Cronobacter bacteriophage and application thereof

A technology of Cronobacter and phage, applied in the field of microorganisms, to achieve the effects of good phage source, good acid-base and temperature tolerance, and good stability

Pending Publication Date: 2022-05-27
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The isolated phage reported in the previous literature has a good inhibitory effect on the growth of Cronobacter sakazakii in milk powder, which has potential application prospects, and has good lysis against Cronobacter malonate and Cronobacter zurichi Effective phages are rarely reported. In order to effectively prevent and control food Cronobacter contamination, it is a technical problem to be solved urgently by those skilled in the art to screen phages that can simultaneously crack Cronobacter malonate and Cronobacter zurichi.

Method used

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  • Cronobacter bacteriophage and application thereof
  • Cronobacter bacteriophage and application thereof
  • Cronobacter bacteriophage and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Isolation and purification of the phage of the present invention

[0026] 1.1 Treatment and enrichment of water samples

[0027] The water samples used for the isolation of phages in the test of the present invention were collected from the urban river water in Guangzhou in 2018.

[0028] Centrifuge the water sample at 10000g for 10min to remove some solid large particles and some bacteria, and collect the supernatant. The supernatant was vacuum filtered and transferred to a clean, sterile container. Add MgSO to a final concentration of 50 mM 4 . Stir well and let stand for 15-20min. After vacuum filtration of the mixture, the supernatant was discarded and the filter membrane was collected. Cut the filter membrane into pieces, put it into a clean and sterile beaker, add an appropriate amount of eluent, and stir and mix well. Transfer the beaker to an ultrasonic cleaner, and ultrasonically elute for 4-5min to fully elute the phage particles from the fil...

Embodiment 2

[0033] Example 2 Whole genome sequencing of phage and new species identification analysis

[0034] DNase I and RNase A were added to 0.5 mL of the purified phage suspension in Example 1, proteinase K, SDS and EDTA were added after incubation at 37°C for 30 minutes, and an equal volume of phenol was added after incubation at 65°C for 30 minutes, and vortexed. Centrifuge at 12000g for 30s for 5min, transfer the upper aqueous phase to a new centrifuge tube. The phage genome was extracted by the phenol-chloroform-isoamyl alcohol method (25:24:1), vortexed for 30 s, centrifuged at 12,000 g for 5 min, and the upper aqueous phase was transferred to a new centrifuge tube; then extracted with an equal volume of chloroform, and repeated Extract until there is no smell of phenol; add an equal volume of isopropanol to the upper extraction solution, mix well, place at -20 °C for 30 min, and then centrifuge at 12000g for 20 min at 4 °C to collect the precipitate; wash the DNA with 70% eth...

Embodiment 3

[0036] Example 3 Determination of the host profile of phage

[0037] After gently mixing 0.1 mL of the bacterial culture solution in Table 1 in the logarithmic phase with 0.7% agar soft agar LB medium (5 mL), pour it on the common LB bottom plate, and dry it naturally for 15 minutes to fix the upper medium. . Then, 5 μL of phage vB_CtuP_B1 purified solution was spotted onto the solidified upper plate, and after natural drying, cultured at 37°C for 6 h or overnight, and the appearance of plaques indicated that the strain was a sensitive host. Results It can be seen from Table 1 that the phage of the present invention can lyse most of Cronobacter malonate and Cronobacter zurich, but cannot lyse other species of Cronobacter. Therefore, the phage obtained by the present invention has certain specificity to Cronobacter malonate and Cronobacter zurich (Table 1).

[0038] Table 1 Phage vB_CtuP_A24 host spectrum

[0039]

[0040]

[0041] In Table 1, "+" indicates that plaq...

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Abstract

The invention provides a Cronobacter bacteriophage, the Cronobacter bacteriophage is Cronobacter bacteriophage vBCtuPB1, the preservation number of the Cronobacter bacteriophage vBCtuPB1 is GDMCC No: 61185-B1, the Cronobacter bacteriophage is a short-tail bacteriophage, the short-tail bacteriophage is of a polyhedral head and short-tail structure, the head is about 51 mm, and the tail is about 11 mm. The bacteriophage can be used for specifically splitting and splitting the cronobacter malonate and the cronobacter suridatensis. The bacteriophage vBCtuPB1 has good stability in the pH range of 4.0-11 and at the temperature of 25-60 DEG C, does not carry any virulence or antibiotic resistance gene, and can be safely applied to prevention and control of food cronobacter.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a Cronobacter phage and its application. Background technique [0002] Cronobacters sakazakii (Cronobacterspp. (Enterobacter sakazakii)) is an important food-borne pathogen that can be infected by people of all ages, especially in premature infants, low birth weight infants or immune-compromised infants. Infant damage can lead to meningitis, bacteremia, and necrotizing enterocolitis, with mortality rates as high as 40 to 80 percent. The International Food and Agriculture Organization and the World Health Organization have listed Cronobacter as a Class A pathogen in infant formula milk powder. There are 7 species of this genus, including C. sakazakii, malonate C. malonaticus and C. turicensis have been reported to cause clinical infection. my country's national food safety standard "GB 10765-2010 National Food Safety Standard Infant Formula" expressly stipulat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A23L5/20A23C7/04A01N63/40A01P1/00C12R1/92
CPCC12N7/00A23L5/28A23C7/04A01N63/40C12N2795/10221C12N2795/10231Y02A50/30
Inventor 曾海燕李程思吴清平张菊梅罗丹丹丁郁陈谋通薛亮王涓吴诗雷涛庞锐杨美艳
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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