Salmonella enteritidis bacteriophage PC571, and bacteriophage composition and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and discloses a salmonella enteritidis bacteriophage PC571, a bacteriophage composition thereof and application of the bacteriophage PC571, the preservation number of the bacteriophage PC571 is CGMCC No.46168, and the bacteriophage PC571 is preserved in China General Microbiological Culture Collection Center on August 16, 2024. The salmonella enteritidis bacteriophage PC571 is wide in splitting spectrum, has good splitting capability on salmonella bacteriophage resistant bacteria, and can cooperate with other existing salmonella bacteriophages for bacteriostasis. The salmonella enteritidis bacteriophage PC571 or the bacteriophage composition thereof can be used as an active ingredient to prepare a pharmaceutical preparation, an environmental disinfectant, a bacteriostatic agent, a detection kit and the like for application. The salmonellosis in a livestock farm can be purified by mixing the phage with water and feed for a long time, and in addition, the phage is relatively high in safety, can effectively prevent and treat infection of salmonella with phage resistance, and has a market application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and particularly relates to a Salmonella enteritidis bacteriophage PC571, a bacteriophage composition thereof and application thereof. BACKGROUND

[0002] Salmonella as a common foodborne pathogen poses a serious threat to human and animal health. However, traditional antibiotic therapy in dealing with Salmonella infection faces many challenges, such as the emergence of drug resistance and drug side effects. Salmonella phage as a natural antibacterial agent has shown great application potential, and the potential of phage in combating Salmonella infection has been confirmed in many studies. With the in-depth study of Salmonella and phage, it is found that there is a complex coevolution process between phage and bacteria. Salmonella constantly evolves its defense mechanisms, such as changing surface receptors, enhancing cell wall thickness or producing new resistance genes, to resist the attack of phage. While phage is not willing to be defeated, they evolve their attack strategies, such as changing adsorption receptors, improving lysozyme activity or producing new attack mechanisms, to break through the defense of Salmonella.

[0003] However, although bacteria can inhibit phage adsorption by changing their structure or deleting phage target receptors, it will affect some important bacterial functions, such as biofilm formation, surface adhesion, motility or antibiotic resistance. On the other hand, the loss or change of cell wall molecules makes the bacteria lose the defense ability to the body's immune system (such as serum complement system or phagocytosis). MARKWITZ P et al. isolated and identified 57 Klebsiella pneumoniae phage-resistant mutants, all of which showed impaired phage adsorption. Among the 57 resistant mutants, many strains showed reduced ability to form biofilm, loss of motility, reduced expression of virulence factors, increased sensitivity to certain antibiotics, and reduced pathogenicity in the model of Galferia mellonella.

[0004] Although some studies have found that phage-resistant bacteria have reduced pathogenicity and virulence, Salmonella with phage resistance still poses an infection risk. However, there is no effective broad-spectrum Salmonella phage that can prevent and treat Salmonella with phage resistance.

[0005] Therefore, the prior art still needs to be further improved. SUMMARY

[0006] In order to solve the above problems, the application provides a Salmonella enteritidis bacteriophage PC571, a bacteriophage composition thereof and application thereof, the Salmonella enteritidis bacteriophage PC571 has broad-spectrum lytic performance on Salmonella with bacteriophage resistance, can be used as an active ingredient to prepare a pharmaceutical preparation, an environmental disinfectant, a bacteriostatic agent and a detection kit, and can effectively prevent and treat Salmonella infection of poultry and realize purification of Salmonella in a farm.

[0007] In a first aspect, the application provides a Salmonella enteritidis bacteriophage PC571, which has a preservation number of CGMCC No. 46168.

[0008] The Salmonella enteritidis bacteriophage PC571 described above is isolated from feces of a broiler farm in Shandong Province, and the bacteriophage is preserved in the China General Microbiological Culture Collection Center on August 16, 2024, and the preservation address is No. 1, Beichen West Road, Haidian District, Beijing, and the preservation number is CGMCC No. 46168.

[0009] In the application, the bacteriophage PC571 includes a mutant strain with a homology higher than 98% or 99% and a basic same bactericidal activity after point mutation, deletion mutation or addition mutation. Since the bacteriophage is very easy to mutate during replication, the mutant of the bacteriophage described above is also within the scope of protection of the application. The sequence of the bacteriophage PC571 can be sequenced by a known method according to the biological material preserved in the application. For those skilled in the art, it does not require creative labor to screen a mutant extremely similar to the bacteriophage in properties according to the bacteriophage provided in the application.

[0010] Under electron microscopy, the bacteriophage PC571 has a polyhedral head structure and a non-contracted tail, the head is 60 nm wide and 68 nm long, the tail is about 180 nm long, according to the classification method of the International Committee on Virus Classification (ICTV), the morphology of the bacteriophage PC571 of the application meets the characteristics of the long-tailed bacteriophage family, and belongs to the long-tailed bacteriophage.

[0011] The bacteriophage PC571 has certain temperature stability and acid-base stability. Within the pH 5.0-10.0 range, the titer can still maintain 10 9 PFU / mL, and the activity is stable. After 60 min under the temperature condition of 60 DEG C, the original activity is still maintained. The bacteriophage can adapt to the environment with a certain temperature range and acid-base range.

[0012] The bacteriophage PC571 has a wide lytic spectrum for the bacteriophage-resistant Salmonella, and the lysis rate reaches 90%. The bacteriophage has high application value in the inhibition of bacteriophage-resistant Salmonella and the prevention and treatment of diseases caused by infection.

[0013] In a second aspect, the present application provides a bacteriophage composition comprising the Salmonella enteritidis bacteriophage PC571.

[0014] In practical applications, in order to further broaden the lysis spectrum of the bacteriophage preparation, fully exert the differences in the lysis spectrum of different bacteriophages, and perform complementary advantages, the PC571 and other bacteriophages can be used in combination, such as being combined with one or two of other existing bacteriophages of Salmonella, so as to expand the bactericidal spectrum and kill as many Salmonella in the environment as possible, for the prevention and treatment of salmonellosis.

[0015] Alternatively, the bacteriophage composition comprises one or more of the following: the bacteriophage PC571, the bacteriophage SP8 with the preservation number of CGMCC No.45256, the bacteriophage SP4 with the preservation number of CGMCC No.14332, and the bacteriophage SPP11 with the preservation number of CGMCC No.18868. Through the combined use of these bacteriophages, not only the lysis spectrum is broadened, but also different types of Salmonella in the breeding environment and Salmonella resistant to bacteriophages can be effectively killed, which can be used for better prevention and treatment of poultry salmonellosis.

[0016] In practical applications, the PC571 can have a synergistic bacteriostatic effect on the combined Salmonella bacteriophage, can reduce the bacterial concentration, and prolong the bacteriostatic time.

[0017] The specific information of the bacteriophage SP8 with the preservation number of CGMCC No.45256 is shown in the patent with the publication number CN116286671A; the specific information of the bacteriophage SP4 with the preservation number of CGMCC No.14332 is shown in the authorized patent with the publication number CN108359644B; and the specific information of the bacteriophage SPP11 with the preservation number of CGMCC No.18868 is shown in the authorized patent with the publication number CN111254121B. The specific characteristics of the three existing bacteriophages will not be described here.

[0018] Preferably, the bacteriophage composition comprises the Salmonella enteritidis bacteriophage PC571 and the bacteriophage SP8 with the preservation number of CGMCC No.45256.

[0019] The experimental results prove that the synergistic bacteriostatic effect of the Salmonella enteritidis bacteriophage PC571 and the bacteriophage SP8 is the best, and the synergistic effect of the two can significantly prolong the bacteriostatic time of the two on multiple strains of clinically isolated Salmonella, and exert a better bacteriostatic effect.

[0020] In addition, the above-mentioned bacteriophage PC571 can be combined with other different kinds of bacteriophages (inhibiting different pathogenic bacteria causing the same disease) for the prevention and treatment of the same disease, such as acute septicemia caused by mixed infection of different pathogenic bacteria.

[0021] In a third aspect, the present application further provides the use of the above-mentioned Salmonella enteritidis bacteriophage PC571 or the above-mentioned bacteriophage composition in the preparation of a pharmaceutical preparation, an environmental disinfectant, a bacteriostatic agent or a detection kit for preventing and treating poultry diseases caused by bacteriophage infection.

[0022] The prevention and treatment include prophylaxis and therapy. The term "prophylaxis" herein refers to all actions for inhibiting or delaying the disease by administering the composition. The term "therapy" herein refers to all actions for improving or ameliorating the disease by administering the composition.

[0023] Optionally, the poultry diseases caused by bacteriophage infection include chicken pullorum disease, fowl typhoid, paratyphoid or oviduct inflammation caused by Salmonella.

[0024] In a fourth aspect, the present application further provides a bacteriophage medicine, wherein the effective component comprises the above-mentioned Salmonella enteritidis bacteriophage PC571 or the above-mentioned bacteriophage composition.

[0025] Optionally, the Salmonella enteritidis bacteriophage PC571 used in the bacteriophage medicine is a bacteriophage proliferation solution or a dosage form further processed.

[0026] Preferably, the bacteriophage medicine further comprises other bacteriostatic or bactericidal active ingredients. The preparation of the medicine is an oral administration dosage form, an external use dosage form or a parenteral administration dosage form.

[0027] The application method of the bacteriophage medicine is to add the bacteriophage or its composition as a therapeutic medicine to drinking water or feed, or to administer by gavage, or by subcutaneous injection, or by intramuscular injection, so as to prevent and treat Salmonella disease and improve the survival rate of poultry.

[0028] Optionally, the bacteriophage pharmaceutical preparation further comprises a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the active components to be administered. In order to formulate the pharmaceutical composition into a liquid preparation, the pharmaceutically acceptable carrier must be suitable for sterility and biocompatibility. Examples include saline, sterile water, Ringer's solution, buffered physiological saline, albumin infusion, glucose solution, maltodextrin solution, glycerol, ethanol, various types of culture medium, etc. They can be used alone or in any combination thereof. Other conventional additives, such as antioxidants, buffers and bacteriostatic agents, etc. can be added as needed. When combined with diluents, dispersants, surfactants, binders and / or lubricants, the composition of the present application can also be prepared into injection and oral dosage forms (e.g. aqueous solutions, suspensions and emulsions, pills, capsules, granules) and other intermediate dosage forms, such as lyophilized agents.

[0029] In a fifth aspect, the present application also provides a bacteriostatic agent, which comprises the Salmonella enteritidis bacteriophage PC571 or the bacteriophage composition as described above.

[0030] In a sixth aspect, the present application also provides an environmental disinfectant, which comprises the Salmonella enteritidis bacteriophage PC571 or the bacteriophage composition as described above.

[0031] Preferably, the concentration of the bacteriophage is 10 8 PFU / mL or more.

[0032] Optionally, the environmental disinfectant further comprises other active ingredients for inhibiting or eliminating bacteria in the poultry breeding environment.

[0033] In a seventh aspect, the present application also provides the use of the environmental disinfectant as described above in the disinfection of the poultry breeding environment or the poultry slaughtering environment, and the application method is that the environmental disinfectant can be used to disinfect Salmonella in the poultry breeding environment or the poultry slaughtering environment by spraying or soaking, etc.

[0034] Specifically, in the application of the poultry breeding environment, the environmental disinfectant can be applied in feed, water and breeding environment, including feeding tools such as cages, waterers, troughs, feces and litters.

[0035] The application method includes but is not limited to liquid soaking, spraying, combined use with aqueous carrier, etc. The water distribution system of the poultry breeding farm, breeding facilities, feeding appliances or other environmental surfaces can be disinfected and decontaminated, and the feed can be disinfected and preserved. This disinfectant can be used to replace antibiotics or traditional disinfectants, and it does not harm humans and poultry.

[0036] Specifically, in the application of the poultry slaughter environment disinfection, the application method is: applying the environment disinfectant to the disinfection of slaughterhouses, meat processing workshops and processing tools to prevent the pollution of Salmonella in the environment.

[0037] In an eighth aspect, the application also provides a detection kit, which comprises the Salmonella enteritidis bacteriophage or the bacteriophage composition.

[0038] Experiments prove that the bacteriophage PC571 has a lytic specificity for host bacteria, and the bacteriophage can be applied to rapid detection of Salmonella, including but not limited to detection of Salmonella in the form of test paper, test paper box and the like, or screening of target pathogenic bacteria in clinical samples.

[0039] The application has the following beneficial effects: 1. The application first isolates a bacteriophage PC571 which can efficiently lyse Salmonella with bacteriophage resistance, and the bacteriophage has strong lytic properties and high lytic rate characteristics for Salmonella bacteriophage-resistant bacteria, can play a synergistic bacteriostatic effect with other existing Salmonella bacteriophages, can inhibit the generation of Salmonella bacteriophage-resistant bacteria in combination with other bacteriophages, prolong the bacteriostatic time, and effectively control the concentration of pathogenic bacteria.

[0040] 2. Based on the excellent lytic performance and environmental stability, the bacteriophage PC571 or the bacteriophage composition thereof can be prepared into a medicine, an environment disinfectant, a bacteriostatic agent or a detection kit for preventing and treating Salmonella disease as an active ingredient, can effectively prevent and treat the spread of Salmonella disease, and can achieve the purpose of purifying Salmonella disease in the breeding farm by adding the bacteriophage into water and feed for a long time while solving the Salmonella infection of poultry.

[0041] 3. The bacteriophage disclosed by the application is obtained from nature and is easy to be industrialized, and the medicine or disinfectant prepared from the bacteriophage can reduce the cost. In addition, the bacteriophage does not contain a coding gene of known antibiotic resistance and virulence factor, and does not have lysogeny-related genes, has high safety, and is green and environmentally friendly. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 Temperature stability test results of the Salmonella enteritidis bacteriophage PC571; Figure 2 Acid and alkali stability test results of the Salmonella enteritidis bacteriophage PC571; Figure 3 One-step growth curve test results of the Salmonella enteritidis bacteriophage PC571; Figure 4 Electron microscope graph of the Salmonella enteritidis bacteriophage PC571; Figure 5Figure for bacteriostatic effect of Salmonella enteritidis bacteriophage PC571 and bacteriophage composition on Salmonella 2411C01; Figure 6 Figure for bacteriostatic effect of Salmonella enteritidis bacteriophage PC571 and bacteriophage composition on Salmonella 2411C02; Figure 7 Figure for bacteriostatic effect of Salmonella enteritidis bacteriophage PC571 and bacteriophage composition on Salmonella 2411C03; Figure 8 Figure for analysis of average weight difference of chicken at the time of slaughter. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings of the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application. In the present application, unless specified, the adopted equipment and raw materials, etc. can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art, unless specified.

[0044] Example 1 Isolation and purification of Salmonella enteritidis bacteriophage PC571 1.1 Experimental method (1) Resuscitation culture of host bacteria and preparation of proliferation liquid Salmonella bacteria preserved in the laboratory were selected, and a sterile inoculation loop was used to streak the frozen stock on SS culture medium, and the culture was incubated at 37°C in a constant temperature incubator for 18-24h to obtain single colonies; a single colony was picked and inoculated into 5mL of NB broth, and the culture was incubated at 37°C with shaking at 170rpm / min for 16h to obtain fresh Salmonella bacterial liquid.

[0045] (2) Bacteriophage isolation and purification An appropriate amount of chicken manure and other samples from Shandong area were taken in a conical flask, and an appropriate amount of broth medium was added, followed by the addition of an appropriate amount of Salmonella bacterial liquid. The mixture was placed in a 37°C, 170rpm / min shaking incubator for 12h, and then centrifuged at 11000rpm for 5min. The supernatant was filtered through a 0.22μm sterile microporous filter to obtain the bacteriophage proliferation liquid.

[0046] The phage stock solution was diluted by 10 times, and the appropriate gradient phage dilution was mixed with Salmonella one by one at a ratio of 1:1. After incubation at 37°C for 5 min, 200 μL of the mixture was placed in the upper agar (agar concentration of 0.7%), mixed well, and then quickly poured into the lower agar (agar concentration of 1.5%) plate, which was shaken and placed until the medium solidified. After being placed in a 37°C incubator for 4-6 h, a double-layer plate with formed plaques was obtained.

[0047] A single plaque was picked from the double-layer agar medium with formed plaques and placed in 1 mL of NB broth for incubation at 37°C and 170 rpm for about 30 min to obtain a phage extract. The phage extract was mixed with the corresponding plaque-forming Salmonella (hereinafter referred to as host bacteria) proliferation solution at a ratio of 1:1 (incubation at 37°C for 5 min), 200 μL of which was placed in the upper agar, mixed well, and then quickly poured into the lower agar plate, which was shaken and placed until the medium solidified. After being placed in a 37°C incubator for 4-6 h, a double-layer plate with formed plaques was obtained again. A single plaque was picked from the double-layer medium with formed plaques using sterilized tweezers and placed in 1 mL of LB broth for incubation at 37°C and 170 rpm for about 30 min to obtain a phage extract. The above steps were repeated three times to obtain a purified phage extract.

[0048] (3) Determination of phage titer An equal amount of the purified phage extract and the host bacteria proliferation solution were taken and placed in 5 mL of liquid NB medium for incubation at 37°C and 170 rpm until the liquid became clear. The clear liquid was centrifuged at 11,000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm sterile microporous filter to obtain a phage proliferation solution. The titer of the newly isolated phage was determined by the double-layer plate method.

[0049] (4) Determination of Salmonella phage lysis spectrum Fifty Salmonella strains preserved in the laboratory were selected, and the lysis effect of the previously isolated Salmonella phage and the existing Salmonella phages SP8, SPP11 and SP4 on the 50 Salmonella strains was determined by the double-layer agar method and the micro-broth culture method to screen Salmonella phages with strong lysis performance.

[0050] 1.2 Experimental results (1) As shown in Table 1, four new Salmonella phages were screened using the isolated strains according to the above experimental method, which were numbered PC571-PC574 in turn. The four Salmonella phages all formed transparent plaques on the double-layer agar medium plate, with no halo around and a clear edge, and the diameter was about 0.5 mm-1 mm. In addition, the titer of the four phages was measured to be 5.00 x 10 8PFU / mL ~ 1.02 x 10 10 PFU / mL, wherein the phage PC571 has the highest titer.

[0051] (2) The results of the determination of the lysis spectrum of the phage are shown in Table 2. Among the four phages, the phage PC571 for Salmonella enteritidis has the highest lysis rate of 98.00% for 50 strains of Salmonella enteritidis, and PC571 shows more excellent lysis performance for Salmonella enteritidis, with a lysis rate of 100.00%. In summary, the phage PC571 for Salmonella enteritidis with the highest titer, the widest lysis spectrum and the best comprehensive performance is selected as the preferred phage, which is used for the determination of subsequent biological characteristics and application effects.

[0052] In addition, compared with other existing phages, the total lysis rate of the phage PC571 and SP8 for 50 strains of Salmonella enteritidis is consistent (98.00%), which is higher than that of SP8 (97.22%), and also higher than that of SP11 (lysis rate of 94.00%) and SP4 (lysis rate of 96.00%).

[0053] Table 14 Phage information of 50 strains of Salmonella enteritidis

[0054] Table 2 Lysis spectrum of Salmonella enteritidis phage

[0055] Example 2 Biological characteristics of Salmonella enteritidis phage PC571 2.1 Experimental method (1) Electron microscope observation of phage Take 20 μL of the sample of the phage PC571 and drop it on a copper mesh with a carbon coating film, and let it naturally precipitate for 15 min. After slightly absorbing it with filter paper, dye it with 2% (W / V) phosphotungstic acid (PTA) for 1-2 min, and slightly absorb it with filter paper. After drying, observe and take pictures under a transmission electron microscope.

[0056] (2) Determination of temperature stability Take 5.0 x 10 9 PFU / mL of the phage PC571 for Salmonella enteritidis is incubated at 40°C, 50°C, 60°C, 70°C, 80°C and 90°C, with three parallel samples for each temperature. After incubation for 20 min, 40 min and 60 min, respectively, the samples are immediately cooled in an ice bath, and then the phage titer at different temperatures is detected by the double-layer plate method. The temperature is taken as the abscissa, and the logarithmic value of the phage titer is taken as the ordinate, and the thermal stability curve of the phage PC571 is drawn. (3) Determination of acid-base stability Take three test tubes containing 4.5 mL of NB broth medium with different pH values (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) respectively, put the test tubes containing the broth with different pH values into a 37°C water bath for 30 min, and after the temperature of the broth in the test tube reaches a stable state, add 0.5 mL of 5.0 x 10 9 PFU / mL of phage, shake well to mix, and incubate in a 37°C constant temperature water bath for 1 h, 2 h, or 3 h. After the specified time, add an appropriate amount of NaOH or HCl to the mixture containing the phage to adjust the pH to about 7.0, and terminate the effect of the acid and alkali on the phage. Take each termination liquid, and determine the change in the titer of the phage under different pH values and different action times by using the double-layer plate method. Plot a curve of the acid and alkali stability of the phage, taking the lg value of the titer of the phage as the vertical coordinate and the pH value as the horizontal coordinate.

[0057] (4) Determination of one-step growth curve Adjust the titer of the phage PC571 and the concentration of the host bacteria (Salmonella enteritidis) to a multiplicity of infection of 10, take 1 mL of the adjusted phage propagation liquid and 1 mL of the bacterial liquid respectively, mix well (at this time, start timing), incubate at 37°C for 5 min, centrifuge at 13000 rpm for 30 s, discard the supernatant, wash once with 5 mL of NB broth, centrifuge and discard the supernatant. Add 5 mL of 37°C preheated NB broth medium, mix the precipitate well, and quickly place it in a 37°C, 170 rpm shaking culture. From 0 time, take out 200 μL each time, centrifuge at 11000 rpm for 5 min, take the supernatant, and determine the titer of the phage at different time points by using the double-layer plate method. Each gradient is repeated 3 times, and the average value is calculated. Plot a one-step growth curve, taking the infection time as the horizontal coordinate and the titer of the phage in the infection system as the vertical coordinate, to obtain the latent period and the burst period of the phage PC571, and calculate the burst size.

[0058] Burst size = total number of phages at the end of the burst period / total number of bacteria at the beginning of the burst period 2.2 Experimental results and analysis (1) The electron microscope observation results of the phage are shown in Figure 4 It is observed that the phage PC571 has a polyhedral head structure and a non-contractile tail. The head is 60 nm wide and 68 nm long, and the tail is about 180 nm long. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), the morphology of the phage PC571 of the present application meets the characteristics of the Long tail phage family, and belongs to the Long tail phage.

[0059] (2) The determination results of the temperature stability of the phage are shown in Figure 1As shown in the figure, the phage PC571 still retains the original activity after 60 min at 60℃; after 20 min at 80℃, the phage still contains 7.50×10 5 PFU / mL of active phage; after 20 min at 90℃, the phage still retains a certain activity; thus, it can be seen that the Salmonella enteritidis phage PC571 has strong high-temperature resistance.

[0060] (3) The determination results of the acid-base stability of the phage are shown in the figure Figure 2 As shown in the figure, the titer of the phage PC571 can still be maintained at 10 9 PFU / mL in the range of pH 5.0~10.0, and the activity is relatively stable. After 3h at pH 4.0, the titer is maintained at 10 4 PFU / mL; after 1h at pH 11.0, the titer is maintained at 10 6 PFU / mL. Thus, it can be seen that the phage PC571 has strong acid-base stability and can resist a certain range of strong acid and strong base environment.

[0061] (4) The determination results of the one-step growth curve of the phage are shown in the figure Figure 3 As shown in the figure, after the phage PC571 infects the host bacteria, the latent period is about 0~30 min, and within 40~90 min after the phage infects the host bacteria, the number of phage increases sharply, and after 90 min, the titer growth tends to be stable, at this time, the titer can reach 10 9 PFU / mL, and further, it is obtained that the burst period of the phage PC571 is about 50 min, and the burst amount is 97 PFU / cell.

[0062] Example 3 Determination of phage lysis spectrum 3.1 Experimental method: (1) Determination of the lysis spectrum of the phage on 30 strains of resistant bacteria Host bacteria: 3 strains of existing Salmonella phage, Salmonella phage SP4 disclosed in the patent CN108359644B authorized by the company, Salmonella phage SPP11 disclosed in the patent CN111254121B authorized, and Salmonella phage SP8 disclosed in the patent CN116286671A, are used to screen 10 strains of resistant bacteria for each of the 3 strains of phage, a total of 30 strains.

[0063] Method: Prepare fresh phage propagation liquid PC571, SP8, SP4, SPP11 respectively, and detect the lysis spectrum of each phage by double-layer plate method by using the 30 strains of Salmonella enteritidis resistant bacteria, and compare the lysis performance of the 4 strains of Salmonella phage.

[0064] (2) Determination of the synergistic antibacterial activity of the bacteriophage composition against three clinically isolated Salmonella enteritidis strains Host bacteria: Three strains of Salmonella enteritidis, namely 2411C01, 2411C02 and 2411C03, were isolated from a chicken farm in Yantai, Shandong.

[0065] Methods: Salmonella phage PC571 was mixed with phage SP4, phage SPP11 and phage SP8 in a 1:1 ratio to form phage compositions. The antibacterial effect of each phage composition proliferation solution on three clinically isolated Salmonella enteritidis strains was detected by the micro-broth method. The synergistic effect of Salmonella phage PC571 with SP8, SP4 and SPP11 was compared and analyzed.

[0066] 3.2 Experimental Results (1) Comparative analysis of the lysis effect of 4 Salmonella phage strains on 30 Salmonella resistant strains As shown in Table 3, Salmonella enteritidis phage PC571 could lyse 27 of the 30 resistant strains, with a total lysis rate of 90.00%. The lysis rates of Salmonella phages SP8, SP4, and SPP11 against the 30 resistant strains were 63.33%, 60.00%, and 56.67%, respectively.

[0067] It is evident that, compared to the three existing Salmonella phages SP8, SP4, and SPP11, the Salmonella enteritidis phage PC571 exhibits a broader lysis spectrum and better lysis performance against the aforementioned 30 resistant strains, reflecting that phage PC571 has a strong antibacterial effect against Salmonella phage-resistant bacteria.

[0068] Table 3. Lysis patterns of 30 Salmonella-resistant strains against 4 bacteriophages

[0069] (2) Analysis of the antibacterial effect of the combination of bacteriophage PC571 and three bacteriophage strains (SP8, SP4, and SPP11) from Figure 5 The results showed that the inhibition durations of the PC571 and SP8 combination, the PC571 and SP4 combination, the PC571 and SPP11 combination, and PC571 against Salmonella 2411C01 were 12h, 8h, 8h, and 6h, respectively. It can be seen that the three phage combinations have a more significant synergistic antibacterial effect than the single phage PC571, and the PC571 and SP8 combination has the longest inhibition time against 2411C01 and the best antibacterial effect.

[0070] from Figure 6The results show that the bacteriostatic time of PC571 and SP8 combination, PC571 and SP4 combination, PC571 and SPP11 combination and PC571 on Salmonella 2411C02 is 5h, 3h, 3h and 3h respectively; it can be seen that the three bacteriophage combinations have certain bacteriostatic effect on Salmonella 2411C02, but only the combination of PC571 and SP8 has synergistic bacteriostatic effect.

[0071] From Figure 7 The results show that the bacteriostatic time of PC571 and SP8 combination, PC571 and SP4 combination, PC571 and SPP11 combination and PC571 on Salmonella 2411C03 is 14h, 12h, 11h and 11h respectively; it can be seen that the two bacteriophage combinations (PC571 and SP8 combination, PC571 and SP4 combination) have synergistic bacteriostatic effect on Salmonella 2411C03, and the combination of PC571 and SP8 has better synergistic bacteriostatic effect on 2411C03.

[0072] In summary, the combination of bacteriophage PC571 and SP8 has longer bacteriostatic time than the combination of PC571 and SP4, PC571 and SPP11, and the OD value is the lowest, which indicates that the combination of PC571 and SP8 has better synergistic bacteriostatic effect.

[0073] Example 4 Whole genome analysis of Salmonella bacteriophage PC571 The genome of bacteriophage PC571 was extracted for whole genome sequencing and sequence analysis, and the results are as follows: (1) The full-length genome of PC571 is 42438 kb, and the G+C content is 49.50%, and the base contents of A, T, G and C are 25.00%, 25.50%, 24.90% and 24.60%, respectively. The RAST online annotation results of the whole genome show that the genome contains 63 open reading frames (ORFs), and the average OFR length is 674 bp, of which 26 ORFs are known functions and 41 ORFs are hypothetical protein expression sequences. Among the 67 open reading frames (ORFs), 15 kinds of structural proteins are found, mainly including structural and packaging proteins of phage (head protein, tail protein, tape protein, tail fiber protein, neck protein, capsid protein, large subunit of terminal enzyme and small subunit of terminal enzyme, etc.), phage lysis-related proteins (lysozyme, perforin), DNA replication and modification-related proteins (restriction endonuclease, DNA binding protein, DNA polymerase, HNH endonuclease and recombination protein, etc.). Among the 63 ORFs, 56 start codons are ATG, 4 start codons are GTG, 2 start codons are TTG, and 1 start codon is AAG. The software tRNAscan-SE analysis shows that the genome does not contain tRNA genes. The online tool CGE server analysis shows that the genome does not contain drug resistance genes and virulence genes. The PHASTEST analysis shows that the genome does not contain lysogen-related genes.

[0074] (2) In the genome of phage PC571, the DNA polymerase protein gene sequence is seen in sequence 1 in the sequence listing, the tail fiber protein gene sequence related to phage host recognition is seen in sequence 3 in the sequence listing, the highly conserved terminase large subunit protein gene sequence is seen in sequence 5 in the sequence listing, and the endolysin protein gene sequence related to lysis ability is seen in sequence 7 in the sequence listing. The relevant information is shown in Table 4 below.

[0075] Table 4 Gene sequence information table

[0076] Example 5 Safety of Salmonella phage PC571 to chicks 5.1 Experimental method Select 90 healthy chicks, and randomly divide them into 3 groups, 30 chicks in each group, respectively, for gavage group, drinking water group, injection group, control group, each chick is given phage PC571, the dose is 5×10 9 PFU / chick, respectively, placed in an isolator for feeding; the chicks are fed to 28 days, and the body weight, mental state, and autopsy are observed, and the heart, liver, spleen, lung, kidney, brain, and intestinal lesions are observed.

[0077] 5.2 Experimental results and analysis There was no significant difference in weight gain between the gavage group, the drinking water group, the injection group and the control group. The chicks were in good condition and ate normally during the feeding process. No disease or toxicity symptoms were observed. The organs and intestines were normal after dissection. This indicates that the use of Salmonella phage according to this method has high safety.

[0078] Example 6 Application of Salmonella phage PC571 in the treatment of Salmonella disease in chicks 6.1 Experimental method 120 healthy chicks were selected and randomly divided into 4 groups, 30 in each group, namely the phage gavage group, the phage feed mixing group, the challenge group and the blank group. They were placed in isolators for feeding.

[0079] Specifically, the phage gavage group, the phage feed mixing group and the challenge group orally administered 5x10 6 CFU of Salmonella enteritidis bacteria solution, and the blank group orally administered the same amount of sterile normal saline. The phage gavage group was administered 0.2 mL (5x10 8 PFU per bird) of phage PC571 proliferation solution 2 hours after the chicks were challenged, and the phage feed mixing group mixed 5x10 8 PFU per bird) of Salmonella phage PC571 evenly into the feed (20 mL per ton of feed) 2 hours after the chicks were challenged. The phage in the gavage group and the feed mixing group was used for 3 consecutive days, and then stopped for 5 days as a course of treatment, and 3 courses were used. The challenge group and the blank group did not use phage.

[0080] The chicks were fed until 28 days old, and the fecal Salmonella positive rate and mortality rate were collected. After the experiment, all the chickens were dissected to observe the Salmonella infection in the liver, and the positive rate of Salmonella infection in the liver was calculated.

[0081] 6.2 Experimental results As shown in Table 5, the mortality rates of the phage gavage group and the phage feed mixing group were 6.67% and 10.00%, respectively, which were much lower than the mortality rate of 66.67% of the challenge group. At the same time, compared with the challenge group, the phage gavage group and the phage feed mixing group reduced the Salmonella positive rate in the feces of chicks by 83.33% and 80.00%, respectively, and reduced the Salmonella positive rate in the liver by 86.67% and 83.33%, respectively. Obviously, these two groups can significantly reduce Salmonella infection. These results show that whether gavage or feed mixing is used to administer phage PC571, it can effectively treat chicks infected with Salmonella.

[0082] Table 5 Statistical table of phage treatment of pigeon Salmonella infection

[0083] Example 7 Application of Salmonella phage PC571 in prevention of Salmonella disease in chicks 7.1 Experimental method A chicken farm in Shandong was selected and divided into four groups, each group containing 1000 chicks, namely the phage SP8 group, the phage PC571 group, the combination of phage PC571 and SP8 group, and a blank group. According to 10 mL / ton, the Salmonella phage was evenly mixed into the drinking water (5 x 10 8 PFU / feather), and the phage was used for 3 days and stopped for 5 days as a course of treatment. Four courses of treatment were used, and the control group did not use phage. The chicks were raised to market, and during this period, the normal medication program was followed according to the other drugs used in the field.

[0084] The mortality rate of the chicks, the detection rate of Salmonella in the liver of the dead chicks, the detection rate of Salmonella resistant bacteria in the feces, and the body weight of the marketable chicks were statistically analyzed.

[0085] 7.2 Experimental results The results are shown in Table 6. Compared with the blank group, the mortality rate of the chicks in the three phage experimental groups was reduced, among which the mortality rate of the chicks in the combination of phage PC571 and SP8 group was the lowest, which was decreased by 17.2% compared with the blank group, and the detection rate of Salmonella in the dead chicks was also the lowest, which was decreased by 50.71% compared with the blank group. The detection rate of Salmonella resistant bacteria in the feces of this group was only 2.20%, which was lower than that of the phage SP8 group (13.70%), the phage PC571 group (5.10%) and the blank group (27.00%).

[0086] From Figure 8 The results showed that the average body weight of the chicks in the three phage groups was higher than that in the blank group, and the difference was extremely significant. The results showed that the use of phage PC571 or the combination of the phage could effectively prevent and control Salmonella infection in chicks, increase the marketable weight of chicks, and reduce the detection rate of clinical Salmonella resistant bacteria. Among them, the combination of phage PC571 and SP8 had better effect.

[0087] Table 6 Data statistics of phage prevention and control of Salmonella infection in chicks

[0088] Example 8 Lysis test of phage PC571 on non-host bacteria 1. Experimental method Ten strains of Escherichia coli, 10 strains of Staphylococcus, 10 strains of Clostridium welchii, and 10 strains of duck Riemerella anatipestifer non-host bacteria were selected for lysis experiment according to the method of double-layer plate lysis spectrum in Example 3.

[0089] 2. Experimental results and analysis PC571 can not form plaque with 10 strains of E. coli, 10 strains of Staphylococcus, 10 strains of Clostridium welchii, 10 strains of duck Riemerella anatipestifer, and can not lyse non-host bacteria. This shows that the tested bacteriophage PC571 has strong host specificity and no damage to microbial community.

[0090] It can be understood that, for those skilled in the art, equivalent replacements or changes can be made according to the technical solutions of the present application and the concept of the present application, and all these changes or replacements shall belong to the protection scope of the appended claims of the present application.

Claims

1. A strain of Salmonella enteritidis phage PC571, characterized in that, Its accession number is CGMCC No.46168.

2. A bacteriophage composition, characterized in that, Includes the Salmonella enteritidis phage PC571 as described in claim 1.

3. The phage composition according to claim 1, characterized in that, Also includes: One or more of the following: bacteriophage SP8 with accession number CGMCC No. 45256, bacteriophage SP4 with accession number CGMCC No. 14332, and bacteriophage SPP11 with accession number CGMCC No. 18868.

4. The phage composition according to claim 3, characterized in that, include: The Salmonella enteritidis phage PC571 as described in claim 1 and the phage SP8 with accession number CGMCC No. 45256.

5. The use of the Salmonella enteritidis phage PC571 as described in claim 1, and the phage composition as described in any one of claims 2 to 4, in the preparation of pharmaceutical preparations, environmental disinfectants, or test kits for preventing and treating poultry diseases caused by Salmonella phage infection.

6. A bacteriophage drug, characterized in that, Its active ingredients include the Salmonella enteritidis phage PC571 as described in claim 1 or the phage composition as described in any one of claims 2 to 4.

7. An antibacterial agent, characterized in that, Its active ingredients include the Salmonella enteritidis phage PC571 as described in claim 1 or the phage composition as described in any one of claims 2 to 4.

8. An environmental disinfectant, characterized in that, Its active ingredients include the Salmonella enteritidis phage PC571 as described in claim 1 or the phage composition as described in any one of claims 2 to 4.

9. The application of the environmental disinfectant as described in claim 8 in the disinfection of poultry farming environments or poultry slaughtering environments, characterized in that, The environmental disinfectant can be applied to feed, water, and aquaculture environments, including cages, drinkers, feed troughs, manure, and bedding; the poultry slaughtering environment includes slaughterhouses, meat processing workshops, and processing equipment.

10. A test kit, characterized in that, Includes the Salmonella enteritidis bacteriophage as described in claim 1 or the bacteriophage composition as described in claim 2.

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