Citrobacter freundii bacteriophage, composition and application thereof

Abstract

The present application relates to the field of microbial technology, in particular to a Citrobacter werkmanii bacteriophage and its composition and application. The Citrobacter werkmanii bacteriophage (Citrobacter werkmanii bacteriophage) is named RDP-CI25012, which was preserved in the China General Microbiological Culture Collection Center on August 25, 2025, and the preservation number is CGMCC No. 46668. A composition comprises the above-mentioned bacteriophage. The above-mentioned bacteriophage or composition is used in (1) preparing a product for killing Citrobacter werkmanii; (2) preparing a product for inhibiting Citrobacter werkmanii; (3) preparing a product for preventing and / or treating Citrobacter werkmanii disease caused by Citrobacter werkmanii. The phage has a lysis rate of 80%, has a very high host range, and has a wide lysis spectrum. The phage has excellent thermal stability, and the titer remains above 10 7 PFU / mL after being treated at 50 DEG C for 4 hours, which lays a solid foundation for its use, storage, transportation and production processing in most actual application scenarios.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Citrobacter wiltii bacteriophage, its composition, and its applications. Background Technology

[0002] The genus *Citrobacter* comprises Gram-negative bacteria, including several species such as *Citrobacter freundii*, *Citrobacter seis*, and *Citrobacter wildmannii*. *Citrobacter wildmannii*, as an opportunistic pathogen, has been frequently detected in aquaculture environments in recent years and has been confirmed as a major pathogen causing outbreaks of various important economic aquatic animals, posing an increasingly serious threat to the global aquaculture industry.

[0003] This bacterium is widely distributed in water, bottom sediment, and the intestines and body surface of aquatic animals. Its pathogenicity is particularly pronounced when the aquaculture environment deteriorates (such as high-density farming, poor water quality, insufficient dissolved oxygen, and excessive ammonia nitrogen and nitrite), the host's immunity is low (such as stress from transportation, pond separation, and sudden temperature changes), or secondary infections exist. Its core threat to aquaculture lies in its strong pathogenicity: Citrobacter wiltii can infect various fish species (such as channel catfish, tilapia, catfish, crucian carp, and carp) and some crustaceans (such as shrimp and crabs) through water, direct contact, or ingestion. After infection, the pathogen can break through the host's skin, gills, or intestinal mucosal barrier, invade the circulatory system, causing bacteremia or septicemia, and invade various internal organs (such as the liver, kidneys, and spleen), leading to severe tissue necrosis and dysfunction. Typical clinical symptoms include extensive congestion, hemorrhage, ulceration, and even perforation on the body surface (especially the abdomen and fin base), ascites, exophthalmos, red and swollen anus, enlarged and discolored liver (yellowish-brown or pale) with necrotic foci, enlarged and blackened spleen, and intestinal inflammation filled with mucus or hemorrhage. This disease has a rapid onset, short course, and high infectivity. Under suitable conditions, it can easily break out rapidly in aquaculture populations, causing high mortality rates in infected fish and shrimp within a short period, sometimes exceeding 50% or even wiping out the entire pond, resulting in devastating direct economic losses for fish farmers.

[0004] Currently, antibiotics are the primary means of controlling *Citrobacter ferruginea* in aquaculture. However, due to the improper use of antibiotics, antibiotic resistance is becoming increasingly prevalent. *Citrobacter ferruginea* commonly exhibits resistance or decreased sensitivity to many commonly used antibiotics, such as sulfonamides, tetracyclines, and β-lactams. This resistance significantly limits the effectiveness of chemical treatments for this disease, leading to poor treatment outcomes, prolonged treatment cycles, and dramatically increased treatment costs. Furthermore, the extensive use of antibiotics also causes drug residue problems, threatening the quality and safety of aquatic products, disrupting the microecological balance of aquaculture areas, and facing increasingly stringent international green barriers to trade. In response to the national policy of reducing and replacing antibiotics, it is necessary to find antibiotic alternatives to control *Citrobacter ferruginea*, reduce antibiotic use, and protect human health and safety.

[0005] Bacteriophages are a general term for viruses that infect microorganisms such as bacteria, fungi, actinomycetes, or spirochetes. They are extremely small, lack a complete cellular structure, and are composed of nucleic acids and proteins. They cannot grow and reproduce independently of a host and therefore must grow within living cells. Bacteriophages must parasitize living bacteria and exhibit strict host specificity, which depends on the molecular structure and complementarity of the phage's adsorption organs and the receptors on the surface of the recipient bacteria. Generally, a single bacterium can be infected by multiple bacteriophages, but a single bacteriophage can only infect one type of bacterium and its closely related species. Bacteriophages are the most common and widely distributed group of viruses, and can usually be found in places rich in bacterial communities, such as soil and the intestines of animals.

[0006] The advantages of phage therapy mainly include:

[0007] ① High specificity: Bacteriophages have high specificity in infecting bacteria and will not kill bacteria other than the target bacteria, thus avoiding the occurrence of diseases caused by dysbiosis due to broad-spectrum antibiotics;

[0008] ② Automatic disappearance after clearing the target bacteria: After the bacteriophage clears the target bacteria in the body, it cannot continue to replicate due to the loss of the host bacteria, and will "automatically disappear" in the body without causing accumulation and poisoning;

[0009] ③Does not disrupt animal metabolism: Bacteriophages only infect bacteria and do not disrupt animal metabolism or cause infectious diseases in animals. At present, no serious toxic side effects have been found in phage treatment.

[0010] ④ It can reduce the use of antibiotics: The application of phage therapy can reduce the use of antibiotics, thus ensuring food safety;

[0011] ⑤ Can identify specific pathogenic bacteria: Bacteriophages can identify specific pathogenic bacteria and have little impact on the environment;

[0012] ⑥ It can use the host to multiply and enter the bacteria: Bacteriophages can use the host to multiply and enter the bacteria, efficiently lysing pathogenic bacteria.

[0013] Therefore, isolating and identifying a bacteriophage that can effectively combat Citrobacter is a pressing problem that needs to be solved.

[0014] The information disclosed in this background section is only intended to enhance the understanding of the background technology of this application and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0015] To address the shortcomings of existing technologies, this invention provides a strain of *Citrobacter werkmanii* bacteriophage, its composition, and its applications. This strain, RDP-CI25012, can broadly lyse multiple *Citrobacter werkmanii* species, exhibits high thermal stability, and can be used to treat and / or prevent *Citrobacter werkmanii* diseases in aquaculture.

[0016] The technical solution of this invention is as follows:

[0017] On the one hand, the present invention provides a strain of Citrobacter werkmaniibacteriophage, named RDP-CI25012, which was deposited at the China General Microbiological Culture Collection Center on August 25, 2025, with the accession number CGMCC No. 46668.

[0018] Furthermore, the optimal multiplicity of infection (MMO) of the *Citrobacter wormi* phage is 0.01, and the highest titer at the optimal MMO is 10. 10 PFU / mL.

[0019] Furthermore, the *Citrobacter wormi* phage maintained a titer of 10 after being exposed to 50°C for 4 hours. 7 With a PFU / mL or higher, it exhibits high temperature stability.

[0020] Furthermore, the *Citrobacter worm* phage exhibits the highest titer (10) at pH 6. 10 The potency of PFU / mL can be maintained at 10 within the pH range of 5-8. 8 -10 10PFU / mL. It exhibits a wide range of acid and alkali tolerance and high acid-base stability. The pH of aquaculture water fluctuates between day and night due to algal photosynthesis (consuming CO2, causing pH to rise) and respiration (releasing CO2, causing pH to fall). The stability of this bacteriophage RDP-CI25012 at pH 5-8 ensures that it remains active in water around the clock and will not be inactivated by pH fluctuations.

[0021] Electron microscopy revealed that the *Citrobacter wiltii* bacteriophage possessed a polyhedral, three-dimensionally symmetrical head encapsulating nucleic acid, approximately 50 nm in diameter, and a tail approximately 16 nm long. The neck connected the head and tail. According to the Ninth Report on Taxonomy of Viruses by the International Organization for Taxonomy of Viruses, this bacteriophage is classified as belonging to the order Tailed Viriformes, family Brachyviridae.

[0022] On the other hand, the present invention provides a composition comprising the above-mentioned Citrobacter worm.

[0023] On the other hand, the above-mentioned Citrobacter gargarizans phage or composition is used in at least one of the following (1) to (3):

[0024] (1) Prepare a product that kills Citrobacter ferruginosa;

[0025] (2) Preparation of products that inhibit Citrobacter ferruginosa;

[0026] (3) Prepare products for the prevention and / or treatment of Citrobacter velutipes disease caused by Citrobacter velutipes.

[0027] Furthermore, the product is a veterinary drug preparation or a disinfectant.

[0028] Furthermore, the veterinary drug formulation also includes a pharmaceutically acceptable carrier, and its dosage form is a powder, solution, emulsion, gel, granule or lyophilized form.

[0029] Furthermore, the application is the preparation of products for the prevention and / or treatment of Citrobacter ferruginosa disease in aquaculture caused by Citrobacter ferruginosa.

[0030] Furthermore, the application is the preparation of products for the prevention and / or treatment of Citrobacter ferruginosa disease in Litopenaeus vannamei caused by Citrobacter ferruginosa.

[0031] The beneficial effects achieved by this invention are as follows:

[0032] (1) This invention isolated a strain of Citrobacter wilteri CI25009, and used this bacterium as a host to isolate a strain of Citrobacter wilteri bacteriophage RDP-CI25012. The bacteriophage has a strong lysis efficiency against Citrobacter wilteri isolated from aquaculture environments. Specifically, the bacteriophage RDP-CI25012 can lyse 16 out of 20 different strains of Citrobacter wilteri, with a lysis rate of 80%, exhibiting an extremely wide host range and a broad lysis spectrum.

[0033] (2) The Citrobacter worm RDP-CI25012 in this invention can still maintain a high titer after 30 passages, indicating that this phage has strong genetic stability, and high stability is of great significance for production and transportation.

[0034] (3) The titer of the Citrobacter worm RDP-CI25012 in this invention remained at 10 after treatment at 50°C for 4 hours. 7 A concentration above PFU / mL indicates that the viral particle (capsid) structure of this bacteriophage is highly stable and resistant to heat denaturation. This also demonstrates the bacteriophage's excellent thermal stability, providing a solid foundation for its use, storage, transportation, and processing in most practical applications. Aquaculture water temperatures can easily reach 30-40°C in summer; the bacteriophage's temperature stability ensures its long-lasting efficacy in aquaculture water.

[0035] (4) By adding the Citrobacter virgaurea phage RDP-CI25012 (≥10) of the present invention to the aquaculture water 8 PFU / mL significantly reduced the mortality rate of Litopenaeus vannamei infected with Citrobacter wilt, effectively controlled the pathogen load in the hepatopancreas and environment, and had no side effects on the host. Prophylactic use of phage RDP-CI25012 provided significant protection in a Litopenaeus vannamei infection model, with highly successful results. It not only significantly reduced mortality but also effectively controlled the number of pathogens in tissues and cleared pathogens from the environment, providing a reliable technical solution for the green control of this disease.

[0036] (5) The Citrobacter worm RDP-CI25012 of this invention can be used for the prevention and treatment of diseases caused by Citrobacter worm and has extremely high safety. The study on the biological characteristics of the Citrobacter worm RDP-CI25012 isolated in this invention provides theoretical guidance for the next step of developing new antibacterial drugs—biocontrol agents. Attached Figure Description

[0037] Figure 1This describes the colony morphology of *Citrobacter wiltii* on LB agar plates in this invention.

[0038] Figure 2 This refers to the plaques of the host bacteria created by the Citrobacter worm RDP-CI25012 in this invention.

[0039] Figure 3 This is an electron microscope image of the Citrobacter worm RDP-CI25012 used in this invention.

[0040] Figure 4 The titer of Citrobacter worm RDP-CI25012 under different infection multiplicity conditions is [not specified].

[0041] Figure 5 This is a one-step growth curve of Citrobacter worm RDP-CI25012 in this invention.

[0042] Figure 6 This is the temperature stability curve of the Citrobacter worm RDP-CI25012 in this invention.

[0043] Figure 7 This is the pH stability curve of Citrobacter worm RDP-CI25012 in this invention.

[0044] Figure 8 This is the genetic stability curve of Citrobacter worm RDP-CI25012 in this invention.

[0045] Figure 9 This is an experimental diagram of the lysis spectrum of Citrobacter worm RDP-CI25012 in this invention. Detailed Implementation

[0046] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] In this invention, unless otherwise specified, the equipment and raw materials used are commercially available or commonly used in the field. The methods in the following embodiments, unless otherwise specified, are conventional methods in the field. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In this invention, *Citrobacter wilteri* CI25009 is referred to simply as "host bacterium" or "host bacterium CI25009" or "CI25009," and *Citrobacter wilteri* bacteriophage RDP-CI25012 is referred to simply as "Citrobacter wilteri* bacteriophage RDP-CI25012" or "RDP-CI25012" or "bacteriophage."

[0048] Example 1: Screening and identification of Citrobacter worm RDP-CI25012

[0049] 1.1 Isolation and Identification of Citrobacter velutipes

[0050] Samples were collected from shrimp farms in Guangdong. The samples were streaked onto LB agar plates and incubated at 37°C for 16 hours. Colony morphology and color were observed. Different colonies were picked and streaked onto LB agar plates. The culture was repeated five times until uniformly morphological colonies were obtained. Figure 1 As shown. Single colonies were picked and streaked onto LB agar medium. Colonies were then identified by 16S rRNA gene sequencing. After identification, the bacterial growth was scraped and placed in 30% glycerol broth and stored at -80℃, numbered CI25009.

[0051] 1.2 Isolation and purification of Citrobacter worm RDP-CI25012

[0052] (1) Treatment of aquaculture water samples: Take sludge water samples from the aquaculture farm, weigh 5g and add 10mL of SM buffer to soak overnight. Then centrifuge the overnight leachate at 10000rpm for 5min, take the supernatant and filter it with a 0.22μm filter. Store the filtrate at 4℃ for later use.

[0053] (2) Phage enrichment: Take 0.1 mL of Citrobacter wilt and 1 mL of filtrate and add it to 5 mL of LB broth. Incubate overnight at 37°C on a shaker. Then centrifuge at 10,000 rpm for 10 min. Filter the supernatant through a 0.22 μm filter and keep the filtrate for later use.

[0054] (3) Phage isolation: Phages were isolated using the double plate method. 0.1 mL of Citrobacter wiltii bacterial suspension was mixed with 0.6% LB soft agar and spread on an LB solid plate. After the soft agar solidified, 0.1 mL of the filtrate was spotted onto the plate and allowed to stand until the mixture was absorbed. After incubation at 37°C for 6 hours, transparent phage plaques were picked up with an inoculation loop and placed in 2 mL of SM buffer. The plaques were stored at 4°C overnight to allow the phages to be completely released, thus obtaining the phages.

[0055] (4) Phage purification: Phages were isolated using the double-plate method. 0.1 mL of the mixed bacterial suspension filtrate and 0.2 mL of the host *Citrobacter wormi* suspension were mixed thoroughly. Soft agar cooled to approximately 50°C was added and mixed again. The mixture was then plated on double plates and incubated at 37°C for 6 hours. A single clear plaque with smooth edges was picked and placed in 1 mL of SM buffer and stored overnight at 4°C. 0.1 mL of the overnight leachate was mixed with 0.1 mL of the bacterial suspension, and soft agar cooled to approximately 50°C was added and mixed thoroughly. The mixture was then plated on double plates and incubated at 37°C for 6 hours. This process was repeated until the phage plaques were uniform in size and had smooth edges, thus obtaining the purified phage. Figure 2 As shown.

[0056] (5) Preparation of bacteriophage: Take 0.2 mL of Citrobacter wilt and 1 mL of purified bacteriophage solution and add them to 50 mL of LB broth. Incubate overnight at 37°C in a shaker. Then centrifuge at 10,000 rpm for 10 min. Filter the supernatant through a 0.22 μm filter and keep the bacteriophage filtrate for later use.

[0057] (6) Preservation method of bacteriophage: The bacteriophage suspension was mixed with 60% glycerol at a ratio of 1:1 and stored at -80℃, named RDP-CI25012. It was deposited at the China General Microbiological Culture Collection Center on August 25, 2025, with the accession number CGMCC No. 46668.

[0058] 1.3 Electron microscopic observation of Citrobacter worm RDP-CI25012

[0059] 1.3.1 Experimental Methods:

[0060] Take 20 μL of liquid containing crude phage particles and drop it onto a copper grid. Allow it to settle naturally for 15 min, then blot away excess liquid with filter paper from the side. Add one drop of 2% phosphotungstic acid (PTA) to the copper grid to stain the phage for 10 min. Then blot away the staining solution with filter paper from the side. After the sample dries, observe the phage morphology using an electron microscope.

[0061] 1.3.2 Experimental Results:

[0062] Electron microscopy observation results as follows Figure 3As shown: The bacteriophage has a polyhedral, three-dimensionally symmetrical head encapsulating nucleic acid, approximately 50 nm in diameter, and a tail about 16 nm long. The neck connects the head and tail. According to the Ninth Report on Taxonomy of Viruses by the International Organization for Taxonomy of Viruses, this bacteriophage is classified as belonging to the family Brachyviridae in the order Twinovirales.

[0063] 1.4 Whole genome sequence sequencing of Citrobacter worm RDP-CI25012

[0064] 1.4.1 Experimental Methods:

[0065] 1) Construct a library starting with 1 μg of phage genomic DNA;

[0066] 2) Covaris M220 ultrasonically breaks down DNA to 300-500 bp;

[0067] 3) Fill in the 3' end with an A and connect the index adapter (TruSeq™ Nano DNA Sample Prep Kit);

[0068] 4) Library enrichment, PCR amplification for 8 cycles;

[0069] 5) 2% agarose gel recovery target band (Certified Low Range Ultra Agarose);

[0070] 6) TBS380 (Picogreen) quantitative analysis: mix according to data ratio and run on the machine;

[0071] 7) Bridged PCR amplification was performed on the cBot solid-phase vector to generate clusters;

[0072] The Illumina Hiseq sequencing platform was used to perform 2×150bp sequencing.

[0073] 1.4.2 Experimental Results:

[0074] The complete genome of phage RDP-CI25012 is shown in SEQ ID NO.1, with a size of 39346 bp. Predictive analysis shows that the genome of phage RDP-CI25012 contains 46 coding sequences and does not contain any virulence genes or lysogenic genes. Among them, the endolysins of RDP-CI25012 (CDS-0019 and CDS-0041) encode a variety of cell wall degrading enzymes. In particular, CDS-0041 contains an endolysin domain and has the ability to lyse Gram-negative bacteria in a broad spectrum.

[0075] Example 2: Characterization of Citrobacter worm RDP-CI25012

[0076] 2.1 Determination of the titer of Citrobacter worm RDP-CI25012

[0077] 2.1.1 Experimental Methods:

[0078] Take 100 μL of the host bacterial culture in the exponential phase, mix it into a 5 mL centrifuge tube, shake well, and place it on an LB semi-solid agar plate, completely covering a 20 mL LB solid agar plate (9 cm in diameter). After semi-solid drying, take 100 μL of the purified phage concentrate and place it on the plate, with three plates for each phage. Incubate overnight at 37°C. Collect phage plaques in 20 mL of SM buffer and incubate at 4°C. The next day, collect the supernatant, filter it through a 0.22 μm filter membrane, and take 100 μL of the filtrate. Dilute it 10-fold with sterile water. Then, mix 100 μL each of the bacterial culture and phage solution on double plates and incubate the plates overnight at 37°C. The phage titer of phage RDP-CI25012 was determined using the above method. Countable plates were selected, and phage plaques were counted.

[0079] Calculate phage titer (PFU / mL) = number of plaques × dilution factor / sample volume (mL).

[0080] 2.1.2 Experimental Results:

[0081] The titer of Citrobacter worm RDP-CI25012 was determined to be 10. 10 PFU / mL.

[0082] 2.2 Determination of the optimal multiple of infection (MOI) of Citrobacter worm RDP-CI25012

[0083] 2.2.1 Experimental Methods:

[0084] MOI refers to the ratio of the number of bacteriophages to the number of host bacteria at the initial infection. The concentration of host bacteria cultured to the exponential phase is adjusted to 10-1. 8 CFU / mL. MOIs were set at 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100. Phage solutions with determined titers were added to the bacterial culture in the specified proportions, 500 μL each. The mixtures were incubated at 37°C with shaking at 200 rpm for 5 h. The combined culture was then centrifuged at 10000 rpm for 10 min, and the phage titer was measured. The highest multiplicity of infection (MLO) was determined as the optimal MLO. The optimal MLO for phage RDP-CI25012 was determined using the above method.

[0085] 2.2.2 Experimental Results:

[0086] The results are as follows Figure 4 As shown, the multiplicity of infection (MOI) of Citrobacter worm RDP-CI25012 was 0.01, resulting in the highest titer. Therefore, the optimal MOI for RDP-CI25012 is 0.01, corresponding to a maximum titer of 10. 10 PFU / mL. Notably, unlike many phages, RDP-CI25012 exhibited the highest yield at a low MOI (0.01).

[0087] 2.3 One-step growth curve of Citrobacter worm RDP-CI25012

[0088] 2.3.1 Experimental Methods:

[0089] Mix 500 μL each of phage fluid and host bacterial fluid at the ratio required for the optimal multiple of infection, and incubate at 37°C for 15 min. Centrifuge at 10000 r / min for 1 min, and wash the precipitate three times with LB broth. Add 10 mL of preheated LB broth at 37°C, mix well, and immediately place in a shaker at 37°C for incubation. Take samples every 10 min to determine the phage titer, perform three replicates, and take the average value. Plot a one-step growth curve with infection time on the x-axis and the logarithm of the phage titer in the infection system on the y-axis to obtain the latency and outbreak phase of phage RDP-CI25012, and calculate the outbreak quantity.

[0090] Outbreak size = Total number of bacteria at the end of the phage outbreak / Total number of bacteria at the beginning of the phage outbreak.

[0091] 2.3.2 Experimental Results:

[0092] The results of the one-step growth curve assay for phage RDP-CI25012 are as follows: Figure 5 As shown, within 80 minutes of infecting the host bacteria with bacteriophage RDP-CI25012, the number of bacteriophages does not increase; this period is called the incubation period. From 80 to 140 minutes after infecting the host bacteria, the number of bacteriophages increases rapidly; this period is the outbreak period of bacteriophages, which is approximately 60 minutes.

[0093] 2.4 Determination of the optimal growth temperature for Citrobacter worm RDP-CI25012

[0094] 2.4.1 Experimental Methods:

[0095] 100 μL of the RDP-CI25012 stock solution was diluted tenfold to a suitable concentration and mixed with an equal volume of bacterial culture. The mixture was then poured into double-layer plates and incubated at 15℃, 20℃, 25℃, 30℃, 37℃, 40℃, 45℃, and 50℃ for 4 hours, respectively. The phage titer was then measured. Growth temperature curves of the phage were plotted with temperature on the x-axis and the logarithm of the phage titer on the y-axis.

[0096] 2.4.2 Experimental Results:

[0097] The results are as follows Figure 6 As shown, phage RDP-CI25012 exhibited the highest titer at 37°C, indicating that 37°C was its optimal growth temperature. Even after treatment at 50°C for 4 hours, the titer remained at 10. 7 A concentration above PFU / mL indicates that the viral particle (capsid) structure of this bacteriophage is highly stable and resistant to heat denaturation. This also demonstrates the bacteriophage's excellent thermal stability, providing a solid foundation for its storage, transportation, and processing in most practical applications.

[0098] 2.5 pH stability determination of Citrobacter worm RDP-CI25012

[0099] 2.5.1 Experimental Methods:

[0100] The pH of LB liquid medium was adjusted to 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, respectively. 100 μL of each medium was mixed with an equal volume of phage stock solution and incubated at 37°C for 2 hours. The titer was then determined using the bilayer plate method, with three replicates for each pH value. pH stability curves of the phages were plotted with pH on the x-axis and the logarithm of the phage titer on the y-axis.

[0101] 2.5.2 Experimental Results:

[0102] like Figure 7 As shown, the pH stability test results of bacteriophage RDP-CI25012 indicate that its highest titer is 10 at pH=6. 10 PFU / mL, and its potency can be maintained at 10 within the pH range of 5-8. 8 -10 10 High levels of PFU / mL.

[0103] The pH of aquaculture water fluctuates between day and night due to algal photosynthesis (consuming CO2, causing pH to rise) and respiration (releasing CO2, causing pH to fall), typically ranging from 6.5 to 9.0. The stability of the bacteriophage RDP-CI25012 at pH 5-8 ensures that it remains active in water around the clock and will not be inactivated by pH fluctuations.

[0104] After oral administration, bacteriophages must overcome the challenge of stomach acid (a highly acidic environment) to reach the intestines and exert their effects.

[0105] Although stomach acid is extremely strong (pH ~1.5-3.5), the high stability of this bacteriophage RDP-CI25012 at pH 5 provides an excellent foundation. Simply combining it with simple enteric-coated capsules or microencapsulation technology (ensuring it is not released in the stomach but only upon entering the higher pH intestines) can greatly guarantee its survival rate. Its stable properties significantly reduce the difficulty of formulation development.

[0106] 2.6 Genetic stability determination of Citrobacter worm RDP-CI25012

[0107] 2.6.1 Experimental Methods:

[0108] The phage RDP-CI25012 was passaged 30 times. In each passage, equal amounts of phage and host bacteria were added to the culture medium. Each passage was cultured for 6 hours. Then, double plates were laid. After passaged 29 times, the titer of the phage was determined.

[0109] 2.6.2 Experimental Results:

[0110] like Figure 8 As shown, the titer of phage RDP-CI25012 remained at 10 throughout the passage process. 10 The PFU / mL indicates that the phage has good biological genetic stability and is an excellent choice for the production process.

[0111] Example 3: Lysis spectrum experiment of Citrobacter worm RDP-CI25012

[0112] 3.1 Experimental Methods:

[0113] The lytic effect of bacteriophage RDP-CI25012 on 20 strains of *Citrobacter wiltans* preserved in the strain library was determined using the double-layer plate spotting method. Different *Citrobacter wiltans* bacterial suspensions were prepared. 200 μL of the bacterial suspension cultured for 6 h was added to 0.6% LB semi-solid medium, mixed thoroughly, poured into Petri dishes, and allowed to solidify. 20 μL of the bacteriophage suspension was then added dropwise to soft agar plates and incubated at 37℃ for approximately 6-8 h, observing for the appearance of phage plaques.

[0114] 3.2 Experimental Results:

[0115] Table 1. Lysis effect of Citrobacter worm RDP-CI25012

[0116]

[0117] Note: +: cleavage; -: non-cleavage

[0118] From Table 1 and Figure 9 It can be seen that phage RDP-CI25012 can lyse 16 out of 20 different strains of Citrobacter wilt, with a lysis rate of 80%, which is high and has a broad lysis spectrum.

[0119] Example 4: Preventive protection experiment of Citrobacter worm RDP-CI25012 against Litopenaeus vannamei.

[0120] The study aimed to verify the effectiveness of the pretreatment of *Citrobacter freundii* bacteriophage isolated in this patented method in aquaculture water and to evaluate its protective efficacy against highly pathogenic *Citrobacter freundii* infection in *Litopenaeus vannamei*.

[0121] 4.1 Experimental Methods:

[0122] ① Grouping of experimental animals: 240 healthy Litopenaeus vannamei shrimp (weight 5.0±0.5 g) were randomly divided into 4 groups (3 replicates per group, 20 shrimp per replicate):

[0123] Group T1 (phage prevention group): Bacteriophage RDP-CI25012 (final concentration 1×10⁻⁶) was added to the aquaculture water. 8 PFU / mL);

[0124] Group T2 (infection control group): An equal volume of sterile PBS buffer was added to the water.

[0125] Group T3 (phage-only treatment group): only phage RDP-CI25012 (1×10⁻⁶) was added. 8 PFU / mL), without challenge;

[0126] Group T4 (blank control group): only sterile PBS buffer was added.

[0127] ②Prevention and treatment and virus challenge:

[0128] Day 0: Bacteriophage RDP-CI25012 was added to the water in groups T1 and T3 at one time, while PBS was added to groups T2 and T4, and aeration was carried out continuously for 2 hours to achieve equilibration.

[0129] Day 1: Groups T1 and T2 were challenged with the virus by immersion (final water concentration 1×10⁻⁶). 8 CFU / mL Citrobacter velutipes CI25009), continuous exposure for 24 hours;

[0130] Experimental period: 7 days (constant temperature of 25±1℃, salinity of 20‰, 50% water change and replenishment of corresponding treatment materials daily).

[0131] Monitoring indicators: Daily records of shrimp mortality rate and typical symptoms (body ulcers, hepatopancreatic turbidity, empty intestines and stomachs).

[0132] Day 7: Collect hepatopancreas tissue from surviving shrimp, homogenize it, spread it on LB agar plates, and count the Citrobacter wilt load (CFU / g); collect water samples to detect phage titer (double-layer plate method) and residual pathogen concentration.

[0133] 5.2 Experimental Results:

[0134] Table 2. Preventive protection experiment of Citrobacter worm RDP-CI25012 against Litopenaeus vannamei.

[0135]

[0136] As can be seen from the data in Table 2, this strain of Citrobacter worm RDP-CI25012 has the following effects:

[0137] ① Significantly reduced mortality: The mortality rate of group T1 (phage prevention) (20%) was 50% lower than that of group T2 (70%), directly demonstrating that prophylactic feeding with phage can effectively protect shrimp and prevent them from dying from pathogen infection. It also proves that the Citrobacter worm phage RDP-CI25012 can effectively block lethal infection by Citrobacter worm worm. After challenge, shrimp in group T1 only showed mild surface congestion, without hepatopancreatic necrosis or large-scale mortality, indicating that phage pretreatment can rapidly inhibit pathogen proliferation.

[0138] ② Highly effective clearance of pathogens from inside and outside the body: The bacterial load in the hepatopancreas of group T1 (10³ CFU / g) was significantly higher than that of group T2 (10³ CFU / g). 7 The CFU / g decreased by four orders of magnitude, and no live bacteria were detected in the aquaculture water, confirming that bacteriophage RDP-CI25012 could eliminate invading and free pathogens through lysis in the early stage of infection. The T3 group (bacteriophage alone) showed no mortality or pathological changes throughout the entire process, indicating that the bacteriophage is safe and non-toxic to the host and also demonstrates that the bacteriophage acted as a "biological disinfectant," cutting off the horizontal transmission route and protecting other individuals in the same pond.

[0139] In summary, this experiment confirms that adding bacteriophages (≥10) to the aquaculture water can effectively control the growth of bacteria. 8 The use of phage RDP-CI25012 significantly reduced mortality in Litopenaeus vannamei infected with Citrobacter wilt, effectively controlled pathogen load in the hepatopancreas and environment, and had no side effects on the host. Prophylactic use of RDP-CI25012 provided significant protection in a Litopenaeus vannamei infection model, with highly successful results. It not only drastically reduced mortality but also effectively controlled the number of pathogens in tissues and eliminated pathogens from the environment, providing a reliable technical solution for the green control of this disease.

[0140] The embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A bacteriophage of Citrobacter freundii (Citrobacter werkmanii bacteriophage) characterized in that, RDP-CI25012, which was deposited with the China General Microbiological Culture Collection Center on August 25, 2025, and has the accession number CGMCC No. 46668.

2. A composition characterized in that: The Citrobacter werkmanii bacteriophage of claim 1.

3. The Citrobacter werkmanii bacteriophage of claim 1 or the composition of claim 2 for use in at least one of the following (1) to (3): (1) preparing a product for killing Citrobacter werkmanii; (2) preparing a product for inhibiting Citrobacter werkmanii; (3) preparing a product for preventing and / or treating Citrobacter werkmanii disease caused by Citrobacter werkmanii.

4. Use according to claim 3, characterized in that: The product is a veterinary preparation or a disinfectant.

5. Use according to claim 4, characterized in that: The veterinary preparation further comprises a pharmaceutically acceptable carrier, and the dosage form is a powder, a solution, an emulsion, a gel, a granule, or a lyophilized agent.

6. Use according to claim 3, characterized in that: The use is for preparing a product for preventing and / or treating Citrobacter werkmanii disease in aquaculture caused by Citrobacter werkmanii.

7. Use according to claim 6, characterized in that: The use is for preparing a product for preventing and / or treating Citrobacter werkmanii disease in Penaeus vannamei caused by Citrobacter werkmanii.

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