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Lytic klebsiella pneumoniae bacteriophage RDP-KP-20005 and application thereof

A technology of RDP-KP-20005 and BK-20005, which is applied in the direction of phage, virus/phage, and medical raw materials derived from virus/phage, can solve the problems of bacterial resistance, untimely treatment, and ineffective medication, etc. High price, good control effect and good application prospect

Active Publication Date: 2021-04-27
RECOM QINGDAO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a strain of lytic Klebsiella pneumoniae phage RDP-KP-20005, aiming to solve the problem of rapid onset of Klebsiella pneumoniae and untimely treatment of poultry in farms due to infection of Klebsiella pneumoniae, and drug resistance of pathogenic bacteria issues such as drug ineffectiveness due to sex

Method used

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  • Lytic klebsiella pneumoniae bacteriophage RDP-KP-20005 and application thereof
  • Lytic klebsiella pneumoniae bacteriophage RDP-KP-20005 and application thereof
  • Lytic klebsiella pneumoniae bacteriophage RDP-KP-20005 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Isolation and preservation of embodiment 1 Klebsiella pneumoniae BK-20005

[0029] The host was sampled from a farm in Tianheng, Jimo, Qingdao, and the diseased chicken liver was taken aseptically, and the chromogenic medium for localization of urethral flora of France was used to isolate the bacteria by streaking. After culturing at 37°C for 18-24 hours, metallic blue colonies with red halos appeared on the culture medium. Pick typical colonies and continue to streak and purify for 3 times, then pick a single colony and inoculate it in 5mL LB broth, 37 Cultivate with shaking at 200rpm for 8 hours to obtain a uniform turbid bacterial suspension. Klebsiella pneumoniae is a Gram-negative bacillus (0.5-0.8μm×1-2μm), without spores, without flagella, oval and short rod-shaped .

[0030] The isolated bacteria were inoculated in various biochemical test media, cultured at 37°C for 24 to 96 hours, and the biochemical reaction results were observed. The biochemical results sho...

Embodiment 2

[0033] Example 2 Isolation and identification of bacteriophage RDP-KP-20005

[0034] (1) The sewage sample used in the test of the present invention is collected from the surrounding sewage of the diseased farm, and is used as a water sample for bacteriophage isolation.

[0035] (2) Separation: Take 10 milliliters of sewage, centrifuge at 10,000 rpm for 5 minutes, remove larger impurities and most of the bacteria, and then use a 0.22 micron microporous membrane to filter and sterilize. Take 3 milliliters of the filtrate and 3 milliliters of the host bacterial suspension, add them together into 20 milliliters of autoclaved LB broth, and then place them in a 37°C incubator for overnight culture. After culturing, take 5 ml, centrifuge at 10,000 rpm for 5 minutes, and then filter and sterilize with a 0.22 micron microporous filter. The filtrate is the stock solution intended to contain phage. Then, the phages were identified using the double-plate method.

[0036] (3) Purificati...

Embodiment 3

[0038] Electron microscope observation of embodiment 3 phage

[0039] Using phosphotungstic acid negative staining method: drop phage liquid (titer 10 10 pfu / m L) 100 μL, place the membrane side of the copper mesh on the phage droplet, take it off after 10 minutes, and let it dry naturally in the air for 2-3 minutes. Then drop a drop of 2% phosphotungstic acid (PTA) aqueous solution on the copper grid for staining, take it off after 10 minutes, dry it in the air for 10-15 minutes, observe with an electron microscope, and select a clear phage image to take pictures.

[0040] Such as figure 1 As shown, the head of bacteriophage RDP-KP-20005 is icosahedral, with a length of 92.281nm and three-dimensional symmetry, and a short tail with a tail length of 107.442nm. It belongs to the Brachyphage family of the tailed bacteriophage order.

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Abstract

The invention discloses a lytic klebsiella pneumoniae bacteriophage RDP-KP-20005, and a host of the bacteriophageis klebsiella pneumoniae BK-20005; the head of the bacteriophage is icosahedron, and is 92.281 nm long, and the bacteriophage is in cubic symmetry and has a short tail, the tail length is 107.442 nm; the bacteriophage belongs to caudovirales podoviridae, the gene full length is48585bp; the bacteriophage does not have a virulence gene or alysogenic gene; and the bacteriophage is collected in China General Microbiological Culture Collection Center on December 17, 2020, and the collection number is CGMCC No. 21408. The bacteriophage can effectively decompose klebsiella pneumoniae, and can effectively and rapidly kill cracking bacteria to prevent and control propagation and proliferation of the klebsiella pneumoniae.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a lytic Klebsiella pneumoniae phage RDP-KP-20005 and its application in poultry disease prevention and control. Background technique [0002] With the development of intensive poultry farming, some common acute infectious diseases have been controlled by vaccines and drugs, while some previously considered unimportant pathogens or opportunistic pathogens have gradually become important pathogens of poultry. , causing mass morbidity and mortality in poultry. For example, chicken colibacillosis and chicken proteus mirabilis disease have been reported more frequently in recent years. Klebsiella pneumoniae (Klebsiella pneumoniae) invading the reproductive and digestive organs of breeder chickens can cause bacterial infection in breeding eggs and lead to dead embryos, bacterial infection in young chicks or direct infection of chicks, causing diarrhea and enteritis sepsis death. The incid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K35/76A61K45/06A61P31/04A23K10/18A23K20/195A23K50/75C11D3/38C11D3/48A01N63/40A01P1/00C12R1/92
CPCC12N7/00A61K35/76A61K45/06A61P31/04A23K10/18A23K20/195A23K50/75C11D3/381C11D3/48A01N63/40C12N2795/10221C12N2795/10231C12N2795/10232A61K2300/00
Inventor 杜新永李先胜王立坤罗成盛赵丹丹张庆刘玉庆盖春云马如霞刘爽
Owner RECOM QINGDAO BIOTECH CO LTD
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