Proteus bacteriophage ppzdss02 and application thereof

Abstract

The application discloses a bacteriophage of Proteus, PpZDSS02, and an application thereof. The bacteriophage has been preserved in the China Center for Type Culture Collection on December 23, 2022, and the preservation number is CCTCC NO: M 20222017. The application screens and separates a bacteriophage of Proteus, PpZDSS02, which has unique morphological and genomic characteristics and is a brand-new bacteriophage capable of efficiently inhibiting Proteus. The bacteriophage can be applied to the preparation of green and inexpensive Proteus inhibitors. The bacteriophage involved in the application belongs to a natural biological material, does not contain any virulence gene in the genome, has no toxic side effects, and can be used as a bacteria-reducing agent in a contaminated environment.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to a Proteus phage PpZDSS02 and its applications. Background Technology

[0002] Proteus is a Gram-negative facultative anaerobic bacterium, an opportunistic pathogen widely distributed in nature (such as soil, water, and decaying organic matter) and in the intestines of humans and animals. It can cause infection in humans and various animals (fish, poultry, etc.), leading to foodborne illnesses. Common foods contaminated with Proteus include animal products, soy products, and cold dishes. Proteus is primarily transmitted through food; ingesting contaminated food is the main route of infection. Therefore, cutting off the source of infection (the excrement of infected animals and humans and their environment) is an effective means of preventing food contamination and food poisoning caused by Proteus. Currently, the main methods for controlling pathogenic and spoilage bacteria are antibiotics and chemical disinfectants. However, problems such as antibiotic resistance and environmental pollution from chemical disinfectants are becoming increasingly prominent. Therefore, finding new antibiotic alternatives is urgently needed.

[0003] Bacteriophages are a type of bacteria-dependent virus that, after infecting host bacteria, rapidly proliferates and lyses host cells, producing a specific antibacterial effect. Furthermore, bacteriophages are widely distributed in nature, exhibit specificity to host bacteria, are inexpensive to prepare, and have good safety profiles. Developing biocontrol agents or microbial agents with Proteus phage as the main component has potential application value for the removal and growth inhibition of Proteus contaminants in the environment. Summary of the Invention

[0004] Purpose of the invention: To address the problems existing in the prior art, this invention provides a bacteriophage that inhibits the growth of Proteus, which can be applied to Proteus in environments contaminated by the excrement of infected animals and humans, providing 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 bacteriophage, which can be used alone or in combination to prepare biological antibacterial agents that can effectively inhibit or reduce pollution and infection caused by Proteus in the ecological environment or in the food production, processing and preservation environment.

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

[0007] The application of the bacteriophage PpZDSS02 described in this invention in inhibiting Proteus.

[0008] The application of the bacteriophage PpZDSS02 in the preparation of Proteus antibacterial agents.

[0009] The application of the bacteriophage PpZDSS02 in the preparation of antibacterial agents that inhibit the reproduction and metabolism of Proteus.

[0010] Among them, the bacteriophage PpZDSS02 is used in the preparation of inhibitors to suppress Proteus in the environment and food.

[0011] Furthermore, the bacteriophage PpZDSS02 is used to inhibit the reproduction and metabolism of Proteus in the excrement of animals and humans infected with Proteus and in their environment.

[0012] The antibacterial agent includes spray liquid, rinsing liquid or biological antibacterial agent.

[0013] Furthermore, the antibacterial agent reduces Proteus contamination by spraying or washing contaminated environments, pollutant collections, transportation environments, food production environments, and equipment.

[0014] Preferably, the purified Proteus phage PpZDSS02 is prepared as a spray or rinsing solution for use in contaminated environments, environmental pollutants, transportation environments, food production environments, and equipment, reducing Proteus contamination in the environment. The Proteus inhibitor of this invention uses phage PpZDSS02, its isolate, or culture as the main component.

[0015] The Proteus phage PpZDSS02, alone or in combination, is used to prepare antibacterial agents that can effectively inhibit or reduce Proteus in environmental pollutants or food environments.

[0016] The Proteus phage PpZDSS02 in this 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 the tail is about 100 nm in length. The morphological characteristics of the obtained bacteriophage are classified into the Myotail Phage family.

[0018] (2) Genomic characteristics: The genome is 42296 bp in length, with a GC content of 37.7% and 79 open reading frames (ORFs). Of these, 62 ORFs encode hypothetical proteins, and 17 ORFs encode known proteins. Whole-genome alignment with the NCBI database revealed that the closest Proteus phage to PpZDSS02, vB_PmiM_ZX7, showed only 84.53% homology and a coverage of only 76%, indicating that PpZDSS02 is a novel phage. Furthermore, no tRANA genes, lysogens, drug resistance genes, or virulence genes were found in the whole genome of phage PpZDSS02, suggesting that this phage can function as a safe and effective antibacterial agent against Proteus.

[0019] (3) It has strong Proteus lysis properties.

[0020] (4) It can effectively inhibit the proliferation of Proteus in culture medium and physical models.

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

[0022] This invention has isolated and screened a Proteus phage, PpZDSS02, which possesses unique morphological and genomic characteristics and is a novel phage capable of efficiently inhibiting Proteus. This phage can be used to prepare green and inexpensive Proteus inhibitors.

[0023] The bio-antibacterial agent prepared from the bacteriophage PpZDSS02 of this invention can effectively control the growth of Proteus in culture media and environment, and can be easily formulated into spray or rinsing solutions for sterilization of environment or equipment, reducing the risk of Proteus in the environment and its contamination of food; the bacteriophage involved in this invention is a natural biological material, does not contain any virulence genes in its genome, has no toxic side effects, and can be used as a bacteriostatic agent in polluted environment. Attached Figure Description

[0024] Figure 1 The morphology of phage PpZDSS02 plaques on LB double-layer agar plates.

[0025] Figure 2 Electron micrograph of bacteriophage PpZDSS02.

[0026] Figure 3 A complete genome sequence diagram of bacteriophage PpZDSS02.

[0027] Figure 4 This is a schematic diagram of the whole genome alignment of bacteriophage PpZDSS02 in the NCBI database.

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

[0029] Figure 6 The inhibitory effect of bacteriophage PpZDSS02 on Proteus in a food model (fish juice). Detailed Implementation

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

[0031] Unless otherwise specified, all raw materials and reagents used in this invention are commercially available.

[0032] The bacteriophage host bacterium used in this invention is *Proteus* (strain number: HZSC 4-1). When preserving bacteriophage PpZDSS02, it was also preserved as a host bacterium. The bacteriophage and host bacterium were treated as a single set of materials with one preservation number. *Proteus* HZSC 4-1 was isolated from spoiled freshwater fish during low-temperature storage using conventional methods. It is a wild-type *Proteus* bacterium, identified by sequencing. Its 16S rDNA is SEQ ID NO. 1. The host bacterium was cultured in 5 mL LB broth at 37°C in a shaker until the logarithmic growth phase, provided by the Zhejiang Provincial Freshwater Fisheries Research Institute. Other wild-type *Proteus* bacters can also be used in this invention.

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

[0034] Example 1

[0035] Phage isolation and purification preparation

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

[0037] 2. Phage enrichment: Add 5 mL of the above filtrate to 5 mL of double-concentration LB liquid medium, and add 100 μL of Proteus vulgaris (HZSC 4-1) in the logarithmic growth phase. Incubate overnight at 37°C with a shaker at 150 rpm. The next day, transfer the culture from the test tube to a sterile centrifuge tube, centrifuge at 8000×g for 10 min at 4°C, and filter the supernatant through a 0.22 μm filter membrane to obtain the phage stock solution. Store at 4°C.

[0038] 3. Spot formation: Take 100 μL of the logarithmic growth phase host bacterial suspension HZSC 4-1 from step 1, mix it with 5 mL of semi-solid culture medium, pour it onto an LB solid plate, and after it solidifies, add 10 μL of the phage stock solution from step 3. After the liquid is absorbed by the culture medium, incubate it in a 37℃ constant temperature incubator for 8 h, and observe whether phage plaques are formed.

[0039] 4. Phage purification: Pick a single clear plaque from the bilayer plate where phage plaques appeared in step 3 and transfer it to 1 mL of sterile SM buffer. Mix well and incubate at 4°C for 24 h. The next day, perform 10-fold serial dilutions of the phage solution using sterile SM buffer (10... -1 ~10 -7 Take 100 μL of phage solution of each dilution and mix it with 100 μL of Proteus vulgaris HZSC 4-1 bacterial solution in logarithmic growth phase at room temperature for 10 min. Then add 5 mL of LB semi-solid medium, mix well, and quickly pour onto pre-prepared LB solid medium to make a double-layer plate. After solidification, invert the plate and incubate at 37°C. Repeat the double-layer plate method three times. When the phage plaques are of uniform size, the phage is considered to have been isolated as pure. The isolated phage is shown in the image. Figure 1 As shown, it is named PpZDSS02.

[0040] 5. Potency Assay: A purified phage plaque was picked and incubated in 5 mL of LB broth containing 100 μL of logarithmic-phase HZSC 4-1 bacterial suspension at 37°C for 8 h. After centrifugation at 8000 rpm for 10 min, the supernatant was filtered through a 0.22 μm filter to obtain the phage purification and separation filtrate. 100 μL of the phage purification and separation filtrate was mixed with 100 μL of logarithmic-phase Proteus suspension and incubated at room temperature for 10 min. This mixture was then added to 10 mL of LB liquid medium and incubated overnight at 37°C with shaking at 150 rpm. The culture was transferred to a sterile centrifuge tube and centrifuged at 5000 × g for 10 min. The supernatant was collected and filtered through a 0.22 μm microporous membrane for sterilization to obtain the phage purification solution. The titer of the phage purification solution was determined using a double-layer plate test. The specific procedure was as follows: the phage purification solution was serially diluted 10-fold with SM buffer (10... -1 -10 -7 Take 100 μL of each dilution of phage and 100 μL of HZSC 4-1 host bacterial culture in logarithmic growth phase, add 5 mL of LB semi-solid medium, mix well, and quickly pour onto pre-prepared LB solid medium to make double-layer plates. After solidification, invert the plates and incubate at 37℃ for 12 h. Manually count the resulting plaques and calculate the titer. The counting results show that the purified PpZDSS02 solution has a titer of 10 against Proteus HZSC 4-1. 9 PFU / mL or higher.

[0041] 6. Phage preservation: Select a uniformly sized and morphologically uniform phage plaque obtained from the last purification and incubate it in 5 mL of LB broth containing 100 μL of logarithmic growth phase HZSC 4-1 bacterial suspension at 37℃ for 8 h. Centrifuge at 8000 r / min for 10 min, and filter the supernatant through a 0.22 μm filter membrane to obtain phage filtrate. Mix 900 μL of phage filtrate and 300 μL of 70% glycerol in a sterilized preservation tube and store at -80℃ to obtain phage stock solution PpZDSS02.

[0042] Example 2

[0043] Morphage morphology observation of PpZDSS02 mainly includes the following steps:

[0044] The morphological characteristics of bacteriophages were observed using transmission electron microscopy. A phosphotungstic acid negative staining method was employed. With the membrane side of a copper mesh facing upwards, 10 μL of the bacteriophage purification solution prepared in Example 1 was dropped onto the copper mesh. After adsorption for 15 min, the moisture was blotted dry with absorbent paper. The copper mesh was then removed and allowed to air dry naturally for 2-3 min. Next, a drop of 2% phosphotungstic acid (PTA) aqueous solution was dropped onto the copper mesh for staining. After 2 min, the mesh was removed, blotted dry with absorbent paper, and allowed to air dry for 5 min. The phage images were then observed using a transmission electron microscope, and clear images were selected for photographic analysis.

[0045] The results are as follows Figure 2 As shown, the head of bacteriophage PpZDSS02 is symmetrical, with a diameter of approximately 50 nm and a tail length of approximately 100 nm, belonging to the Myotail Phage family.

[0046] Example 3

[0047] The genome sequencing of bacteriophage PpZDSS02 mainly includes the following steps:

[0048] The phage purification solution prepared in Example 1 was sent to Shanghai Lingen Biotechnology Co., Ltd. for whole-genome sequencing. Genomic DNA samples were first extracted using a bacterial genomic DNA extraction kit manufactured by OMEGA, and the genomic DNA was quantified using a TBS-380 fluorometer (Turner Biosystems, Sunnyvale, California, USA). High-quality DNA samples (OD260 / 280 ratio between 1.8 and 2.0, concentration greater than 6 μg) were selected to construct fragment libraries, and sequencing was performed using the Illumina NovaSeq 6000 platform. The raw paired-end sequencing reads were trimmed and quality-controlled using Trimmomatic software according to parameters (sliding window: 4:15, minimum length: 75) (version 0.36, website: http: / / www.usadellab.org / cms / uploads / supplementary / Trimmomatic). Genome assembly was further performed using ABySSv2.2.0 (http: / / www.bcgsc.ca / platform / bioinfo / software / abyss) software with multiple K-value parameters. Subsequently, GapCloserv 1.12 (https: / / sourceforge.net / projects / soapdenovo2 / files / GapCloser / ) was used to fill remaining local gaps and correct for single-base polymorphisms in the final assembly. Genome annotation used GeneMark v4.17 (http: / / topaz.gatech.edu / GeneMark / ) to identify gene models. All gene models were then aligned using BLASTP with 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), and functional annotation was performed using the BLASTP module.

[0049] The results are as follows Figure 3As shown, the PpZDSS02 gene has a total length of 42296 bp (SEQ ID NO.2), a GC content of 37.7%, and 79 open reading frames (ORFs). Of the 79 ORFs encoding proteins, 19 ORFs had no homologous genes found in the database, and 60 ORFs encoded proteins with homologous sequences found in the database. Among these, 44 ORFs encoded hypothetical proteins, and 16 protein sequences had clearly defined functions. Whole-genome alignment with the NCBI database revealed that the closest bacteriophage to PpZDSS02 (Proteus phage ph vB_PmiM_ZX7) showed only 84.53% homology and a coverage of only 76% (e.g., ...). Figure 4 As shown in the figure, according to current nomenclature, genomic data shows that PpZDSS02 is a novel bacteriophage. Comparison of all gene and protein sequences with the CARD database revealed no pathogenic factors or resistance genes, indicating its safety at the gene level. Based on the physiological and biochemical characteristics of the bacteriophage, it was identified as Proteus phage PpZDSS02 and deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China; accession number: CCTCC NO: M 20222019; deposit date: December 23, 2022.

[0050] Example 4

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

[0052] Logarithmic growth phase of Proteus HZSC 4-1 was diluted with 0.9% physiological saline to obtain a final concentration of 1×10⁻⁶. 7 CFU / mL bacterial suspension, Example 1 preparation of bacteriophage PpZDSS02 purification solution (10 9 The PFU / mL solution was diluted with SM buffer to obtain 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 A suspension of PFU / mL and CFU / mL was used to determine the inhibitory effect of different concentrations of phage PpZDSS02 on Proteus mirabilis. Experimental groups: 800 μL of LB liquid medium and 100 μL of bacterial suspension (10...) were added to 1.5 mL centrifuge tubes, respectively. 7CFU / mL) and 100 μL of phage PpZDSS02 purification buffer (concentrations of 10 CFU / mL) were diluted with 100 μL of the diluted solution. 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 a mixture of bacteria and bacteriophage to each well of a 96-well plate; control group: add 900 μL of LB liquid medium and 100 μL of bacterial suspension (10 PFU / mL) to each 1.5 mL centrifuge tube. 7 CFU / mL), add 200 μL of bacterial culture (in a 1.5 mL centrifuge tube) to each well of a 96-well plate. Incubate at 37℃ for 24 h, and measure the OD value at 600 nm using an automated growth curve analyzer. Each group was measured in triplicate. Results are as follows. Figure 5 As shown, in the experimental group, the OD value of the experimental group with added bacteriophage PpZDSS02 suspension was measured. 600nm The titer was significantly lower than that of the control group, indicating that phage PpZDSS02 can effectively reduce the number of Proteus in liquid LB medium. Specifically, when the ratio of phage titer to bacterial concentration was 0.0001 (phage concentration 10...), the titer was significantly lower than that of the control group. 3 PFU / mL showed the best antibacterial effect, with the experimental group showing the highest OD within 0–6 hours. 600 The nm value did not change much, indicating that the growth of Proteus was completely inhibited.

[0053] Example 5

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

[0055] Fresh sea bass fillets were cut into chunks and weighed. Water was added at a 1:1 mass ratio and boiled for 10 minutes. The mixture was filtered through cheesecloth. The fillets were then rehydrated with an equal amount of water and boiled for another 5 minutes. The fish broth was filtered through filter paper, and the pH was adjusted to 7 with NaOH. The broth was then dispensed into Erlenmeyer flasks and sterilized at 121°C for 15 minutes. Logarithmic growth phase Proteus mirabilis HZSC 4-1 was diluted with 0.9% physiological saline to obtain a final concentration of 1×10⁻⁶. 6 The bacterial suspension at CFU / mL was diluted with SM buffer to form a final concentration of 10 CFU / mL. (Note: The original text contains some formatting errors and inconsistencies. A more accurate translation would require the full context.) 6 PFU / mL, 10 4 PFU / mL, 10 2 A suspension of PFU / mL. Experimental group: 800 μL of fish juice and 100 μL of bacterial suspension (10...) were added to a 1.5 mL centrifuge tube. 6CFU / mL) and 100 μL of phage PpZDSS02 suspension (concentrations of 10 ... were respectively. 6 PFU / mL, 10 4 PFU / mL, 10 2 PFU / mL); Control group: 900 μL fish juice and 100 μL bacterial culture (10 PFU / mL) were added to a 1.5 mL centrifuge tube. 6 CFU / mL). Incubate at 28℃ for 24 h, and measure bacterial colony count (CFU / mL) every 2 h.

[0056] The results are as follows Figure 6 As shown, the bacterial concentration in the experimental group with added phage PpZDSS02 suspension was significantly lower than that in the control group at 4 hours, and decreased by nearly 3 log values ​​compared with the original bacterial suspension. This indicates that phage PpZDSS02 can effectively reduce the number of Proteus in fish juice and inhibit its growth for a certain period of time.

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

[0058]

Claims

1. A bacteriophage of Proteus, PpZDSS02, characterized by, The bacteriophage has been preserved in China Center for Type Culture Collection, and the preservation time is December 23, 2022, and the preservation number is CCTCC NO: M 20222017.

2. A Proteeus bacteriostatic agent, characterized by comprising: The bacteriostatic agent takes the bacteriophage PpZDSS02 or its culture as the main component of the bacteriostatic agent.

3. The Proteeae bacteriostatic agent of claim 2, wherein, The bacteriophage PpZDSS02 of the Proteus bacteriophage is used alone or in combination to prepare the bacteriostatic agent.

Images