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An imine-resistant bacillus pyocyaneus bacteriophage and its use for treating infection therefrom

A technology for Pseudomonas aeruginosa infection and Pseudomonas aeruginosa, which is applied in the directions of medical raw materials derived from viruses/phages, viruses/phages, antibacterial drugs, etc., and can solve problems such as the feasibility of hindering the treatment of phage preparations.

Inactive Publication Date: 2008-01-23
TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Of course, the mechanism of phage damage to host bacteria is still debated, but this does not prevent the feasibility of phage preparations for treatment

Method used

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  • An imine-resistant bacillus pyocyaneus bacteriophage and its use for treating infection therefrom
  • An imine-resistant bacillus pyocyaneus bacteriophage and its use for treating infection therefrom
  • An imine-resistant bacillus pyocyaneus bacteriophage and its use for treating infection therefrom

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Take 1L of sewage from the sewage treatment center of Tongji Hospital, add 58g of NaCl, and centrifuge at 10,000×g for 10min. Take the supernatant, add PEG-8000 to a final concentration of 10% (w / v), put it in a refrigerator at 4°C overnight, and centrifuge at 10,000×g for 20min at 4°C. Resuspend the pellet with 5ml SM Buffer. Extract with an equal volume of chloroform once. Mix 300 μl of treated sewage with 200 μl of overnight cultured host bacteria, incubate at 37°C for 20 minutes, mix with 3ml of melted top layer agar at 50°C, spread the plate, cool and invert at 37°C for overnight culture. Remove the largest plaque with a tip, dissolve it in 2ml LB culture solution, add imipenem-resistant Pseudomonas aeruginosa φA392 cultivated overnight to the culture solution at a ratio of 1:100, shake at 250 rpm at 37°C 4.5 to 5 hours. Centrifuge at 12,000 rpm for 5 minutes, and take 80% of the supernatant and store it at 4°C. Take this supernatant and co-culture with φA392 b...

Embodiment 2

[0035] The overnight cultured host bacteria φA392 was diluted 1:100, and cultured to the early logarithmic growth phase. Add 10 μl of the concentrated phage in Example 1, continue to incubate for 5 hours, add 1 / 10 volume of chloroform, continue shaking for 10 minutes, then add DNase I and RNase A to a final concentration of 1 μg / ml [3] , placed at room temperature for 30 minutes, and centrifuged to remove bacterial debris. Add 1 / 6 volume of PEG / NaCl, overnight at 4°C. The next day, centrifuge at 12,000×g for 20 minutes at 4°C. Resuspend the pellet with 1ml SM Buffer. Add 1 / 6 volume of PEG / NaCl to precipitate again, incubate on ice for 1 hour, centrifuge at 12,000×g for 20 minutes at 4°C. Resuspend the pellet with 200 μl SM Buffer to obtain amplified phage, which can reach the desired titer after repeated amplification.

Embodiment 3

[0037] Use 30 strains of diluted imipenem-resistant Pseudomonas aeruginosa to make a uniform lawn on LB medium. Divide and mark 16-20 squares on the back of the culture medium, then drop phage φA392 solution into the corresponding squares, and after the droplets are dry, place them upside down at 37°C for 12-16 hours to observe the results (Figure 4). It can be seen that φA392 forms plaques on 22 strains, as follows: IMR-Pa 91, 15, 9915, 9472, 9986, 9747, 9484, 8095, 8145, 505, 9431, 7827, 1014, 1092, A392, A399 , 1423, 1505, 1612, 1182, 1578, 2024.

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Abstract

Disclosed is an imine-resistant bacillus pyocyaneus bacteriophage and its use for treating infection, wherein a stain of broad-spectrum drug-resistant bacillus pyocyaneus bacteriophage phiA392, which can effectively kill drug-resistant bacillus pyocyaneus in vivo and in vitro, the bacteriophage can decompose multiple strains of clinically segregated imipenem-resistant bacillus pyocyaneus in vivo. The invention proves the superiority and feasibility of bacteriophage as a therapeutic substance in the clinical application to the treatment of bacillus pyocyaneus resistant affection, and provides a novel therapeutic means for clinical imipenem-resistant bacillus pyocyaneus affection.

Description

【Technical field】 [0001] The present invention relates to an isolated wide phage and its therapeutic application 【Background technique】 [0002] Infection is a disease that has plagued human health for a long time. Although many infectious diseases that were previously unheard of have been effectively controlled since the advent of antibiotics, due to the emergence of drug resistance, clinical infection control has become a difficult problem again, especially at present, the emergence of clinical drug-resistant bacteria has greatly accelerated. Beyond the update speed of antibiotics, human beings are already facing a situation where no antibiotics are available. Moreover, some antibiotics themselves have high toxicity and side effects, and their selectivity is not strong, which can cause damage to the body or lead to superinfection. Therefore, it is necessary to study new treatments. [0003] In recent years, phage therapy has gradually gained people's attention as a biol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01A61K35/76A61P31/04
Inventor 胡俊波王晶胡北徐敏超
Owner TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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