Pseudomonas stutzeri strain and use thereof

Abstract

The application belongs to the technical field of microorganisms, and discloses a parapeptococcus shigelloides bacteriophage with cross-species lysis capability and application thereof. Plesiomonas shigelloides virus The parapeptococcus shigelloides bacteriophage is classified and named as Plesiomonas shigelloides virus , and was preserved in the China General Microbiological Culture Collection Center on March 21, 2025, with a preservation number of CGMCC No. 46409. The parapeptococcus shigelloides bacteriophage has lysis effects on parapeptococcus shigelloides and aeromonas veronii. The bacteriophage has a high lysis rate on parapeptococcus shigelloides and also has lysis effects on aeromonas veronii, and is a bacteriophage with cross-species lysis, good alkali resistance and stability, and good thermal stability. The bacteriophage can be prepared into medicines for preventing and treating parapeptococcus shigelloides disease and / or aeromonas veronii disease of cultured fish, environmental disinfectants and water additives, and the like, so as to reduce the use of antibiotics.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a Shiga-like phage with cross-genus lysis capability and its application. Background Technology

[0002] *Shigella spp.* and *Aeromonas vesiculosus* are serious pathogens in aquaculture and important zoonotic pathogens. *Shigella spp.* can cause acute hemorrhagic septicemia, intestinal ulcers, and systemic infections in fish, with mortality rates exceeding 60%. *Aeromonas vesiculosus* is widespread in the aquatic environment and is closely associated with fish skin ulceration, hepatopancreatic necrosis, and ascites. Diseases caused by both pathogens are prone to outbreaks during hot seasons. These two bacteria often co-infect in farmed aquatic animals. Co-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, with particularly significant harm in immunocompromised populations. Related statistics show that numerous diarrhea cases each year are related to aquatic pathogen infections. Among them, *Shigella spp.*, as an important aquatic pathogen, places a heavy burden on public health.

[0004] Currently, aquaculture relies heavily on antibiotics to prevent bacterial diseases. However, long-term overuse leads to frequent outbreaks of multidrug-resistant strains, which can even be transmitted to humans through the food chain, exacerbating the difficulty of clinical treatment. Bacteriophages are natural enemies of bacteria and are abundant in nature. Phage therapy, with its advantages of strong targeting and eco-friendliness, is considered an alternative to antibiotic treatment. In aquaculture, bacteriophages can be used to reduce or prevent bacterial infections in aquatic animals and to control bacterial density in the aquaculture environment. However, existing bacteriophages cannot simultaneously lyse *Shigella* and *Aeromonas vesiculosus*. Summary of the Invention

[0005] To address the above problems, this invention provides a Shiga-like phage with cross-genus lysis capability and its applications.

[0006] This invention is achieved through the following technical solution:

[0007] A *Shigella*-like bacteriophage with cross-genus lytic ability, the *Shigella*-like bacteriophage being classified and named... Plesiomonas virus It was deposited on March 21, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46409.

[0008] The Shigella-like phage against Shigella-like ( Plesiomonas shigelloides ) and Aeromonas versicolor ( Aeromonas veronii All of them have a pyrolysis effect.

[0009] The application of the aforementioned Shigella-like phage in the preparation of drugs for treating diseases caused by Shigella or Aeromonas vesiculosus in fish.

[0010] The application of the aforementioned Shiga-like phage in the preparation of biological bactericides, cleaning agents or disinfectants for aquaculture water.

[0011] The application of the aforementioned Shigella phage in the preparation of reagents with cross-genus lysis capability, wherein the reagent uses the aforementioned Shigella phage as the sole active ingredient.

[0012] The application of the aforementioned Shigella phage in the preparation of a kit with cross-genus lysis capability, wherein the kit uses the aforementioned Shigella phage as the sole active ingredient.

[0013] The application of the aforementioned Shigella-like phage in the preparation of aquatic feed additives, wherein the aquatic feed additives include the aforementioned Shigella-like phage and excipients.

[0014] Preferably, the excipient is one or more of trehalose, skim milk powder, and glycerin.

[0015] Preferably, the Shiga-like phage is inoculated at a multiplicity of infection of 0.001 to 10.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention provides a *Shigella*-like bacteriophage with cross-genus lytic ability and its applications. The *Shigella*-like bacteriophage is classified and named... Plesiomonas virus This bacteriophage was deposited on March 21, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46409. Classified as belonging to the order Cauvirales, family Brachyviridae, it exhibits cross-genera lytic ability and lytic activity against pathogenic *Shigella spp.* and *Aeromonas vesiculosus*. It is highly alkali-resistant and possesses some acid resistance, exhibits certain temperature stability, and is easily proliferated and accumulated. The phage genome does not contain drug resistance or virulence genes. This provides a phage source for the industrial production of bacteriophages for the prevention, treatment, and purification of water bodies in aquaculture environments caused by pathogenic *Shigella spp.* and *Aeromonas vesiculosus*. Furthermore, it can be formulated into drugs for the prevention and treatment of *Shigella spp.* and / or *Aeromonas vesiculosus* diseases in farmed fish, as well as environmental disinfectants and water additives, thereby reducing the use of antibiotics.

[0018] bacteriophage vB_PshP_C1, classified and named Plesiomonas virus The depositary institution is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 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. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The plaques formed by the bacteriophage vB_PshP_C1 of this invention.

[0021] Figure 2 This is an electron microscope image of the bacteriophage vB_PshP_C1 of this invention.

[0022] Figure 3 This is the optimal MOI for the bacteriophage vB_PshP_C1 of this invention.

[0023] Figure 4 The acid-base stability of the bacteriophage vB_PshP_C1 of this invention.

[0024] Figure 5 Temperature stability of the bacteriophage vB_PshP_C1 of this invention.

[0025] Figure 6 This is a one-step growth curve of the bacteriophage vB_PshP_C1 of this invention.

[0026] Figure 7 This is a circled diagram of the genome of the bacteriophage vB_PshP_C1 of this invention.

[0027] Figure 8 This is a graph verifying the lysis ability of the bacteriophage vB_PshP_C1 of this invention; Figure 8 In the image, A represents the phage plaques formed by vB_PshP_C1 on a Shigella-like Osmotherium JRPC1 culture plate; B represents the phage plaques formed by vB_PshP_C1 on a Aeromonas versicolor JRCV1 culture plate.

[0028] Figure 9 The growth inhibition curve of the bacteriophage vB_PshP_C1 of this invention against *Shigella*-like bacteria is shown.

[0029] Figure 10 The growth inhibition curve of bacteriophage vB_PshP_C1 of this invention against Aeromonas vilvoferi is shown. Detailed Implementation

[0030] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. 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 thorough and complete understanding of the disclosure of the present invention.

[0031] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0032] The beneficial effects of the present invention will be illustrated below through specific embodiments.

[0033] Unless otherwise specified, the following embodiments are all conventional methods.

[0034] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0035] Samples and sources: The sea bass and water samples came from an aquaculture farm in Zhuhai, Guangdong Province, and the sampling time was September 2023.

[0036] Shigella-like enomonas ( Plesiomonas shigelloides JRPC1 and Aeromonas verrucosa ( Aeromonas veronii JRCV1 was purchased from Shandong Xinde Technology Co., Ltd.

[0037] Example 1

[0038] Isolation, purification, and preservation of bacteriophage vB_PshP_C1:

[0039] (1) Isolation of bacteriophages using *Shigella spp.* JRPC1 as the host bacteriophage: Bacteriophages were isolated using double-layer plates. Approximately 2 mL of culture 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. 1 mL of *Shigella spp.* JRPC1 bacterial suspension was added, and the mixture was incubated at 37°C for 4 hours using a shaker. The supernatant was then filtered through a 0.22 μm filter membrane and temporarily stored at 4°C. 200 μL of *Shigella spp.* was used to isolate bacteriophages with a viable count of 1 × 10⁻⁶ cells. 8Mix 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 LB solid agar plates, and allow it to solidify to form a double-layer plate. Incubate overnight at 37°C and observe plaque formation.

[0040] (2) Phage purification: Using a sterile inoculation needle, a single phage plaque was collected, placed in SM buffer, and shaken to mix. The mixture was then filtered through a 0.22 μm filter membrane. The filtrate was used to prepare a double-layer plate with the host bacteria. This purification process was repeated 6 times, collecting single phage plaques and forming double-layer plates to create phage plaques of uniform morphology and size on the plates. Figure 1 As shown, the purified phage formed plaques with a diameter of 3mm~4mm, which were round, transparent, and without halos. According to international nomenclature rules, it was named vB_PshP_C1.

[0041] (4) Preservation of bacteriophages: Mix bacteriophage fluid with sterile glycerol in a ratio of 7:3, dispense into 2 mL cryovials, and store at -80°C, or prepare lyophilized powder and store at 4°C.

[0042] Example 2

[0043] Electron microscopy observation of bacteriophages: Take 20 μL of liquid containing bacteriophages, the liquid concentration is 1.5 × 10⁻⁶. 9 PFU / mL was added to a copper grid and allowed to settle naturally for 15 min. Then, 50 μL of phosphotungstic acid (pH 7.0) was added to the copper grid for negative staining of the phages for 10 min. The morphology of the phages was observed under a transmission electron microscope. The EM image of vB_PshP_C1 is shown below. Figure 2 As shown.

[0044] Depend on Figure 2 It is known that the head of bacteriophage vB_PshP_C1 is icosahedral, with a head length 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 bacteriophage can be identified as belonging to the order Tailed bacteriophages and the family Brachypophagidae.

[0045] Example 3

[0046] 1. Optimal multiplicity of infection (MOI) for bacteriophage vB_PshP_C1:

[0047] Shigella-like O. genus JRPC1 was inoculated into 5 mL of LB liquid medium and cultured at 37°C with shaking at 200 rpm until mid-log phase, yielding approximately 10 viable cells. 8 CFU / mL. Phage fluid was filtered through a 0.22 μm filter membrane and then serially diluted to 10⁻⁶ CFU / mL with SM buffer. 6PFU / mL. Phage concentrations were mixed according to the bacterial culture concentration ratios of 10, 1, 0.1, 0.01, 0.001, and 0.0001. The mixtures were allowed to stand at 37°C for 15 minutes for adsorption. 5 mL of preheated LB medium was added to each mixture, and the mixtures were incubated at 37°C with shaking at 200 rpm for 4 hours. The culture was then filtered through a 0.22 μm filter. The titer was determined using the double-layer plate method, with three replicates and the average value taken. The results showed that the highest titer (3.2 × 10⁻⁶) was observed at MOI = 0.0001. 10 PFU / mL was used to determine the optimal MOI, such as Figure 3 As shown.

[0048] Example 4

[0049] Acid-base stability of bacteriophage vB_PshP_C1: Prepare 1 mol / L HCl and 1 mol / L NaOH solutions. Take 50 mL of LB liquid medium, calibrate with a pH meter, and adjust the pH to the range of 1.0–14.0 respectively. Take 900 μL of LB medium at each pH value, add 100 μL of bacteriophage solution to each, resulting in a concentration of 10. 8 PFU / mL, vortex for 10 seconds. Incubate at 37°C for 2 hours, then immediately stop the reaction by placing the mixture on ice for 5 minutes. The cell titer was determined using the double-layer plate method at different pH values, repeated three times. Results showed that the titer was stable within the pH range of 4.0–12.0, with a value greater than 10. 8 PFU / mL, the potency decreases to less than 10 when pH < 4.0 or pH > 12.0. 3 PFU / mL, such as Figure 4 As shown.

[0050] Example 5

[0051] Temperature stability of bacteriophage vB_PshP_C1: The bacteriophage solution was prepared at a concentration of 2×10⁻⁶. 8 PFU / mL was aliquoted into centrifuge tubes, 1 mL per tube. The tubes were incubated in constant temperature water baths at 40℃, 50℃, 60℃, and 70℃, respectively, while the control group was placed on ice. Each group had three replicates. Incubation at each temperature was 20 min, 40 min, and 60 min, followed immediately by 5 min on ice. 100 μL of each sample was then used to determine the potency using the bilayer plate method. Results are as follows: Figure 5 As shown: Treatment at 40℃ and 50℃ for 60 min resulted in no significant change in potency, which remained at 10. 8 At approximately PFU / mL, treatment at 60℃ for 20 min maintained a potency of 3.5 × 10⁻⁶ PFU / mL. 6 PFU / mL, treated for 40 min, titer 2.8 × 10⁻⁶ 4 PFU / mL, treated for 60 min, titer 2.5 × 10⁻⁶ 2PFU / mL.

[0052] Example 6

[0053] This example illustrates the determination of the one-step growth curve of bacteriophage vB_PshP_C1: the host bacterial culture was mixed at the optimal MOI of 0.0001, with a concentration of 10. 8 CFU / mL and phage fluid, concentration 10 4 PFU / mL, total volume 1mL. Incubate at 37℃ for 5 minutes with gentle shaking 3 times during this period. Centrifuge at 12000rpm for 5 minutes at 4℃, discard the supernatant, and gently pipette the precipitate twice with preheated LB medium at 37℃. Finally, resuspend in 1mL LB medium. Transfer the host bacteria-phage mixture to 100mL of LB medium at 37℃ and incubate with shaking at 37℃ and 200rpm. From the mixing time point t=0, take 1mL of sample every 10 minutes, immediately stop the reaction in an ice bath, filter through a 0.22μm filter membrane, and store at -80℃. After thawing the samples, determine the phage titer using the double-layer plate method, with 3 replicates per group. Plot the one-step growth curve of the phage with time on the x-axis and phage titer on the y-axis. The results are shown below. Figure 6 As shown, the incubation period of bacteriophage vB_PshP_C1 is 20 minutes, followed by a steady increase in titer. The burst period lasts for 150 minutes, followed by a plateau period.

[0054] Example 7

[0055] High-titer phages obtained by the double-layer plate amplification method were centrifuged at 10000g, 4℃ for 10 min. The supernatant was collected, filtered for sterilization, and the viral genome was extracted using a centrifugation column method and sent to a sequencing company for sequencing. The full-length genome was 43419 bp. The genome diagram is shown below. Figure 7 The annotation results showed that the phage genome had 44 open reading frames, and the sequence alignment of protein-coding genes was performed using Diamond software, annotating 31 proteins.

[0056] Example 8

[0057] Validation of the cross-genus lysis ability of bacteriophage vB_PshP_C1

[0058] Mix 200 μL of *O. shigella* JRPC1 and 200 μL of *Aeromonas vegetans* JRCV1 separately with LB semi-solid medium cooled to approximately 55°C, then pour the mixtures onto LB solid agar plates and allow them to solidify to create double-layer plates. Take 10 μL of each mixture... 6 PFU / mL phage broth was added dropwise to the culture medium, allowed to air dry, and then incubated at 37℃ for 8 hours. Plaque formation was then observed. Results are as follows: Figure 8As shown, the bacteriophage vB_PshP_C1 exhibits lytic activity against both Shigella-like Osmonica JRPC1 and Aeromonas versicolor JRCV1.

[0059] Example 9

[0060] Detection of in vitro antibacterial activity of bacteriophage vB_PshP_C1

[0061] *O. shigella* JRPC1 and *Aeromonas vesiculosus* JRCV1 were inoculated into LB medium and cultured at 37°C to the logarithmic phase. The bacterial concentration was adjusted to 10 CFU / mL. Bacteriophages were inoculated into *O. shigella* JRPC1 and *Aeromonas vesiculosus* JRCV1 at MOIs of 10, 1, 0.1, 0.01, and 0.001, respectively, in equal volumes of LB broth. Both phage and bacterial cultures were 100 μL. The cultures were incubated at 37°C with shaking at 200 rpm for 12 hours. Each group had three replicates, with a control group consisting of bacterial cultures without phages. The OD of the bacteria was measured every hour. 600 Value, drawing Figure 9 and Figure 10 The results showed that bacteriophage vB_PshP_C1 had a strong inhibitory effect on the growth of both *O. shigella* JRPC1 and *Aeromonas vesiculosus* JRCV1. In vitro, the OD value of the control group *O. shigella* JRPC1 continuously increased, stabilizing at around 0.7 after 6 hours. The inhibitory effect on *O. shigella* JRPC1 was manifested in that, at all MOI values, the OD value remained below 0.23 from 0 to 13 hours, without any upward trend.

[0062] In vitro, the OD value of Aeromonas vesiculosus JRCV1 in the control group continuously increased, stabilizing at around 0.92 after 11 hours. The inhibitory effect on Aeromonas vesiculosus JRCV1 was manifested in the following ways: at all MOI values, the OD value slowly increased from 2 to 11 hours, but remained significantly lower than the OD value of the control group. Particularly at MOI=10 and MOI=1, it effectively inhibited Aeromonas vesiculosus JRCV1.

[0063] Example 10: A biological bactericide

[0064] This embodiment provides a biocidal agent. The bacteriophage vB_PshP_C1 screened in Example 1 is mixed with a protectant and a synergist in any proportion, and then dispensed into sterile spray bottles at 10 mL / bottle. The protectant is gelatin, and the synergist is 0.1 mM EDTA.

[0065] The antibacterial activity of the bio-fungicide was tested, and the results showed that the bio-fungicide had an inhibitory effect on both Shigella-like bacteria and Aeromonas verrucosa.

[0066] Example 11: A reagent with cross-genus cleavage capability

[0067] This embodiment provides a reagent with cross-genus lysis capability. The bacteriophage vB_PshP_C1 obtained in Example 1 was diluted with SM buffer to a final concentration of 1×10⁻⁶. 9 The reagent, containing PFU / mL, was aliquoted and stored at 4°C to obtain a reagent with cross-genera lysis capability. The SM buffer formulation was 50 mM Tris-HCl, 100 mM NaCl, 8 mM MgSO₄, pH 7.5. Cross-genera lysis capability was tested using this reagent, and the results showed that it had lytic activity against both *Shigella* and *Aeromonas vesiculosus*.

[0068] Example 12: An aquatic feed additive

[0069] This embodiment provides an aquatic feed additive, which is made by mixing the bacteriophage vB_PshP_C1 screened in Example 1 with trehalose and skim milk powder in any proportion. After pre-freezing at -80℃ and vacuum freeze-drying, it is mixed with puffed corn flour in any proportion, granulated, with a particle size of 1 mm, and packaged in a light-proof and sealed container.

[0070] The cross-genera lysis ability of this feed additive was tested, and the results showed that the reagent had a lysis effect on both Shigella-like *Oximonas oryzae* and *Aeromonas viride*.

[0071] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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.

[0072] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention should be determined by the appended claims.

Claims

1. A Shigella-like phage with cross-genus lytic ability, characterized in that, The Shigella-like phage is classified as... Plesiomonas virus It was deposited at the China General Microbiological Culture Collection Center on March 21, 2025, with accession number CGMCC No. 46409; The Shigella-like phage against Shigella-like ( Plesiomonas shigelloides ) and Aeromonas versicolor ( Aeromonas veronii All of them have a pyrolysis effect.

2. The use of the Shigella-like phage according to claim 1 in the preparation of a medicament for treating diseases caused by Shigella or Aeromonas vesiculosus in fish.

3. The application of the Shigella-like phage according to claim 1 in the preparation of biological bactericides, cleaning agents or disinfectants for aquaculture water, characterized in that, The biological bactericide, cleaning agent, or disinfectant is used to prevent and control Shigella spp. and / or Aeromonas versicolor.

4. The use of the *Shigella*-like bacteriophage according to claim 1 in the preparation of reagents for lysing *Shigella*-like bacteria and / or *Aeromonas vesiculosus*, characterized in that, The reagent uses the aforementioned Shigella phage as its sole active ingredient.

5. The use of the *Shigella*-like bacteriophage according to claim 1 in the preparation of a kit for lysing *Shigella*-like bacteria and / or *Aeromonas vesiculosus*, characterized in that, The kit uses the aforementioned Shigella phage as its sole active ingredient.

6. The application of the Shigella-like phage according to claim 1 in the preparation of aquatic feed additives, characterized in that, The aquatic feed additive is used to prevent or treat diseases in aquatic animals caused by Shigella-like bacteria and / or Aeromonas verrucosa.

7. The application according to claim 6, characterized in that, The aquatic feed additive also includes excipients, which are one or more of trehalose, skim milk powder, and glycerin.

8. The application according to any one of claims 2 to 7, characterized in that, The Shiga-like phages were inoculated at a multiplicity of infection of 0.001 to 10.

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