Novel listeria bacteriophage LPLM20 and application thereof

Abstract

The invention belongs to the field of microbial technology and food safety, and discloses a novel listeria bacteriophage LPLM20 and application thereof. The consistency between the genome of the bacteriophage LPLM20 and the genome sequence of the existing known bacteriophage is lower than 70%, and the bacteriophage LPLM20 is a novel listeria monocytogenes bacteriophage with the preservation number of CCTCC NO: M 20241865. The bacteriophage LPLM20 is a long-tailed bacteriophage, keeps stable activity at the pH value of 3-12 and the temperature of 30-50 DEG C, is wide in splitting spectrum, can split 53 strains of listeria monocytogenes including 5 serotypes, listeria ininocytogenes and listeria wisteria, can inhibit pollution caused by the listeria monocytogenes, and can be used as an antibacterial substance for preventing and controlling listeria pollution.

Description

Technical Field

[0001] This invention belongs to the fields of microbial technology and food safety, specifically relating to a novel Listeria bacteriophage LPLM20 and its applications. Background Technology

[0002] Listeria monocytogenes (hereinafter referred to as Listeria) is an important zoonotic foodborne pathogen. Listeria monocytogenes is widely distributed in nature, highly tolerant of adverse environments, and can survive at temperatures ranging from 0°C to 45°C, even growing at refrigerated temperatures, thus easily persisting in the environment for extended periods. Livestock and poultry are the primary hosts of Listeria monocytogenes, which can contaminate food through raw material transport, food processing, and cross-contamination. Foods easily contaminated with this bacterium include raw milk, cheese, meat and meat products, eggs, vegetable salads, and seafood. More than 90% of human cases of Listeria monocytogenes infection are caused by consuming food contaminated with Listeria. The three most prevalent pathogenic Listeria serotypes are 1 / 2a, 1 / 2b, and 4b. Listeriosis has a hospitalization rate as high as 91%, ranking first among all foodborne illnesses. Therefore, the prevention and control of Listeria monocytogenes is of paramount importance for food safety and public health security.

[0003] Bacteriophages are among the most abundant organisms in the ecosystem, capable of lysing bacteria and achieving antibacterial effects. Bacteriophages typically only infect specific species or strains of bacteria and are harmless to animals and humans, making them a safe and highly effective potential for harmful bacteria control. Patent CN 101220350 B discloses a Listeria monocytogenes phage, BPH-LM-1, which is sensitive to heat and pH; its titer drops to 19% after incubation at 50°C for 2 hours, and decreases rapidly at pH < 5 or pH > 11. 5 PFU / mL phage was completely inactivated within 12 hours. 3 CFU / mL Listeria. Patent publication CN101955916 B discloses the Listeria monocytogenes phage LipG2-5, which is stable in activity at pH 5-8 and completely inactivated at ≤ pH 4 and ≥ pH 9; 10 8 PFU / mL phage treatment for artificial contamination 10 4 Milk containing CFU / mL Listeria monocytogenes reduced the Listeria monocytogenes count to 50% of the control group without phage administration after 72 hours, but Listeria monocytogenes was still detectable throughout the experimental testing period. Summary of the Invention

[0004] The purpose of this invention is to provide a novel Listeria phage LPLM20, the preservation number of which is: CCTCC NO:M 20241865.

[0005] Another objective of this invention is to provide the application of Listeria phage LPLM20 in inhibiting Listeria in the fields of food, environment, medical devices, and clinical applications.

[0006] The present invention also includes the use of bacteriophage LPLM20 in the preparation of medicaments for the treatment or prevention of Listeria monocytogenes infection.

[0007] To achieve the above objectives, the present invention adopts the following technical measures:

[0008] This invention screened a broad-spectrum bacteriophage, LPLM20, from samples from a farm in Wuhan, which exhibits lytic activity against three species of Listeria. This bacteriophage was deposited at the China Center for Type Culture Collection (CCTC C) on August 28, 2024, at Wuhan University, Wuhan, China; accession number: CCTCC NO:M 20241865; classification and naming: Listeria monocytogenes phage LPLM20.

[0009] The Listeria monocytogenes phage LPLM20 in this invention has the following biological characteristics:

[0010] Bacteriophage LPLM20 has a polyhedral head with a length of 59.12±1.71 nm (n=5) and a width of 52.25±2.58 nm (n=5). Its tail has a length of 282.71±2.16 nm (n=5) and a width of 10.19±2.11 nm (n=5), consistent with the characteristics of a long-tailed bacteriophage.

[0011] This bacteriophage exhibits good pH and temperature tolerance. LPLM20 maintains stable activity at pH 3–12, but its activity decreases sharply at pH < 3 and pH > 12. At temperatures of 4℃, 30℃, and 40℃, LPLM20 maintains stable activity; however, incubation at 50℃ for 30 min and 60 min reduces the phage titer by 2 log10 PFU / mL and 4 log10 PFU / mL, respectively. This broad acid-base and temperature tolerance indicates that LPLM20 is suitable for sterilization applications in various scenarios, including food, environment, medical devices, and clinical settings.

[0012] The genome of bacteriophage LPLM20 is approximately 40,000 bp in length. Its alignment with Listeria phage A118 (accession number NC_003216.1), which has the highest similarity published by NCBI, shows a coverage of 61% and a similarity of 89.79%. Its alignment with Listeria phage 184 (accession number PP601405.1) shows a coverage of 60% and a similarity of 91.18%. Its alignment with Listeria phage LP-030-2 (NC_021539.2) shows a coverage of 1% and a similarity of 96.03%. The similarity to other bacteriophage nucleic acid sequences is even lower. This indicates that this bacteriophage is a novel Listeria monocytogenes bacteriophage. The specific sequences of bacteriophage LPLM20 provided by this invention are shown in SEQ ID NO. 1-2.

[0013] Analysis of its genome for virulence and resistance genes revealed no genes encoding virulence and resistance, indicating that the phage LPLM20 poses no potential safety risk as an antibacterial agent.

[0014] In this application, bacteriophage LPLM20 includes mutant strains with point mutations, deletion mutations, or addition mutations exhibiting homology higher than 98% and substantially the same bactericidal activity. Since bacteriophages are highly susceptible to mutation during replication, the aforementioned mutant strains of bacteriophages are also within the scope of protection claimed in this application. The gene sequence of a bacteriophage can be obtained through sequencing using known methods. For those skilled in the art, obtaining highly similar mutant strains of bacteriophages according to the present invention requires no inventive effort.

[0015] The scope of protection of this invention also includes:

[0016] The specific sequence of bacteriophage LPLM20 is shown in SEQ ID NO.1-2.

[0017] A compound comprising Listeria phage with accession number CCTCC NO: M20241865.

[0018] The above-mentioned bacteriophages or their compounds are used in the preparation of drugs for the treatment or prevention of Listeria infection.

[0019] The application of the above-mentioned bacteriophages or their compounds in the preparation of Listeria monocytogenes inhibitors.

[0020] The application of the above-mentioned bacteriophages or their complexes in the non-therapeutic in vitro inhibition of Listeria.

[0021] The application of the above-mentioned bacteriophages or their compounds in the preparation of disinfectants to prevent Listeria contamination.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0023] Phage LPLM20 has a sequence similarity to existing known phages that is less than 70% according to the International Committee on Taxonomy of Viruses (ICTV) standard for species classification (if the genome sequence similarity of two phages is less than 70%, they are assigned to different genera), making it a novel Listeria monocytogenes phage.

[0024] Phage LPLM20 lysed three Listeria species, including Listeria monocytogenes, Listeria innocense, and Listeria wiltii; the lysed Listeria monocytogenes contained 53 strains of Listeria monocytogenes from 5 serotypes.

[0025] Bacteriophage LPLM20 has excellent pH tolerance and can maintain stable activity in environments with pH 3-12, making it applicable to a wide range of environmental pH levels.

[0026] Bacteriophage LPLM20 has excellent heat resistance. It can maintain high activity for 1 hour at 50℃, but its activity decreases after 30 minutes at 60℃. It has a wide applicable temperature range.

[0027] The bacteriophage LPLM20 has strong lytic ability and inhibits the growth of Listeria monocytogenes in 90% of the tested strains. At an MOI of 1000, it can completely inhibit the growth of Listeria monocytogenes within 12 hours.

[0028] Bacteriophage LPLM20 has a short onset time. When added to liquid milk contaminated with Listeria monocytogenes, it takes only 9 hours at 4°C for Listeria monocytogenes to be undetectable by plate counting.

[0029] Phage LPLM20 can effectively treat Listeria monocytogenes infection and significantly reduce the bacterial load in the tissues of infected mice. Attached Figure Description

[0030] Figure 1 This is a photograph of the Listeria monocytogenes phage LPLM20 of this invention on a double-layer agar plate.

[0031] Figure 2 This is an electron microscope image of Listeria monocytogenes phage LPLM20 of the present invention.

[0032] Figure 3 The graph shows the adsorption rate of Listeria monocytogenes phage LPLM20 according to the present invention.

[0033] Figure 4 This is a one-step growth curve result of Listeria monocytogenes phage LPLM20 of the present invention.

[0034] Figure 5This is a schematic diagram of the inhibition curve of the bacteriophage LPLM20 of the present invention against Listeria monocytogenes LLM1.

[0035] Figure 6 This is a graph showing the temperature stability results of the bacteriophage LPLM20 of this invention.

[0036] Figure 7 This is a graph showing the pH stability results of the bacteriophage LPLM20 of this invention.

[0037] Figure 8 This is a VIRIDIC heatmap showing the genome similarity of the bacteriophage LPLM20 of this invention.

[0038] Figure 9 Phylogenetic tree constructed for the large subunit of the LPLM20 terminal enzyme of the bacteriophage of this invention.

[0039] Figure 10 The graph shows the bactericidal effect of bacteriophage LPLM20 on Listeria monocytogenes-contaminated fresh milk at 4℃.

[0040] Figure 11 The results show the bactericidal ability of bacteriophage LPLM20 against Listeria monocytogenes-contaminated fresh milk at 25°C. Detailed Implementation

[0041] The present invention will be further illustrated below with reference to embodiments, but the scope of protection of the present invention is not limited to the scope described in the embodiments. Unless otherwise specified, the technical solutions described in the present invention are conventional techniques; the reagents or materials described, unless otherwise specified, are all from commercial sources.

[0042] Example 1:

[0043] Isolation and preparation of bacteriophages from Listeria monocytogenes

[0044] (1) Isolation of bacteriophages

[0045] The samples used in this invention were obtained from a poultry farm in Wuhan. Listeria monocytogenes LLM1 was used as the host bacterium to isolate bacteriophages. The sample was added to PBS solution and allowed to stand at room temperature for a period of time. Then, it was centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was filtered through a 0.22 μm filter membrane; this filtrate was used as the initial sample for bacteriophage isolation and was stored at 4°C. The host bacterium was inoculated into 5 mL of TSB-YE (tryptone soybean broth containing 0.6% yeast extract) medium and cultured at 30°C to reach the logarithmic growth phase. The host bacterium, sample, and TSB-YE medium were mixed at a ratio of 1:2:4 (1 mL bacterial culture, 2 mL initial sample, 4 mL TSB-YE medium) and cultured at 30°C with shaking at 200 rpm for 8-12 h to allow the bacteriophages to proliferate. The cultured suspension was centrifuged at 10,000 r / min for 10 min at 4 °C. The supernatant was filtered through a 0.22 μm filter membrane, and the filtrate was collected. The spot method was used to determine whether the above steps had isolated bacteriophages.

[0046] (2) Proliferation and purification of bacteriophages

[0047] A single phage plaque was picked from the upper agar layer and placed in 10 mL of TSB-YE medium containing 100 μL of host bacteria. The medium was incubated at 30°C with shaking at 200 rpm for 8–12 h to allow phage proliferation. The resulting suspension was centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was filtered through a 0.22 μm filter, and the filtrate was collected as the proliferated phage stock solution. Then, a double-layer plate method was used to obtain double-layer plates containing phage, ultimately yielding phage LPLM20, whose plaques are shown below. Figure 1 As shown.

[0048] This bacteriophage was deposited at the China Center for Type Culture Collection (CCTCC) on August 28, 2024, and is classified as Listeria monocytogenes phage LPLM20. The deposit address is Wuhan University, Wuhan, China; the accession number is CCTCC NO: M 20241865.

[0049] Example 2:

[0050] Morphage morphology observation

[0051] Morphological observation of bacteriophages under transmission electron microscopy: Bacteriophages were proliferated to obtain sufficient titer phage liquid, which was then centrifuged to obtain phage precipitate. SM buffer solution was added to obtain phages with a titer ≥10. 10The phage concentrate was prepared at a concentration of PFU / mL. The specific steps were as follows: First, the cleaned copper mesh was immersed in the phage concentrate, then stained with phosphotungstic acid for 30 seconds. After air drying, it was observed and photographed using a transmission electron microscope. Length was measured using ImageJ image processing software.

[0052] The results are as follows Figure 2 As shown, phage LPLM20 conforms to the characteristics of long-tailed phage.

[0053] Example 3:

[0054] LPLM20 phage lysis profile determination

[0055] The experiment used Listeria monocytogenes phage LPLM20 to determine the lysis profiles of 59 strains of Listeria monocytogenes, 57 strains of Listeria monocytogenes, 1 strain of Listeria innocense, and 1 strain of Listeria wiltii, with different serotypes.

[0056] The above-mentioned strains were cultured to the logarithmic growth phase. 200 μL of the bacterial culture was mixed with 8 mL of 0.7% TSA-YE semi-solid medium. After thorough mixing, the mixture was poured onto a pre-prepared TSA-YE solid agar plate and allowed to stand for approximately 10 minutes until the upper layer of medium solidified. Then, 50 μL of the culture was added from a 10⁻⁶ agar plate. 2 -10 9 A phage suspension of PFU / mL, serially diluted tenfold, was spotted onto a semi-solid culture medium and incubated at 30°C for 6-8 hours. The phage plaque formation was then observed to determine the phage's host lysis activity against the test bacteria.

[0057] Phage LPLM20 was able to lyse 53 strains of Listeria monocytogenes, including 5 serotypes: 1 / 2a, 1 / 2b, 3a, 4a, and 4b. It was also able to lyse Listeria innocense and Listeria wiltii, with a lysis rate of 93.22% (55 / 59).

[0058] Example 4:

[0059] Optimal Multiple of Infection Assay for Bacteriophage LPLM20

[0060] Phage solution was added at different dilutions and 10 mg / L of phage solution according to multiplicity of infection ratios of 100, 10, 1, 0.1, 0.01, and 0.001. 6 100 μL of the host bacterium LLM1 (CFU / mL) culture was added to 800 μL of TSB-YE medium. The mixture was vortexed and incubated at 30°C with shaking for 4 h. After incubation, the mixture was centrifuged at 11000 rpm for 10 min at 4°C. 100 μL of the supernatant was collected and serially diluted 10-fold with TSB-YE liquid medium. The phage titer was determined using the double-layer agar plate method. If the phage titer was highest at a certain MOI, then that MOI represents the optimal multiplicity of infection for that phage.

[0061] The results showed that the highest potency (5.86 x 10⁻⁶) was observed when MOI = 0.001. 9 PFU / mL, i.e., the optimal MOI for LPLM20 is 0.001.

[0062] Example 5:

[0063] Adsorption rate of bacteriophage LPLM20

[0064] At the optimal MOI value (0.001), 500 μL each of fresh phage fluid and host bacterium LLM1 bacterial culture were mixed in a sterile 2 mL centrifuge tube and incubated on a shaker at 30 °C. Every 1 min, 100 μL of the liquid was aspirated, centrifuged at 12000 rpm for 5 min, and then serially diluted 10-fold. The titer of the phage in the upper layer was determined using the double-layer plate method.

[0065] The results are as follows Figure 3 As shown, after incubation at 30°C for 10 min, 80% of the phage particles of LPLM20 were adsorbed onto the host bacteria, and after incubation for 15 min, 99% of the phage particles were completely adsorbed onto the host bacteria.

[0066] Example 6:

[0067] One-step growth curve determination of bacteriophage LPLM20

[0068] Add 500 μL of 10 at the optimal MOI ratio (0.001). 4 PFU / mL phage and 500μL 10 7 CFU / mL host bacterium LLM1 culture was mixed and incubated at 30°C for 15 min. Then, it was centrifuged at 10000 rpm for 2 min at 4°C, the supernatant was discarded, and the culture was resuspended twice in 1 mL TSB medium. The mixture was then combined with 9 mL TSB medium and incubated at 30°C with shaking. Two 200 μL tubes of the mixed culture were collected every 10 min. The phage titer was determined using the double-layer plate method.

[0069] The results of the one-step growth curve assay for phage LPLM20 are as follows: Figure 4 As shown, the latency period of LPLM20 is about 40 min, the burst period is about 80 min, and the average pyrolysis rate is 63.5 PFU / Cell.

[0070] Example 7:

[0071] The lysis curves of phage LPLM20 against Listeria monocytogenes LLM1 at MOIs of 100, 10, 1, 0.1, and 0.01.

[0072] Dilute the phage stock solution to different dilutions and set aside. Add 100 μL of a 10-1 concentration to each well of a 96-well plate.6 The host bacterial culture was prepared at a concentration of CFU / mL. Then, 100 μL of phage at different dilutions were added according to MOIs of 100, 10, 1, 0.1, 0.01, and 0.001, and mixed thoroughly. Positive control group: 100 μL of phage at a concentration of CFU / mL was added. 6 CFU / mL bacterial culture and 100μL TSB-YE medium; microplate reader settings: wavelength 600nm, temperature 30℃, OD value measured every 1 hour.

[0073] The lysis curve of bacteriophage LPLM20 against Listeria monocytogenes LLM1 is as follows: Figure 5 As shown, compared with the positive control group without phage, the OD values ​​of the test bacteria measured at different MOIs remained stable at a low level within 12 hours, indicating that phage LPLM20 has a good and sustained inhibitory effect on the growth of Listeria monocytogenes LLM1.

[0074] Example 8:

[0075] Effect of temperature on the stability of bacteriophage LPLM20

[0076] Dilute the phage stock solution to 10. 9 PFU / mL, 100 μL of phage, and 900 μL of TSB-YE were added. The mixture was then placed in a constant temperature water bath at 4℃, 30℃, 40℃, 50℃, and 60℃, and the titer was measured at 30 min and 60 min, respectively.

[0077] Temperature stability test results are as follows Figure 6 As shown, the potency remained basically stable at 10 at 4℃, 30℃, and 40℃. 9 The PFU / mL titer remained at 10 after treatment at 50℃ for 30 min and 60 min. 7 PFU / mL and 10 5 PFU / mL. The above results indicate that phage LPLM20 maintains stable activity at 4℃ and 30℃-50℃, demonstrating good temperature tolerance.

[0078] Example 9:

[0079] Effect of pH on the stability of bacteriophage LPLM20

[0080] Using TSB-YE liquid medium as the medium, the pH value was adjusted to (2-13) with NaOH and HCl. 100 μL of phage stock solution with known titer was added to 900 μL of TSB-YE medium with different pH values. After incubation at 30℃ for 60 min, the titer of the treated phage was determined by the double-layer plate method.

[0081] The results are as follows Figure 7As shown, phage LPLM20 exhibits excellent acid and alkali tolerance, maintaining stable activity within a pH range of 3-12.

[0082] Example 10:

[0083] Phage LPLM20 genome sequencing and analysis

[0084] The genome of bacteriophage LPLM20 was extracted using the ZnCl2 precipitation method, and sequencing was performed by a sequencing company. The bacteriophage genome contains approximately 67 open reading frames (ORFs). The complete genome sequence of bacteriophage LPLM20 was aligned to the NCBI database. Eleven bacteriophages with the highest sequence similarity were selected, and pairwise comparisons of their genomes were performed online at http: / / rhea.icbm.uni-oldenburg.de / VIRIDIC / . The results are as follows: Figure 8 As shown. A phylogenetic tree was constructed using the terminal enzyme large subunits of bacteriophages to identify taxonomic relationships between bacteriophages. The terminal enzyme large subunit sequences of Listeria bacteriophages were downloaded from NCBI, multiple sequence alignment was performed using MUSCLE, and a phylogenetic tree was constructed using MEGA (v11.0.10).

[0085] The results are as follows Figure 8 As shown, phage LPLM20 shares the highest sequence identity (56.3%) with the published Listeria phage A118 (NC_003216.1). The phylogenetic tree shows that phage LPLM20 shares the highest sequence identity with phage A118 in different evolutionary branches ( Figure 9 According to the International Committee on Taxonomy of Viruses (ICTV) standards for species classification: if the genome sequence similarity of two bacteriophages is less than 70%, they are assigned to different genera; at the same time, the results of the phylogenetic tree can prove that bacteriophage LPLM20 is a novel Listeria bacteriophage.

[0086] Furthermore, analysis of its genome for virulence and resistance genes revealed no genes encoding virulence and resistance, indicating that the bacteriophage LPLM20 poses no potential safety risk when used as an antibacterial agent in food.

[0087] BLASTN alignment analysis revealed that phage LPLM20 has multiple specific sequences, and the nucleotide sequences of two specific genes are shown in SEQ ID NO.1-2.

[0088] Example 11:

[0089] Investigation on the antibacterial effect of bacteriophage LPLM20 against Listeria monocytogenes in liquid milk

[0090] The liquid milk was purchased from a local supermarket in Wuhan. 100 μL of Listeria monocytogenes LLM1 cultured to the logarithmic growth phase was added to 9.8 mL of fresh milk, resulting in a final viable cell concentration of 10. 3 CFU / mL. Then add 100 μL of diluted phage suspension, resulting in a final titer of 10. 6 PFU / mL (MOI = 10) 3 ) and 10 7 PFU / mL (MOI = 10) 4 PBS buffer was added to the fresh milk samples as a negative control. After incubating the samples in a shaker at 4°C for 0, 2, 4, 6, 9, 12, 24, and 48 hours, 200 μL of the sample was taken and counted on LA plates. Similarly, after incubating the samples in a shaker at 25°C for 0, 2, 4, 6, 9, and 12 hours, 200 μL of the sample was taken and counted on LA plates. Each group was set up in triplicate, and the average value was used for analysis.

[0091] The experimental results of the application of bacteriophage LPLM20 in liquid milk at 4℃ are as follows: Figure 10 As shown: the viable bacterial count in the control group remained essentially unchanged from 0 to 48 hours, while the count after adding an MOI of 10... 3 and 10 4 In the phage experimental group, after 9 hours of treatment, the viable count of Listeria monocytogenes was reduced to below the detection limit (<10 CFU / mL), and no viable bacteria were detected until 24 hours of treatment.

[0092] The experimental results of the application of bacteriophage LPLM20 in liquid milk at 25℃ are as follows: Figure 11 As shown: the viable bacterial count in the control group increased continuously from 0 to 24 hours, while the count increased after adding an MOI of 10. 3 and 10 4 The number of viable bacteria in the LPLM20 phage experimental group was consistently suppressed. Although the number of viable bacteria increased slowly, it was significantly lower than that in the control group (p<0.01).

[0093] The above results indicate that phage LPLM20 has a good antibacterial effect against Listeria monocytogenes LLM1 in liquid milk at both 4℃ and 25℃.

[0094] Example 12:

[0095] Application of bacteriophage LPLM20 in the preparation of drugs for the prevention or treatment of Listeria monocytogenes infections:

[0096] Six-week-old Kunming rats were divided into an untreated group and a treatment group, with 10 rats in each group. The untreated group was administered Listeria monocytogenes by gavage daily for days 1-3 of the experiment. 7 CFU / animal, 1 mL bacterial culture, 1 mL PBS / animal administered by gavage daily from day 4 to 6; phage treatment group administered Listeria monocytogenes 10 mg / animal by gavage daily from day 1 to 3 of the experiment.7 CFU / animal, 1mL bacterial suspension, LPLM2010 bacteriophage administered by gavage daily from day 4 to 6. 9 Five mice were euthanized by cervical dislocation on days 7 and 10 of the experiment, with PFU / mouse and 1 ml of phage solution. Small intestine, spleen, and liver were collected, and tissue bacterial load was determined using the plate count method. The therapeutic effect of phage therapy was evaluated based on… The value represents the ratio of the average bacterial load in the tissues of mice in the phage treatment group to the average bacterial load in the tissues of mice in the control group. The smaller the value, the less bacterial load the mice have after phage treatment, and the more obvious the treatment effect.

[0097] The results showed that, compared with the untreated group, on days 7 and 10, the total number of bacteria in the small intestine of mice in the phage-treated group was 60.99% and 54.84% of that in the control group, respectively; the total number of bacteria in the spleen was 89.48% and 47.02% of that in the control group, respectively; and the total number of bacteria in the liver was 94.64% and 22.62% of that in the control group, respectively.

[0098] This indicates that phage LPLM20 can effectively treat Listeria monocytogenes infection.

Claims

1. An isolated Listeria phage LPLM20, the preservation number of which is: CCTCC NO: M20241865.

2. The specific sequence of phage LPLM20 according to claim 1 is shown in SEQ ID NO.1~2.

3. A compound comprising Listeria phage with accession number CCTCC NO: M20241865.

4. The use of the bacteriophage of claim 1 or the compound of claim 3 in the preparation of a medicament for treating or preventing Listeria infection.

5. The use of the bacteriophage of claim 1 or the compound of claim 3 in the preparation of Listeria monocytogenes inhibitors.

6. The use of the bacteriophage of claim 1 or the compound of claim 3 in non-therapeutic in vitro inhibition of Listeria.

7. The use of the bacteriophage of claim 1 or the compound of claim 3 in the preparation of a disinfectant to prevent Listeria contamination.

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