CN116410935A - A multivalent Vibrio parahaemolyticus phage vB_VpaP_G1 capable of cross-families infection and its application - Google Patents

Abstract

The invention belongs to the technical field of biology, and discloses a cross-family infectious multivalent vibrio parahaemolyticus phage vB_VpaP_G1 and application thereof, wherein G1 is a cross-family bacterial multivalent phage, and can crack bacteria such as Cronobacter, salmonella, escherichia coli, yersinia small intestine and Aeromonas caviae from the Enterobacteriaceae besides the Vibrio parahaemolyticus of the Vibrio, can be used as an antibacterial agent for preventing and treating various bacteria, effectively solves the problem of drug resistance faced by the current antibiotic treatment, and has good utilization value and application prospect.

Description

A multivalent Vibrio parahaemolyticus phage vB_VpaP_G1 capable of cross-family infection and its application

technical field

The invention belongs to the field of biotechnology, and more specifically relates to a strain of multivalent Vibrio parahaemolyticus phage vB_VpaP_G1 capable of transfamilies infection and its application.

Background technique

Vibrio parahaemolyticus (Vibrio parahaemolyticus) is a halophilic Gram-negative bacterium belonging to the family Vibrio, which is widely distributed in fish, shrimp and shellfish in estuaries and oceans around the world. Diarrhea, fever, gastroenteritis, etc. are often caused by eating fresh, undercooked or cross-contaminated aquatic products contaminated by Vibrio parahaemolyticus. According to existing reports, food poisoning incidents caused by Vibrio parahaemolyticus occupy the first place in food poisoning incidents, seriously endangering people's health and causing huge economic losses. Antibiotic treatment is the most important means to prevent and treat bacterial infections. However, the long-term and irregular abuse of antibiotics in recent years has caused antibiotic resistance to bring a huge burden to clinical treatment and social economy, and because of the broad-spectrum killing characteristics of antibiotics, The native microbial ecosystem will cause irreversible damage, which will eventually pose a threat to human health and become a global public health security problem. Therefore, it is urgent to find alternative treatments for antibiotics to deal with bacterial resistance.

Bacteriophages exist widely in nature and can specifically cause the lysis of host cells and cause bacterial death, which provides a new method for the control of food-borne pathogens. Because of their host specificity, bacteriophages will not disrupt the balance of the micro-ecological system, are safe for humans and animals, and are not easy to develop tolerance. They have advantages over antibiotics in the control of pathogenic bacteria, and gradually become a substitute for antibiotics. , applied to the market. Phages are highly specific, and in recent years, academic circles have found that some phages also have a certain broad-spectrum lytic ability for host bacteria between different species, so this kind of phages are called "multivalent phages", and multivalent phages are used to kill bacteria simultaneously. The technology of living a variety of pathogenic bacteria has the advantages of high broad-spectrum, safety and efficiency, and environmental friendliness. This discovery has laid a solid theoretical foundation for the wide application of phages.

There are some limitations in phage applications and phage therapy.

First of all, the reason why phages are expected to replace antibiotics in the treatment of bacterial infections is because of their specificity to the host bacteria, so that they will not harm other bacteria while killing the host bacteria. However, there are many kinds of bacteria in nature, and the narrow spectrum of phages It is very difficult to prevent and control the mixed infection of various bacteria, because this makes it difficult to make a breakthrough in its application.

Secondly, some phages have lysogenic ability. In the life cycle of lysogenic phages, that is, temperate phages, their DNA is mainly integrated into the chromosomes of bacteria to reproduce together with bacteria. Only in the case of adversity stress will enter the cracking cycle. This leads to two problems, one is that the pathogenic bacteria cannot be quickly killed; the other is that it will cause horizontal gene transfer, which may integrate the bacterial pathogenic gene into the phage genome, thereby causing the pathogenic gene to spread among strains.

Finally, the stability of phage preparations will affect the important factors of its practical application. During the transportation and sales of phage preparations, the titer of phages must be kept within the effective range. In addition, the phages must ensure that they can survive stably in the environment where they are placed and applied. play an effective role.

Therefore, on the one hand, the key to solving these limitations is to find a phage that has a wide cleavage spectrum and can lyse a variety of different serotypes of Vibrio parahaemolyticus. Force factor, and can produce better disinfecting and preventive effects, can be safely applied in actual production.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the narrow deficiency of phage lysis spectrum in the prior art, and provide a novel Vibrio parahaemolyticus phage vB_VpaP_G1 (hereinafter referred to as G1), which is a strain that can infect hosts across families. The multivalent phage has a broad host spectrum, and has good killing and preventive effects on bacteria of Vibrio, Aeromonas, Enterobacteriaceae, and Yersiniaceae, and does not contain lysogeny genes and virulence genes, which can be applied to the research and development of antibacterial agents.

The second object of the present invention is to provide the application of the above-mentioned phage G1.

The purpose of the present invention is achieved through the following technical solutions:

The present invention firstly provides a strain of Vibrio parahaemolyticus phage vB_VpaP_G1, which is classified as Vibriophage. The phage was deposited in the Guangdong Microbial Culture Collection Center on January 5, 2023, with the preservation number GDMCCNO: 63110-B1, and the preservation address Address: 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City.

The phage was isolated from the sewer water samples of Huangsha Aquatic Market in Guangzhou by sedimentation-filtration method. Its host is O11-56. The pH tolerance range of the phage is pH 5-11, and it can withstand high temperature of 60°C. It is named vB_VpaP_G1 (hereinafter abbreviated as G1).

Specifically, the composition may be a pharmaceutical composition or a bactericidal composition.

The present invention also provides the application of the hemolytic Vibrio phage vB_VpaP_G1, and the specific application includes:

(1) Inhibit the reproduction of Vibrio parahaemolyticus, Aeromonas, Salmonella, Streptococcus pneumoniae, Escherichia coli, Cronobacter, and Yersinia enteritidis;

(2) Kill Vibrio parahaemolyticus, Aeromonas, Salmonella, Streptococcus pneumoniae, Escherichia coli, Cronobacter, Yersinia enteritidis;

(3) Prevention and control of pollution caused by Vibrio parahaemolyticus, Aeromonas, Salmonella, Streptococcus pneumoniae, Escherichia coli, Cronobacter, and Yersinia enteritidis;

(4) Preparation of medicines for treating diseases caused by Vibrio parahaemolyticus, Aeromonas, Salmonella, Streptococcus pneumoniae, Escherichia coli, Cronobacter, and Yersinia enteritidis infection.

Preferably, the disease is selected from acute diarrhea, sepsis, salmonellosis, bronchitis, pneumonia, urinary system trauma infection, meningitis, peritonitis, bacteremia, and necrotizing enterocolitis. The invention provides an environment-friendly new drug for preventing and controlling the diseases caused by the above-mentioned bacterial infection.

Preferably, in the above application, the bactericidal composition may be a water body disinfectant, so as to be applied in the process of breeding, transporting and storing aquatic products. More preferably, its concentration is ≥ 10 ⁵ pfu/mL. At this concentration, the disinfection effect of water disinfectant is the best. Of course, it is foreseeable that the water body disinfectant also contains other compatible bactericidal active ingredients or auxiliary agents.

Preferably, in the above-mentioned application, the bactericidal composition or/and pharmaceutical composition can also be a feed additive, and adding the phage or the composition containing the phage to the feed of aquatic animals such as Penaeus vannamei can effectively improve the survival of shrimp seedlings rate, in feed additives, the content of the bacteriophage ≥ 10 ⁵ pfu/mL.

The present invention also provides a method for cultivating the Vibrio parahaemolyticus phage vB_VpaP_G1, which comprises inoculating the Vibrio parahaemolyticus phage vB_VpaP_G1 in a liquid medium, culturing it to the logarithmic growth phase, and then inserting the phages for phage culture.

Compared with the prior art, the present invention has the following advantages:

The invention provides a strain of Vibrio parahaemolyticus phage, the phage is named vB_VpaP_G1, the Vibrio parahaemolyticus phage G1 has a short incubation period (10min) to Vibrio parahaemolyticus, and can be amplified by a specific host , and the phage can withstand high temperature of 60°C, pH 5.0-pH 11.0, and be inactivated after 30 minutes of ultraviolet light irradiation. It has strong stability, low requirements for transportation and storage, and can be used under different conditions applicable in the environment. The most notable point is that G1 is a multivalent phage that can lyse bacteria across families. In addition to lysing Vibrio parahaemolyticus from the Vibrio family, it can also lyse Cronobacter, Salmonella, Escherichia coli from the Enterobacteriaceae Bacteria, Yersinia enterica and Aeromonas caviae from the Aeromonas family can be used as antibacterial agents for the prevention and control of various bacteria, effectively solving the problem of drug resistance faced by current antibiotic treatment, and having relatively Good utilization value and application prospect.

Description of drawings

Figure 1 is an electron micrograph of phage G1;

Figure 2 is a sequence alignment diagram of phage G1 VIDIRIC;

Figure 3 is an analysis diagram of the phage G1 protein sharing network;

Figure 4 is a phylogenetic tree of phage G1;

Figure 5 is the MOI of phage G1;

Figure 6 is the temperature stability of phage G1;

Fig. 7 is the pH stability of phage G1;

Figure 8 is the ultraviolet resistance of phage G1;

Figure 9 is a one-step growth curve of phage G1.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings in the embodiments of the present invention, and the content described will not limit the present invention described in the claims. Apparently, the described embodiments are only key embodiments, not all embodiments, and the content is easily understood by those skilled in the art.

The isolation and cultivation of embodiment 1 phage

1. Activation and cultivation of host bacteria

The food-borne Vibrio parahaemolyticus, Cronobacter, Salmonella, Yersinia enteritidis, Escherichia coli, Listeria, Staphylococcus aureus, Aeromonas, Bacillus, etc. Provided by the Institute of Microbiology, take the dry powder tube out of the refrigerator at 4°C, smash the mouth of the tube, take it out with a sterile pipette tip, add it to 3% sodium chloride alkaline peptone water, and place it in a shaker at 37°C, 200r/min. After 24 hours, after being identified by streaking on a chromogenic plate, pick a single colony and inoculate it in 5 mL of liquid medium, culture at 37°C and 200 r/min for 24 hours to obtain a single suspension.

2. Isolation and purification of phage

On June 30, 2020, water samples were collected from the sewer of Huangsha Aquatic Products Trading Market in Guangzhou City, Guangdong Province. The sewage samples were centrifuged at 8000×g for 10 minutes at room temperature, and the supernatant was filtered through a 0.45 μm filter. After adding solid anhydrous Magnesium sulfate to a final concentration of 50mM, mix well, let stand at room temperature for 10-20min, use a suction filtration device, pass the mixed solution through a 0.45μm cellulose filter membrane, take the filter membrane and put it into the phage eluent for ultrasonic elution, and then The eluate was filtered through a 0.45 filter, and the filtrate was stored at 4°C for future use. Take a certain amount of the above-mentioned filtrate and the suspension of the host in the logarithmic phase, mix them into the double-material TSB containing 4mM CaCl ₂ , and pass through a 0.45 μm filter after culturing for 24 hours to obtain the phage filtration culture solution. Then use the double-layer plate method to identify whether the phage filter culture medium contains phage, pick the filter culture medium containing the target host phage, and use the double-layer plate method to perform multiple purifications, and finally form phage plaques with the same size and shape on the medium That is, the purification is completed. This phage was named vB_VpaP_G1 (hereinafter abbreviated as G1).

The phage identification of embodiment 2 phage

1. Morphological identification of phage

Take 15 μl of 1×10 ¹⁰ pfu/mL phage sample dropwise onto the microporous copper grid, let it settle naturally for 15 minutes, absorb the excess liquid with filter paper, add 10 μl of phosphotungstic acid (2%) for staining for 5 minutes, and absorb the excess liquid with filter paper Liquid, after natural drying, use a transmission electron microscope to find phage particles, observe its shape, and take pictures for records. For specific pictures, see Figure 1. The phage G1 has an icosahedral head and a short non-shrinking tail. The diameter of the head is 34±2nm , tail length 12 ± 2nm. According to its morphological characteristics combined with the formal taxonomic code of ICTV, phage G1 fits the typical characteristics of Caudoviricetes Podoviridae.

2. Molecular biological identification of phage

(1) Genome extraction of phage G1

The phages were concentrated using the PEG-NaCl method and allowed to stand overnight at 4°C. The next day, centrifuge at 12000×g for 20 minutes. Resuspend the pellet with SMbuffer. The concentrated phages were extracted with phenol, chloroform and isoamyl alcohol, and stored at -20°C for future use.

(2) Whole-genome sequencing of bacteriophage G1 and analysis of its whole-genome characteristics

The concentration of the extracted DNA was measured using a fluorescence quantification instrument Qubit 3.0 Fluorometer, and 100 ng of the above DNA sample was taken, and a phage DNA library was constructed using the QIAseq FX DNA Library Kit kit, and sequenced on the IlluminaNextSeq 550 (Illumina, USA) sequencing platform. Sequence assembly and splicing of the sequencing library was performed using SPAdes software. The final splicing results were compared on NCBI. Phage G1 is a short-tailed virus with RNA polymerase. Therefore, G1 was preliminarily classified as Autographiviridae, and phage G1 showed the highest homology (73.01%) with phage phiR8-01 (NC_047951.1), but the genome coverage was very low (19%). The results of BLASTn and ANI indicated that the coverage rate between the G1 genome and the phiR8-01 genome was too low, and the similarity results were not reliable. Therefore, the genome similarity between phage G1 and other phages was reanalyzed using VIRIDIC. The results of VIRIDIC showed that the maximum similarity between G1 and other known sequences was 47.1% (Fig. 2). The International Committee on Taxonomy of Viruses (ICTV) describes a genus as a group of highly similar viruses sharing at least 60–70% of nucleotides across the length of the genome, with G1 similarity to other sequences well below this threshold. In order to further clarify the taxonomic status of G1, the phage protein sharing network was analyzed. The results of vConTACT analysis showed that vB_VpaP_G1 and Melnykvirinae subfamily phages formed a VC cluster subset (Figure 3), and further obtained the terminator enzyme large subunit The support of the phylogenetic tree (Fig. 4). Therefore, it is suggested that phage G1 establishes a Youngvirus genus under Melonykvirinae subfamily, and G1 is designated as a new species in this new genus.

Table 1 briefly illustrates the characteristics of phage G1. A total of 50 ORFs have been annotated in phage G1, of which 74.0% ORFs (37/50) can be annotated to proteins with known functions. These ORFs can be divided into structural functional domains, nucleic acid metabolism Functional domains, packaging functional domains, lytic functional domains, DNA injection functional domains, anti-defense and other functional domains.

Structural and functional domains include: outer capsid protein, DNA maturation enzyme A, tail-forming protein, tail fiber protein, structural protein, caudal tube protein B, caudal tube protein A, major capsid protein, scaffold protein, head-to-tail junction protein, etc.

Nucleic acid metabolism functional domains include: DNA helicase, DNA primer enzyme, ribosome-like regulatory factor protein.

N-acyltransferase superfamily proteins, RNA polymerases, ATP-binding proteins, DNA endonucleases.

DNA exonuclease, DNA polymerase, DNA ligase, etc.

Packaging functional domain includes: terminal enzyme large subunit and terminal enzyme small subunit, it is generally believed that terminal enzyme large subunit participates in DNA cleavage, while terminal enzyme small subunit participates in recognizing and binding concatemer DNA.

Cleavage domains include: phage lysins, perforins and spanin proteins. Lysin is a lytic enzyme encoded by phage, which helps the release of progeny phage from bacterial cells by cleaving the peptidoglycan of the bacterial cell wall. The role of perforin is to form poly-perforin tubular channels on the cell membrane, leading to lytic destruction of the cell.

The functional domain of DNA injection includes: cleavage transglycosidase activity protein, internal core protein A, internal core protein.

Anti-defenses include: silencing protein inhibitor, S-adenosylmethionine lyase, and S-adenosylmethionine lyase

Table 1 Functional classification of open reading frames of bacteriophage G1 whole genome

Example 3 Detection of biological characteristics of bacteriophage G1

1. Determination of phage G1 titer

The determination of the phage titer was determined by the double-layer plate method. First, the purified phage expansion medium was serially diluted 10 times, and an appropriate dilution was selected, and 100 μL of the phage dilution and 100 μL of the parasite cultured to the logarithmic phase were drawn respectively. Vibrio hemolyticus O11-56 bacteria liquid, at the same time, add it to TSB (containing 0.4% technical agar, 2mM CaCl ₂ ) cooled to about 45°C, mix well, pour it on a TSA plate, and do 3 parallel repetitions for each dilution . After the soft agar had solidified, culture it in a constant temperature incubator at 37°C for 5-8 hours, observe and count the plates with 30-300 plaques, and calculate the titer of phage G1 to be 2.57×10 ⁹ pfu/mL.

2. Determination of optimal multiplicity of infection (MOI) of phage G1

Propagate phage G1 and its Vibrio parahaemolyticus O11-56 according to conventional methods, determine the titer of phage and host bacteria, and configure different ratios (100, 10, 1, 0.1, 0.01, 0.001 and 0.0001) of phage-logarithmic phase Vibrio parahaemolyticus bacteria liquid, in which the concentration of the host bacteria is 10 ⁸ cfu/mL, is then added to the TSB culture medium (containing 2mM CaCl ₂ ) according to the above ratio, and cultured in a shaker at 37°C and 200rpm/min After 10 hours, the culture solution was filtered through a 0.45 μm filter, and finally an appropriate gradient of phage dilution was selected, and the titer of the phage was determined by the double-layer plate method, and three independent experiments were carried out. The results in Fig. 5 show that the optimal MOI of phage vB_VpP_G1 was 1, and the phage titer was 8×10 ⁷ pfu/mL.

3. The host bacteria of phage G1

Take 100 μL logarithmic phase bacteria solution and add it to 5mL 0.4% TSA soft agar, mix well and pour it on a 1.5% TSA solid plate, then drop 2 μL phage G1 culture solution on the soft agar, put it on each host bacteria plate, do Five gradient phage G1 dilutions (10 ¹⁰ , 10 ⁹ , 10 ⁸ , 10 ⁷ , 10 ⁶ , 10 ⁵ pfu/mL) were marked, placed in a 37°C incubator for cultivation, observed and recorded after 5 hours Whether there are plaques, so as to confirm the host spectrum of phage G1. As shown in Table 2, the host spectrum identification of strains from 11 genera and 15 species from 7 families showed that G1 could lyse 17 strains of Vibrio parahaemolyticus (17/124), and the lysis rate was 13.7%. G1 can also lyse 3 strains of Aeromonas (3/20) of Aeromonas, with a lysis rate of 15%; 19 strains of Salmonella (19/35) of Enterobacteriaceae, with a lysis rate of 54.3%; 9 strains of Streptococcus pneumoniae, the lysis rate was 30%; 12 strains of Escherichia coli of Enterobacteriaceae, the lysis rate was 42.8%; 56 strains of Cronobacter (56/69) of Enterobacteriaceae, the lysis rate was 81.2%; 7 strains Yersinia enteritidis (7/186), the lysis rate was 3.8%.

Table 2 Host range of phage vB_VpaP_G1

4. Determination of the stability of phage G1 to temperature, pH and ultraviolet

Add 1 mL of phage suspension (1×10 ⁸ pfu/mL) to each of the 1.5 mL EP tubes, place them in different water bath temperatures (25, 37, 50, 60 and 70°C) and incubate for 1 hour, and cool immediately after the end To room temperature, the titer of the phage suspension was detected by the double-layer plate method, and three independent experiments were performed. Refer to Figure 6 for specific results. After phage G1 was treated at 25°C to 50°C for 1 hour, the titer remained basically unchanged, and after 1 hour at 60°C, the titer decreased by about 3 lg values, and after 45 minutes in a water bath at 70°C, the titer was completely unchanged. Inactivate. Therefore, the thermal stability of phage G1 is better.

For the determination of phage pH, filter through TSB liquid medium configured to different pH values (3, 4, 5, 6, 7, 8, 9, 10, 11, 12) with a disposable syringe and a 0.45 μm filter head Sterilize. Add 0.1 mL of phage lysate (1×10 ⁸ pfu/mL) to 0.9 mL of TSB with different pH, take it out after incubating at 37°C for 1 hour, and detect the phage titer by double-layer plate method, and conduct three independent experiments. Referring to Figure 7 for specific results, phage G1 can exist stably in the pH range of 5 to 11. When pH=4, when it is too acidic, the phage titer is reduced by 3 lg values, and when pH=3, the phage is completely inactivated. When pH=12, over alkali, all phages are inactivated.

In the test of phage UV 245 tolerance, add phage suspension (1×10 ⁸ pfu/mL) into a sterile petri dish, and use a UV lamp in a biological safety cabinet. The sample is 15cm away from the UV lamp, starting from 0min , 100 μL sample solution was drawn every 5 minutes, and the phage titer was determined by the double-layer plate method, and three independent experiments were carried out. Refer to Figure 8 for specific results. Phage G1 is more sensitive to ultraviolet light, and the titer of phage decreased significantly every 5 minutes. When irradiated with ultraviolet light for 5 minutes, the titer decreased significantly by 2 lg values, and decreased by 6 lg values in total at 20 minutes, and the phages were completely killed after 30 minutes.

5. One-step growth curve of phage G1

Inoculate 100 μL of the overnight culture of Vibrio parahaemolyticus into 5 mL of TSB, place in a constant temperature shaker at 37°C, and culture at 200 rpm/min until OD ₆₀₀ =0.2, and dilute the bacterial cell suspension to 1×10 ⁸ pfu/mL, Aspirate 1mL of bacterial culture solution with a pipette gun, centrifuge at 8000g for 5min, absorb the supernatant, pay attention to avoid absorbing the bacterial pellet, resuspend in 0.9mL SM buffer, and mix with 100μL 1× ¹⁰⁸ pfu/mL phage suspension (MOI=0.1), add _CaCl2 to a final concentration of 2mM, mix well, let the mixture stand at 37°C for 15min, centrifuge at 12000g for 2min, remove the free phage in the supernatant, and resuspend the bacterial host pellet in 10mL TSB (2mM CaCl ₂ ), then place the mixture on a constant temperature shaker, shake and culture at 37°C and 200rpm/min, start from 0min, draw 100μL sample solution every 5min, and determine the phage by the double-layer plate method Titer. Refer to Figure 9 for specific results. The G1 incubation period is 10 minutes, the lysis period is 25 minutes, and the lysis volume is 29 pfu/cell. Although the incubation period of phage G1 is relatively short, it may be able to have a certain inhibitory effect on the initial growth of V. parahaemolyticus.

The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principle and spirit of the present invention, various changes, modifications, substitutions and modifications to these embodiments still fall within the protection scope of the present invention.

Claims (4)

1. the application of vibrio parahaemolyticus phage vB_VpaP_G1, it is characterized in that, described application comprises: (1) Inhibit the reproduction of Vibrio parahaemolyticus, Aeromonas, Salmonella, Streptococcus pneumoniae, Escherichia coli, Cronobacter, and Yersinia enteritidis; (2) Kill Vibrio parahaemolyticus, Aeromonas, Salmonella, Streptococcus pneumoniae, Escherichia coli, Cronobacter, Yersinia enteritidis; (3) Prevention and control of pollution caused by Vibrio parahaemolyticus, Aeromonas, Salmonella, Streptococcus pneumoniae, Escherichia coli, Cronobacter, and Yersinia enteritidis; (4) Preparation of medicines for treating diseases caused by Vibrio parahaemolyticus, Aeromonas, Salmonella, Streptococcus pneumoniae, Escherichia coli, Cronobacter, and Yersinia enteritidis infection; The phage vB_VpaP_G1 was deposited in the Guangdong Provincial Microbial Culture Collection Center on January 5, 2023, with the preservation number GDMCC NO: 63110-B1, and the preservation address is: 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City. 2. application according to claim 1, is characterized in that, described disease is selected from acute diarrhea, septicemia, salmonellosis, bronchitis, pneumonia, urinary system wound infection, meningitis, peritonitis, bacteremia, necrotic small intestine colitis. 3. The application according to claim 1, wherein the composition comprises a pharmaceutical composition and a bactericidal composition. 4. The application according to claim 3, characterized in that, the pharmaceutical composition is a feed additive, and the bactericidal composition is a water body disinfectant.

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