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Staphylococcus aureus phage SapYZUalpha and application thereof

A staphylococcus, golden yellow technology, applied in the field of bioengineering, can solve the problem that the incubation period is not as good as other bacteriophages, phages are not widely used, and the outbreak amount is low, so as to inhibit the formation of Staphylococcus aureus biofilm, reduce the Contamination and spread risk, effect of efficient lysis activity

Active Publication Date: 2022-07-29
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when phages are used as antibacterial agents, they have high requirements for the required phages, not only require the phage itself to have high-speed reproduction characteristics, but also have a wide range of lytic properties for host bacteria. Therefore, phages have not been widely used.
Although many documents have reported that phages are used to control contaminated food, food equipment, etc. of Staphylococcus aureus, these phages still have many deficiencies, such as Staphylococcus aureus phage VB_SauS_SH-St 15644 has a shorter incubation period (12min), but The outbreak amount is very low, only 13PFU / cell; Staphylococcus aureus phage PALS2 has a wide range of host spectrum characteristics, but its incubation period (30min) and burst amount (12PFU / cell) are far inferior to other phages; in addition, Staphylococcus aureus Phage Stab21 has a higher burst (130PFU / cell), but its titer is only about 10 8 PFU / cell cannot achieve the effect of high-speed reproduction and antibacterial

Method used

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  • Staphylococcus aureus phage SapYZUalpha and application thereof
  • Staphylococcus aureus phage SapYZUalpha and application thereof
  • Staphylococcus aureus phage SapYZUalpha and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Phage isolation and purification

[0047] Phage isolation

[0048] The sewage samples in the present invention were collected from Yangzhou farmers market in Jiangsu. Take 500 mL of sewage sample and place it in a high-speed centrifuge at 10,000 rpm for 10 min to remove impurities in the sewage. Take 3 mL of supernatant and add it to a test tube of 3 mL of 2×LB liquid medium, and add 100 μL of logarithmic growth phase Staphylococcus aureus at the same time. ATCC29213 was cultured overnight at 37°C with shaking at 130 rpm. The next day, all the culture in the test tube was transferred to a 10mL sterile centrifuge tube, centrifuged in a 8,000rpm centrifuge for 10min to remove the precipitate, and the supernatant was passed through a 0.22μm sterile microporous membrane, and the filtrate was the phage stock solution. , stored in a 4°C refrigerator for later use.

[0049] A 10-fold serial dilution by volume of the phage stock solution (10 -1 -10 -7 ), take 100 μL of pha...

Embodiment 2

[0055] Characterization of bacteriophage SapYZUalpha

[0056] Phage morphological features

[0057] The microscopic morphological characteristics of the phage SapYZUalpha isolated in the Example were observed by transmission electron microscope. The bacteriophage purified solution obtained in Example 1 (10 9 PFU / mL) concentrated to 10 10 PFU / mL, take 20uL of purified phage (10 10 pfu / mL) suspension was added dropwise to a 200 mesh carbon-coated copper mesh, adsorbed for 15 minutes, and then taken out of the copper mesh, naturally dried for 2-3 minutes, dyed with 2% sodium phosphotungstate (pH 7.6) solution, and dried after 2 minutes. Naturally dried for 10 min, observed by transmission electron microscope (TEM, Hitachi H600A) at a voltage of 100 kV, and a clear phage image was selected for photographic analysis. SapYZUalpha microscopic morphology such as figure 2 As shown, the phage SapYZUalpha has a symmetrical head with a diameter of about 88.68 nm and a tail of about ...

Embodiment 3

[0067] Bacteriostatic effect of bacteriophage SapYZUalpha in culture medium

[0068] Staphylococcus aureus ATCC 29213 was cultured to the logarithmic growth phase, centrifuged at 9000g for 2min, washed with PBS (pH 7.4) to remove the medium, and adjusted to a bacterial concentration of 10 6 CFU / mL, the bacterial suspension (100uL) was mixed with phage according to different MOIs (0.0001, 0.01, 1 and 100) in equal volume ratios, and 2uL of the mixture was placed in a 96-well plate containing 198uL of TSB medium. Incubate at 37 °C for 24 h, and measure the OD value at OD590 nm every 3 h. The experiment was performed in parallel for 5 times.

[0069] After selecting 5 strains of porcine-derived Staphylococcus aureus (M7, M10, M13, M18 and S14, Table 2) in the host spectrum to the logarithmic growth phase, centrifuged at 9000g for 2min, and washed with PBS (pH 7.4) to remove the medium, And adjust the concentration of each bacterial strain to 10 6 CFU / mL, mixed according to 1:1:...

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Abstract

The invention discloses a staphylococcus aureus bacteriophage SapYZUalpha and application thereof, the staphylococcus aureus bacteriophage SapYZUalpha is identified to have unique genome structural characteristics, belongs to a novel bacteriophage and is preserved in the China Center for Type Culture Collection, the preservation time is January 5, 2022, and the preservation number is CCTCC NO: M2022023. The bacteriophage capable of efficiently cracking staphylococcus aureus can effectively inhibit staphylococcus aureus in various matrixes, and can be applied to preparation of a bacteriostatic agent for controlling staphylococcus aureus pollution. The bacteriostatic agent prepared by the invention can effectively control the growth of staphylococcus aureus in a culture medium and a food matrix, can effectively inhibit the formation of staphylococcus aureus pellicles on the surfaces of equipment, vessels and raw material solids, is simple to prepare and convenient to use, and can reduce the propagation of staphylococcus aureus and the risk of causing food-borne diseases.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to Staphylococcus aureus bacteriophage SapYZUalpha and applications thereof. Background technique [0002] Staphylococcus aureus (S. aureus, SA) is a Gram-positive bacterium and is an important pathogenic bacteria commonly found in clinical and food processing environments, which can cause skin infections, respiratory infections and food poisoning in animals and humans. In recent decades, the misuse of antibiotics has led to the emergence and rapid development of multidrug-resistant S. aureus, especially methicillin-resistant S. aureus (MRSA), which makes S. aureus infection clinical treatment is very challenging. According to reports, MRSA is frequently detected in contaminated dairy and meat products around the world, with detection rates reaching 2.5% and 3.9%, respectively. However, the use of antibiotics and artificial preservatives in food and related products is no...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A01N63/40A01P1/00A23L3/3571C12R1/92
CPCC12N7/00A01N63/40A23L3/3571C12N2795/10121C12N2795/10131A23V2002/00A23V2200/10A01N63/20Y02A50/30
Inventor 周文渊高亚军温花李雅洁杨振泉李华祥赵尹蕾吉婷婷
Owner YANGZHOU UNIV
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