CN117802053A - Pseudo-intermediate staphylococcus phage and application thereof in preventing and treating canine superficial pyodermatosis - Google Patents

Abstract

The invention discloses a pseudo-intermediate staphylococcus phage and application thereof in preventing and treating canine superficial pyoderma, belonging to the technical field of biology. The phage has been deposited in China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms at 10 and 10 of 2023 with the deposit number: cgmccno.45679. The bacteriophage SP914 has good biological characteristics, strong capability of cracking pseudointermediate staphylococci, no virulence factors, capability of removing the biological membrane of host bacteria, and potential and value of being applied to clinical treatment of canine pyoderma.

Description

Pseudo-intermediate staphylococcus phage and application thereof in preventing and treating canine superficial pyodermatosis

Technical Field

The invention relates to the technical field of biology, in particular to a pseudo-intermediate staphylococcus phage and application thereof in preventing and treating canine superficial pyoderma.

Background

Canine superficial pyoderma is a superficial bacterial infection of canine epidermis and hair follicles, including impetigo, mucocutaneous pyoderma, and superficial bacterial folliculitis (Byukusenge et al 2021). Impetigo is characterized by the development of pustules in skin with sparse hair, and mucocutaneous pyoderma occurs primarily in dogs' lips and perioral skin (Boehm et al 2017), with superficial bacterial folliculitis being an infection localized to the superficial parts of the hair follicle.

Common pathogenic bacteria for canine superficial pyoderma include staphylococci, proteus, escherichia coli, and pseudomonas aeruginosa, with pseudointermediate staphylococci (also known as "pseudointermediate staphylococci") being one of the major pathogenic bacteria. The pseudo-intermediate staphylococcus is a new coagulase positive staphylococcus (Annie, 2021) reported and named by Devrie et al in 2005, is the most commonly separated bacteria in clinical canine specimens, is mainly related to skin and ear infections, has the characteristics of difficult healing and quick recurrence and transmission, belongs to zoonotic pathogens, has research and shows that the pseudo-intermediate staphylococcus can cause postoperative urosepsis (Subedi et al, 2021), and brings great health threat to pets and pets. Studies have shown that 50% of pseudo-intermediate staphylococci harvested from canine skin are MRSP and 97.8% of MPSP are multi-resistant to 3 or more antibiotics commonly used in clinic (Feng Yong et al 2012). Pseudo-staphylococcus intermedium has a high rate of biofilm formation (Arima et al, 2018), which is one of the important causes of MRSP acquired multiple drug resistance. The biofilm refers to a biofilm formed by embedding organized microorganism aggregates in extracellular polysaccharide during the growth of bacteria, and after the microorganisms are attached to the surface of an object, the extracellular polysaccharide is released through interaction to wrap the microorganisms. The bacteria are coated by biological film, and the resistance to disinfectants, host defense systems, cytophagocytosis and antibiotics is obviously enhanced, so that the bacteria are not easy to clear, and the repeated and continuous infection is caused. The pseudointer-type staphylococci can produce a variety of exotoxins, including enterotoxins and leukotoxins, which cause severe damage to host tissues and adversely affect host defenses (abouelkhalir et al 2020).

Antibiotics are the first choice for treating canine superficial pyoderma, but the therapeutic effect is not ideal due to the enhancement of bacterial resistance. Phage is a virus with high specificity, no antibody production, short development cycle, no side effects, etc. (zithrombic et al, 2018). Phage have become an important tool for biotechnology professionals in recent years, including drug design, synthesis of new proteins, protein and DNA vaccines, detection of pathogenic bacteria, screening of proteins, polypeptides or antibodies, phage display technology, and the like (Rehman et al, 2019). Furthermore, most important is phage therapy, which is a therapeutic means for treating pathogenic bacterial infections by phage lysis of bacteria (Luo Tingting, et al, 2016). However, few phages currently available for pseudo-intermediate staphylococcal treatment have been reported; and some phage genomes contain drug resistance genes and genes encoding virulence factors, so that the risk of transmitting the drug resistance genes and the virulence genes is caused, and the application of phage in the treatment of canine superficial pyoderma caused by pseudo-intermediate staphylococci is further limited.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a pseudo-intermediate staphylococcus phage and application thereof in preventing and treating canine superficial pyoderma. The invention separates and identifies a pseudo-intermediate staphylococcus phage SP914, the biological characteristics of the SP914 are good, the genome does not contain virulence factors, and the biological membrane of host bacteria can be removed, so the invention has potential and value for being applied to clinical treatment of canine pyoderma.

Specifically, the invention relates to the following technical scheme:

in a first aspect of the present invention, there is provided a pseudo-middle staphylococcal phage (Staphylococcus pseudintermedius phage) SP914 which has been deposited at the China general microbiological culture collection center (CGMCC, address: national institute of microorganisms at national academy of sciences of China, national center for culture Collection of microorganisms, 1 st edition, 3 rd edition, of the Korean region of Beijing city) with a deposit number of: CGMCC No.45679.

The pseudo-intermediate staphylococcal phage SP914 has the following characteristics:

phage SP914 belongs to the order coccyphaidae, the family myophaidae, with a regular polyhedron with a small head and a contractile long tail. Biological characteristics and resistance detection results show that phage SP914 can lyse pseudo-staphylococcus intermedius with a measured titer of 1.24X10 ¹² PFU/mL, optimal multiplicity of infection is 1; the bactericidal kinetics of phage showed that when SP914 titer was at 10 ¹⁰ ～10 ¹² PFU/mL can continuously inhibit for 10 h ⁶ CFU/mL bacteria grow at an optimal growth temperature of 37 ℃, and have stronger tolerance to low temperatures than high temperatures, are insensitive to pH, and are sensitive to ultraviolet rays.

The DNA library was sequenced using an Illumina HiSeq high throughput sequencing platform, and the encoding genes were predicted using GeneMarkS, which showed that phage SP914 was double-stranded DNA with a full genome length of 135,892bp and a GC content of 46.61%, and that the ORFs annotation showed that phage SP914 contained no virulence genes, with safety of application.

Phage SP914 has a clearance effect on the biofilm produced by the pseudo-intermediate staphylococci, and the clearance efficiency is the highest after the two are contacted for 4 hours.

In a second aspect of the invention, there is provided the use of the pseudo-intermediate staphylococcal phage SP914 described above in (1) or (2) as follows:

(1) Preparing a product for inhibiting or killing pseudo-intermediate staphylococci;

(2) Preparing the medicine for preventing and/or treating the canine superficial pyoderma.

Preferably, the canine superficial pyoderma is caused by a pseudointermediate staphylococcal infection.

In a third aspect of the present invention, there is provided a phage preparation comprising the above-described pseudo-intermediate staphylococcal phage SP914 as an active ingredient.

Preferably, the phage preparation has a dosage of pseudointermediate staphylococcal phage SP914 of greater than or equal to 10 ⁷ PFU/mL。

The phage preparation described above may have the pseudomiddle staphylococcal phage SP914 of the invention as the sole active ingredient; other phages or antibiotics besides the phages of the invention can be contained, and the combined use of the phage with the phages of the invention can obtain the effect of preventing and treating the canine superficial pyoderma equivalent to or better than the phage.

The phage preparation can be prepared into a preparation form suitable for the object, such as a liquid preparation, a gel preparation, a freeze-dried preparation or an oral solid preparation, and can be used for preventing or treating superficial pyoderma caused by the staphylococcus pseudointermedia by spraying, smearing, injection or oral administration.

Examples of the auxiliary components include aqueous media such as physiological saline, buffers, and distilled water, nonaqueous media such as polyethylene glycol and propylene glycol, surfactants, vegetable oils, emulsifiers, excipients, binders, lubricants, cosolvents, and antioxidants.

In a fourth aspect of the invention, there is provided the use of a phage preparation as described above in the manufacture of a medicament for the prevention and/or treatment of canine superficial pyoderma.

In the above application, the canine superficial pyoderma is caused by a pseudointermediate staphylococcal infection.

The invention has the beneficial effects that:

(1) The invention separates 1 bacteriophage SP914 from the surrounding hair and scab of the affected dog focus, the bacteriophage SP914 belongs to the myotail bacteriophage family, can specifically lyse pseudo-middle staphylococcus, and has high potency; furthermore, the bacterial SP914 of the present invention has excellent pH resistance and is excellent in environments of strong acids and strong basesCan still keep 10 ⁷ The higher potency of PFU/mL can be kept in the normal temperature range of the canine skin, and has the potential of developing pharmaceutical preparations.

(2) The bacteriophage SP914 of the invention does not contain virulence genes and is safe to use.

(3) The bacteriophage SP914 provided by the invention has the capability of clearing the biomembrane of the pseudo-intermediate staphylococcus, so that the drug resistance of the pseudo-intermediate staphylococcus to antibiotics is reduced, and the capability of treating the canine superficial pyoderma in a synergistic way with the antibiotics is provided.

Drawings

Fig. 1: phage morphology observations.

Fig. 2: electron microscopy of phage SP914.

Fig. 3: results of potency test of phage SP914.

Fig. 4: optimal multiplicity of infection with phage SP914.

Fig. 5: one-step growth curve of phage SP914.

Fig. 6: uv stability of phage SP914.

Fig. 7: pH stability of phage SP914.

Fig. 8: effect of temperature on phage SP914 titer.

Fig. 9: phage SP914 kinetic assay results.

Fig. 10: phage SP914 genomic circle map.

Fig. 11: phage SP914 genomic analysis GC content map.

Fig. 12: pseudo-intermediate staphylococcal biofilm formation ability results.

Fig. 13: the clearance of the pseudomiddle staphylococcal biofilm by phage SP914 results.

Detailed Description

As described above, the main pathogenic bacteria of the canine superficial pyoderma is pseudointermediate staphylococci, which has strong drug resistance and can generate a biological film, and after the pseudointermediate staphylococci generate the biological film, the pseudointermediate staphylococci can be planted on the skin to evade the immune system, limit the action of antibiotics and resist disinfectants such as iodophor, thereby further increasing the difficulty in preventing and treating the pseudointermediate staphylococci.

Phage therapy is a novel pathogenic bacteria control mode and has unique advantages. However, the bacteriolytic activity of the existing phage pseudointermedia staphylococci is not ideal. Moreover, the action relationship between the phage and the pathogenic bacteria is complex, and even some phages can establish symbiotic relationship with the pathogenic bacteria to induce and strengthen the biological film. Therefore, it is still difficult to control pseudointermedia staphylococci which are resistant and can produce biofilm by using phage.

In view of the above, the present invention has conducted intensive studies on phage for preventing and treating pseudo-intermediate staphylococci, by collecting hair, scab and wet plantar hair around the lesion of skin of a canine, and performing a large number of separation and screening by a double-layer flat plate method, and finally separating a phage SP914 which can specifically lyse pseudo-intermediate staphylococci, has high lysis activity and measured titer of 1.24×10 ¹² PFU/mL, optimal multiplicity of infection is 1; the bactericidal kinetics of phage showed that when SP914 titer was at 10 ¹⁰ ～10 ¹² PFU/mL can continuously inhibit for 10 h ⁶ CFU/mL bacterial growth. Furthermore, the phage has a clearing effect on the biofilm produced by the pseudointermedia.

Further, the ORFs annotation result shows that phage SP914 of the invention does not contain virulence genes and has application safety.

In conclusion, the bacteriophage SP914 has good biological characteristics, strong capability of cracking pseudo-intermediate staphylococci, no virulence factors, capability of removing biological membranes of host bacteria, and potential and value of being applied to clinical treatment of canine pyoderma, thereby providing the invention.

In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with reference to specific embodiments. If experimental details are not specified in the examples, the conditions are generally conventional or recommended by the reagent company; reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified. The composition of the medium and buffer used in the present invention is as follows:

LB solid medium: 10g of sodium chloride, 5g of yeast powder, 10g of tryptone and 15g of agar powder are added into 1000mL of deionized water, and the pH value is adjusted to 7.4.

LB liquid medium: 10g of sodium chloride, 5g of yeast powder and 10g of tryptone are added into 1000mL of deionized water, and the pH value is adjusted to 7.4.

LB semisolid culture medium: 10g of sodium chloride, 5g of yeast powder, 10g of tryptone and 7.5g of agar powder are added into 1000mL of deionized water, and the pH value is adjusted to 7.4.

SM buffer: to 950mL of deionized water was added 11.7g of sodium chloride, 1.4g of magnesium sulfate heptahydrate, 50mL of Tirs-HCl and the pH was adjusted to 7.5.

The pseudointermediate staphylococci used in the examples of the present invention were isolated from skin lesions of dogs and were present pseudointermediate staphylococci.

Example 1: isolation and purification of pseudo-intermediate staphylococcal phage

1. Enrichment of phages:

hair, crusts around canine skin lesions, and sewage in the base were taken from a taway breeding base. The hair and crust were placed in 10mL of physiological saline, shaken with a shaker for 2min, then placed in a centrifuge, centrifuged at 12,000rmp for 5min, and the supernatant was taken. After the sewage is centrifuged, the supernatant is taken and filtered and stored for standby by a filter membrane with the diameter of 0.22 mu m.

6mL of LB liquid medium and 100 mu L of pseudo-middle staphylococcus liquid are added into a 10mL sterilization centrifuge tube, finally 1mL of supernatant after centrifugation is added, the temperature is 37 ℃, 200rmp shaking culture is carried out for 12h,12,000rmp centrifugation is carried out for 5min, the supernatant is taken, and a filter membrane with the thickness of 0.22 mu m is used for filtering to obtain phage amplification liquid. Phage amplification and isolation solutions can be stored at 4 ℃.

2. Separation by a double-layer plate method:

preparing LB solid culture medium and LB semisolid culture medium, sterilizing under high pressure, spreading LB solid culture medium on bottom layer, and waiting for cooling. When the LB semisolid culture medium is at about 60 ℃, 8mL of semisolid culture medium and 100 mu L of bacterial liquid are added into a 10mL centrifuge tube, and the mixture is shaken upside down and poured into the LB solid culture medium to wait for cooling. 100. Mu.L of phage amplification solution was pipetted onto a double-layered plate and incubated in an incubator at 37℃for 24h. If a patch of irregularly shaped transparent spots or individual transparent spots appears on the dish, it is indicated that phage are present.

3. Purification of phage:

adding 5mL of LB liquid medium and 100 mu L of pseudo-middle staphylococcal bacteria liquid into a 10mL centrifuge tube, stamping the bacterial plaque 3 times by using a pipette gun head, pouring the bacterial plaque into the centrifuge tube, culturing overnight by using a shaking table to obtain a stock phage liquid, filtering the stock phage liquid by using a 0.22 mu m filter membrane to obtain a phage liquid, preparing a double-layer flat plate according to the steps, slightly different, adding 100 mu L of pseudo-middle staphylococcal bacteria liquid and 100 mu L of phage liquid into the 10mL centrifuge tube after adding the semi-solid LB medium, mixing the stock phage liquid with the 100 mu L of pseudo-middle staphylococcal bacteria liquid in an upside down manner, pouring the mixture onto solid LB, and culturing the solid LB in an incubator at 37 ℃ for 24 hours. Adding 1mL of sterilized SM buffer solution into a 2mL centrifuge tube, taking 3 plaques with similar sizes by using a gun head, adding the plaques into the SM buffer solution, oscillating for 2min by using a shaker, filtering to obtain a first purified phage solution, and repeating the steps for 3 times until the sizes and the shapes of the plaques are basically consistent (figure 1). Finally, a phage was isolated and designated SP914.

10. Mu.L of phage SP914 was dropped on a copper net and left standing for 1min, the excess liquid was sucked off with filter paper, stained with 2% phosphotungstic acid for 1min, the excess liquid was sucked off, and observed by electron microscopy after drying, as shown in FIG. 2, phage SP914 had a regular polyhedron small head followed by a long and retractable tail with a head length of 44.47nm, a width of 38.99nm, a tail length of 109.78nm and a width of 6.20nm.

The phage SP914 was subjected to the biological preservation of the patent procedure for the following period: 2023, 10 months and 10 days; preservation unit: china general microbiological culture Collection center (CGMCC, address: china academy of sciences of China, including national institute of microbiology, no. 3, north Chen West Lu No. 1, chaoyang district, beijing, city); the preservation number is: CGMCC No.45679.

Example 2: biological characterization of the pseudo-intermediate staphylococcal phage SP914

1. Phage titer assay:

phage titer (PFU/mL) is the number of phage particles per 1mL volume. 1mL of pseudo-middle staphylococcus liquid and phage liquid which are cultivated to a logarithmic phase are added into 20mLLB liquid medium, shaking cultivation is carried out for 12 hours at 37 ℃, filtration is carried out by a filter membrane, SM buffer solution is used for continuous 10-fold ratio dilution, 3 double-layer plates are made for each concentration according to the method, cultivation is carried out for 12 hours at 37 ℃ in an incubator, the number of plaques is calculated, average value is taken, and the titer is calculated according to a formula.

Phage titer (PFU/mL) =number of plaques x dilution x 10.

As a result, as shown in FIG. 3, when the phage stock was diluted to 10 ⁹ 124 plaques were found at the fold, and the potency of SP914 was 1.24X10 according to the formula ¹² PFU/mL。

2. Determination of phage lysis spectra:

3 strains of pseudo-middle staphylococci and 7 strains of escherichia coli are adopted as objects, a double-layer flat plate is manufactured, 50 mu L of phage SP914 liquid is dripped on the surface of the double-layer flat plate, the double-layer flat plate is cultured overnight in a 37 ℃ incubator, and if plaque appears on the surface of the double-layer flat plate, the double-layer flat plate has a cracking effect on the surface of the double-layer flat plate. The results are shown in Table 1.

Table 1: phage lysis profiling

Note that: "+" represents cleavage; "-" means not cleaved.

The results show that: phage SP914 only has a lytic effect on 3 pseudo-intermediate staphylococci, and has strong specificity.

3. Complete lytic titers of phages:

fixing the concentration of the staphylococcus pseudointermedia bacterial liquid to be 10 ⁶ 2 mu L of CFU/mL is dripped on a solid agar detection plate for 12 times, the distance is controlled, and the solid agar detection plate is dried. The phage solution was diluted 10-fold in gradient, and 2. Mu.L of each concentration was added to the original bacterial solution and dried. Three replicates were each observed after culturing in an incubator at 37℃for 12-18 hours.

The concentration of the fixed bacterial liquid is 10 ⁶ CFU/mL, phage SP914 stock solution titer was 10 ⁹ PFU/mL, when the titer is diluted to 10 ⁷ The PFU/mL can completely inhibit bacterial growth when the potency is diluted to 10 ⁶ The bacteria grow well when PFU/mL, so the concentration of the bacterial liquid is 10 ⁶ Complete lysis titers of phage at CFU/mL were 10 ⁷ PFU/mL。

4. Optimal multiplicity of infection of phage:

the multiplicity of infection (MOI) of phage is the ratio of phage to bacterial numbers. The optimal multiplicity of infection (OMOI) is the ratio of the number of phage that most effectively kills bacteria when they produce more progeny phage. The concentration of the pseudo-middle staphylococcus liquid in the logarithmic phase is adjusted to 10 ⁸ CFU/mL, phage titers were measured, MOI was set to 0.001, 0.01, 0.1, 1, 10, 100, and bacterial and phage solutions were 1mL each, and shake cultured at 37℃for 8 hours. The titer was measured after filtration, and the multiplicity of infection with the highest titer, i.e. the optimal multiplicity of infection, was repeated in three groups each.

As a result, as shown in FIG. 4, the titer of SP914 was highest when the multiplicity of infection was 1, indicating that the optimum multiplicity of infection of the pseudo-intermediate staphylococcal phage was 1.

5. One-step growth curve of phage:

the one-step growth curve is a test curve for quantitatively describing the growth rule of the phage, and the incubation period and the cracking amount of the phage are obtained. Mixing 1mLSP914 phage solution and 1mL pseudo-intermediate staphylococci at optimal infection ratio, incubating at 37deg.C with 200rmp shaker for 30min, centrifuging at 12,000rmp for 1min, discarding supernatant, re-suspending with LB liquid, centrifuging again, repeating for 3 times, adding 10mL preheated LB liquid, shaking at 37deg.C, sampling once every 10min for measuring titer, and setting three groups of replicates each.

As a result, as shown in FIG. 5, 30 minutes before SP914 contacts the host bacteria, the number of phages did not increase, indicating that this period of time is a incubation period. The number of phages increases in bursts at 30-90 min, indicating that the period is burst period, the length is about 60min, and the burst amount is about 10 ⁵ PFU/mL. After 90min the phage titer was no longer increased, indicating a walk-inStationary phase.

6. Influence of ultraviolet light on phage:

subpackaging the phage solution with the measured titer into 2mL centrifuge tubes, selecting test points, uniformly irradiating the centrifuge tubes with sunlight, respectively measuring the titers after 10, 20, 30, 40, 50, 60 and 70min irradiation, respectively setting three groups of replicates, and carrying out light-shielding treatment on the control groups.

As a result, as shown in FIG. 6, phage titers were increased significantly (P < 0.05) when SP914 was irradiated with ultraviolet light for 10min and 20 min. When irradiated by ultraviolet rays for 30min, phage titers began to decrease, with little difference from the control group. Phage titers exhibited a very significant decrease (P < 0.001) after 40min of uv exposure. These results indicate that SP914 has some resistance to UV light for the first 20min, but after more than 20min of UV exposure, the phage resistance to UV light gradually decreases until complete inactivation.

7. Effect of pH on phage:

the pH of the sterilized SM buffer solution is respectively adjusted to 3-12 by NaOH and HCl, the SM buffer solution with the pH of 7.4 is set as a control group, 500 mu L of phage solution with determined titers is mixed with the SM buffer solution with equal volumes of each pH, the titers are determined after shaking culture for 2 hours at 37 ℃, and three groups of replicates are respectively set.

The results are shown in FIG. 7, where SP914 was in a pH 3, 4, 5, 12 environment, the potency was extremely significantly reduced (P<0.001 At a pH of 3, but at a potency of 10 ⁷ PFU/mL. At pH 6, 11, the potency was significantly reduced (P<0.05). The titers were not significantly different from the control group at pH 7, 7.4, 8, 9, 10, with the highest titers at pH 7.4. The SP914 was shown to be more pH tolerant, less resistant to strong acids than strong bases, and most suitable for phage survival at pH 7.4.

8. Influence of temperature on phage:

separately packing the phage solution with measured titer into 2mL centrifuge tubes, standing at-20deg.C, 4deg.C, 25deg.C, 37deg.C, 28deg.C, 65deg.C, 70deg.C and 95deg.C for 1 hr, respectively, setting 37 deg.C group as control group, measuring titer by double-layer plate method, and repeating three groups.

As a result, as shown in FIG. 8, the phage titer was extremely remarkably reduced (P < 0.001) when SP914 was at-20 ℃, 46 ℃, 54 ℃, 65 ℃, 70 ℃ and 95 ℃, and 0 when the temperature reached 54 ℃ or higher. Phage titers decreased significantly when at 4 ℃ (P < 0.05). When the temperature is 25-37 ℃, the phage titer is not greatly different from that of a control group, and the efficiency is highest at 37 ℃. The optimum survival temperature of SP914 was 37℃and the tolerance to low temperatures was stronger than the tolerance to high temperatures.

9. Bacteriocidal kinetics of phages:

the concentration of the fixed pseudo-middle staphylococcus bacteria liquid is 10 ⁶ Diluting phage solution with CFU/mL ratio of 10 times, collecting 100 μL of phage solution in 96-well plate, adding 100 μL of phage solution, and measuring OD every 15min with dynamic enzyme-labeling instrument ₆₂₀ The values were measured for a total of 24h.

The results are shown in FIG. 9 when phage titer was at 10 ¹² PFU/mL～10 ¹⁰ PFU/mL, phage was inhibited for 24h for 10 hours ⁶ Pseudostaphylococcus intermedia growth at CFU/mL concentration. When the potency is at 10 ⁷ PFU/mL～10 ⁹ At PFU/mL, phage inhibited the pseudointermedial staphylococcal proliferation to some extent, and eventually the bacterial count increased exponentially, but the bacterial count was much lower than in the control. When the potency is at 10 ² PFU/mL～10 ⁶ At PFU/mL, the growth trend of the pseudo-intermediate staphylococci is not much different from that of the control group, which indicates that the bacteria inhibition effect is not generated.

Example 3: analysis of the toxicity Gene of pseudo-intermediate staphylococcal phage SP914

1. The test method comprises the following steps:

extracting DNA of phage SP914 from the purified phage with bacterial DNA kit (OMEGA), constructing library by Illumina TruSeqTMNano DNA Sample Prep Kit method, cutting DNA into proper size, modifying DNA tail, connecting adapter, and performing PCR amplification.

Based on sequencing-by-synthesis (Sequencing By Synthesis, SBS) technology, DNA libraries were sequenced using Illumina Hiseq high throughput sequencing platform, sequencing was done by fries biotechnology limited, singapore.

And (3) splicing the optimized sequence by using AByss (http:// www.bcgsc.ca/platfor/bioinfo/software/AByss) splicing software to obtain the optimal assembly result. Next, the assembly results were subjected to local hole filling and base correction using GapClosser (https:// sourceforge. Net/projects/sodden ovo 2/files/GapClosser /) software (Zeman et al, 2019).

The coding genes are predicted by the GeneMarkS technique, and the accuracy of the predicted gene start positions is improved by combining a coding region model, a non-coding region model and a regulatory region model near the gene start positions (Peng Yuan, 2019). The protein sequences of the predicted genes were aligned with databases such as NR, swiss-Prot, eggNOG, KEGG, GO, respectively, to annotate the genes.

2. Test results:

the second generation sequencing of phage SP914 produced effective data of approximately 29.83Mb in size, 11955 fragments, SP914 having a full genome length of 135,892bp, which is linear double stranded DNA, with genome circles as shown in FIG. 10 and a (G+C) content of 46.61% in the genome as shown in FIG. 11.

Phage genomes were read using GeneMarks software, and the results showed that the SP914 genome contained 65 Open Reading Frames (ORFs), 33 being hypothetical proteins, and 32 having specific functions. By annotating the genome to obtain results, we split these functional ORFs into several modules: 1. related proteins consisting of phage structures: main capsid protein, tail tape protein, tail tube protein, tail sheath protein, and basal plate J-like protein. 2. Proteins associated with replication and metabolism: transposases, DNA polymerase I structures, DNA priming enzymes, DNA helicases, RNA polymerases, HNH endonuclease, ribo-diphosphate reductase subunits, ribonucleotide reductase large subunits, recombinant endonucleases, DNA methyltransferases, recombinant exonucleases, adenine-specific DNA methylases, repressor proteins, dutpases, portal proteins. 3. Protein associated with phage packaging: a large terminal enzyme subunit and a small terminal enzyme subunit. 4. Protein related to sterilization and bacteriostasis: perforin, ig domain-containing protein, ORFs annotated for known functions are shown in table 2.

Table 2: ORF function annotation of phage SP914

ORFs annotation results showed that SP914 does not contain virulence genes and has application safety.

Example 4: cleaning action of bacteriophage SP914 on pseudo-intermediate staphylococcal biofilm

1. The test method comprises the following steps:

1.1 biofilm-producing ability of pseudointermediate staphylococci:

1mL of liquid LB was added to a 2mL centrifuge tube, and a single colony of pseudomiddle staphylococci (strains 914, 308, 718) was picked and inoculated into the centrifuge tube, and shaken by a 180rmp shaker at 37℃for 8h. The shaken bacteria were centrifuged for 3min at 12,000rmp, the supernatant discarded and diluted to 108CFU/mL with sterile PBS. 150. Mu.L of liquid LB medium and 50. Mu.L of diluted bacterial liquid were added to a sterile 96-well plate, and three replicates were performed for each sample. Covering with sealing film, and culturing in incubator at 37deg.C for 24 hr. The liquid in the hole is sucked by a liquid transfer device, and the hole wall and the hole bottom can not be touched. After addition of 1mL of sterile PBS, the solution was pipetted off and repeated 3 times. Drying the sterilized 96-well plate in an oven for 5min, dripping 200 μl of dye liquor for dyeing, standing for 10min, adding sterile PBS, washing for 3 times, and waiting for drying. 200. Mu.L of 95% ethanol was added dropwise thereto, and the mixture was incubated for 15 minutes, and the absorbance at 620nm was measured by an enzyme-labeled instrument.

1.2 Capacity of phage SP914 to clear pseudo-middle staphylococcal biofilm:

150. Mu.L of liquid LB medium was added to the sterilized 96-well plate, and 50. Mu.L of 10 was inoculated ⁷ And (5) placing the CFU/mL bacterial liquid into an incubator for culturing for 24 hours. Washing with sterile PBS for 5 times, washing free bacteria, standing in a 64 deg.C oven for 5min, and drying. Setting blank control group, dripping 10 into each hole ⁹ PFU/mL phage solution 200. Mu.L, incubator culture. Is sucked and discarded at 1, 2, 4, 6 and 8 hours respectivelyWashing the supernatant with sterile PBS three times, drying in an oven for 5min, staining with 200 μl dye, standing for 10min, washing 96-well plate with PBS solution for 5 times, and naturally air-drying. 200. Mu.L of 95% ethanol was added dropwise thereto, and the mixture was allowed to stand for 15 minutes, whereby the absorbance at 620nm was measured by an enzyme-labeled instrument. The calculation was performed using the following formula.

Percent inhibition = [ (OD blank-OD treatment)/OD blank ] ×100

2. Test results:

2.1 biofilm-producing ability of pseudointermediate staphylococci:

as shown in FIG. 12, the absorbance of each of 3 pseudo-middle staphylococci was extremely remarkably increased (P < 0.001), indicating that the 3 host bacteria were able to produce a biofilm, but the ability to produce a biofilm was different, the ability of strain 308 to produce a biofilm was strongest, and the ability of strain 718 to produce a biofilm was weakest.

2.2 Capacity of phage SP914 to clear pseudo-middle staphylococcal biofilm:

as a result, as shown in FIG. 13, phage SP914 cleared the biofilm of three pseudo-intermediate staphylococci to varying degrees, but at varying efficiencies. When the time is 1 h-4 h, the cleaning efficiency of the biological film is gradually enhanced along with the time, and the highest cleaning efficiency is reached at the 4 th h.

The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the same, but rather, various modifications and variations may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims (8)

1. A pseudomiddle staphylococcal phage (Staphylococcus pseudintermedius phage) SP914 with accession number: cgmccno.45679.

2. Use of the pseudomiddle staphylococcal phage SP914 of claim 1 in (1) or (2) as follows:

(1) Preparing a product for inhibiting or killing pseudo-intermediate staphylococci;

(2) Preparing the medicine for preventing and/or treating the canine superficial pyoderma.

3. The use of claim 2 wherein the canine superficial pyoderma is caused by a pseudointermediate staphylococcal infection.

4. A phage preparation comprising the pseudostaphylococcus intermedia phage SP914 of claim 1.

5. The phage preparation of claim 4, wherein the phage preparation has a dosage of pseudos.intermedia phage SP914 of greater than or equal to 10 ⁷ PFU/mL。

6. The phage preparation of claim 4, further comprising an adjuvant selected from one or more of physiological saline, buffer, distilled water, polyethylene glycol, propylene glycol, surfactant, vegetable oil, emulsifier, excipient, binder, lubricant, co-solvent, and antioxidant.

7. The phage preparation of claim 4, wherein the phage preparation is in the form of a liquid preparation, a gel preparation, a lyophilized preparation or an oral solid preparation.

8. Use of a phage preparation according to any one of claims 4-7 for the manufacture of a medicament for the prevention and/or treatment of canine superficial pyoderma.