Proteus phage PpZDSS02 and application thereof

Abstract

The invention discloses a proteusbacillus vulgaris bacteriophage PpZDSS02 and application thereof, the bacteriophage is preserved in China Center for Type Culture Collection, the preservation time is December 23, 2022, and the preservation number of the bacteriophage is CCTCC NO: M 20222017. The proteusbacillus vulgaris bacteriophage PpZDSS02 is separated and screened, has unique form and genome characteristics, and is a brand-new bacteriophage capable of efficiently inhibiting proteusbacillus vulgaris. The phage can be applied to preparation of a green and cheap proteusbacillus vulgaris inhibitor. The bacteriophage provided by the invention belongs to a natural biological material, does not contain any virulence gene in a genome, has no toxic or side effect, and can be used as a bacteria reducing agent in a polluted environment.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and particularly relates to a Proteus phage PpZDSS02 and its application. Background Art

[0002] Proteus is a Gram-negative facultative anaerobe and a conditional pathogen, which is widely distributed in nature (such as soil, water, decaying organic matter) and the intestines of humans and animals. It can cause infections in humans and various animals (such as fish, poultry, etc.), leading to foodborne diseases. Common foods contaminated by Proteus mainly include animal foods, soy products, and cold dishes, etc. Proteus is mainly transmitted through food, and ingesting contaminated food is the main route of infection. Therefore, cutting off the sources of infection (excreta of infected animals and humans and their environment, etc.) is an effective means to prevent food contamination and prevent food poisoning caused by Proteus. Currently, the most important means for preventing and controlling pathogenic bacteria and spoilage bacteria are still antibiotics and chemical disinfectants, but problems such as antibiotic-induced drug resistance and environmental pollution caused by chemical disinfectants are becoming increasingly prominent. Therefore, it is urgent to find new alternatives to antibiotics.

[0003] Phages are a type of bacteria-dependent virus. After infecting host bacteria, they can rapidly proliferate and lyse cells in the host cells, producing specific antibacterial effects. In addition, phages are widely present in nature, have specificity for host bacteria, have low preparation costs, and good safety. Developing a biological control agent or bacteriostatic agent with Proteus phage as the main component has potential application value for the removal and growth inhibition of contaminated Proteus in the environment. Summary of the Invention

[0004] Object of the Invention: Aiming at the problems existing in the prior art, the present invention provides a phage that has an inhibitory effect on the growth of Proteus, which can be applied to Proteus in the environment contaminated by the excreta of infected animals and humans, and provides a safe, efficient, and inexpensive biological antibacterial product for the ecological environment or the production, processing, and preservation environment of food.

[0005] The present invention also provides the application of the phage. The phage can be used alone or in combination to prepare a biological bacteriostatic agent, which can effectively inhibit or reduce the pollution and infection caused by Proteus in the ecological environment or the production, processing, and preservation environment of food.

[0006] Technical Solution: To achieve the above object, the present invention provides a Proteus phage PpZDSS02. The phage has been deposited with the China Center for Type Culture Collection on December 23, 2022, and its deposit number is CCTCC NO: M 20222017, and the deposit address is Wuhan University, Wuhan.

[0007] Application of the phage PpZDSS02 of the present invention in inhibiting Proteus

[0008] Among them, application of the phage PpZDSS02 in preparing an antibacterial agent against Proteus

[0009] Among them, application of the phage PpZDSS02 in preparing an antibacterial agent for inhibiting the reproduction and metabolism of Proteus

[0010] Among them, application of the phage PpZDSS02 in preparing an inhibitor against Proteus in the environment and food

[0011] Furthermore, application of the phage PpZDSS02 in inhibiting the reproduction and metabolism of Proteus in the excreta of animals and humans infected with Proteus and their environment

[0012] Among them, the antibacterial agent includes a spraying solution, a rinsing solution or a biological bacteria control agent

[0013] Furthermore, the antibacterial agent reduces the contamination of Proteus therein by spraying or washing the polluted environment, pollutant collectors, transportation environment, and food production environment and utensils

[0014] Preferably, the purified Proteus phage PpZDSS02 is made into a spraying solution or a rinsing solution for use in the polluted environment, environmental pollutants and transportation environment, and food production environment and utensils to reduce the contamination of Proteus in the environment. The antibacterial agent against Proteus of the present invention, the antibacterial agent uses the phage PpZDSS02 or an isolate or culture as the main component of the antibacterial agent

[0015] Among them, the Proteus phage PpZDSS02 is used alone or in combination to prepare an antibacterial agent, which can effectively inhibit or reduce Proteus in environmental pollutants or food environments

[0016] The Proteus phage PpZDSS02 in the present invention has the following biological characteristics:

[0017] (1) Morphological characteristics: Observed by transmission electron microscopy, the head is symmetrical, with a diameter of about 50 nm and a tail length of about 100 nm. The morphological characteristics of the obtained phage belong to the Myoviridae family

[0018] (2)Genomic characteristics: The full length of the genome is 42,296 bp, the GC content is 37.7%, and there are 79 open reading frames (ORFs). Among them, 62 ORFs encode hypothetical proteins, and 17 ORFs encode known proteins. Through whole-genome alignment in the NCBI database, it was found that the homology with a Proteus phage vB_PmiM_ZX7, which is the closest to phage PpZDSS02, is only 84.53% and the coverage rate is only 76%. The genomic data indicates that PpZDSS02 is a novel phage. In addition, no tRANA genes, lysogenic genes, drug resistance genes, or virulence genes were found in the whole genome of phage PpZDSS02, suggesting that this phage can be used as an inhibitor against Proteus and is safe and effective.

[0019] (3)It has strong Proteus-lysing performance.

[0020] (4)It can effectively inhibit the growth of Proteus in culture media and food models.

[0021] Advantages: Compared with the prior art, the present invention has the following advantages:

[0022] The present invention isolated and screened a Proteus phage PpZDSS02, which has unique morphological and genomic characteristics and is a brand-new phage that can effectively inhibit Proteus. This phage can be applied to the preparation of green and inexpensive Proteus inhibitors.

[0023] The biological bacteriostatic agent prepared from the phage PpZDSS02 of the present invention can effectively control the growth of Proteus in culture media and the environment, and is easily formulated into a spraying solution or a rinsing solution to sterilize the environment or utensils, reducing the risk of Proteus in the environment and its contamination of food; the phage involved in the present invention belongs to natural biological materials, and there are no virulence genes in the genome, without toxic side effects, and can be used as a bacteriostatic agent in polluted environments. Description of the Drawings

[0024] Figure 1 It shows the plaque morphology of phage PpZDSS02 on the LB double-layer agar plate.

[0025] Figure 2 It is the electron micrograph of phage PpZDSS02.

[0026] Figure 3 It is the circular map of the whole genome sequence of phage PpZDSS02.

[0027] Figure 4 It is the schematic diagram of whole-genome alignment of phage PpZDSS02 in the NCBI database;

[0028] Figure 5The inhibitory effect of phage PpZDSS02 on Proteus in liquid LB medium.

[0029] Figure 6 The inhibitory effect of phage PpZDSS02 on Proteus in a food model (fish juice). Specific implementation mode

[0030] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0031] The raw materials and reagents in the present invention are all commercially available without special instructions.

[0032] The test phage host bacterium Proteus (strain number: HZSC 4-1) in the present invention was preserved as the host bacterium when phage PpZDSS02 was preserved. The phage and the host bacterium are stored as a set of materials under one preservation number. Proteus HZSC 4-1 was isolated from spoiled freshwater fish during low-temperature storage by a conventional method and is a wild-type Proteus. It was identified as Proteus through sequencing, and its 16S rDNA is SEQ ID NO.1. The host bacterium was cultured in 5 mL of LB broth at 37 °C on a shaker until the logarithmic phase and was provided by the Zhejiang Institute of Freshwater Fisheries. Other wild-type Proteus can be used in the present invention.

[0033] SM buffer: It contains 5.8 g of NaCl, 2.0 g of MgSO4·7H2O, 1 M Tris-HCl, pH 7.4 in 1 L, and deionized water is used as the solvent.

[0034] Example 1

[0035] Isolation and purification preparation of phage

[0036] 1. Sample collection and treatment: 30 mL of sewage from a farmers' market in Yangzhou, Jiangsu Province was collected in a 50 mL centrifuge tube and centrifuged at 5000×g for 10 min. 5 mL of the supernatant was passed through a 0.22 μm filter membrane to remove miscellaneous bacteria, and the filtrate was collected.

[0037] 2. Phage enrichment: 5 mL of the above filtrate was added to 5 mL of double-concentration LB liquid medium, and at the same time, 100 μL of Proteus (HZSC 4-1) in the logarithmic growth phase was added. It was placed on a shaker at 37 °C at 150 rpm and cultured overnight. The next day, the culture in the test tube was transferred to a sterile centrifuge tube, centrifuged at 4 °C and 8000×g for 10 min, and the supernatant was passed through a 0.22 μm filter membrane to obtain the phage stock solution, which was stored in a 4 °C refrigerator.

[0038] 3. Spotting: Take 100 μL of the host bacterium HZSC 4-1 bacterial solution in the logarithmic growth phase from step 1, mix it into 5 mL of semi-solid medium, pour it onto an LB solid plate. After it solidifies, add 10 μL of the phage stock solution from step 3. After the liquid is absorbed by the medium, place it in a constant temperature incubator at 37°C for 8 h, and observe whether plaque formation occurs.

[0039] 4. Phage purification: Pick a single clear plaque on the double-layer plate with plaques in step 3 and transfer it to 1 mL of sterile SM buffer, mix well, and place it at 4°C for 24 h; the next day, perform a 10-fold serial dilution (10 -1 ~10 -7 ) of the phage solution with sterile SM buffer. Take 100 μL of each dilution of the phage solution and mix it with 100 μL of the Proteus HZSC 4-1 bacterial solution in the logarithmic growth phase at room temperature for 10 min, then add 5 mL of LB semi-solid medium, mix well, and quickly pour it onto the previously prepared LB solid medium to make a double-layer plate. After solidification, invert it and place it in a constant temperature incubator at 37°C for cultivation. Repeat the operation of the double-layer plate method 3 times. After the sizes of the plaques obtained are consistent, it is considered that the isolated phage is pure. The isolated phage is as shown in Figure 1 and is named PpZDSS02.

[0040] 5. Titer determination: Pick a plaque after purification and culture it in 5 mL of LB broth containing 100 μL of the HZSC 4-1 bacterial solution in the logarithmic growth phase in a constant temperature incubator at 37°C for 8 h, centrifuge at 8000 r / min for 10 min, take the supernatant and filter it through a 0.22 μm filter membrane to obtain a purified and separated phage filtrate. Take 100 μL of the purified and separated phage filtrate and mix it with 100 μL of the Proteus solution in the logarithmic growth phase, place it at room temperature for 10 min, then add it to 10 mL of LB liquid medium, and incubate it at 37°C with constant shaking at 150 rpm overnight; transfer the culture in the test tube to a sterilized centrifuge tube, centrifuge at 5000×g for 10 min, collect the supernatant, filter and sterilize it through a 0.22 μm microporous filter membrane to obtain a purified phage solution. Use the double-layer plate to detect the titer of the purified phage solution. The specific procedure is as follows: Perform a 10-fold serial dilution (10 -1 -10 -7 ) of the purified phage solution with SM buffer. Take 100 μL of each dilution of the phage dilution and mix it with 100 μL of the HZSC 4-1 host bacterial solution in the logarithmic growth phase for 10 min, then add 5 mL of LB semi-solid medium, mix well, and quickly pour it onto the previously prepared LB solid medium to make a double-layer plate. After solidification, invert it and place it in a constant temperature incubator at 37°C for 12 h, manually count the formed plaques, and calculate the titer. The counting results show that the titer of the purified solution of PpZDSS02 against Proteus HZSC 4-1 reaches 10 9 PFU / mL or more.

[0041] 6. Preservation of phage: Pick a plaque with uniform size and morphology obtained after the last purification and inoculate it into 5 mL of LB broth containing 100 μL of HZSC 4-1 bacterial liquid in the logarithmic growth phase. Incubate it in a constant temperature incubator at 37 °C for 8 h, centrifuge at 8000 r / min for 10 min, take the supernatant and filter it through a 0.22 μm filter membrane to obtain a phage filtrate. Take 900 μL of the phage filtrate and 300 μL of glycerol with a concentration of 70% and mix them in a sterilized preservation tube, and store them at -80 °C. This is the original solution of phage PpZDSS02.

[0042] Example 2

[0043] Observation of the morphology of phage PpZDSS02 mainly includes the following steps:

[0044] Observe the morphological characteristics of the phage with a transmission electron microscope. Adopt the negative staining method with phosphotungstic acid. Place the side of the copper grid with a film facing up, take 10 μL of the phage purification solution prepared in Example 1 and drop it on the copper grid. After adsorption for 15 min, blot the moisture with absorbent paper, take out the copper grid, and let it dry naturally in the air for 2 - 3 min. Then, drop a drop of 2% phosphotungstic acid (PTA) aqueous solution on the copper grid for staining. After 2 min, remove it, blot the moisture with absorbent paper, and dry it in the air for 5 min. Observe with a transmission electron microscope and select clear phage images for photographing and analysis.

[0045] The results are as Figure 2 shown. The head of phage PpZDSS02 is symmetric, with a diameter of about 50 nm and a tail length of about 100 nm, belonging to the family Myoviridae.

[0046] Example 3

[0047] Genome sequencing of phage PpZDSS02 mainly includes the following steps:

[0048] The purified phage solution prepared in Example 1 was sent to Shanghai Ling'en Biotechnology Co., Ltd. for whole-genome sequencing. First, a bacterial genomic DNA extraction kit produced by OMEGA was used to extract genomic DNA samples, and a TBS-380 fluorometer (Turner Biosystems, Sunnyvale, California, USA) was used to quantify the genomic DNA. High-quality DNA samples (OD260 / 280 ratio between 1.8 and 2.0, content greater than 6 micrograms) were selected to construct fragment libraries, and the Illumina NovaSeq 6000 platform was used for sequencing. The original paired-end sequencing reads were trimmed and quality-controlled using the Trimmomatic software according to the parameters (sliding window: 4:15, minimum length: 75) (version 0.36, website http: / / www.usadellab.org / cms / uploads / supplementary / Trimmomatic). Further, the ABySS v2.2.0 (http: / / www.bcgsc.ca / platform / bioinfo / software / abyss) software was used to perform genome assembly with multiple k-mer parameters. Subsequently, GapCloser v1.12 (https: / / sourceforge.net / projects / soapdenovo2 / files / GapCloser / ) was used to fill the remaining local internal gaps, and single-nucleotide polymorphisms in the final assembly results were corrected. Genome annotation used GeneMark v4.17 (http: / / topaz.gatech.edu / GeneMark / ) to identify gene models. Then, all gene models were aligned against the non-redundant databases (NR in NCBI), SwissProt (http: / / uniprot.org), KEGG (http: / / www.genome.jp / kegg / ), and COG (http: / / www.ncbi.nlm.nih.gov / COG) using BLASTP, and functional annotation was performed using the BLASTP module.

[0049] The results are as Figure 3As shown, the total length of the PpZDSS02 gene is 42,296 bp (SEQ ID NO.2), the GC content is 37.7%, and there are 79 open reading frames (ORFs); among the proteins encoded by the 79 ORFs, 19 ORFs do not have homologous genes found in the database, and homologous sequences are found for the proteins encoded by 60 ORFs. Among them, the proteins encoded by 44 ORFs are hypothetical proteins, and the functions of 16 protein sequences are clear. Through whole-genome alignment in the NCBI database, it is found that the homology with the closest phage to PpZDSS02 (Proteus phage ph vB_PmiM_ZX7) is only 84.53% and the coverage rate is only 76% (as Figure 4 shown). According to the current naming rules, the genomic data shows that PpZDSS02 is a novel phage. Comparing all gene protein sequences with the CARD database, no pathogenic factors and resistance genes are found, indicating its safety at the gene level. Combining the physiological and biochemical characteristics of the phage, it is identified as Proteus phage PpZDSS02, and this phage is deposited in the China Center for Type Culture Collection (abbreviated as CCTCC). Deposit address: Wuhan University, Wuhan; Deposit number: CCTCC NO: M 20222019; Deposit date: December 23, 2022.

[0050] Example 4

[0051] Inhibitory effect of phage PpZDSS02 on Proteus in liquid LB medium

[0052] Take Proteus HZSC 4-1 in the logarithmic growth phase and dilute it with 0.9% normal saline to form a bacterial suspension with a final concentration of 1×10 7 CFU / mL. The purified phage PpZDSS02 solution prepared in Example 1 (10 9 PFU / mL) is diluted with SM buffer to form suspensions with final concentrations of 10 9 PFU / mL, 10 8 PFU / mL, 10 7 PFU / mL, 10 6 PFU / mL, 10 5 PFU / mL, 10 4 PFU / mL, 10 3 PFU / mL CFU / mL. Set the following experimental groups to measure the inhibitory effect of phage PpZDSS02 at different concentrations on Proteus. Test group: Add 800 μL of LB liquid medium and 100 μL of bacterial liquid (10 7(CFU / mL) and 100 μL of the diluted phage PpZDSS02 purification solution (concentrations of 10 9 PFU / mL, 10 8 PFU / mL, 10 7 PFU / mL, 10 6 PFU / mL, 10 5 PFU / mL, 10 4 PFU / mL, 10 3 PFU / mL). Add 200 μL of the mixture of bacteria and phage to each well of a 96-well plate; Control group: Add 900 μL of LB liquid medium and 100 μL of bacterial solution (10 7 CFU / mL) to a 1.5 mL centrifuge tube, and add 200 μL of the bacterial solution (from the 1.5 mL centrifuge tube) to each well of the 96-well plate. Incubate at 37 °C for 24 h, and measure the OD value at 600 nm using an automatic growth curve analyzer. Each group is measured in parallel 3 times. The results are as Figure 5 shown. In the experimental groups, the OD 600nm measured in the experimental groups with the addition of the phage PpZDSS02 suspension was significantly lower than that of the control group, indicating that phage PpZDSS02 can effectively reduce the number of Proteus in the liquid LB medium. Among them, when the ratio of the titer of phage PpZDSS02 to the bacterial solution concentration is 0.0001 (phage concentration 10 3 PFU / mL), the antibacterial effect is the best. The OD 600 nm value in the experimental group did not change much within 0 - 6 h, indicating that the growth of Proteus was completely inhibited.

[0053] Example 5

[0054] Inhibitory effect of phage PpZDSS02 on Proteus in fish juice

[0055] Cut fresh perch fish meat into pieces, weigh it, add water at a mass ratio of 1:1, boil for 10 min, filter with gauze, take the meat block, add an equal mass of water again, continue to boil for 5 min, take the fish juice, filter it with filter paper, adjust the pH to 7 with NaOH, dispense it into conical flasks, and sterilize at 121 °C for 15 min for standby. Dilute Proteus HZSC 4-1 in the logarithmic growth phase with 0.9% normal saline to form a bacterial suspension with a final concentration of 1×10 6 CFU / mL. Dilute the phage PpZDSS02 purification solution in Example 1 with SM buffer to form suspensions with final concentrations of 10 6 PFU / mL, 10 4 PFU / mL, 10 2 PFU / mL. Experimental group: Add 800 μL of fish juice and 100 μL of bacterial solution (10 6(CFU / mL) and 100 μL of phage PpZDSS02 suspension (concentrations are 10 6 PFU / mL, 10 4 PFU / mL, 10 2 PFU / mL); Control group: Add 900 μL of fish juice and 100 μL of bacterial solution (10 6 CFU / mL) into a 1.5 mL centrifuge tube. Incubate at 28 °C for 24 h, and measure the number of bacterial colonies (CFU / mL) every 2 h.

[0056] The results are as Figure 6 shown. The bacterial concentration measured at 4 h in the experimental group with the addition of phage PpZDSS02 suspension was significantly lower than that in the control group, and decreased by nearly 3 log values compared to the original bacterial solution, indicating that phage PpZDSS02 can effectively reduce the number of Proteus in fish juice and inhibit its growth within a certain period of time.

[0057] The total length of the PpZDSS02 gene is 42296 bp (SEQ ID NO.2):

[0058]

Claims

1. A Proteus phage PpZDSS02, characterized in that The phage has been deposited in the China Center for Type Culture Collection on December 23, 2022, and its deposit number is: CCTCC NO: M 20222017.

2. Use of the bacteriophage PpZDSS02 according to claim 1 in inhibiting Proteus.

3. The use according to claim 2, characterized in that: The application of the bacteriophage PpZDSS02 in the preparation of a Proteus antibacterial agent.

4. The use according to claim 2, characterized in that: The bacteriophage PpZDSS02 is used in preparing an antibacterial agent for inhibiting the reproduction and metabolism of Proteus.

5. The use according to claim 3, characterized in that: The bacteriophage PpZDSS02 is used in preparing an inhibitor for inhibiting Proteus in the environment and food.

6. The use according to claim 3, characterized in that: The antibacterial agent includes a spray liquid, a rinse liquid or a biological bacteriostatic agent.

7. The use according to claim 6, characterized in that: The antibacterial agent is sprayed or washed on the polluted environment, pollutant collection, transportation, food production environment and utensils to reduce the contamination of Proteus therein.

8. A Proteus antibacterial agent, characterized in that The antibacterial agent preferably uses bacteriophage PpZDSS02 or its isolate or culture as the main component of the antibacterial agent.

9. The Proteus antibacterial agent according to claim 8, characterized in that The Proteus phage PpZDSS02 can be used alone or in combination to prepare an antibacterial agent, which can effectively inhibit or reduce environmental pollutants or Proteus in the food environment.

Images