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Mutant staphylococcus aureus phage and application thereof

A technology for staphylococcus and staphylococcus infection, applied in the field of mutant Staphylococcus aureus bacteriophage, can solve problems such as antibiotic resistance crisis, and achieve the effects of good in vivo treatment effect, wide host spectrum and strong bacteriostatic ability

Pending Publication Date: 2022-03-25
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The current crisis of antimicrobial resistance, with the overuse and misuse of antibiotics in agriculture, medicine and food

Method used

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  • Mutant staphylococcus aureus phage and application thereof
  • Mutant staphylococcus aureus phage and application thereof
  • Mutant staphylococcus aureus phage and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Isolation and Screening of Staphylococcus aureus Phage 4PHSA25

[0033] The detailed steps are as follows:

[0034] (1) Sample collection

[0035] Pig endometritis swab samples were collected from a pig farm in Guangxi Zhuang Autonomous Region.

[0036] (2) Isolation of Staphylococcus aureus phage

[0037] The pig endometritis swab samples were soaked in physiological saline overnight at 4°C, centrifuged at 8000r for 10min, and the supernatant was filtered through a 0.22μm filter. This filtrate was used as a pretreatment sample for phage isolation, and was stored overnight at 4°C. In 10mL TSB liquid medium (tryptone 17g, sodium chloride 5g, soytone 3g, glucose 2.5g, dipotassium hydrogen phosphate 2.5g, pH7.3±0.2), add 100ul host in a volume ratio of 1:100 Bacteria (Staphylococcus aureus SA25), cultured at 37°C to proliferate to the logarithmic phase. Add 10 mL of the pretreated fecal water sample and 40 mL of TSB liquid medium to the aforementioned logarith...

Embodiment 2

[0040] Electron Microscopic Observation of Example 2 Phage 4PHSA25

[0041] Phage staining was observed by negative staining with phosphotungstic acid (Clokie and Kropinski 2009). Firstly, the phage was concentrated, and the phage 4PHSA25 was proliferated in large quantities, and 1 mL of the phage stock solution was added to 100 mL of the logarithmic growth phase host bacterial liquid (Staphylococcus aureus SA25) for 3-4 hours until the bacterial liquid became clear. Centrifuge at 8000r for 10min at 4°C, take the supernatant and filter it with a 0.22μm filter to sterilize. The filtered phage was further balanced by adding 5 mL of 30% sucrose solution with a long needle, centrifuged at 4°C and 30,000r for 2 h to concentrate the phage, discarded the supernatant, and added 200 μL of TEN solution to the precipitate to resuspend. The concentrated and resuspended phages were sent to the transmission electron microscope platform, and the samples were stained with conventional phosph...

Embodiment 3

[0043] Example 3: Extraction and Whole Genome Sequencing of Phage Genome

[0044] The phage genome was extracted using proteinase K / SDS method. Take 1 mL of the concentrated phage stock solution after ultracentrifugation, add 20ul of RNaseI, 2ul of DNaseI, and 10ul of Buffer, mix well and lyse at 37°C for 1h. Add 10% SDS to 0.5% according to 1 / 5 of the volume of the phage concentrate, mix 50ul proteinase K by inversion and then bathe in 56°C water bath for 1h. Add 1 mL of protein extract with a volume ratio of 25:24:1 phenol:chloroform:isoamyl alcohol (prepared for use, stored away from light) and mix it upside down. Centrifuge at 13000r for 10 min. Pipette the supernatant into a new 2mLEP tube with a 200uL broken pipette tip, and repeat 3-4 times. For the last time, transfer the collected supernatant to a 10mL EP tube, add it to pre-cooled absolute ethanol at a ratio of 1:2 by volume, and let it stand overnight at -20°C. The next day, use a broken pipette tip to put the fl...

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Abstract

The invention belongs to the technical field of animal infectious disease prevention and treatment, and particularly relates to a mutant staphylococcus aureus bacteriophage and application thereof. The staphylococcus aureus bacteriophage 4PHCISA25 separated in the invention is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M20211354. The invention provides an effect of the bacteriophage as a bactericide for preventing and treating staphylococcus aureus and a biofilm of the staphylococcus aureus, and provides a bactericide product with application potential for clinically treating staphylococcus aureus infection diseases. The staphylococcus aureus phage disclosed by the invention is wide in splitting range and strong in splitting capacity, and can be used for strongly killing methicillin-resistant staphylococcus aureus.

Description

technical field [0001] The invention belongs to the technical field of prevention and treatment of animal infectious diseases, and in particular relates to a mutant Staphylococcus aureus phage and its application. Background technique [0002] Staphylococcus aureus (S.aureus) is a G+ bacteria that can cause various infectious diseases in humans and livestock, such as local abscess, sepsis and sepsis. S. aureus can attach and persist on host tissues such as bone and heart valves, causing osteomyelitis and endocarditis. This bacterium is also one of the common pathogens that cause bacterial food poisoning and nosocomial infection, and is closely related to the hygiene and safety of the food industry and the medical field. In recent years, due to the use of antibiotics, Staphylococcus aureus is resistant to methicillin to form "super bacteria" (MRSA), which causes a large number of patients infected with "super bacteria" to become ill every year and causes 21.8% of infected pa...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K35/76A61P17/02A61P31/04A01N63/40A01N63/20A01P1/00C12R1/92
CPCC12N7/00A61K35/76A61P31/04A61P17/02A01N63/40A01N63/20C12N2795/10321Y02A50/30
Inventor 吴斌王霜彭忠杨丹华琳宋文博陈焕春
Owner HUAZHONG AGRI UNIV
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