Cronobacter sakazakii phage csp-tjdf01 and applications thereof

Abstract

The application relates to the field of biotechnology, and discloses a Cronobacter sakazakii phage Csp-TJDF01 and application thereof. The phage has been preserved in the China Center for Type Culture Collection (CCTCC) on November 20, 2025, the preservation address is Wuhan University, Wuhan, China, the preservation number is CCTCC NO: M 20252610, the full length of the genome is 143081 bp, and the phage does not carry virulence and drug resistance genes. The phage has high efficient lytic activity on the Cronobacter sakazakii, can significantly inhibit the formation of a biofilm of the Cronobacter sakazakii on a solid surface, and can effectively inhibit bacterial growth in a food matrix such as infant formula milk powder. The phage has high safety, is suitable for preparing bacteriostatic agents or killing agents, and is used for pathogenic bacteria control in medical and food environments.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to a Cronobacter akazakii bacteriophage Csp-TJDF01 and its application in the preparation of antibacterial agents, disinfectants, biological control agents and drugs. Background Technology

[0002] *Cronobacter sakazakii* is a Gram-negative, facultative anaerobic, opportunistic pathogen belonging to the genus *Cronobacter* in the family Enterobacteriaceae. This bacterium can survive in a variety of environments, including in low-moisture-activity foods such as dried infant formula. *Cronobacter sakazakii* infection can cause serious illnesses in newborns and infants, including necrotizing enterocolitis, meningitis, and sepsis, with a mortality rate as high as 40%–80%, making it a significant foodborne pathogen threatening the health of infants and young children.

[0003] Powdered infant formula (PIF) is a primary carrier of Cronobacter sakazakii contamination. Because this bacterium is highly resistant to dry environments, it can survive and multiply during the production, storage, and preparation of infant formula. Furthermore, Cronobacter sakazakii can form biofilms on solid surfaces such as medical devices and food processing equipment, increasing the difficulty of removal and posing a serious challenge to food and medical safety.

[0004] Currently, control strategies for Cronobacter sakazakii contamination mainly include chemical disinfectants, heat treatment, and antibiotics. However, chemical disinfectants may leave residues and affect product quality; heat treatment has limited effectiveness on contaminated dried foods; and the overuse of antibiotics may lead to the emergence and spread of drug-resistant strains. Therefore, there is an urgent need to develop new, safe, efficient, and highly specific antimicrobial strategies.

[0005] Bacteriophages are a class of viruses that obligately infect bacteria. They possess advantages such as strong host specificity, self-replication ability, no impact on normal flora, and low likelihood of developing drug resistance, making them an ideal alternative or supplement to traditional antibacterial methods. In recent years, research on the application of bacteriophages in the field of food safety has been increasing, and several commercially available bacteriophage preparations have been approved for use in food preservation and pathogen control.

[0006] However, phage resources for *Cronobacter sakazakii* remain scarce. The number of reported *Cronobacter sakazakii* phages is limited, and most phages have narrow host ranges and low lysis efficiency, making them insufficient for practical applications. Therefore, isolating and identifying novel, highly efficient *Cronobacter sakazakii* phages and developing their applications in food safety, healthcare, and other fields has significant theoretical and practical value. Summary of the Invention

[0007] The purpose of this application is to provide a Cronobacter sakazakii bacteriophage Csp-TJDF01 and its application to solve the problems mentioned in the background art.

[0008] This application discloses a Cronobacter sakazakii bacteriophage Csp-TJDF01, which was deposited on November 20, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20252610.

[0009] In a preferred embodiment, the bacteriophage Csp-TJDF01 is a double-stranded DNA bacteriophage with a full-length genome of 143081 bp and a GC content of 46.60%.

[0010] This application provides the use of the bacteriophage Csp-TJDF01 described above in the preparation of antibacterial agents, disinfectants, preservatives or biocontrol agents for inhibiting Cronobacter sakazakii.

[0011] In a preferred embodiment, the antibacterial agent, disinfectant, preservative, or biocontrol agent is used for any of the following purposes: (1) Remove Cronobacter sakazakii from culture media, food matrices, medical environments, or food processing environments; (2) Inhibits Cronobacter sakazakii on medical devices, food processing facilities, storage and transport equipment, containers or solid surfaces; or (3) Prevent the metabolism and reproduction of Cronobacter sakazakii carried by medical devices or food ingredients.

[0012] In a preferred embodiment, the antibacterial agent, disinfectant, preservative, or biological control agent is a spray liquid or rinsing liquid.

[0013] This application provides the use of the bacteriophage Csp-TJDF01 described above in inhibiting the formation of biofilms in Cronobacter sakazakii.

[0014] In a preferred embodiment, the application is to inhibit the formation of Cronobacter sakazakii biofilms on the surfaces of medical devices, utensils, solid surfaces, food processing facilities, or storage and transport equipment.

[0015] This application provides the use of the bacteriophage Csp-TJDF01 described above in inhibiting the growth of Cronobacter sakazakii in infant formula, special medical purpose formula, regular milk powder or mixed milk powder.

[0016] This application provides the use of the bacteriophage Csp-TJDF01 described above in the preparation of a medicament for the prevention or treatment of Cronobacter sakazakii infection.

[0017] In a preferred embodiment, the dosage form of the drug is a liquid formulation or a lyophilized formulation.

[0018] In a preferred embodiment, the antibacterial agent, disinfectant, preservative, biocontrol agent, or drug uses the isolate or culture of the bacteriophage Csp-TJDF01 as the active ingredient, either alone or in combination with other drugs.

[0019] In a preferred embodiment, the multiplicity of infection (MOI) of the bacteriophage Csp-TJDF01 with Cronobacter sakazakii is 0.01 to 100; and / or, the titer of the bacteriophage Csp-TJDF01 is 10. 5 ~10 9 PFU / mL.

[0020] In a preferred embodiment, the *Cronobacter sakazakii* is *Cronobacter sakazakii* D17 and / or *Cronobacter sakazakii* ATCC 29544.

[0021] The Cronobacter sakazakii bacteriophage Csp-TJDF01 and its applications provided in this application have the following beneficial effects: (1) Novel phage resource: The phage Csp-TJDF01 provided in this application is a novel Cronobacter sakazakii phage isolated from sewage samples. Comparison with the GenBank database showed no reports of its complete genome sequence and related genes, thus classifying it as a novel phage resource. This phage is a double-stranded DNA (dsDNA) phage with a genome length of 143081 bp and a GC content of 46.60%. Morphologically, it belongs to the Myocaudidae family.

[0022] (2) High safety: The results of genomic analysis show that bacteriophage Csp-TJDF01 does not contain any known virulence genes, lysogen-related genes or drug resistance genes, which meets the safety requirements as a biological antimicrobial agent and can be safely applied in the fields of food processing and medical and health care.

[0023] (3) High lysis efficiency and wide host range: Bacteriophage Csp-TJDF01 has high lysis activity against both Cronobacter sakazakii D17 and ATCC29544 strains, and the titer of the purified solution can reach 10. 9 The presence of PFU / mL or higher, along with bright, clear, and uniformly sized plaques, indicates good lysis characteristics.

[0024] (4) Effective inhibition of biofilm formation: In the prior art, chemical disinfectants and most reported bacteriophages are difficult to effectively remove biofilms formed by bacteria. However, the bacteriophage Csp-TJDF01 of this application can still significantly inhibit the formation of biofilms by Cronobacter sakazakii on solid surfaces even under extremely low multiplicity of infection (MOI=0.01) conditions (p<0.001, as shown in Figure 5). This effect is unexpected and provides a new approach to solving the problem of biofilm contamination on the surfaces of medical devices and food processing facilities.

[0025] (5) Significant antibacterial activity in culture medium: Bacteriophage Csp-TJDF01 showed significant inhibitory effects on mixed and single strains of Cronobacter sakazakii in nutrient broth medium (p<0.01), and reduced bacterial OD within 12 h of culture. 600 The fact that it was kept at a low level (<0.5) demonstrates that it has a good antibacterial effect.

[0026] (6) Significant antibacterial effect in food matrix: Bacteriophage Csp-TJDF01 can significantly inhibit the growth of Cronobacter sakazakii D17 and / or ATCC 29544 in infant formula (PIF), special medical purpose formula, ordinary milk powder and mixed milk powder matrix (p<0.01), providing a new biological means for the safety control of milk powder and other dairy products.

[0027] (7) Wide range of applications: The isolates or cultures of bacteriophage Csp-TJDF01 can be used as active ingredients to prepare antibacterial agents, disinfectants, preservatives or biological control agents, suitable for disinfection in medical environments and food processing environments, as well as surface treatment of medical devices, food processing facilities, storage and transportation equipment, etc. At the same time, it can also be used to prepare pharmaceutical preparations (liquid preparations or lyophilized preparations) for the prevention or treatment of Cronobacter sakazakii infection.

[0028] (8) High flexibility of use: Bacteriophage Csp-TJDF01 can be used alone or in combination with other antibacterial drugs. It has good compatibility and application flexibility, and the usage parameters can be adjusted according to the actual application scenario and target bacterial load.

[0029] (9) Genome uniqueness: After whole genome sequencing, the NCBI BLAST tool was used to compare the GenBank database (threshold: E-value < 1e-10). The results showed that the genome sequence of phage Csp-TJDF01 did not have a highly homologous sequence in the database, and the whole genome coverage of the phage in the database was 89%, confirming that it is a new phage resource.

[0030] The specification of this application contains numerous technical features distributed across various technical solutions. Listing all possible combinations of these technical features (i.e., technical solutions) would make the specification excessively lengthy. To avoid this problem, the various technical features disclosed in the above-described invention, the various technical features disclosed in the following embodiments and examples, and the various technical features disclosed in the accompanying drawings can be freely combined to form various new technical solutions (all of which are considered to have been described in this specification), unless such a combination of technical features is technically infeasible. For example, one example discloses feature A+B+C, and another example discloses feature A+B+D+E. Features C and D are equivalent technical means that serve the same function, and technically only one needs to be used; they cannot be used simultaneously. Feature E can technically be combined with feature C. Therefore, the solution A+B+C+D should not be considered as described because it is technically infeasible, while the solution A+B+C+E should be considered as described. Attached Figure Description

[0031] Figure 1 The morphology of the phage plaque of phage Csp-TJDF01 in this application is shown.

[0032] Figure 2 This is a transmission electron microscope image of the bacteriophage Csp-TJDF01 from this application.

[0033] Figure 3 This is the whole genome sequence characteristic of the bacteriophage Csp-TJDF01 of this application.

[0034] Figure 4 This is a schematic diagram of the whole genome alignment of bacteriophage Csp-TJDF01 in the NCBI database.

[0035] Figure 5 shows the inhibitory effect of the bacteriophage Csp-TJDF01 of this application on the biofilm of Cronobacter sakazakii on a solid surface. Figure 5A Results of treatment with a mixture of Cronobacter sakazakii bacteria (D17 and ATCC 29544); Figure 5B Results of treatment with Cronobacter sakazakii D17; Figure 5C The results of treatment with *Cronobacter sakazakii* ATCC 29544 are shown in the figure. The control group bar chart represents the control group not treated with phage Csp-TJDF01; the blank group bar chart represents the blank control group without *Cronobacter sakazakii*; the remaining bars are plotted with the MOI of phage Csp-TJDF01 set at 100 (10⁻⁶). 9 PFU / mL), 10 (10 8 PFU / mL), 1 (10 7 PFU / mL), 0.1 (10 6PFU / mL), 0.01 (10 5 The experimental group was treated with PFU / mL.

[0036] Figure 6 shows the inhibitory effect of the bacteriophage Csp-TJDF01 of this application on Cronobacter sakazakii in nutrient broth medium. Figure 6A Results of treatment with a mixture of Cronobacter sakazakii bacteria (D17 and ATCC 29544); Figure 6B Results of treatment with Cronobacter sakazakii D17; Figure 6C The results of treatment with *Cronobacter sakazakii* ATCC 29544 are shown. The square dots (control group) in the figure represent the OD values ​​of *Cronobacter sakazakii* without the addition of phage Csp-TJDF01 under 37°C incubation conditions. 600 Changes; the remaining points were calculated with phage Csp-TJDF01 MOI as 100 (10) 6 PFU / mL), 10 (10 5 PFU / mL), 1 (10 4 PFU / mL), 0.1 (10 3 PFU / mL), 0.01 (10 2 Cronobacter sakazakii treated with PFU / mL was cultured at 37°C and the OD 600 change.

[0037] Figure 7 shows the growth inhibitory effect of the bacteriophage Csp-TJDF01 of this application on Cronobacter sakazakii in infant formula. Figure 7A Results of treatment with a mixture of Cronobacter sakazakii bacteria (D17 and ATCC 29544); Figure 7B Results of treatment with Cronobacter sakazakii D17; Figure 7C The results are for Cronobacter sakazakii ATCC 29544 treatment. The square dots in the figure represent the OD values ​​of untreated infant formula (0 PFU / mL) incubated at 37°C. 600 Change; the number of circular dots is 10. 6 OD of infant formula treated with PFU / mL bacteriophage Csp-TJDF01 600 change.

[0038] Figure 8 shows the growth inhibitory effect of the bacteriophage Csp-TJDF01 of this application on Cronobacter sakazakii in mixed milk powder. Figure 8A Results of treatment with a mixture of Cronobacter sakazakii bacteria (D17 and ATCC 29544); Figure 8B Results of treatment with Cronobacter sakazakii D17; Figure 8CThe results are for Cronobacter sakazakii ATCC 29544 treatment. The square dots (0 PFU / mL) in the figure represent the viable bacterial load (OD) in the mixed milk powder that was not treated with phage disinfectant. 600 Changes; the circular dots represent meridians 10. 6 Changes in viable bacterial load in mixed milk powder treated with PFU / mL Csp-TJDF01 phage disinfectant. Detailed Implementation

[0039] In the following description, many technical details are presented to help the reader better understand this application. However, those skilled in the art will understand that the technical solutions claimed in this application can be implemented even without these technical details and various variations and modifications based on the following embodiments.

[0040] Explanation of some concepts: "Cronobacter sakazakii" refers to a Gram-negative, facultative anaerobic, opportunistic pathogen belonging to the family Enterobacteriaceae. It is an important foodborne pathogen, particularly prevalent in infant formula, where it can cause diseases such as necrotizing colitis, sepsis, and meningitis. In this application, the term encompasses both wild-type strains (such as D17) and standard strains (such as ATCC 29544).

[0041] "Bacteriophage" (or Phage) refers to a class of viruses that obligately infect bacteria. They have typical virological characteristics, are tiny, lack cellular structure, and strictly depend on live host bacteria for replication and reproduction.

[0042] "Double-stranded DNA phage" (dsDNA phage) refers to a bacteriophage whose genome is double-stranded DNA.

[0043] Myoviridae is a family within the order Myoviridae, characterized by its retractable tail sheath.

[0044] "Isolate or culture of phage Csp-TJDF01" refers to any preparation containing the phage virus particles obtained through isolation, purification, or amplification, including but not limited to phage purification solution, phage amplification solution (lysis buffer), phage concentrate, phage suspension, culture supernatant containing the phage, or lyophilized powder thereof. In this application, they can all serve as sources of active ingredients for antibacterial or disinfectant agents.

[0045] "Titer" refers to the concentration of infectious phages in a phage suspension, usually expressed as plaque-forming units per milliliter (PFU / mL). In this application, the titer is determined using the bilayer plate method.

[0046] "PFU / mL" (Plaque Forming Unit per mL) refers to the number of phages that can form plaques per milliliter of solution, and is a unit of measurement for phage concentration.

[0047] The "Multiplicity of Infection" (MOI) refers to the ratio of the number of bacteriophages to the number of host bacteria in an infection system (i.e., PFU / CFU), representing the average number of bacteriophages infected per bacterial cell.

[0048] "CFU / mL" (Colony Forming Unit per mL) refers to the number of viable bacteria that can form colonies per milliliter of solution; it is a unit of measurement for bacterial concentration.

[0049] "Biofilm" refers to a three-dimensional microbial community formed when bacteria adhere to a solid surface (such as medical devices, food processing equipment, pipes, etc.) and secrete extracellular polymers (such as polysaccharides, proteins, DNA, etc.) to encapsulate themselves. This biofilm can protect bacteria from the bactericidal effects of disinfectants and antibiotics.

[0050] An "open reading frame" (ORF) is a nucleotide sequence in a genome that runs from the start codon to the stop codon and may encode a protein.

[0051] "Infant formula" (PIF) refers to a powdered product made primarily from milk and milk protein products, with the addition of appropriate amounts of vitamins, minerals and / or other ingredients, produced and processed using only physical methods, and is suitable for consumption by normal infants and young children.

[0052] "Special medical purpose formula milk powder" refers to formula milk powder specially processed and formulated to meet the special nutritional or dietary needs of people with restricted food intake, digestive and absorption disorders, metabolic disorders, or specific disease states. It includes, but is not limited to, low lactose formula, high protein, low fat, and low sugar formula, and complete nutrition formula.

[0053] In this application, "bacteriostatic agent, disinfectant, preservative, or bio-control agent" refers to a formulation with bacteriophage Csp-TJDF01 as the core active ingredient, designed to inhibit the growth of *Cronobacter sakazakii*, reduce its viable bacterial load, remove its contamination, or inhibit its biofilm formation. These terms can be used interchangeably depending on the application scenario (e.g., environmental disinfection, food preservation, biocontrol), but their core technical essence is the use of the lytic activity of bacteriophages to control target bacteria. Their use does not include direct administration to humans or animals for the purpose of treating diseases.

[0054] The present application will be further described below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the scope of protection of the present application. Without departing from the concept of the present application, those skilled in the art can make conventional changes or substitutions to the steps and conditions in the following embodiments, all of which shall fall within the scope of protection of the present application.

[0055] Unless otherwise specified, all raw materials and reagents used in this application are commercially available products.

[0056] I. Information on the Preservation of Biological Materials The Cronobacter sakazakii phage Csp-TJDF01 of this application was deposited on November 20, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, accession number: CCTCC NO: M 20252610.

[0057] II. Source of Materials The bacteriophage host bacterium used in this application, Cronobacter sakazakii (strain number: D17), was isolated from a milk powder sample using conventional methods and preserved by the Department of Nutrition, Shanghai East Hospital. It was a wild-type Cronobacter sakazakii strain. Cronobacter sakazakii ATCC 29544 was purchased from Beijing Bio-Bio Biotechnology Co., Ltd.

[0058] Wastewater samples were collected from Shanghai Oriental Hospital.

[0059] III. Terminology Definitions Unless otherwise stated in this application: "Isolates or cultures of phage Csp-TJDF01" refers to phage particles, phage amplification solutions, phage concentrates, phage purification solutions, and culture supernatants containing the phage obtained through isolation and purification, which can be used as active ingredients in formulations.

[0060] "Titer (PFU / mL)" was calculated by counting plaque-forming units (PFU) using the double-layer plate method.

[0061] "Multiple of Infection (MOI)" is the ratio of the number of bacteriophages to the number of host bacteria.

[0062] Example 1: Isolation and purification preparation of bacteriophage Csp-TJDF01 I. Phage Isolation Take 50 mL of wastewater sample and place it in a 100 mL centrifuge tube. Resuspend the sample and centrifuge at 8000 rpm for 10 min. Collect the supernatant and sterilize it through a 0.22 μm filter membrane to obtain a sterile filtrate. Take 5 mL of the filtrate and 100 μL of Cronobacter sakazakii D17, add 5 mL of 2× nutrient broth liquid medium, vortex to mix, and incubate overnight at 37°C. Centrifuge the culture medium at 8000 rpm for 10 min at 4°C. Sterilize the supernatant through a 0.22 μm filter membrane to obtain a phage enrichment solution.

[0063] The phage enrichment solution was serially diluted 10-fold with sterile SM buffer [1 L: NaCl 5.8 g, MgSO4·7H2O 2.0 g, 1 M Tris-HCl (pH 7.4) 50 mL] (10-fold serial dilution). -1 ~10 -7 Take 200 μL of phage dilution and 200 μL of Cronobacter sakazakii D17, mix at room temperature for 5 min, then add to 10 mL of nutrient broth semi-solid culture medium, mix well, pour into nutrient broth solid plates, let stand for 5 min until solidified, make double-layer plates, and incubate at 37℃ for 8 h.

[0064] Pick plaques from the above bilayer plates and place them in 900 μL of SM buffer, vortex to mix, and incubate at 4°C for 24 h. Dilute the supernatant 10-fold with SM buffer. Take 200 μL of the phage dilution and 200 μL of logarithmic-phase Cronobacter sakazakii D17, mix at room temperature for 5 min, then add to 10 mL of nutrient broth semi-solid medium, mix well, pour into nutrient broth solid plates, and incubate for 5 min until solidified to prepare bilayer plates. Incubate at 37°C for 8 h. Repeat the above steps 3–5 times to obtain the phage isolate, which is stored at 4°C for later use.

[0065] The lysis efficiency of phage isolates was assessed using a double-layer agar plate. 100 μL of logarithmic-phase *Cronobacter sakazakii* D17 was mixed with 5 mL of semi-solid nutrient broth, vortexed, and poured onto a solid nutrient broth agar plate. The plate was allowed to stand for 5 min until solidified, creating a double-layer plate. 10 μL of the phage isolate was added to each defined area, and the plate was allowed to stand and dry under sterile conditions. After drying, the plate was inverted and incubated at 37°C for 8 h. Phages exhibiting clear and bright lysis zones on both *Cronobacter sakazakii* D17 and ATCC 29544 plates were selected and named Csp-TJDF01.

[0066] II. Purification and Preparation of Bacteriophages Take 100 μL of *Cronobacter sakazakii* phage Csp-TJDF01 separation solution (prepared as above) and mix with 100 μL of *Cronobacter sakazakii* in logarithmic growth phase (D17 or ATCC 29544 bacterial solution). Incubate at room temperature for 10 min, then add 10 mL of nutrient broth liquid medium and incubate overnight at 37°C with constant temperature shaking at 120 rpm. Transfer the culture to a sterile centrifuge tube, centrifuge at 8000 rpm for 10 min, collect the supernatant, and filter through a 0.22 μm microporous membrane for sterilization. The filtrate is the phage amplification solution. Add DNase I (final concentration 1 μg / mL) and RNase A (final concentration 1 μg / mL) to the phage amplification solution and vortex to mix. Then add NaCl (final concentration 1 mol / L), vortex to mix, and incubate on ice for 2 h. Centrifuge at ×g and discard the precipitate. Add PEG-8000 (final concentration 10%) to the supernatant, incubate on ice for 5 h, centrifuge at 10000 ×g and discard the supernatant. Resuspend the phage precipitate in 1 mL of SM buffer to obtain phage concentrate. Add 3 mL of 1.7 g / mL, 1.5 g / mL and 1.45 g / mL cesium chloride to an ultracentrifuge tube, respectively. Add 2 mL of phage concentrate, centrifuge at 30000 rpm for 2 h, and aspirate the band between 1.5 g / mL and 1.45 g / mL cesium chloride to obtain phage purification solution.

[0067] The titer of phage purification solution was determined using a double-layer plate assay. The specific procedure was as follows: the phage purification solution was serially diluted 10-fold with SM buffer (10... -1 ~10 -7 Take 200 μL of each phage dilution and 200 μL of logarithmic growth phase Cronobacter sakazakii D17, mix at room temperature for 5 min, then add to 10 mL of nutrient broth semi-solid medium, mix well, pour into nutrient broth solid plates, let stand for 5 min until solidified, and invert in a 37℃ incubator for 8 h. Manually count the number of phage plaques and calculate the titer (PFU / mL).

[0068] Results: The Csp-TJDF01 purification solution achieved a titer of 10 against both Cronobacter sakazakii D17 and ATCC 29544. 9 PFU / mL or higher, and the phage plaques formed on the plate are bright, clear, and uniform in size (e.g., Figure 1 (As shown).

[0069] It should be noted that the bacteriophage Csp-TJDF01 isolate or culture obtained in this embodiment can be used as a source of active ingredients for the subsequent preparation of antibacterial agents, disinfectants, preservatives, biological control agents or pharmaceutical preparations.

[0070] Example 2 Characterization of bacteriophage Csp-TJDF01 I. Morphological characteristics The microscopic morphological characteristics of the bacteriophages were observed using transmission electron microscopy. The bacteriophages were negatively stained with phosphotungstic acid. With the membrane-covered side of the copper mesh facing upwards, 30 μL of the Csp-TJDF01 purification solution obtained in Example 1 (10 μL) was taken. 9 PFU / mL was dropped onto a 200-mesh carbon-coated copper mesh. After adsorption for 10 min, the copper mesh was removed and allowed to air dry for 2–3 min. Then, 10 μL of 2% phosphotungstic acid (PTA, pH 7.6) was added to the copper mesh, and staining was performed for 10 min. The negative staining solution was blotted dry with filter paper, and the mesh was baked under an infrared lamp for 30 min. The microscopic morphology of the bacteriophages was observed using a Tecnai-12 transmission electron microscope, and clear images were selected for photographic analysis.

[0071] Results: The microstructure of bacteriophage Csp-TJDF01 is as follows Figure 2 As shown, bacteriophage Csp-TJDF01 exhibits a symmetrical head with a diameter of approximately 65.75 nm and a tail length of 96.62 nm. It possesses a retractable tail sheath and its morphological characteristics classify it into the Myotail Phage family.

[0072] II. Genomic Characteristics The bacteriophage Csp-TJDF01 purification solution prepared in Example 1 (10 9Add DNase I (final concentration 1 μg / mL) and RNase A (final concentration 1 μg / mL) to PFU / mL, vortex to mix; then add NaCl (final concentration 1 mol / L), vortex to mix, and incubate on ice for 2 h; centrifuge at 10000 ×g and discard the precipitate, add PEG-8000 (final concentration 10%) to the supernatant, incubate on ice for 5 h, centrifuge at 10000 ×g and discard the supernatant, resuspend the phage precipitate in 1 mL of SM buffer to obtain phage concentrate; add 3 mL of 1.7 g / mL, 1.5 g / mL and 1.45 g / mL cesium chloride to ultracentrifuge tubes respectively, add 2 mL of phage concentrate, centrifuge at 30000 rpm for 2 h, aspirate the band between 1.5 g / mL and 1.45 g / mL cesium chloride, and add 1 / 10 3 mol / L NaAc (pH 10). 5.2) Add two volumes of anhydrous ethanol to precipitate the nucleic acid, and incubate overnight at -20°C; centrifuge at 12000 ×g for 10 min at 4°C; wash the precipitate once with 70% ethanol and once with anhydrous ethanol, and air dry the precipitate for 10 min; suspend the precipitate with an appropriate amount of TE (pH 8.0), and quantify the phage DNA using a GeneQuant nucleic acid quantification instrument, and store at -20°C; the extracted phage DNA was sent to Shanghai Lingen Bio-Gene Sequencing Co., Ltd. for Illumina Hiseq sequencing.

[0073] Results: Phage DNA sequencing results (e.g.) Figure 3 As shown in the image: (1) Nucleic acid type: Csp-TJDF01 is a double-stranded DNA (dsDNA) bacteriophage.

[0074] (2) Genome structure characteristics: The Csp-TJDF01 genome is 143081 bp in length, of which the total length of coding genes is 130317 bp, with an average length of 536 bp, accounting for 91.10% of the total length. The GC content is 46.60%, and it has 243 open reading frames (ORFs), including 166 hypothetical protein coding sequences and 77 sequences with known protein functions. The homology is between 41.1% and 100%.

[0075] (3) Novelty: The comparison results showed that the complete genome sequence and related genes of Cronobacter sakazakii phage Csp-TJDF01 were not found in the GenBank database, indicating that Csp-TJDF01 is a novel phage (e.g., Figure 4 (As shown).

[0076] (4) Safety: The results of genomic analysis showed that Csp-TJDF01 does not contain any known virulence genes, lysogen-related genes or drug resistance genes, and meets the safety requirements as an antibacterial agent.

[0077] III. Security Analysis The safety analysis of the genome of bacteriophage Csp-TJDF01 was performed using the following methods: (1) Virulence gene detection: The phage genome was compared and analyzed using a virulence factor database. The results showed that no known virulence factor encoding genes were detected in the phage Csp-TJDF01 genome.

[0078] (2) Detection of drug resistance genes: Screening was performed using a drug resistance gene database. The analysis results showed that no known drug resistance genes were detected in the genome of bacteriophage Csp-TJDF01.

[0079] (3) Lysogenicity detection: Lysogenic genes were predicted in the genome, and lysogenic marker genes such as integrase and CI inhibitory protein were detected. The analysis results showed that no lysogenic genes were found in the genome of phage Csp-TJDF01, confirming that it is a strictly lytic phage.

[0080] Example 3: Inhibitory effect of bacteriophage Csp-TJDF01 on biofilm formation of Cronobacter sakazakii This embodiment verifies the inhibitory effect of bacteriophage Csp-TJDF01 on the formation of Cronobacter sakazakii biofilm on solid surfaces under different multiples of infection (MOI) conditions.

[0081] I. Experimental Methods Take 100 μL of Cronobacter sakazakii D17 and ATCC 29544 in the logarithmic growth phase, vortex to mix, and dilute with nutrient broth to a final concentration of 10. 7 CFU / mL. Similarly, 100 μL of phage Csp-TJDF01 was diluted to a final concentration of 10. 9 PFU / mL, 10 8 PFU / mL, 10 7 PFU / mL, 10 6 PFU / mL and 10 5 PFU / mL.

[0082] Take the above-mentioned mixed bacterial culture of Cronobacter sakazakii (D17 and ATCC 29544, 10) 7 CFU / mL) were respectively compared with bacteriophage Csp-TJDF01 (10 9 PFU / mL, 10 8 PFU / mL, 10 7 PFU / mL, 10 6 PFU / mL and 10 5PFU / mL was mixed into 96-well plates at multiples of infection (MOI) of 100, 10, 1, 0.1, and 0.01, and set as phage Csp-TJDF01 (different MOI) treatment groups. Mixed bacterial suspensions of Cronobacter sakazakii (D17 and ATCC 29544, 10...) were added. 7 Equal volumes of CFU / mL SM buffer were mixed into 96-well plates and used as the negative control (Control). A 96-well plate containing only nutrient broth was used as the blank control (Blank). All groups were incubated at 37°C for 24 h.

[0083] Remove the 96-well plate, aspirate the bacterial culture from the wells, and wash three times with PBS to thoroughly remove airborne bacteria. Dry at room temperature. Add 200 μL of 0.1% (w / v) crystal violet solution to each well and stain for 10 min. Discard the crystal violet solution and rinse the 96-well plate with PBS until the wash buffer is colorless. Dry at room temperature, add 200 μL of 33% (v / v) acetic acid destaining agent to each well, and shake to dissolve for 10 min. Measure the absorbance (OD) at 600 nm using a microplate reader. 600 Each group should be tested in triplicate, and each parallel test should be repeated three times.

[0084] II. Experimental Results (1) Biofilm inhibition results of mixed bacteria (D17 and ATCC 29544) like Figure 5A As shown, add 10 9 PFU / mL, 10 8 PFU / mL, 10 7 PFU / mL, 10 6 PFU / mL, 10 5 OD50 of PFU / mL phage Csp-TJDF01 experimental groups (MOI 100, 10, 1, 0.1, 0.01 treatment groups) 600 All values ​​were significantly lower than those of the negative control group (p < 0.001), indicating that phage Csp-TJDF01 can significantly inhibit the formation of mixed Cronobacter sakazakii biofilm on the surface of solid porous plates in the MOI range of 0.01 to 100.

[0085] (2) Results of biofilm inhibition of Cronobacter sakazakii D17 Similarly, using the above method, a single Cronobacter sakazakii D17 (10 7 CFU / mL) and the above-mentioned bacteriophage Csp-TJDF01 (10 9 PFU / mL, 10 8 PFU / mL, 10 7 PFU / mL, 106 PFU / mL, 10 5 PFU / mL) mixed. Results as follows Figure 5B As shown, the OD of each experimental group 600 All values ​​were significantly lower than those of the negative control group (p < 0.001), indicating that phage Csp-TJDF01 can significantly inhibit the formation of Cronobacter sakazakii D17 biofilm on the surface of solid porous plates.

[0086] (3) Results of biofilm inhibition of Cronobacter sakazakii ATCC 29544 The above method was used to employ a single Cronobacter sakazakii ATCC 29544 (10 7 (CFU / mL) was mixed with the above-mentioned phage Csp-TJDF01. The results are as follows: Figure 5C As shown, the OD of each experimental group 600 All values ​​were significantly lower than those of the negative control group (p < 0.001), indicating that phage Csp-TJDF01 can significantly inhibit the formation of Cronobacter sakazakii ATCC 29544 biofilm on the surface of solid porous plates.

[0087] III. Conclusion The above results indicate that phage Csp-TJDF01 has a titer of 10 within a multiplicity of infection (MOI) range of 0.01–100. 5 ~10 9 At PFU / mL, both can effectively inhibit the formation of biofilms by Cronobacter sakazakii D17 and / or ATCC 29544 on solid surfaces.

[0088] Furthermore, the solid surface of the 96-well plate in this embodiment can represent surfaces such as medical devices, utensils, solid surfaces, food processing facilities, or storage and transportation equipment in practical application scenarios. Therefore, bacteriophage Csp-TJDF01 can be used to inhibit the formation of Cronobacter sakazakii biofilms on the aforementioned surfaces.

[0089] Example 4: Antibacterial effect of bacteriophage Csp-TJDF01 in nutrient broth medium This embodiment verifies the inhibitory effect of bacteriophage Csp-TJDF01 on Cronobacter sakazakii in culture medium, providing support for the application of bacteriophages as antibacterial agents, disinfectants, or biological control agents.

[0090] I. Experimental Methods Take 100 μL of a mixture containing Cronobacter sakazakii (D17 and ATCC 29544, 10) 4 Artificially contaminated culture medium (CFU / mL) was mixed with 100 μL of bacteriophage Csp-TJDF01 (10 CFU / mL). 6 PFU / mL, 105 PFU / mL, 10 4 PFU / mL, 10 3 PFU / mL, 10 2 PFU / mL were mixed with multiples of infection (MOI) of 100, 10, 1, 0.1, and 0.01. A phage-free control group was included. The samples were incubated at 37°C for 10 h. The absorbance of *Cronobacter sakazakii* at 600 nm was measured every 2 h. Each group was performed in triplicate, with each replicate measured three times.

[0091] II. Experimental Results (1) Antibacterial results of mixed bacteria (D17 and ATCC 29544) like Figure 6A As shown, with prolonged culture time, the OD of the negative control group decreased. 600 Rapid growth, reaching 1.09 at 10 h, while the addition of Csp-TJDF01 treatment group (10 6 PFU / mL, 10 5 PFU / mL, 10 4 PFU / mL in three treatment groups) OD 600 The OD level remained low throughout the 10 h of incubation. 600 < 0.5), indicating that the bacteriophage Csp-TJDF01 in this application significantly inhibited the growth of mixed Cronobacter sakazakii (D17 and ATCC 29544) in the culture medium (p < 0.01).

[0092] (2) Antibacterial results of Cronobacter sakazakii D17 Using the method described above, single Cronobacter sakazakii D17 (10 4 (CFU / mL) was mixed with the above-mentioned phage Csp-TJDF01. The results are as follows: Figure 6B As shown, the phage Csp-TJDF01 significantly inhibited the growth of Cronobacter sakazakii D17 in the culture medium (p < 0.01).

[0093] (3) Antibacterial results of Cronobacter sakazakii ATCC 29544 Using the method described above, a single Cronobacter sakazakii ATCC 29544 (10 4 (CFU / mL) was mixed with the above-mentioned phage Csp-TJDF01. The results are as follows: Figure 6C As shown, the phage Csp-TJDF01 significantly inhibited the growth of Cronobacter sakazakii ATCC 29544 in the culture medium (p < 0.01).

[0094] III. Conclusion The above results indicate that bacteriophage Csp-TJDF01 can be used to eliminate Cronobacter sakazakii D17 and / or ATCC 29544 in culture media. More specifically, its isolates or cultures can be used as active ingredients to prepare antibacterial agents, disinfectants, or biocontrol agents for eliminating Cronobacter sakazakii in culture media, medical environments, or food processing environments.

[0095] Example 5: Antibacterial effect of bacteriophage Csp-TJDF01 in infant formula This embodiment verifies the inhibitory effect of bacteriophage Csp-TJDF01 on Cronobacter sakazakii in powdered infant formula (PIF) matrix.

[0096] I. Experimental Methods Add 12 g of infant formula (PIF) to 100 mL of deionized water, vortex to mix, autoclave at 115°C for 20 min, and cool for later use to obtain a 12% PIF solution. Take 30 mL of the above sterile 12% (w / v) PIF solution and test it against a mixture of Cronobacter sakazakii (D17 and ATCC 29544, 10...). 6 The concentration (CFU / mL) was serially diluted 10-fold to a final concentration of 10. 6 CFU / mL, resulting in artificially contaminated PIF.

[0097] Add 30 μL of phage Csp-TJDF01 (10) to the above PIF. 6 The PFU / mL concentration was designated as the phage Csp-TJDF01 treatment group. A negative control group (0 PFU / mL) was prepared by adding only 30 μL of SM buffer to the PFU. The samples were stored at 37°C for 10 h. The absorbance of *Cronobacter sakazakii* at 600 nm was measured every 2 h. Each group was performed in triplicate, with each replicate measured three times.

[0098] II. Experimental Results (1) Antibacterial results of mixed bacteria (D17 and ATCC 29544) like Figure 7A As shown, within 10 h of constant temperature storage, the OD of the negative control group was... 600 It rapidly rose to around 0.5 within 4 hours, while the phage Csp-TJDF01 (10 6 PFU / mL) can reduce OD within 10 h. 600 The concentration was controlled below 0.4, and the results showed that the Csp-TJDF01 phage in this application had a significant inhibitory effect on Cronobacter sakazakii in the PIF matrix (p < 0.01).

[0099] (2) Antibacterial results of Cronobacter sakazakii D17 The above method was used to employ a single Cronobacter sakazakii D17 (10) 6 Artificially contaminated PIFs were prepared using bacteriophage Csp-TJDF01 (10 CFU / mL). 6 PFU / mL was added to the artificially contaminated PIF and stored at 37°C for 10 h. Results were as follows: Figure 7B As shown, the OD of the experimental group 600 The levels were significantly lower than those in the negative control group (p < 0.01), indicating that phage Csp-TJDF01 can significantly inhibit Cronobacter sakazakii D17 in the PIF matrix.

[0100] (3) Antibacterial results of Cronobacter sakazakii ATCC 29544 The above method was used to employ a single Cronobacter sakazakii ATCC 29544 (10 6 Artificial contamination PIF was prepared using CFU / mL. Results are as follows: Figure 7C As shown, the OD of the experimental group 600 The levels were significantly lower than those in the negative control group (p < 0.01), indicating that phage Csp-TJDF01 can significantly inhibit Cronobacter sakazakii ATCC 29544 in the PIF matrix.

[0101] III. Conclusion The above results indicate that bacteriophage Csp-TJDF01 can be used to inhibit the growth of Cronobacter sakazakii D17 and / or ATCC 29544 in infant formula. For example, bacteriophage Csp-TJDF01 can be used as an active ingredient to prepare antibacterial agents, disinfectants, preservatives, or biocontrol agents for use in food matrices.

[0102] Example 6: Antibacterial effect of bacteriophage Csp-TJDF01 in mixed milk powder This embodiment verifies the inhibitory effect of bacteriophage Csp-TJDF01 on Cronobacter sakazakii in a mixture of special medical purpose formula milk powder and regular milk powder.

[0103] I. Experimental Methods Three types of milk powder were selected from special medical purpose formula milk powder [low lactose formula, high protein (>80%) low fat and low sugar formula, and complete nutrition formula] and ordinary milk powder (infant formula PIF, adult full-fat formula, and adult skimmed formula) and mixed in equal proportions. 12 g of the mixed milk powder was added to 100 mL of deionized water, vortexed to mix, autoclaved at 115℃ for 20 min, and cooled for later use to obtain a 12% mixed milk powder solution.

[0104] Take 30 mL of the above sterile 12% (w / v) mixed milk powder solution and test it against a mixture of Cronobacter sakazakii (D17 and ATCC29544, 10). 6 The concentration (CFU / mL) was serially diluted 10-fold to a final concentration of 10. 6 CFU / mL, resulting in an artificially contaminated mixed milk powder solution.

[0105] Add 30 μL of bacteriophage Csp-TJDF01 (10 μL) to the above mixed milk powder solution. 6 The treatment group was designated as phage Csp-TJDF01 (PFU / mL). A negative control group (0 PFU / mL) was prepared by adding only 30 μL of SM buffer to the above mixed milk powder solution. The solution was stored at 37℃ for 10 h. The absorbance of *Cronobacter sakazakii* at 600 nm was measured every 2 h. Each group was performed in triplicate, with each replicate measured three times.

[0106] II. Experimental Results (1) Antibacterial results of mixed bacteria (D17 and ATCC 29544) like Figure 8A As shown, the absorbance at 600 nm was 0.31 and 0.26 for the negative control group and the Csp-TJDF01 treatment group during the initial stage of constant temperature storage. With the increase of storage time, the total number of bacteria in the mixed milk powder solution of the Csp-TJDF01 treatment group was significantly lower than that of the control group throughout the storage period, and decreased by 0.46 compared with the control group at 10 h. This indicates that the Csp-TJDF01 phage treatment in this application has a significant inhibitory effect on the growth of Cronobacter sakazakii in mixed milk powder (p < 0.01).

[0107] (2) Antibacterial results of Cronobacter sakazakii D17 The above method was used to employ a single Cronobacter sakazakii D17 (10) 6 Prepare an artificially contaminated mixed milk powder solution using CFU / mL. Add bacteriophage Csp-TJDF01 (10... 6 (PFU / mL) was added to the above artificially contaminated mixed milk powder solution, and stored at 37°C for 10 h. The results were as follows: Figure 8B As shown, the OD of the experimental group 600 The levels were significantly lower than those in the negative control group (p < 0.01), indicating that phage Csp-TJDF01 can significantly inhibit Cronobacter sakazakii D17 in the mixed milk powder matrix.

[0108] (3) Antibacterial results of Cronobacter sakazakii ATCC 29544 The above method was used to employ a single Cronobacter sakazakii ATCC 29544 (10 6Artificially contaminated mixed milk powder solution was prepared using CFU / mL. Results are as follows: Figure 8C As shown, the OD of the experimental group 600 The levels were significantly lower than those in the negative control group (p < 0.01), indicating that phage Csp-TJDF01 could significantly inhibit Cronobacter sakazakii ATCC 29544 in the mixed milk powder matrix.

[0109] III. Conclusion The above results indicate that phage Csp-TJDF01 can be used to inhibit the growth of Cronobacter sakazakii D17 and / or ATCC 29544 in special medical purpose formula milk powder, regular milk powder and their mixed milk powder.

[0110] Example 7: Preparation and application of bacteriophage Csp-TJDF01 antibacterial agent, disinfectant, and biocontrol agent. This embodiment provides a method for preparing bacteriophage Csp-TJDF01 antibacterial agent, disinfectant, preservative or biological microbial control agent and its application in medical and food processing environments.

[0111] I. Preparation of bacteriophage disinfectant stock solution The purified phage Csp-TJDF01 solution prepared in Example 1 was mixed with SM buffer or other suitable buffer to prepare a phage disinfectant stock solution. For example, the potency of the phage disinfectant stock solution can be 10. 5 ~10 9 Any value within the range of PFU / mL.

[0112] II. Preparation of spraying or rinsing solutions The phage disinfectant stock solution can be diluted with sterile water, SM buffer, or physiological saline to prepare a spray or rinsing solution. Optionally, the potency of the spray or rinsing solution can be adjusted according to the actual application requirements.

[0113] Furthermore, the spray or rinse solution can be used alone; alternatively, it can be used in combination with other drugs without altering the bacteriophage as the active ingredient.

[0114] III. Application The antibacterial agent, disinfectant, preservative, or biological microbial control agent may be used for any of the following purposes: (1) Spray or rinse the medical or food processing environment to remove Cronobacter sakazakii contamination from the environment; (2) Spray or rinse medical devices, food processing facilities, storage and transportation equipment, utensils or solid surfaces to reduce the load of Cronobacter sakazakii and inhibit biofilm formation; (3) As a food ingredient additive, it is used to prevent the metabolism and reproduction of Cronobacter sakazakii carried by medical devices or food ingredients.

[0115] IV. Usage Parameters Based on the experimental results of Examples 3-6, the recommended usage parameters for the antibacterial agent, disinfectant, preservative, or biological microbial control agent are as follows: Phage titer: 10 5 ~10 9 PFU / mL; Multiple of infection (MOI): 0.01–100.

[0116] It should be noted that the above parameter ranges are illustrative examples, and those skilled in the art can make appropriate adjustments based on the actual application scenario and the target bacterial load.

[0117] Example 8 Preparation of bacteriophage Csp-TJDF01 drug formulation This embodiment provides a method for using bacteriophage Csp-TJDF01 to prepare drugs for the prevention or treatment of Cronobacter sakazakii infection.

[0118] I. Preparation of Liquid Formulations Take the purified solution (isolated material or culture) of phage Csp-TJDF01 prepared in Example 1, use phage Csp-TJDF01 as the active ingredient, add an appropriate amount of pharmaceutically acceptable liquid carrier, and prepare a liquid formulation.

[0119] Optionally, the liquid carrier may be, but is not limited to, physiological saline, phosphate buffer, SM buffer, etc.

[0120] Furthermore, optionally, the liquid formulation can be used alone or in combination with other drugs for the prevention or treatment of Cronobacter sakazakii infection.

[0121] II. Preparation of Lyophilized Formulations Take the purified solution (isolated material or culture) of phage Csp-TJDF01 prepared in Example 1, use phage Csp-TJDF01 as the active ingredient, add an appropriate amount of freeze-drying protectant, and prepare a freeze-dried formulation by freeze-drying process.

[0122] Optionally, the lyophilization protectant can be a commonly used lyophilization protectant in the art.

[0123] Furthermore, optionally, the lyophilized formulation is reconstituted with a suitable solvent before use. The lyophilized formulation can be used alone or in combination with other drugs for the prevention or treatment of Cronobacter sakazakii infection.

[0124] It should be noted that the efficacy verification provided in this application specification mainly focuses on in vitro antibacterial activity, biofilm inhibition, and food matrix antibacterial activity. The above-mentioned "preparation of a drug for the prevention or treatment of Cronobacter sakazakii infection" is used as a Swiss-type use statement to meet the requirements of claim drafting.

[0125] The above embodiments have the following technical effects: (1) Novel Phage Resource: The phage Csp-TJDF01 provided in the above embodiments is a novel Cronobacter sakazakii phage isolated from sewage samples. Comparison with the GenBank database showed no reports of its complete genome sequence and related genes, classifying it as a novel phage resource. This phage is a double-stranded DNA (dsDNA) phage with a genome length of 143,081 bp and a GC content of 46.60%. Morphologically, it belongs to the Myocaudidae family.

[0126] (2) High safety: The results of genomic analysis show that bacteriophage Csp-TJDF01 does not contain any known virulence genes, lysogen-related genes or drug resistance genes, which meets the safety requirements as a biological antimicrobial agent and can be safely applied in the fields of food processing and medical and health care.

[0127] (3) High lysis efficiency and wide host range: Bacteriophage Csp-TJDF01 has high lysis activity against both Cronobacter sakazakii D17 and ATCC29544 strains, and the titer of the purified solution can reach 10. 9 The presence of PFU / mL or higher, along with bright, clear, and uniformly sized plaques, indicates good lysis characteristics.

[0128] (4) Effective inhibition of biofilm formation: In the prior art, chemical disinfectants and most reported bacteriophages are difficult to effectively remove biofilms formed by bacteria. However, the bacteriophage Csp-TJDF01 in the above embodiment can still significantly inhibit the formation of biofilms by Cronobacter sakazakii on solid surfaces even under extremely low multiplicity of infection (MOI=0.01) conditions (p<0.001, as shown in Figure 5). This effect is unexpected and provides a new approach to solving the problem of biofilm contamination on the surfaces of medical devices and food processing facilities.

[0129] (5) Significant antibacterial activity in culture medium: Bacteriophage Csp-TJDF01 showed significant inhibitory effects on mixed and single strains of Cronobacter sakazakii in nutrient broth medium (p<0.01), and reduced bacterial OD within 12 h of culture. 600 The fact that it was kept at a low level (<0.5) demonstrates that it has a good antibacterial effect.

[0130] (6) Significant antibacterial effect in food matrix: Bacteriophage Csp-TJDF01 can significantly inhibit the growth of Cronobacter sakazakii D17 and / or ATCC 29544 in infant formula (PIF), special medical purpose formula, ordinary milk powder and mixed milk powder matrix (p<0.01), providing a new biological means for the safety control of milk powder and other dairy products.

[0131] (7) Wide range of applications: The isolates or cultures of bacteriophage Csp-TJDF01 can be used as active ingredients to prepare antibacterial agents, disinfectants, preservatives or biological control agents, suitable for disinfection in medical environments and food processing environments, as well as surface treatment of medical devices, food processing facilities, storage and transportation equipment, etc. At the same time, it can also be used to prepare pharmaceutical preparations (liquid preparations or lyophilized preparations) for the prevention or treatment of Cronobacter sakazakii infection.

[0132] (8) High flexibility of use: Bacteriophage Csp-TJDF01 can be used alone or in combination with other antibacterial drugs. It has good compatibility and application flexibility, and the usage parameters can be adjusted according to the actual application scenario and target bacterial load.

[0133] (9) Genome uniqueness: After whole genome sequencing, the NCBI BLAST tool was used to compare the GenBank database (threshold: E-value < 1e-10). The results showed that the genome sequence of phage Csp-TJDF01 did not have a highly homologous sequence in the database, and the whole genome coverage of the phage in the database was 89%, confirming that it is a new phage resource.

[0134] It should be noted that in this patent application, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one" does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this patent application, if it refers to performing an action according to an element, it means performing the action at least according to that element, including two cases: performing the action only according to that element, and performing the action according to that element and other elements. Expressions such as "multiple," "repeatedly," and "various" include two, two times, two kinds, and more than two, more than two times, and more than two kinds.

[0135] All documents mentioned in this application are considered to be incorporated in their entirety into the disclosure of this application so that they can serve as a basis for modifications if necessary. Furthermore, it should be understood that after reading the foregoing disclosure of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope of protection claimed in this application.

Claims

1. A Cronobacter sakazakii bacteriophage Csp-TJDF01, characterized in that, The bacteriophage was deposited on November 20, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20252610.

2. The bacteriophage Csp-TJDF01 according to claim 1, characterized by, The bacteriophage Csp-TJDF01 is a double-stranded DNA bacteriophage with a full-length genome of 143081 bp and a GC content of 46.60%.

3. The use of the bacteriophage Csp-TJDF01 according to claim 1 in the preparation of antibacterial agents, disinfectants, preservatives or biological control agents for inhibiting Cronobacter sakazakii.

4. Use according to claim 3, characterized in that, The antibacterial agent, disinfectant, preservative, or biological microbial control agent is used for any of the following purposes: (1) Remove Cronobacter sakazakii from culture media, food matrices, medical environments, or food processing environments; (2) Inhibits Cronobacter sakazakii on medical devices, food processing facilities, storage and transport equipment, containers or solid surfaces; or (3) Prevent the metabolism and reproduction of Cronobacter sakazakii carried by medical devices or food ingredients.

5. Use according to claim 3, characterized in that, The antibacterial agent, disinfectant, preservative, or biological antibacterial agent is a spray or rinsing solution.

6. The use of the bacteriophage Csp-TJDF01 according to claim 1 in inhibiting the formation of biofilms in Cronobacter sakazakii.

7. Use according to claim 6, characterized in that, The application is to inhibit the formation of Cronobacter sakazakii biofilms on the surfaces of medical devices, utensils, solid surfaces, food processing facilities, or storage and transport equipment.

8. The use of the bacteriophage Csp-TJDF01 according to claim 1 in inhibiting the growth of Cronobacter sakazakii in infant formula, special medical purpose formula, ordinary milk powder or mixed milk powder.

9. The use of the bacteriophage Csp-TJDF01 according to claim 1 in the preparation of a medicament for the prevention or treatment of Cronobacter sakazakii infection.

10. Use according to claim 9, characterized in that, The drug is in the form of a liquid preparation or a lyophilized preparation.

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