A virulent Shigella flexneri phage capable of secreting depolymerase and resistant to high temperature, and its isolation method and application

Abstract

The present application provides a virulent Shigella flexneri phage that can secrete depolymerase and is resistant to high temperatures, named Shigella flexneri phage PGSH‑20417, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Committee on August 24, 2022, with a deposit number of CGMCC No. 45260, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The isolation method of Shigella flexneri phage provided in the present application provides a source for large-scale production of phages; the present application also provides the use of Shigella flexneri PGSM‑20417 in the preparation of prevention and or treatment of diseases caused by Shigella flexneri.

Description

Technical Field

[0001] The present application belongs to the field of microbial technology, and specifically relates to a virulent Shigella flexneri phage that can secrete depolymerase and is resistant to high temperatures, and a separation method and application thereof. Background Art

[0002] Shigella flexneri is a common pathogen of poultry, which can be transmitted to the next generation through vertical transmission. When poultry are infected with Shigella flexneri, they will experience symptoms such as diarrhea, which will lead to growth retardation, decreased egg production, and even death, causing great economic losses to the poultry industry. At present, the prevention and control of salmonellosis mainly relies on antibiotics. The long-term and large-scale use of antibiotics has also led to an increase in the number of drug-resistant strains of Shigella flexneri. Veterinary clinics are facing a severe situation where no drugs are available. Therefore, finding alternatives to antibiotics or studying biological control methods for diseases has become a problem to be solved in the current prevention and control of bacterial diseases. Bacteriophages have the advantages of strong bactericidal effect, high utilization rate, high specificity, and are not restricted by bacterial resistance.

[0003] In view of this, this application is hereby filed. Summary of the invention

[0004] In order to solve one of the above technical defects, the present invention provides a high temperature resistant Shigella flexneri phage that can secrete depolymerase, and the titer can still reach 1*10 under high temperature conditions of 70°C for 50 minutes. 6 , has good tolerance to temperature; can effectively and efficiently infect Shigella flexneri; is intended to replace antibiotics for the treatment and prevention of diseases caused by Shigella flexneri.

[0005] According to the first aspect of the embodiment of the present application, a virulent Shigella flexneri phage that can secrete depolymerase and is resistant to high temperatures is provided, named Shigella flexneri phage PGSH-20417, which was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms on August 24, 2022, with a deposit number of CGMCC No. 45260, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0006] According to the second aspect provided by the embodiment of the present application, a depolymerase is provided, which is secreted and expressed by the above-mentioned Shigella flexneri phage. Further, the DNA sequence of the Shigella flexneri phage is as shown in SEQ ID NO.1; the DNA sequence of the depolymerase is as shown in SEQ ID NO.2; and the amino acid sequence of the depolymerase is as shown in SEQ ID NO.3.

[0007] According to the third aspect provided in the embodiments of the present application, there is provided a use of a virulent Shigella flexneri phage that secretes a depolymerase and is resistant to high temperatures in any one of the following (1)-(4):

[0008] (1) For the preparation of drugs for the prevention and / or treatment of diseases caused by Shigella flexneri in poultry;

[0009] (2) additives for the prevention and / or treatment of diseases caused by Shigella flexneri in poultry;

[0010] (3) Used in the preparation of cleaning agents or disinfectants for controlling Shigella flexneri in poultry breeding environments;

[0011] (4) Used to control Shigella flexneri in drinking water, feed and the environment.

[0012] According to a fourth aspect provided in an embodiment of the present application, a method for isolating a virulent Shigella flexneri phage that can secrete a depolymerase and is resistant to high temperatures is provided, comprising the following steps:

[0013] (1) Isolation of Shigella flexneri phage: The filtrate obtained by filtering sewage is added to LB liquid culture medium together with the logarithmic phase host bacteria suspension, and then cultured in an incubator. The filtrate after culturing is the Shigella flexneri phage stock solution; then, the phage plaques are cultured by the double plate method;

[0014] (2) Purification of Shigella flexneri phage:

[0015] A single plaque is picked from the double-layer plate where plaques appear in step (1), cultured in PBS buffer, and the supernatant is filtered. The filtered filtrate is cultured on the double-layer plate, and the above steps are repeated until the size, morphology and clarity of the plaques on the double-layer plate are consistent, thereby obtaining purified Shigella flexneri phage.

[0016] Further, the separation of Shigella flexneri phage comprises the following steps:

[0017] (1.1) Take sewage, centrifuge it, and initially filter it to remove larger impurities and most bacteria, then filter it through a microporous membrane to sterilize it to obtain filtrate A;

[0018] (1.2) Add filtrate A and an equal volume of a host bacterial suspension in the logarithmic phase into a high-pressure sterilized LB liquid culture medium, and then culture it in an incubator. After the culture, filter it to obtain filtrate B;

[0019] (1.3) Cultivate by double plate method until plaques appear.

[0020] Furthermore, the culture conditions in the incubator are: 37°C constant temperature shaking incubator, culture for 10-12 hours.

[0021] Further, the purification of Shigella flexneri phage comprises the following steps:

[0022] (2.1) Pick a single, independent, uniform, clear and transparent plaque from the double-layer plate where plaques appeared in step (1), place it in PBS buffer, and crush it with a sterile inoculation loop. After soaking, take the supernatant and filter it with a microporous filter membrane, and then dilute the filtrate;

[0023] (2.2) Spread the diluted filtrate from step (2.1) and the host bacterial suspension on a double-layer plate, and repeat the above steps 3-5 times until the plaque size, morphology and clarity on the double-layer plate are consistent to obtain purified Shigella flexneri phage.

[0024] Furthermore, the microporous filter membrane is a 0.22um microporous filter membrane.

[0025] Beneficial effects of this application:

[0026] 1. The present application discloses a lytic Shigella flexneri phage isolated from a fermented mattress environment of dairy cows. The phage is named Shigella flexneri phage PGSM-20417, which has a strong lytic effect on pathogenic Salmonella SA-20417 in the environment and has good application prospects in preventing and controlling diseases caused by Shigella flexneri. The method for isolating Shigella flexneri phage provided in the present application provides a source for large-scale production of phages. The present application also provides the use of Shigella flexneri PGSM-20417 in the preparation of a preparation for preventing and or treating diseases caused by Shigella flexneri.

[0027] 2. The Shigella flexneri phage provided in the present application can secrete depolymerase and is resistant to high temperatures. It can be widely used in the infection of pathogenic Shigella flexneri phage in poultry and in the control of Shigella flexneri phage in drinking water, feed and the environment; it has good tolerance to temperature and is conducive to long-term storage; it has a high potency and a good control effect.

[0028] 3. The Shigella flexneri phage provided by the present invention, wherein the 12th open reading frame of the genome of the phage encodes a depolymerase. The Shigella flexneri phage encodes a depolymerase consisting of 735 amino acids; the depolymerase has the property of degrading biofilms. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0030] Figure 1 The phage plaque formed by the Shigella flexneri phage of the present application;

[0031] Figure 2 Electron microscopic photograph of Shigella flexneri phage of the present application;

[0032] Figure 3 This is a one-step production curve diagram of Shigella flexneri phage of the present application;

[0033] Figure 4 This is a temperature tolerance curve of Shigella flexneri bacteriophage of the present application;

[0034] Figure 5 This is a pH tolerance curve of Shigella flexneri phage of the present application;

[0035] Figure 6 This is the map of the spike protein gene located on a separate evolutionary branch;

[0036] Figure 7 Figure 2 shows the hydrolase genes located on separate evolutionary branches. DETAILED DESCRIPTION

[0037] In order to make the technical solutions and advantages in the embodiments of the present application more clearly understood, the exemplary embodiments of the present application are further described in detail below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present application, rather than an exhaustive list of all the embodiments. It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0038] The present application provides a virulent Shigella flexneri phage that can secrete depolymerase and is resistant to high temperatures, named Shigella flexneri phage PGSH-20417, which was deposited in the General Microbiology Center of the China Microbiological Culture Collection Committee on August 24, 2022, with a deposit number of CGMCC No.45260, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The head of the phage is a regular hexagonal icosahedron, with a head diameter of about 63nm and a tail length of 27nm. It is a brachyura phage; the gene is 39090bp long, does not have virulence genes and lysogenic genes, and is used to prevent and / or treat diseases caused by Shigella flexneri; for example, its application in pathogenic Shigella flexneri infection in poultry and its application in controlling Shigella flexneri in drinking water, feed and the environment. It is worth noting that the Shigella flexneri phage and its host bacteria in the examples of the present application were isolated by our unit from wastewater and diseased materials in farms.

[0039] Example 1

[0040] The method for isolating Shigella flexneri bacteriophage comprises sampling, step (1) isolating Shigella flexneri bacteriophage and step (2) purifying Shigella flexneri bacteriophage.

[0041] The samples used in the experiment of the present invention are collected from sewage around diseased breeding farms in Qingdao City and used as water samples for phage separation.

[0042] Step (1) The separation of Shigella flexneri bacteriophage is specifically the following steps:

[0043] (1.1) Take 10 mL of sewage, centrifuge at 12000 rpm for 5 min, filter out large impurities and most bacteria with filter paper, and then filter with a 0.22 um microporous filter membrane to obtain the filtrate;

[0044] (1.2) Take 3 mL of the filtrate prepared in step (1.1) and 3 mL of the logarithmic phase host bacteria suspension, add them together into 20 mL of autoclaved LB liquid culture medium, and then place in a 37°C constant temperature shaking incubator and culture for 11 hours.

[0045] (1.3) After the culture, take 5 ml of the solution, centrifuge at 12000 rpm for 5 min, and then filter and sterilize using a 0.22 μm microporous filter. The resulting filtrate is the stock solution of Shigella flexneri phage;

[0046] (1.4) Spot the stock solution prepared in step (1.3) on a double plate, and culture plaques using the double plate method. The obtained plaques are the isolated Shigella flexneri cells.

[0047] Step (2) purification of Shigella flexneri phage specifically comprises the following steps:

[0048] (2.1) Pick a single, independent, uniform, clear and transparent plaque from the double-layer plate with plaques, place it in 1 mL of PBS buffer, and crush it with a sterile inoculation loop, then soak it for 15 minutes, take the supernatant, and filter it with a 0.22 μm microporous filter to obtain a filtrate;

[0049] (2.2) Dilute the filtrate filtered in step (2.1) (a single plaque will appear when spreading on two plates); spread the diluted filtrate and the host bacterial suspension on two plates, repeat 3-5 times, and when the plaque size, morphology and clarity on the two plates are consistent, purified Shigella flexneri phage is obtained.

[0050] The isolated and purified Shigella flexneri phage was negatively stained with phosphotungstic acid and the size and shape of the phage were observed under an electron microscope.

[0051] Figure 2 This is an electron microscope photo of Shigella flexneri phage; Figure 2 As shown, electron microscopy showed that the phage head was a regular hexagon, with a head diameter of about 63nm and a tail length of 27nm, belonging to the Brachyura phage family.

[0052] Example 2

[0053] Determination of the titer of Shigella flexneri phage

[0054] The purified Shigella flexneri phage was spread on a double-layer plate, and then a single plaque was taken and added to LB liquid culture medium, and the host bacterial suspension was added at the same time. After the Shigella flexneri phage was proliferated, the proliferated liquid was centrifuged at 12000rpm for 5 minutes and then filtered with a 0.22um microporous filter. The filtrate was diluted 10 times with PBS buffer to 10 8 Take 0.1 mL of the last three dilutions of phage dilution and 0.1 mL of host bacterial suspension, spread on double-layer plates, and determine the phage titer. Wherein, phage titer (pfu / mL) = number of plaques * dilution times * 10.

[0055] The results showed that the plaques had consistent morphology, size and clarity, and had typical lytic phage characteristics. The titer of the Shigella flexneri phage could reach 10 12 pfu / mL or above.

[0056] Example 3

[0057] Determination of the optimal multiplicity of infection of Shigella flexneri phage

[0058] The Shigella flexneri phage proliferation liquid and the host were added to the LB liquid culture medium at a multiplicity of infection of 10:1, 1:1, 1:10, 1:100, and 1:1000, and the total volume of the culture system was ensured to be the same. After shaking culture at 37°C and 170 rpm for 6 hours, centrifuge at 12000 r / min for 5 minutes at room temperature, and take the supernatant to spread on two plates to determine its titer.

[0059] The results are shown in Table 1; Shigella flexneri phage PGSM-20417 efficiently infects Shigella flexneri.

[0060] Table 1 Optimal infection multiplicity titer of Shigella flexneri phage

[0061]

[0062] Example 4

[0063] One-step growth curve assay of Shigella flexneri phage

[0064] The Shigella flexneri phage and host bacteria were mixed evenly according to the optimal infection multiplicity, incubated at 37°C for 5 minutes, centrifuged at 7500rpm for 10 minutes, and the supernatant was discarded; the precipitate was resuspended in LB liquid culture medium, and cultured on a shaker at 37°C and 170rpm. At the same time, the time was set, and samples were taken every 10 minutes, and the supernatant was taken after centrifugation at 4°C and 12000rpm for 2 minutes. The phage titer was determined by the double-layer plate method, the phage one-step growth curve was drawn, and the burst volume was calculated. The burst volume = the number of phages at the end of the outbreak / the number of host bacteria at the beginning of the infection.

[0065] like Figure 3 As shown, the phage incubation period is 20 min, the burst period is 60 min, and the burst volume is 115 PFU / cell.

[0066] Example 5

[0067] Determination of thermal stability of Shigella flexneri bacteriophage

[0068] Take 100uL of pure culture solution of Shigella flexneri phage and transfer it to a sterile EP tube. Incubate it at 30℃, 40℃, 50℃, 60℃, 70℃ and 80℃ for 30min respectively. Take out the EP tube immediately after the incubation, cool it in an ice bath, dilute it 10 times with sterile PBS buffer, and spread it on double plates for counting.

[0069] Depend on Figure 4 It can be seen that the titer of the Shigella flexneri phage is stable in the temperature range of 30-60°C and remains at 10 10 pfu / mL, and the titer can still reach 1*10 6 , after being exposed to high temperature of 80℃ for 30min, it still has biological activity, indicating that the phage has good tolerance to temperature.

[0070] Example 6

[0071] Determination of acid-base stability of Shigella flexneri phage

[0072] 100uL of pure culture solution of Shigella flexneri phage was inoculated into LB medium with pH values ​​of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, respectively, and cultured at 37°C for 6-8h. The phage titer was determined by the double-layer plate method, and the test was repeated 3 times.

[0073] like Figure 5 As shown, the titer of Shigella flexneri phage is relatively stable between pH 4 and 10. When the pH value is lower than 3 or greater than 11, the titer level is significantly reduced.

[0074] Example 7

[0075] Whole genome sequencing of Shigella flexneri bacteriophage PGSM-20417

[0076] Performed by Borodovsky Laboratory, School of Biological Sciences, Georgia Institute of Technology, Atlanta.

[0077] The sequencing results showed that the full length of the Shigella flexneri phage genome was 39090bp, the GC base content was 50.8%, and 38 read open regions were predicted, including 32 functional protein genes and 6 hypothetical protein genes, without virulence genes and lysogeny genes.

[0078] By comparing with the GenBank database, analyzing the conserved functional protein genes spike protein and hydrolase genes, and constructing phylogenetic trees based on the gene information of these phages, the evolutionary relationship of Shigella flexneri phage PGSM-20417 was analyzed, and it was found that Shigella flexneri phage PGSM-204173 was located in a separate evolutionary branch in terms of the main spike protein gene and hydrolase gene. The above results indicate that phage PGSM-20417 is a new type of phage.

[0079] Although the preferred embodiments of the present application have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0080] Obviously, those skilled in the art can make various changes and modifications to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. A virulent Shigella flexneri phage that can secrete depolymerase and is resistant to high temperatures, named Shigella flexneri phage PGSH-20417, was deposited in the General Microbiology Center of the China Microbiological Culture Collection on August 24, 2022, with the deposit number CGMCC No.45260, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

2. Use of the virulent Shigella flexneri phage capable of secreting depolymerase and resistant to high temperature as claimed in claim 1 in any one of the following (1) to (4): (1) For the preparation of drugs for preventing and / or treating diseases caused by Shigella flexneri in poultry; (2) For the production of additives for the prevention and / or treatment of diseases caused by Shigella flexneri in poultry; (3) Used in the preparation of cleaning agents or disinfectants for controlling Shigella flexneri in poultry breeding environments; (4) Used to control Shigella flexneri in drinking water, feed and the environment.

Images