Staphylococcus aureus bacteriophage and application

A technology for staphylococcus and staphylococcus infection, which is applied to Staphylococcus aureus bacteriophages and application fields, and can solve the problems of high specificity requirements of host bacteria and unpopularized bacteriophages.

Active Publication Date: 2018-12-25
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of phages has not been promoted. In addition to the discovery and application of antibiot

Method used

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  • Staphylococcus aureus bacteriophage and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Isolation and screening of phage SH-St 15644

[0027] Add a small amount of chloroform to the sewage samples from Ruijin Hospital Affiliated to Shanghai Jiaotong University and Shanghai Huashan Hospital, centrifuge at 4°C and 3500rpm for 10 minutes, take the supernatant, dilute it with SM Buffer appropriately, and take 10μl and 400μl of methicillin-resistant Staphylococcus aureus bacteria liquid (OD 600 =0.6) to mix, add the upper medium to mix, and pour the bottom medium to make a double-layer LB plate. Inverted after solidification, and incubated overnight in a 37°C incubator. If there are phage plaques on the plate, use a syringe needle to pick a typical, clear single phage plaque, add it to 1mL SM Buffer and crush it, place it on a shaker at 4°C for more than 4 hours, add 0.1mL chloroform to the tube, and centrifuge Take the supernatant and store it at 4°C. After repeated purification at least 3 times by the double-layer plate method (the shape and size...

Embodiment 2

[0028]Example 2 Purification of phage SH-St 15644

[0029] The phage SH-St 15644 culture fluid (10 6 PFU / mL) and methicillin-resistant Staphylococcus aureus (OD 600 =0.6,10 8 CFU / mL) were mixed and cultured at 37°C. On the next day, add 2% chloroform and let stand at 4°C for 1 hour, then centrifuge at 6500r / min for 15min at 4°C. DNase1 enzyme and RNase1 enzyme were added to the supernatant at a final concentration of 1 μg / mL, and left at room temperature for 30 minutes. Add NaCl to a final concentration of 0.5 M, and after 1 hour of ice-bath, centrifuge at 11,000 g for 10 minutes at 4°C. Take the supernatant, add solid PEG8000 to a final concentration of 10% (w / v), mix well and let stand at 4°C overnight. Take the mixture from the previous day, centrifuge at 4°C, 8500r / min for 20min, discard the supernatant, and resuspend the pellet with SM Buffer. Add an equal amount of chloroform and mix lightly for 30 seconds, centrifuge at 4000g for 10 minutes, and take the supernatan...

Embodiment 3

[0030] Example 3 Determination of phage SH-St 15644 titer

[0031] The titer of phage refers to the number of live phage contained in 1 mL of culture medium. The phage SH-St 15644 prepared in Example 2 was diluted to 10 with SM Buffer -7 、10 -8 、10 -9 After three gradients, take 10 μl and 400 μl of methicillin-resistant Staphylococcus aureus bacteria solution (OD 600 =0.6,10 8 CFU / mL) were mixed and added to the upper medium, then poured into the bottom medium to form a double-layer plate, and placed in a 37°C incubator overnight after it solidified. On the second day, count the number of plaques (calculation formula: titer (pfu / mL)=number of plaques×dilution factor×100).

[0032] The diameter of plaques formed by SH-St 15644 infected with methicillin-resistant Staphylococcus aureus was about 0.5-1mm. Spots are round and transparent. see figure 1 In A, the titer of phage SH-St 15644 prepared in Example 2 was 2×10 11 pfu / mL.

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Abstract

The invention discloses staphylococcus aureus bacteriophage and application. The staphylococcus aureus bacteriophage and the application have the advantages that staphylococcus aureus can be specifically effectively eliminated by the staphylococcus aureus bacteriophage, excellent in-vivo and in-vitro antibacterial effects can be realized by the staphylococcus aureus bacteriophage for medicine-resistant staphylococcus aureus, experimental foundations can be provided to clinically developing preparations for preventing and treating medicine-resistant staphylococcus aureus infection, and the staphylococcus aureus bacteriophage has great clinical application potential; the skin abscess average areas of nude mice of bacteriophage treatment groups are 47.32 mm<2> in skin abscess models when MOI(multiplicity of infection) is equal to 10, and the average bacterium load is 3.553*10<7> CFU/g; the skin abscess average areas of nude mice of MRSA (methicillin-resistant staphylococcus aureus) infection groups are 150.4 mm<2>, and the average bacterium load is 2.284*10<8> CFU/g; the skin abscess areas of the mice of the bacteriophage treatment groups are smaller than the skin abscess areas of the mice of the MRSA infection groups, the colony count of the mice of the bacteriophage treatment groups is lower than the colony count of the mice of the MRSA infection groups, and the difference of the skin abscess areas and the colony count has statistical significance.

Description

(1) Technical field [0001] The invention relates to a staphylococcus aureus phage and its application. (2) Background technology [0002] Staphylococcus aureus (SAU) is a common and highly pathogenic Gram-positive coccus clinically. Since the advent of penicillin, the infection caused by Staphylococcus aureus has been controlled, but with the widespread use of penicillin, new drug-resistant bacteria continue to emerge. In 1959, methicillin was used in clinical treatment, and it effectively controlled the infection of penicillin-resistant Staphylococcus aureus. However, in 1961, the first strain of methicillin-resistant Staphylococcus aureus (MRSA) was isolated in the UK. ), which has become one of the important pathogens of nosocomial and community infections. In recent years, the main clonal type of hospital-acquired Staphylococcus aureus commonly seen in China is ST239. The main clonal types of community-acquired S. aureus were ST398 and ST59. [0003] MRSA can cause s...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K35/76A61P31/04C12R1/92
CPCA61K35/76A61P31/04C12N7/00C12N2795/00021C12N2795/00032
Inventor 华允芬何平罗婷婷徐梦莎
Owner ZHEJIANG UNIV OF TECH
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