Acid-resistant and high-temperature-resistant staphylococcus aureus bacteriophage as well as composition, kit and application thereof

A staphylococcus, golden yellow technology, applied in the field of bacteriophage, can solve problems such as poor bactericidal effect, and achieve the effect of strong acid resistance, good high temperature resistance, and no side effects

Pending Publication Date: 2022-04-29
PHAGELUX (NANJING) BIO-TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to improve the problem that the existing Staphylococcus aureus phage has poor sterilization effect under high temperature and acidic conditions, the present invention isolates and cultivates an acid-resistant and high-temperature resistant Staphylococcus aureus phage

Method used

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  • Acid-resistant and high-temperature-resistant staphylococcus aureus bacteriophage as well as composition, kit and application thereof
  • Acid-resistant and high-temperature-resistant staphylococcus aureus bacteriophage as well as composition, kit and application thereof
  • Acid-resistant and high-temperature-resistant staphylococcus aureus bacteriophage as well as composition, kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Screening and Purification of Phage

[0043] (1) Sample collection and processing

[0044] The sample in the present invention is obtained from the water body near the Zhongcai Logistics Market in Jiangning District, Nanjing City, Jiangsu Province.

[0045] (2) Enrichment of phages targeting the target Staphylococcus aureus in the sample Mix the above filtrate with 2 times LB liquid medium at a ratio of 1:1, inoculate 100 μL of the target Staphylococcus aureus strain, and enrich overnight.

[0046] (3) Phage screening

[0047] Centrifuge the above-mentioned enrichment solution to get the supernatant to pass through a 0.22μm membrane, mix 1mL with 5mL LB semi-solid medium containing the target Staphylococcus aureus, pour it on a petri dish containing LB solid medium, and wait until the semi-solid medium After the medium solidified, culture overnight at 37°C, observe whether there are plaques the next day, and record the experimental results.

[0048] (4) Pha...

Embodiment 2

[0052] Example 2 Determination of the optimal multiplicity of infection (MOI) of Staphylococcus aureus phage Sa88P1 (Staphylococcus aureus phageSa88P1) to Staphylococcus aureus Pick a single colony of Staphylococcus aureus and inoculate it into a test tube filled with 3 mL of LB culture solution, 37 Cultivate with shaking at 150 rpm for 12 hours to obtain a suspension of the host bacteria. The bacterial suspension was transferred to 10 mL LB culture medium at a ratio of 1:100, and cultured at 37°C with shaking at 150 rpm to the pre-logarithmic phase. Add the pure culture solution of Staphylococcus aureus phage SA88P1 (prepared by Example 1) and host bacteria (MOI=number of phages / number of bacteria) according to the multiplicity of infection ratio, and add LB liquid medium to make the total volume of each tube the same. Shake overnight at 150 rpm in a shaker at 37°C. After the culture was completed, centrifuge at 5000 g for 10 min and collect the supernatant to measure the ph...

Embodiment 3

[0057] Example 3 Deletion detection test of virulence genes or adverse genes of Staphylococcus aureus phage Sa88P1 In this example, 103 virulence genes identified as originating from lysogenic phages in pathogenic bacteria were selected as shown in Table 2. The complete genome of staphylococcal phage Sa88P1 was analyzed by bioinformatics to determine whether it contained the following virulence genes. The results show that Staphylococcus aureus phage Sa88P1 does not contain the following virulence genes or adverse genes, so it cannot encode proteins that may cause potential health risks. Therefore, Staphylococcus aureus phage Sa88P1 will not affect the health of humans or animals.

[0058] Table 2 The main known virulence genes of lysogenic phages in pathogenic bacteria

[0059]

[0060]

[0061]

[0062]

[0063]

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Abstract

The invention relates to the technical field of bacteriophages, in particular to an acid-resistant and high-temperature-resistant staphylococcus aureus bacteriophage as well as a composition, a kit and application thereof. The staphylococcus aureus phage is a staphylococcus aureus phage Sa88P1 (Staphylococcus aureus phage Sa88P1), and the staphylococcus aureus phage is a staphylococcus aureus phage Sa88P1 (Staphylococcus aureus phage Sa88P1). The staphylococcus aureus bacteriophage has excellent acid resistance, has specific killing activity on staphylococcus aureus, and can provide a new bacteriophage strain source for industrial production of bacteriophage bactericides. The staphylococcus aureus bacteriophage also has high temperature resistance, has a survival rate of 60-70% after being subjected to a water bath at 65 DEG C for 2 h, has a titer of 106-107 PFU / mL after being subjected to a water bath at 75 DEG C for 2 h, can be prepared into various forms of products such as a feed additive, a drinking water additive, a feed, drinking water, a cleaning agent or a disinfectant, and can kill staphylococcus aureus in a relatively wide temperature range and pH range.

Description

technical field [0001] The invention relates to the technical field of phages, in particular to an acid-resistant and high-temperature-resistant Staphylococcus aureus phage and its composition, kit and application. Background technique [0002] 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%. [0003] Phage is a virus that kills specific bacteria by binding to specific sites on the surface of bacterial cells, and it exists widely on the earth. Since the Pasteur Laboratory (Paris) proposed the use of phage therapy for the treatment of bacterial infections in the 1930s, more and more studies have proved that phages have high antibacterial activity and specificity, and can prevent and treat drug-resist...

Claims

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Application Information

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IPC IPC(8): C12N7/00A01N63/40A01N33/12A01P1/00
CPCC12N7/00A01N63/40A01N33/12C12N2795/00021C12N2795/00031Y02A50/30
Inventor 许小康华雨田李涛乔欢何四龙徐旭凌丛郁肖逍丁良
Owner PHAGELUX (NANJING) BIO-TECH CO LTD
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