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A drug-resistant Klebsiella pneumoniae phage rdp-kp-20004 and its application

A technology of RDP-KP-20004 and Klebsiella pneumoniae, which is applied to viruses/phages, medical raw materials derived from viruses/phages, applications, etc., can solve the problems of drug resistance of bacteria, untimely treatment, and ineffective medication, etc. Achieve the effect of high potency, good control effect and good application prospect

Active Publication Date: 2021-11-26
山东润达检测技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a strain of drug-resistant Klebsiella pneumoniae phage RDP-KP-20004, aiming to solve the problem of rapid onset of poultry in farms due to Klebsiella pneumoniae infection and untimely treatment, and the pathogenic bacteria are resistant to Drug properties lead to problems such as ineffectiveness of medication

Method used

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  • A drug-resistant Klebsiella pneumoniae phage rdp-kp-20004 and its application
  • A drug-resistant Klebsiella pneumoniae phage rdp-kp-20004 and its application
  • A drug-resistant Klebsiella pneumoniae phage rdp-kp-20004 and its application

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Effect test

Embodiment 1

[0029] Isolation and preservation of embodiment 1 Klebsiella pneumoniae BK-20004

[0030] The host was sampled from a farm in Tianheng, Jimo, Qingdao, and the diseased chicken liver was taken aseptically, streaked on the selective medium, and cultured at 37°C for 18-24h. The bacteria formed thick, off-white, round Convex, smooth colonies with moist surface; thick and rich pink viscous colonies formed on MacConkey agar medium, with diffusivity; white viscous precipitates formed in LB broth, which became uniform and turbid after shaking; pick Take typical colonies and continue to streak and purify for 3 to 5 times until the colonies have the same shape, then pick a single colony and inoculate it in 5 mL of LB broth medium, shake it at 200 rpm for 12 hours at 37°C, and obtain a uniform turbid bacterial suspension. Then through 16sRNA molecular identification and serotype identification, it was determined to be pathogenic Klebsiella pneumoniae, and one of the strains was named BK-...

Embodiment 2

[0031] Embodiment 2 host bacterium BK-20004 drug susceptibility test

[0032] According to the implementation standard M100-S17CLSI of antibiotic susceptibility test, the susceptibility test of six commonly used clinical antibiotics, ampicillin, ceftazidime, cefoperazone, kanamycin, cefepime and ciprofloxacin, was carried out with Escherichiacoli ATCC 25922 as the test Quality control strains for resistance spectrum testing. The specific method is as follows:

[0033] (1) Preparation of MHA medium: Melt the sterilized MHA medium, cool it to about 50°C and pour it into a flat plate with a thickness of about 4 mm, and let it stand for half an hour to solidify the medium.

[0034] (2) Preparation of bacterial liquid: 10 μL of host BK-20004 and quality control strain were pipetted into 5 mL of liquid LB, and incubated at 37° C. and 200 rpm for 12 hours with shaking.

[0035] (3) Take 6-6.5mL of heated and melted liquid agar powder, add it to a 10mL sterilized EP tube, then draw ...

Embodiment 3

[0044] Example 3 Isolation and identification of bacteriophage RDP-KP-20004

[0045] (1) Disease material treatment: take diseased chickens from the farm, dissect the liver, cut 2g of liver tissue by aseptic operation, add 2mL of sterilized LB liquid, grind the liver fully, then centrifuge at 10000rpm for 30min, and take the supernatant for filtration. 0.22μm filter, the filtrate is used for later use;

[0046] (2) Preparation of phage enrichment solution: Take 0.2mL of bacterial suspension and 1mL of filtrate and add it to 5mL of LB broth, culture overnight at 37°C with shaking at 100rpm, then centrifuge at 10000rpm for 5min, take the supernatant and pass it through a 0.22μm filter, filter out of liquid standby;

[0047] (3) Separation of phage: Separation of phage by the double-layer plate method, take 0.1mL of the filtrate of the phage enrichment liquid and 0.2mL of the host Klebsiella suspension, mix evenly, bathe in 37°C water for 10min, and spread double plates After c...

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Abstract

The invention discloses a drug-resistant Klebsiella pneumoniae phage RDP-KP-20004 and its application. The phage preservation number is CGMCC No.21407. After sequencing the whole gene, it is found that it does not have virulence genes and lysogens. The drug-resistant Klebsiella pneumoniae phage RDP-KP-20004 provided by the present invention has higher temperature tolerance and wider acid-base tolerance range, and is effective for pathogenic Klebsiella pneumoniae Klebsiella in the poultry breeding environment. Klebsiella pneumoniae BK-20004 has a strong lysis effect, and provides a phage source for industrial production of phages for the prevention and treatment of pathogenic Klebsiella pneumoniae BK-20004 in poultry breeding environments; Klebsiella pneumoniae BK-20004 provided by the present invention 20004 has good advantages in the treatment of diseases after infection with Klebsiella pneumoniae, with high potency and good control effect; it has a good application prospect in the prevention and control of poultry pathogenic Proteus.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a drug-resistant Klebsiella pneumoniae phage RDP-KP-20004 and its application in poultry animal pathogenic Klebsiella pneumoniae infection. Background technique [0002] The discovery and application of antibiotics is a great revolution for mankind. Since its appearance, it has been playing an important role. Its special curative effect on bacterial infection has promoted the rapid leap of human medical level. However, the non-standard use of antibiotics has led to bacterial resistance and even multi-drug resistance has become an increasingly prominent problem. Medical researchers point out that more than 50% of antibiotics are misused in the world every year, and the proportion in China is even close to 80%. In addition to the medical field, the excessive use of antibiotics in planting and breeding is also serious, which will directly affect food safety and threaten the health of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00A61K35/76A61K45/06A61P31/04A23K10/18A23K20/195A23K50/75C11D3/38C11D3/48A01N63/40A01P1/00C12R1/92
Inventor 杜新永李先胜盖春云赵丹丹罗成盛张庆刘玉庆王立坤马如霞刘爽
Owner 山东润达检测技术有限公司
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