Plesiomonas shigelloides bacteriophage with cross-genus splitting capability and application of plesiomonas shigelloides bacteriophage

Abstract

The invention belongs to the technical field of microorganisms, and discloses a plesiomonas shigelloides bacteriophage with cross-genus splitting decomposition capacity and application of the plesiomonas shigelloides bacteriophage, the plesiomonas shigelloides bacteriophage is classified and named as Plesiomonas virus, the plesiomonas shigelloides bacteriophage is preserved in China General Microbiological Culture Collection Center on March 21, 2025, and the preservation number is CGMCC No.46409; the plesiomonas shigelloides bacteriophage has a splitting decomposition effect on both plesiomonas shigelloides and aeromonas veronii. The bacteriophage has a high cracking rate on plesiomonas shigelloides and also has a cracking effect on aeromonas veronii, is a bacteriophage with cross-genus-species cracking, and is good in alkali resistance stability and good in thermal stability. The compound can be prepared into drugs, environment disinfectants, water additives and the like for preventing and treating plesiomonas shigelloides diseases and / or aeromonas veronii diseases of cultured fishes, so that the use of antibiotics is reduced.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and in particular relates to a Shigella-like phage with cross-genus lysis ability and application thereof. Background Art

[0002] Pleiomonas shigelloides and Aeromonas vernix are serious pathogens in aquaculture and are also important zoonotic pathogens. Pleiomonas shigelloides can cause acute hemorrhagic septicemia, intestinal ulcers, and systemic infections in fish, with mortality rates exceeding 60%. Aeromonas vernix is widely present in aquatic environments and is closely associated with surface ulcers, hepatopancreatic necrosis, and ascites in fish. Diseases caused by both pathogens are prone to outbreaks during hot weather. These two bacteria often cause mixed infections in aquaculture animals. Mixed infections of these two bacteria have been detected in outbreaks of mortality in southern catfish, sweetfish, and yellow catfish, causing severe economic losses to the aquaculture industry.

[0003] Furthermore, these two pathogens can enter the human food chain through contaminated aquatic products, such as fish and shrimp, causing acute diarrhea, abdominal pain, and even sepsis, particularly in immunocompromised individuals. Statistics show that numerous diarrheal cases are associated with infections from aquatic pathogens each year. Among them, Shigella-like bacteria, a significant aquatic pathogen, poses a heavy burden on public health.

[0004] Currently, aquaculture relies heavily on antibiotics to prevent and treat bacterial diseases. However, long-term abuse can lead to the frequent occurrence of multidrug-resistant strains, which can even be transmitted to humans through the food chain, making clinical treatment more difficult. Bacteriophages are natural enemies of bacteria and are highly abundant in nature. Phage therapy has the advantages of strong targeting and being eco-friendly, and is considered an alternative to antibiotic treatment. In the field of aquaculture, phages can be used to reduce or prevent bacterial infections in aquatic animals, and can also be used to control bacterial density in aquaculture water environments. However, existing phages cannot simultaneously lyse Shigella-like bacteria and Aeromonas vernix. Summary of the Invention

[0005] To solve the above problems, the present invention provides a Shigella-like phage with cross-genus lysis ability and its application.

[0006] The present invention is achieved through the following technical solutions: A Shigella phage-like bacteriophage having cross-genus lysis ability, the Shigella phage-like bacteriophage is classified and named Plesiomonas virus , and was deposited in the General Microbiology Center of China Culture Collection Administration on March 21, 2025, with the deposit number CGMCC No.46409.

[0007] The Shigella-like Pleistomonas phage is effective against Shigella-like Pleistomonas ( Plesiomonas shigelloides ) and Aeromonas wilkeri ( Aeromonas veronii ) have a cracking effect.

[0008] The invention relates to an application of the Shigella shigella-like phage in the preparation of medicines for treating fish diseases caused by Shigella shigella or Aeromonas welchii.

[0009] The Shigella-like bacteriophage is used in the preparation of biological fungicides, cleaners or disinfectants for aquaculture water bodies.

[0010] The invention relates to an application of the Shigella-like bacteriophage in the preparation of a reagent with cross-genus lysis ability, wherein the reagent uses the Shigella-like bacteriophage as the only active ingredient.

[0011] The invention relates to an application of the Shigella-like bacteriophage in preparing a kit with cross-genus lysis ability, wherein the kit uses the Shigella-like bacteriophage as the only active ingredient.

[0012] The invention discloses an application of the Shigella-like bacteriophage in the preparation of an aquatic feed additive. The aquatic feed additive comprises the Shigella-like bacteriophage and auxiliary materials.

[0013] Preferably, the auxiliary material is one or more of trehalose, skim milk powder and glycerol.

[0014] Preferably, the Shigella-like phage is inoculated at a multiplicity of infection of 0.001-10.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a Shigella phage-like bacteriophage with cross-genus lysis ability and its application. The Shigella phage-like bacteriophage is classified and named Plesiomonas virus , was deposited in the General Microbiology Center of the China National Committee for the Preservation of Microorganisms on March 21, 2025, with the deposit number CGMCC No. 46409; the phage is classified as the Caudovirales, Breviviridae, and has cross-genus lysis ability, and is lytic against pathogenic Shigella-like Pleistomonas and Aeromonas vernix. It has strong alkali resistance and certain acid resistance, has a certain temperature stability, is easy to proliferate and enrich, and the phage genome does not contain drug resistance genes and virulence genes. It provides a source of phages for the industrial production of phages for the prevention and treatment of pathogenic Shigella-like Pleistomonas and Aeromonas vernix in aquaculture environments and the purification of seedling aquaculture water bodies. At the same time, it can be prepared into drugs, environmental disinfectants, and water additives for the prevention and treatment of Shigella-like Pleistomonas and / or Aeromonas vernix in farmed fish, thereby reducing the use of antibiotics.

[0016] Bacteriophage vB_PshP_C1, classified and named Plesiomonas virus The depository is the General Microbiology Center of China Culture Collection of Microorganisms, abbreviated as CGMCC. The depository address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit number is CGMCC No. 46409, and the deposit date is March 21, 2025. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 These are plaques formed by the bacteriophage vB_PshP_C1 of the present invention.

[0019] Figure 2 This is an electron microscope photograph of the bacteriophage vB_PshP_C1 of the present invention.

[0020] Figure 3 This is the optimal MOI of the bacteriophage vB_PshP_C1 of the present invention.

[0021] Figure 4 It is the acid-base stability of the bacteriophage vB_PshP_C1 of the present invention.

[0022] Figure 5 This is the temperature stability of the bacteriophage vB_PshP_C1 of the present invention.

[0023] Figure 6 This is the one-step growth curve of the bacteriophage vB_PshP_C1 of the present invention.

[0024] Figure 7 This is a circle map of the genome of the bacteriophage vB_PshP_C1 of the present invention.

[0025] Figure 8 This is a diagram verifying the lysis ability of the bacteriophage vB_PshP_C1 of the present invention; Figure 8 In the figure, A shows the plaques formed by vB_PshP_C1 on the culture plate of Pseudomonas shigelloides JRPC1; B shows the plaques formed by vB_PshP_C1 on the culture plate of Aeromonas vernix JRCV1.

[0026] Figure 9 This is the growth inhibition curve of the bacteriophage vB_PshP_C1 of the present invention against Pseudomonas shigelloides.

[0027] Figure 10 This is the growth inhibition curve of the bacteriophage vB_PshP_C1 of the present invention on Aeromonas veronii. DETAILED DESCRIPTION

[0028] To facilitate understanding of the present invention, the present invention will be described more fully below, along with preferred embodiments of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present invention.

[0029] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0030] The beneficial effects of the present invention are described below through specific examples.

[0031] In the following examples, unless otherwise specified, all methods are conventional.

[0032] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0033] Samples and sources: The sea bass and water samples came from an aquaculture farm in Zhuhai, Guangdong, and the sampling time was September 2023.

[0034] Pleistomonas shigelloides ( Plesiomonas shigelloides ) JRPC1 and Aeromonas vernix ( Aeromonas veronii ) JRCV1 was purchased from Shandong Xinde Technology Co., Ltd.

[0035] Example 1 Isolation, purification and storage of bacteriophage vB_PshP_C1: (1) Isolation of phage using Shigella spp. JRPC1 as host bacteria: Phage was isolated using a double-layer plate. About 2 mL of aquaculture water collected from a sea bass farm in Zhuhai, Guangdong Province was filtered and sterilized, then added to a test tube containing 5 mL of LB broth, and 1 mL of Shigella spp. JRPC1 bacterial solution was added. The culture was incubated at 37°C for 4 hours in a constant temperature shaker. The supernatant was sterilized by filtering with a 0.22 μm filter membrane and temporarily stored at 4°C. 200 μL of Shigella spp., with a viable count of 1×10 8Mix the CFU / mL with 100 μL of filtered supernatant and 5 mL of LB semi-solid medium cooled to approximately 55°C. Pour the mixture onto a solid LB plate and allow it to solidify to form a double-layer plate. Incubate the plate overnight at 37°C and observe the formation of plaques.

[0036] (2) Purification of phage: Use a sterile inoculation needle to remove a single phage plaque, place it in SM buffer and shake to mix, filter with a 0.22 μm filter membrane, take the filtrate and prepare a double-layer plate with the host bacteria again, repeat the removal of a single phage plaque, spread the double-layer plate and perform the purification operation 6 times, forming phage plaques of uniform shape and size on the plate. Figure 1 As shown, the purified phage was obtained, and the plaques formed were 3 mm to 4 mm in diameter, round and clear, without a halo. According to the international nomenclature rules, it was named vB_PshP_C1.

[0037] (4) Preservation of phage: Mix the phage solution with sterilized glycerol in a ratio of 7:3, dispense into 2 mL cryovials, and store in a -80°C refrigerator, or make into lyophilized powder and store in a 4°C refrigerator.

[0038] Example 2 Electron microscopic observation of phages: Take 20 μL of liquid containing phages, the concentration of which is 1.5×10 9 PFU / mL, drop it on the copper grid, let it precipitate naturally for 15 minutes, add 50 μL of phosphotungstic acid with a pH of 7.0 on the copper grid to negatively stain the phage for 10 minutes, and observe the morphology of the phage under a transmission electron microscope. The electron microscope photo of vB_PshP_C1 is shown in the figure. Figure 2 shown.

[0039] Depend on Figure 2 The vB_PshP_C1 phage has an icosahedral head with a major diameter of 61 nm, a transverse diameter of 58 nm, and a tail length of 13 nm. According to the 9th report of the International Committee on Taxonomy of Viruses, this phage belongs to the order Caudovirales, family Brachyuraviridae.

[0040] Example 3 1. Optimal multiplicity of infection (MOI) of bacteriophage vB_PshP_C1: The Shigella-like bacteria JRPC1 was inoculated into 5 mL of LB liquid medium and cultured at 37°C with shaking at 200 rpm until the mid-logarithmic phase, and the effective viable count was about 10 8 CFU / mL. The phage solution was filtered through a 0.22 μm filter membrane and then diluted with SM buffer to 10 6PFU / mL. According to the phage concentration: bacterial solution concentration = 10, 1, 0.1, 0.01, 0.001, 0.0001, the mixture was allowed to stand at 37°C for 15 minutes for adsorption. 5mL of preheated LB culture medium was added respectively, the preheating temperature was 37°C, and the culture was shaken at 37°C and 200rpm for 4 hours. The culture solution was filtered with a 0.22μm filter membrane. The titer was determined by the double-layer plate method, and the average value was obtained after three repetitions. The results showed that the titer was the highest at MOI = 0.0001, which was 3.2×10 10 PFU / mL, determined as the optimal MOI, such as Figure 3 shown.

[0041] Example 4 Acid-base stability of phage vB_PshP_C1: Prepare 1 mol / L HCl and 1 mol / L NaOH solutions. Take 50 mL of LB liquid medium and adjust the pH to 1.0-14.0 after calibration with a pH meter. Take 900 μL of LB medium at each pH value and add 100 μL of phage solution to each of them at a concentration of 10 8 PFU / mL, vortex mix for 10 seconds. Incubate in a 37℃ water bath for 2 hours. Immediately after incubation, place the mixture in an ice bath for 5 minutes to terminate the reaction. The double-layer plate method was used to determine the titer of the bacteria under different pH conditions, and the results were repeated three times. The results showed that the titer was stable in the pH range of 4.0-12.0, and was greater than 10 8 PFU / mL, when pH < 4.0 or pH > 12.0, the titer dropped to less than 10 3 PFU / mL, such as Figure 4 shown.

[0042] Example 5 Temperature stability of bacteriophage vB_PshP_C1: The phage solution was diluted to a concentration of 2×10 8 PFU / mL, divided into centrifuge tubes, 1mL per tube. Place incubators at 40℃, 50℃, 60℃, and 70℃ respectively, and place the control group on ice. Set up 3 replicates for each group. Incubate at each temperature for 20min, 40min, and 60min, respectively, and immediately ice bath for 5min after taking out. Take 100μL sample respectively and determine the titer by double-layer plate method. The results are as follows Figure 5 As shown: When treated at 40℃ and 50℃ for 60min, the titer did not change significantly and remained at 10 8 PFU / mL, treated at 60℃ for 20min, the titer was maintained at 3.5×10 6 PFU / mL, treated for 40 min, the titer was 2.8×10 4 PFU / mL, treated for 60 min, titer was 2.5×10 2 PFU / mL.

[0043] Example 6 This example is used to illustrate the determination of the one-step growth curve of bacteriophage vB_PshP_C1: the host bacterial solution was mixed at an optimal MOI of 0.0001, and the concentration was 10 8 CFU / mL and phage solution, the concentration was 10 4 PFU / mL, total volume 1mL. Stand at 37℃ for adsorption for 5 minutes, shaking gently 3 times during the period. Centrifuge at 12000rpm at 4℃ for 5 minutes to discard the supernatant, gently blow the precipitate twice with preheated 37℃ LB medium, and finally resuspend in 1mL LB. Transfer the host bacteria-phage mixture to 100mL, 37℃ LB medium, and shake and culture at 37℃ and 200rpm. From the mixing time point t=0, take 1mL sample every 10 minutes, immediately terminate the reaction in an ice bath, filter with 0.22μm filter membrane and store at -80℃. After thawing the sample, the phage titer was determined by the double-layer plate method, with 3 replicates for each group. The one-step growth curve of the phage was drawn with time as the horizontal axis and the phage titer as the vertical axis. The results are shown as follows. Figure 6 As shown: The incubation period of phage vB_PshP_C1 is 20 minutes, followed by a steady increase in titer, a burst period lasting 150 minutes, and then a plateau period.

[0044] Example 7 The high titer phage obtained by double-layer plate amplification method was centrifuged at 10,000 g and 4°C for 10 min. The supernatant was collected and sterilized by filtration. The viral genome was extracted using a centrifugal column method and sent to a sequencing company for sequence determination. The full length of the genome was 43,419 bp. The genome circle map is shown in Figure 7 The annotation results showed that the phage genome had 44 open reading frames. Sequence alignment of protein-coding genes was completed using Diamond software, and 31 proteins were annotated.

[0045] Example 8 Verification of the cross-genus lysis ability of bacteriophage vB_PshP_C1 Mix 200 μL of Shigella-like Pleistomonas JRPC1 and 200 μL of Aeromonas vernix JRCV1 with LB semi-solid medium cooled to about 55°C, pour them onto LB solid plate medium, and let them stand until solidified to make double-layer plates. 6 PFU / mL phage liquid was added dropwise to the culture medium, allowed to air dry, and then incubated at 37°C for 8 hours to observe the formation of phage plaques. Figure 8 As shown, the phage vB_PshP_C1 has a lytic effect on both Shigella-like bacteria JRPC1 and Aeromonas vermiformis JRCV1.

[0046] Example 9 Detection of antibacterial ability of bacteriophage vB_PshP_C1 in vitro The Shigella-like Pseudomonas JRPC1 and Aeromonas vernix JRCV1 were inoculated into LB medium respectively and cultured at 37°C until the logarithmic phase. The bacterial solution concentration was adjusted to 10 CFU / mL. The phage was inoculated into the same volume of LB broth with the Shigella-like Pseudomonas JRPC1 and Aeromonas vernix JRCV1 at MOI = 10, 1, 0.1, 0.01, and 0.001, respectively. The volume of the phage solution and the bacterial solution was 100uL. The culture was shaken at 37°C and 200rpm for 12 hours. Each group was repeated 3 times, and the control group was a bacterial solution without phage. The OD of the bacteria was measured every 1 hour. 600 Value, draw Figure 9 and Figure 10 The results showed that phage vB_PshP_C1 had a strong inhibitory effect on the growth of both P. shigelloides JRPC1 and Aeromonas vernix JRCV1. In vitro, the OD value of P. shigelloides JRPC1 in the control group continued to rise, stabilizing at around 0.7 after 6 hours. The inhibitory effect on P. shigelloides JRPC1 was demonstrated by the fact that at all MOI values, the OD value remained below 0.23 from 0 to 13 hours, with no upward trend.

[0047] In vitro, the OD value of Aeromonas vermiformis JRCV1 in the control group continued to rise, stabilizing at around 0.92 after 11 hours. The inhibitory effect on Aeromonas vermiformis JRCV1 was demonstrated by a slow increase in OD values from 2 to 11 hours at all MOI values, but remained significantly lower than that of the control group. The inhibitory effect was particularly strong at MOIs of 10 and 1.

[0048] Example 10: A biocide This example provides a biocide. The bacteriophage vB_PshP_C1 screened in Example 1 was mixed with a protective agent and a synergist in any proportion, and the mixture was dispensed into sterile spray bottles at 10 mL per bottle. The protective agent was gelatin, and the synergist was 0.1 mM EDTA.

[0049] The antibacterial ability of the biofungicide was tested, and the results showed that the biofungicide had an inhibitory effect on both Shigella-like bacteria and Aeromonas vernix.

[0050] Example 11: A reagent with cross-genus lysis ability This example provides a reagent with cross-genus lysis ability. The phage vB_PshP_C1 screened in Example 1 was diluted with SM buffer to a final concentration of 1×10 9PFU / mL, aliquot and store at 4°C to obtain a reagent with cross-genus lysis ability. SM buffer is formulated with 50mM Tris-HCl, 100mM NaCl, 8mM MgSO, pH 7.5. This reagent was tested for cross-genus lysis ability, and the results showed that it lysed both Pseudomonas shigelloides and Aeromonas vernix.

[0051] Example 12: Aquatic feed additive This embodiment provides an aquatic feed additive, comprising: mixing the bacteriophage vB_PshP_C1 screened in Example 1 with trehalose and skim milk powder in any proportion; pre-freezing at -80°C, vacuum freeze-drying, and then mixing with puffed corn flour in any proportion; granulating the mixture into 1 mm particle size; and sealing the mixture in a light-proof package.

[0052] The cross-genus lysis ability test of the feed additive was carried out, and the results showed that the reagent had a lysis effect on both Shigella-like bacteria and Aeromonas vernix.

[0053] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0054] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. A person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims.

Claims

1. A Shigella-like phage with cross-genus lysis ability, characterized in that: The Shigella-like phage is classified as Plesiomonas virus , deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms on March 21, 2025, with the deposit number CGMCC No.46409; The Shigella-like Pleistomonas phage is effective against Shigella-like Pleistomonas ( Plesiomonas shigelloides ) and Aeromonas wilkeri ( Aeromonas veronii ) have a cracking effect.

2. Use of the Shigella-like phage according to claim 1 in the preparation of a drug for treating diseases caused by Shigella shigella or Aeromonas welchii in fish.

3. Use of the Shigella-like phage according to claim 1 in the preparation of biocides, cleaners or disinfectants for aquaculture water bodies.

4. The use of the Shigella-like phage according to claim 1 in preparing a reagent with cross-genus lysis ability, characterized in that The reagent uses the Shigella-like bacteriophage as the only active ingredient.

5. Use of the Shigella-like phage according to claim 1 in preparing a kit having cross-genus lysis ability, characterized in that The kit uses the Shigella-like bacteriophage as the only active ingredient.

6. The use of the Shigella-like bacteriophage according to claim 1 in the preparation of an aquatic feed additive, characterized in that: The aquatic feed additive comprises the Shigella-like bacteriophage and auxiliary materials.

7. The use according to claim 6, characterized in that The auxiliary material is one or more of trehalose, skim milk powder and glycerol.

8. The use according to any one of claims 2 to 7, characterized in that The Shigella-like phage is inoculated at a multiplicity of infection of 0.001-10.

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