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Novel staphylococcus aureus phage as well as composition and application thereof

A staphylococcus and phage technology, applied in the field of microorganisms, can solve the problem of single phage bactericidal ability and other issues

Active Publication Date: 2021-05-28
PHAGELUX (NANJING) BIO-TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, most of the Staphylococcus aureus phage literature is limited to the research on the bactericidal ability of a single phage (Wang, Z.F., et al., SLPW: A Virulent Bacteriophage Targeting Methicillin-Resistant Staphylococcus aureus In vitro and In vivo. Frontiers in Microbiology, 2016.7: p.10.; Jia, H.Y., et a1., Characterization and completegenome sequence analysis of Staphylococcus aureus bacteriophage JS01. VirusGenes, 2015.50(2): p.345-348. Jiangnan University, a phage of Staphylococcus aureus and its application, application number CN201110380587), but there are few reports on the characteristics of the cocktail mixture composed of several phages

Method used

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  • Novel staphylococcus aureus phage as well as composition and application thereof
  • Novel staphylococcus aureus phage as well as composition and application thereof
  • Novel staphylococcus aureus phage as well as composition and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Isolation, preparation and purification of phage

[0050] In the present invention, the source samples of brachyphage BP-13, Staphylococcus aureus BP-13A, brachyphage BP-14 and brachyphage AHJD-like phage genus BP-39 were collected from the sewage treatment plant in Montreal, Canada, and passed through double-layer filter paper. After filtration, centrifuge at low speed and normal temperature, and then filter the supernatant with a 0.22 μm filter membrane.

[0051] Isolation of phage: Take 10mL of filtered supernatant, add it to 10mL 2 times TSB medium, add 1mL of phage host bacteria logarithmic phase bacteria liquid at the same time, place it at 37°C for 16h, take the above culture, and put it under the condition of 8000rpm Centrifuge for 10 min, filter the supernatant with a 0.22 μm filter membrane, and set aside. Take 0.5mL of phage host bacteria logarithmic phase bacteria liquid, add 5mL, 40 ℃ semi-solid TSB medium, mix well, pour on the TSA plate, and p...

Embodiment 2

[0053] Embodiment 2: Electron microscope observation of phage

[0054] Take the supernatant of each phage culture obtained in Example 1 for electron microscope observation: take 20 μ L of sample and drop it on the copper grid, wait for its natural precipitation for 15 minutes, absorb excess liquid from the side with filter paper, add 1 drop of 2% phosphotungstic acid (PTA ) on a copper grid, dyed for 10 minutes, sucked the dye solution from the side with filter paper, and observed with an electron microscope after drying: the results are as follows: figure 1 As shown, the BP-13 phage morphology was found to have a regular polyhedral head structure and a shorter tail, the head diameter was about 150nm, and the tail length was about 10nm ( figure 1 -A); The morphology of BP-13A bacteriophage was found to have a regular polyhedral head structure and a shorter tail, the diameter of the head was about 100nm, the length of the tail was about 10nm, and the end of the tail had six sho...

Embodiment 3

[0055] Example 3: Extraction and sequencing of phage genome

[0056] Take 100 mL of each phage prepared in Example 1, add DNaseI and RNaseA at a final concentration of 1 μg / mL, incubate at 37°C for 60 min, add 5.84 g NaCl (final concentration 1 mol / L), dissolve and place in an ice bath for 1 h. Centrifuge at 11,000 rpm for 10 min at 4°C, and transfer the supernatant to a new centrifuge tube. Add solid PEG8000 (final concentration 10%, that is, add 10 g to 100 mL), and after complete dissolution, ice bath for at least 1 h. Centrifuge at 11,000 rpm for 20 min at 4°C, and resuspend the pellet with a small amount of SM solution. Add an equal volume of chloroform and isoamyl alcohol for extraction, shake gently for 30 s, centrifuge at 8000 rpm for 1 min, absorb the supernatant, and repeat the extraction until clarification. Add DNase I and RNase A again to a final concentration of 1 μg / mL, and react at 37°C for 30-60 minutes. Add EDTA to a final concentration of 20 mmol / L (that ...

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Abstract

The invention relates to the field of biology, in particular to novel staphylococcus aureus bacteriophages as well as a composition and application thereof, and provides four staphylococcus aureus bacteriophages including: podoviridae BP-13 with the preservation number of CCTCC NO: M 2015142; staphylococcus aureus BP-13A with the preservation number of CCTCC M2016535; podoviridae BP-14 with the preservation number of CCTCC NO: M 2015143; and podoviridae AHJD-like phage BP-39 with the preservation number of CCTCC NO: M 2015144. The four bacteriophages are strict virulent bacteriophages, can effectively kill various staphylococcus aureus such as MRSA, MSSA, BORSA and the like, have a wide host range, but have no toxic effect on normal microbial flora, and the DNA of the bacteriophages cannot encode virulence genes. Therefore, the bacillus subtilis can be used for preventing or treating human and livestock bacterial infection caused by the staphylococcus aureus, provides an excellent strain resource for developing a novel antibacterial preparation, and has a good application and development prospect. Besides, the host range and the antibacterial efficiency of the cocktail mixture disclosed by the invention are further greatly improved.

Description

[0001] This application is a divisional application of the invention patent application with the application number CN201710048705.4, the application date is January 22, 2017, and the invention title is "Novel Staphylococcus aureus phage and its composition and application". technical field [0002] The invention belongs to the technical field of microbes, and in particular relates to novel staphylococcus aureus phages, their composition, their preparation method and application. Background technique [0003] Staphylococcus aureus (Staphylococcus aureus) is an important zoonotic pathogen, which widely exists in nature and has a wide range of infection and strong adaptability. It can cause wound infection, endocarditis, osteomyelitis, and toxic shock Syndrome and other diseases, resulting in a mortality rate ranging from 11% to 48%. Methicillin-sensitive Staphylococcus aureus (MSSA) can be killed by penicillin and methicillin, but more than half of Staphylococcus aureus in th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K35/76A61P31/04A61P17/02A61P15/14C12R1/92
CPCA61K35/76C12N7/00A61P31/04A61P17/02A61P15/14C12N2795/10221C12N2795/10232Y02A50/30
Inventor 费文斌肖逍苏胜兵乔欢丛郁徐旭凌丁良许文建周思翔熊剑胜沈禅娟王艳赵伟王卫斌霍茨蒙德·曼德维尔马克·恩格尔
Owner PHAGELUX (NANJING) BIO-TECH CO LTD
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