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Pathogenic Escherichia coli phage oral preparation and preparation method thereof

A technology for oral preparations of Escherichia coli, which is applied in the field of oral preparations of pathogenic coliphage phages and its preparation, can solve the problems of insufficient products, and achieve the effect of avoiding evolution and the generation of drug-resistant strains

Active Publication Date: 2020-04-24
HENGYANG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are not many phages and related phage preparations that can lyse pathogenic Escherichia coli, and it is difficult to meet market demand

Method used

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  • Pathogenic Escherichia coli phage oral preparation and preparation method thereof
  • Pathogenic Escherichia coli phage oral preparation and preparation method thereof
  • Pathogenic Escherichia coli phage oral preparation and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Isolation and purification of enterotoxigenic Escherichia coli K88 phage

[0026] 1. Isolation of phage:

[0027] In July 2016, in a medium-sized farm in the suburbs of Hengyang, severe diarrhea occurred in multiple pens of piglets within 2 months, and the disease could not be controlled after antibiotic treatment. The mortality rate was as high as 20%. After isolation, a highly toxic strain of Enterotoxigenic Escherichia coli E.coli-HuNHY19 was deposited on April 24, 2019 in the General Microbiology Center of the China Committee for the Collection of Microorganisms (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), and its preservation number It is CGMCC No.17622. After typing and identification, E.coli-HuNHY19 was O8: K91, K88ac, indicating that the main prevalent pathogenic Escherichia coli was enterotoxigenic Escherichia coli K88.

[0028] In this regard, sewage samples were further collected at the discharge port of the sewage tre...

Embodiment 2

[0046] 1. Temperature Sensitivity of Phage-HY19

[0047] Take 600 µL for a titer of 10 8 The phage liquid of pfu / mL was placed in 5 sterile EP tubes, respectively, at 50°C, 60°C, 70°C, 80°C, and 90°C for 20 min, 40 min, and 60 min, and each temperature was set for 3 replicates , after the action time was over, samples were taken, immediately placed in a water bath to cool down, and then the titer of the phage was determined, and the experiment was repeated 3 times. The temperature sensitivity of Phage-HY19 as image 3 As shown, the activity of phage Phage-HY19 did not change significantly after being treated at 50-70°C for 1 hour; after being treated at 80°C for 40 minutes, all phages were inactivated; after being treated at 90°C for 20 minutes, all phages were inactivated.

[0048] Determination of the host spectrum of phage Phage-HY19

[0049] 2.1 Test material:

[0050] E.coli-HuNHY19 (preservation number is CGMCC No.17622, ​​O8: K91, K88ac, self-isolated and identified...

Embodiment 3

[0056] Embodiment 3: Activity test of bacteriophage Phage-HY19

[0057] 3.1 Toxicity test

[0058] Take a total of 20 5-week-old female experimental Kunming mice, and randomly divide them into two groups, each with 10 mice, wherein group 1 is the test group, and the oral concentration is 10 9 Pfu / ml phage culture solution 2mL, group 2 was the control group, the same amount of PBS was orally administered for 14 days, the mice were killed by neck dislocation, and the changes of viscera, digestive tract and mucous membrane were observed. The results showed that the phage Phage-HY19 had no effect on the daily behavior of the mice, and there was no abnormality in the anatomical examination.

[0059] In vitro bactericidal activity

[0060] Escherichia coli E.coli-HuNHY19 (preservation number: CGMCC No. 17622), CVCC1524 (O8: K91, K88ac) cultured overnight was 1.0 mL each (the bacteria content was 1×10 9 cfu / mL) mixed and OD it with LB 600nm The value is adjusted to 1.0 (the bacte...

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Abstract

A phage Phage-HY19 with a strong cracking effect on pathogenic Escherichia coli ETEC-K88ac is separated from a farm environment, and is processed into an oral preparation, the oral preparation has thecharacteristic of wide host range for the main pathogen ETEC-K88ac causing animal diarrhea, and the phage product is nontoxic, so the phase product can be safely applied to prevention and treatment of animal diarrhea; and the ETEC-K88ac is specifically cracked through phage, so ETEC-K88ac evolution and drug-resistant strains caused in the use process of antibiotics can be avoided.

Description

Technical field: [0001] The invention relates to an oral preparation of pathogenic colibacillus phage and a preparation method thereof, belonging to the field of biotechnology. Background technique: [0002] Pathogenic Escherichia coli is a common pathogen that causes animal diarrhea, and antibiotics are the main treatment in clinical treatment. However, in recent years, the use of antibiotics in the clinical treatment of animal diarrhea is often careless and unreasonable, making Escherichia coli resistant And drug resistance is becoming more and more serious, and the prevention and treatment of colibacillosis is becoming more and more difficult. With the emergence of multiple drug-resistant strains and the emergence of disadvantages such as drug resistance transfer, it is imperative to find effective, green, safe, and pollution-free antibacterial or bacteriostatic agents to replace antibiotics to control pathogenic Escherichia coli. Phages are a type of bacteria-dependent ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/76A61K9/14A61K47/36A61P31/04A61P1/12C12N7/00C12N7/02
CPCA61K35/76A61K9/146C12N7/00A61P31/04A61P1/12C12N2795/00051Y02A50/30
Inventor 唐青海杨海阳坤滕威杨灿王芳宇易诚何丽芳刘会敬唐姣玉曹丽敏唐斯萍
Owner HENGYANG NORMAL UNIV
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