the bacterial count of chicken meat was reduced by 10.5-fold after LP4H1 was applied to the chicken meat surface at 25°C for 12 hours, and by 12-fold after LP4H1 was applied to the chicken meat surface at 4°C for 12 hours.

[0084] Example 5: Bacteriophage sterilization in liquid food

[0085] Listeria monocytogenes XYSN cultured to the logarithmic growth phase was mixed with bacteriophage LP4H1 at an MOI of 10. 200 μL of each was added to 5 mL of pasteurized milk and shaken to mix. The mixture was placed in a static culture at 180 rpm / min and 4°C. 100 μL of milk was aspirated at 0 hour, 6 hours, 12 hours, and 24 hours for gradient dilution spot plate detection of the host bacteria count.

[0086] The results are as follows Figure 13 As shown in the results, phage LP4H1 treatment for 12 hours significantly reduced the number of Lm in milk, and the number of Lm host bacteria decreased by 12.7 times.

[0087] Example 6: Bacteriophage sterilization effect of spraying phage on whole chicken and cut chicken samples

[0088] After the phage spray preparation is prepared in large quantities in the laboratory, the phage preparation is placed in the sprayer. 9PFU / mL) was added to the sprayer. The spray pressure was adjusted to 0.2-0.3 MPa. The nozzle was positioned approximately 50 cm from the chicken samples on the factory conveyor belt, with a coverage angle of 90°. The conveyor speed was approximately 0.2 m / s, and each cut and whole chicken sample was sprayed for approximately 2 seconds, with a spray volume of approximately 500 μL. The chicken samples subjected to spray reduction included 50 whole chickens and 50 cut chickens. All phage-treated chicken samples and an equal number of untreated chicken samples were placed in sterile sampling bags and brought back to the laboratory. An appropriate amount of UVM selective enrichment solution was poured into the sterile sampling bag and incubated at 30°C for 24 hours. 100 μL of the culture medium was added to 10 mL of Fraser broth and incubated at 37°C for 24 ± 2 hours. 50 μL of the culture medium was streaked onto three Listeria monocytogenes chromogenic plates and incubated at 37°C for 24 hours. Single colonies with a blue-green color and a white halo on the Listeria chromogenic plate were picked and purified on BHI solid medium. After purification, colony PCR was performed to identify whether they were Lm.

[0089] The results are as follows Figure 14 As shown, only three Lm strains were isolated from 100 phage-treated samples, a 3% isolation rate. Twenty-five Lm strains were isolated from 100 control samples, a 25% isolation rate. Phage treatment reduced the isolation rate of Listeria monocytogenes by 88%.

Claims

1. A Listeria phage, characterized in that The phage is Listeria monocylogenesphage LP4H1, which is deposited in the China Center for Type Culture Collection on April 22, 2025, with a deposit number of CCTCCNO: M 2025849.

2. The Listeria phage according to claim 1, characterized in that The phage LP4H1 is a muscle-tailed phage with a symmetrical head and a diameter of about 72±2 nm, a tail length of about 80±2 nm, and a tail diameter of 16±1 nm.

3. The Listeria phage according to claim 1, characterized in that The bacteriophage LP4H1 has good acid and alkali tolerance, and the optimum pH value is 3-11.

4. The Listeria phage according to claim 1, wherein The bacteriophage LP4H1 has good thermal stability, and the optimum temperature is 30-50°C.

5. The Listeria phage according to claim 1, characterized in that When the infection multiplicity of the bacteriophage LP4H1 and the host bacteria is 1, the titer is 6×10 9 PFU / mL.

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

7. The use according to claim 6, characterized in that The serotypes of Listeria include 1 / 2a, 1 / 2b, 4b and 4h.

8. A feed additive or food additive comprising the Listeria phage according to any one of claims 1 to 5 as an active ingredient.

9. A cleaner or disinfectant for aquaculture and slaughtering environments, comprising the Listeria phage according to any one of claims 1 to 5 as an active ingredient.

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

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