New klebsiella pneumoniae phage and application thereof

A Klebsiella and phage technology, applied in the direction of phage, virus/phage, application, etc., can solve the problems of the increase in the isolation rate of highly virulent Klebsiella pneumoniae-resistant bacteria and the difficulty of treatment, and achieve less toxic and side effects. , strong disinfecting effect, good disinfecting effect

Active Publication Date: 2019-11-12
南宁鑫创生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To make matters worse, the large-scale use of broad-spectrum antibiotics in recent years has led to an increase in the isolation rate of highly virulent Klebsiella pneumoniae drug-resistant bacteria year by year, and even the emergence of fully drug-resistant strains, which brings greater difficulties to the treatment of this bacterial infection. Human life safety and economic benefits have brought great threats, so it is urgent to find a new antibacterial drug

Method used

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  • New klebsiella pneumoniae phage and application thereof
  • New klebsiella pneumoniae phage and application thereof
  • New klebsiella pneumoniae phage and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Isolation of Klebsiella pneumoniae Phage vB_KpnM_Bp5

[0034] The samples were collected from the sewage in a septic tank of a pig farm in Wuming, Guangxi. The samples were centrifuged at 4°C and 12,000 rpm for 20 minutes, and the supernatants were filtered with 0.45 μm and 0.25 μm membranes, respectively. Take 5mL of the filtrate, add 0.1mL of the host bacteria preserved in the China Center for Type Culture Collection, add sterile CaCl 2 After mixing the mother solution to a final concentration of 1mM, add 5mL of 2×LB liquid medium and place it at 37°C for 12-16h. The next day, the above culture was centrifuged at 4°C and 12000 rpm for 10 min, and the supernatant was sterilized by filtration with a 0.25 μm filter membrane to form a stock solution containing phage, that is, a phage suspension.

[0035] Streak-inoculate the host bacteria preserved in the China Center for Type Culture Collection on the agar medium. After culturing overnight, pick a single clone and inocu...

Embodiment 2

[0040] Amplification and purification of Klebsiella pneumoniae phage vB_KpnM_Bp5

[0041] Take 0.1ml of the spare phage of Example 1 and 0.1ml of the spare host bacteria culture of Example 1 to act in a test tube for 15min, add 10ml of LB liquid medium, cultivate at 37°C for 6h, centrifuge at 4°C and 12000rpm for 20min, take the supernatant, Filter through a 0.25 μm membrane filter, and the filtrate is the phage lysate.

[0042] PEG purification: add DNaseI and RNase A to the phage lysate to a final concentration of 1 μg / ml, incubate at 37°C for 30 minutes, add NaCl with a final concentration of 1M in an ice bath for 1 hour, centrifuge at 12,000 rpm at 4°C for 10 minutes, take the supernatant and add PEG8000 with a final concentration of 10%, overnight at 4°C, centrifuge at 12,000rpm for 10min at 4°C, discard the supernatant, invert for 5min, add SM buffer to resuspend, add an equal volume of chloroform for gentle shaking for 30s, and centrifuge at 5,000rpm for 15min at 4°C. ...

Embodiment 3

[0045] Transmission electron microscope observation of Klebsiella pneumoniae phage vB_KpnM_Bp5

[0046] The phage suspension purified in Example 2 was observed with an electron microscope, and the phage suspension purified in Example 2 was dropped on a copper sheet, and naturally precipitated for 2 to 3 minutes, and the excess liquid was absorbed with filter paper, and a drop of 2% phosphotungstic acid was dropped (PTA, 2%w / v) staining, use transmission electron microscope observation after room temperature drying; Observation result is as follows figure 2 As shown, the phage has an icosahedral head and a retractable tail. The diameter of the head is about 53nm, and the length of the tail is about 56nm. Classification - The Eighth Report of the International Committee on Taxonomy of Viruses", the phage belongs to Myoviridae.

[0047] The applicant self-named the phage vB_KpnM_BP5, and it was deposited in the China Center for Type Culture Collection (CCTCC) on June 13, 2019. ...

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Abstract

The invention discloses new klebsiella pneumoniae phage vB_KpnM_Bp5. The klebsiella pneumoniae phage (klebsiella pneumoniae phage) phage ) vB_KpnM_Bp5 is preserved in the China Center for Type CultureCollection (CCTCC) on June 13, 2019, and the preservation number is CCTCC NO:M 2019452. The phage has better disinfecting and sterilizing effects on host multidrug resistant klebsiella pneumoniae separated from piggery sewage, also has good disinfecting and sterilizing effects on other two multidrug resistant klebsiella pneumoniae separated from hospitals, is wide in antimicrobial spectrum, and can be applied to preparation of medicines for preventing and treating multidrug resistant klebsiella pneumoniae.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a new Klebsiella pneumoniae phage and its application. Background technique [0002] Klebsiella pneumoniae (Klebsiella pneumoniae, KP) is a Gram-negative bacterium belonging to the Enterobacteriaceae (Enterobacteriaceae) Klebsiella genus (Klebsiella). Klebsiella pneumoniae is a common zoonotic pathogen that can cause pneumonia, meningitis, liver abscess, wound infection, sepsis and other diseases in humans and animals. It is one of the common opportunistic pathogens in human medicine , but also caused considerable economic losses to the aquaculture industry. [0003] Antibiotics such as β-lactams, tetracyclines, sulfonamides, and quinolones are the most important drugs for the prevention and treatment of bacterial infections in veterinary medicine. Due to long-term drug selection pressure, pathogenic bacteria have shown serious resistance to these drugs, and even The emergence of m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K35/76A61P31/04A01N63/00A01P1/00C12R1/92
CPCA01N63/00A61K35/76A61P31/04C12N7/00C12N2795/10121C12N2795/10131C12N2795/10132
Inventor 王晓晔张聪李珣韦德源葛晨玲胡传活司红彬
Owner 南宁鑫创生物科技有限公司
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