Acinetobacter baumannii phage and its application

A technology of Acinetobacter baumannii and bacteriophage, applied in the field of microbial engineering, can solve the problem of too high specificity requirements of host bacteria, and achieve the effects of high clinical application value, small toxic and side effects, and high safety.

Active Publication Date: 2017-09-12
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, thousands of clinical cases of phage therapy have accumulated in humans, but phage therapy has not been widely used in the past few decades. In addition to the discovery and widespread use of antibiotics, the fundamental reason is that phages require too much specificity for host bacteria. High, it is impossible for people to find so many type-specific phages to adapt to so many types and different types of bacterial infections

Method used

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  • Acinetobacter baumannii phage and its application
  • Acinetobacter baumannii phage and its application
  • Acinetobacter baumannii phage and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1 Isolation and preparation of Acinetobacter baumannii phage

[0017] (1) Sewage collection and treatment

[0018] The sewage was collected from the untreated sewage station of the First Affiliated Hospital of the Third Military Medical University, centrifuged at 10,000 r / min at 4°C for 10 minutes, and filtered with a 0.22 μm filter membrane to obtain the supernatant.

[0019] (2) Specific amplification of Acinetobacter baumannii phage

[0020] Get 100mL of the supernatant obtained in step (1), add 50mL LB liquid medium and mix well, add Acinetobacter baumannii host bacterial liquid (OD 600nm (0.6) 2mL. After mixing, place in a constant temperature shaker at 37°C and shake at 160r / min for overnight culture. The next day, centrifuge at 10,000r / min for 10min at 4°C, and take the supernatant. This step is phage amplification culture.

[0021] (3) Determination of the presence or absence of specific phages by spot assay

[0022] The supernatant that step (2) obta...

Embodiment 2

[0025] Embodiment 2 Purification of Acinetobacter baumannii phage

[0026] The phage stock solution that obtains in embodiment 1 is diluted to 10 with LB liquid culture medium 7 Double layer agar plate detection was carried out according to the method of step (3) in Example 1. Use an inoculation loop to pick a single phage plaque with regular shape and smooth edges, and inoculate it into 2 mL of Acinetobacter baumannii host bacterial solution (OD 600nm 0.6), shake culture at 37°C at 160rpm / min for 4-6h, the solution becomes clear, indicating that the bacteria are lysed, centrifuge at 10000r / min for 10min at 4°C. The finally obtained supernatant was sterilized by filtration with a 0.22 μm microporous membrane. Repeat the above process 3 to 5 times to obtain the purified phage, which is named SWH-Ab-1, and is preserved in the China Center for Type Culture Collection. The address of the preservation unit is: Wuhan University, Wuhan, China, and the preservation number is CCTCC M...

Embodiment 3

[0027] Example 3. Titer determination and preservation of phage

[0028] The purified phage sample in Example 2 was diluted to 10 with LB liquid medium 7 times, take 10 μL sample, add to 0.2mL Acinetobacter baumannii host bacterial solution (OD 600nm 0.6), then adopt the double-layer agar plate culture method of step (3) in Example 1 to cultivate the phage, and get a plate with an appropriate density to calculate the number of plaques. Do 3 repetitions, and the calculation of phage titer takes the average value obtained from 3 repetitions. The titer of phage (pfu / mL)=the number of plaques on the plate with appropriate density×dilution×100. The results were measured: the titer of the phage was 8×10 9 pfu / mL. Add sterile glycerol to the final phage to a final concentration of 30%, and store at -80°C for future use.

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Abstract

The invention relates to an Acinetobacter baumannii (Ab) phage and application thereof, belonging to the field of microbial engineering. The isolated phage monomer was named SWH‑Ab‑1, which has a broad-spectrum bactericidal ability against Acinetobacter baumannii. Phage SWH‑Ab‑1 was deposited in China Center for Type Culture Collection (CCTCC) (Address: China Type Culture Collection Center in Wuhan University Campus, Luojia Mountain, Wuchang City, Wuhan City), the date of preservation was October 23, 2014, and the preservation number was CCTCC M2014507.

Description

technical field [0001] The invention relates to a phage capable of specifically lysing Acinetobacter baumannii strains and its application in sterilization and antibacterial, belonging to the field of microbial engineering. Background technique [0002] Acinetobacter baumannii (Ab) is a non-fermenting, oxidase-negative, catalase-positive Gram-negative bacterium that widely exists in water, soil and hospital environments in nature and is the main conditional cause of hospital-acquired infections. One of the bacteria that can cause respiratory tract, urinary tract and wound infections in hospitalized patients, and is also a pathogenic bacteria that causes burn infections. The bacterium is widely distributed in the hospital environment and can survive for a long time. It is very easy to cause infection in critically ill patients. It is often isolated from the blood, urine, pus, and respiratory secretions of infected patients and infected in non-fermenting bacteria. second only...

Claims

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Application Information

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IPC IPC(8): C12N7/00A01N63/00A01P1/00A61K35/76A61P31/04C12R1/92
Inventor 张波罗娟
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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