Application of bacteriophage in reduction of cholera propagation

Abstract

According to the application of the bacteriophage to reduction of cholera transmission, the bacteriophage phi YSZKA is preserved in the China Center for Type Culture Collection on August 1, 2018, the preservation number is CCTCC NO: M 2018513, and the bacteriophage phi YSZKA is classified and named as klebsiella pneumoniae bacteriophage phi YSZKA. The method solves the problems of secondary pollution, wide transmission of vibrio cholerae and the like of the traditional split bacteriophage. The application of the bacteriophage can reduce the toxin production efficiency of vibrio cholerae in oceans, soil and sewers, thereby providing more possibilities for prevention and control of cholera epidemic situation and alleviation of cholera symptoms.

Description

Technical Field

[0001] The invention belongs to the technical field of bacteriophages, and in particular relates to the application of a bacteriophage in reducing the spread of cholera. Background Art

[0002] Currently, cholera patients are mainly treated with oral rehydration, which does not fundamentally deal with the large number of Vibrio cholerae in the cholera epidemic environment. It is known that Vibrio cholerae encodes the cholera toxin gene, which has an important impact on the pathogenicity of Vibrio cholerae. Specifically, the microscopic Vibrio cholerae encodes the cholera toxin gene ctx and ctxB , causing intestinal mucosal cells to lose water, and ultimately leading to diarrhea, vomiting and death from dehydration on a macro scale. Therefore, it is necessary and urgent to fundamentally reduce the synthesis of Vibrio cholerae toxins at the micro level and further develop technological inventions to prevent the risk of cholera epidemic transmission.

[0003] Interactions between microbial groups can regulate gene expression in bacteria. Bacteriophages (phages) and host bacteria are typical examples of cross-kingdom interactions. Bacteriophages are organisms that survive by infecting living host bacteria. They are widely distributed in water, soil, and air. It is estimated that their total amount reaches 10 31 orders of magnitude. At the same time, phages have two lifestyles: lytic and lysogenic. Lysogenic phages integrate genetic material into the genome of host bacteria, allowing the phage genome to maintain a "prophage" state and stably exist in the host bacterial genome, and replicate with the host bacteria. Lysogenic phages carry auxiliary metabolic genes (AMGs), which can supplement the metabolism of host bacteria after successful integration. Therefore, lysogenic phages can serve as stable genetic carriers to compensate for the expression of multiple physiological functions of the host. In addition, prophages release phage particles through proliferation, infect other host bacteria in the environment, and promote horizontal gene transfer. Therefore, lysogenic phages carry toxins to inhibit auxiliary metabolic genes, assist host bacteria in weakening the synthetic expression function of cholera toxin genes, and ultimately achieve the purpose of inhibiting the spread of cholera epidemics.

[0004] Phage synthesis refers to the use of synthetic biology methods to design artificial phages that carry functional genes. By synthesizing artificial phages, we can quantitatively identify the role and function of phages in environmental microbiomes, facilitate artificial intervention in the design of lysogenic phages that carry toxins to inhibit AMGs, and use the auxiliary metabolic capacity of lysogenic phages and the characteristics of not producing lytic DNA to cause secondary pollution to further improve the original phages in reducing the pathogenicity of Vibrio cholerae and inhibiting the spread of cholera epidemics.

[0005] Through relevant literature review and patent search, no public publication or acceptance of biotechnology related to regulating the toxin production capacity of Vibrio cholerae by bacteriophage AMGs to inhibit the spread of cholera epidemics has been found. The existing method closest to the present invention is to use lytic phages to lyse harmful Vibrio cells, thereby controlling the growth of multiple Vibrio in water bodies. For example, invention publications CN114561363A, CN115786277A, CN115717128A and CN117778333B are to solve the problem of multiple bacterial diseases and infections in aquaculture by screening and separating broad-spectrum lytic phages to lyse multiple Vibrio species in water bodies or aquaculture environments. Invention publication CN118028249A prepares an aquatic feed additive containing a high-temperature resistant Vibrio cholerae phage RDP-VC23062, which is used to replace antibiotics to prevent and control bacterial diseases in aquaculture animals. At the same time, the invention also covers the preparation method and application strategy of the additive. In addition to using lytic phages for the prevention and control of Vibrio in water, there are also inventions that screen for antagonistic strains of Vibrio cholerae, Bacillus subtilis ( Bacillus subtills ) YJY20-01, which competitively excludes Vibrio cholerae, provides nutrients, and promotes the growth of farmed animals, thereby reducing the risk of aquatic animals being infected with Vibrio cholerae. These methods indicate that lytic phages or antagonistic strains have the ability to inhibit the growth of Vibrio in aquaculture environments, but generally lack the risk control of secondary contamination of toxin gene DNA products after Vibrio lysis, and lack the potential for application in a wider range of environments other than aquaculture environments.

[0006] The main defects of the existing technology are: Most of the existing phage control technologies for Vibrio cholerae are aimed at Vibrio cholerae and other Vibrio species in aquaculture environments, ignoring the pathogenicity regulation of O1 pathogenic Vibrio cholerae in various environments. In addition, after the artificially screened lytic phage lyses Vibrio cholerae, its lytic toxin gene DNA products can be absorbed by other microorganisms in the water, posing a risk of secondary pollution. And oral rehydration treatment of cholera patients generally cannot solve the problem of environmental Vibrio cholerae transmission.

[0007] The main reasons for the defects are: Since bacteriophages can be used as substitutes for antibiotics to kill pathogens in the environment, the application cases of lytic phages are far more than those of phage AMGs. However, there is a risk that the lysis products of lytic phages after lysing pathogens will be absorbed by environmental microorganisms, and it is difficult to assess the long-term negative ecological impact. Therefore, it is of great significance to add lysogenic phages with toxin inhibition function to reduce the proportion of pathogenic Vibrio cholerae and reduce the risk of cholera epidemics. Summary of the invention

[0008] Technical problem solved: The invention aims to solve the above-mentioned defects of the prior art and provides a bacteriophage for use in reducing the spread of cholera. The method releases bacteriophage into a variety of cholera outbreak environments, and reduces the risk of cholera transmission by injecting toxins into the phage to inhibit AMGs and weaken the virulence of the host Vibrio cholerae, while avoiding the risk of secondary toxin gene DNA contamination caused by the lysis of Vibrio by the lytic phage.

[0009] Technical solution: Application of bacteriophage φYSZKA in the preparation of a gene product that inhibits Vibrio cholerae toxins. The bacteriophage φYSZKA was deposited in the China Center for Type Culture Collection on August 1, 2018, with a deposit number of CCTCC NO: M2018513, and was classified and named Klebsiella pneumoniae phage φYSZKA ( Klebsiella phage φYSZKA).

[0010] The bacteriophage φYSZKA is used for preparing a DNA product that reduces the toxin gene released after Vibrio lysis in the environment.

[0011] The bacteriophage φYSZKA is used in the preparation of a product for reducing the spread of cholera by reducing the toxin gene DNA released after Vibrio lysis in the environment.

[0012] The above Vibrio cholerae toxin gene is ctx and ctxB.

[0013] The above products may also contain other biological or chemical substances that inhibit the toxin production of Vibrio cholerae.

[0014] The above product also contains at least one carrier or auxiliary component for improving the stability, storage life or application effect of the bacteriophage.

[0015] A method for reducing the spread rate of cholera epidemics by injecting the product into the ocean, soil or urban water bodies.

[0016] The above method for reducing the transmission rate of cholera epidemic also includes monitoring changes in the content of Vibrio cholerae toxin in the environment and adjusting the application amount of the product as needed.

[0017] The working principle of the present invention is: 1. Bacteriophages are the most species-rich type of life on earth, and they exclusively infect living host bacteria; 2. Bacteriophages are a type of bacterial virus composed of a protein capsid (60%) and nucleic acid (40%). They are stable in the environment, with a length of about 20 to 200 μm, which is equivalent to hundreds or thousands of times the length of bacteria. They can migrate in the environment, which is beneficial to saving application costs; 3. Auxiliary metabolic genes (AMGs) encoded by bacteriophages can be transferred to host bacteria through horizontal gene transfer to inhibit the expression of key host functions, such as the production process of cholera toxin; 4. The pathogenicity of Vibrio cholerae depends on the production of cholera toxin. The bacteriophage provided by the present invention inhibits the production of cholera toxin after being integrated into the host genome.

[0018] Beneficial effects: 1. Weaken the pathogenicity of Vibrio cholerae. Phages inhibit toxin gene expression through AMGs, reduce the content of cholera toxin in the environment, and stabilize the growth of pathogenic Vibrio cholerae in the environment; 2. Reduce the use of oral rehydration. Phage transplantation reduces the pathogenicity of Vibrio cholerae, inhibits the manifestation of cholera symptoms, and reduces the amount of oral rehydration in the medical treatment of cholera patients; in short, phage transplantation reduces the pathogenicity of Vibrio cholerae in the environment and combines modern biosynthesis and ecological principles to provide an efficient and environmentally friendly way to prevent and control cholera, which is of great significance for reducing the spread of cholera. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the inhibition of pathogenicity of Vibrio cholerae by bacteriophage φYSZKA transplantation.

[0020] Figure 2 This is a transmission electron micrograph of bacteriophage φYSZKA.

[0021] Figure 3 This is the one-step growth curve of bacteriophage φYSZKA.

[0022] Figure 4 The toxin gene of Vibrio cholerae / bacteriophage mixture injected into the South China Sea water sample 12 hours later ctx Expression amount.

[0023] Figure 5 The toxin gene of Vibrio cholerae / bacteriophage mixture injected into the South China Sea water sample 12 hours later ctxB Expression amount.

[0024] Figure 6 Biofilm formation in water samples from the South China Sea after injection of a mixed sample of Vibrio cholerae / bacteriophage for 12 hours.

[0025] Figure 7It is the toxin gene of a mixed sample of Vibrio cholerae / bacteriophage injected into a sewer 12 hours later. ctx Expression amount.

[0026] Figure 8 It is the toxin gene of a mixed sample of Vibrio cholerae / bacteriophage injected into a sewer 12 hours later. ctxB Expression amount.

[0027] Fig. 9 This is the biofilm formation in a sewer 12 hours after the injection of a mixed sample of Vibrio cholerae / bacteriophage.

[0028] Fig.10 It is the toxin gene of Vibrio cholerae / bacteriophage mixed sample injected into the soil of a certain urban green space 12 hours later ctx Expression amount.

[0029] Fig.11 It is the toxin gene of Vibrio cholerae / bacteriophage mixed sample injected into the soil of a certain urban green space 12 hours later ctxB Expression amount.

[0030] Fig.12 The lytic phage and the lytic toxin gene of Vibrio in the environment after the administration of φYSZKA phage ctx The DNA product content.

[0031] Fig.13 The lytic phage and the lytic toxin gene of Vibrio in the environment after the administration of φYSZKA phage ctxB The DNA product content. DETAILED DESCRIPTION

[0032] The following specific implementation methods do not limit the technical solutions of the present invention in any form. Any technical solutions obtained by equivalent replacement or equivalent transformation fall within the protection scope of the present invention. The above-mentioned bacteriophage φYSZKA was deposited in the China Center for Type Culture Collection on August 1, 2018, with the deposit number: CCTCC NO: M 2018513, and the classification name is Klebsiella pneumoniae phage φYSZKA (Klebsiella phage φYSZKA). The deposit address is Luojia Mountain, Wuchang, Wuhan City, Hubei Province, China Center for Type Culture Collection, Wuhan University.

[0033] The above genes ctxA, ctxB Refers to the cholera toxin gene carried by Vibrio cholerae detected in the sample.

[0034] Example 1: Take 8 100 mL fresh 3% LB liquid culture media, and add 1 mL of 10% LB liquid culture media to the 3% LB liquid culture media. 8 cfu / mL Vibrio cholerae 919T suspension and 1mL with 1010 pfu / mLφYSZKA phage suspension, ensuring that the infection multiplicity is 100:1. The remaining treatments 2, 3, 4, and 5 were treated with infection multiplicity of 10:1, 1:1, 1:10, and 1:100, respectively. The phage titer was measured after culturing at 32°C and 150rpm for 12 hours. The results are shown in Table 1: When the optimal infection multiplicity is 1:10, the φYSZKA phage titer is the highest.

[0035] Table 1 Determination of the optimal multiplicity of infection

[0036] Example 2: The test water samples were collected from the surface water of the bay. Two groups of treatments were set up in the indoor experiment: ① Control group (CK): Each water sample was added with 100 mL of a single 7 CFU·mL -1 ② Phage treatment (V): based on the control group, 100 mL of 10 5 PFU·mL -1 φYSZKA phage suspension. Water samples were collected 12 hours after mixing to determine the cholera toxin gene in the water samples under the CK and V treatments. ctx The expression levels were: 4.56×10 3 ± 2.29×10 2 copies·μL -1 , 3.65×10 3 ± 1.14×10 2 copies·μL -1 ( Figure 4 ). Cholera toxin gene in water samples under CK and V treatments ctxB The relative expression levels were: 4.48×10 3 ±2.16×10 2 copies·μL -1 , 3.32×10 3 ± 1.24×10 2 copies·μL -1 ( Figure 5 ). The biofilm formation ability of Vibrio cholerae in water samples (OD 595 nm) was measured. The phage control group and the treatment group were 3.29± 0.35 and 2.03± 0.17 ( Figure 6 ). This indicates that the addition of φYSZKA phage suspension has a significant effect on weakening the cholera toxin gene and inhibiting the formation of Vibrio cholerae biofilm.

[0037] Example 3: Sewers are key monitoring targets in cholera outbreak areas. Test water samples were collected from sewer samples. Two groups of treatments were set up in the indoor experiment: ① Control group (CK): Each water sample was added with 100 mL of a single 10 7 CFU·mL -1 ② Phage treatment (V): based on the control group, 100 mL of 10 5 PFU·mL -1 φYSZKA phage suspension. Water samples were collected 12 hours after mixing to determine the cholera toxin gene in the water samples under the CK and V treatments. ctx The expression levels were: 7.25×10 3 ± 1.98×10 2 copies·μL -1 4.98×10 3 ± 1.14×10 2 copies·μL -1 ( Figure 7 ). Cholera toxin gene in water samples under CK and V treatments ctxB The relative expression levels were: 6.99×10 3 ±3.58×10 2 copies·μL -1 4.70×10 3 ± 2.41×10 2 copies·μL -1 ( Figure 8 ). Cholera toxin gene ctx and ctxB The expression levels of phage control and treatment groups were reduced by 31.3% and 32.8%, respectively. The biofilm formation ability of Vibrio cholerae in water samples (OD 595nm) was determined as follows: 3.28± 0.41, 1.79± 0.29 ( Fig. 9 ). This indicates that the addition of φYSZKA bacteriophage suspension can significantly regulate the pathogenicity of Vibrio cholerae in the key monitoring areas of cholera outbreaks.

[0038] Example 4: Although soil is not the main habitat of Vibrio cholerae, in areas where sanitation measures are lacking, soil is the main receiving environment for feces and can also contain enteric pathogens such as Vibrio cholerae. The test soil samples were collected from urban green space soil. Basic physical and chemical properties of soil: pH 6.1, total nitrogen 1.6 g kg -1 , water-soluble nitrogen 1.8 g·kg -1 , total phosphorus 1.2 g kg -1 , total potassium 15.5 g·kg -1 , CEC 17.2 cmol·kg -1 .

[0039] Two treatment groups were set up in the indoor microcosm experiment: ① Control group (CK): 100 mL of 10 7 CFU·mL -1 ② Phage treatment (V): based on the control group, 100 mL of 10 5 PFU·mL -1 Soil samples were collected 12 hours after mixing to determine the cholera toxin gene in soil samples under the CK and V treatments. ctx The expression levels were: 2.65×10 3 ± 1.73×10 2 copies·μL -1 , 2.03×10 3 ± 1.81×10 2 copies·μL -1 ( Fig.10 ). Cholera toxin gene in soil samples under CK and V treatments ctxB The relative expression levels were: 2.77×10 3 ± 2.05×10 2 copies·μL -1 , 2.32×10 3 ± 2.21×10 2 copies·μL -1 ( Fig.11 ). Cholera toxin gene ctx and ctxB The expression levels of α, β, and β were decreased by 23.4% and 16.3%, respectively.

[0040] Example 5: Monitoring of DNA concentration in the environment Three treatment groups were set up: ① Control group (CK): Each sample was added with 100 mL of 10 7 CFU·mL -1 2. Lytic phage treatment (LV): 100 mL of 10 5 PFU·mL -1 Lytic phage KVP40 ③ Phage treatment (V): Inoculate 100 mL of 10 5 PFU·mL -1 φYSZKA phage suspension. Water samples were collected 12 hours after mixing and centrifuged to separate bacterial precipitates and supernatant containing lysed DNA products. The speed was set to 5000 rpm for 300 seconds. qPCR was used to determine the cholera toxin gene in the supernatant under the treatment of CK, LV, and V. ctxand ctxB content 。 CK, LV, V in process ctx The contents are: 1.05×10 2 ± 1.23×10 1 copies·μL -1 , 2.03×10 2 ±1.79×10 1 copies·μL -1 , 1.09×10 2 ± 1.08×10 1 copies·μL -1 ( Fig.12 ). CK, LV, V three groups of treatment ctxB The contents are: 1.34×10 2 ± 1.19×10 1 copies·μL -1 , 2.16×10 2 ± 1.43×10 1 copies·μL -1 , 1.11×10 2 ± 1.02×10 1 copies·μL -1 ( Fig.13 Compared with the lytic phage KVP40 treatment (LV), the cholera toxin gene in the DNA product supernatant of the φYSZKA phage treatment ctx and ctxB The contents of φYSZKA and φYSZKA were reduced by 46.3% and 48.6% respectively. This indicates that the addition of φYSZKA bacteriophage suspension can significantly reduce the toxin gene DNA products released after Vibrio lysis in the environment.

Claims

1. Use of bacteriophage φYSZKA in the preparation of a gene product that inhibits Vibrio cholerae toxins, wherein the bacteriophage φYSZKA was deposited in the China Center for Type Culture Collection on August 1, 2018, with a deposit number of CCTCC NO: M 2018513, and is classified as Klebsiella pneumoniae phage φYSZKA ( Klebsiella phage φYSZKA).

2. Use of the bacteriophage φYSZKA according to claim 1 in the preparation of a DNA product that reduces toxin gene release after Vibrio lysis in the environment.

3. Use of the bacteriophage φYSZKA according to claim 1 in the preparation of a product for reducing the spread of cholera by reducing the toxin gene DNA released after Vibrio lysis in the environment.

4. The use according to claim 1, characterized in that: The Vibrio cholerae toxin gene is ctx and ctxB.

5. The use according to claim 1, characterized in that: The product may also contain other biological or chemical substances that inhibit toxin production by Vibrio cholerae.

6. The use according to claim 1, characterized in that: The product also contains at least one carrier or auxiliary component for improving the stability, storage life or application effect of the bacteriophage.

7. A method for reducing the spread rate of cholera epidemic, characterized in that: Injecting the product of claim 2 into the ocean, soil or urban water.

8. The method for reducing the spread rate of cholera epidemic according to claim 7, characterized in that: It also involves monitoring changes in the levels of Vibrio cholerae toxin in the environment and adjusting the application rate of the product as needed.

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