Citrobacter freundii phage and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to a citrobacter freundii phage and application thereof. The citrobacter freundii phage is a citrobacter freundii phage CFP7ACFGC5 (Citrobacter freundii phage CFP7ACFGC5), the citrobacter freundii phage CFP7ACFGC5 is preserved in the China Center for Type Culture Collection on July 21, 2025, and the number of the citrobacter freundii phage CFP7ACFGC5 is CCTCC (China Center for Type Culture Collection) NO: M20251640. The bacteriophage has a relatively broad-spectrum host range, and can still keep relatively high activity while splitting target bacteria; meanwhile, the bacteriophage is excellent in acid resistance and thermal stability, can meet the application requirements of multiple temperature nodes in production, and has a good application prospect.

Description

Technical Field

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

[0002] *Citrobacter werkman and Gillen* is a Gram-negative, short rod-shaped bacterium belonging to the genus *Citrobacter* in the family Enterobacteriaceae. This bacterium is flagellated, non-capsulated, and exhibits strong growth capacity, making it a typical opportunistic pathogen causing zoonotic diseases in humans, livestock, and fish. In recent years, with the overuse of antibiotics, this bacterium has developed increasing resistance to multiple antibiotics, posing a serious challenge to clinical treatment and disease control in aquaculture. In humans, *Citrobacter werkman* can cause urinary tract infections and septicemia; in aquatic animals, it can cause septicemia in red swamp crayfish, dropsy in fish, skin rot, and ascites in amphibians, seriously threatening the healthy development of the aquaculture industry.

[0003] Traditional prevention and control methods mainly rely on antibiotics, but long-term use has not only led to the emergence of a large number of drug-resistant strains, but also caused water pollution and ecological imbalance. Therefore, it is urgent to find safe and effective alternatives. Bacteriophages, as a type of virus that specifically infects bacteria, have advantages such as strong host specificity, no disruption of normal flora, and no residual toxicity, making them an important candidate to replace antibiotics.

[0004] Currently, several *Citrobacter freundii* phages have been isolated and identified. For example, *Citrobacter freundii* phage PRF01, disclosed in CN114763539B, belongs to the Myocaudidae family and has a broad lytic spectrum, making it suitable for multidrug-resistant bacterial infections. However, it becomes inactive within 1 hour at pH < 2 and pH > 12, and inactivated within 20 minutes at around 70°C. Another example is *Citrobacter freundii* phage RDP-CF-21001, disclosed in CN115873808B, which exhibits strong lytic ability against *Citrobacter freundii* in water, but becomes inactive within 30 minutes at around 60°C.

[0005] Although some progress has been made in the study of Citrobacter freundii bacteriophage, there is still room for further research on the stability and heat resistance of Citrobacter freundii bacteriophage. Summary of the Invention

[0006] The purpose of this invention is to provide a Citrobacter freundii phage to improve the source diversity, stability and heat resistance of the Citrobacter freundii phage, which has broad application prospects.

[0007] The present invention achieves the above objectives through the following technical solutions:

[0008] First, the present invention provides a Citrobacter freundii phage CFP7ACFGC5, which was deposited at the China Center for Type Culture Collection on July 21, 2025, with the number CCTCC NO: M20251640.

[0009] The morphology of Citrobacter freundii phage CFP7ACFGC5 is a polyhedral symmetry head and a very short tail. The head diameter is 50-60 nm, the tail length is 2-5 nm, and the tail diameter is 2-4 nm. The nucleotide sequence of the Citrobacter freundii phage is shown in SEQ ID No. 1.

[0010] As a further improvement of the present invention, the titer of the *Citrobacter freundii* phage CFP7ACFGC5 reaches 7.1 × 10⁻⁶. 10 When the concentration is PFU / mL, the MOI is 1:1000.

[0011] Second, the present invention provides a phage composition containing Citrobacter freundii phage CFP7ACFGC5.

[0012] As a further improvement of the present invention, the composition includes Klebsiella pneumoniae phage P6AKPJS20.

[0013] Third, the present invention provides an application of the above-mentioned Citrobacter freundii phage CFP7ACFGC5 and the above-mentioned phage composition, the application including any one of the following:

[0014] (1) A disinfectant for preparing Citrobacter freundii lysate;

[0015] (2) Drugs, kits or medical devices used to treat and / or prevent diseases caused by Citrobacter freundii;

[0016] (3) Used to prepare food, feed or additives for the prevention of diseases caused by Citrobacter freundii.

[0017] Fourth, the present invention provides a disinfectant for lysing Citrobacter freundii, the active ingredient of which includes the above-mentioned Citrobacter freundii phage CFP7ACFGC5 or the above-mentioned phage composition.

[0018] Fifth, the present invention provides an application of the above-mentioned disinfectant in environmental disinfection.

[0019] Sixth, the present invention provides a medicament for treating and / or preventing diseases caused by Citrobacter freundii, the active ingredient of which includes the above-mentioned Citrobacter freundii phage CFP7ACFGC5 or the above-mentioned phage composition.

[0020] As a further improvement of the present invention, the concentration of Citrobacter freundii phage CFP7ACFGC5 in the drug is 10. 5 -10 6 At the PFU / mL level, the sterilization rate is >90%.

[0021] As a further improvement of the present invention, the drug also includes a pharmaceutically acceptable carrier, and the form of the drug includes, but is not limited to, application to the surface, mouth, rectum, pleura and other parts of the host being treated by means of carrier delivery, concentrated injection or drug immersion.

[0022] As a further improvement of the present invention, the carrier delivery form includes, but is not limited to, oral aqueous carriers, oral anhydrous carriers, cream preparations, etc.; the concentrated injection form includes, but is not limited to, vaccine injection, intrapleural injection, intravenous injection, etc.; the drug immersion form includes, but is not limited to, aerosols, rinsing agents, etc.

[0023] Seventh, the present invention provides a kit for treating and / or preventing diseases caused by Citrobacter freundii, comprising the above-mentioned Citrobacter freundii phage CFP7ACFGC5 or the above-mentioned phage composition.

[0024] As a further improvement of the present invention, the kit includes, but is not limited to, a biosensor kit.

[0025] Eighth, the present invention provides a food for preventing diseases caused by Citrobacter freundii, comprising the above-mentioned Citrobacter freundii phage CFP7ACFGC5 or the above-mentioned phage composition.

[0026] As a further improvement of the present invention, the food includes foods for special medical purposes, health foods or functional foods, and ordinary foods, including but not limited to solid beverages, liquid beverages, dairy products, biscuits, pastries, canned foods, alcoholic beverages, confectionery products, and frozen drinks.

[0027] Ninth, the present invention provides an aquatic feed containing the above-mentioned Citrobacter freundii phage CFP7ACFGC5 or the above-mentioned phage composition.

[0028] Tenth, the present invention provides a feed additive containing the above-mentioned Citrobacter freundii phage CFP7ACFGC5 or the above-mentioned phage composition.

[0029] Eleventh, the present invention provides a broad-spectrum antibacterial agent, comprising an antibacterial agent, and further comprising the above-mentioned Citrobacter freundii phage CFP7ACFGC5 or the above-mentioned phage composition.

[0030] The beneficial effects of this invention are as follows:

[0031] The Citrobacter freundii phage CFP7ACFGC5 of this invention is a virulent phage isolated from nature. Toxicological experiments have proven that it is safe and has no side effects. The DNA of the test phage does not encode proteins that may cause potential health risks and does not contain virulence genes or harmful genes. This invention does not involve any genetic modification of the test phage.

[0032] The Citrobacter freundii phage CFP7ACFGC5 sample of this invention is readily available and can be highly purified using standard virus purification methods, making it suitable for industrial production. Phage CFP7ACFGC5 can be specifically amplified by the host bacterium and exhibits high toxicity to the host bacterium, with an MOI of 0.001. Only a small amount of initial phage is required for large-scale proliferation, providing a high-quality phage strain source for the industrial production of phage bactericides and demonstrating excellent prospects for widespread application.

[0033] The *Citrobacter freundii* phage CFP7ACFGC5 of this invention exhibits excellent heat resistance; its titer decreases by only two orders of magnitude after treatment at 65°C for 24 hours, and it still retains 10% titer after a 2-hour water bath at 75°C. 4 PFU / mL potency.

[0034] The present invention, Citrobacter freundii phage CFP7ACFGC5, at a concentration of 10 2 -10 3 10 PFU / mL Citrobacter freundii culture medium 5 -10 6 The PFU / mL phage achieved a kill rate of over 90% against this concentration of Citrobacter freundii, demonstrating excellent bactericidal ability against Citrobacter freundii.

[0035] The bacteriophage of the present invention has a strong lytic ability, with a lytic rate of up to 97% against Citrobacter freundii.

[0036] The Citrobacter freundii phage CFP7ACFGC5 and its compositions of the present invention can be prepared by those skilled in the art into biological agents applicable to the prevention and control of diseases caused by, but not limited to, Citrobacter freundii, according to the description of the present invention and common knowledge in the art. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the phage plaque of *Citrobacter freundii* described in this invention;

[0038] Figure 2 This is a schematic diagram of the morphological structure of the *Citrobacter freundii* bacteriophage described in this invention under a transmission electron microscope;

[0039] Figure 3 This is a map showing the distribution of unique endonuclease cleavage sites in the genome of the *Citrobacter freundii* bacteriophage described in this invention.

[0040] Figure 4 This is a phylogenetic tree diagram of the *Citrobacter freundii* bacteriophage described in this invention;

[0041] Figure 5 This is a sequence difference comparison diagram between the *Citrobacter freundii* phage described in this invention and 12 *Citrobacter freundii* phages;

[0042] Figure 6The stability of the *Citrobacter freundii* phage described in this invention under different pH conditions;

[0043] Figure 7 The stability of the *Citrobacter freundii* phage described in this invention before and after preparation under different powder preparation conditions;

[0044] Figure 8 The lytic ability of the *Citrobacter freundii* phage against *Citrobacter freundii* as described in this invention;

[0045] Figure 9 The result of the lysis of non-host pathogenic bacteria by the Citrobacter freundii bacteriophage described in this invention; Figure 9 The horizontal axis represents: A1. Citrobacter koseri, A2. Citrobacter sedlakii, A3. Vibrio parahaemolyticus, A4. Vibrio alginolyticus, A5. Vibrio harveyi, A6. Escherichia coli, A7. Salmonella, A8. Staphylococcus aureus, A9. Pseudomonas aeruginosa.

[0046] Figure 10 The result of the lysis of non-pathogenic beneficial bacteria by the Citrobacter freundii phage described in this invention; Figure 10 The horizontal axis represents B1. Non-pathogenic Rhizobia, B2. Non-pathogenic Bacillus licheniformis, B3. Non-pathogenic Bacillus subtilis, B4. Non-pathogenic Bacillus megaterium de Bary, and B5. Clostridium butyricum.

[0047] Figure 11 This invention describes the protective effect of Citrobacter freundii bacteriophage and its composition on crab larvae. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to experiments and accompanying drawings. It should be noted that the specific embodiments described below are only for further illustration of the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0049] I. Biological Preservation Information

[0050] 1. Citrobacter freundii bacteriophage

[0051] Preservation name: Citrobacter freundii phage CFP7ACFGC5

[0052] Preservation Institution: China Center for Type Culture Collection

[0053] Abbreviation for depository institution: CCTCC

[0054] Address: School of Life Sciences, Wuhan University

[0055] Deposit date: July 21, 2025

[0056] Registration number at the Preservation Center: CCTCC NO: M20251640.

[0057] 2. Klebsiella pneumoniae bacteriophage;

[0058] Preservation name: Klebsiella pneumoniae phage P6AKPJS20

[0059] Preservation Institution: China Center for Type Culture Collection

[0060] Abbreviation for depository institution: CCTCC

[0061] Address: School of Life Sciences, Wuhan University

[0062] Deposit date: December 31, 2024

[0063] Registration number at the Preservation Center: CCTCC NO: M20242965.

[0064] II. Explanation

[0065] The following appears in this invention:

[0066] The formula for LB liquid culture medium is: 10g tryptone, 5g yeast extract, 10g sodium chloride, 1000mL distilled water, pH 7.0.

[0067] The formula for LB solid medium is: 10g tryptone, 5g yeast extract, 10g sodium chloride, 15g agar, 1000mL distilled water, pH 7.0.

[0068] The semi-solid agar medium formula is as follows: 10g tryptone, 5g yeast extract, 10g sodium chloride, 7g agar, 1000mL distilled water, pH 7.0.

[0069] The SM buffer solution is formulated as follows: 5.8g sodium chloride, 2g magnesium sulfate, 50mL 1mol / L Tris-HCl, 0.25g gelatin, and 1000mL distilled water.

[0070] Unless otherwise specified, all methods used in this invention are conventional methods known to those skilled in the art, and all reagents and materials used are commercially available products, and all instruments used are conventional instruments known to those skilled in the art.

[0071] III. Methods

[0072] 3.1 Phage screening and purification

[0073] (1) Sample collection and processing

[0074] The samples used in this invention were collected from urban sewage in Nanjing, Jiangsu Province.

[0075] The collected samples were centrifuged at 5000 r / min for 10 min and then passed through a 0.22 μm filter membrane.

[0076] (2) Citrobacter freundii strain ACFGC5 was isolated from urban sewage in Nanjing, Jiangsu Province.

[0077] (3) Enrichment of phages targeting Citrobacter freundii in the sample

[0078] Take 10 mL of the above filtrate and mix it with 2 times LB liquid medium at a ratio of 1:1. At the same time, add 1 mL of the logarithmic phase bacterial culture of the phage host bacterium Citrobacter freundii strain ACFGC5 and enrich overnight at 30°C.

[0079] (4) Phage screening and purification

[0080] Centrifuge the above enrichment solution, collect the supernatant, and pass it through a 0.22 μm membrane. Take 0.5 mL of the logarithmic growth phase phage host bacteria suspension, add it to 5 mL of LB medium at 48 °C, mix well, and pour onto an LB plate to prepare a double-layer plate containing the host bacteria. Take 10 μL of the filtered supernatant and drop it onto the solidified double-layer plate. After air-drying under aseptic conditions, incubate overnight at 30 °C. Observe whether phage droplets form the next day and record the experimental results.

[0081] Pick the above-mentioned plaques and place them in 1 mL of SM buffer. Shake at 150 rpm for 15 min, then perform serial dilutions. Add each dilution to 0.5 mL of logarithmic-phase phage host bacterial suspension, mix thoroughly, and then add to 5 mL of LB semi-solid medium and mix again. Pour the mixture onto petri dishes containing LB solid medium. After the semi-solid medium solidifies, incubate overnight at 30°C. Repeat this step 3-5 times to finally obtain single phage plaques of uniform morphology and size on a double-layer plate. Figure 1 The purified phage, named *Citrobacter freundii* phage CFP7ACFGC5, was deposited at the China Center for Type Culture Collection (CCTCC) on July 21, 2025, with the accession number CCTCC NO: M20251640. A single plaque of phage CFP7ACFGC5 was picked and placed in 50 mL of LB medium containing 1 mL of logarithmic-phase host bacterial culture, and incubated at 30°C for 8 h. The culture was centrifuged at 8000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane to obtain the pure culture medium of *Citrobacter freundii* phage CFP7ACFGC5.

[0082] The morphology of Citrobacter freundii phage CFP7ACFGC5 under a transmission electron microscope is as follows: Figure 2 As shown, the morphology is a polyhedral symmetrical head and an extremely short tail. The head diameter is 50-60 nm, the tail length is 2-5 nm, and the tail diameter is 2-4 nm. Figure 2 The head diameter of Citrobacter freundii phage CFP7ACFGC5 is 57 nm, the tail length is 3 nm, and the tail diameter is 2 nm.

[0083] (5) Preparation of bacteriophage particles

[0084] The host bacteriophage CFP7ACFGC5 of *Citrobacter freundii* was amplified using the host bacterium. After the culture medium clarified, it was centrifuged at 8000g for 10 min to remove impurities. Solid polyethylene glycol (PEG8000) was added to a final concentration of 10% (w / v), stirred to dissolve, and incubated overnight at 4°C. The mixture was then centrifuged at 8000g for 20 min at 4°C, and the precipitate was resuspended in SM buffer. The resulting phage suspension was stored at 4°C for later use.

[0085] 3.2 Preparation of Citrobacter freundii phage particles and extraction, sequencing and whole-genome analysis of the genome

[0086] (1) Particle preparation and genome extraction and sequencing of Citrobacter freundii bacteriophage

[0087] Take 100 mL of pure culture medium of Citrobacter freundii phage CFP7ACFGC5 prepared in section 3.1, add DNase I and RNase A to a final concentration of 1 μg / mL, incubate at 37℃ for 60 min, then add 5.84 g NaCl (final concentration 1 mol / L), dissolve, and place in an ice bath for 1 h. Centrifuge at 11000 rpm for 10 min at 4℃, and transfer the supernatant to a new centrifuge tube. Add solid polyethylene glycol (PEG8000) to a final concentration of 10% (w / v), and after complete dissolution, incubate on ice for at least 1 h. Centrifuge at 11000 rpm for 20 min at 4℃, resuspend the precipitate with a small amount of SM solution to obtain the concentrated phage particle solution, which can be stored at 4℃ for later use.

[0088] Phage nucleic acid was extracted and sequenced using a λ phage genomic DNA kit. The nucleotide sequence of the Citrobacter freundii phage CFP7ACFGC5 is shown in SEQ ID No. 1 after nucleotide sequencing.

[0089] Sequencing of the Citrobacter freundii phage CFP7ACFGC5 revealed that the phage genome is a linear double-stranded DNA with a total length of 39598 bp and a G+C content of 45.5%.

[0090] (2) Biological characteristics of genomic restriction sites of Citrobacter freundii bacteriophage

[0091] The unique endonuclease species and sites of the whole genome of *Citrobacter freundii* phage CFP7ACFGC5 were analyzed using SnapGene software. The distribution of unique endonuclease cleavage sites in this genome is as follows: Figure 3 As shown.

[0092] (3) Genetic evolution analysis of Citrobacter freundii bacteriophage

[0093] Phylogenetic tree analysis was performed using the genome of bacteriophage CFP7ACFGC5 in NCBI. Figure 4 It can be seen that the phage closest in genetic distance to CFP7ACFGC5 is the Citrobacter phage vB_CfrS_K1M (PP537791.1); the genome of phage CFP7ACFGC5 was compared with the sequences of 12 Citrobacter phages in NCBI for differential diagnosis. Figure 5The genome of bacteriophage CFP7ACFGC5 showed significant differences from the sequences of 12 bacteriophage strains.

[0094] Based on the unique genomic information of the phage CFP7ACFGC5 and its morphological characteristics under transmission electron microscopy, and according to the classification criteria established by the International Committee on Taxonomy of Viruses (ICTV), the phage CFP7ACFGC5 is a strain of Citrobacter freundii phage, belonging to the class Caudoviricetes. Furthermore, this phage is highly likely to be a novel phage.

[0095] 3.3 Detection test for deletion of virulence genes or adverse genes in Citrobacter freundii bacteriophage

[0096] In this embodiment, 103 virulence genes identified as originating from lysogenic bacteriophages within pathogenic bacteria were selected, as shown in Table 1. The whole genome of *Citrobacter freundii* phage CFP7ACFGC5 was determined and subjected to bioinformatics analysis to ascertain whether it contains the following virulence genes. The results showed that *Citrobacter freundii* phage CFP7ACFGC5 does not contain the following virulence genes or harmful genes, and therefore cannot encode proteins that may pose potential health risks. Therefore, *Citrobacter freundii* phage CFP7ACFGC5 does not affect the health of humans or animals.

[0097] Table 1. Major known virulence genes of lysogenic bacteriophages within pathogenic bacteria.

[0098]

[0099] 3.4 Determination of the optimal multiple of infection (MOI) of Citrobacter freundii bacteriophage against Citrobacter freundii

[0100] (1) Determination of the phage titer of Citrobacter freundii

[0101] Using SM solution as a diluent, the stock solution of Citrobacter freundii phage CFP7ACFGC5 (prepared from 3.1) was serially diluted 10-fold to 10-fold. 8 Times. Take l0 respectively. 5 l0 6 l0 7 and l0 8Mix 1000 μL of the diluted phage culture with 300 μL of the host bacterial culture, and let stand for 15 min to allow for full binding to the receptors on the bacterial surface. Add the mixture to 5 mL of semi-solid agar medium cooled to 50°C, mix well, and immediately spread onto solidified agar plates. After the agar has solidified, incubate upside down at 30°C for 6-8 h. Three replicates are required for each dilution, and the average of the three replicates for each dilution is used for counting. The phage titer (PFU / mL) is calculated as: average number of plaques × dilution factor.

[0102] (2) Determination of MOI of Citrobacter freundii bacteriophage

[0103] A single colony of *Citrobacter freundii* was picked and inoculated into a test tube containing 3 mL of LB broth. The culture was incubated at 30°C and 150 rpm for 8 hours with shaking to obtain a host bacterial suspension. The bacterial suspension was transferred to 10 mL of LB broth at a 1:100 ratio and incubated at 30°C and 150 rpm until the early logarithmic growth phase. Pure culture medium of *Citrobacter freundii* phage CFP7ACFGC5 (prepared from step 3.1) and host bacteria (MOI = phage count / bacterial count) were added according to the multiplicity of infection ratio. LB broth was added to ensure the total volume of each tube was the same. The tubes were incubated at 30°C and 150 rpm for 8 hours with shaking. After incubation, the tubes were centrifuged at 5000g for 10 minutes, and the supernatant was collected to determine the phage titer. Double replicates were performed at each point, and the average value was taken. The MOI that produced the highest phage titer was considered the optimal multiplicity of infection. The experiment was repeated three times.

[0104] Table 2. Titer of Citrobacter freundii phage CFP7ACFGC5 at different multiplicity of infection.

[0105]

[0106] The results are shown in Table 2. The highest titer (7.1 × 10⁻⁶) was achieved by Citrobacter freundii phage CFP7ACFGC5. 10 When the concentration is PFU / mL, its MOI is 1:1000. It requires a small initial input, reproduces rapidly, and can be applied in industrial production.

[0107] 3.5 Toxicological Experiments

[0108] Eighty healthy, SPF (Specific Pathogen Free) zebrafish, approximately 3-4 cm in length, were randomly divided into two groups (phage group and control group) after a 3-day acclimatization period, with 40 fish in each group and a stocking density of 20 fish / 50L. The phage group was supplemented with a final concentration of 1×10⁻⁶. 8The zebrafish were diluted with sterile physiological saline to obtain pure culture medium of Citrobacter freundii phage CFP7ACFGC5 (prepared in 3.4) at a concentration of PFU / mL. An equal volume of sterile physiological saline was added to the control group. Each group was fed twice daily, with a 20% water change per day. After each water change, the CFP7ACFGC5 filtrate and sterile physiological saline were added proportionally to maintain the final concentration. The zebrafish were fed continuously for 15 days, and their condition was observed and recorded.

[0109] One hundred and eighty healthy, SPF (Specific Pathogen Free) Litopenaeus vannamei shrimp, each weighing approximately 1g, were randomly divided into two groups (phage group and control group) after 3 days of acclimatization, with 90 shrimp in each group and a stocking density of 30 shrimp / 50L. The phage group was supplemented with a final concentration of 1×10⁻⁶. 8 The control group received a sterile saline solution containing a pure culture medium of Citrobacter freundii phage CFP7ACFGC5 (prepared at 3.4 PFU / mL). The control group received an equal volume of sterile saline solution to the CFP7ACFGC5 phage. Each group was fed twice daily, with a 20% water change per day. After each water change, the CFP7ACFGC5 filtrate and sterile saline solution were added proportionally to maintain the final concentration. The group was fed continuously for 15 days, and the condition of the Litopenaeus vannamei was observed and recorded.

[0110] Twenty healthy, age- and weight-matched SPF (Specific Pathogen Free) mice (half male and half female) were randomly divided into two groups (phage group and control group) after three days of acclimatization, with 10 mice in each group (5 males and 5 females). The phage group received a tail vein injection of 100 μL (1×10⁻⁶) of sterile physiological saline diluted with pure Citrobacter freundii phage CFP7ACFGC5 culture medium (prepared at 3.4°C). 10 The mice were administered sterile saline solution via tail vein injection (PFU / mL). The control group was also treated with this solution. Food intake, activity levels, body temperature, weight changes, and survival were observed. Ten days later, the mice were euthanized by neck dislocation, and their internal organs were examined.

[0111] Forty healthy, age- and weight-matched SPF (Specific Pathogen Free) mice (half male and half female) were randomly divided into four groups (phage group 1, phage group 2, phage group 3, and control group) after three days of acclimatization, with 10 mice in each group (5 males and 5 females). Phage group 1, phage group 2, and phage group 3 were administered oral 0.2 mL / mouse, 0.5 mL / mouse, and 1 mL / mouse, respectively, with a titer of 3 × 10⁻⁶. 9 The mice were given a pure culture medium of Citrobacter freundii phage CFP7ACFGC5 (prepared at 3.4) at a concentration of PFU / mL. The control group was given an equal volume of physiological saline. The mice were observed for 15 consecutive days. The mice were then euthanized by cervical dislocation and their internal organs were examined.

[0112] The results showed that, in all the above treatment groups, immersion in high concentrations of Citrobacter freundii phage CFP7ACFGC5 had no effect on the survival of zebrafish and Litopenaeus vannamei, with no morbidity or mortality. Intravenous injection of a large dose of Citrobacter freundii phage CFP7ACFGC5 into the tail vein had no effect on the daily behavior of mice; during the experiment, mice did not exhibit any clinical symptoms such as lethargy, rough coat, decreased appetite, weight loss, or death, and no abnormalities were found in tissues or internal organs upon anatomical examination. Oral administration of different doses of Citrobacter freundii phage had no effect on the daily behavior of mice, with no symptoms such as depression, fever, vomiting, or diarrhea. No abnormalities were found in the internal organs of mice in any of the treatments upon anatomical examination. The Citrobacter freundii phage CFP7ACFGC5 of this invention is biosafety and can be used as a feed additive. Of course, Citrobacter freundii phage CFP7ACFGC5 can also be used as a health product or a pharmaceutical, and this toxicology experiment has also demonstrated that it is equally safe.

[0113] 3.6 Thermal stability test of Citrobacter freundii phage CFP7ACFGC5

[0114] Pure phage culture (prepared in 3.4) was aliquoted into sterile EP tubes and incubated in water baths at 55℃, 65℃, and 75℃ for 2h, 24h, and 48h, respectively. After the incubation period, the sample tubes were removed and immediately placed in an ice bath to cool. After appropriate dilution, the phage titer was determined using the double-layer plate method. The experiment was repeated three times.

[0115]

[0116] The results are shown in Table 3. In the experimental group, the activity of *Citrobacter freundii* phage CFP7ACFGC5 was relatively stable at 55℃. *Citrobacter freundii* phage CFP7ACFGC5 exhibited good tolerance to high temperatures; compared to the control, its titer decreased by only two orders of magnitude after treatment at 65℃ for 24 hours, and it still retained 10% titer after a 2-hour water bath at 75℃. 4 PFU / mL potency.

[0117] 3.7 pH stability test of Citrobacter freundii phage CFP7ACFGC5

[0118] The valence is 7.1 × 10 8PFU / mL Citrobacter freundii phage CFP7ACFGC5 samples (prepared in 3.4) were adjusted to pH 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, and 14.0, respectively, and the titers were measured after 1 h, 4 h, 8 h, 24 h, and 96 h.

[0119] The test results are as follows Figure 6 As shown, Citrobacter freundiiphage CFP7ACFGC5 retained a titer of 300 PFU / mL after treatment at pH 2.0 for 24 h, and maintained a stable high titer after treatment at pH 4.0-9.0 for 96 h and at pH 4.0-10.0 for 4 h. Citrobacter freundiiphage CFP7ACFGC5 exhibits good acid and alkali tolerance, especially acid tolerance.

[0120] 3.8 Bioactivity Tests of Citrobacter freundii Bacteriophage Spray Powder and Lyophilized Powder

[0121] (1) Bioactivity test of Citrobacter freundii phage spray powder

[0122] Dissolve 2% starch (w / v) and 4% sorbitol (w / v) in a solution with a potency of 2.35 × 10⁻⁶. 10 Phage was coated by incubating PFU / mL *Citrobacter freundii* phage CFP7ACFGC5 pure culture medium (prepared in 3.4) at 30℃ and 180 r / min for 2 h. The coated phage solution was then sprayed onto defatted rice bran carrier at a ratio of 1:4 (w / w), stirred to ensure uniform mixing, dried at 50℃ for 2 h (humidity <10%), and passed through a 40-mesh sieve. After preparation, samples were taken and the phage titer was determined using the double-layer plate method, with the experiment repeated three times. The phage spray powder was stored at room temperature.

[0123] (2) Bioactivity test of lyophilized powder of Citrobacter freundii bacteriophage

[0124] Dissolve 2% starch (w / v) and 4% sorbitol (w / v) in a solution with a potency of 2.35 × 10⁻⁶. 10Phage was coated by incubating PFU / mL *Citrobacter freundii* phage CFP7ACFGC5 pure culture medium (prepared in 3.4) at 30℃ and 180 r / min for 2 h. The coated phage solution was then added to a material tray and freeze-dried under vacuum for 50±2 h, followed by pulverization through a 40-mesh sieve. The moisture content of the freeze-dried powder was <10%. After preparation, samples were taken and the phage titer was determined using the double-layer plate method, with the experiment repeated three times. The freeze-dried phage powder was stored at room temperature.

[0125] The results are as follows Figure 7 As shown, there was no significant difference in titer between the Citrobacter freundii phage CFP7ACFGC5 before and after preparation as a spray powder and a lyophilized powder, indicating that the phage has good tolerance and stability to the preparation conditions of both spray powder and lyophilized powder.

[0126] 3.9 Lysis experiment of Citrobacter freundii bacteriophage on Citrobacter freundii

[0127] The lysis profile of bacteriophages was determined using the double-layer plate drop method. A total of 153 single colonies of *Citrobacter freundii* from different isolation sources were selected and inoculated into test tubes containing 3 mL of LB liquid medium. The cultures were incubated at 30°C for 4 h to obtain bacterial suspensions for each strain. 300 μL of the bacterial suspension was mixed with LB semi-solid medium and plated onto ordinary agar plates. 5 μL of the suspension with a titer of 6 × 10⁻⁶ was then used as the plate sample. 7 PFU / mL pure culture medium of Citrobacter freundii phage (prepared from 3.4) was dropped onto each plate, and after air drying, it was incubated at 30℃ for 8 hours. The results were then observed.

[0128] The results are as follows Figure 8 As shown, the Citrobacter freundii phage described in this invention has a wide host range and can recognize Citrobacter freundii isolated from different animals. Its lysis rate is above 97%, and it can be well applied in the preparation of biocides for lysing Citrobacter freundii.

[0129] 3.10 Lysis test of Citrobacter freundii phage against non-host pathogenic bacteria

[0130] The lysis spectrum of bacteriophages was determined using the double-layer plate drop method.

[0131] Seventy-seven single colonies of non-host pathogenic bacteria were collected, including 5 strains of *Citrobacter koseri*, 4 strains of *Citrobacter sedlakii*, 10 strains of *Vibrio parahemolyticus*, 11 strains of *Vibrio alginolyticus*, 6 strains of *Vibrio harveyi*, 8 strains of *Escherichia coli*, 10 strains of *Salmonella sp.*, 12 strains of *Staphylococcus aureus*, and 11 strains of *Pseudomonas aeruginosa*. Each of these tested strains was inoculated into test tubes containing 3 mL of TSB and cultured under optimal conditions until the logarithmic growth phase to obtain bacterial suspensions. Take 300 μL of bacterial suspension and mix it separately with semi-solid culture medium, then spread it on ordinary agar plates. Take 5 μL of the culture medium with a titer of 6 × 10⁻⁶. 7 PFU / mL of pure phage culture of *Citrobacter freundii* (prepared from 3.4) was dropped onto each plate. After air drying, the plates were incubated overnight under the optimal culture conditions for each tested bacterium. The results were observed as follows: Figure 9 As shown.

[0132] Depend on Figure 9 It is evident that the *Citrobacter freundii* bacteriophage described in this invention cannot recognize the tested non-host pathogenic bacteria. This indicates that the tested bacteriophage possesses extremely high specificity.

[0133] 3.11 Lysis test of Citrobacter freundii phage against non-pathogenic beneficial bacteria

[0134] Sixty-one single colonies of non-pathogenic beneficial bacteria were selected, including 13 non-pathogenic rhizobia, 14 non-pathogenic Bacillus licheniformis, 15 non-pathogenic Bacillus subtilis, 11 non-pathogenic Bacillus megaterium, and 8 Bacillus coagulans. Each of these tested strains was inoculated into test tubes containing 3 mL of TSB and cultured to the logarithmic growth phase under optimal conditions to obtain bacterial suspensions. 300 μL of the bacterial suspension was mixed with semi-solid culture medium and plated onto ordinary agar plates. 5 μL of the suspension with a titer of 6 × 10⁻⁶ was then collected. 7 PFU / mL of pure phage culture of *Citrobacter freundii* (prepared from 3.4) was dropped onto each plate. The plates were incubated overnight under the optimal culture conditions for each tested bacterium, and the results were observed. The results are as follows: Figure 10 As shown.

[0135] Depend on Figure 10It is evident that the *Citrobacter freundii* bacteriophage described in this invention failed to recognize any of the 61 tested non-pathogenic beneficial bacteria strains. This indicates that the tested bacteriophages did not damage the beneficial bacterial microbial community.

[0136] 3.12 Preparation of the composition of Citrobacter freundii bacteriophage CFP7ACFGC5

[0137] The potency is 5×10 9 Pure culture medium of Citrobacter freundii phage CFP7ACFGC5 (prepared in 3.4) and pure culture medium of Klebsiella pneumoniae phage P6AKPJS20 were prepared by uniformly mixing equal volumes of the two phages to prepare a 1:1 composition of Citrobacter freundii phage CFP7ACFGC5 and Klebsiella pneumoniae phage P6AKPJS20 (Composition 1).

[0138] The potency is 5×10 9 A pure culture medium of PFU / mL Citrobacter freundii phage CFP7ACFGC5 (prepared in 3.4) and an aqueous solution of hydrogen peroxide with a final concentration of 200 mg / L were mixed in equal volumes at a 1:1 ratio to prepare a composition (composition 2).

[0139] 3.13 Bactericidal effect of Citrobacter freundii bacteriophage CFP7ACFGC5 and its composition in liquids

[0140] (1) Citrobacter freundii counting method

[0141] LB solid medium was used, and the colony count was performed by dilution and spread. The culture was carried out at 30°C for 24 hours, and the number of colonies was counted.

[0142] (2) Experiment on the bactericidal effect of Citrobacter freundii bacteriophage CFP7ACFGC5 in liquid

[0143] Cultivate *Citrobacter freundii* ACFGC5 to the logarithmic growth phase, aliquot the culture into different test tubes, and dilute the bacterial culture with an equal volume of liquid LB medium until the final concentration of *Citrobacter freundii* ACFGC5 is 3.8 × 10⁻⁶. 3CFU / mL, inoculated with different concentrations of pure Citrobacter freundii phage CFP7ACFGC5 culture medium (prepared at 3.4), so that the concentration of Citrobacter freundii phage CFP7ACFGC5 in the samples was 5×10⁻⁶. 2 PFU / mL, 5×10 3 PFU / mL, 5×10 4 PFU / mL, 5×10 5 PFU / mL, 5×10 6 PFU / mL. A control group and a blank group (CK) were also set up. The control group was given a final concentration of 3.8 × 10⁻⁶ PFU / mL. 3 The control group received CFU / mL of Citrobacter freundii; the control group received an equal volume of physiological saline. The residual amount of Citrobacter freundii was measured after 4 hours. Results are shown in Table 4.

[0144]

[0145] Table 4 shows that the concentration of Citrobacter freundii phage CFP7ACFGC5 was 10. 5 -10 6 At a concentration of PFU / mL, C. freundii phage CFP7ACFGC5 showed the best bactericidal effect against C. freundii in liquid LB medium, with a kill rate exceeding 90%. This indicates that C. freundii phage CFP7ACFGC5 has the potential to be used as a biocide.

[0146] (3) Experiment on the bactericidal effect of the composition containing high concentration of Citrobacter freundii bacteriophage CFP7ACFGC5 in liquid.

[0147] The preparation method of the Citrobacter freundii phage CFP7ACFGC5 composition is described in 3.12.

[0148] Cultivate *Citrobacter freundii* ACFGC5 to the logarithmic growth phase, aliquot into different test tubes, and dilute the bacterial culture with liquid LB medium to a final concentration of 3.8 × 10⁻⁶. 3 CFU / mL, respectively inoculated with a final concentration of 5×10 6 Compositions 1 and 2 were prepared using *Citrobacter freundii* phage CFP7ACFGC5 at a concentration of PFU / mL. A control group and a blank group (CK) were established. The control group was given a final concentration of 3.8 × 10⁻⁶ PFU / mL. 3 The control group received CFU / mL of Citrobacter freundii; an equal volume of physiological saline was administered. The residual amount of Citrobacter freundii was measured after 4 hours, using the method described in step 1 of this embodiment. Results are shown in Table 5.

[0149]

[0150] Table 5 shows the test results: the concentration of Citrobacter freundii phage CFP7ACFGC5 was a high concentration of 5×10⁻⁶. 6 At PFU / mL, compositions containing it not only exhibit good bactericidal effects but also show no antagonistic effect on other components. Compositions containing Citrobacter freundii phage CFP7ACFGC5 have the potential to be used as biocides.

[0151] 3.14 Protective effect of Citrobacter freundii bacteriophage CFP7ACFGC5 and its composition on crab larvae

[0152] Healthy crab larvae weighing approximately 1-2g were selected and acclimatized for 7 days. They were then divided into 5 groups (single phage CFP7ACFGC5 group, composition 1, composition 2, control group, and blank group), with 100 crabs in each group. A final concentration of 3.8 × 10⁻⁶ was added to the water in each of the experimental groups (single phage CFP7ACFGC5 group, composition 1, and composition 2) and the control group. 5 Citrobacter freundii CFU was added to the CFP7ACFGC5 group after 3 hours, with a final concentration of 5 × 10⁻⁶. 5 Pure culture medium of phage CFP7ACFGC5 (prepared at 3.4°C) at PFU / mL was added to composition group 1 to achieve a final phage concentration of 5 × 10⁻⁶. 5 Composition 1 (prepared in 3.12) with PFU / mL was added to composition 2, with a final phage concentration of 5 × 10⁻⁶. 5 Composition 2 (prepared in 3.12) with PFU / mL and a final hydrogen peroxide concentration of 200 mg / L was used; the control group was replaced with an equal volume of sterile SM solution. The water in the blank group was not treated in any way. Each group was fed twice a day, and the water was changed on the 5th day of the experiment, with a 20% water change volume. After the water change, the corresponding phage or composition filtrate and sterile SM solution were added to each group in proportion to maintain the final concentration. The feeding was continued for 10 days, and the condition of the crab larvae was observed and recorded.

[0153] The results are as follows Figure 11 As shown: By day 10 of the experiment, 2 crab larvae in the blank group died; only 12 crab larvae survived in the control group, with a mortality rate of 88%; the number of surviving crab larvae in the single phage CFP7ACFGC5 group, combination 1 and combination 2 groups were all above 73, and their mortality rates were all below 27%. Among them, the number of surviving crab larvae in the combination 1 treatment was the highest, at 79, with a survival rate of 79%.

[0154] This indicates that Citrobacter freundii bacteriophage CFP7ACFGC5 and its composition have a significant protective effect on crab larvae and can be used as a biological bactericide to effectively kill Citrobacter freundii in aquaculture water and protect aquaculture animals, thereby improving their survival rate.

[0155] 3.15 Preparation and use of the kit for Citrobacter freundii phage CFP7ACFGC5

[0156] The kit contains 5-10 mL of a potency of 1×10⁻⁶. 9 Citrobacter freundii phage CFP7ACFGC5, 1 L LB semi-solid medium, 1 L LB solid medium.

[0157] The method of using the Citrobacter freundii phage CFP7ACFGC5 kit is as follows: Take a titer of 1×10⁻⁶. 9 PFU / mL of *Citrobacter freundii* phage CFP7ACFGC5 was used to determine the lysis profile of the test phage using a double-layer plate drop method. A single colony of the test strain was picked and inoculated into the target liquid medium. The culture was then incubated with shaking at the target temperature, taking into account the growth characteristics of the test strain, to prepare the test strain suspension. 300 μL of the test strain suspension was mixed with 5 mL of LB semi-solid medium and plated onto LB solid plates. 10 μL of *Citrobacter freundii* phage CFP7ACFGC5 was then dropped onto the plate. After air drying, the plates were incubated at the target temperature according to the growth characteristics of the test strain. The results were then observed to determine whether the target strain was present.

[0158] The biosensor kit contains 20-50 mL of material with a potency of 1×10⁻⁶. 8 The phage of Citrobacter freundii CFP7ACFGC5.

[0159] The method for using the Citrobacter freundii phage CFP7ACFGC5 biosensor kit is as follows: Take the sample to be tested, add sterile physiological saline to just cover the sample, and prepare a homogenate. The titer is 1×10⁻⁶. 8 PFU / mL of Citrobacter freundii phage CFP7ACFGC5 liquid was used to immobilize the phage on the surface of a magnetoelastic sensor and incubated in a humid environment at 30°C for 30 min. The magnetoelastic sensor was then placed in the sample to be tested and left to stand at the target temperature for bacterial growth for 30 min. The resonant frequency shift before and after sensor detection was measured by an electromagnetic coil, thus obtaining the number or concentration of target bacteria in the sample.

[0160] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A Citrobacter freundii phage CFP7ACFGC5, characterized in that, The Citrobacter freundii phage CFP7ACFGC5 was deposited at the China Center for Type Culture Collection on July 21, 2025, with the accession number CCTCC NO: M20251640.

2. The Citrobacter freundiiphage CFP7ACFGC5 according to claim 1, characterized in that, When the titer of the Citrobacter freundii phage CFP7ACFGC5 reaches 7.1×10¹⁰ PFU / mL, its MOI is 1:1000.

3. A bacteriophage composition, characterized in that, Contains Citrobacter freundii phage CFP7ACFGC5 as described in any one of claims 1-2.

4. The phage composition according to claim 3, characterized in that, The composition includes Klebsiella pneumoniae phage P6AKPJS20.

5. The application of the Citrobacter freundii phage CFP7ACFGC5 as described in any one of claims 1-2 and the phage composition as described in any one of claims 3-4, characterized in that, The application includes any of the following: (1) Used to prepare disinfectant and bactericide for lysing Citrobacter freundii; (2) Used to prepare medicines, kits or medical devices for the treatment and / or prevention of diseases caused by Citrobacter freundii; (3) Used to prepare food, feed or additives for the prevention of diseases caused by Citrobacter freundii.

6. A disinfectant for lysing Citrobacter freundii, characterized in that, Its active ingredients include Citrobacter freundii phage CFP7ACFGC5 as described in any one of claims 1-2 or the phage composition as described in any one of claims 3-4.

7. The application of the disinfectant and bactericide according to claim 6 in environmental disinfection.

8. A medicine for treating and / or preventing diseases caused by Citrobacter freundii, characterized in that, Its active ingredients include Citrobacter freundii phage CFP7ACFGC5 as described in any one of claims 1-2 or the phage composition as described in any one of claims 3-4.

9. The medicament according to claim 8, characterized in that, When the concentration of Citrobacter freundii phage CFP7ACFGC5 in the drug is in the range of 10⁵-10⁶ PFU / mL, the bactericidal rate is >90%.

10. The medicament according to claim 8, characterized in that, The drug also includes a pharmaceutically acceptable carrier, and the form of the drug includes being carried by a carrier, concentrated for injection, or immersed in a drug solution.

11. A kit for treating and / or preventing diseases caused by Citrobacter freundii, characterized in that, The kit includes, but is not limited to, the Citrobacter freundii phage CFP7ACFGC5 as described in any one of claims 1-2 or the phage composition as described in any one of claims 3-4, and the kit includes, but is not limited to, a biosensor kit.

12. A food product for preventing diseases caused by Citrobacter freundii, characterized in that, Includes the Citrobacter freundii phage CFP7ACFGC5 as described in any one of claims 1-2 or the phage composition as described in any one of claims 3-4.

13. An aquatic feed, characterized in that, The feed contains Citrobacter freundii phage CFP7ACFGC5 as described in any one of claims 1-2 or the phage composition as described in any one of claims 3-4.

14. A feed additive, characterized in that, The feed additive contains Citrobacter freundii phage CFP7ACFGC5 as described in any one of claims 1-2 or the phage composition as described in any one of claims 3-4.

15. A broad-spectrum antibacterial preparation, comprising an antibacterial agent, characterized in that, It also includes the Citrobacter freundii phage CFP7ACFGC5 as described in any one of claims 1-2 or the phage composition as described in any one of claims 3-4.

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