A bacteriophage targeting enterotoxin A-producing Staphylococcus aureus and its application

Abstract

The present invention provides a bacteriophage targeting enterotoxin A-producing Staphylococcus aureus and an application thereof, wherein the bacteriophage is Staphylococcus aureus phage (Staphylococcus aureusphage) GRNSAP30, and the deposit number is CCTCC NO: M 2024537. The bacteriophage GRNSAP30 of the present invention has a good killing effect on clinical enterotoxin A-producing Staphylococcus aureus isolated from all over the country, with a lysis rate of up to 98%. The bacteriophage can be used to disinfect Staphylococcus aureus on the surfaces of various foods, medical devices, dining utensils, toiletries, etc., so as to reduce the incidence of clinical bacterial infection.

Description

Technical Field

[0001] The present invention relates to the technical field of bacteriophages, and in particular to a bacteriophage targeting enterotoxin A-producing Staphylococcus aureus and application thereof. Background Art

[0002] Staphylococcus aureus is one of the most important foodborne pathogens in the world. Humans can cause food poisoning by ingesting one or more staphylococcal enterotoxins (SEs) produced by it. Various SEs interact with the enteric nervous system, causing symptoms such as vomiting, diarrhea, or intestinal inflammation by stimulating afferent neurons or inducing neurotransmitters released from chromaffin cells. There are currently 23 confirmed enterotoxins, including classic SEs (SEA-SEE) and novel SEs (SEG-SEX). Among SEs, SEA (enterotoxin A) is considered to be the most critical enterotoxin factor causing staphylococcal food poisoning (SFP), and about 80% of food poisoning incidents caused by staphylococci are related to SEA. SEA has extremely strong tolerance and remains active after high heat treatment, pepsin and trypsin digestion.

[0003] Bacteriophages are natural enemies of bacteria. They were discovered earlier than antibiotics and were used to treat bacterial diseases as soon as they were discovered. The discovery of antibiotics caused people to gradually ignore them. As bacterial resistance became more serious, phage therapy once again entered people's field of vision. However, bacteriophages are viral microorganisms and will be affected by the residual disinfectants in farms, thus affecting the effectiveness of their use. Therefore, screening out disinfectant-resistant bacteriophages against enterotoxin A-producing Staphylococcus aureus has great clinical significance. The replacement of bacteriophages with disinfectants is more in line with farm disinfection procedures and prevents the generation of drug-resistant bacteria, which has a high clinical application value. Summary of the invention

[0004] In view of this, the present invention proposes a bacteriophage targeting enterotoxin A-producing Staphylococcus aureus and application thereof.

[0005] The technical solution of the present invention is implemented as follows: In the first aspect, the present invention provides a phage against enterotoxin A-producing Staphylococcus aureus, wherein the phage is Staphylococcus aureus phage (Staphylococcusaureusphage) GRNSAP30, and the deposit number is CCTCC NO: M 2024537.

[0006] In a second aspect, the present invention provides a phage composition against enterotoxin A-producing Staphylococcus aureus, wherein the composition comprises Staphylococcus aureus phage GRNSAP30.

[0007] In a third aspect, the present invention provides a use of a bacteriophage targeting enterotoxin A-producing Staphylococcus aureus in the preparation of an enterotoxin A-producing Staphylococcus aureus inhibitor.

[0008] In a fourth aspect, the present invention provides a use of a bacteriophage targeting enterotoxin A-producing Staphylococcus aureus in a bactericide.

[0009] On the basis of the above technical solution, preferably, the bactericide is used to kill enterotoxin A-producing Staphylococcus aureus.

[0010] In a fifth aspect, the present invention provides a use of a bacteriophage targeting enterotoxin A-producing Staphylococcus aureus in the preparation of a drug for treating or preventing a disease caused by infection with enterotoxin A-producing Staphylococcus aureus.

[0011] The bacteriophage of the present invention against enterotoxin A-producing Staphylococcus aureus has the following beneficial effects compared with the prior art:

[0012] The bacteriophage GRNSAP30 of the present invention has a good killing effect on clinical enterotoxin A-producing Staphylococcus aureus isolated from all over the country, with a lysis rate of up to 98%. The bacteriophage can be used to disinfect Staphylococcus aureus on the surfaces of various foods, medical equipment, dining utensils, toiletries, etc., so as to reduce the incidence of clinical bacterial infection.

[0013] The bacteriophage GRNSAP30 of the present invention has strong acid and alkali resistance stability and extremely high fermentation efficiency, which greatly reduces the production cost and has high clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0015] Figure 1 This is an electron micrograph of Staphylococcus aureus phage GRNSAP30;

[0016] Figure 2 is the pH stability of Staphylococcus aureus phage GRNSAP30; DETAILED DESCRIPTION

[0017] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] The technical solutions described in the present invention, unless otherwise specified, are all conventional technologies; the reagents or materials described, unless otherwise specified, are all from commercial channels.

[0019] The formula of the TSB liquid culture medium used in the present invention is: 17g of casein pancreatic digest, 3g of soy flour papain digest, 5g of sodium chloride, 2.5g of dipotassium hydrogen phosphate, 2.5g of glucose, 1000mL of purified water, pH 7.3±0.2, and it is used after being sterilized by pressure steam at 121°C.

[0020] The formula of the TSB semi-solid culture medium used in the present invention is: 17g of casein pancreatic digest, 3g of soy flour papain digest, 5g of sodium chloride, 2.5g of dipotassium hydrogen phosphate, 2.5g of glucose, 7.5g of agar, 1000mL of purified water, pH 7.3±0.2, and it is used after being sterilized by pressure steam at 121°C.

[0021] Example 1 Phage Isolation and Purification

[0022] 16 water samples were collected from a chicken farm in Jiangxi Province where Staphylococcus aureus was found. Each sample was 25 mL. The water sample was centrifuged at 10,000 rpm for 30 min, and 20 mL of the supernatant was filtered and sterilized. 10 mL of the filtrate was mixed with 10 mL of 2×TSB liquid culture medium and 1 mL of Staphylococcus aureus culture medium in the logarithmic phase (10 8cfu / mL) and cultured overnight at 37℃180rpm to enrich phages. Centrifuge the sample enrichment solution at 10000rpm for 15min, take the supernatant and sterilize it through a 0.22μm microporous filter membrane to obtain a filtrate containing phages. Take 1mL of the filtrate and mix it evenly with 500μL of its host Staphylococcus aureus solution, let it stand for 15min to allow it to adsorb to the bacterial surface. Add 7mL of TSB semi-solid agar medium cooled to 50℃ to the above mixture, mix well and immediately spread it on the solidified NA plate. After the agar solidifies, invert and culture it at 37℃ for 8-12h to observe the growth of phage plaques. On the double-layer plate where plaques are formed, pick up large and transparent plaques with a sterile pipette tip, desorb them by shaking in 1mL SM buffer, and sterilize them through a 0.22μm microporous filter to obtain a phage filtrate. Inoculate the filtrate in 10mL TSB liquid culture medium, add 100μL of host Staphylococcus aureus culture activated overnight and mix well, culture overnight at 37℃ and 180rpm, centrifuge at 5000rpm for 10min, take the supernatant and pass it through a 0.22μm microporous filter, and observe the morphology of plaques using the double-layer plate method. After repeating the operation 3-5 times, plaques of the same shape and size can be obtained, the center of the plaque is transparent, and the diameter is about 1mm. The phage was sent to the China Center for Type Culture Collection for preservation on March 21, 2024, and its classification name is Staphylococcus aureus phage GRNSAP30. The address is Wuhan University, Wuhan, China. The preservation number is CCTCC NO: M2024537.

[0023] Example 2 Screening of Staphylococcus aureus phages against enterotoxin A-producing Staphylococcus aureus

[0024] 600 μL of bacterial suspension of 52 clinically isolated and identified enterotoxin A-producing Staphylococcus aureus were mixed with TSB semi-solid medium and spread on the prepared NA plates, 30 strains of Staphylococcus aureus phage were activated, and 5 μL of the above 30 phages were dripped on the plates. After natural air drying, they were cultured at 37°C for 8-12 hours and the results were observed. The experiment was repeated three times, and the results are shown in Table 1.

[0025] Table 1 Lysis of enterotoxin A-producing Staphylococcus aureus by bacteriophage

[0026] Phage number Lysis rate Phage number Lysis rate Phage number Lysis rate GRNSAP1 58％ GRNSAP19 54％ GRNSAP35 88％ GRNSAP2 52％ GRNSAP20 75％ GRNSAP36 83％ GRNSAP3 60％ GRNSAP21 58％ GRNSAP37 46％ GRNSAP4 87％ GRNSAP23 62％ GRNSAP38 65％ GRNSAP5 88％ GRNSAP24 67％ GRNSAP39 40％ GRNSAP11 87％ GRNSAP25 65％ GRNSAP42 48％ GRNSAP13 65％ GRNSAP28 52％ GRNSAP43 44％ GRNSAP14 63％ GRNSAP30 98％ GRNSAP45 87％ GRNSAP15 69％ GRNSAP32 75％ GRNSAP46 88％ GRNSAP16 71％ GRNSAP33 87％ GRNSAP47 83％

[0027] Table 2 Lysis of enterotoxin A-producing Staphylococcus aureus by bacteriophage GRNSAP30

[0028] Bacteria No. Cracking Bacteria No. Cracking Bacteria No. Cracking Bacteria No. Cracking GRNJP29 Cleavable GRNJP50 Cleavable GRNJP67 Cleavable GRNJP91 Cleavable GRNJP34 Cleavable GRNJP52 Cleavable GRNJP68 Cleavable GRNJP93 Cleavable GRNJP35 Cleavable GRNJP53 Cleavable GRNJP69 Cleavable GRNJP95 Cleavable GRNJP37 Cleavable GRNJP55 Cleavable GRNJP70 Cleavable GRNJP96 Cleavable GRNJP39 Cleavable GRNJP56 Cleavable GRNJP81 Cleavable GRNJP97 Cleavable GRNJP42 Cleavable GRNJP58 Cleavable GRNJP82 Cleavable GRNJP98 Cleavable GRNJP43 Cleavable GRNJP59 Cleavable GRNJP83 Cleavable GRNJP101 Cleavable GRNJP44 Cleavable GRNJP60 Cleavable GRNJP84 Cleavable GRNJP102 Cleavable GRNJP45 Cleavable GRNJP62 Cleavable GRNJP85 Cleavable GRNJP104 Cleavable GRNJP46 Cleavable GRNJP63 Cleavable GRNJP86 Cleavable GRNJP106 Cleavable GRNJP47 Cleavable GRNJP64 Cleavable GRNJP88 Cleavable GRNJP108 Cleavable GRNJP48 Cleavable GRNJP65 Cleavable GRNJP89 Cleavable GRNJP109 Cleavable GRNJP49 Cleavable GRNJP66 Cleavable GRNJP90 Cleavable GRNJP120 Cleavable

[0029] As shown in Tables 1 and 2, the Staphylococcus aureus phage GRNSAP30 can kill enterotoxin A-producing Staphylococcus aureus from different regions, with a lysis rate of up to 98%.

[0030] The phage was sent to the China Center for Type Culture Collection for preservation on March 21, 2024, and the classification name is Staphylococcus aureus phage GRNSAP30, address: Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M2024537. The collection center tested the activity of the phage on March 28, 2024, and the result was in a viable state.

[0031] Example 3 Safety test of Staphylococcus aureus bacteriophage GRNSAP30

[0032] Virulence gene or adverse gene detection test of Staphylococcus aureus phage GRNSAP30. In this example, a variety of virulence genes identified as lysogenic phages derived from pathogenic bacteria were selected, and the whole genome of Staphylococcus aureus phage GRNSAP30 was measured and bioinformatics analysis was performed to determine whether it contained the following virulence genes.

[0033] The results showed that Staphylococcus aureus phage GRNSAP30 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 GRNSAP30 will not affect the health of humans or animals.

[0034] Table 3 Major known toxic genes of lysogenic phages in pathogenic bacteria

[0035]

[0036]

[0037] Example 4 Determination of the optimal fermentation conditions for bacteriophage GRNSAP30

[0038] A single colony of the host Staphylococcus aureus was picked and inoculated into a test tube containing 3 mL of TSB culture medium. The culture was shaken at 180 rpm at 37 ° C for 12 hours to obtain a host bacterial suspension. The bacterial suspension was transferred to 10 mL of TSB culture medium at a ratio of 1:100 and cultured at 37 ° C and 180 rpm to the early logarithmic period. The phage GRNSAP30 and its host Staphylococcus aureus were diluted to a certain concentration and counted, mixed and cultured in different proportions, and TSB liquid culture medium was added to make the total volume of each tube the same. Four copies were prepared for each concentration ratio and cultured at 180 rpm for 12 hours at 25 ° C, 28 ° C, 32 ° C and 37 ° C respectively. After the culture was completed, the fermentation broth was centrifuged at 6000g for 10 minutes and the supernatant was collected. The supernatant was passed through a 0.22 μm microporous filter membrane and counted by double-layer plate method. Each point was cultured in duplicate tubes and the average value was taken. The condition that produced the highest phage titer was the optimal fermentation condition. The experiment was repeated 3 times and the results are shown in Table 4.

[0039] Table 4 The titer of phage GRNSAP30 at different infection multiplicity

[0040]

[0041]

[0042] As shown in Table 4, when the concentration of phage GRNSAP30 was 2.5×10 5 PFU / mL, host bacteria concentration is 3.3×10 8 CFU / mL, when the fermentation temperature was 30℃, the fermentation titer of bacteriophage GRNSAP30 reached the highest level of 5.6×10 11 PFU / mL.

[0043] Example 5 Temperature stability test of Staphylococcus aureus bacteriophage GRNSAP30

[0044] Take several sterile 50mL centrifuge tubes, add 20mL TSB to each tube and place it in a constant temperature water bath at the corresponding temperature. After the temperature is balanced, add 1mL of pure phage culture solution to each tube and act at 4℃, 25℃, 37℃, 45℃, 55℃, 65℃, 75℃, and 85℃ for 1h, 2h, 12h, 24h, 48h, and 72h, respectively. After the action time is over, take out the sample tube and immediately cool it in a room temperature water bath. After appropriate dilution, use the double-layer plate method to determine the phage titer. Each point is cultured in duplicate tubes to take the average value. The experiment was repeated 3 times. The results are shown in Table 5.

[0045] Table 5 The titer of phage GRNSAP30 after storage at different temperatures for different time periods

[0046]

[0047] As shown in Table 5, the activity of bacteriophage GRNSAP30 remained basically unchanged at a temperature below 55°C, and it still had a high titer after being placed in a 65°C water bath for 12 hours. It had good stability at 4-55°C and could be stored for a long time.

[0048] Example 6 pH stability test of Staphylococcus aureus bacteriophage GRNSAP30

[0049] Take 9 mL of TSB liquid culture medium and adjust it to different pH values ​​(2, 3, 4, 5, 6, 7, 8, 9, 10, 11), place it in a constant temperature water bath at 25°C, and add 1 mL of 1×10 9 PFU / mL Staphylococcus aureus phage GRNSAP30 pure culture solution was placed in a 25℃ environment for 4 hours. After diluting some samples, the phage titer was determined by the double-layer plate method. Duplicate culture was performed at each point to obtain the average value. The experiment was repeated 3 times. The results are shown in the figure. Figure 1 .

[0050] Figure 1 As shown, the Staphylococcus aureus phage GRNSAP30 is completely inactivated at pH = 2, and can maintain a high activity between pH 3-11. This shows that the Staphylococcus aureus phage GRNSAP30 can adapt to the environment of pH 3-11.

[0051] Example 7 Determination of the removal effect of enterotoxin A-producing Staphylococcus aureus in food

[0052] In order to explore the effect of this bacteriophage in removing enterotoxin A-producing Staphylococcus aureus, this example conducted an experiment. The specific method and results are as follows:

[0053] Under sterile conditions, 5 portions of milk were weighed, each 98 mL, and divided into 5 groups.

[0054] Group 1: Blank group: No treatment was performed and the cells were placed in a 37°C constant temperature incubator.

[0055] Group 2: Virus-challenging group: 1 mL of enterotoxin A-producing Staphylococcus aureus SA202405 (1×10 7 pfu / mL) and then placed in a 37°C constant temperature incubator.

[0056] Group 3 Treatment 1: Add 1 mL of enterotoxigenic Staphylococcus aureus SA202405 (1×10 7 pfu / mL) and added 1 mL of 1×10 7pfu / mL Staphylococcus aureus phage GRNSAP30, and then placed in a 37°C constant temperature incubator.

[0057] Group 4 Treatment 2: Add 1 mL of enterotoxigenic Staphylococcus aureus SA202405 (1×10 7 pfu / mL), and amoxicillin solution was added according to the minimum inhibitory concentration of SA202405 (16 μg / mL), and then placed in a 37°C constant temperature incubator.

[0058] Group 5 Treatment 3: Add 1 mL of enterotoxigenic Staphylococcus aureus SA202405 (1×10 7 pfu / mL) and added 1 mL of 1×10 7 pfu / mL phage GRNSAP46, and then placed in a 37°C constant temperature incubator, and the bacterial OD600 was measured every 4 hours. The results are shown in Table 6.

[0059] Table 6 Bacterial concentration statistics

[0060]

[0061] As shown in Table 6, it can be seen from treatment group 1 that the effect of Staphylococcus aureus phage GRNSAP30 in removing enterotoxin A-producing Staphylococcus aureus is equivalent to that of the high-dose antibiotic group (treatment group 2), and is safe and has no residue.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A bacteriophage against enterotoxin A-producing Staphylococcus aureus, characterized in that: The bacteriophage is Staphylococcus aureus phage GRNSAP30, and its deposit number is CCTCC NO: M2024537.

2. A phage composition against enterotoxin A-producing Staphylococcus aureus, characterized in that: The composition comprises the Staphylococcus aureus phage GRNSAP30 according to claim 1.

3. Use of a bacteriophage targeting enterotoxin A-producing Staphylococcus aureus as claimed in claim 1 in the preparation of an enterotoxin A-producing Staphylococcus aureus inhibitor.

4. Use of a bacteriophage targeting enterotoxin A-producing Staphylococcus aureus as claimed in claim 1 in a bactericide.

5. The use according to claim 4, characterized in that: Fungicides are used to kill enterotoxigenic Staphylococcus aureus.

6. Use of a bacteriophage targeting enterotoxin A-producing Staphylococcus aureus as claimed in claim 1 in the preparation of a drug for treating or preventing a disease caused by infection with enterotoxin A-producing Staphylococcus aureus.

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