Broad-spectrum phage capable of efficiently lysing cronobacter sp., bactericide, and use

Abstract

Provided are a broad-spectrum phage Cronobacter phage vB_CsaM_CBT2 capable of efficiently lysing Cronobacter sp., a bactericide and the use thereof, the accession number of the broad-spectrum phage Cronobacter phage vB_CsaM_CBT2 being CCTCC NO:M 2023524. The phage only specifically lyses Cronobacter sp., has a broad lysis spectrum, and can cover four species (involving multi-drug resistant bacteria) of Cronobacter genus. The phage has good stability under a pH value of 3-11 and a temperature of 25-70℃, reaches a sterilization effect of up to 80.55-99.97% within 12 h in milk powder samples, and does not carry any virulence and antibiotic drug-resistant genes, thereby satisfying safety requirements in practical application.

Description

A broad-spectrum bacteriophage capable of efficiently lysing Cronobacter, a bactericide and its application Technical Field

[0001] The present invention belongs to the field of microbial technology, and in particular relates to a broad-spectrum bacteriophage capable of efficiently lysing Cronobacter genus, a bactericide and applications thereof. Background Art

[0002] Cronobacter is a major foodborne pathogen that can cause meningitis, sepsis, and necrotizing enterocolitis in neonates and infants, with severe neurological sequelae. The mortality rate in neonates infected with Cronobacter is as high as 40% to 80%. Powdered infant formula (PIF) is considered the primary route of transmission for Cronobacter infection. Cronobacter is highly resistant to environmental stress and can survive in PIF for up to 2.5 years. Therefore, consuming contaminated PIF carries a serious risk of fatal meningitis and enteric infections. In 2004, the Food and Agriculture Organization of the United Nations and the World Health Organization, following a risk assessment, listed Cronobacter, along with Salmonella, as Category A pathogens in infant formula. However, Cronobacter contamination incidents continue to occur, making effective prevention and control of Cronobacter a common challenge that the dairy industry urgently needs to address.

[0003] Currently, antibiotics are the most effective treatment for Cronobacter infections. In recent years, research reports have confirmed a growing trend of increasing drug resistance in Cronobacter. Some studies have indicated that commonly used hospital antibiotics, such as penicillin and first- and second-generation cephalosporins, have lost their effectiveness against nosocomial Cronobacter infections, and Cronobacter isolates with multidrug resistance have been increasingly identified. As bacterial resistance becomes increasingly serious, the country has strengthened its efforts to regulate the misuse of antibiotics, highlighting the importance of identifying alternative antimicrobial options.

[0004] Bacteriophages are viruses that rely on specific bacterial cells as their host cells. They can inject their genetic material into the host bacteria through their tail organelles, lysing them and harmlessly killing them. Phages offer advantages such as abundant sources, strong specificity, and high safety, making them excellent alternatives to antibiotics used in the prevention and control of food biocontamination. To effectively control Cronobacter contamination in food, it is imperative to identify Cronobacter phages with a broad lysis spectrum.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a broad-spectrum phage, a fungicide and an application thereof that can efficiently lyse Cronobacter genus. The phage Cronobacter phage vB_CsaM_CBT2 can efficiently lyse multidrug-resistant Cronobacter and has a broad lysis spectrum.

[0007] The invention provides a Cronobacter phage vB_CsaM_CBT2, with a preservation number of CCTCC NO: M 2023524.

[0008] The present invention provides use of the Cronobacter phage vB_CsaM_CBT2 described in the above technical solution in preparing a preparation for inhibiting and / or killing Cronobacter.

[0009] Preferably, the preparation comprises Cronobacter phage vB_CsaM_CBT2 in the form of a proliferation liquid or a concentrated solution of the proliferation liquid.

[0010] Preferably, the method for preparing the proliferation fluid comprises the following steps:

[0011] The logarithmic phase bacterial liquid of Cronobacter, Cronobacter phage vB_CsaM_CBT2 and LB broth medium were mixed and cultured to obtain a culture solution, the culture solution was centrifuged to obtain a supernatant, and the supernatant was filtered to obtain a Cronobacter phage vB_CsaM_CBT2 proliferation liquid.

[0012] Preferably, the Cronobacter comprises one or more of Cronobacter sakazakii, Cronobacter turicensis, Cronobacter muytjensii and Cronobacter condimenti.

[0013] Preferably, the working titer of Cronobacter phage vB_CsaM_CBT2 in the Cronobacter inhibitory preparation is ≥2.95×10 8 pfu / mL, and the working titer of Cronobacter phage vB_CsaM_CBT2 in the Cronobacter killing preparation is ≥1×10 10 pfu / mL.

[0014] The present invention provides a Cronobacter inhibitor or bactericide, comprising the Cronobacter phage vB_CsaM_CBT2 described in the above technical solution and pharmaceutically acceptable excipients.

[0015] Preferably, the working pH value of the Cronobacter inhibitor or bactericide is 3-11.

[0016] Preferably, the working potency of the Cronobacter inhibitor is ≥2.95×10 8 pfu / mL; the working potency of Cronobacter bactericide is ≥1×10 10 pfu / mL.

[0017] Preferably, the pharmaceutically acceptable excipients include one or more of a dispersant, a stabilizer, a filler and a solvent.

[0018] The present invention provides use of the Cronobacter phage vB_CsaM_CBT2 described in the above technical solution or the Cronobacter inhibitor or bactericide described in the above technical solution in preparing a reagent for preventing and controlling Cronobacter contamination.

[0019] Preferably, the multiplicity of infection of the reagent is 1:10 to 1:1000.

[0020] Preferably, the controlling Cronobacter contamination comprises antagonizing Cronobacter contamination in the product and / or the environment.

[0021] Preferably, the product comprises a food product.

[0022] Beneficial effects of the present invention: The present invention provides a Cronobacter phage vB_CsaM_CBT2, with a deposit number of CCTCC NO: M 2023524. The Cronobacter phage vB_CsaM_CBT2 of the present invention can maintain a titer of 10 at a temperature of 25-60°C. 10 pfu / mL, the titer of Cronobacter phage vB_CsaM_CBT2 can be maintained at 10 under the condition of pH value of 4-11. 8 pfu / mL or above, the titer of Cronobacter phage vB_CsaM_CBT2 was high, the temperature and pH stability was good, and the antibacterial effect was good.

[0023] The Cronobacter phage vB_CsaM_CBT2 described in the present invention has a broad host spectrum and can target and lyse four strains of the genus Cronobacter: C. sakazakii, C. turicensis, C. muytjensii, and C. condimenti. The Cronobacter phage vB_CsaM_CBT2 provided by the present invention can lyse the multidrug-resistant Cronobacter sakazakii clinical strain GZcsf-1, which is resistant to antibiotics such as ampicillin, cefazolin, ceftriaxone, aztreonam, gentamicin, tetracycline, chloramphenicol, and trimethoprim / sulfamethoxazole.

[0024] Biological deposit certificate

[0025] [Corrected 08.09.2023 according to Rule 91] Cronobacter phage vB_CsaM_CBT2 was deposited in the China Center for Type Culture Collection on April 12, 2023, with the deposit number CCTCC NO:M 2023524. The depository address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0027] Figure 1 is a plaque image of Cronobacter phage vB_CsaM_CBT2;

[0028] Figure 2 is an electron micrograph of Cronobacter phage vB_CsaM_CBT2;

[0029] Figure 3 is a heat map of Cronobacter phage vB_CsaM_CBT2 genome analysis;

[0030] Figure 4 is a phylogenetic tree analysis diagram of the Cronobacter phage vB_CsaM_CBT2 terminal enzyme large subunit;

[0031] Figure 5 is a schematic diagram of the one-step growth curve of Cronobacter phage vB_CsaM_CBT2;

[0032] Figure 6 is a schematic diagram of the temperature tolerance of Cronobacter phage vB_CsaM_CBT2;

[0033] Figure 7 is a schematic diagram of pH tolerance of Cronobacter phage vB_CsaM_CBT2. DETAILED DESCRIPTION

[0034] The invention provides a Cronobacter phage vB_CsaM_CBT2, with a preservation number of CCTCC NO: M 2023524.

[0035] The present invention screened and isolated a virulent Cronobacter phage Cronobacter phage vB_CsaM_CBT2 (hereinafter referred to as Cronobacter phage vB_CsaM_CBT2) that efficiently lyses multidrug-resistant bacteria from sewage from a sewage treatment plant in Hefei City, Anhui Province. The present invention performed a whole-genome analysis on Cronobacter phage vB_CsaM_CBT2. Cronobacter phage vB_CsaM_CBT2 belongs to a member of the Strabovirida genus and is classified as Cronobacter phage. The genome of Cronobacter phage vB_CsaM_CBT2 described in the present invention is 179,308bp in length and has 278 coding sequences. The Cronobacter phage vB_CsaM_CBT2 described in the present invention has a polyhedral head and tail structure, with a head length of about 105nm, a width of about 72nm, and a tail of about 110nm.

[0036] Through the performance study of the Cronobacter phage vB_CsaM_CBT2 described in the present invention, it was found that the phage has a wide host spectrum, good temperature and pH stability, and good antibacterial effect. The host spectrum of the Cronobacter phage vB_CsaM_CBT2 described in the present invention is wide, and it can target and lyse 17 strains of four species in the genus Cronobacter, such as Cronobacter sakazakii (C.sakazakii), Cronobacter zurich (C.turicensis), Cronobacter muytjensii, and Cronobacter condimenti (C.condimenti), and can provide resource guarantees for the prevention and control of multidrug-resistant Cronobacter clinical strains. The Cronobacter phage vB_CsaM_CBT2 described in the present invention does not contain bacterial resistance and virulence genes, has high safety, and can be applied to the prevention and control of Cronobacter in food.

[0037] The working pH value of the Cronobacter phage vB_CsaM_CBT2 of the present invention is preferably 3 to 11, more preferably 5 to 10, and more preferably 6 to 9. The working temperature of the Cronobacter phage of the present invention is preferably 25°C to 60°C, more preferably 25 to 50°C, even more preferably 25 to 40°C, and more preferably 25°C.

[0038] The pH value and temperature of the present invention are set to improve the lysis effect of Cronobacter phage on Cronobacter.

[0039] The present invention provides the use of the Cronobacter phage described in the above technical solution in preparing a preparation for inhibiting and / or killing Cronobacter.

[0040] The Cronobacter phage vB_CsaM_CBT2 inhibitory and / or Cronobacter killing agent of the present invention preferably comprises a Cronobacter phage vB_CsaM_CBT2 inhibitory or Cronobacter phage vB_CsaM_CBT2 inhibitor prepared in the form of a Cronobacter phage vB_CsaM_CBT2 proliferation solution or a concentrated solution of the Cronobacter phage vB_CsaM_CBT2 proliferation solution. The method for preparing the Cronobacter phage vB_CsaM_CBT2 proliferation solution of the present invention preferably comprises the following steps:

[0041] Cronobacter was first cultured in LB broth medium, and the Cronobacter logarithmic phase bacterial liquid was collected;

[0042] The Cronobacter logarithmic phase bacterial liquid, Cronobacter phage vB_CsaM_CBT2 and LB broth medium are mixed for a second culture to obtain a culture solution, the culture solution is centrifuged to obtain a supernatant, and the supernatant is filtered to obtain a Cronobacter phage vB_CsaM_CBT2 proliferation solution.

[0043] In the present invention, Cronobacter is preferably cultured in LB broth to collect a logarithmic phase culture of Cronobacter. The temperature of the first culture is preferably 35-40°C, more preferably 33-39°C, and even more preferably 37°C. The Cronobacter of the present invention is preferably Cronobacter GZcsf-1.

[0044] After the first culture, the Cronobacter logarithmic phase bacterial solution is collected; the present invention preferably mixes the Cronobacter logarithmic phase bacterial solution and Cronobacter phage vB_CsaM_CBT2 with LB broth medium for a second culture to obtain a culture solution. The temperature of the second culture of the present invention is preferably 30-40°C, more preferably 32-38°C, and more preferably 37°C. The duration of the second culture of the present invention is preferably 2-6 hours, more preferably 4 hours. The volume ratio of the Cronobacter logarithmic phase bacterial solution of the present invention, the Cronobacter phage described in the above technical solution, and the LB broth medium is preferably (1-2): (1-2): 50, more preferably 1:1:50. The ratio of the viable cell count of the Cronobacter phage of the present invention to the Cronobacter bacterial solution is preferably 1:10-1:1000, more preferably 1:70-1:500, even more preferably 1:80-1:200, and more preferably 1:100.

[0045] After obtaining the culture fluid, the present invention preferably centrifuges the culture fluid to obtain the supernatant; the supernatant is filtered to obtain the Cronobacter phage proliferation fluid. The centrifugal force of the centrifugation of the present invention is preferably 3800-4200g, more preferably 4000g, and the centrifugation time is preferably 15-20min, more preferably 18min. The filtration of the present invention is preferably achieved by a filter membrane, and the pore size of the filter membrane is preferably 0.45μm. In the present invention, the working titer of the Cronobacter phage in the Cronobacter phage proliferation fluid is preferably ≥2.95×10 8 pfu / mL, more preferably 6.40×10 9 ~1.18×10 10 pfu / mL, and more preferably 1.13×10 10 ~1.18×10 10 pfu / mL, more preferably 1.18×10 10 pfu / mL.

[0046] After obtaining the Cronobacter phage proliferation liquid, the present invention preferably adds NaCl and polyethylene glycol 8000 to the Cronobacter phage proliferation liquid and then centrifuges to obtain a precipitate, which is then mixed with SM buffer to obtain a Cronobacter phage concentrate.

[0047] The LB broth culture medium of the present invention preferably uses distilled water as a solvent, includes the following components at concentrations: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride, and has a pH value of 6.9-7.1.

[0048] The double LB broth culture medium of the present invention preferably uses distilled water as a solvent, includes the following components at concentrations: 20 g / L tryptone, 10 g / L yeast extract, and 20 g / L sodium chloride, and has a pH value of 6.9-7.1.

[0049] After obtaining the Cronobacter phage proliferation liquid or phage concentrated liquid, a preparation for inhibiting or killing Cronobacter is prepared. The working titer of Cronobacter phage vB_CsaM_CBT2 in the Cronobacter inhibition preparation of the present invention is ≥2.95×10 8 pfu / mL, more preferably ≥1×10 9 pfu / mL.

[0050] The working potency of Cronobacter phage vB_CsaM_CBT2 in the Cronobacter killing preparation of the present invention is ≥1×10 10 pfu / mL, more preferably 1.18×10 10 pfu / mL.

[0051] In the present invention, the Cronobacter species inhibited and / or killed by the Cronobacter phage preferably include one or more of Cronobacter sakazakii, Cronobacter zurichensis, Cronobacter muytjensii and Cronobacter condimenti, more preferably Cronobacter sakazakii, Cronobacter zurichensis, Cronobacter muytjensii and Cronobacter condimenti.

[0052] The present invention provides a Cronobacter inhibitor or bactericide comprising the Cronobacter phage vB_CsaM_CBT2 described in the above technical solution and a pharmaceutically acceptable excipient. The working pH value of the Cronobacter inhibitor or bactericide of the present invention is preferably 3 to 11, more preferably 5 to 10, and even more preferably 6 to 9. The working titer of the Cronobacter phage of the present invention is preferably ≥ 2.95 × 10 8 pfu / mL, more preferably 1×10 9 pfu / mL; the working potency of Cronobacter bactericide is preferably ≥1×10 10 pfu / mL, more preferably 1.18×10 10 pfu / mL.

[0053] In the present invention, the pharmaceutically acceptable excipients preferably include one or more of a dispersant, a stabilizer, a filler and a solvent.

[0054] The present invention provides use of the Cronobacter phage vB_CsaM_CBT2 described in the above technical solution or the Cronobacter inhibitor or bactericide described in the above technical solution in preparing a reagent for preventing and controlling Cronobacter contamination.

[0055] The application of the present invention preferably includes the following steps: mixing the Cronobacter phage vB_CsaM_CBT2 described in the above technical solution or the Cronobacter inhibitor or bactericide described in the above technical solution with a sample containing Cronobacter for inhibition or killing; more preferably, mixing the Cronobacter phage proliferation liquid or concentrated liquid described in the above technical solution with a sample containing Cronobacter for inhibition or killing.

[0056] The method for preparing the Cronobacter phage proliferation solution or concentrated solution of the present invention has been discussed above and will not be repeated here.

[0057] In the present invention, the mixing method is not particularly limited and conventional methods may be used.

[0058] In the present invention, the prevention and control of Cronobacter contamination includes antagonizing Cronobacter contamination in products and / or environments. The product of the present invention is preferably food, and the food of the present invention preferably includes infant food, and more preferably includes infant formula.

[0059] In the present invention, the multiplicity of infection of the reagent is preferably 1:10 to 1:1000, more preferably 1:70 to 1:500, further preferably 1:80 to 1:200, and more preferably 1:100.

[0060] The multiplicity of infection of the Cronobacter phage of the present invention is set to improve the lysis effect of the Cronobacter phage on Cronobacter.

[0061] The titer of the Cronobacter phage vB_CsaM_CBT2 of the present invention does not change significantly under different conditions of temperature of 25-70°C and pH value of 3-11. The titer of the Cronobacter phage vB_CsaM_CBT2 can be maintained at 10 10 pfu / mL, the titer of Cronobacter phage vB_CsaM_CBT2 can be maintained at 10 at pH 4-11. 8 pfu / mL or more. After incubation at pH 3 for 1 h, the titer of Cronobacter phage vB_CsaM_CBT2 was 4.30×10 7pfu / mL, after incubation at pH 11 for 1 h, the titer was 3.27×10 8 pfu / mL; after incubation at 65°C for 1 h, the titer of Cronobacter phage vB_CsaM_CBT2 was 2.827×10 8 pfu / mL. After incubation at 70℃ for 1h, the titer of Cronobacter phage vB_CsaM_CBT2 was 2.60×10 8 pfu / mL.

[0062] The Cronobacter phage vB_CsaM_CBT2 provided by the present invention specifically lyses Cronobacter and can target strains of four multidrug-resistant Cronobacter species, namely Cronobacter sakazakii, Cronobacter zurichensis, Cronobacter muytjensii, and Cronobacter condimenti. Cronobacter phage vB_CsaM_CBT2 has good stability at a pH of 3 to 11 and a temperature of 25 to 70°C, exhibits excellent bactericidal effects, and does not carry any virulence or antibiotic resistance genes, meeting safety requirements in practical applications. The present invention provides an innovative strategy and valuable resource for preventing and controlling Cronobacter contamination in infant formula, its industrial chain, and the environment.

[0063] Cronobacter phage vB_CsaM_CBT2 is used to kill Cronobacter GZcsf-1 in milk powder, with a sterilization effect of up to 80.55-99.97% in milk powder samples within 12 hours. This invention provides a new strategy and resource guarantee for preventing and controlling Cronobacter contamination in infant formula milk powder, its industrial chain and environment, and the development of bacterial agents.

[0064] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0065] The culture medium used in the following examples is:

[0066] The LB broth medium is composed of: distilled water as solvent, 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride, with a pH value of 6.9-7.1.

[0067] The double LB broth medium is composed of: distilled water as solvent, 20 g / L tryptone, 10 g / L yeast extract and 20 g / L sodium chloride, with a pH value of 6.9-7.1.

[0068] When using double LB broth medium, add CaCl2 to a final concentration of 2mM.

[0069] 0.7% LB-Ca soft agar medium is prepared by adding CaCl2 with a final concentration of 2 mM to LB soft agar with an agar mass concentration of 0.7%.

[0070] 1.5% LB agar medium is an LB agar medium with an agar mass concentration of 1.5%.

[0071] 0.4% LB-Ca soft agar medium is prepared by adding CaCl2 with a final concentration of 2 mM to LB soft agar with an agar mass concentration of 0.4%.

[0072] Example 1 Isolation and purification of bacteriophage

[0073] 1. Water sample treatment

[0074] (1) The water sample was sewage collected from the Hefei sewage treatment plant in Anhui Province in 2022.

[0075] (2) Centrifuge the water sample at 10,000 g for 10 minutes. Remove large solid particles and some bacteria by centrifugation to obtain a supernatant. Use a 0.45 μm water filter membrane to vacuum filter the supernatant into a clean, sterile conical flask. Add MgSO4 to the mixture in the conical flask to a final concentration of 50 mM, stir evenly, and let it stand for 15 to 20 minutes to obtain a first mixed solution. Vacuum filter the first mixed solution, discard the supernatant, and collect the filter membrane. Cut the filter membrane into pieces and place it in a beaker filled with an appropriate amount of eluent. Ultrasonicate for 4 to 5 minutes to elute the phage particles from the filter membrane to obtain a second mixed solution. Use a 0.45 μm filter head to filter the second mixed solution into another dry, sterile conical flask. This liquid is the phage mixed solution. Store at 4°C for later use.

[0076] (3) Cronobacter GZcsf-1 was inoculated aseptically into 5 mL of LB broth and cultured overnight to obtain an activated bacterial suspension. The resulting bacterial suspension was inoculated into a fresh 5 mL of LB broth at a volume ratio of 1% and cultured until the logarithmic phase, obtaining a logarithmic phase bacterial suspension of Cronobacter GZcsf-1.

[0077] (4) Take 500 μL of the phage mixture obtained in step (2), 500 μL of double LB broth medium (containing CaCl2 at a final concentration of 2 mM), and 50 μL of the logarithmic phase bacterial solution of Cronobacter GZcsf-1 obtained in step (3), mix them evenly, and culture them at 37°C with shaking for 8 hours to obtain a culture solution. Centrifuge the culture solution at 4000g for 15-20 minutes to obtain a supernatant, which is filtered through a 0.45 μm filter into a sterile centrifuge tube to obtain the phage lysate. Store at 4°C for later use.

[0078] 2. Phage purification

[0079] Pour 1.5% LB agar onto a clean, sterile Petri dish. Add 100 μL of logarithmic-phase Cronobacter GZcsf-1 culture and 100 μL of phage lysate to 5 mL of 0.7% LB-Ca soft agar. Mix thoroughly and spread onto the surface of dry 1.5% LB agar. Incubate at 37°C for 8 h until clearly visible plaques are formed.

[0080] Use a sterile pipette to pick a single, transparent plaque and dissolve it in an appropriate amount of SM buffer. Mix thoroughly, centrifuge at 4000g for 15-20 minutes, and collect the supernatant. Filter the supernatant through a 0.45μm filter to obtain the purified phage solution. Repeat the purification process until the plaques are single, transparent, and distinct (see Figure 1). The isolated Cronobacter phage was designated vB_CsaM_CBT2.

[0081] 3. Phage proliferation and concentration

[0082] To a sterile centrifuge tube, add 1 mL of LB-Ca broth to a sterile centrifuge tube, add 20 μL of Cronobacter GZcsf-1 cultured to logarithmic phase and 20 μL of purified phage solution. Incubate at 37°C, 200 rpm for 4 hours. Centrifuge the resulting culture at 4000 g for 15-20 minutes. Filter the supernatant through a 0.45 μm filter into a new sterile centrifuge tube to obtain the phage growth medium.

[0083] Add NaCl and polyethylene glycol 8000 to the resulting phage growth medium to a final concentration of 0.5 M NaCl and 10% polyethylene glycol 8000, respectively. Incubate at 4°C overnight to allow phage particles to settle. Centrifuge at 12,000 g for 10 minutes, remove the supernatant, collect the precipitate, and redissolve it in SM buffer to obtain the phage concentrate. Store at 4°C until needed. (For long-term storage, add glycerol to a final concentration of 30% and store at -20°C.)

[0084] Example 2 Observation of phage morphology

[0085] The purified liquid of bacteriophage vB_CsaM_CBT2 obtained in Example 1 was dropped onto a copper mesh and absorbed with filter paper after 3 to 5 minutes. A drop of phosphotungstic acid (PTA) was added and stained for 2 to 3 minutes. The excess liquid was then absorbed and dried in air. The mixture was then observed using a field emission transmission electron microscope. The head of bacteriophage vB_CsaM_CBT2 was approximately 105 nm long, 72 nm wide, and 110 nm in tail (see Figure 2).

[0086] Example 3 Phage genome sequencing and analysis

[0087] 1. Phage DNA extraction

[0088] (1) DNase I and RNase A were added to 500 μL of the phage concentrate obtained in Example 1, and the mixture was incubated at 37°C for 60 min (inverted every 30 min). Then, 10% SDS, proteinase K, and EDTA were added, and the mixture was incubated at 65°C for 60 min (inverted every 30 min). An equal volume of Tris-saturated phenol was added, the mixture was vortexed for 30 s, and centrifuged at 12,000 g for 10 min to obtain the upper aqueous phase.

[0089] (2) Pipette the upper aqueous phase into a new centrifuge tube; add a phenol-chloroform-isoamyl alcohol mixture with an equal volume to the aqueous phase, where the volume ratio of phenol, chloroform, and isoamyl alcohol in the mixture is 25:24:1. Vortex and oscillate for 30 seconds, then centrifuge at 12,000 g for 10 minutes to obtain the upper aqueous phase.

[0090] (3) Pipette the upper aqueous phase into a new centrifuge tube, add an equal volume of chloroform, and vortex for 30 seconds to mix the liquid evenly. Centrifuge at 12,000 g for 10 minutes to obtain the upper aqueous phase. Repeat the chloroform extraction process twice to obtain the upper aqueous phase.

[0091] (4) Add an equal volume of isopropanol to the upper aqueous phase, mix well, place at -20°C for 30 min, centrifuge at 12000g for 20 min at 4°C, and collect the precipitate.

[0092] (5) Wash the precipitate with 200 μL of 70% ethanol, centrifuge at 12,000 g for 5 min, discard the supernatant, and retain the precipitate. Repeat the ethanol wash twice.

[0093] (6) Dry the DNA pellet at room temperature, add 20 μL of sterile water preheated at 65°C, and store at -20°C until use.

[0094] 2 Whole-genome analysis

[0095] Whole-genome sequencing of phage DNA was performed using an Illumina NextSeq sequencer. The Illumina sequencing results revealed that phage vB_CsaM_CBT2 is a linear, double-stranded DNA (dsDNA) phage. The complete genome sequence of phage vB_CsaM_CBT2 is 179,308 base pairs long, with a GC content of 44.83% and containing 279 coding sequences. The similarity between phage vB_CsaM_CBT2 and Enterobacteriaceae phage vB_EkoM5VN and Cronobacter phage vB_CsaM_leN is 95.4% and 95.3%, respectively.

[0096] Comparison with the NCBI database revealed that phages vB_EkoM5VN and vB_CsaM_leN belong to the family Straboviridae, while vB_CsaM_CBT2 belongs to a member of the family Straboviridae and is classified as a Cronobacter phage, namely, Cronobacter phage vB_CsaM_CBT2. This conclusion is supported by the Cronobacter phage vB_CsaM_CBT2 genome analysis heat map (Figure 3) and the phylogenetic tree analysis of the terminase large subunit (Figure 4), indicating that vB_CsaM_CBT2 is a Cronobacter phage.

[0097] Compared with the virulence and antibiotic resistance gene databases, bacteriophage vB_CsaM_CBT2 did not contain virulence genes and antibiotic genes, indicating that it can be safely used to control Cronobacter.

[0098] Example 4 Determination of the host spectrum of Cronobacter phage vB_CsaM_CBT2

[0099] 1.5% LB agar medium was spread onto a dry, sterile culture dish and allowed to dry. Prepare the bacterial logarithmic phase solutions numbered 1 to 60 in Table 1, and add 100 μL of the bacterial solution cultured to the logarithmic phase to 5 mL of 0.4% LB-Ca soft agar medium, mix well, spread onto a dried plate, and allow to dry naturally to solidify the soft agar medium. 2 μL of the Cronobacter phage vB_CsaM_CBT2 concentrate prepared in Example 1 was added to the soft agar using the spotting method, dried naturally, and cultured at 37°C for 4 to 6 hours. The culture results were divided into clear spotting areas (+) and no plaques (-) in the spotting areas.

[0100] The results are shown in Table 1. 19 strains of six species of Cronobacter and 41 non-Cronobacter strains were selected. Among them, Cronobacter phage vB_CsaM_CBT2 specifically lysed Cronobacter, and lysed 17 strains of four species of Cronobacter, including Cronobacter sakazakii, Cronobacter zurichensis, Cronobacter muytjensii and Cronobacter condimenti. Among them, serial numbers 1 to 13 belonged to Cronobacter sakazakii, serial numbers 14 to 15 belonged to Cronobacter zurichensis, serial number 16 belonged to Cronobacter muytjensii, and serial number 17 belonged to Cronobacter condimenti. It has no lytic effect on non-Cronobacter species.

[0101] The present invention does not particularly limit the source of the strain for verifying the effect of Cronobacter phage vB_CsaM_CBT2, and conventional strains can be used.

[0102] In this embodiment, there are 60 strains in Table 1, 19 of which are Cronobacter selected from the laboratory of Hefei University of Technology, of which LMG 26250 and NCTC 9529 are standard strains; 10 strains of Burkholderia gladiolus selected, of which ATCC 33664 is the standard strain, and the remaining 9 strains are Burkholderia gladiolus isolated and identified from black fungus, enoki mushrooms, and shiitake mushrooms purchased in various vegetable markets in Hefei from 2021 to 2022; 10 strains of Vibrio parahaemolyticus were isolated from food by the Guangdong Institute of Microbiology; the remaining 21 strains are preserved by Hefei University of Technology.

[0103] Taking the strains LMG 26250, NCTC 9529, ATCC 33664, cro2451A1, G362, and PAO1 in Table 1 as examples, their specific sources are described as follows:

[0104] LMG 26250 and NCTC 9529 are standard strains purchased from Beijing Biobowei Biotechnology Co., Ltd.;

[0105] ATCC 33664 was purchased from Beina Chuanglian Biotechnology Co., Ltd.;

[0106] Strain cro2451A1 can be found in the prior art (Zeng Haiyan, Li Chengsi, Luo Dandan, et al, Novel phage vB_CtuP_B1 for controlling Cronobacter malonaticus and Cronobacter turicensis in ready-to-eat lettuce and powered infant formula. [J]. Food Research International, 2021, 143: 110255);

[0107] Strain G362 refers to the prior art (Ye YingWang, Gao Jina, Jiao Rui, et al, The Membrane Proteins Involved in Virulence of Cronobacter sakazakii Virulent G362 and Attenuated L3101 Isolates. [J]. Frontiers in microbiology, 2015, 6: 01238);

[0108] Strain PAO1 is described in the prior art (Klockgether Jens, Munder Antje, Neugebauer Jens, et al. Genome diversity of Pseudomonas aeruginosa PAO1 laboratory strains. [J]. Journal of bacteriology, 2010, 192(4): 1113-1121.); the sources of the remaining strains are not described here.

[0109] Table 1 Host spectrum of bacteriophage vB_CsaM_CBT2

[0110] Example 5 Determination of the optimal multiplicity of infection of phage

[0111] Cronobacter GZcsf-1 was cultured to the early logarithmic stage, so that the bacterial concentration was 10 8cfu / mL. Different treatment groups were treated with Cronobacter phage vB_CsaM_CBT2 phage growth medium prepared in Example 1 and host Cronobacter GZcsf-1 bacterial culture at multiplicity of infection (MOI) ratios of 10:1, 1:1, 1:10, 1:100, 1:1000, and 1:10,000. The cultures were incubated at 37°C and 200 rpm for 4 hours, centrifuged at 12,000 rpm for 10-15 minutes, and filtered through a 0.45 μm filter. The filtrate was titered using a double-layer agar plate method. Three replicates were performed for each treatment group. The optimal MOI was the one that produced the highest titer.

[0112] The results are shown in Table 2. When the multiplicity of infection (MOI) was 1:100, the titer was the highest, which was 1.18×10 10 pfu / mL, that is, the optimal MOI of bacteriophage vB_CsaM_CBT2 is 1:100.

[0113] Table 2 Optimal multiplicity of infection of phage

[0114] Example 6 Determination of one-step growth curve of phage

[0115] Cronobacter GZcsf-1 was cultured to the early logarithmic stage, so that the bacterial concentration was 10 8 cfu / mL, according to the optimal infection multiplicity of 1:100, the Cronobacter phage vB_CsaM_CBT2 phage growth liquid prepared in Example 1 and the host Cronobacter GZcsf-1 were added, and after 5 minutes of water bath at 37°C, the supernatant was discarded to obtain a precipitate. The precipitate was washed twice with LB medium. 30mL of LB medium preheated at 37°C was added to the obtained precipitate and shaken at 37°C. Samples were taken at 0min, 5min, 10min, 15min, and 20min, and then sampled every 10min. Each sample was centrifuged at 12000rpm for 30s, filtered with a 0.45μm filter head, and the phage titer at each time point was determined. Three parallel experiments were set up. The titer was measured by the double-layer agar plate method, with the infection time as the horizontal axis and the phage titer as the vertical axis, and a one-step growth curve was drawn, as shown in Table 3 and Figure 5.

[0116] Table 3 Phage titer (pfu / mL)

[0117] According to Figure 5 and Table 3, 0–20 min was the incubation period of Cronobacter phage vB_CsaM_CBT2, 20–100 min was the lysis period of Cronobacter phage vB_CsaM_CBT2, and 100–120 min was the stationary period of Cronobacter phage vB_CsaM_CBT2.

[0118] Example 7 Determination of Temperature Stability of Cronobacter phage vB_CsaM_CBT2

[0119] Take 1mL and the titer is 10 10 Place Cronobacter phage vB_CsaM_CBT2 proliferation medium at 1 pfu / mL in a 1.5 mL centrifuge tube and incubate in a 25°C water bath for 1 hour. After incubation, remove the tube and cool on ice. Measure the titer using the double-layer agar plate method. Set up three parallel experiments.

[0120] Example 8

[0121] Take 1mL and the titer is 10 10 Place Cronobacter phage vB_CsaM_CBT2 proliferation medium at 1 pfu / mL in a 1.5 mL centrifuge tube and incubate in a 30°C water bath for 1 hour. After incubation, remove the tube and cool on ice. Measure the titer using the double-layer agar plate method. Set up three parallel experiments.

[0122] Example 9

[0123] Take 1mL and the titer is 10 10 Place Cronobacter phage vB_CsaM_CBT2 proliferation medium at 1 pfu / mL in a 1.5 mL centrifuge tube and incubate in a 37°C water bath for 1 hour. After incubation, remove the tube and cool on ice. Measure the titer using the double-layer agar plate method. Set up three parallel experiments.

[0124] Example 10

[0125] Take 1mL and the titer is 10 10 Place Cronobacter phage vB_CsaM_CBT2 proliferation medium at 1 pfu / mL in a 1.5 mL centrifuge tube and incubate in a 40°C water bath for 1 hour. After incubation, remove the tube and cool on ice. Measure the titer using the double-layer agar plate method. Set up three parallel experiments.

[0126] Example 11

[0127] Take 1mL and the titer is 10 10Place Cronobacter phage vB_CsaM_CBT2 proliferation medium at 1 pfu / mL in a 1.5 mL centrifuge tube and incubate in a 50°C water bath for 1 hour. After incubation, remove the tube and cool on ice. Measure the titer using the double-layer agar plate method. Set up three parallel experiments.

[0128] Example 12

[0129] Take 1mL and the titer is 10 10 Place Cronobacter phage vB_CsaM_CBT2 proliferation medium at 1 pfu / mL in a 1.5 mL centrifuge tube and incubate in a 60°C water bath for 1 hour. After incubation, remove the tube and cool on ice. Measure the titer using the double-layer agar plate method. Set up three parallel experiments.

[0130] Example 13

[0131] Take 1mL and the titer is 10 10 Place Cronobacter phage vB_CsaM_CBT2 proliferation medium at 1 pfu / mL in a 1.5 mL centrifuge tube and incubate in a 65°C water bath for 1 hour. After incubation, remove the tube and cool on ice. Measure the titer using the double-layer agar plate method. Set up three parallel experiments.

[0132] Example 14

[0133] Take 1mL and the titer is 10 10 Place Cronobacter phage vB_CsaM_CBT2 proliferation medium at 1 pfu / mL in a 1.5 mL centrifuge tube and incubate in a 70°C water bath for 1 hour. After incubation, remove the tube and cool on ice. Measure the titer using the double-layer agar plate method. Set up three parallel experiments.

[0134] Comparative Example 1

[0135] Take 1mL and the titer is 10 10 Place Cronobacter phage vB_CsaM_CBT2 proliferation medium at 1 pfu / mL in a 1.5 mL centrifuge tube and incubate in a 75°C water bath for 1 hour. After incubation, remove the tube and cool on ice. Measure the titer using the double-layer agar plate method. Set up three parallel experiments.

[0136] Comparative Example 2

[0137] Take 1mL and the titer is 10 10 Place Cronobacter phage vB_CsaM_CBT2 proliferation medium at 10 pfu / mL in a 1.5 mL centrifuge tube and incubate in an 80°C water bath for 1 hour. After incubation, remove the tube and cool on ice. Measure the titer using the double-layer agar plate method. Set up three parallel experiments.

[0138] The titer determination results of phages incubated for 1 h at different temperatures in Examples 7 to 14 and Comparative Examples 1 to 2 are shown in Table 4 and Figure 6 .

[0139] Table 4 Phage titer determination results (pfu / mL) after 1 h incubation at different temperatures

[0140] According to Table 4 and Figure 6, the titer of Cronobacter phage vB_CsaM_CBT2 remained almost unchanged after being exposed to 25-70°C for 1 h. The titer of phage vB_CsaM_CBT2 decreased by about 8.4 log pfu / mL after being exposed to 65°C and 70°C for 1 h. After being exposed to 75°C and 80°C for 1 h, phage vB_CsaM_CBT2 was completely inactivated.

[0141] Example 15

[0142] Take 100 μL of the titer of 10 10 Place Cronobacter phage vB_CsaM_CBT2 growth medium at 10 pfu / mL in a 1.5 mL centrifuge tube. Add 900 μL of LB broth (pH 3). Place the tube in a 37°C water bath for 1 hour. After incubation, remove the tube and measure the titer using a double-layer agar plate. Set up three replicates.

[0143] Example 16

[0144] Same as Example 15, except that 900 μL of LB broth medium with a pH value of 4 was added.

[0145] Example 17

[0146] Same as Example 15, except that 900 μL of LB broth medium with a pH value of 5 was added.

[0147] Example 18

[0148] Same as Example 15, except that 900 μL of LB broth medium with a pH of 6 was added.

[0149] Example 19

[0150] Same as Example 15, except that 900 μL of LB broth medium with a pH value of 7 was added.

[0151] Example 20

[0152] Same as Example 15, except that 900 μL of LB broth medium with a pH of 8 was added.

[0153] Example 21

[0154] Same as Example 15, except that 900 μL of LB broth medium with a pH of 9 was added.

[0155] Example 22

[0156] Same as Example 15, except that 900 μL of LB broth medium with a pH value of 10 was added.

[0157] Example 23

[0158] Same as Example 15, except that 900 μL of LB broth medium with a pH of 11 was added.

[0159] Comparative Example 3

[0160] Same as Example 15, except that 900 μL of LB broth medium with a pH value of 2 was added.

[0161] Comparative Example 4

[0162] Same as Example 15, except that 900 μL of LB broth medium with a pH of 12 was added.

[0163] The titer determination results of phages incubated for 1 h at different temperatures in Examples 15 to 23 and Comparative Examples 3 to 4 are shown in Table 5 and Figure 7 .

[0164] Table 5 Phage titer determination results (pfu / mL) after 1 h incubation at different pH values

[0165] According to Table 5 and Figure 7, the titer of Cronobacter phage vB_CsaM_CBT2 remained almost unchanged in an environment with a pH value of 3 to 11. At pH 2 and 12, the phage vB_CsaM_CBT2 was completely inactivated.

[0166] Example 24 Bactericidal Effect of Bacteriophage in Prepared Milk Powder

[0167] Three brands of sterile infant formula were used for the experiment. The milk powder was dissolved in sterile water to prepare a milk powder sample with a mass concentration of 10%. Cronobacter GZcsf-1 bacterial solution was added to the milk powder sample to construct an experimental treatment model. Cronobacter GZcsf-1 is described in the prior art (Zeng Haiyan, Lei Tao, He Wenjing, et al. Novel Multidrug-Resistant Cronobacter sakazakii Causing Meningitis in Neonate, China, 2015. [J]. Emerging infectious diseases, 2018, 24(11): 2121.).

[0168] Experimental group: 500 μL 10 5 cfu / mL of Cronobacter GZcsf-1 bacterial solution and 500 μL of 10 10 The phage growth solution prepared in Example 1 at a concentration of 10 pfu / mL was added to 25 mL of milk powder sample and mixed thoroughly. The mixture was then incubated at 4°C and 37°C at 200 rpm. Samples were taken at 0 h, 2 h, 4 h, 6 h, and 12 h, and the bacterial count was determined using the plate count method to determine the bactericidal efficacy. Three replicates were performed for each treatment at 4°C and 37°C.

[0169] Control group: Sterile physiological saline was used instead of phage proliferation solution as the control group, and three parallel experiments were set up for the control group.

[0170] The bactericidal rate of bacteriophage vB_CsaM_CBT2 against Cronobacter was calculated according to the following formula:

[0171] The bactericidal rate (%) of bacteriophage vB_CsaM_CBT2 against Cronobacter is (the bacterial number of host in control group - the bacterial number of host in experimental group) / the bacterial number of host in control group.

[0172] The results are shown in Tables 6 and 7. When cultured at 4°C for 6 h, the sterilization rate of Cronobacter phage vB_CsaM_CBT2 against Cronobacter in brand 1 milk powder sample was about 98.60%; the sterilization rate against Cronobacter in brand 2 milk powder sample was about 77.86%; and the sterilization rate against Cronobacter in brand 3 milk powder sample was about 71.87%. When cultured at 37°C and 200 rpm for 2 h, the sterilization rate of Cronobacter phage vB_CsaM_CBT2 against Cronobacter in brand 1 milk powder sample was about 99.96%; the sterilization rate against Cronobacter in brand 2 milk powder sample was about 99.84%; and the sterilization rate against Cronobacter in brand 3 milk powder sample was about 99.97%. In general, Cronobacter phage was more effective at 37°C than at 4°C. vB_CsaM_CBT2 showed a better killing effect against Cronobacter, especially when cultured at 37°C for 2 h, where the killing rate of Cronobacter phage vB_CsaM_CBT2 against Cronobacter in milk powder samples reached over 99.9%. This indicates that Cronobacter phage vB_CsaM_CBT2 can effectively kill Cronobacter in infant formula.

[0173] Table 6 Number of Cronobacter in different treatment groups of different brands of milk powder (unit: CFU / mL)

[0174] Table 7 Bactericidal rate of bacteriophage vB_CsaM_CBT2 against Cronobacter

[0175] In summary, the Cronobacter phage vB_CsaM_CBT2 provided by the present invention exhibits excellent stability and bactericidal efficacy at pH values ​​of 3-11 and temperatures of 25-70°C. It also lacks any virulence or antibiotic resistance genes, meeting safety requirements for practical applications. Its bactericidal efficacy in milk powder samples reached 80.55-99.97% within 12 hours. This invention provides an innovative strategy and valuable resource for preventing and controlling Cronobacter contamination in infant formula, its supply chain, and the environment.

[0176] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A Cronobacter phage vB_CsaM_CBT2, with the deposit number being: CCTCC NO:M 2023524.

2. Use of the Cronobacter phage vB_CsaM_CBT2 according to claim 1 in preparing a preparation for inhibiting and / or killing Cronobacter.

3. The use according to claim 2, characterized in that: The preparation comprises Cronobacter phage vB_CsaM_CBT2 in the form of a proliferation liquid or a concentrated solution of the proliferation liquid.

4. The use according to claim 3, characterized in that: The preparation method of the proliferation liquid comprises the following steps: The logarithmic phase bacterial liquid of Cronobacter, Cronobacter phage vB_CsaM_CBT2 and LB broth medium are mixed and cultured to obtain a culture liquid, the culture liquid is centrifuged to obtain a supernatant, and the supernatant is filtered to obtain a Cronobacter phage vB_CsaM_CBT2 proliferation liquid.

5. The use according to claim 2, characterized in that: The Cronobacter includes one or more of Cronobacter sakazakii, Cronobacter turicensis, Cronobacter muytjensii and Cronobacter condimenti.

6. The use according to claim 2 or 3, characterized in that: The working titer of Cronobacter phage vB_CsaM_CBT2 in the Cronobacter inhibition preparation is ≥2.95×10 8 pfu / mL, and the working titer of Cronobacter phage vB_CsaM_CBT2 in the preparation for killing Cronobacter is ≥1×10 10 pfu / mL.

7. A Cronobacter inhibitor or bactericide, characterized in that Contains the Cronobacter phage vB_CsaM_CBT2 described in claim 1 and pharmaceutically acceptable excipients.

8. The Cronobacter inhibitor or bactericide according to claim 7, characterized in that The working pH value of the Cronobacter inhibitor or bactericide is 3-11.

9. The Cronobacter inhibitor or bactericide according to claim 7, characterized in that The working titer of the Cronobacter inhibitor is ≥2.95×10 8 pfu / mL; the working potency of Cronobacter bactericide is ≥1×10 10 pfu / mL.

10. The Cronobacter inhibitor or bactericide according to claim 7, characterized in that: The pharmaceutically acceptable excipients include one or more of a dispersant, a stabilizer, a filler and a solvent.

11. Use of the Cronobacter phage vB_CsaM_CBT2 according to claim 1 or the Cronobacter inhibitor or bactericide according to any one of claims 7 to 10 in the preparation of an agent for preventing and controlling Cronobacter contamination.

12. The use according to claim 11, characterized in that: The multiplicity of infection of the reagent is 1:10 to 1:1000.

13. The use according to claim 11, characterized in that: The controlling of Cronobacter contamination includes antagonizing Cronobacter contamination in products and / or the environment.

14. The use according to claim 13, characterized in that The products include food products.

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